Accepted Manuscript Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold Martina Hrast, Marko Anderluh, Damijan Knez, Christopher P. Randall, Hélène Barreteau, Alex J. O’Neill, Didier Blanot, Stanislav Gobec PII:
S0223-5234(13)00773-3
DOI:
10.1016/j.ejmech.2013.11.031
Reference:
EJMECH 6577
To appear in:
European Journal of Medicinal Chemistry
Received Date: 11 July 2013 Revised Date:
15 November 2013
Accepted Date: 24 November 2013
Please cite this article as: M. Hrast, M. Anderluh, D. Knez, C.P. Randall, H. Barreteau, A.J. O’Neill, D. Blanot, S. Gobec, Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold, European Journal of Medicinal Chemistry (2014), doi: 10.1016/ j.ejmech.2013.11.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold
Barreteau3, Alex J. O’Neill2, Didier Blanot3 and Stanislav Gobec1*
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Martina Hrast1, Marko Anderluh1, Damijan Knez1, Christopher P. Randall2, Hélène
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
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School of Molecular and Cellular Biology and Antimicrobial Research Centre, University of
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1
3
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Leeds, Leeds LS 9JT, U.K.
Univ Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS,
91405 Orsay, France
University of Ljubljana
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*Corresponding author: Stanislav Gobec
Faculty of Pharmacy, Aškerčeva 7
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1000 Ljubljana, Slovenia
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Tel: +386-1-4769500
Fax: +386-1-4258031
E-mail: [email protected]
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ABSTRACT MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug
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discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best
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inhibitors displayed promising antibacterial activities against selected gram-positive and gram-negative strains. These results represent an important step towards the development of
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new antibacterial agents targeting peptidoglycan biosynthesis.
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Abbreviations: DAP,
2,6-diaminopimelic
acid;
GlcNAc,
N-acetyl
glucosamine;
MurC,
UDP-N-
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acetylmuramate:L-Ala ligase; MurD, UDP-N-acetylmuramoyl-L-Ala:D-Glu ligase; MurE, UDP-N-acetylmuramoyl-L-Ala-D-Glu:meso-DAP ligase; MurF, UDP-N-acetylmuramoyl-LAla-γ-D-Glu-meso-DAP (or
L-Lys):D-Ala-D-Ala
ligase; MurNAc, N-acetyl muramic acid;
UDP, uridine 5’-diphosphate; UMtri-L-Lys, UDP-N-acetylmuramoyl-L-Ala-D-Glu-L-Lys;
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UMtri-mDAP, UDP-N-acetylmuramoyl-L-Ala-γ-D-Glu-meso-DAP.
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1. INTRODUCTION The increasing emergence of pathogenic bacteria resistant to all known antibiotics has created an urgent need for development of new antibacterial agents [1-3]. Cell wall biosynthesis in
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bacteria is well characterised and thus represents an attractive target for the development of novel antibacterial drugs [4-6]. The biosynthetic pathway of peptidoglycan encompasses cytoplasmic, membrane and extracellular stages [4, 6, 7].
Mur ligases (MurC-F) are cytoplasmic ATP-dependent enzymes which catalyse the sequential
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addition of L-Ala, D-Glu and meso-DAP in Gram-negative or L-Lys in Gram-positive bacteria, and the dipeptide D-Ala-D-Ala to UDP-MurNAc, to form UDP-MurNAc-pentapeptide [7, 8].
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All Mur ligases have similar mechanisms of action, and share the same three-domain topology [9]. They are essential for the survival of bacteria and lack human counterparts; consequently, they represent interesting targets for the discovery of new antibacterials with selective toxicity [8]. There are only a few known classes of MurF inhibitors. The first classes were pseudo-tripeptide and pseudo-tetrapeptide aminoalkylphosphinic acids [10], followed by sulphonamides developed by Abbott Laboratories [11, 12]. Thiazolylaminopyrimidines [13],
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8-hydroxyquinolines [14] and diarylquinolines [15] were discovered by Johnson & Johnson. Recently, a triazine derivative was identified through structure-based virtual screening [16]. Additionally, ligand-based virtual screening was successfully employed by our group and one new compound with micromolar inhibitory activities against S. pneumoniae MurF (MurFSp)
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and E. coli MurF (MurFEc) was identified [17]. In our previous research, we also reported a series of cyanothiophene-based inhibitors of MurF enzymes from different pathogenic bacteria, where systematic structural modifications
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of the lead compound resulted in new potent nanomolar inhibitors of MurFSp and micromolar inhibitors of MurFEc and S. aureus MurF (MurFSa). The crystal structures of MurFSp in complex with two new inhibitors (PDB codes 3zm5 and 3zm6) revealed the binding modes of compounds, which allowed us to undertake structure-based design of novel, improved analogues with optimized physicochemical properties. Although some of the compounds from the first series showed good MurF inhibitory potency and moderate antibacterial activity against S. pneumoniae, the solubility of the majority of assayed compounds in water was low. We postulated that the large planar lipophilic portion of the molecules associated with rather
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high molecular mass led to their high tendency for precipitation, making them inadequate drug candidates [18]. In order to present further insight into the structure-activity relationship of cyanothiophenebased MurF inhibitors, we report the design, synthesis and biological evaluation of a new
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series of inhibitors. Different structural modifications of the parent compounds resulted in a focused library of 37 new inhibitors, providing low micromolar inhibitors of MurF from E. coli and S. pneumoniae.
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2. RESULTS AND DISCUSSION
2.1. Design. According to the results from Abbott Laboratories [11, 12] and our previous research [18], 2-aminothiophene-3-carbonitrile and 5-sulfamoyl-2-chlorobenzoic acid linked
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together via an amide bond, are essential for good MurF inhibitory potency (i.e., compound I, Figure 1.). Additionally, 3D similarity search revealed micromolar inhibitor II, where the cyclohexene ring in compound I was replaced by cyclopentene ring, sulfamoyl substituent was replaced by the 1-pyrazolylmethyloxy moiety and the chloro substituents of benzoic acid part were replaced by hydrogens [17].
Due to promising potencies of compounds I and II, the structural features of both inhibitors
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were combined. Overlapping both compounds yielded the key scaffold (in red, Fig. 1.), which was used as a starting point in the design of novel MurF inhibitors. The main problem observed with compounds I and II was their low aqueous solubility due to quite high lipophilicity (ClogP(I) = 2.82, and ClogP(II) = 3.40). It is well known that antibacterial
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agents have slightly different physiochemical properties than the rest of the drugs [19, 20]. Average molecular weights are usually higher for antibacterials, with a defined cut-off at 600
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Da. In addition, the lipophilicity of the Gram-positive and especially of Gram-negative antibacterials compared to “normal” drugs is reduced. These unique properties were taken into account during the design of the second generation of MurF inhibitors, where more polar compounds were targeted. Two steps were performed in designing second-generation MurF inhibitors, as depicted in Figure 1. In the first step, various alkyl or aryl substituents bearing mostly polar functional groups (R1) were attached to the main scaffold in order to examine the inhibitory activities of compounds with different replacements of the 4-chloropyrazole moiety. In the second step, various phenyl and benzyl substituents were appended to position 6 (R2 substituent) of the
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4,5,6,7-tetrahydrobenzo[b]thiophene
and
4,5,6,7-tetrahydrothieno[2,3-c]pyridine,
respectively. It is known from our previous study that extension of these ring systems significantly improves the potency of compounds against S. pneumoniae MurF and gains the potency against other MurF orthologes [18]. Although this extension increases lipophilicity,
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the presence of the protonated amine or guanidine moiety outweighs the contribution of these lipophilic residues so that the overall ClogP does not exceed the value of 2.8. (Table S1)
To predict the binding affinity of the designed compounds to MurF, a docking study was performed using the crystal structure of MurF enzyme from S. pneumoniae (PDB code: 3zm5)
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and OEDocking software (Release 3.0.1, OpenEye Scientific Software, Inc.). This revealed the compounds with simple amine or guanidine at position R1 as the ones with the highest predicted affinities (Fig. 2a-d). Furthermore, the replacement of H with Cl on the benzoic acid
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moiety was proposed to be beneficial for potent inhibition of MurF.
As all predicted binding poses were plausible and with good docking scores, we decided to incorporate both amino and guanidine groups into the structures of potential inhibitors. The design presented herein proved to be successful, as majority of amines/guanidines demonstrated low micromolar inhibiton of MurF from E. coli and S. pneumoniae.
chlorosulfonated
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2.2. Chemistry. Compound 4 was synthesized as outlined in Scheme 1. In the first step, the 2,4-dichlorobenzoic
acid
(1)
reacted
with
the
tert-butyl
(2-
aminoethyl)carbamate to yield compound 2. Then, the free acid was coupled with 2aminothiophene through a two-stage process, including the conversion of the acid into acyl
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chloride in the presence of oxalyl chloride and catalytic amount of DMF, followed by the coupling in the presence of pyridine as a base, to obtain 3. Compound 4 was prepared by the cleavage of the Boc protective group in the presence of CF3COOH, and was subsequently
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transformed into hydrochloride salt with HCl in ethyl acetate. The synthesis of target compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a and 27b is presented in Scheme 2. First, the hydroxy group of compounds 5 and 6 was acetylated with acetic anhydride in pyridine. Next, compounds 9 and 10 were obtained using the same synthetic strategy as described for 3. The acetoxy protective groups of compounds 9 and 10 were cleaved in the presence of K2CO3 in methanol. The diverse alkyl or aryl substituents were attached to the hydroxyl group of compounds 11 and 12 with two different approaches. In the first method, compounds 14, 17-19, 20a, 20b, 21a and 21b were obtained using K2CO3 or Cs2CO3 and different benzyl or alkyl halides. An alternative method (the Mitsunobu reaction 6
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using PPh3, diisopropyl azodicarboxylate (DIAD) and the corresponding alcohol) was employed to give compounds 15 and 16. As (1H-pyrazol-1-yl)methanol (13) was not commercially available, we prepared it by the already reported procedure [21]. Compounds 22a, 22b, 23a and 23b were obtained after the cleavage of the Boc protective group using
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CF3COOH. Amine groups of compounds 22a, 23a and 23b were converted into di-Boc protected guanidines in the presence of mercuric chloride and triethylamine. Finally, the cleavage of both Boc protective groups with CF3COOH, followed by direct transformation to hydrochloride salts with HCl in ethyl acetate, gave target compounds 26a, 27a and 27b.
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The synthesis of target compounds 33-36, 37a-f, 38a-f, 39c, 42a-e and 43 is outlined in Scheme 3. 2-Chloro-5-hydroxybenzoic (6) acid was first protected as methyl ester and then alkylated using tert-butyl (2-bromoethyl)carbamate and K2CO3 to yield 28. Compound 31
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was obtained after alkaline hydrolysis of the methyl ester with aqueous sodium hydroxide, followed by cleavage of Boc protective group using CF3COOH. The amino group was further protected as trifluoroacetamide with trifluoroacetic anhydride and pyridine as a base to give 32. Compounds 33a-b were obtained from 32 and the corresponding 2-aminothiophenes using the same procedure as described for compound 3. Compound 34 was obtained after the cleavage of the Boc group of 33a with the CF3COOH. Final compounds 35 and 36 were
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obtained by alkaline hydrolysis of the trifluoroacetamide group of 33b and 34, respectively. The variously substituted benzyl fragments were attached to compound 34 via two different strategies. The first method was a nucleophilic substitution, where K2CO3 and different benzyl bromides were used to yield compounds 37a-c; in the second method, compounds
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37d-f were synthesized by sodium triacetoxyborohydride-promoted reductive amination of 34 with various benzaldehydes. In the next step, the removal of the trifluoroacetamide group of 37a-f was achieved through alkaline hydrolysis, which led to target compounds 38a-f. Finally,
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target compounds 42a-e and 43 were obtained using the same strategy as described for 26a. 2.3. Inhibition of MurF. All target compounds were assayed for inhibition of MurF from E. coli and S. pneumoniae, using the Malachite green assay [22], which detects the orthophosphate generated during the enzymatic reaction. To avoid possible non-specific inhibition, all of the compounds were tested in the presence of detergent (0.005% Triton X114) [23]. Results are presented as residual activities (RAs) of the respective enzyme in the presence of 250 or 100 µM of each compound. For the most active compounds, IC50 values were also determined.
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First, a small series of compounds (Table 1, compounds 14-19) was synthesized where the 4chloropyrazole moiety was replaced with different aryl and alkyl fragments. None of these compounds significantly inhibited MurF. However, compounds with aminoethyl (23a) and aminopropyl substituents (23b) showed modest inhibitory activities against both MurF
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enzymes. As expected, the introduction of chlorine ortho to the amide bond was essential for the activity while the length of alkyl chain had little influence on the potency. Furthermore, compounds with the guanidino moiety (26a, 27a and 27b) were prepared and their potencies were 2- to 3-fold higher than potencies for the corresponding amine.
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If we compare the compound I with the compounds 23a and 27a, we can notice that the morpholinosulfonyl fragment and chlorine next to sulfonamide linker are rather important for good inhibition of MurFSp. This can be seen also in the co-crystal structure, where one of the
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sulfonyl oxygens of compound I makes two hydrogen bonds with the protein and the chlorine forms hydrophobic interactions [18]. These interactions are not present in our new inhibitors, resulting in their higher IC50 values against MurFSp. However, this interaction pattern is not necessary for inhibition of MurFEc, as compound I showed no inhibition of this ortholog. Furthermore, to examine the importance of the sulfonamido group, compound 4 was synthesized. By the comparison of compounds 4 and 23a, we can see that the inhibition is in
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the same range as far as MurFSp is concerned (4, IC50 = 219 µM; 23a, IC50 = 170 µM). However, in the case of MurFEc compound 4 was 2-fold less potent than 23b, which makes sulfonamido group less favorable for inhibition of this enzyme. In the next step, compounds with 4-hydroxyphenyl and different substituted benzyl
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substituents attached to position 6 of the 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile or 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile moiety were synthesized (Table 2), as it is known that substitution of this ring leads to greater potency [11, 12, 18]. First,
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compounds with the 4-hydroxyphenyl fragment appended to the saturated ring (33b and 35) were prepared. The protected compound (33b) showed no inhibition against MurF while the compound with free amino and hydroxyl groups (35) inhibited both MurF enzymes in the low micromolar range (IC50 = 58 µM and 81 µM for MurFSp and MurFEc, respectively). Compounds with different benzyl substituents and trifluoracetamide group (37a-f) showed modest inhibitory activities against MurF. The most potent inhibitor of MurFSp and MurFEc within this set was 37f with the p-tetrazole group attached to the benzyl fragment, with IC50 values of 103 and 152 µM, respectively. Furthermore, inhibitors with a free amino group (38a-f and 49c) were synthesized and most of them showed improved activity against both
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MurF enzymes. Exceptions were 38c and 38f with carboxylic acid and tetrazole on para position, respectively, which showed significantly lower inhibition of MurFSp activity. The introduction of substituents to either para or meta position on the benzyl ring led to 2-fold lower potency against MurFSp compared to 38a (IC50 = 51 µM), while the p-substituents on
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the benzyl ring did not influence the activity against MurFEc within this series. Finally, compounds with the guanidine substituent (42a-e and 43) were prepared. The most potent inhibitor within this set was compound 43, with the p-hydroxyphenyl group attached to the saturated ring: IC50 values were 20 and 25 µM against MurFSp and MurFEc, respectively. Compounds 42a and 42d had similar inhibitory activities against both MurF orthologs as the
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corresponding amine precursors, while inhibitors 42b, 42c and 42e showed about 2-fold better activity against MurFSp than their corresponding amines (38b, 38c and 38e, respectively).
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Unfortunately, the introduction of the guanidine moiety does not improve the activity of inhibitors (42a-e) against MurFEc compared to their respective amine precursors (38a-e and 39c).
2.4. Antibacterial activity. For antibacterial evaluation of selected compounds, typical representatives of Gram-positive (S. aureus) and Gram-negative bacteria (E. coli) were chosen (Table 3). In the case of E. coli, compounds were further evaluated against efflux-
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deficient strains (E. coli SM1411 and ES100), and those in which the outer membrane had been artificially permeabilized with polymyxin B (PMBN). Compounds 23a-b, 26a and 27ab showed modest antibacterial activities against S. aureus and E. coli, which increased against the latter species upon deletion of the AcrAB-TolC efflux transporter. Compounds 33a, 34, 36
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and 37a-f showed no activity against E. coli and S. aureus; this was expected since none of these compounds showed potent inhibition of MurFEc and MurFSp. Compound 35 was inactive
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against S. aureus and E. coli, although antibacterial activity was observed against PMBNpermeabilized and/or efflux-deficient strains of E. coli. Compounds 38a-f and 39c showed modest antibacterial activities against both bacterial species. 38c showed limited inhibition of both MurF enzymes (IC50 ~ 500 µM), and no antibacterial activity was observed. On the other hand, compounds 38b, 38d, 38e and 39c demonstrated antibacterial activities against S. aureus and E. coli strains, and inhibited isolated enzymes more potently (IC50 around or below 100 µM). The most potent inhibitor of MurF within this series was 38a, which has 2-fold lower MIC values against all strains compared to its analogues. Finally, compounds with the guanidine moiety 42a-e and 43 showed promising antibacterial activities against S. aureus with MICs ranging between 4 and 32 µg/mL. The latter compounds also demonstrated a 9
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modest antibacterial activity against E. coli. All compounds with antibacterial activity against E. coli exhibited increased activity against efflux-deficient strains and strains with increased membrane permeability. Additional studies were performed to determine whether the antibacterial activities of the
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compounds are the consequence of peptidoglycan biosynthesis inhibition or non-specific effect. The BacLightTM assay was used to assess the membrane integrity of S. aureus SH1000 at 4 × MIC values of inhibitors [24]. Unfortunately, all of the tested compounds (38b, 42a, 42c, 42e and 43) significantly decreased the membrane integrity thus showing non-specific
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3. CONCLUSION
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We have designed, synthesized and evaluated a second generation of 37 new MurF inhibitors with cyanothiophene scaffold. The major improvement of the present compounds over the first-generation inhibitors is their reduced lipophilicity and increased solubility, with conserved enzyme inhibitory potency in micromolar range. The most potent inhibitors have well balanced inhibitory properties against both MurF from S. pneumonie and MurF from E. coli (the best inhibitor 42 inhibits MurFSp and MurFEc with IC50 values of 20 µM and 25 µM,
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respectively). Furthermore, the most potent compounds within this series demonstrate antibacterial activities. As these compounds achieve part of their antimicrobial action by damaging the bacterial cytoplasmic membrane, they are unlikely to be developed as antibacterial drug candidates until this nonspecific effect is diminished, without
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compromising their inhibitory effect on MurF.
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4. EXPERIMENTAL SECTION 4.1. Inhibition assay. The inhibition of MurF ligases was determined using the Malachite green assay with slight modifications. The mixture with final volume of 50 µL contained: S. pneumoniae MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114, 100 µM D-Ala-D-Ala,
50 µM UMtri-L-Lys, 250 µM ATP, purified MurFSp [17], and 250 or 100 µM of
each tested compound dissolved in DMSO. E. coli MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114, 600 µM D-AlaD-Ala,
100 µM UMtri-mDAP, 500 µM ATP, purified MurFEc [25], and 250 or 100 µM of
each tested compound dissolved in DMSO.
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In both cases, the final concentration of DMSO was 5% (v/v). After incubation for 15 min at 37 °C, the enzyme reaction was stopped by adding Biomol®reagent and the absorbance was measured at 650 nm. All of the experiments were run in duplicate. Residual activities (RAs) were calculated with respect to similar assays without the tested compounds and with 5%
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DMSO. The IC50 values, which were determined by measuring the residual activities at seven different compound concentrations, represented the concentration for which the RA was 50%.
4.2. Microbiological evaluation. Minimum inhibitory concentrations (MICs) for selected compounds were determined by broth microdilution in Mueller-Hinton broth (MHB), against
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S. aureus SH1000 [26], and E. coli strains 1411, SM1411 (acrAB derivative of 1411) [27], AB734 and ES100 (tolC derivative of AB734) [28], according to CLSI guidelines [29]. To
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assess the impact of outer-membrane permeability on antimicrobial activity against E. coli, MICs were also determined against strains 1411 and AB734 in the presence of 4 µg/mL polymyxin B nonapeptide (PMBN) [30]. The BacLightTM assay was used to measure membrane damage in S. aureus SH1000 induced by compounds at 4 × MIC, compared to a drug free control [24].
4.3. Chemistry. All of the chemicals used were obtained from commercial sources (Acros,
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Sigma-Aldrich, Alfa Aeser, Apollo Scientific, Fluka and Merck), and were used without further purification. Solvents were used without purification or drying, unless otherwise stated. Reactions were monitored using analytical thin-layer chromatography plates (Merck, silica gel 60 F254, 0.25 mm), and compounds were visualized with ultraviolet light and
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ninhydrin or ferric chloride. Silica gel grade 60 (particle size 0.040–0.063 mm, Merck, Germany) was used for flash column chromatography. 1H and
13
C NMR spectra were
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recorded on a Bruker AVANCE III 400 MHz spectrometer in acetone-d6, CDCl3, DMSO-d6, CD3OD or pyridine-d5, with TMS as the internal standard. Mass spectra were obtained with a VG-Analytical Autospec Q mass spectrometer (Centre for Mass Spectrometry, Institute Jožef Stefan, Ljubljana). Infrared spectra were recorded on a Perkin-Elmer FTIR 1600 spectrometer. Melting points were determined using a Reichert hot-stage microscope and are uncorrected. Microanalyses were performed on a 240 C Perkin Elmer elemental analyzer. Analyses indicated by the symbols of the elements were within 0.4% of the theoretical values. HPLC analyses were performed on an Agilent Technologies HP 1100 instrument with a G1365B UV-VIS detector (220 and 254 nm), using a Luna C18 column (4.6 × 250 mm) at a
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flow rate of 1 mL/min. The eluent was a mixture of 0.1% CF3COOH in water (A) and acetonitrile (B). The gradient was 10% to 90% B in 19 min. 4.3.1. 5-(N-(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)-2,4-dichlorobenzoic acid (2). To a solution of tert-butyl (2-aminoethyl)carbamate (0.450 g, 2.81 mmol) in DCM (15 mL), Et3N
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(0.59 mL, 4.21 mmol) was added. The reaction mixture was cooled on an ice bath and 1 (0.813 g, 2.81 mmol) was added portion wise. The reaction mixture was stirred for 16 h, diluted with 30 mL of DCM, and washed with 1 M HCl (2 × 40 mL) and brine (1 × 50 mL), and dried with Na2SO4. The solvent was evaporated under reduced pressure and the crude
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product was purified by flash chromatography.
Yield = 15%; white needles, mp = 192-193 °C; 1H NMR (400 MHz, CDCl3): δ 1.34 (s, 9H, 3
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× CH3), 2.90 (q, J = 5.6 Hz, 2H, SO2-NH-CH2), 2.96 (q, J = 6.4 Hz,2H, CO-NH-CH2), 6.72 (t, J = 5.6 Hz, 1H, SO2-NH), 8.01 (s, 1H, Ar-H), 8.17 (t, J = 6.4 Hz, 1H, O-CO-NH), 8.34 (s, 1H, Ar-H) ppm. 13C NMR (100 MHz, CD3OD): δ 28.77, 28.83, 40.74, 41.08, 41.23, 43.57, 80.26, 80.35, 132.19, 133.93, 134.67, 136.52, 137.01, 138.40, 158.46, 158.60, 167.88 ppm. ESI HRMS calcd. for [M+(H)]+ 555.1447, found 555.1448. IR (KBr) νmax: 3377, 2980, 2935, 1694, 1586, 1526, 1454, 1385, 1367, 1336, 1277, 1253, 1163, 1086 cm-1. Tert-butyl
(2-(2,4-dichloro-5-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
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4.3.2.
yl)carbamoyl) phenylsulfonamido)ethyl)carbamate (3). To a suspension of 2 (0.305 g, 0.74 mmol) in anhydrous DCM (15 mL), catalytic amounts of DMF and (COCl)2 (0.190 mL, 2.21 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at room temperature. The
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solvent was evaporated to dryness and the residue was dissolved in DCM (20 mL), the mixture of 2-aminothiophene (0.125 g, 0.70 mmol) and pyridine (0.180 mL, 2.21 mmol) in DCM (15 mL) was added drop-wise, and the reaction was stirred overnight at room
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temperature. The reaction mixture was washed with 1 M HCl (2 × 40 mL), saturated aqueous NaHCO3 (1 × 40 mL) and brine (1 × 40 mL), and dried (Na2SO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography. Yield = 24%; white crystals, mp = 196–198 °C; 1H NMR (400 MHz, CDCl3): δ 1.43 (s, 9H, 3 × CH3), 1.85–1.90 (m, 4H, CH2-CH2-CH2-CH2), 2.63 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 2.70 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 3.06 (q, J = 4.8 Hz, 2H, SO2-NH-CH2), 3.23 (q, J = 4.8 Hz, 2H, SO2-NH-CH2-CH2), 4.95 (t, J = 4.8 Hz, 1H, SO2-NH), 6.21 (t, J = 4.8 Hz, 1H, SO2-NH-CH2-CH2-NH), 7.61 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 10.04 (bs, 1H, NH-CO-Ar) ppm. 13C NMR (100 MHz, CDCl3): δ 22.10, 23.05, 24.01, 24.06, 28.35, 40.24, 43.44, 79.99, 12
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94.88, 114.59, 129.62, 131.57, 132.12, 132.61, 132.90, 134.42, 136.12, 136.57, 145.83, 156.34, 161.00 ppm. ESI HRMS calcd. for [M-(H)]- 571.0643, found 571.0644. IR (KBr) νmax: 3414, 3077, 2974, 2227, 1683, 1637, 1616, 1579, 1481, 1447, 1400, 1383, 1369, 1347, 1327, 1286, 1259, 1167, 1105, 1078 cm-1. 5-[(2-aminoethyl)sulfamoyl]-2,4-dichloro-N-(3-cyano-4,5,6,7-tetrahydro-1-
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4.3.3.
benzothiophen-2-yl)benzamide hydrochloride (4). To a solution of 3 (0.095 g, 0.166 mmol) in DCM (10 mL), CF3COOH (0.6 mL, 8.0 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure and the product
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was dissolved in 1 M HCl in EtOAc (3 mL). EtOAc was removed and the product was dried in a desiccator overnight.
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Yield = 100%; pale brown crystals, mp = 204–206 °C; 1H NMR (400 MHz, MeOD): δ 1.85– 1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.62 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 2.72 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 3.10 (t, J = 6.0 Hz, 2H, SO2-NH-CH2), 3.22 (t, J = 6.0 Hz, 2H, SO2-NH-CH2-CH2), 7.91 (s, 1H, Ar-H), 8.33 (s, 1H, Ar-H) ppm, NH3+ and CONH are exchanged. 13C NMR (100 MHz, MeOD): δ 23.31, 24.23, 24.91, 25.09, 40.77, 41.13, 97.21, 114.61, 131.01, 133.05, 133.15, 134.25, 134.82, 135.66, 137.55, 137.99, 146.59, 164.12 ppm. ESI HRMS calcd. for [M+(H)]+ 473.0276, found 473.0268. IR (KBr) νmax: 3437, 2928, 2361, 220 nm, 100% at 254 nm).
TE D
2228, 1671, 1582, 1448, 1334, 1295, 1174, 1084, 1034 cm-1. HPLC tR = 11.977 min (100% at
4.3.4. General procedure for synthesis of compounds 7 and 8.
EP
To a solution of 5-hydroxybenzoic acid (5) (17.4 mmol) in pyridine (10 mL), acetic anhydride was added dropwise (6.6 mL, 70.0 mmol). The reaction mixture was stirred at room temperature for 1 h and was subsequently poured into water (50 mL). The water phase was
AC C
acidified to pH 2 and extracted with EtOAc (3 × 40 mL). The combined organic phases were washed with 0.1 M HCl (1 × 100 mL), brine (1 × 100 mL), and dried with Na2SO4. The solvent was evaporated under reduced pressure and the crude product was used without further purification.
4.3.4.1. 3-acetoxybenzoic acid (7). Yield = 89%; white crystals, mp = 127–129 °C (lit. [31] 128.7–131.3 °C); 1H NMR (400 MHz, DMSO-d6): δ 2.28 (s, 3H, CO-CH3), 7.39 (ddd, J1 = 8.1, J2 = 2.4, J3 = 1.1 Hz, 1H, Ar-H), 7.52–7.56 (m, 1H, Ar-H), 7.65–7.69 (m, 1H, Ar-H), 7.80–7.86 (m, 1H, Ar-H), 13.18 (bs, 1H, COOH) ppm.
13
ACCEPTED MANUSCRIPT
4.3.5. General procedure for synthesis of 9 and 10. To a solution of 7 (14.0 mmol) in anhydrous DCM (30 mL), DMF (catalytic amount) and (COCl)2 (3.60 mL, 42 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at room temperature. The solvent was evaporated to dryness and the residue was dissolved in
RI PT
DCM (20 mL). A mixture of 2-aminothiophene (2.245 g, 12.6 mmol) and pyridine (3.30 mL, 42 mmol) in DCM (30 mL) was added drop-wise, and the reaction was stirred overnight at room temperature. The reaction mixture was washed with 1 M HCl (40 mL), saturated aqueous NaHCO3 (40 mL) and brine (40 mL), and dried (Na2SO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography
SC
and recrystallized from ethanol.
4.3.5.1. 3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenyl acetate (9).
M AN U
Yield = 65%; white crystals, mp = 161–162 °C; 1H NMR (400 MHz, CDCl3): δ 1.82–1.88 (m, 4H, CH2-CH2-CH2-CH2), 2.34 (s, 3H, OCO-CH3), 2.59–2.71 (m, 4H, CH2-CH2-CH2-CH2), 7.35 (ddd, J1 = 8.1, J2 = 2.3, J3 = 1,0 Hz, 1H, Ar-H), 7.54 (t, J = 8.0 Hz, 1H, Ar-H), 7.65 (t, J = 2.0 Hz, 1H, Ar-H), 7.78 (ddd, J1 = 7.8, J2 = 1.7, J3 = 1.0 Hz, 1H, Ar-H), 8.99 (s, 1H, CONH) ppm. 13C NMR (100 MHz, CDCl3): δ 21.11, 22.12, 23.09, 23.98, 94.26, 114.54, 121.12, 124.93, 126.26, 128.94, 130.07, 131.25, 133.41, 146.53, 150.95, 162.87, 169.30 ppm. ESI
TE D
HRMS calcd. for [M+(H)]+ 341.0960, found 341.0966. IR (KBr) νmax: 3237, 3202, 3074, 2936, 2842, 2365, 2345, 2223, 2212, 1765, 1671, 1572, 1545, 1458, 1369, 1325, 1291, 1272, 1206, 1146, 1094,1010 cm-1. Anal. Calcd. for C18H16N2O3S × 0.1 H2O: C, 63.18; H, 4.77; N, 8.19. Found C, 62.90; H, 4.45; N, 8.05.
EP
4.3.6. General procedure for synthesis of 11 and 12. To a suspension of 9 (3.0 g, 8.0 mmol) in methanol (20 mL), K2CO3 (1.69 g, 12.0 mmol) was
AC C
added. The reaction mixture was stirred at room temperature for 1 h, and the solvent was evaporated under reduced pressure. The residue was dissolved in 1 M HCl (40 mL) and extracted with EtOAc (3 × 40 mL). The combined organic phases were washed with brine, dried with Na2SO4, and the solvent was removed under reduced pressure. The product was used without further purification. 4.3.6.1.
N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-hydroxybenzamide
(11).
1
Yield = 87%; small white crystals, mp = 249–250°C; H NMR (400 MHz, acetone-d6): δ 1.80–1.90 (m, 4H, CH2-CH2-CH2-CH2), 2.53–2.71 (m, 4H, CH2-CH2-CH2-CH2), 7.07–7.13 (m, 1H, Ar–H), 7.35–7.42 (m, 1H, Ar–H), 7.45–7,54 (m, 2H, 2 × Ar–H), 8.85 (s, 1H, CO–
14
ACCEPTED MANUSCRIPT
NH), 10.52 (s, 1H, Ar–OH) ppm.
13
C NMR (100 MHz, acetone-d6): δ 22.92, 23.84, 24.46,
24.67, 96.20, 114.52, 115.62, 119.87, 120.44, 129.47, 130.70, 132.17, 134.96, 147.33, 158.47, 165,45 ppm. ESI HRMS calcd. for [M+(H)]+ 299.0854, found 299.0856. IR (KBr) νmax: 3380, 3244, 3077, 2992, 2943, 2922, 2840, 2345, 2222, 1660, 1616, 1587, 1576, 1559, 1490, 1458,
C, 64.41; H, 4.73; N, 9.39. Found C, 64.19; H,4.56; N, 9.27. 4.3.7. General procedure for synthesis of 14-29, 20a-b and 21a-b.
RI PT
1399, 1327, 1306, 1283, 1260, 1216, 1147, 1078 , 1029 cm-1. Anal. Calcd. for C16H14N2O2S:
Method A: To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-
SC
hydroxybenzamide (12) (0.270 g, 0.90 mmol) in DMF (5 mL), K2CO3 or Cs2CO3 (1.35 mmol) was added. After 15 min, the corresponding bromide or chloride (1.35 mmol) was added and the reaction mixture was stirred for 16 h at 50 °C. The solvent was evaporated under reduced
M AN U
pressure and the residue was dissolved in EtOAc (40 mL). The organic phase was washed with water (2 × 30 mL), 1 M HCl (1 × 30 mL), brine (1 × 30 mL), and dried with Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography. 4.3.7.1.
3-((4-chlorobenzyl)oxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)benzamide (14). Yield = 44%; white crystals, mp = 195–196 °C; 1H NMR (400 MHz,
TE D
CDCl3): δ 1.79–1.90 (m, 4H, CH2-CH2-CH2-CH2), 2.58–2.70 (m, 4H, CH2-CH2-CH2-CH2), 5.11 (s, 2H, O-CH2-Ar), 7.17–7.21 (m, 1H, Ar-H), 7.34–7.42 (m, 4H, 4 × Ar-H), 7.42–7.48 (m, 2H, 2 × Ar-H), 7.51–7.55 (m, 1H, Ar-H), 8.85 (s, 1H, CO-NH) ppm.
13
C NMR (100
MHz, CDCl3): δ 22.12, 23.10, 24.00, 69.46, 94.08, 113.64, 114.49, 119.42, 120.07, 128.87,
EP
128.92, 130.26, 131.12, 133.29, 134.06, 134.74, 146.57, 159.04, 163.27 ppm. ESI HRMS calcd. for [M+(H)]+ 429.0934, found 423.0939. IR (KBr) νmax: 3446, 3252, 3200, 3076, 2932,
AC C
2855, 2366, 2217, 1889, 1670, 1602, 1595, 1573, 1558, 1490, 1470, 1456, 1411, 1399, 1378, 1327, 1296, 1279, 1229, 1164, 1153, 1116, 1095, 1082, 1054, 1032, 1014 cm-1. Anal. Calcd. for C23H19ClN2O2S × 0.3 H2O: C, 64.49; H, 4.61; N, 6.54. Found C, 64.29; H, 4.32; N, 6.44. Method B: To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3hydroxybenzamide (12) (0.250 g, 0.838 mmol) in anhydrous THF (5 mL), the corresponding alcohol (0.838 mmol) and PPh3 (0.330 g, 1.25 mmol) were added. The reaction mixture was cooled on ice bath and DIAD (0.25 mL, 1.25 mmol) was added. The reaction was stirred overnight at room temperature. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography.
15
ACCEPTED MANUSCRIPT
4.3.7.2.
3-((1H-pyrazol-1-yl)methoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)benzamide (16). Yield = 26%; pale yellow crystals, mp = 86–88 °C; 1H NMR (400 MHz, CDCl3): δ 1.69–1.79 (m, 4H, CH2-CH2-CH2-CH2), 2.44 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2CH2), 2.56 (t, J = 4.8 Hz, 2H, CH2-CH2-CH2-CH2), 6.09 (s, 2H, CH2-O), 6.37 (t, J = 2.4 Hz,
RI PT
1H, CH-CH-CH-pyrazol), 6.84 (ddd, J1 = 8.0, J2 = 2.8, J3 = 0.8 Hz, 1H, Ar-H), 6.92 (dt, J1 = 7.6, J2 = 1.6 Hz, 1H, Ar-H), 6.98 (dd, J1 = 2.4, J2 = 1.6 Hz, 1H, Ar-H), 7.10 (t, J = 8.0 Hz, 1H, Ar-H), 7.54 (dd, J1 = 1.6, J2 = 0.4 Hz, 1H, CHA-CH-CHB-pyrazole), 7.62 (bs, 1H, CO-NH), 7.84 (d, J = 2.4 Hz, 1H, CHA-CH-CHB-pyrazole) ppm. 13C NMR (100 MHz, CDCl3): δ 21.62, 22.62, 24.12, 24.62, 64.25, 107.30, 110.01, 112.66, 115.25, 118.54, 119.74, 129.46, 131.28,
SC
134.42, 134.79, 136.96, 140.30, 148.79, 156.04, 170.75 ppm. ESI HRMS calcd. for [M+(H)]+ 379.1229, found 379.1220. IR (KBr) νmax: 3418, 2937, 2225, 1668, 1599, 1449, 1376, 1291,
M AN U
1248, 1194, 1128, 1086, 1054 cm-1. HPLC tR = 12.696 min (100% at 220 nm, 100% at 254 nm).
4.3.8. General procedure for synthesis of 22a-b and 23a-b.
To a solution of Boc-protected amine (20a-b and 21a-b) (0.1 mmol) in DCM, CF3COOH (3– 5 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. Solvent was evaporated and some products were converted into hydrochloride salts with HCl
4.3.8.1.
TE D
in EtOAc.
3-(2-aminoethoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide
(22a). Yield = 76%; white crystals, mp = 158–160 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.73–1.84 (m, 4H, CH2-CH2-CH2-CH2), 2.54–2.67 (m, 4H, CH2-CH2-CH2-CH2), 3.25–3.31
EP
(m, 2H, NH-CH2-CH2-O), 4.22–4.30 (m, 2H, NH-CH2-CH2-O), 7.25–7.31 (m, 1H, Ar-H), 7.50-7.57 (m, 2H, 2 × Ar–H), 7.58–7.64 (m, 1H, Ar-H), 7.93–8.14 (bs, 3H, NH3+) ppm.
13
C
AC C
NMR (100 MHz, DMSO-d6): δ 21.67, 22.57, 23.45, 23.59, 38.32, 64.63, 96.29, 114.18, 114.30, 118.81, 121.07, 128.84, 129.82, 131.45, 133.80, 146.16, 157.74, 164.79 ppm. ESI HRMS calcd. for [M+(H)]+ 342.1276, found 342.1277. IR (KBr) νmax: 3434, 3235, 3195, 3080, 2936, 2220, 1669, 1576, 1559, 1511, 467, 1450, 1326, 1297, 1282, 1230, 1203, 1183, 1128, 1076, 1025 cm-1. Anal. Calcd. for C18H19N3O2S × CF3COOH: C, 52.74; H, 4.43; N, 9.23. Found C, 52.56; H, 4.22; N, 9.13. 4.3.9. General procedure for synthesis of compounds 24a, 25a-b, 40a-e and 41. To a solution of amine (22a, 23a-b, 35, 38a-e or 39c) (0.150 mmol) in anhydrous DMF (5 mL), di-Boc-S-methylisothiourea (0.150 mmol), Et3N (0.300 mmol) and HgCl2 (0.150 mmol)
16
ACCEPTED MANUSCRIPT
were added. The reaction mixture was stirred for 2 h at room temperature. The formed precipitate was filtered off and the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (30 mL), washed with water (3 × 30 mL), saturated NaHCO3 (30 mL), brine (30 mL), and dried with Na2SO4. The crude product was purified by flash 4.3.9.1.
RI PT
chromatography. Tert-butyl(tert-butoxycarbonylamino)(2-(3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]
thiophen-2-ylcarbamoyl)phenoxy)ethylamino)methylencarbamate (24a). Yield = 51%; white crystals, mp = 165–166 °C; 1H NMR (400 MHz, CDCl3): δ 1.53 (dd, J1 = 6.8, J2 = 1.2 Hz, 16H, 2 × COO-(CH3)3), 1.81–1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.61–2.74 (m, 4H, CH2-CH2-
SC
CH2-CH2), 3.90 (q, J = 5.3 Hz, 2H, NH-CH2-CH2-O), 4.20 (t, J = 5.2 Hz, 2H, NH-CH2-CH2O), 7.18–7.27 (m, 1H, Ar-H), 7.41–7.50 (m, 2H, 2 × Ar-H), 7.50–7.56 (m, 1H, Ar-H), 8.78 (t, 13
C
M AN U
J = 5.3 Hz, 1H, C-NH-CH2), 8.90 (s, 1H, CO-NH-C), 11.20 (s, 1H, C-NH-Boc) ppm.
NMR (100 MHz, acetone-d6): δ 14.37, 22.92, 23.30, 23.83, 24.46, 24.68, 28.14, 28.47, 32.32, 40.70, 67.21, 79.02, 83.84, 96.29, 114.50, 114.93, 119.77, 121.46, 129.56, 130.78, 132.27, 135.00, 159.79 ppm. ESI HRMS calcd. for [M+(H)]+ 584.2543, found 584.2545. IR (KBr) νmax: 3341, 3285, 3080, 2976, 2934, 2362, 2344, 2219, 1741, 1656, 1623, 1576, 1546, 1493, 1459, 1434, 1415, 1397, 1362, 1330, 1300, 1276, 1223, 1146, 1086, 1062, 1046, 1027 cm-1. Anal. Calcd. for C29H37N5O6S × 0.9 H2O: C, 58.06; H, 6.52; N, 11.67. Found C, 58.33; H,
TE D
6.44; N, 11.27.
4.3.10. General procedure for synthesis of compounds 26a, 27a-b, 42a-e and 43. To a solution of Boc-protected guanidine (24a, 25a-b, 40a-e and 41) (0.095 mmol) in DCM
EP
(5 mL), CF3COOH (0.210 mL, 2.84 mmol) was added, and the mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and 1 M HCl in EtOAc (3 ml) was added to the residue. The formed precipitate was collected by filtration. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2-guanidinoethoxy)
AC C
4.3.10.1.
benzamide (26a). Yield = 86%; white crystals, mp = 179–181 °C; 1H NMR (400 MHz, acetone-d6): δ 1.84–1.89 (m, 4H, CH2-CH2-CH2-CH2), 2.57–2.61 (m, 2H, CH2-CH2-CH2CH2), 2.68–2.72 (m, 2H, CH2-CH2-CH2-CH2), 3.78–3.83 (m, 2H, NH-CH2-CH2-O), 4.33 (t, J = 5.2 Hz, 2H, NH-CH2-CH2-O), 7.25–7,29 (m, 1H, Ar-H), 7.48–7.53 (m, 1H, Ar-H), 7.62– 7.69 (m, 2H, 2 × Ar-H), 7.79 (bs, 3H, C–NH3+), 8.68 (bs, 1H, NH), 10.72 (bs, 1H, NH) ppm. 13
C NMR (100 MHz, MeOD): δ 22.91, 23.82, 24.47, 24.68, 41.68, 41.79, 67.71, 96.50,
114.50, 114.94, 119.87, 121.62, 129.64, 130.74, 132.31, 135.02, 147.20, 159.52, 165.34 ppm. ESI HRMS calcd. for [M+(H)]+ 384.1494, found 384.1493. IR (KBr) νmax: 3425, 3366, 3179,
17
ACCEPTED MANUSCRIPT
2936, 2855, 2363, 2344, 2225, 1674, 1579, 1558, 1454, 1397, 1326, 1304, 1272, 1206, 1186, 1134, 1063, 1028 cm-1. HPLC tR = 11,836 min, (98.2% at 220 nm, 100% at 254 nm). 4.3.11. Methyl 2-chloro-5-hydroxybenzoate (28). To a cooled (0°C) solution of 2-chloro-5hydroxybenzoic acid (6) (4 g, 23.6 mmol) in methanol (50 mL), thionyl chloride (2.5 mL, 35
RI PT
mmol) was added drop-wise. The reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (100 ml). The organic phase was washed with saturated NaHCO3 (3 × 50 mL), water (50 mL) and brine (50 mL), dried with Na2SO4 and evaporated under reduced pressure.
SC
Yield = 99%; white crystals, mp = 96–97 °C; 1H NMR (400 MHz, CDCl3): δ 3.91 (s, 3H, OCH3), 6.91 (dd, J1 = 8.7, J2 = 3.1 Hz, 1H, Ar-H), 7.27 (d, J = 8.7 Hz, 1H, Ar-H), 7.31 (d, J = 3.1 Hz, 1H, Ar-H).
13
C NMR (100 MHz, CDCl3): δ 52.79, 118.15, 120.33, 124.67, 130.25,
M AN U
132.14, 154.51, 166.77 ppm. ESI HRMS calcd. for [M+(H)]+ 187.0162, found 187.0155. HPLC tR = 10.618 min (100% at 220 nm, 100% at 254 nm).
4.3.12. Methyl 5-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-chlorobenzoate (29). To a solution of 28 (1.00 g, 5.36 mmol) in DMF (15 mL), K2CO3 (1.11 g, 8.04 mmol) was added. After 10 min at room temperature, tert-butyl 2-bromoethylcarbamate (1.56 g, 6.97 mmol) was added
TE D
and the reaction mixture was stirred at 80 °C for 16 h. DMF was evaporated, the crude residue was dissolved in EtOAc (50 mL), washed with water (2 × 30 mL), 1 M NaOH (2 × 30 mL), water (1 × 50 mL), brine (10 mL), and dried with Na2SO4. The crude residue was purified by flash chromatography.
EP
Yield = 73%; colourless oil; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, 3 × CH3), 3.56 (q, J = 5.6 Hz, 2H, O-CH2-CH2-NH), 3.95 (s, 3H, O-CH3), 4.05 (t, J = 4.8 Hz, 2H, O-CH2-CH2-
AC C
NH), 4.98 (bs, 1H, NH-CO), 6.98 (dd, J1 = 8.8, J2 = 2.8 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H), 7.37 (d, J = 6.0 Hz, 1H, Ar-H) ppm.
13
C NMR (100 MHz, CDCl3): δ 28.38, 39.95, 52.53,
67.71, 79.72, 116.72, 119.22, 125.39, 130.56, 131.95, 155.83, 156.88, 165.87 ppm. ESI HRMS calcd. for [M+(H)]+ 330.1108, found 330.1103. 4.3.13. 5-(2-(tert-butoxycarbonylamino)ethoxy)-2-chlorobenzoic acid (30). To a solution of 29 (1.00 g, 3.03 mmol) in a mixture of dioxane and water (1/1, 30 mL), NaOH (0.24 g, 6.00 mmol) was added. The reaction mixture was stirred at room temperature for 4 h. Dioxane was evaporated under reduced pressure and the remaining water was acidified with 1 M HCl to pH 3. The water phase was extracted with EtOAc (3 × 30 mL). The combined organic phases
18
ACCEPTED MANUSCRIPT
were washed with brine (1 × 50 mL), dried with Na2SO4 and evaporated under reduced pressure. Yield = 98%; white crystals, mp = 117–119 °C; 1H NMR (400 MHz, CDCl3): δ 1.48 (s, 9H, 3 × CH3), 3.58 (q, J = 5.6 Hz, 2H, O-CH2-CH2-NH), 4.07 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH),
RI PT
5.03 (bs, 1H, NH-CO), 7.03 (dd, J1 = 8.0, J2 = 2.4 Hz, 1H, Ar-H), 7.40 (d, J = 8.8 Hz, 1H, ArH), 7.52 (d, J = 2.8 Hz, 1H, Ar-H), 7.64 (bs, 1H, COOH) ppm. 13C NMR (100 MHz, acetoned6): δ 28.63, 40.51, 68.35, 78.98, 117.93, 119.83, 124.93, 132.34, 132.64, 156.77, 158.37, 166.56 ppm. ESI HRMS m/z calcd. for [M-(H)]+ 314.0795, found 314.0791. IR (KBr) νmax:
SC
3403, 2981, 2576, 1717, 1669, 1605, 1572, 1527, 1478, 1464, 1430, 1394, 1366, 1275, 1252, 1207, 1162, 1110, 1077, 1053, 1037 cm-1.
M AN U
4.3.14. 2-(3-carboxy-4-chlorophenoxy)ethan aminium 2,2,2-trifluoroacetate (31). To a solution of 30 (1.00 g, 3.17 mmol) in DCM, CF3COOH (7.0 mL, 91.4 mmol) was added and stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and the crude product was used without further purification.
Yield = 100%; mp = 147–149 °C; 1H NMR (400 MHz, acetone-d6): δ 4.29 (t, J = 5.6 Hz, 2H, O-CH2-CH2-NH2), 4.51 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH2), 7.17 (dd, J1 = 8.8, J2 = 3.2 Hz,
TE D
1H, Ar-H), 7.42 (d, J = 3.2 Hz, 1H, Ar-H), 7.43 (d , J = 8.8 Hz, 1H, Ar-H) ppm. 4.3.15. 2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzoic acid (32). To a suspension of 31 (1.00 g, 4.64 mmol) in anhydrous DCM (15 mL), anhydrous pyridine (1.12 mL, 13.9 mmol) was added and the reaction mixture was cooled to 0°C. (CF3CO)2O (0.72 mL, 5.10
EP
mmol) was added drop-wise and the mixture was stirred for 2 h at 0 °C then allowed to warm to room temperature. The reaction mixture was diluted with DCM (30 mL), washed with 1 M
AC C
HCl (2 × 30 mL), water (30 mL), and saturated NaHCO3 (3 × 30 ml). The combined alkaline water phases were acidified with concentrated HCl to pH = 1 and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with water (50 mL), brine (50 mL) and dried with Na2SO4. The solvent was evaporated under reduced pressure. Yield = 76%; white crystals, mp = 141–143°C; 1H NMR (400 MHz, acetone-d6): δ 3.79 (q, J = 5.6 Hz, 2H, O-CH2-CH2-NH), 4.28 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 7.15 (dd, J1 = 8.8, J2 = 3.2 Hz, 1H, Ar-H), 7.43 (d, J = 2.8 Hz, 1H, Ar-H), 7.45 (d, J = 8.4 Hz, 1H, Ar-H), 8.79 (bs, 1H, NH-CO) ppm. 13C NMR (100 MHz, acetone-d6): δ 39.98 (d, 4JF,C = 12 Hz, CH2-NH), 67.01, 117.08 (q, 1JF,C = 285.8 Hz, CF3), 117.89, 119.86, 125.21, 132.46, 132.70, 157.93 (qd,
19
ACCEPTED MANUSCRIPT
2
JF,C = 36.5 Hz, 2JF,C = 8 Hz, CO-CF3), 158.07, 166.52 ppm. ESI HRMS m/z calcd. for [M-
(H)]+ 310.0094, found 310.0088. IR (KBr) νmax: 3414, 3316, 3110, 2927, 2633, 1710, 1597, 1560, 1486, 1469, 1432, 1414, 1384, 1374, 1349, 1321, 1277, 1260, 1235, 1206, 1182, 1116,
4.3.16. General procedure for synthesis of compounds 33a and 33b.
RI PT
1061, 1052, 1039 cm-1. HPLC tR = 10.996 min (100% at 220 nm, 100% at 254 nm).
To a solution of 32 (2.15 g, 6.88 mmol) in anhydrous DCM (30 mL), DMF (catalytic amount) and (COCl)2 (1.77 mL, 20.6 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at room temperature. The solvent was evaporated to dryness and the residue was dissolved
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in DCM (20 mL). A mixture of the corresponding 2-aminothiophene (1.73 g, 6.19 mmol) and pyridine (1.63 mL, 20.6 mmol) in DCM (20 mL) was added drop-wise, and the reaction was stirred overnight at room temperature. The reaction mixture was washed with 1 M HCl (30
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mL), saturated aqueous NaHCO3 (30 mL) and brine (30 mL), and dried (Na2SO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography.
4.3.16.1. Tert-butyl 2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano4,5-dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (33a). Yield = 79%; orange crystals, mp = 89–91 °C; 1H NMR (400 MHz, CDCl3): δ 1.46 (s, 9H, 3 × CH3), 2.67 (t, J = 5.6 Hz,
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2H,CH2-CH2-N), 3.68 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.78 (q, J = 5.4 Hz, 2H, O-CH2-CH2NH), 4.13 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 4.51 (s, 2H, CH2-N-CO), 6.98 (bs, 1H, NHCO-CF3), 7.01 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.37 (d, J = 8.7 Hz, 1H, Ar-H), 7.48 (d, J = 3.1 Hz, 1H, Ar-H), 9.84 (s, 1H, NH-CO-thiophene) ppm.
13
C NMR (100 MHz, CDCl3): δ
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24.07, 28.39, 31.45, 36.56, 39.18, 66.27, 80.65, 94.18, 113.65, 114.32, 116.95, 117.18, 119.95, 122.99, 131.64, 131.98, 147.04, 154.54, 157.21, 157.57 (q, 2JC,F = 37.4 Hz), 161.82, 162.62 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 573.1186, found 573.1201. IR (KBr) νmax
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3261, 2982, 2935, 2217, 1666, 1545, 1459, 1416, 1367, 1287, 1237, 1210, 1153, 1118, 1063, 1038, 1005 cm-1. HPLC tR = 16.299 min (100% at 220 nm, 100% at 254 nm). 4.3.17. 2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium trifluoroactetate (34). To a solution of 33a (2.13 g, 3.72 mmol) in DCM (30 mL), CF3COOH (8.60 mL, 112 mmol) was added and stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and the residue was dried in a desiccator overnight.
20
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Yield = 96%; orange crystals, mp = 90–92 °C; 1H NMR (400 MHz, CDCl3): δ 3.07 (t, J = 6.2 Hz, 2H, CH2-CH2-N), 3.60 (t, J = 6.2 Hz, 2H, CH2-CH2-N), 3.81 (q, J = 5.1 Hz, 2H, O-CH2CH2-NH), 4.16 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 4.39 (s, 2H, CH2-NH2+), 6.71 (t, J = 5.7 Hz, 1H, NH-CO-CF3), 7.07 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.44 (d, J = 8.8 Hz, 1H, Ar13
C NMR (100
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H), 7.56 (d, J = 3.1 Hz, 1H, Ar-H), 10.00 (s, 1H, NH-CO-thiophene) ppm.
MHz, MeOD): δ 22.26, 27.78, 40.29, 42.35, 42.76, 67.46, 95.33, 113.83, 116.08, 116.57, 118.94, 119.78, 121.38, 124.03, 130.43, 132.32, 135.79, 149.83, 158.83, 161.78 (q, J = 36.8 Hz), 166.53 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 473.0662, found 473.0668. IR (KBr)
= 9.914 min (96.6% at 220 nm, 97.1% at 254 nm).
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νmax 2992, 2842, 2221, 1711, 1667, 1547, 1462, 1419, 1292, 1135, 1067, 1039 cm-1. HPLC tR
4.3.18. General procedure for synthesis of compounds 35, 36 and 38a-f.
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To a solution of 33b, 34 or 37a-f (0.17 mmol) in methanol (2 mL), 1 M NaOH (2 mL) was added. The reaction mixture was stirred overnight. The methanol was evaporated and the residue was diluted with water (20 mL). The water phase was extracted with EtOAc (3 × 20 mL) and the combined organic phases were washed with water (30 mL), brine (30 mL) and dried with Na2SO4. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography.
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-tetrahydro
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4.3.18.1.
benzo[b]thiophen-2-yl)benzamide (35). Yield = 100%; white crystals, mp = 162–164 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.81–1.90 (m, 1H, CH-CHAHB-CH2), 1.97–2.02 (m, 1H, CHCHAHB-CH2), 2.61–2.68 (m, 3H, CH2-CH-CH2-CH2), 2.80–2.92 (m, 2H, CH2-CH), 3.22 (t, J
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= 4.8 Hz, 2H, O-CH2-CH2-NH2), 4.22 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH2), 6.72 (d, J = 8.4 Hz, 2H, Ar-H), 7.09–7.13 (m, , 3H, 3 × Ar-H), 7.24 (d, J = 3.2 Hz, 1H, Ar-H), 7.45 (d, J = 8.8 Hz, 1H, Ar-H), 9.25 (bs, 1H, NH-CO) ppm.
13
C NMR (100 MHz, pyridine-d6): δ 26.76,
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32.06, 34.27, 41.92, 42.18, 69.31, 97.49, 116.97, 117.96, 118.38, 120.29, 125.17, 130.44, 130.72, 133.03, 133.68, 138.26, 138.39, 149.53, 159.40, 159.57, 167.05 ppm. ESI HRMS calcd. for [M+(H)]+ 468.1149, found 468.1143. IR (KBr) νmax: 3549, 3475, 3413, 3237, 3084, 3012, 2922, 2224, 2032, 1677, 1656, 1638, 1617, 1574, 1556, 1514, 1460, 1408, 1384, 1317, 1296, 1234, 1201, 1178, 1156, 1139, 1074, 1017 cm-1. HPLC tR = 11.363 min (100% at 220 nm, 100% at 254 nm). 4.3.19. General procedure for synthesis of compounds 37a-f. Method A: To a solution of 34 (0.30 g, 0.511 mmol) in DMF (3 mL), Et3N (1.5 mmol) was added. After 10 min of stirring at room temperature, the corresponding benzyl bromide (0.766 21
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mmol) was added. The reaction mixture was stirred overnight at room temperature. DMF was then evaporated, the crude residue was dissolved in EtOAc (20 mL) and washed with water (3 × 10 mL), brine (10 mL) and dried with Na2SO4. The crude product was purified by flash chromatography.
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Method B: To a solution of 34 (0.50 g, 0.852 mmol) in THF (5 mL), the corresponding benzaldehyde (1.28 mmol) and Na(OAc)3BH (0.45 g, 2.13 mmol) were added, and the reaction mixture was stirred overnight at room temperature. To quench the reaction, saturated aqueous NaHCO3 (10 mL) was used, and the water phase was extracted with EtOAc (3 × 20 The crude residue was purified by flash chromatography. 4.3.19.1.
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mL). The combined organic phases were washed with brine (30 mL) and dried with Na2SO4.
N-(6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2-chloro-5-(2-
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(2,2,2-trifluoroacetamido)ethoxy)benzamide (37a). Yield = 35%; pale brown crystals, mp = 71–73 °C; 1H NMR (400 MHz, acetone-d6): δ 2.68 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 2.85 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.76–3.79 (m, 4H, N-CH2-Ar+ O-CH2-CH2NH ), 4.26 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 7.11 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.25–7.29 (m, 2H, 2 × Ar-H), 7.33–7.37 (m, 2H, 2 × Ar-H), 7.39-7.44 (m, 3H, 3 × Ar-H), 8.79 (bs, 1H, NH-CO-CF3), 11.01 (s, 1H, NH-CO-thiophene) ppm.
13
C NMR (100 MHz,
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acetone-d6): δ 24.98, 40.04, 50.15, 51.41, 62.04, 67.18, 95.27, 114.08, 115.69, 116.38, 118.54, 119.28, 132.30, 127.55, 128.05, 129.21, 129.77, 131.14, 131.85, 135.97, 139.53, 147.26, 158.29, 164.51 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 563.1132, found 563.1137. IR νmax 3319, 3081, 2923, 2807, 2214, 1714, 1667, 1545, 1456, 1405, 1366, 1288, 1209, 1154,
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1057, 1036, 1007 cm-1. HPLC tR = 11.907 min (100% at 220 nm, 100% at 254 nm).
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4.4. Molecular docking studies
A docking study was performed using the crystal structure of MurF enzyme from S. pneumoniae (PDB code: 3zm5) and OEDocking software (Release 3.0.1, OpenEye Scientific Software, Inc.).
4.4.1. Ligand preparation The molecules were built with ChemBioDraw Ultra 12.0 (CambridgeSoft). The ligand geometries were optimized with ChemBio 3D Ultra 12.0 (CambridgeSoft) using MM2 force field until a minimum 0.100 root mean square (RMS) gradient was reached. The optimized structure was refined with GAMESS interface using the semi-empirical AM1 method, QA 22
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optimization algorithm and Gasteiger Hückel charges for all atoms for 100 steps. Amines and guanidines were kept in their ionized state, corresponding to pH 7.4. FRED requires a set of input conformers for each ligand, which were generated with OMEGA 2.4.6, with maximum number of conformations set to 1000, and using the RMS cut-off value (Root Mean Square
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(RMS) cartesian distance below which two conformers are duplicates) of 0.3 Å. All the other options were left as default values. 4.4.2. Receptor preparation & docking protocol
The crystal structure of MurF enzyme from S. pneumoniae in complex with the ligand (PDB
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code: 3zm5) was taken as a starting-point. The ligand (2,4-dichloro-N-[3-cyano-6-[(4hydroxyphenyl)methyl]-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium-2-yl]-5-
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morpholinosulfonyl-benzamide) was taken as a reference structure and a grid box including all active-site atoms (including hydrogens) with a volume of 32562 Å, dimensions of 36.22 Å × 36.33 Å × 24.67 Å, and outer contour of 2953 Å, was created around it as a “docking receptor” using Make Receptor 3.0.1.
The docking software FRED (OEDocking 3.0.1) was used for docking studies with the default settings, except for dock resolution, which was set to “high”, and number of poses, which was
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set to 50. The proposed 10 binding modes with the highest rank of the docked inhibitors were evaluated using final score and root mean square deviation (RMSD) as a tool to explore relative structural differences between proposed binding modes. The graphical representations of the proposed binding positions of all the molecules were obtained using Accelrys
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Discovery Studio 2.5 (Accelrys Software Inc.). 4.4.3. Validation of the docking protocol
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The docking procedure should be able to correctly predict the binding poses of the molecules in the PDB database i.e., the crystal structures of ligands in complex with proteins. We have attempted to reproduce the pose of the ligand as seen in its X-ray complex with the target MurF enzyme from S. pneumoniae (PDB code: 3zm5). The top 10 docking poses of the docked ligand were within 1.5 Å RMSD of the ligand crystal structure. The predicted top score pose was the one that best fitted the crystal structure of the ligand, overlapping it almost completely, which offers the proof of the protocol validation (Fig. 3).
ACKNOWLEDGEMENTS
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We thank OpenEye Scientific Software, Inc. for free academic licenses of their software, and the Ministry of Higher Education, Science and Technology of the Republic of Slovenia for financial support. This study was partially supported by a Young Researcher grant to MH, and
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an L1-4039 grant to SG, both from the Slovenian Research Agency.
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REFERENCES [1] A.J. Alanis, Resistance to antibiotics: are we in the post-antibiotic era?, Arch. Med. Res. 36 (2005) 697–705.
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[2] P.C. Appelbaum, 2012 and beyond: potential for the start of a second pre-antibiotic era?, J. Antimicrob. Chemoth. 67 (2012) 2062–2068.
[3] I. Chopra, The 2012 Garrod Lecture: Discovery of antibacterial drugs in the 21st century, J. Antimicrob. Chemoth., (2012).
[4] T.D.H. Bugg, D. Braddick, C.G. Dowson, D.I. Roper, Bacterial cell wall assembly: still an
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attractive antibacterial target, Trends Biotechnol. 29 (2011) 167–173.
[5] A. Gautam, R. Vyas, R. Tewari, Peptidoglycan biosynthesis machinery: A rich source of
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drug targets, Cr. Rev. in Biotechn. 31 (2011) 295–336.
[6] D.W. Green, The bacterial cell wall as a source of antibacterial targets, Expert Opin. Ther. Tar. 6 (2002) 1–20.
[7] H. Barreteau, A. Kovač, A. Boniface, M. Sova, S. Gobec, D. Blanot, Cytoplasmic steps of peptidoglycan biosynthesis, FEMS Microbiol. Rev. 32 (2008) 168–207. [8] A. El Zoeiby, F. Sanschagrin, R.C. Levesque, Structure and function of the Mur enzymes:
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development of novel inhibitors, Mol. Microbiol. 47 (2003) 1–12. [9] C.A. Smith, Structure, function and dynamics in the mur family of bacterial cell wall ligases, J. Mol. Biol. 362 (2006) 640–655. [10] D. J. Miller, S. M. Hammond, D. Anderluzzi, T. D. H. Bugg, Aminoalkylphosphinate
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inhibitors of D-Ala-D-Ala adding enzyme, J. Chem. Soc., Perk. T. 1. (1998) 131–142. [11] Y.G. Gu, A.S. Florjancic, R.F. Clark, T. Zhang, C.S. Cooper, D.D. Anderson, C.G. Lerner, J.O. McCall, Y. Cai, C.L. Black-Schaefer, G.F. Stamper, P.J. Hajduk, B.A.
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Beutel, Structure–activity relationships of novel potent MurF inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 267–270. [12] G.F. Stamper, K.L. Longenecker, E.H. Fry, C.G. Jakob, A.S. Florjancic, Y.G. Gu, D.D. Anderson, C.S. Cooper, T. Zhang, R.F. Clark, Y. Cia, C.L. Black-Schaefer, J. Owen McCall, C.G. Lerner, P.J. Hajduk, B.A. Beutel, V.S. Stoll, Structure-based optimization of MurF inhibitors, Chem. Biol. Drug Des. 67 (2006) 58–65. [13] E.Z. Baum, S.M. Crespo-Carbone, D. Abbanat, B. Foleno, A. Maden, R. Goldschmidt, K. Bush, Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF, Antimicrob. Agents Ch. 50 (2006) 230–236.
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[14] E.Z. Baum, S.M. Crespo-Carbone, A. Klinger, B.D. Foleno, I. Turchi, M. Macielag, K. Bush, A MurF inhibitor that disrupts cell wall biosynthesis in Escherichia coli, Antimicrob. Agents Ch. 51 (2007) 4420–4426. [15] E.Z. Baum, S.M. Crespo-Carbone, B.D. Foleno, L.D. Simon, J. Guillemont, M.
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Macielag, K. Bush, MurF inhibitors with antibacterial activity: Effect on muropeptide levels, Antimicrob. Agents Ch. 53 (2009) 3240–3247.
[16] S. Turk, A. Kovač, A. Boniface, J.M. Bostock, I. Chopra, D. Blanot, S. Gobec, Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening, Bioorg. Med. Chem. 17 (2009) 1884–
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[17] S. Turk, M. Hrast, I. Sosič, H. Barreteau, D. Mengin-Lecreulx, D. Blanot, S. Gobec,
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Biochemical characterization of MurF from Streptococcus pneumoniae and the identification of a new MurF inhibitor through ligand-based virtual screening, Acta Chim. Slov. 60 (2013) 294–299.
[18] M. Hrast, S. Turk, I. Sosič, D. Knez, C.P. Randall, H. Barreteau, C. Contreras-Martel, A. Dessen, A.J. O’Neill, D. Mengin-Lecreulx, D. Blanot, S. Gobec, Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF, Eur. J. Med. Chem. 66 (2013) 32–45.
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[19] P.D. Leeson, A.M. Davis, Time-related differences in the physical property profiles of oral drugs. J. Med. Chem. 47 (2004) 6338–6348. [20] R. O’Shea, H.E. Moser, Physicochemical Properties of Antibacterial Compounds: Implication for Drug Discovery, J. Med. Chem. 51 (2008) 2871–2878.
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[21] I. Dvoretzky, G.H. Richter, Formaldehyde condensation in the pyrazole series, J. Org. Chem. 15 (1950) 1285–1288.
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[22] P.A. Lanzetta, L.J. Alvarez, P.S. Reinach, O.A. Candia, An improved assay for nanomole amounts of inorganic phosphate, Anal. Biochem. 100 (1979) 95–97. [23] S.L. McGovern, B.T. Helfand, B. Feng, B.K. Shoichet, A specific mechanism of nonspecific inhibition, J. Med. Chem. 46 (2003) 4265–4272. [24] J.K. Hobbs, K. Miller, A.J. O’Neill et al. Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus. J. Antimicrob. Chemother. 62 (2008) 1003–1008. [25] S. Dementin, A. Bouhss, G. Auger, C. Parquet, D. Mengin-Lecreulx, O. Dideberg, J. van Heijenoort, D. Blanot, Evidence of a functional requirement for a carbamoylated lysine
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residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments, Eur. J. Biochem. 268 (2001) 5800–5807. [26] A.J. O'Neill, Staphylococcus aureus SH1000 and 8325-4: comparative genome sequences of key laboratory strains in staphylococcal research. Lett. Appl. Microbiol. 51
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(2010) 358–361. [27] A.J. O'Neill, J.M. Bostock, A.M. Moita, I. Chopra, Antimicrobial activity and mechanisms of resistance to cephalosporin P1, an antibiotic related to fusidic acid. J. Antimicrob. Chemother. 50 (2002) 839–848.
[28] E. Shapiro, F. Baneyx, Stress-based identification and classification of antibacterial
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agents: second-generation Escherichia coli reporter strains and optimization of detection, Antimicrob. Agents Chemother. 46 (2002) 2490–2497.
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[29] P.A. Wayne, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-Ninth Edition. Clinical and Laboratory Standards Institute (2012) CLSI Document M07–A9.
[30] R.A. Dixon, I. Chopra, Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide, Antimicrob. Agents Chemother. 29 (1986) 781–788. [31] E.R. Marshall, J.A. Kuck, R.C. Elderfield, Studies on lactones related to the cardiac aglycones. X. Synthesis of simple hydroxylated β-substituted ∆α,β-butenolides, J. Org.
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Chem. 7 (1942) 444–456.
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List of table and figure captions:
Table 1. Inhibitory activities of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a and 27b
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against MurF from S. pneumoniae and E. coli.
Table 2. Inhibitory activities of compounds 33-38f, 39c, 42a-e and 43 against MurF from S. pneumoniae and E. coli.
and 43 against S. aureus and E. coli bacterial strains.
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Table 3. Antibacterial activities of compounds 23a-b, 26a, 27a-b, 33a, 34-38f, 39c, 42a-e
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Figure 1. Two-step design of new cyanothiophene MurF inhibitors. Overlapping scaffold essential for good inhibitory potency is shown in red.
Figure 2. New cyanothiophene MurF inhibitors (sticks rendering, coloured by atom) docked to MurFSp (PDB code: 3zm5) with a co-crystallized inhibitor (sticks rendering, in green). The best ranked predicted binding mode can be seen for a) amine, and b) guanidine of truncated
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analogue of II; guanidine is predicted to fit into the “morpholine” pocket (binds morpholine moiety from the co-crystallized inhibitor) by forming 4 H-bonds with Asn134, Asn137 and Asn326 (in green, demonstrated by the distances between guanidine protons and aminoacid carbonyl oxygens), while the rest of the molecule overlaps the structure of a co-crystallized
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cyanothiophene scaffold. Virtually the same binding mode is predicted for c) amine with benzyl substituent attached to position 6 of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine (c), while its guanidine analogue (d) moves towards the interior of the binding site to form close contact
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with Asp323 (in green, ionic bond and two H-bonds).
Figure
3.
Crystal
structure
of
the
ligand
(2,4-dichloro-N-[3-cyano-6-[(4-
hydroxyphenyl)methyl]-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium-2-yl]-5-morpholino sulfonyl-benzamide, shown as green sticks) and top docked pose of the ligand, as predicted by FRED (shown as stick colored by atom type). The top docked ligand pose had an RMSD of 1.15 Å.
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Scheme 1: Reagents and conditions: (a) tert-butyl (2-aminoethyl)carbamate, Et3N, DCM, 0 °C to rt, 16 h; (b) 1. (COCl)2, DMF, DCM, 0 °C to rt, 0.5 h; 2. 2-aminothiophene, pyridine, DCM, 0 °C to rt, 16 h; (c) 1. CF3COOH, DCM, rt, 2 h; 2. 1 M HCl in EtOAc.
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Scheme 2: Reagents and conditions: (a) Ac2O, pyridine, rt, 1 h; (b) 1. (COCl)2, DMF, DCM, 0 °C to rt, 0.5 h; 2. amine, pyridine, DCM, 0 °C to rt, 16 h; (c) K2CO3, MeOH, rt, 1 h; (d) A: K2CO3 or Cs2CO3, corresponding halogenide, 50 °C, 16 h; B: (1H-pyrazol-1-yl)methanol (13) or cyclopentylmethanol, PPh3, DIAD, THF, 0 °C to rt, 16 h; (e) CF3COOH, DCM, rt, 2 h, 1 M HCl in EtOAc; (f) di-Boc-methyl-thiourea, HgCl2, Et3N, DMF, rt, 4 h; (g) 1. CF3COOH,
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DCM, rt, 2 h; 2. 1 M HCl in EtOAc.
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Scheme 3: Reagents and conditions: (a) SOCl2, MeOH, rt, 3 h; (b) K2CO3, DMF, 80 °C, 16 h; (c) NaOH, dioxane/water, rt, 16 h; (d) CF3COOH, DCM, rt, 2 h; (e) (CF3CO)2O, pyridine, DCM, 0 °C to rt, 2 h; (f) 1. (COCl)2, DMF, DCM, 0 °C to rt, 1 h; 2. corresponding 2aminothiophene, pyridine, DCM, 0 °C to rt, 16 h; (g) CF3COOH, DCM, rt, 2 h; (h) 1 M NaOH, MeOH, rt, 16 h; (i) A: K2CO3, corresponding benzyl bromide, DMF, rt, 16 h; B: corresponding benzaldehyde, Na(OAc)3BH, AcOH, THF, rt, 16 h; (j) di-Boc-methyl-thiourea,
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HgCl2, Et3N, DMF, rt, 4 h; (k) 1. CF3COOH, DCM, rt, 2 h; 2. 1 M HCl in EtOAc.
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RAa (%) or IC50b (µM) Compound
R1
X
MurFEc
-H
15
-H
16
-H
17
-Cl
18
-Cl
19
-Cl
22a
102%
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14
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MurFSp
97%
99%
89%
85%
81%
82%
102%
97%
87% c
74%c
-H
98%
93%
22b
-H
53%c
75%c
23a
-Cl
170 µM
208 µM
23b
-Cl
128 µM
272 µM
26a
-H
167 µM
258 µM
27a
-Cl
75 µM
56 µM
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81%
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27b
a
78 µM
-Cl
102 µM
Residual activity (%) of the enzyme at 100 µM of the tested compound. Data are means of
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two independent experiments. Standard deviations were within ±10% of the means; b
Concentration of the inhibitor for which the residual activity of the enzyme is 50%; cResidual
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activity (%) of the enzyme at 250 µM of the tested compound.
N
33b
C
34
N
35
C
-H2+CF3COO-
-NH2
N N
37b
N
37c
N
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37a
-NH2
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36
R
RAa (%) or IC50b (µM) MurFSp
MurFEc
96%
102%
98%
101%
99%
91%
58 µM
81 µM
85%
65%
172 µM
46%
154 µM
81%
55%
255 µM
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33
R
3
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Y
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Compound
2
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-H
37d
N
71%
56%
37e
N
55%
45%
37f
N
103 µM
152 µM
ACCEPTED MANUSCRIPT
N
-NH2
51 µM
84 µM
38b
N
-NH2
125 µM
73 µM
38c
N
-NH2
663 µM
336 µM
38d
N
-NH2
119 µM
150 µM
38e
N
-NH2
107 µM
76 µM
38f
N
-NH2
454 µM
83 µM
39c
N
-NH2
109 µM
88 µM
42a
N
55 µM
57 µM
42b
N
61 µM
78 µM
42c
N
37 µM
80 µM
42d
N
117 µM
143 µM
SC M AN U TE D
EP
AC C
a
RI PT
38a
42e
N
61 µM
66 µM
43
C
20 µM
25 µM
Residual activity (%) of the enzyme at 250 µM of the tested compound. Data are means of
two independent experiments. Standard deviations were within ±10% of the means; b
Concentration of the inhibitor for which the residual activity of the enzyme is 50%.
ACCEPTED MANUSCRIPT
MIC (µg/ml) S. aureus
Compound SH1000
E. coli 1411a
1411+
AB734+
PMBNd AB734a
PMBNd
>256
128
>256
128
128
64
23a
128
128
32
128
32
32
32
23b
128
64
32
64
32
32
32
26a
64
64
64
64
>256
16
16
27a
64
16
8
16
8
8
8
27b
>256
32
32
32
32
16
8
33a
>256
>256
>256
>256
>256
>256
>256
34
>256
>256
>256
>256
>256
>256
>256
35
>256
>256
16
>256
16
32
8
36
>256
>256
>256
>256
>256
>256
>256
37a
>256
>256
>256
>256
>256
>256
>256
37b
>256
>256
>256
>256
>256
>256
>256
37c
>256
>256
>256
>256
>256
>256
>256
37d
>256
>256
>256
>256
>256
>256
>256
37e
>256
>256
>256
>256
>256
>256
>256
37f
>256
>256
>256
>256
>256
>256
64
38a
64
64
32
64
32
32
16
38b
128
128
64
128
64
64
32
38c
>256
>256
>256
>256
>256
>256
>256
38d
>256
128
64
128
64
64
64
38e
128
256
>256
>256
>256
>256
>256
39c
128
>256
64
>256
64
64
32
42a
8
32
16
32
16
32
8
42b
32
32
32
32
32
32
8
42c
8
64
32
64
32
16
8
42d
32
128
64
128
64
64
32
42e
16
128
32
256
32
16
16
M AN U
TE D
EP
AC C
RI PT
>256
SC
4
SM1411b ES100c
ACCEPTED MANUSCRIPT
43 a
4
64
16
64
16
16
4
E. coli 1411 and AB734 are parental strains of SM1411 and ES100, respectively; bSM1411
is 1411 deleted for acrAB; cES100 is AB734 deleted for tolC; dPMBN, polymyxin B
AC C
EP
TE D
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SC
RI PT
nonapeptide.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
RI PT
ACCEPTED MANUSCRIPT
TE D
M AN U
SC
a)
AC C
EP
b)
RI PT
ACCEPTED MANUSCRIPT
TE D
M AN U
SC
c)
AC C
EP
d)
ACCEPTED MANUSCRIPT
O
O OH
HO
O
HO
a X
S
O O
X 5 (X = H) 6 (X = Cl)
O
N H
b
7 (X = H) 8 (X = Cl)
O
X
CN
9 (X = H) 10 (X = Cl) c
O R1
O
N H
S d
11 (X = H) 12 (X = Cl) O N H
O
SC
NHBoc
n
S
e
NH3+Cl-
n
X CN 22a (X = H, n = 1) 22b (X = H, n = 2) 23a (X = Cl, n = 1) 23b (X = Cl, n = 2)
M AN U
O X 20a (X = H, n = 1) 20b (X = H, n = 2) 21a (X = Cl, n = 1) 21b (X = Cl, n = 2)
CN
X
CN
O N H
OH
N H
X CN 14-19, 20a,b, 21a,b
S
RI PT
O
S
f
O
NH
S
O
N H CN
n
N H
X
HCl
AC C
EP
TE D
26a (X = H, n = 1) 27a (X = Cl, n = 1) 27b (X = Cl, n = 2)
NH2
NBoc
O
g
S
N H
O
X CN 24a (X = H, n = 1) 25a (X = Cl, n = 1) 25b (X = Cl, n = 2)
n
N H
NHBoc
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
Design, synthesis and evaluation of new series of cyanothiophene-based inhibitors of MurF. Low micromolar inhibitors of MurF from Escherichia coli and Streptococcus pneumoniae
RI PT
were obtained.
AC C
EP
TE D
M AN U
SC
The inhibitors show antibacterial activity against strains of S. aureus and E. coli.
ACCEPTED MANUSCRIPT
SUPPORTING INFORMATION ON: Manuscript Title: Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold
Barreteau3, Alex J. O’Neill2, Didier Blanot3 and Stanislav Gobec1*
RI PT
Authors: Martina Hrast1, Marko Anderluh1, Damijan Knez1, Christopher P. Randall2, Hélène
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
2
School of Molecular and Cellular Biology and Antimicrobial Research Centre, University of
SC
1
Leeds, Leeds LS 9JT, U.K.
Univ Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS,
91405 Orsay, France
M AN U
3
*Corresponding author: Stanislav Gobec University of Ljubljana
1000 Ljubljana, Slovenia Tel: +386-1-4769500 Fax: +386-1-4258031.
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Faculty of Pharmacy, Aškerčeva 7
AC C
Table of Contents
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E-mail: [email protected]
Compound Characterization.………………......................................................….………...S2 References………………………………………………………………………………......S17 1
H NMR spectra for compounds 23a-b, 27b, 33, 35, 36, 37a-c, 37e-f, 38a-f, 39c, 40c, 42a,
42c-e and 43…………………………………………………………… …………………..S18 13
C NMR spectra for compounds 23b, 27b, 33, 35, 36, 37a-c, 37e-f, 38a, 38c, 38e-f, 39c, 40c,
42a-c, 42e and 43…………………………………………………………………………...S31 Table S1: ClogP values of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a, 27b, 33-38f, 39c, 42a-e and 43………………………………………………………………………..……....S42
S1
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5-acetoxy-2-chlorobenzoic acid (8). Yield = 95%; white crystals, mp = 117–120 °C, 1H NMR (400 MHz, CDCl3): δ 2.35 (s, 3H, CO-CH3), 7.28 (dd, J1 = 8.4, J2 = 2.8 Hz, 1H, Ar-H), 7.52 (d, J = 8.7 Hz, 1H, Ar-H), 7.79 (d, J = 2.3 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, CDCl3):
RI PT
δ 21.01, 125.62, 127.06, 129.12, 131.81, 132.39, 148.85, 168.91, 169.20 ppm. ESI HRMS calcd. for [M+(H)]+ 212.9955, found 212.9950. IR (KBr) νmax: 3070, 1762, 1709, 1602, 1576, 1474, 1412, 1374, 1308, 1262, 1217, 1112, 1049, 1014, 941, 907, 871, 833 cm-1.
4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenyl
acetate
SC
(10). Yield = 60%; pale yellow crystals, mp = 169–176 °C; 1H NMR (400 MHz, CDCl3): δ 1.81–1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.35 (s, 3H, O-CH3), 2.62–2.74 (m, 4H, CH2-CH2CH2-CH2), 7.27 (dd, J1 = 8.7, J2 = 2.8 Hz, 1H, Ar-H), 7.53 (d, J = 8.7 Hz, 1H, Ar-H), 7.70 (d, 13
C NMR (100 MHz, CDCl3): δ 20.99,
M AN U
J = 2.8 Hz, 1H, Ar-H), 9.82 (s, 1H, CO-NH) ppm.
22.10, 23.07, 24.00, 24.03, 94.81, 114.13, 125.07, 126.50, 127.78, 129.32, 131.38, 131.85, 132.15, 145.77, 149.69, 160.91, 168.77 ppm. ESI HRMS calcd. for [M+(H)]+ 375.0570, found 375.0558. IR (KBr) νmax: 3504, 3248, 3193, 3081, 3000, 2953, 2228, 1762, 1683, 1575, 1560, 1464, 1438, 1409, 1371, 1348, 1329, 1292, 1269, 1238, 1206, 1150, 1116, 1051, 1014,
TE D
969, 951, 914, 876, 842, 820 cm-1.
2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-hydroxybenzamide
(12).
Yield = 100%; pale brown powder, mp = 175–180 °C; 1H NMR (400 MHz, CDCl3): δ 1.82– 1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.61–2.74 (m, 4H, CH2-CH2-CH2-CH2), 6.80 (s, 1H, ArOH), 7.02 (dd, J1 = 8.7, J2 = 3.0 Hz, 1H, Ar-H), 7.35 (d, J = 8.8 Hz, 1H, Ar-H), 7.62 (d, J =
EP
3.0 Hz, 1H, Ar-H), 9.86 (s, 1H, CO-NH) ppm. 13C NMR (100 MHz, CDCl3): δ 22.07, 23.06, 24.01, 24.07, 94.99, 114.12, 118.56, 121.00, 121.89, 129.50, 131.13, 131.50, 132.22, 145.75,
AC C
155.48, 161.95 ppm. ESI HRMS calcd. for [M+(H)]+ 333.0465, found 333.0460. IR (KBr) νmax: 3430, 3321, 2936, 2840, 2209, 1647, 1602, 1573, 1557, 1484, 1466, 1423, 1310, 1259, 1217, 1118, 1036, 876, 822, 811 cm-1. 1H-pyrazol-1-ylmethanol (13).To a solution of 1H-pyrazol (0.750 g, 11.0 mmol) in diethyl ether (5 ml), 37% formaldehyde (0.9 mL, 11.0 mmol) was added. The reaction mixture was stirred over night at room temperature. The solvent was evaporated under reduced pressure and crude product was used without further purification [1].
S2
ACCEPTED MANUSCRIPT
Yield = 99%; white cubical crystals, mp = 84–86°C; 1H NMR (400 MHz, DMSO-d6): δ 5.37 (d, J = 7.6 Hz, 2H, CH2), 6.28 (t, J = 2.0 Hz, 1H, Ar-H), 6.72 (t, J = 7.6 Hz, 1H, OH), 7.48 (dd, J1 = 2.0, J2 = 0.8 Hz, 1H, Ar-H), 8.79 (dd, J1 = 2.0, J2 = 0.8 Hz, 1H, Ar-H) ppm. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(cyclopentylmethoxy)benzamide (15).
RI PT
Yield = 32%; yellow crystals, mp = 128–130 °C; 1H NMR (400 MHz, CDCl3): δ 1.35–1.40 (m, 2H), 1.59–1.69 (m, 4H), 1.79–1.86 (m, 6H), 2.38–2.45 (m, 1H), 2.54-2.58 (m, 4H), 4.27 (d, J = 7.1 Hz, 2H, O-CH2-CH), 6.96 (dd, J1 = 8.0, J2 = 2.6 Hz, 1H, Ar-H), 7.00–7.06 (m, 2H, 2 × Ar-H), 7.26 (d, J = 8.0 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, CDCl3): δ 21.07, 22.11,
SC
23.23, 23.49, 24.35, 28.47, 37.73, 71.82, 99.35, 114.31, 114.82, 117.30, 119.31, 119.92, 128.74, 131.05, 131.95, 154.98, 157.19, 161.43 ppm. ESI HRMS calcd. for [M+(H)]+ 381.1637, found 381.1634. IR (KBr) νmax: 3340, 3026, 2943, 2867, 2479, 2365, 2217, 2164,
M AN U
1942, 1869, 1610, 1586, 1488, 1458, 1402, 1385, 1352, 1301, 1262, 1242, 1214, 1172, 1100, 1082, 1026 cm-1. Anal. Calcd. for C22H24N2O2S × 0.7 H2O: C, 67.22; H, 6.51; N, 7.13. Found C, 67.15; H, 6.37; N, 7.12.
5-(benzyloxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide (17). Yield = 6%; white needles, mp = 162–164 °C; 1H NMR (400 MHz, CDCl3): δ 1.83–1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.66–2.72 (m, 4H, CH2-CH2-CH2-CH2), 5.14 (s, 2H, CH2-O), 7.12
TE D
(dd, J1= 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.33-7.49 (m, 6H, 6 × Ar-H), 7.65 (dd, J1 = 3.2, J2 = 1.5 Hz, 1H, Ar-H), 9.68 (s, 1H, CO-NH) ppm.
13
C NMR (100 MHz, CDCl3): δ 21.67, 22.61,
24.09, 24.48, 53.99, 70.44, 109.71, 113.12, 118.68, 121.87, 127.53, 128.16, 128.20, 128.65, 129.05, 130.49, 134.05, 135.54, 135.90, 136.19 , 148.55, 156.98, 167.95 ppm. ESI HRMS
EP
calcd. for [M+(H)]+ 423.0934, found 423.0937. IR (KBr) νmax: 3436, 3341, 2946, 2206, 1661, 1550, 1458, 1312, 1272, 1236, 1024, 879, 824 cm-1.
AC C
2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-((4-cyanobenzyl)oxy) benzamide (18). Yield = 30%; yellow powder, mp = 239–243 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.70–1.78 (m, 4H, CH2-CH2-CH2-CH2), 2.36–2.53 (m, 4H, CH2-CH2-CH2-CH2), 5.24 (s, 2H, CH2-O), 6.92 (dd, J1 = 8.7, J2 = 3.1 Hz,1 H, Ar-H), 7.20 (d, J = 3.0 Hz, 1H, ArH), 7.25 (d, J = 8.8, 1H, Ar-H), 7.67 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 7.87 (d, J = 8.2 Hz, 2H, 2 × Ar-H) ppm.
13
C NMR (100 MHz, DMSO-d6): δ 22.49, 23.35, 23.80, 24.17, 68.41, 90.28,
110.40, 115.04, 115.71, 118.76, 118.94, 121.43, 122.51, 128.06, 128.23, 130.21, 132.40, 142.44, 142.81, 156.05, 168.13 ppm. ESI HRMS calcd. for [M+(H)]+ 448.0887, found
S3
ACCEPTED MANUSCRIPT
448.0877. IR (KBr) νmax: 3431, 2929, 2845, 2228, 2197, 1592, 1533, 1470, 1392, 1360, 1304, 1257, 1228, 1153, 1130, 1106, 1049, 1026, 985, 889, 871, 817 cm-1. 5-(2-amino-2-oxoethoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2yl)benzamide (19). Yield = 57%; pale orange crystals, mp = 217–218 °C; 1H NMR (400 MHz,
RI PT
DMSO-d6): δ 1.75–1.81 (m, 4H, CH2-CH2-CH2-CH2), 2.53 (t, J = 6.4 Hz, 2H, CH2-CH2-CH2CH2), 2.64 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 4.51 (s, 2H, O-CH2-CO-NH2), 7.13 (dd, J1 = 8.8, J2 = 2.4 Hz, 1H, Ar-H), 7.21 (d, J = 3.2 Hz, 1H, Ar-H), 7.45 (bs, 1H, CO-NHAHB), 7.50 (d, J = 8.8 Hz, 1H, Ar-H), 7.61 (bs, 1H, CO-NHAHB), 12.32 (bs, 1H, NH-CO) ppm. 13C
SC
NMR (100 MHz, DMSO-d6): δ 21.68, 22.57, 23.39, 23.54, 66.98, 94.51, 113.94, 115.70, 117.93, 121.86, 128.20, 130.48, 131.30, 135.28, 145.55, 156.33, 164.02, 169.36 ppm. ESI HRMS calcd. for [M+(H)]+ 390.0679, found 390.0680. IR (KBr) νmax: 3413, 2927, 2205,
M AN U
1668, 1640, 1616, 1564, 1549, 1458, 1404, 1320, 1287, 1271, 1237, 1125, 1061, 1040 cm-1. HPLC tR = 13.462 min (96.4% at 220 nm, 98.9% at 254 nm). Tert-butyl
(2-(3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenoxy)
ethyl)carbamate (20a). Yield = 14%; white crystals, mp = 181–182 °C; 1H NMR (400 MHz, CDCl3): δ 1.48 (s, 9H, COO-(CH3)3), 1.83–1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.62–2.73 (m, 4H, CH2-CH2-CH2-CH2), 3.59 (q, J = 5.1 Hz, 2H, NH-CH2-CH2-O), 4.12 (t, J = 5.1 Hz, 2H,
TE D
NH-CH2-CH2-O), 5.03 (s, 1H, CO-NH-CH2), 7.13–7.18 (m, 1H, Ar-H), 7.44–7.50 (m, 3H, 3 × Ar-H), 8.86 (s, 1H, CO-NH) ppm.
13
C NMR (100 MHz, CDCl3): δ 22.12, 23.10, 23.99,
28.40, 39.97, 67.49, 79.67, 94.14, 113.68, 114.54, 119.20, 119.46, 128.84, 130.17, 131.13, 133.30, 146.64, 155.89, 159.09, 163.43 ppm. ESI HRMS calcd. for [M+(H)]+ 442.1801,
EP
found 442.1812. IR (KBr) νmax: 3358, 3245, 3077, 2979, 2939, 2880, 2223, 1684, 1670, 1576, 1539, 1465, 1395, 1365, 1330, 1295, 1280, 1268, 1254, 1228, 1172, 1135, 1111, 1051, 1001
9.42.
AC C
cm-1. Anal. Calcd. for C23H27N3O4S: C, 62.56; H, 6.16; N, 9.52. Found C, 62.34; H, 6.23; N,
Tert-butyl
(3-(3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenoxy)
propyl)carbamate (20b). Yield = 69%; white crystals, mp = 164–166 °C; 1H NMR (400 MHz, CDCl3): δ 1.44 (s, 9H, COO-(CH3)3), 1.81–1.89 (m, 4H, CH2-CH2-CH2-CH2), 1.98–2.03 (m, 2H, O-CH2-CH2-CH2), 2.61 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 2.67 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 3.35 (q, J = 6.4 Hz, 2H, CH2-NH-CO), 4.09 (t, J = 6.0 Hz, 2H, O-CH2CH2), 4.83 (t, J = 5.6 Hz, 1H, CH2-NH-COO), 7.13 (ddd, J1 = 8.4, J2 = 2.4, J3 = 1.2 Hz, 1H, Ar-H), 7.41 (t, J = 8.0 Hz, 1H, Ar-H), 7.44–7.48 (m, 2H, 2 × Ar-H), 9.09 (bs, 1H, NH-CO) S4
ACCEPTED MANUSCRIPT
ppm.
13
C NMR (100 MHz, acetone-d6): δ 22.93, 23.84, 24.48, 24.69, 28.66, (peak for
CH2CH2CH2 under solvent peak), 38.15, 66.64, 78.54, 96.34, 114.51, 114.77, 119.64, 120.95, 129.57, 130.62, 132.25, 134.88, 147.25, 156.75, 160.17, 165.39 ppm. ESI HRMS calcd. for [M+(H)]+ 456.1957, found 456.1960. IR (KBr) νmax: 3545, 3417, 3234, 2965, 2928, 2222,
RI PT
1616, 1544, 1441, 1384, 1287, 1268, 1224, 1170, 1099, 1046 cm-1. Anal. Calcd. for C24H29N3O4S: C, 63.27; H, 6.42; N, 9.22. Found C, 63.28; H, 6.34; N, 9.18. Tert-butyl
(2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)
phenoxy)ethyl)carbamate (21a). This compound was used for further reaction without
Tert-butyl
SC
purification.
(2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)
M AN U
phenoxy)propyl)carbamate (21a). This compound was used for further reaction without purification.
3-(3-aminopropoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide
(22b).
Yield = 73%; pale brown crystals, mp = 194–196 °C; 1H NMR (400 MHz, MeOD): δ 1.86– 1.93 (m, 4H, CH2-CH2-CH2CH2), 2.21 (p, J = 6.8 Hz, 2H, CH2-CH2-CH2), 2.64 (t, J = 4.8 Hz, 2H, CH2-CH2-CH2-CH2), 2.72 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 3.22 (p, J = 7.6
TE D
Hz, 2H, CH2-NH2), 4.23 (t, J = 5.6 Hz, 2H, CH2-CH2-O), 7.25 (ddd, J1 = 8.4, J2 = 2.8, J3 = 1.2 Hz, 1H, Ar-H), 7.50 (t, J = 8.4 Hz, 1H, Ar-H), 7.55 (dd, J1 = 2.4, J2 = 1.6 Hz, 1H, Ar-H), 7.64 (ddd, J1 = 7.6, J2 = 1.6, J3 = 1.2 Hz, 1H, Ar-H) ppm.
13
C NMR (100 MHz, pyridine-d5): δ
24.26, 25.20, 26.03, 26.22, 29.77, 39.19, 67.74, 99.12, 117.02, 117.25, 121.03, 123.34,
EP
131.25, 131.87, 133.98, 136.89, 149.51, 160.97, 168.03 ppm. ESI HRMS calcd. for [M+(H)]+ 356.1433, found 356.1424. IR (KBr) νmax: 3549, 3472, 3412, 3233, 2925, 2215, 2028, 1616, nm).
AC C
1544, 1464, 1383, 1271, 1148 cm-1. HPLC tR = 11.563 min (97.1% at 220 nm, 96.9% at 254
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) benzamide (23a). Yield = 12%; pale yellow crystals, mp = 198–202 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.75–1.80 (m, 4H, CH2-CH2-CH2-CH2), 2.50–2.55 (m, 2H, CH2-CH2-CH2-CH2, under solvent peak), 2.63 (t, 2H, J = 4.8 Hz, H, CH2-CH2-CH2-CH2), 3.24 (t, J = 5.2 Hz, 2H, H2NCH2-CH2-O), 4.22 (t, J = 7.0 Hz, 2H, H2N-CH2-CH2-O), 7.16 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.21 (d, J = 3.1 Hz, 1H, Ar-H), 7.51 (d, J = 8.7, 1H, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 22.31, 23.18, 23.74, 24.00, 55.99, 66.27, 91.15, 115.34, 115.86, 116.60, 117.91, 122.46, 122.89, 128.86, 130.32, 140.95, 156.26, 167.26 ppm. ESI HRMS calcd. for [M+(H)]+ S5
ACCEPTED MANUSCRIPT
376.0887, found 376.0875. IR (KBr) νmax: 3371, 2934, 2363, 2216, 1719, 1660, 1578, 1462, 1398, 1326, 1289, 1262, 1229, 1121, 1025, 976, 896, 826 cm-1. HPLC tr = 11.650 min (94.7% at 220 nm, 95.0% at 25 4 nm). 5-(3-aminopropoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)
RI PT
benzamide (23b). Yield = 16%; yellow crystals, mp = 87–90°C; 1H NMR (400 MHz, CDCl3): δ 1.59–2.44 (bs, 2H, -NH2), 1.83–1.90 (m, 4H, CH2-CH2-CH2-CH2), 1.94 (q, J = 6.5 Hz, 2H, H2N-CH2-CH2-CH2-O), 2.62–2.73 (m, 4H, CH2-CH2-CH2-CH2), 2.85 (t, J = 6.8 Hz, 2H, H2NCH2-CH2-CH2-O), 4.08 (t, J = 6.1 Hz, 2H, H2N-CH2-CH2-CH2-O), 7.02 (dd, J1 = 8.8, J2 = 3.1
SC
Hz, 1H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.46 (d, J = 3.1 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 22.40, 23.26, 23.77, 24.09, 27.30, 36.54, 64.95, 90.65, 114.98, 115.46, 118.45, 122.12, 128.50, 130.14, 134.34, 141.74, 156.43, 167.81, 173.26 ppm. ESI
M AN U
HRMS calcd. for [M+(H)]+ 390.1043, found 390,1047. IR (KBr) νmax: = 3436, 2930, 2196, 1508, 1466, 1370, 1289, 1223, 1025, 815 cm-1. HPLC tr = 12.038 min (93.9% at 220 nm, 93.5% at 25 4nm).
Tert-butyl(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-4,5,6,7-tetrahydrobenzo [b]thiophen-2-yl)carbamoyl)phenoxy)ethylamino)methylencarbamate (25a). Yield = 96%; pale yellow crystals; mp = 103–106 °C; 1H NMR (400 MHz, CDCl3): δ 1.52 (s, 9H, COO-
TE D
(CH3)3), 1.54 (s, 9H, COO-(CH3)3), 1.80–1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.65 (d, J = 5.7 Hz, 2H, CH2-CH2-CH2-CH2), 2.70 (d, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 3.89 (q, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 4.17 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 7.13 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.42 (d, J = 8.8 Hz, 1H, Ar-H), 7.56 (d, J = 3.1 Hz, 1H, Ar-H), 8.82 (s,
EP
1H, NH-CH2-CH2-O), 9.72 (s, 1H, NH-CO), 11.48 (s, 1H, NH-Boc) ppm. Tert-butyl(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-4,5,6,7-tetrahydrobenzo
AC C
[b]thiophen-2-yl)carbamoyl)phenoxy)propylamino)methylencarbamate (25b). Yield = 71%, white powder; mp = 109–113 °C; 1H NMR (400 MHz, CDCl3): δ 1.52 (s, 18H, 2 × COO(CH3)3), 1.81–1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.82 (q, J = 6.3 Hz, 2H, O-CH2-CH2-CH2NH), 2.61–2.75 (m, 4H, CH2-CH2-CH2-CH2), 3.68 (q, J = 6.4 Hz, 2H, O-CH2-CH2-CH2-NH), 4.11–4.16 (m, 2H, O-CH2-CH2-CH2-NH), 7.14 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.40 (d, J = 8.9 Hz, 1H, Ar-H), 7.59 (d, J = 3.1 Hz, 1H, Ar-H), 8.58–8.77 (m, 1H, NH-CH2-CH2-CH2O), 9.70 (s, 1H, NH-CO), 11.53 (s, 1H, NH-Boc) ppm.
13
C NMR (100 MHz, DMSO-d6): δ
21.69, 22.58, 23.38, 23.53, 27.55, 27.94, 31.28, 53.60, 55.88, 67.20, 78.08, 82.85, 94.31,
S6
ACCEPTED MANUSCRIPT
113.19, 115.47, 117.31, 121.32, 128.07, 130.42, 131.19, 133.93, 135.38, 152.00, 155.13, 156.95, 163.02 ppm Amino((2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl) phenoxy)ethyl)amino)methaniminium chloride (27a). Yield = 93%; yellow crystals, mp = 99–
RI PT
103 °C; 1H NMR (400 MHz, MeOD): δ 1.80–1.96 (m, 4H, CH2-CH2-CH2-CH2), 2.58–2.75 (m, 4H, CH2-CH2-CH2-CH2), 3.63–3.69 (m, 2H, O-CH2-CH2-NH), 4.20 (t, J = 5.0 Hz, 2H, OCH2-CH2-NH), 7.15 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.23 (d, J = 3.0 Hz, 1H, Ar-H), 7.48 (d, J = 8.9 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 21.67, 22.56, 23.38,
SC
23.53, 66.70, 94.49, 113.95, 115.26, 118.07, 121.80, 128.21, 130.26, 131.29, 135.41, 145.56, 156.60, 156.98, 163.98 ppm. (1 C covered by the signal of the solvent). ESI HRMS calcd. for [M+(H)]+ 418.1104, found 418.1108. IR (KBr) νmax: 3403, 2934, 2214, 1654, 1550, 1458,
M AN U
1290, 1218, 1065, 819 cm-1. HPLC tR = 12.342 min (95.3% at 220 nm, 97.7% at 254 nm). Amino((2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl) phenoxy)propyl)amino)methaniminium chloride (27b). Yield = 99%; yellow solid, mp = 94– 97 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.72–1.83 (m, 4H, CH2-CH2-CH2-CH2), 1.94 (p, J = 6.4 Hz, 2H, O-CH2-CH2-CH2-NH), 2.50–2.55 (m, 2H, CH2-CH2-CH2-CH2, under solvent peak), 2.59–2.67 (m, 2H, CH2-CH2-CH2-CH2), 3.28 (q, J = 6.5 Hz, 2H, O-CH2-CH2-CH2-
TE D
NH), 4.08 (t, J = 6.1 Hz, 2H, O-CH2-CH2-CH2-NH), 7.13 (dd, J1 = 8.8, J2 = 3.0 Hz, 1H, ArH), 7.19 (d, J = 3.0 Hz, 1H, Ar-H), 7.49 (d, J = 8.8 Hz, 1H, Ar-H), 7.74 (t, J = 5.7 Hz, 1H, NH-Boc), 12.30 (s, 1H, NH-CO) ppm.
13
C NMR (100 MHz, DMSO-d6): δ 21.67, 22.56,
23.38, 23.53, 28.00, 37.73, 65.49, 94.48, 113.95, 115.18, 117.87, 121.41, 128.18, 130.52,
EP
131.28, 135.38, 145.56, 156.82, 156.92, 164.07 ppm. HPLC tR = 12.821 min (95.2% at 220 nm, 96.8% at 254 nm).
AC C
4-(2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5,6,7tetrahydrobenzo[b]thiophen-6-yl)phenyl acetate (33b). Yield = 70%; yellow crystals, mp = 194–196 °C; 1H NMR (400 MHz, CDCl3): δ 1.99 (qd, J1 = 11.2, J2 = 5.6 Hz, 1H, CHCHAHB-CH2), 2.18–2.24 (m, 1H, CH-CHAHB-CH2), 2.33 (s, 3H, CO-CH3), 2.72–2.89 (m, 3H, CH2CH-CH2-CH2), 2.98–3.12 (m, 2H, CH2-CH), 3.85 (q, = 5.6 Hz, 2H, O-CH2-CH2-NH), 4.19 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 6.77 (bs, 1H, NH-CO-CF3), 7.07 (dd, J1 = 8.8, J2 = 3.2 Hz, 1H, Ar-H), 7.08 (d, J = 8.8 Hz, 2H, Ar-H), 7.30 (d, J = 8.0 Hz, 2H, Ar-H), 7.45 (d, J = 8.8 Hz, 1H, Ar-H), 7.58 (d, J = 3.2 Hz, 1H, Ar-H), 9.77 (bs, 1H, NH-CO) ppm. 13C NMR (100 MHz, CDCl3): δ 21.18, 24.28, 29.38, 31.83, 39.18, 40.22, 66.27, 94.41, 114.12, 115.73 S7
ACCEPTED MANUSCRIPT
(q, 1JC,F =285.8 Hz, CF3), 116.95, 120.01, 121.70, 123.02, 127.85, 128.63, 131.10, 131.68, 132.06, 142.53, 146.41, 149.26, 157.20, 157.52 (q, 2JC,F = 37.1 Hz, CO-CF3), 161.57, 169.75 ppm. ESI HRMS calcd. for [M+(H)]+ 606.1077, found 606.1081. IR (KBr) νmax: 3414, 3198, 3078, 2927, 2222, 1730, 1685, 1560, 1507, 1461, 1384, 1297, 1242, 1204, 1186, 1155, 1039,
RI PT
1020 cm-1. HPLC tR = 16.721 min (96.3% at 220 nm, 100% at 254 nm). 5-(2-aminoethoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)
benzamide (36). Yield = 76%; pale brown solid, mp = 128–130 °C; 1H NMR (400 MHz, MeOD): δ 2.79 (t, J = 5.8 Hz, 2H, CH2-CH2-N), 3.30 (t, J = 5.8 Hz, 2H, CH2-CH2-N), 3.35 (t,
SC
J = 5.0 Hz, 2H, O-CH2-CH2-NH2), 4.09 (s, 2H, CH2-N), 4.26 (t, J = 5.0 Hz, 2H, O-CH2-CH2NH2), 7.15 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.27 (d, J = 3.1 Hz, 1H, Ar-H), 7.46 (d, J = 8.9 Hz, 1H, Ar-H) ppm.
13
C NMR (100 MHz, MeOD): δ 23.11, 40.33, 43.03, 43.73, 66.43,
M AN U
96.06, 114.73, 116.77, 119.43, 124.81, 125.42, 131.14, 132.42, 137.16, 150.59, 158.36 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 377.0839, found 377.0833. IR (KBr) νmax 3068, 2949, 2718, 2547, 2202, 1642, 1549, 1508, 1463, 1401, 1366, 1327, 1295, 1261, 1217, 1180, 1153, 1123, 1083, 1020, 1003 cm-1. HPLC tR = 6.518 min (100% at 220 nm, 100% at 254 nm). 2-chloro-N-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2(2,2,2-trifluoroacetamido)ethoxy)benzamide (37b). Yield = 53%; white crystals, mp = 88–90
TE D
°C; 1H NMR (400 MHz, CDCl3): δ 2.72 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 2.81 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 3.55 (s, 2H, CH2-N), 3.65 (s, 2H, N-CH2-Ar), 3.77–3.81 (m, 5H, O-CH3 + O-CH2-CH2-NH), 4.13 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 6.75 (bs, 1H, NH-CO-CF3), 6.86 (d, J = 8.6 Hz, 2H, Ar-H), 7.02 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.25 (d, J = 8.6 Hz, 2H,
EP
Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.51 (d, J = 3.1 Hz, 1H, Ar-H), 9.73 (s, 1H, NH-COthiophen) ppm. 13C NMR (100 MHz, acetone-d6): δ 24.99, 40.04, 50.02, 51.29, 55.53, 61.47,
AC C
67.18, 95.26, 114.09, 114.54, 115.70, 116.38, 118.55, 119.28, 123.30, 127.62, 130.97, 131.16, 131.24, 131.85, 135.96, 147.21, 158.29, 159.99, 164.48 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 593.1237, found 593.1251. IR (KBr) νmax 3326, 2214, 1715, 1668, 1546, 1511, 1459, 1366, 1290, 1242, 1210, 1158, 1059, 1034, 1007 cm-1. HPLC tR = 12.107 min (98.9% at 220 nm, 100% at 254 nm). Methyl
4-((2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5-
dihydrothieno[2,3-c]pyridin-6(7H)-yl)methyl)benzoate (37c). Yield = 59%; yellow crystals, mp = 109–111 °C; 1H NMR (400 MHz, acetone-d6): δ 2.70 (t, J = 5.7 Hz, 2H, CH2-CH2-N), 2.87 (t, J = 5.7 Hz, 2H, CH2-CH2-N), 3.65 (s, 2H, CH2-N), 3.77 (q, 2H, J = 5.4 Hz, 2H, OS8
ACCEPTED MANUSCRIPT
CH2-CH2-NH-CO), 3.85 (s, 2H, N-CH2-Ar), 3.88 (s, 3H, COO-CH3), 4.26 (t, J = 5.4 Hz, 2H, O-CH2-CH2-NH-CO), 7.11 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.28 (d, J = 3.0 Hz, 1H, ArH), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 7.56 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 7.99 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 8.77 (bs, 1H, NH-CO-CF3), 11.02 (s, 1H, NH-CO-thiophene) ppm. 13C NMR
RI PT
(100 MHz, acetone-d6): δ 24.93, 40.04, 50.23, 51.45, 52.34, 61.50, 67.19, 95.24, 114.06, 115.69, 116.38, 118.55, 119.28, 123.30, 127.37, 129.74, 130.11, 130.33, 131.11, 131.85, 135.96, 145.27, 147.33, 158.29, 164.52, 167.19 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 621.1186, found 621.1190. IR (KBr) νmax 3308, 3088, 2957, 2911, 2754, 2214, 1733, 1697, 1666, 1611, 1577, 1459, 1434, 1416, 1372, 1330, 1283, 1220, 1183, 1140, 1112, 1057, 1037,
SC
1011 cm-1. HPLC tR = 12.004 min (100% at 220 nm, 100% at 254 nm).
2-chloro-N-(3-cyano-6-(3-hydroxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2-
M AN U
(2,2,2-trifluoroacetamido)ethoxy)benzamide (37d). Yield = 60%; pale yellow crystals, mp = 228–230 °C; 1H NMR (400 MHz, acetone-d6): δ 2.68 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 2.85 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.69 (s, 2H, N-CH2-Ar), 3.78 (q, J = 5.4 Hz, 2H, O-CH2-CH2-NH), 4.26 (t, J = 5.4 Hz, 2H, O-CH2-CH2-NH), 6.68-6.87 (m, 3H, 2 × Ar-H + NH-CO-CF3), 6.92 (s, 1H, Ar-H), 7.10–7.18 (m, 2H, 2 × Ar-H), 7.28 (d, J = 3.0 Hz, 1H, Ar-H), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 8.78 (s, 1H, NH-CO-thiophen), 11.01 (s, 1H, OH)
TE D
ppm. ESI HRMS m/z calcd. for [M+(H)]+ 579.1081, found 579.1076. IR (KBr) νmax 3323, 2216, 1772, 1730, 1656, 1581, 1552, 1481, 1457, 1400, 1345, 1310, 1262, 1211, 1182. 1137, 1121, 1084, 1062, 1043 cm-1. HPLC tR = 11.233 min (100% at 220 nm, 100% at 254 nm). 2-chloro-N-(3-cyano-6-(4-cyanobenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2-
EP
(2,2,2-trifluoroacetamido)ethoxy)benzamide (37e). Yield = 74%; yellow crystals, mp = 170– 172 °C; 1H NMR (400 MHz, CDCl3): δ 2.77 (t, J = 5.0 Hz, 2H, CH2-CH2-N), 3.85 (t, J = 5.0
AC C
Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2-Ar), 3.82 (q, J = 4.8 Hz, 2H, O-CH2-CH2-NH-CO), 4.16 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH-CO), 6.75 (bs, 1H, NH-COCF3), 7.0 (d, J = 8.9 Hz, 1H, Ar-H), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 7.49–7.55 (m, 3H, 3 × ArH), 7.65 (d, J = 7.3 Hz, 1H, Ar-H), 9.79 (s, 1H, NH-CO-thiophene) ppm.
13
C NMR (100
MHz, CDCl3): δ 24.03, 39.37, 49.70, 51.08, 61.25, 66.52, 94.41, 111.51, 114.00, 117.24, 117.33, 119.04, 120.42, 123.27, 126.61, 129.57, 130.14, 131.60, 132.39, 132.57, 143.91, 146.90, 157.41, 157.82, 161.64 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 588.1084, found 588.1081. IR νmax 3315, 3053, 2835, 2219, 1703, 1654, 1585, 1554, 1481, 1467, 1455, 1434, 1394, 1339, 1319, 1299, 1276, 1230, 1202, 1178, 1161, 1112, 1065, 1050, 1034, 1010 cm-1. HPLC tR = 11.750 min (97.4% at 220 nm, 100% at 254 nm). S9
ACCEPTED MANUSCRIPT
N-(6-(4-(1H-tetrazol-5-yl)benzyl)-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamide (37f). Yield = 45%; pale yellow crystals, mp = 137–139 °C; 1H NMR (400 MHz, MeOD): δ 2.89 (t, J = 5.5 Hz, 2H, CH2-CH2N), 3.29 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 3.69 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 4.03 (s,
RI PT
2H, CH2-N), 4.15 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 4.19 (s, 2H, N-CH2-Ar), 7.10 (d, J = 8.9 Hz, 1H, Ar-H), 7.19 (d, J = 3.0 Hz, 1H, Ar-H), 7.42 (d, J = 8.9 Hz, 1H, Ar-H), 7.62 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 8.08 (d, J = 8.4 Hz, 2H, 2 × Ar-H) ppm. 2 × NH exchanged. 13C NMR (400 MHz, MeOD): δ 24.11, 40.39, 50.61, 51.30, 61.46, 67.54, 95.65, 114.21, 116.18, 116.63, 119.75, 124.14, 124.54, 128.59, 129.03, 131.16, 132.21, 132.38, 136.11, 136.97, 149.19,
SC
158.93, 159.26, 159.63, 166.49 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 631.1254, found 631.1249. IR (KBr) νmax 2942, 2218, 1712, 1673, 1546, 1460, 1429, 1368, 1288, 1209, 1184,
M AN U
1154, 1062, 1036, 1012 cm-1. HPLC tR = 10.862 min (100% at 220 nm, 100% at 254 nm). 5-(2-aminoethoxy)-N-(6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2chlorobenzamide (38a). Yield = 71%; yellow crystals, mp = 122–124 °C; 1H NMR (400 MHz, MeOD): δ 2.72 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 2.88 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.13 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.59 (s, 2H, CH2-N), 3.76 (s, 2H, N-CH2-Ar), 4.12 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 7.07 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.22 (d, J = 13
C NMR (100 MHz, DMSO-d6):
TE D
3.1 Hz, 1H, Ar-H), 7.28–7.41 (m, 6H, 6 × Ar-H) ppm.
24.01, 35.80, 49.46, 51.09, 60.95, 65.33, 90.72, 115.66, 115.98, 117.56, 121.18, 122.64, 127.03, 127.68, 128.27, 128.46, 128.80, 129.04, 130.43, 138.46, 140.54, 156.17, 162.31, 167.19 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 467.1309, found 467.1298. IR (KBr) νmax
EP
2926, 2880, 2804, 2762, 2199, 1657, 1574, 1512, 1463, 1406, 1365, 1318, 1275, 1224, 1165, 1132, 1080, 1053 cm-1. HPLC tR = 8.527 min (100% at 220 nm, 100% at 254 nm). [2,3-
AC C
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-tetrahydrothieno
c]pyridin-2-yl)benzamide (38b). Yield = 75%; yellow crystals, mp = 123–125 °C; 1H NMR (400 MHz, MeOD): δ 2.71 (t, J = 5.8 Hz, 2H, CH2-CH2-N), 2.86 (t, J = 5.4 Hz, 2H, CH2-CH2N), 3.12 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH2), 3.56 (s, 2H, CH2-N), 3.69 (s, 2H, N-CH2-Ar), 3.79 (s, 3H, O-CH3), 4.11 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH2), 6.91 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.06 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.21 (d, J = 3.0 Hz, 1H, Ar-H), 7.30 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.38 (d, J = 8.9 Hz, 1H, Ar-H) ppm. ESI HRMS m/z calcd. for [M+(H)]+ 497.1414, found 497.1409. IR νmax 3354, 3298, 2820, 2363, 2217, 1736, 1662, 1586, 1512, 1458, 1418, 1365, 1312, 1276, 1244, 1226, 1169, 1132, 1070, 1049, 1029 cm-1. HPLC tR = 8.794 min (100% at 220 nm, 100% at 254 nm). IR νmax 3354, 3298, 2920, 2820, S10
ACCEPTED MANUSCRIPT
2363, 2217, 1736, 1662, 1586, 1512, 1458, 1418, 1365, 1312, 1276, 1244, 1226, 1169, 1132, 1070, 1049, 1029 cm-1. HPLC tR = 8.794 min (100% at 220 nm, 100% at 254 nm). 4-((2-(5-(2-aminoethoxy)-2-chlorobenzamido)-3-cyano-4,5-dihydrothieno[2,3-c]pyridin6(7H)-yl)methyl)benzoic acid (38c). Yield = 79 %; pale yellow crystals, mp = 175–177 °C; 1H
RI PT
NMR (400 MHz, DMSO-d6): δ 2.53 (t, J = 5.2 Hz, 2H, CH2-CH2-N), 2.73 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 3.19 (t, 2H, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.43 (s, 2H, CH2-N), 3.73 (s, 2H, N-CH2-Ar), 4.15 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 6.95 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.23 (d, J = 3.1 Hz, 1H, Ar-H), 7.33(d, J = 8.8 Hz, 1H, Ar-H), 7.46 (d, J = 8.3 Hz,
SC
2H, 2 × Ar-H), 7.991 (d, J = 8.3 Hz, 2H, 2 × Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 23.99, 38.56, 49.56, 51.16, 60.50, 65.39, 90.61, 115.60, 115.99, 117.66, 120.95, 122.64, 127.59, 128.65, 129.33, 130.36, 130.42, 140.70, 143.46, 145.45, 156.18, 167.31, 167.51 ppm.
M AN U
ESI HRMS m/z calcd. for [M+(H)]+ 511.1207, found 511.1203. IR (KBr) νmax 2927, 2804, 2215, 2198, 1640, 1584, 1568, 1516, 1462, 1366, 1306, 1275, 1224, 1164, 1131, 1078, 1053, 1022 cm-1. HPLC tR = 7.718 min (100% at 220 nm, 100% at 254 nm). 5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(3-hydroxybenzyl)-4,5,6,7-tetrahydrothieno
[2,3-
c]pyridin-2-yl)benzamide (38d). Yield = 89%; pale yellow crystals, mp = 113–115 °C; 1H NMR (400 MHz, MeOD): δ 2.72 (t, J = 5.2 Hz, 2H, CH2-CH2-N), 2.87 (t, J = 5.2 Hz, 2H,
TE D
CH2-CH2-N), 3.12 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.59 (s, 2H, CH2-N), 3.68 (s, 2H, NCH2-Ar), 4.11 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 6.72 (d, J = 8.0 Hz, 1H, Ar-H), 6.83– 6.86 (m, 2H, 2 × Ar-H), 7.06 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.15–7.22 (m, 2H, 2 × ArH), 7.39 (d, J = 8.9 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, MeOD): δ 25.16, 41.34, 50.75,
EP
54.96, 62.79, 69.42, 95.69, 115.70, 116.68, 117.52, 118.89, 121.91, 124.39, 126.74, 129.08, 130.62, 131.12, 131.60, 132.17, 138.17, 139.91, 158.84, 167.99 ppm. ESI HRMS m/z calcd.
AC C
for [M+(H)]+ 483.1258, found 483.1245. IR νmax 3063, 2925, 2809, 2210, 1663, 1584, 1545, 1457, 1405, 1365, 1278, 1225, 1154, 1120, 1054, 1008 cm-1. HPLC tR = 7.930 min (99.2% at 220 nm, 99.5% at 254 nm). 5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-cyanobenzyl)-4,5,6,7-tetrahydrothieno[2,3c]pyridin-2-yl)benzamide (38e). Yield = 83%; yellow solid, mp = 125–127 °C; 1H NMR (400 MHz, acetone-d6): δ 2.69 (t, J = 5.7 Hz, 2H, CH2-CH2-N), 3.87 (t, J = 5.7 Hz, 2H, CH2-CH2N), 3.56 (t, J = 5.8 Hz, 2H, O-CH2-CH2-NH2), 3.66 (s, 2H, CH2-N), 3.86 (s, 2H, N-CH2-Ar), 4.25 (t, J = 5.8 Hz, 2H, O-CH2-CH2-NH2), 7.09 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.27 (d, J = 3.0 Hz, 1H, Ar-H), 7.40 (d, J = 8.9 Hz, 1H, Ar-H), 7.64 (d, J = 8.3 Hz, 2H, 2 × Ar-H), S11
ACCEPTED MANUSCRIPT
7.76 (d, J = 8.3 Hz, 2H, 2 × Ar-H) ppm.
13
C NMR (100 MHz, acetone-d6): δ 24.87, 50.22,
51.19, 51.48, 61.27, 70.02, 95.08, 111.73, 114.26, 116.43, 119.02, 119.50, 122.76, 126.97, 130.49, 130.97, 131.71, 133.08, 136.05, 145.66, 147.98, 158.83, 164.77 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 492.1261, found 492.1271. IR (KBr) νmax 2926, 1823, 2215, 1668, 1553,
RI PT
1458, 1413, 1363, 1293, 1218, 1167, 1134, 1116, 1067, 1012 cm-1. HPLC tR = 8.375 min (94.5% at 220 nm, 95.6% at 254 nm).
N-(6-(4-(1H-tetrazol-5-yl)benzyl)-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2aminoethoxy)-2-chlorobenzamide (38f). Yield = 64%; yellow crystals, mp = 221–223 °C; 1H
SC
NMR (400 MHz, DMSO-d6): δ 2.61 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 2.78 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 3.25 (t, 2H, J = 4.9 Hz, 2H, O-CH2-CH2-NH2), 3.53 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2-Ar), 4.21 (t, J = 4.9 Hz, 2H, O-CH2-CH2-NH2), 7.10 (dd, J1 = 8.8, J2 = 3.0 Hz,
M AN U
1H, Ar-H), 7.24 (d, J = 3.0 Hz, 1H, Ar-H), 7.37 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 7.46 (d, J = 8.9 Hz, 1H, Ar-H), 7.95 (d, J = 8.2 Hz, 2H, 2 × Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 23.78, 38.36, 49.00, 50.42, 60.47, 65.14, 93.22, 115.61, 117.66, 122.24, 125.90, 128.99, 129.54, 130.37, 130.61, 137.33, 156.34, 159.82, 164.88 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 535.1431, found 535.1428. IR νmax 3633, 2837, 2217, 2116, 2022, 1646, 1566, 1583, 1476, 1451, 1403, 1365, 1312, 1289, 1233, 1166, 1122, 1056, 1023, 1011 cm-1. HPLC
Methyl
TE D
tR = 7.689 min (97.1% at 220 nm, 100% at 254 nm).
4-((2-(5-(2-aminoethoxy)-2-chlorobenzamido)-3-cyano-4,5-dihydrothieno[2,3-
c]pyridin-6(7H)-yl)methyl)benzoate (39c). Yield = 71%; pale yellow crystals, mp = 136–138 °C; 1H NMR (400 MHz, MeOD): δ 2.73 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 2 .87 (t, J = 5.4 Hz,
EP
2H, CH2-CH2-N), 3.33 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.62 (s, 2H, CH2-N), 3.83 (s, 2H, N-CH2-Ar), 3.91 (s, 3H, COO-CH3), 4.24 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 7.15 (dd,
AC C
J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.26 (d, J = 3.1 Hz, 1H, Ar-H), 7.46 (d, J = 8.9 Hz, 1H, ArH), 7.52 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 8.01 (d, J = 8.9 Hz, 2H, 2 × Ar-H) ppm.
13
C NMR
(100 MHz, MeOD): δ 25.20, 40.43, 50.70, 51.90, 52.77, 62.20, 66.72, 96.16, 114.74, 116.73, 119.56, 124.74, 127.70, 130.64, 130.85, 131.65, 132.44, 136.81, 144.69, 148.83, 158.43, 166.54, 168.52, 178.99 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 525.1363, found 525.1354. IR νmax 2950, 2911, 2797, 2211, 1718, 1608, 1574, 1530, 1459, 1434, 1401, 1311, 1275, 1194, 1175, 1105, 1017 cm-1. HPLC tR = 8.752 min (96.9% at 220 nm, 95.2% at 254 nm). Tert-butyl
(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(benzyl)-4,5,6,7-tetra-
hydrothieno[2,3-c]pyridine-2-ylcarbamoyl)phenoxy)ethylamino)methylenecarbamate
(40a). S12
ACCEPTED MANUSCRIPT
Yield = 56%; yellow solid, mp = 92–94 °C; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, O(CH3)3), 1.49 (s, 9H, O-(CH3)3), 2.77 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 2 .88 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.58 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2-Ar), 3.88 (q, J = 5.3 Hz, 2H, O-CH2CH2-NH), 4.16 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 7.13 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, ArCH2-NH), 9.76 (s, 1H, NH-Boc), 11.46 (s, NH-CO-thiophene) ppm.
RI PT
H), 7.27–7.38 (m, 6H, 6 × Ar-H), 7.56 (d, J = 3.1 Hz, 1 H, Ar-H), 8.74 (t, J = 5.3 Hz, O-CH213
C NMR (100 MHz,
CDCl3): δ 24.24, 28.25, 28.47, 40.08, 49.57, 50.87, 61.85, 67.13, 79.69, 83.51, 94.21, 114.07, 117.28, 120.61, 122.69, 126.74, 127.74, 128.72, 129.34, 130.06, 131.56, 132.09, 137.62, 146.96, 153.25, 156.54, 157.86, 161.86, 163.55 ppm. ESI HRMS m/z calcd. for [M+(H)]+
SC
709.2575, found 709.2565. IR νmax 3334, 2977, 2221, 1720, 1682, 1638, 1617, 1547, 1478, 1452, 1412, 1361, 1322, 1285, 1253, 1229, 1137, 1048, 1023 cm-1.
(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-
M AN U
Tert-butyl
tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylene-carbamate (40b). Yield = 74%; pale yellow crystals, mp = 107–109 °C; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, O-(CH3)3), 1.48 (s, 9H, O-(CH3)3), 2.73 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 2 .84 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 3.57 (s, 2H, CH2-N), 3.67 (s, 2H, N-CH2-Ar), 3.79 (s, 3H, OCH3), 3.83 (q, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 4.11 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH),
TE D
6.86 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.08 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.26 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.37 (d, J = 8.9 Hz, 2H, Ar-H), 7.51 (d, J = 3.0 Hz, 1 H, Ar-H),8.69 (t, J = 5.2 Hz, O-CH2-CH2-NH), 9.75 (s, 1H, NH-Boc), 11.46 (s, NH-CO-thiophene) ppm. 13C NMR (100 MHz, CDCl3): δ 24.26, 28.26, 28.48, 40.09, 49.44, 50.77, 55.49, 61.23, 67.15, 79.68,
EP
83.52, 94.24, 114.06, 117.33, 020.66, 122.70,126.21, 126.84, 129.67, 130.11, 130.56, 131.54, 132.13, 146.94, 153.28, 156.56, 157.91, 159.24, 161.84, 163.60 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 739.2654, found 739.2672. IR νmax 3332, 2976, 2933, 2216, 1721, 1638, 1613,
AC C
1546, 1512, 1457, 1411, 1364, 1320, 1284, 1244, 1134, 1048, 1024 cm-1. Methyl
4-((2-(5-(2-(2,3-bis(tert-butoxycarbonyl)guanidino)ethoxy)-2-chlorobenzamido)-3-
cyano-4,5-dihydrothieno[2,3-c]pyridin-6(7H)-yl)methyl)benzoate (40c). Yield = 54%; yellow crystals, mp = 106–108 °C; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, O-(CH3)3), 1.49 (s, 9H, O-(CH3)3), 2.75 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 2 .87 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.79 (s, 2H, N-CH2-Ar), 3.84 (q, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 3.90 (s, 3H, COO-CH3), 4.12 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 7.09 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.37 (d, J = 8.9 Hz, 1H, Ar-H), 7.44 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 7.52 (d, J = 3.1 Hz, 1 H, Ar-H), 8.01 (d, J = 8.3 Hz, 2H, 2 × Ar-H),8.72 (t, J = 5.3 Hz, O-CH2-CH2-NH), 9.77 S13
ACCEPTED MANUSCRIPT
(s, 1H, NH-Boc), 11.45 (s, NH-CO-thiophene) ppm.
13
C NMR (100 MHz, CDCl3): δ 24.23,
28.27, 28.49, 40.15, 49.69, 51.01, 52.25, 61.44, 67.15, 79.87, 83.60, 94.24, 114.04, 117.38, 120.72, 122.73, 126.51, 129.15, 129.67, 130.07, 131.48, 132.16, 143.12, 147.07, 153.28, 156.60, 157.91, 161.87, 163.47, 167.11 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 767.2630,
RI PT
found 767.2632. Tert-butyl (tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(3-hydroxybenzyl)-4,5,6,7 tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylene-carbamate
(40d).Yield = 36%; yellow solid, mp = 117–119 °C; 1H NMR (400 MHz, CDCl3): δ 1.45 (s,
SC
9H, O-(CH3)3), 1.47 (s, 9H, O-(CH3)3), 2.63 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 2.78 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 3.47 (s, 2H, CH2-N), 3.59 (s, 2H, N-CH2-Ar), 3.71 (q, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 3.90 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 6.80–6.83 (m, 3H, 3 × Ar-H),
M AN U
6.93 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.13-7.15 (m, 2H, 2 × Ar-H), 7.28 (d, J = 8.9 Hz, 1H, Ar-H), 8.71 (t, J = 5.5Hz, 1H, O-CH2-CH2-NH), 10.00 (bs, 1H, NH-Boc), 11.46 (bs, NHCO-thiophene). ESI HRMS m/z calcd. for [M+(H)]+ 725.2524, found 725.2503. Tert-butyl
(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(4-cyanobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylenecarbamate
Tert-butyl
TE D
(40e). Yield = 23%.This compound was used for further reaction without purification. (tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylene
carbamate
(41). Yield = 63%; yellow crystals, mp = 133–135 °C; 1H NMR (400 MHz, MeOD): δ 1.49
EP
(s, 9H, O-(CH3)3), 1.53 (s, 9H, O-(CH3)3), 1.97 (qd, J1 = 10.8, J2 = 5.6 Hz, 1H, CH-CHAHBCH2), 2.10–2.16 (m, 1H, CH-CHAHB-CH2), 2.67–2.80 (m, 3H, CH2-CH-CH2-CH2), 2.91–3.03 (m, 2H, CH2-CH), 3.79 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 4.20 (t, J = 5.2 Hz, 2H, O-CH2-
AC C
CH2-NH2), 6.76 (d, J = 8.8 Hz, 2H, Ar-H), 7.14 (d, J = 8.4 Hz, 2H, Ar-H), 7.17 (dd, J1 = 8.8, J2 = 3.2 Hz, 1H, Ar-H), 7.24 (d, J = 2.8 Hz, 1H, Ar-H), 7.44 (d, J = 8.8 Hz, 1H, Ar-H) ppm. 13
C NMR (100 MHz, acetone-d6): δ 25.14, 28.15, 28.49, 30.61, 31.63, 32.61, 40.83, 67.56,
79.09, 83.87, 95.40, 114.31, 116.16, 116.55, 119.26, 123.24, 128.69, 129.33, 131.83, 132.02, 136.01, 137.21, 147.09, 153.72, 156.85, 157.15, 158.34, 164.46, 164.53 ppm. ESI HRMS calcd. for [M+(H)]+ 710.2415, found 710.2419. IR (KBr) νmax: 3549, 3412, 2976, 2930, 2213, 1786, 1724, 1636, 1617, 1575, 1549, 1516, 1456, 1411, 1384, 1368, 1324, 1285, 1230, 1140, 1051, 1024 cm-1. Anal. Calcd. for C35H40ClN5O7S × 0.4 H2O: C, 58.59; H, 5.73; N, 9.76. Found C, 58.29; H, 5.32; N, 10.11. S14
ACCEPTED MANUSCRIPT
1-(2-(3-((6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)carbamoyl)-4chlorophenoxy)ethyl)guanidinium chloride (42a). Yield = 63%; white solid, mp = 150– 152°C; 1H NMR (400 MHz, MeOD): δ 3.09 (t, J = 5.1 Hz, 2H, CH2-CH2-N), 3.63 (t, J = 4.7 Hz, 1H, O-CH2-CH2-NH), 3.78 (t, J = 4.7 Hz, 1H, O-CH2-CH2-NH), 4.18 (t, J = 4.8 Hz, 1H,
RI PT
O-CH2-CH2-NH), 4.25 (t, J = 4.8 Hz, 1H, O-CH2-CH2-NH), 4.45 (s, 2H, CH2-N), 4.66 (s, 2H, N-CH2-Ar), 7.16 (dt, J1 = 8.8, J2 = 3.0 Hz, 1H, Ar-H), 7.23 (t, J = 3.3 Hz, 1H, Ar-H), 7.47 (dd, J1 = 9.0, J2 = 2.1 Hz, 1H, Ar-H), 7.53–7.59 (m, 3H, 3 × Ar-H), 7.60-7.62 (m, 2H, 2 × ArH) ppm. (1 × CH2 under solvent peak). 13C NMR (100 MHz, MeOD): δ 22.85, 28.27, 42.19, 50.46, 60.95, 68.21, 95.27, 113.79, 116.66, 116.76, 119.85, 119.89, 120.90, 124.51, 124.59,
SC
130.30, 130.57, 130.74, 131.69, 132.53, 132.58, 136.04, 150.34, 158.75, 166.58 ppm. IR νmax 3117, 2944, 2677, 2553, 2221, 1738, 1670, 1591, 1543, 1456, 1398, 1375, 1275, 1248, 1212,
M AN U
1150, 1067, 1038 cm-1. HPLC tR = 8.957 min (98.9% at 220 nm, 98.9% at 254 nm). 1-(2-(4-chloro-3-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2ylcarbamoyl)phenoxy)ethyl)guanidinium chloride (42b).Yield = 87%; pale yellow crystals, mp =75–77 °C; 1H NMR (400 MHz, MeOD): δ 3.08 (t, J = 4.7 Hz, 2H, CH2-CH2-N), 3.78 (t, J = 4.7 Hz, 2H, CH2-CH2-N), 3.85 (s, 3H, Ar-O-CH3), 4.25 (t, J = 5.1 Hz, 2H, O-CH2-CH2NH), 4.39 (s, 2H, CH2-N), 4.42 (s, 2H, N-CH2-Ar), 4.49 (t, 2H, J = 4.9 Hz, O-CH2-CH2-NH),
TE D
7.07 (d, J = 8.5 Hz, 2H, 2 × Ar-H), 7.16 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.22 (t, J = 3.0 Hz, 1H, Ar-H), 7.47 (d, J = 8.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.5 Hz, 2H, 2 × Ar-H) ppm. 13C NMR (100 MHz, MeOD): δ 22.87, 28.27, 42.37, 50.27, 56.08, 60.57, 67.72, 95.26, 113.80, 115.98, 116.76, 116.66, 119.89, 120.99, 121.91, 124.59, 130.60, 132.53, 134.09, 136.07,
EP
150.31, 158.60, 162.92, 166.53 ppm. HRMS m/z calcd. for [M+(H)]+ 539.1632, found 539.1630. IR νmax 3146, 2939, 2838, 2576, 2361, 2218, 1735, 1673, 1612, 1585, 1545, 1515, 1458, 1398, 1370, 1249, 1180, 1147, 1065, 1028 cm-1. HPLC tR = 9.256 min (98.9% at 220
AC C
nm, 99.2% at 254 nm).
1-(2-(4-chloro-3-((3-cyano-6-(4-(methoxycarbonyl)benzyl)-4,5,6,7-tetrahydrothieno[2,3c]pyridin-2-yl)carbamoyl)phenoxy)ethyl)guanidinium chloride (42c). Yield = 85%; pale yellow solid, mp = 112–114 °C; 1H NMR (400 MHz, MeOD): δ 3.10 (t, J = 4.9 Hz, 2H, CH2CH2-N), 3.63 (t, J = 4.8 Hz, 2H, CH2-CH2-N), 3.83 (s, 3H, COO-CH3), 4.18 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 4.45 (s, 2H, CH2-N), 4.65 (s, 2H, N-CH2-Ar), 7.15 (dd, J1 = 8.8, J2 = 2.9 Hz, 1H, Ar-H), 7.22 (d, J = 2.9 Hz, 1H, Ar-H), 7.46 (d, J = 8.8 Hz, 1H, Ar-H), 7.75 (d, J = 8.1 Hz, 2H, 2 × Ar-H), 8.16 (d, J = 8.1 Hz, 2H, 2 × Ar-H) ppm. 1 × CH2 under solvent peak. 13
C NMR (100 MHz, MeOD): δ 22.86, 42.16, 50.73, 50.75, 53.09, 60.22, 68.20, 95.24, S15
ACCEPTED MANUSCRIPT
113.79, 116.65, 119.85, 120.80, 124.40, 130.53, 131.58, 132.50, 132.85, 133.35, 135.19, 136.03, 150.38, 158.76, 159.17, 166.58, 167.76 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 567.1581, found 567.1592. IR νmax 3156, 2950, 2568, 2218, 1721, 1675, 1586, 1545, 1457, 1436, 1371, 1281, 1248, 1187, 1148, 1111, 1064, 1039, 1022 cm-1. HPLC tR = 9.219 min
RI PT
(100% at 220 nm, 100% at 254 nm). 1-(2-(4-chloro-3-((3-cyano-6-(3-hydroxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-
yl)carbamoyl)phenoxy)ethyl)guanidinium chloride (42d). Yield = 76%; white solid, mp = 159–161 °C; 1H NMR (400 MHz, MeOD): δ 3.07 (bs, 2H, CH2-CH2-N), 3.63 (bs, 2H, O-
SC
CH2-CH2-NH), 4.17 (t, J = 4.7 Hz, 2H, CH2-CH2-N), 4.42 (s, 2H, N-CH2-Ar), 4.46 (s, 2H, OCH2-CH2-NH), 6.93–7.03 (m, 3H, 3 × Ar-H), 7.14–7.21 (m, 2H, 2 × Ar-H), 7.33 (t, J = 7.4 Hz, 1H, Ar-H), 7.46 (d, J = 8.6 Hz, 1H, Ar-H) ppm. (1 × CH2 under solvent peak, NH, OH
M AN U
exchanged). 13C NMR (100 MHz, MeOD): δ 22.82, 42.16, 50.52, 60.91, 65.28, 68.20, 95.16, 113.70, 116.55, 118.38, 119.02, 119.73, 120.80, 123.06, 124.29, 130.43, 131.41, 131.72, 132.39, 135.92, 150.21, 158.64, 159.07, 159.63, 166.47 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 525.1476, found 525.1468. IR (KBr) νmax 3319, 3161, 2720, 2582, 2219, 1737, 1664, 1590, 1546, 1481, 1457, 1421, 1371, 1284, 1243, 1149, 1065, 1039, 1023 cm-1. HPLC tR = 8.355 min (99.1% at 220 nm, 99.2% at 254 nm).
TE D
1-(2-(4-chloro-3-((3-cyano-6-(4-cyanobenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2yl)carbamoyl)phenoxy)ethyl)guanidinium chloride (42e). Yield = 82%; white solid, mp = 169–171 °C; 1H NMR (400 MHz, MeOD): δ 3.12 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.65 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.74 (s, 2H, O-CH2-CH2-NH), 4.20 (t, J = 5.0 Hz, 2H, O-CH2-CH2-
EP
NH), 4.45 (s, 2H, CH2-N), 4.66 (s, 2H, N-CH2-Ar), 7.17 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.24 (d, J = 3.0 Hz, 1H, Ar-H), 7.48 (d, J = 8.9 Hz, 1H, Ar-H), 7.83 (d, J = 8.2 Hz, 2H, 2 ×
AC C
Ar-H), 7.92 (d, J = 8.3 Hz, 2H, 2 × Ar-H) ppm.13C NMR (100 MHz, MeOD): δ 22.92, 28.26, 42.16, 50.76, 60.03, 68.20, 95.27, 113.80, 115.44, 116.65, 119.13, 119.85, 120.96, 124.42, 130.55, 132.51, 133.53, 134.38, 135.74, 136.04, 150.33, 158.76, 159.18, 166.59 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 534.1479, found 534.1475. IR (KBr) νmax 3329, 3154, 2545, 2221, 1736, 1661, 1587, 1546, 1458, 1419, 1370, 1290, 1200, 1173, 1124, 1065, 1039 cm-1. HPLC tR = 8.796 min (98.4% at 220 nm, 98.8% at 254 nm). Amino((2-(4-chloro-3-((3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2yl)carbamoyl)phenoxy)ethyl)amino)methaniminium chloride (43). Yield = 79%; pale brown crystals, mp = 205–206 °C; 1H NMR (400 MHz, MeOD): δ 1.98 (m, 1H, CH-CHAHB-CH2), S16
ACCEPTED MANUSCRIPT
2.11–2.17 (m, 1H, CH-CHAHB-CH2), 2.69–2.82 (m, 3H, CH-CH2-CH2), 2.93–3.02 (m, 2H, CH2-CH-CH2-CH2), 3.65 (t, J = 4.8 Hz, 2H, CH2-NH-C), 4.20 (t, J = 4.8 Hz, 2H, CH2-CH2O), 6.77 (d, J = 8.8 Hz, 2H, 2 × Ar-H), 7.14 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.16 (dd, J1 = 5.6, J2 = 3.2 Hz, 1H, Ar-H), 7.24 (d, J = 3.2 Hz, 1H, Ar-H), 7.48 (d, J = 8.8 Hz, 1H, Ar-H) ppm. 13
RI PT
C NMR (100 MHz, MeOD): δ 25.59, 31.06, 33.06, 41.43, 42.07, 68.10, 96.56, 114.89,
116.34, 116.52, 119.62, 124.29, 128.88, 130.55, 132.35, 132.77, 136.33, 137.65, 147.35, 156.99, 158.60, 159.05, 166.15 ppm. ESI HRMS calcd. for [M+(H)]+ 510.1367, found 510.1361. IR (KBr) νmax: 3414, 2224, 1652, 1616, 1575, 1511, 1458, 1387, 1305, 1239, 1123,
SC
1061, 1040 cm-1. HPLC tR = 11.881 min (98.1% at 220 nm, 97.2% at 254 nm). REFERENCES:
M AN U
[1] K. Sundaravel, E. Suresh, M. Palaniandavar, Iron(III) complexes of tridentate N3 ligands as models for catechol dioxygenases: Stereoelectronic effects of pyrazole coordination,
AC C
EP
TE D
Inorg. Chim. Acta. 363 (2010) 2768–2777.
S17
ppm
7
6,5
% 50 100150
23b (1D 1H) CDCl3 400MHz
6
5,5
5
%
2,21
1,04 1,00
1,00
0
10
20
30
4,5
4,5
4
4
3,5
3,5
3
3
4,16
2,08 1,58
2,39
5,88
M AN U
5
2,866 2,849 2,832 2,712 2,701 2,686 2,660 2,649 2,646 2,635 1,965 1,949 1,932 1,916 1,907 1,900 1,893 1,878 1,870 1,863 1,852 1,848 1,842 1,832
5,5
4,093 4,077 4,062
TE D
ppm 7,465 7,458 7,398 7,376 7,285 7,039 7,031 7,017 7,009
100
6
7,48
EP
80
6,5
2,41 2,12
60
AC C
40
7
2,14
20 7,5
2,15
0 ppm
1,00
1,00 1,00
50
SC
40
70
2,5
2,5
1,79 1,78 1,77 1,76 1,75
2,63 2,54 2,51 2,50 2,50 2,50 2,49
3,24
3,37
4,22
ppm 7,52 7,50 7,22 7,21 7,17 7,16 7,15 7,14
80
% 50 100150
RI PT
60
1
%
ACCEPTED MANUSCRIPT
H NMR spectra for compounds 23a-b, 27b, 33, 35, 36, 37a-c, 37e-f , 38a-f, 39c, 40c, 42a,
42c-e, 43
23a (1D 1H) DMSO 400MHz
2 1,5
2
S18
ppm
0,92
0
5
9
8
7
6
5
4
3 3
1,463
1,725
4
9,05
2,674
5
2,00
6
2,00 2,01
7
4,144 4,131 4,118 3,786 3,772 3,682
8
1,97
9
33 (1D 1H) CDCl3 400MHz
%
0,96
0,77
0
1,98 3,16
1,89
1,98
2,00
1,00 1,07
1,06
M AN U
% 50 100150
10
4,505
7,483 7,475 7,385 7,363 7,240 7,021 7,014 6,999 6,992
ppm 9,841
25
11
2,00
20
TE D
15
12
1,94
EP
10 ppm
0,99 0,99
AC C
%
SC
10
30
40
RI PT
20
50
60
4,081 3,670 3,306 3,290 3,275 3,258 2,651 2,639 2,625 2,516 2,512 2,507 2,502 2,498 2,090 1,972 1,956 1,940 1,924 1,913 1,908 1,779
7,754 7,740 7,726 7,496 7,474 7,188 7,181 7,146 7,139 7,124 7,117
12,295
ppm
% 50 100150
ACCEPTED MANUSCRIPT
27b (1D 1H) DMSO 400MHz
2
2
S19
AC C EP TE D
ppm 9 8 7 6
2,00
2,05
0,88 3,03
0,88
0,82
0
5
4
3
0,93 0,95
3,37
2,27
2,06
M AN U
%
4
SC
2
8
10
12
RI PT
6
14
16
7,485 7,463 7,233 7,225 7,130 7,115 7,106 7,094 6,720 6,699 4,224 4,212 4,199 3,344 3,321 3,229 3,216 3,216 3,204 2,914 2,886 2,884 2,865 2,830 2,829 2,817 2,683 2,681 2,678 2,677 2,674 2,669 2,664 2,656 2,643 2,635 2,623 2,509 2,505 2,500 2,495 2,491 2,010 2,008 2,004 2,002 1,997 1,995 1,976 1,974 1,973 1,907 1,891 1,883 1,882 1,877 1,873 1,871 1,869 1,861 1,860 1,852 1,849 1,844 1,841 1,840 1,836 1,830 1,811
9,251
ppm
% 50 100150
ACCEPTED MANUSCRIPT
35 (1D 1H) DMSO 400MHz
2
S20
%
1,0
10
20
ppm
10
9,5
9
8,5
8
7,5
7
6,5
6
5
5,5
5
4
4,5
4
3,5
2,1 2,0
6
2,829 2,814 2,800 2,732 2,719
37b (1D 1H) CDCl3 400MHz
%
2,00
4,01
4,07 2,02
2,06
1,05
3,02 2,13 2,15
0,68
0,64
0
M AN U
% 50 100150
7
5,3 2,0 2,0
4,142 4,129 4,116 4,106 4,088 3,812 3,796 3,785 3,772 3,650 3,548
7,517 7,403 7,381 7,265 7,260 7,249 7,244 7,240 7,031 7,023 7,009 7,001 6,875 6,870 6,858 6,853
9,733
ppm
8
2,8
70
9
2,1 1,0
60
TE D
50
10
1,0
40
EP
30 ppm
1,0 1,0
AC C
0
SC
20
60
80
RI PT
40
100
4,274 4,261 4,247 3,794 3,781 3,767 3,759 3,618 3,614 3,609 2,863 2,854 2,848 2,834 2,696 2,692 2,682 2,678 2,673 2,663 2,061 2,055 2,050 2,044 2,039
8,779 7,442 7,442 7,420 7,416 7,415 7,398 7,398 7,368 7,365 7,365 7,361 7,347 7,344 7,343 7,332 7,328 7,297 7,297 7,294 7,290 7,283 7,275 7,261 7,257 7,254 7,127 7,119 7,105 7,097
ppm 11,011
% 50 100150
ACCEPTED MANUSCRIPT
37a (1D 1H) Acetone 400MHz
3 2
3
2,5
S21
ppm
0,96
0
50
10
9,5
9
8,5
8
7,5
7
6,5
6
5,5
5
4,5
4
1,98 2,01
4
3,5
2,854 2,842 2,765
3,829 3,817 3,788 3,608
5
4,162
6
2,01
7
37e (1D 1H) CDCl3 400MHz
%
2,01 0,96 1,01 1,00
2,04
0,70
0,69
0
1,98
3,78
1,95
3,04 2,13
2,05
M AN U
7,661 7,642 7,548 7,518 7,499 7,470 7,439 7,417 7,264 7,066 7,044 6,755 6,752
9,786
% 50 100150
8
1,99 1,89
400 ppm
9
2,05
350
10
0,93
300
TE D
250
11
1,00
200 ppm
2,31 3,08 1,06
150
EP
100
AC C
%
SC
20
60
80
RI PT
40
4,275 4,262 4,248 3,883 3,850 3,781 3,768 3,654 3,650 3,646 2,886 2,871 2,856 2,710 2,700 2,696 2,692 2,061 2,055 2,050 2,044 2,039
8,004 7,999 7,988 7,983 7,566 7,562 7,545 7,443 7,421 7,284 7,277 7,129 7,121 7,106 7,099
8,776
11,023
ppm
100
% 50 100150
ACCEPTED MANUSCRIPT
37c (1D 1H) Acetone 400MHz
3 2
3
2,5
S22
ppm
6,09
0
7
6,5
6
5,5
5
4,5
4
4
3,5
3,5
1,91
4,5
1,87
5
3,763 3,593 3,334 3,329 3,318 3,314 3,310 3,306 3,302 3,146 3,133 3,125 3,120 2,890 2,875 2,868 2,861 2,737 2,723 2,712 2,710
5,5
4,131 4,119 4,106
4,888
6
38a (1D 1H) MeOD 400MHz
%
2,01 2,13
0,94 0,98
1,03
2,07
2,05
0
2,00
6,69
2,09
1,96
M AN U
% 50 100150
6,5
1,89
10 ppm 7,413 7,409 7,404 7,392 7,389 7,381 7,376 7,372 7,359 7,355 7,340 7,319 7,315 7,311 7,305 7,298 7,291 7,284 7,281 7,277 7,220 7,212 7,087 7,080 7,065 7,058 7
2,12
8
TE D
6
7,5
2,00
EP
4
8
2,14
1,00
2 ppm
1,03
AC C
%
SC
20
60
80
RI PT
40
100
3,700 3,687 3,674 3,318 3,314 3,310 3,306 3,302 3,288 3,273 2,905 2,891 2,877
4,190 4,168 4,154 4,141 4,025
4,909 4,903
ppm 8,091 8,070 7,641 7,635 7,620 7,614 7,435 7,430 7,413 7,407 7,196 7,188 7,121 7,116 7,108 7,094 7,086
% 50 100150
ACCEPTED MANUSCRIPT
37f (1D 1H) MeOD 400MHz
3 2,5
3
S23
%
ppm
8
7,5
7
50
7,470 7,449 7,338 7,316 7,231 7,223 6,969 6,961 6,947 6,939
7,919 7,902 7,898
ppm
6,5
6
5,5
5
4,5
4
3,5
2,50
2,01
3,207 3,194 3,181
4
2,741 2,727 2,714 2,545 2,534 2,534 2,509 2,504 2,500 2,495 2,491
3,426
4,5
2,60
5
3,731
5,5
4,165 4,152 4,139
6
38c (1D 1H) DMSO 400MHz
%
2,11
2,18
0,99
1,01
1,01 2,01
0
3,5
3
2,00
2,01
1,89
2,03
2,08
3,13
M AN U
% 50 100150
TE D
40
6,5
2,57
2,00
EP
30
7
0,99
0,99 0,91
20
AC C
10 ppm
1,97
2,00
0
4
SC
2
8
10
12
14
RI PT
6
16
18
3,798 3,696 3,568 3,318 3,314 3,310 3,306 3,302 3,137 3,129 3,117 3,104 2,874 2,859 2,845 2,729 2,724 2,715 2,703 2,701
4,120 4,107 4,094
4,888
ppm 7,393 7,371 7,314 7,293 7,216 7,208 7,074 7,066 7,052 7,044 6,924 6,920 6,903 6,895
% 50 100150
ACCEPTED MANUSCRIPT
38b (1D 1H) MeOD 400MHz
3 2,5
2,5
S24
AC C EP TE D
ppm 7,5 7 6,5 6 5,5
2,09
0,99
2,21
1,06
2,13
0,95
0
5
4,5
4
3,5 3
2,00
3,30
2,03
2,01
2,63
M AN U
%
SC
5
15
20
RI PT
10
25
30 % 50 100150
3,684 3,585 3,318 3,314 3,310 3,306 3,302 3,137 3,124 3,111 2,886 2,879 2,867 2,850 2,733 2,720
4,124 4,111 4,098
4,892
ppm 7,398 7,376 7,218 7,211 7,185 7,164 7,145 7,079 7,071 7,057 7,049 6,859 6,842 6,839 6,735 6,731 6,717 6,713 6,710
ACCEPTED MANUSCRIPT
38d (1D 1H) MeOD 400MHz
2,5
S25
ppm
2,09
0
8
7,5
7
4,220 4,208
6,5
6
5,5
5
4,5
4
3
3,5
3
2,00
3,5
3,260 3,248 3,235 2,798 2,784 2,770 2,620 2,608 2,509 2,505 2,500 2,496 2,492
4
3,530
4,5
2,24
5
2,00
5,5
2,14
6
38f (1D 1H) DMSO 400MHz
%
2,01
0,95
0,96
0,94
2,00
2,04
0
2,05
2,11
2,20
2,17
2,73
M AN U
% 50 100150
6,5
3,705
TE D
7,473 7,451 7,384 7,363 7,242 7,234 7,113 7,106 7,091 7,084
7,956 7,935
ppm
100
7
2,26
2,03
0,99
80
7,5
0,98
60 ppm
1,00 1,95
40
EP
20
AC C
%
SC
20
60
RI PT
40
80
2,885 2,870 2,856 2,703 2,693 2,689 2,685 2,674 2,586 2,568 2,551 2,533
3,869 3,659 3,655 3,650 3,577 3,563 3,548
4,262 4,247 4,233
ppm 7,771 7,766 7,755 7,750 7,654 7,649 7,633 7,412 7,390 7,274 7,267 7,108 7,100 7,086 7,078
% 50 100150
ACCEPTED MANUSCRIPT
38e (1D 1H) Acetone 400MHz
2,5
2,5
S26
%
ppm
11
10
9
8
7
6
5
4
3
10,02 9,00
3,5
1,487 1,480 1,468
4
2,26 2,01
4,5
2,933 2,881 2,867 2,856 2,764 2,751
5
3,06 2,09 1,93 1,94
4,118 4,104 4,086 3,899 3,843 3,829 3,789 3,617
5,5
40c (1D 1H) CDCl3 400MHz
%
2,01
1,03
0,88
2,03
2,04
0
2,00
2,00
1,92
1,94
3,19 2,12
M AN U
% 50 100150
6
2,78
8,016 7,995 7,523 7,515 7,452 7,431 7,385 7,363 7,240 7,102 7,094 7,080 7,072
8,730 8,716 8,704
9,769
ppm 11,452
6,5
0,96
60
TE D
50
7
0,97 2,05 1,03
40
EP
30
7,5
2,20
20
8
0,94
10
AC C
0 ppm
0,89
0,97
SC
20
60
80
RI PT
40
3,620 3,344 3,331 3,318 3,314 3,310 3,306 3,302 2,888 2,873 2,860 2,740 2,726 2,713
3,907 3,829
4,257 4,245 4,232
4,903
7,535 7,515 7,468 7,446 7,261 7,254 7,169 7,161 7,147
ppm 8,021 8,001
100
% 50 100150
ACCEPTED MANUSCRIPT
39c (1D 1H) MeOD 400MHz
3 2,5
2
S27
ppm
2,00
0
10
8
7,5
50 % 50 100150
7 6
6,5 5,5
42c (1D 1H) MeOD 400MHz
%
2,00
1,06 1,03
2,27 3,25 1,13
0
6
5,5
4,078 4,065 4,053 3,832
3,534 3,522 3,511
1,65
3,00
2,07
1,55
4,5
3,5
3
4,5
4
4
3,208 3,204 3,200 3,196 3,192 2,992
4,344
5
1,58
5
2,13
1,34
1,33
1,11 1,12
2,02
M AN U
4,780
7,364 7,342 7,112 7,105 7,058 7,051 7,036 7,029
7,646 7,626
ppm 8,061 8,041
6,5
4,538
TE D
40
7
1,92
EP
30
7,5
0,73 0,83
20 ppm
0,91
1,99
AC C
%
SC
5
15
RI PT
10
20
3,090
3,318 3,314 3,310 3,306 3,302
3,793 3,782 3,771 3,645 3,634 3,622
4,437 4,260 4,249 4,238 4,189 4,177 4,165
4,567
4,887
ppm 7,622 7,615 7,606 7,598 7,558 7,548 7,548 7,542 7,534 7,481 7,476 7,459 7,454 7,235 7,226 7,218 7,177 7,170 7,163 7,155 7,148 7,141
% 50 100150
ACCEPTED MANUSCRIPT
42a (1D 1H) MeOD 400MHz
3,5 3
S28
ppm
2,05
0
5
10
8
7,5
7
6,5
6
5,5
5
4,5
4,5
3,738 3,665 3,652 3,640 3,338 3,334 3,330 3,326 3,321 3,131 3,117 3,103
4,208 4,196 4,183
5
4,447
4,660
5,5
4
4
1,91
42e (1D 1H) MeOD 400MHz
%
2,09 1,64
3,06
2,12
1,07
1,22
0
2,00
2,64
2,01
M AN U
4,907
7,496 7,474 7,243 7,236 7,189 7,181 7,167 7,159
7,912 7,841 7,820
% 50 100150
6
1,83 1,93
40 ppm
6,5
2,05
35
TE D
30
7
2,00
EP
25
7,5
2,02
0,98 1,00
20
8
1,00
15 ppm
2,00
AC C
%
SC
10
30
40
RI PT
20
50
60
3,5
3,089
3,326
3,641
4,184 4,116 4,098
4,476 4,435 4,431
4,912
7,483 7,461 7,348 7,225 7,174 7,153 7,041 7,018 6,963 6,943
ppm
% 50 100150
ACCEPTED MANUSCRIPT
42d (1D 1H) MeOD 400MHz
3,5 3
3
S29
AC C EP TE D
ppm 8 7,5 7 6,5 6 5,5
2,01
2,00
0,99 3,02
1,00
0
5
4,5
4
3,5 3 2,5
0,99
1,01
2,85
2,06
2,06
M AN U
%
2
SC
1
4
5
6
RI PT
3
7
8
7,493 7,471 7,241 7,234 7,168 7,161 7,151 7,146 7,139 7,135 7,130 6,774 6,753 4,904 4,209 4,197 4,184 3,662 3,650 3,638 3,336 3,332 3,328 3,324 3,319 3,060 3,018 3,016 3,011 3,002 2,986 2,985 2,970 2,938 2,925 2,813 2,783 2,777 2,776 2,771 2,764 2,755 2,749 2,742 2,739 2,733 2,731 2,727 2,725 2,724 2,720 2,719 2,165 2,164 2,163 2,160 2,158 2,155 2,151 2,145 2,131 2,127 2,124 2,000 1,999 1,987 1,985 1,973 1,970 1,965 1,958
ppm
% 50 100150
ACCEPTED MANUSCRIPT
43 (1D 1H) MeOD 400MHz
2
S30
%
0
2
6
AC C
4
10
EP
8
ppm
160
150
27b (1D 13C) DMSO 400MHz
140
TE D
130
120
110
100 90
90 80
80 70
70 60
60 50
50
40
40
30
28,023 23,554 23,404 22,577 21,687
100
40,107 39,899 39,692 39,480 39,272 39,065 38,857 37,749
%
0
1
2
M AN U
110
65,512
120
94,503
130
117,895 115,205 113,973
140
121,431
150
131,306 130,544 128,200
160
135,403
% 50 100150 ppm
145,592
156,940 156,871
164,096
ppm
SC
3
5
40,111 39,903 39,692 39,484 39,276 39,068 38,857 38,591 36,564 27,312 24,104 23,783 23,280 22,416
64,968
90,675
122,109 118,478 115,752 115,726 115,485 115,008
130,169 128,521
141,765
156,459
162,434
167,836
ppm
% 50 100150
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4
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13
C NMR spectra for compounds 23b, 27b, 33, 35, 36, 37a-c, 37e-f, 38a, 38c, 38e-f, 39c, 40c,
42a-c, 42e,43
23b (1D 13C) DMSO 400MHz
30 20
20
S31
ppm
0
2
4
AC C
%
8
10
EP
6
14
16
18
TE D
12 ppm
170 150
160 140
150 130
140 120
130
120 110 100
35 (1D 13C) Pyr 400MHz
%
110
100
90 70
80
60
70
60
50 40
50
40
26,766
80
34,279 32,067
90
42,197 41,931
69,315
M AN U
97,500
160
152,122 151,852 151,582 149,537 138,397 138,270 137,785 137,537 137,293 133,684 133,035 130,731 130,443 125,762 125,515 125,267 120,298 118,384 117,969 116,981
159,580 159,413
% 50 100150 ppm
167,049
20
10
SC
0
30
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20
40
30
24,143
31,448 28,390 28,047
36,559
39,180
66,265
80,646 77,379 77,062 76,745
94,181
122,993 119,953 117,179 116,949 114,321 113,647
131,983 131,640
147,038
ppm 162,622 161,820 157,759 157,383 157,212 154,536
% 50 100150
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33 (1D 13C) CDCl3 400MHz
30 20
20
S32
ppm
160
150
140
130
120
110
100
90
80
70
5
10
AC C
0
EP
15
25
62,039
67,183
95,266
139,528 135,970 131,851 131,144 129,773 129,211 129,047 129,011 128,049 127,553 123,302 119,278 119,161 116,376 114,080
147,260
158,294
164,506
50
40
30,496 30,303 30,110 29,920 29,727 29,534 29,340 24,981
37a (1D 13C) Acetone 400MHz
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30 % 50 100150 ppm
TE D
20
40,039
60
51,409 50,148
%
SC
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ACCEPTED MANUSCRIPT
30
20
S33
ppm
0
2
AC C
%
6
EP
4
10
12
14
TE D
8
160
150
160 140
140 120
130
120 100
37c (1D 13C) Acetone 400MHz
110
%
100
90
80
70
60
50
40
30,496 30,303 30,110 29,920 29,727 29,534 29,340 24,926
60
40,043
80
52,342 51,453 50,228
61,503
67,186
95,244
M AN U
135,955 131,854 131,107 130,331 130,112 129,744 127,374 123,302 119,282 118,545 116,387 116,384 115,687 114,058
% 50 100150 ppm
147,329 145,266
158,294
ppm 167,189 164,517
2
4
SC
0
8
10
12
14
RI PT
6
16 ppm
30,496 30,303 30,110 29,920 29,727 29,534 29,340 24,992
40,036
51,285 50,020
55,532
61,470
67,179
95,259
135,959 131,851 131,238 131,235 131,162 131,158 130,972 127,622 127,618 123,302 119,282 118,549 116,376 115,695 114,535 114,087
147,212
159,985 158,287
164,480
18
% 50 100150
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37b (1D 13C) Acetone 400MHz
40 20
30
20
S34
AC C EP TE D
ppm 160 140 120 100
M AN U
%
5
15
20
25
RI PT
10
SC
0
30
80
60
40
0,211
24,303
39,370
51,082 49,704
61,253
66,524
77,547 77,230 76,913
94,410
132,566 132,387 131,596 130,138 129,570 126,613 123,267 120,424 119,038 117,329 117,241 114,000 111,514
143,907
146,903
161,638 157,821 157,413
ppm
35
% 50 100150
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37e (1D 13C) CDCl3 400MHz
20 0
S35
ppm
0
2
AC C
%
6
EP
4
160
140
10 ppm
120
100
80
60
40
23,999
51,164 49,560 40,133 39,926 39,718 39,510 39,299 39,091 38,883 38,664 38,562
60,503
65,385
90,607
% 50 100150
38c (1D 13C) DMSO 400MHz
M AN U
130,417 130,363 129,331 128,657 128,653 127,585 122,638 120,954 117,659 115,993 115,599
145,454 143,460 140,697
156,179
167,508 167,312
TE D
8
SC
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ACCEPTED MANUSCRIPT
20
S36
ppm
0
2
AC C
%
6
EP
4
10
12
TE D
8
14 % 50 100150
170
160
150
140 120
130 110
120 100
38f (1D 13C) DMSO 400MHz
110
%
100
90
80
60
70
50
60
40
50
30
40
30
23,780
70
50,417 48,999 40,133 39,926 39,718 39,510 39,299 39,091 38,883 38,358
80
60,474
90
65,144
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93,220
130
117,655 115,606
140
130,610 130,370 129,535 128,985 125,901 122,244
150
137,329 137,325
156,339
160
159,820
ppm
164,884
ppm
2
SC
0
6
8
10
RI PT
4
12
14 % 50 100150
11,867
30,470 30,276 30,087 29,894 29,700 29,507 29,318 24,845
51,448 51,168 50,198 46,986
61,243
69,992
95,054
136,020 133,053 131,686 131,682 130,946 130,465 126,940 122,736 119,470 118,996 116,405 116,401 114,236 111,698
145,636
158,803
ppm 164,745
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38e (1D 13C) Acetone 400MHz
20 10
20
S37
ppm
0
5
AC C
%
15
20
EP
10
30
35
40
160
150
140
140
130
120
120
40c (1D 13C) CDCl3 400MHz
%
0
110
100
90
80
70
60
50
40
28,579 28,492 28,372 28,270 28,189 24,231
60
40,146
52,351 51,013 49,690
80
61,435
100
67,151
83,595 79,866 77,879 77,551 77,438 77,230 76,913
94,235
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114,044
160
117,376
132,161 131,476 130,073 129,668 129,154 126,515 122,727 120,722
143,134
% 50 100150
180
147,067
153,279
157,905 156,603
163,471 161,867
167,109
ppm
ppm
TE D
25
SC
2
6
8
RI PT
4
10
25,204
62,197 52,773 51,902 50,703 49,792 49,577 49,365 49,150 48,939 48,727 48,512 40,434
66,720
96,164
136,806 132,446 132,442 131,651 130,853 130,714 130,638 127,700 124,736 119,560 116,738 116,734 114,744
144,694
148,828
158,426
168,524 166,544
178,993
ppm
% 50 100150
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39c (1D 13C) MeOD 400MHz
40 20
30
S38
ppm
0
1
AC C
%
3
EP
2
5
6
TE D
4 ppm
160 140
140 130 120
120 110
42b (1D 13C) MeOD 400MHz
%
100 80
80
70
60
60 50 40
40
22,871
90
28,270
67,719 60,574 56,076 50,645 50,422 50,368 50,265 50,145 49,788 49,577 49,365 49,150 48,939 48,723 48,512 42,370
100
86,176
95,263
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124,594 121,911 120,992 119,888 116,756 116,658 115,976 113,796
150
136,069 134,086 132,526 130,598
160
150,312
158,597
% 50 100150 ppm
162,917
166,530
7
2
SC
0
6
RI PT
4
8
10
22,853
68,211 60,946 50,710 50,670 50,553 50,382 50,313 50,240 50,204 50,167 50,094 50,021 49,792 49,577 49,365 49,150 48,939 48,727 48,512 42,169 28,273
86,176
95,267
136,044 132,581 132,526 131,691 130,736 130,565 130,299 124,586 120,897 119,891 119,851 116,760 116,658 113,793
150,341
158,754
166,581
ppm
% 50 100150
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42a (1D 13C) MeOD 400MHz
30 20
20
S39
ppm
0
1
AC C
%
3
EP
2
5
6
160
150
% 50 100150
160 140
140
120
130
120
100
42e (1D 13C) MeOD 400MHz
110
%
0
100
90
80
70
60
40
50
40
30
22,922
60
28,262
50,761 49,792 49,577 49,365 49,150 48,939 48,727 48,512 42,155
80
60,028
68,200
95,267
M AN U
136,040 135,741 134,564 134,378 133,718 133,532 132,508 130,550 124,590 124,415 120,956 119,848 119,129 116,749 116,647 115,437 113,804
150,333
159,217 159,177 158,761
166,592
ppm
ppm
TE D
4
SC
1
3
4
5
RI PT
2
6
22,856
60,217 53,091 50,754 50,736 49,788 49,573 49,361 49,150 48,935 48,723 48,508 42,158
68,204
95,238
136,029 135,187 133,353 132,854 132,497 131,575 130,528 124,404 120,799 119,851 116,651 113,789
150,381
159,170 158,758
167,762 166,577
ppm
% 50 100150
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42c (1D 13C) MeOD 400MHz
20
20
S40
AC C EP TE D
ppm 160 140 120
0
M AN U
%
SC
5
15
20
RI PT
10
25
30 % 50 100150
100
80
60
40
25,595
33,067 31,066
49,716 49,501 49,289 49,078 48,863 48,651 48,436 42,086 41,444
68,110
96,573
119,637 116,531 116,349 114,898
124,303
137,663 136,343 132,778 132,363 130,555 128,885
147,363
159,054 158,609 157,001
166,158
ppm
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43 (1D 13C) MeOD 400MHz
20
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Structure
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Compound
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Table S1: ClogP values of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a, 27b, 33-38f, 39c, 42a-e and 43.
I
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II
2.83
3.40
2.42
AC C
EP
4
ClogP
5.87
15
5.48
16
3.16
14
S42 17
5.07
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4.50
RI PT
18
1.80
19
2.59
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SC
22a
22b
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23a
AC C
26a
EP
23b
2.99
2.50
2.90
1.96
27a
1.88
27b
2.23
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3.52
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33
4.26
SC
33b
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34
35
EP
AC C
37a
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36
2.04
3.24
0.38
3.82
37b
3.74
37c
3.79
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3.15
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37d
3,25
SC
37e
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37f
38a
TE D
38b
2.82
2.74
0.29
AC C
EP
38c
3.31
38d
2.15
38e
2.25
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2.31
RI PT
38f
2.79
39c
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SC
42a
42b
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42c
AC C
42e
EP
42d
43
2.19
2.11
2.16
1.53
1.63
2.62
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S0223-5234(13)00773-3
DOI:
10.1016/j.ejmech.2013.11.031
Reference:
EJMECH 6577
To appear in:
European Journal of Medicinal Chemistry
Received Date: 11 July 2013 Revised Date:
15 November 2013
Accepted Date: 24 November 2013
Please cite this article as: M. Hrast, M. Anderluh, D. Knez, C.P. Randall, H. Barreteau, A.J. O’Neill, D. Blanot, S. Gobec, Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold, European Journal of Medicinal Chemistry (2014), doi: 10.1016/ j.ejmech.2013.11.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold
Barreteau3, Alex J. O’Neill2, Didier Blanot3 and Stanislav Gobec1*
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Martina Hrast1, Marko Anderluh1, Damijan Knez1, Christopher P. Randall2, Hélène
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
2
School of Molecular and Cellular Biology and Antimicrobial Research Centre, University of
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1
3
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Leeds, Leeds LS 9JT, U.K.
Univ Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS,
91405 Orsay, France
University of Ljubljana
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*Corresponding author: Stanislav Gobec
Faculty of Pharmacy, Aškerčeva 7
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1000 Ljubljana, Slovenia
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Tel: +386-1-4769500
Fax: +386-1-4258031
E-mail: [email protected]
1
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ABSTRACT MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug
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discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best
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inhibitors displayed promising antibacterial activities against selected gram-positive and gram-negative strains. These results represent an important step towards the development of
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new antibacterial agents targeting peptidoglycan biosynthesis.
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Abbreviations: DAP,
2,6-diaminopimelic
acid;
GlcNAc,
N-acetyl
glucosamine;
MurC,
UDP-N-
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acetylmuramate:L-Ala ligase; MurD, UDP-N-acetylmuramoyl-L-Ala:D-Glu ligase; MurE, UDP-N-acetylmuramoyl-L-Ala-D-Glu:meso-DAP ligase; MurF, UDP-N-acetylmuramoyl-LAla-γ-D-Glu-meso-DAP (or
L-Lys):D-Ala-D-Ala
ligase; MurNAc, N-acetyl muramic acid;
UDP, uridine 5’-diphosphate; UMtri-L-Lys, UDP-N-acetylmuramoyl-L-Ala-D-Glu-L-Lys;
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UMtri-mDAP, UDP-N-acetylmuramoyl-L-Ala-γ-D-Glu-meso-DAP.
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1. INTRODUCTION The increasing emergence of pathogenic bacteria resistant to all known antibiotics has created an urgent need for development of new antibacterial agents [1-3]. Cell wall biosynthesis in
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bacteria is well characterised and thus represents an attractive target for the development of novel antibacterial drugs [4-6]. The biosynthetic pathway of peptidoglycan encompasses cytoplasmic, membrane and extracellular stages [4, 6, 7].
Mur ligases (MurC-F) are cytoplasmic ATP-dependent enzymes which catalyse the sequential
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addition of L-Ala, D-Glu and meso-DAP in Gram-negative or L-Lys in Gram-positive bacteria, and the dipeptide D-Ala-D-Ala to UDP-MurNAc, to form UDP-MurNAc-pentapeptide [7, 8].
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All Mur ligases have similar mechanisms of action, and share the same three-domain topology [9]. They are essential for the survival of bacteria and lack human counterparts; consequently, they represent interesting targets for the discovery of new antibacterials with selective toxicity [8]. There are only a few known classes of MurF inhibitors. The first classes were pseudo-tripeptide and pseudo-tetrapeptide aminoalkylphosphinic acids [10], followed by sulphonamides developed by Abbott Laboratories [11, 12]. Thiazolylaminopyrimidines [13],
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8-hydroxyquinolines [14] and diarylquinolines [15] were discovered by Johnson & Johnson. Recently, a triazine derivative was identified through structure-based virtual screening [16]. Additionally, ligand-based virtual screening was successfully employed by our group and one new compound with micromolar inhibitory activities against S. pneumoniae MurF (MurFSp)
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and E. coli MurF (MurFEc) was identified [17]. In our previous research, we also reported a series of cyanothiophene-based inhibitors of MurF enzymes from different pathogenic bacteria, where systematic structural modifications
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of the lead compound resulted in new potent nanomolar inhibitors of MurFSp and micromolar inhibitors of MurFEc and S. aureus MurF (MurFSa). The crystal structures of MurFSp in complex with two new inhibitors (PDB codes 3zm5 and 3zm6) revealed the binding modes of compounds, which allowed us to undertake structure-based design of novel, improved analogues with optimized physicochemical properties. Although some of the compounds from the first series showed good MurF inhibitory potency and moderate antibacterial activity against S. pneumoniae, the solubility of the majority of assayed compounds in water was low. We postulated that the large planar lipophilic portion of the molecules associated with rather
4
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high molecular mass led to their high tendency for precipitation, making them inadequate drug candidates [18]. In order to present further insight into the structure-activity relationship of cyanothiophenebased MurF inhibitors, we report the design, synthesis and biological evaluation of a new
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series of inhibitors. Different structural modifications of the parent compounds resulted in a focused library of 37 new inhibitors, providing low micromolar inhibitors of MurF from E. coli and S. pneumoniae.
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2. RESULTS AND DISCUSSION
2.1. Design. According to the results from Abbott Laboratories [11, 12] and our previous research [18], 2-aminothiophene-3-carbonitrile and 5-sulfamoyl-2-chlorobenzoic acid linked
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together via an amide bond, are essential for good MurF inhibitory potency (i.e., compound I, Figure 1.). Additionally, 3D similarity search revealed micromolar inhibitor II, where the cyclohexene ring in compound I was replaced by cyclopentene ring, sulfamoyl substituent was replaced by the 1-pyrazolylmethyloxy moiety and the chloro substituents of benzoic acid part were replaced by hydrogens [17].
Due to promising potencies of compounds I and II, the structural features of both inhibitors
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were combined. Overlapping both compounds yielded the key scaffold (in red, Fig. 1.), which was used as a starting point in the design of novel MurF inhibitors. The main problem observed with compounds I and II was their low aqueous solubility due to quite high lipophilicity (ClogP(I) = 2.82, and ClogP(II) = 3.40). It is well known that antibacterial
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agents have slightly different physiochemical properties than the rest of the drugs [19, 20]. Average molecular weights are usually higher for antibacterials, with a defined cut-off at 600
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Da. In addition, the lipophilicity of the Gram-positive and especially of Gram-negative antibacterials compared to “normal” drugs is reduced. These unique properties were taken into account during the design of the second generation of MurF inhibitors, where more polar compounds were targeted. Two steps were performed in designing second-generation MurF inhibitors, as depicted in Figure 1. In the first step, various alkyl or aryl substituents bearing mostly polar functional groups (R1) were attached to the main scaffold in order to examine the inhibitory activities of compounds with different replacements of the 4-chloropyrazole moiety. In the second step, various phenyl and benzyl substituents were appended to position 6 (R2 substituent) of the
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4,5,6,7-tetrahydrobenzo[b]thiophene
and
4,5,6,7-tetrahydrothieno[2,3-c]pyridine,
respectively. It is known from our previous study that extension of these ring systems significantly improves the potency of compounds against S. pneumoniae MurF and gains the potency against other MurF orthologes [18]. Although this extension increases lipophilicity,
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the presence of the protonated amine or guanidine moiety outweighs the contribution of these lipophilic residues so that the overall ClogP does not exceed the value of 2.8. (Table S1)
To predict the binding affinity of the designed compounds to MurF, a docking study was performed using the crystal structure of MurF enzyme from S. pneumoniae (PDB code: 3zm5)
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and OEDocking software (Release 3.0.1, OpenEye Scientific Software, Inc.). This revealed the compounds with simple amine or guanidine at position R1 as the ones with the highest predicted affinities (Fig. 2a-d). Furthermore, the replacement of H with Cl on the benzoic acid
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moiety was proposed to be beneficial for potent inhibition of MurF.
As all predicted binding poses were plausible and with good docking scores, we decided to incorporate both amino and guanidine groups into the structures of potential inhibitors. The design presented herein proved to be successful, as majority of amines/guanidines demonstrated low micromolar inhibiton of MurF from E. coli and S. pneumoniae.
chlorosulfonated
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2.2. Chemistry. Compound 4 was synthesized as outlined in Scheme 1. In the first step, the 2,4-dichlorobenzoic
acid
(1)
reacted
with
the
tert-butyl
(2-
aminoethyl)carbamate to yield compound 2. Then, the free acid was coupled with 2aminothiophene through a two-stage process, including the conversion of the acid into acyl
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chloride in the presence of oxalyl chloride and catalytic amount of DMF, followed by the coupling in the presence of pyridine as a base, to obtain 3. Compound 4 was prepared by the cleavage of the Boc protective group in the presence of CF3COOH, and was subsequently
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transformed into hydrochloride salt with HCl in ethyl acetate. The synthesis of target compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a and 27b is presented in Scheme 2. First, the hydroxy group of compounds 5 and 6 was acetylated with acetic anhydride in pyridine. Next, compounds 9 and 10 were obtained using the same synthetic strategy as described for 3. The acetoxy protective groups of compounds 9 and 10 were cleaved in the presence of K2CO3 in methanol. The diverse alkyl or aryl substituents were attached to the hydroxyl group of compounds 11 and 12 with two different approaches. In the first method, compounds 14, 17-19, 20a, 20b, 21a and 21b were obtained using K2CO3 or Cs2CO3 and different benzyl or alkyl halides. An alternative method (the Mitsunobu reaction 6
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using PPh3, diisopropyl azodicarboxylate (DIAD) and the corresponding alcohol) was employed to give compounds 15 and 16. As (1H-pyrazol-1-yl)methanol (13) was not commercially available, we prepared it by the already reported procedure [21]. Compounds 22a, 22b, 23a and 23b were obtained after the cleavage of the Boc protective group using
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CF3COOH. Amine groups of compounds 22a, 23a and 23b were converted into di-Boc protected guanidines in the presence of mercuric chloride and triethylamine. Finally, the cleavage of both Boc protective groups with CF3COOH, followed by direct transformation to hydrochloride salts with HCl in ethyl acetate, gave target compounds 26a, 27a and 27b.
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The synthesis of target compounds 33-36, 37a-f, 38a-f, 39c, 42a-e and 43 is outlined in Scheme 3. 2-Chloro-5-hydroxybenzoic (6) acid was first protected as methyl ester and then alkylated using tert-butyl (2-bromoethyl)carbamate and K2CO3 to yield 28. Compound 31
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was obtained after alkaline hydrolysis of the methyl ester with aqueous sodium hydroxide, followed by cleavage of Boc protective group using CF3COOH. The amino group was further protected as trifluoroacetamide with trifluoroacetic anhydride and pyridine as a base to give 32. Compounds 33a-b were obtained from 32 and the corresponding 2-aminothiophenes using the same procedure as described for compound 3. Compound 34 was obtained after the cleavage of the Boc group of 33a with the CF3COOH. Final compounds 35 and 36 were
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obtained by alkaline hydrolysis of the trifluoroacetamide group of 33b and 34, respectively. The variously substituted benzyl fragments were attached to compound 34 via two different strategies. The first method was a nucleophilic substitution, where K2CO3 and different benzyl bromides were used to yield compounds 37a-c; in the second method, compounds
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37d-f were synthesized by sodium triacetoxyborohydride-promoted reductive amination of 34 with various benzaldehydes. In the next step, the removal of the trifluoroacetamide group of 37a-f was achieved through alkaline hydrolysis, which led to target compounds 38a-f. Finally,
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target compounds 42a-e and 43 were obtained using the same strategy as described for 26a. 2.3. Inhibition of MurF. All target compounds were assayed for inhibition of MurF from E. coli and S. pneumoniae, using the Malachite green assay [22], which detects the orthophosphate generated during the enzymatic reaction. To avoid possible non-specific inhibition, all of the compounds were tested in the presence of detergent (0.005% Triton X114) [23]. Results are presented as residual activities (RAs) of the respective enzyme in the presence of 250 or 100 µM of each compound. For the most active compounds, IC50 values were also determined.
7
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First, a small series of compounds (Table 1, compounds 14-19) was synthesized where the 4chloropyrazole moiety was replaced with different aryl and alkyl fragments. None of these compounds significantly inhibited MurF. However, compounds with aminoethyl (23a) and aminopropyl substituents (23b) showed modest inhibitory activities against both MurF
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enzymes. As expected, the introduction of chlorine ortho to the amide bond was essential for the activity while the length of alkyl chain had little influence on the potency. Furthermore, compounds with the guanidino moiety (26a, 27a and 27b) were prepared and their potencies were 2- to 3-fold higher than potencies for the corresponding amine.
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If we compare the compound I with the compounds 23a and 27a, we can notice that the morpholinosulfonyl fragment and chlorine next to sulfonamide linker are rather important for good inhibition of MurFSp. This can be seen also in the co-crystal structure, where one of the
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sulfonyl oxygens of compound I makes two hydrogen bonds with the protein and the chlorine forms hydrophobic interactions [18]. These interactions are not present in our new inhibitors, resulting in their higher IC50 values against MurFSp. However, this interaction pattern is not necessary for inhibition of MurFEc, as compound I showed no inhibition of this ortholog. Furthermore, to examine the importance of the sulfonamido group, compound 4 was synthesized. By the comparison of compounds 4 and 23a, we can see that the inhibition is in
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the same range as far as MurFSp is concerned (4, IC50 = 219 µM; 23a, IC50 = 170 µM). However, in the case of MurFEc compound 4 was 2-fold less potent than 23b, which makes sulfonamido group less favorable for inhibition of this enzyme. In the next step, compounds with 4-hydroxyphenyl and different substituted benzyl
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substituents attached to position 6 of the 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile or 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile moiety were synthesized (Table 2), as it is known that substitution of this ring leads to greater potency [11, 12, 18]. First,
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compounds with the 4-hydroxyphenyl fragment appended to the saturated ring (33b and 35) were prepared. The protected compound (33b) showed no inhibition against MurF while the compound with free amino and hydroxyl groups (35) inhibited both MurF enzymes in the low micromolar range (IC50 = 58 µM and 81 µM for MurFSp and MurFEc, respectively). Compounds with different benzyl substituents and trifluoracetamide group (37a-f) showed modest inhibitory activities against MurF. The most potent inhibitor of MurFSp and MurFEc within this set was 37f with the p-tetrazole group attached to the benzyl fragment, with IC50 values of 103 and 152 µM, respectively. Furthermore, inhibitors with a free amino group (38a-f and 49c) were synthesized and most of them showed improved activity against both
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MurF enzymes. Exceptions were 38c and 38f with carboxylic acid and tetrazole on para position, respectively, which showed significantly lower inhibition of MurFSp activity. The introduction of substituents to either para or meta position on the benzyl ring led to 2-fold lower potency against MurFSp compared to 38a (IC50 = 51 µM), while the p-substituents on
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the benzyl ring did not influence the activity against MurFEc within this series. Finally, compounds with the guanidine substituent (42a-e and 43) were prepared. The most potent inhibitor within this set was compound 43, with the p-hydroxyphenyl group attached to the saturated ring: IC50 values were 20 and 25 µM against MurFSp and MurFEc, respectively. Compounds 42a and 42d had similar inhibitory activities against both MurF orthologs as the
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corresponding amine precursors, while inhibitors 42b, 42c and 42e showed about 2-fold better activity against MurFSp than their corresponding amines (38b, 38c and 38e, respectively).
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Unfortunately, the introduction of the guanidine moiety does not improve the activity of inhibitors (42a-e) against MurFEc compared to their respective amine precursors (38a-e and 39c).
2.4. Antibacterial activity. For antibacterial evaluation of selected compounds, typical representatives of Gram-positive (S. aureus) and Gram-negative bacteria (E. coli) were chosen (Table 3). In the case of E. coli, compounds were further evaluated against efflux-
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deficient strains (E. coli SM1411 and ES100), and those in which the outer membrane had been artificially permeabilized with polymyxin B (PMBN). Compounds 23a-b, 26a and 27ab showed modest antibacterial activities against S. aureus and E. coli, which increased against the latter species upon deletion of the AcrAB-TolC efflux transporter. Compounds 33a, 34, 36
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and 37a-f showed no activity against E. coli and S. aureus; this was expected since none of these compounds showed potent inhibition of MurFEc and MurFSp. Compound 35 was inactive
AC C
against S. aureus and E. coli, although antibacterial activity was observed against PMBNpermeabilized and/or efflux-deficient strains of E. coli. Compounds 38a-f and 39c showed modest antibacterial activities against both bacterial species. 38c showed limited inhibition of both MurF enzymes (IC50 ~ 500 µM), and no antibacterial activity was observed. On the other hand, compounds 38b, 38d, 38e and 39c demonstrated antibacterial activities against S. aureus and E. coli strains, and inhibited isolated enzymes more potently (IC50 around or below 100 µM). The most potent inhibitor of MurF within this series was 38a, which has 2-fold lower MIC values against all strains compared to its analogues. Finally, compounds with the guanidine moiety 42a-e and 43 showed promising antibacterial activities against S. aureus with MICs ranging between 4 and 32 µg/mL. The latter compounds also demonstrated a 9
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modest antibacterial activity against E. coli. All compounds with antibacterial activity against E. coli exhibited increased activity against efflux-deficient strains and strains with increased membrane permeability. Additional studies were performed to determine whether the antibacterial activities of the
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compounds are the consequence of peptidoglycan biosynthesis inhibition or non-specific effect. The BacLightTM assay was used to assess the membrane integrity of S. aureus SH1000 at 4 × MIC values of inhibitors [24]. Unfortunately, all of the tested compounds (38b, 42a, 42c, 42e and 43) significantly decreased the membrane integrity thus showing non-specific
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mechanism of the antibacterial activity.
3. CONCLUSION
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We have designed, synthesized and evaluated a second generation of 37 new MurF inhibitors with cyanothiophene scaffold. The major improvement of the present compounds over the first-generation inhibitors is their reduced lipophilicity and increased solubility, with conserved enzyme inhibitory potency in micromolar range. The most potent inhibitors have well balanced inhibitory properties against both MurF from S. pneumonie and MurF from E. coli (the best inhibitor 42 inhibits MurFSp and MurFEc with IC50 values of 20 µM and 25 µM,
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respectively). Furthermore, the most potent compounds within this series demonstrate antibacterial activities. As these compounds achieve part of their antimicrobial action by damaging the bacterial cytoplasmic membrane, they are unlikely to be developed as antibacterial drug candidates until this nonspecific effect is diminished, without
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compromising their inhibitory effect on MurF.
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4. EXPERIMENTAL SECTION 4.1. Inhibition assay. The inhibition of MurF ligases was determined using the Malachite green assay with slight modifications. The mixture with final volume of 50 µL contained: S. pneumoniae MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114, 100 µM D-Ala-D-Ala,
50 µM UMtri-L-Lys, 250 µM ATP, purified MurFSp [17], and 250 or 100 µM of
each tested compound dissolved in DMSO. E. coli MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114, 600 µM D-AlaD-Ala,
100 µM UMtri-mDAP, 500 µM ATP, purified MurFEc [25], and 250 or 100 µM of
each tested compound dissolved in DMSO.
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In both cases, the final concentration of DMSO was 5% (v/v). After incubation for 15 min at 37 °C, the enzyme reaction was stopped by adding Biomol®reagent and the absorbance was measured at 650 nm. All of the experiments were run in duplicate. Residual activities (RAs) were calculated with respect to similar assays without the tested compounds and with 5%
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DMSO. The IC50 values, which were determined by measuring the residual activities at seven different compound concentrations, represented the concentration for which the RA was 50%.
4.2. Microbiological evaluation. Minimum inhibitory concentrations (MICs) for selected compounds were determined by broth microdilution in Mueller-Hinton broth (MHB), against
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S. aureus SH1000 [26], and E. coli strains 1411, SM1411 (acrAB derivative of 1411) [27], AB734 and ES100 (tolC derivative of AB734) [28], according to CLSI guidelines [29]. To
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assess the impact of outer-membrane permeability on antimicrobial activity against E. coli, MICs were also determined against strains 1411 and AB734 in the presence of 4 µg/mL polymyxin B nonapeptide (PMBN) [30]. The BacLightTM assay was used to measure membrane damage in S. aureus SH1000 induced by compounds at 4 × MIC, compared to a drug free control [24].
4.3. Chemistry. All of the chemicals used were obtained from commercial sources (Acros,
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Sigma-Aldrich, Alfa Aeser, Apollo Scientific, Fluka and Merck), and were used without further purification. Solvents were used without purification or drying, unless otherwise stated. Reactions were monitored using analytical thin-layer chromatography plates (Merck, silica gel 60 F254, 0.25 mm), and compounds were visualized with ultraviolet light and
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ninhydrin or ferric chloride. Silica gel grade 60 (particle size 0.040–0.063 mm, Merck, Germany) was used for flash column chromatography. 1H and
13
C NMR spectra were
AC C
recorded on a Bruker AVANCE III 400 MHz spectrometer in acetone-d6, CDCl3, DMSO-d6, CD3OD or pyridine-d5, with TMS as the internal standard. Mass spectra were obtained with a VG-Analytical Autospec Q mass spectrometer (Centre for Mass Spectrometry, Institute Jožef Stefan, Ljubljana). Infrared spectra were recorded on a Perkin-Elmer FTIR 1600 spectrometer. Melting points were determined using a Reichert hot-stage microscope and are uncorrected. Microanalyses were performed on a 240 C Perkin Elmer elemental analyzer. Analyses indicated by the symbols of the elements were within 0.4% of the theoretical values. HPLC analyses were performed on an Agilent Technologies HP 1100 instrument with a G1365B UV-VIS detector (220 and 254 nm), using a Luna C18 column (4.6 × 250 mm) at a
11
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flow rate of 1 mL/min. The eluent was a mixture of 0.1% CF3COOH in water (A) and acetonitrile (B). The gradient was 10% to 90% B in 19 min. 4.3.1. 5-(N-(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)-2,4-dichlorobenzoic acid (2). To a solution of tert-butyl (2-aminoethyl)carbamate (0.450 g, 2.81 mmol) in DCM (15 mL), Et3N
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(0.59 mL, 4.21 mmol) was added. The reaction mixture was cooled on an ice bath and 1 (0.813 g, 2.81 mmol) was added portion wise. The reaction mixture was stirred for 16 h, diluted with 30 mL of DCM, and washed with 1 M HCl (2 × 40 mL) and brine (1 × 50 mL), and dried with Na2SO4. The solvent was evaporated under reduced pressure and the crude
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product was purified by flash chromatography.
Yield = 15%; white needles, mp = 192-193 °C; 1H NMR (400 MHz, CDCl3): δ 1.34 (s, 9H, 3
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× CH3), 2.90 (q, J = 5.6 Hz, 2H, SO2-NH-CH2), 2.96 (q, J = 6.4 Hz,2H, CO-NH-CH2), 6.72 (t, J = 5.6 Hz, 1H, SO2-NH), 8.01 (s, 1H, Ar-H), 8.17 (t, J = 6.4 Hz, 1H, O-CO-NH), 8.34 (s, 1H, Ar-H) ppm. 13C NMR (100 MHz, CD3OD): δ 28.77, 28.83, 40.74, 41.08, 41.23, 43.57, 80.26, 80.35, 132.19, 133.93, 134.67, 136.52, 137.01, 138.40, 158.46, 158.60, 167.88 ppm. ESI HRMS calcd. for [M+(H)]+ 555.1447, found 555.1448. IR (KBr) νmax: 3377, 2980, 2935, 1694, 1586, 1526, 1454, 1385, 1367, 1336, 1277, 1253, 1163, 1086 cm-1. Tert-butyl
(2-(2,4-dichloro-5-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
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4.3.2.
yl)carbamoyl) phenylsulfonamido)ethyl)carbamate (3). To a suspension of 2 (0.305 g, 0.74 mmol) in anhydrous DCM (15 mL), catalytic amounts of DMF and (COCl)2 (0.190 mL, 2.21 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at room temperature. The
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solvent was evaporated to dryness and the residue was dissolved in DCM (20 mL), the mixture of 2-aminothiophene (0.125 g, 0.70 mmol) and pyridine (0.180 mL, 2.21 mmol) in DCM (15 mL) was added drop-wise, and the reaction was stirred overnight at room
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temperature. The reaction mixture was washed with 1 M HCl (2 × 40 mL), saturated aqueous NaHCO3 (1 × 40 mL) and brine (1 × 40 mL), and dried (Na2SO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography. Yield = 24%; white crystals, mp = 196–198 °C; 1H NMR (400 MHz, CDCl3): δ 1.43 (s, 9H, 3 × CH3), 1.85–1.90 (m, 4H, CH2-CH2-CH2-CH2), 2.63 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 2.70 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 3.06 (q, J = 4.8 Hz, 2H, SO2-NH-CH2), 3.23 (q, J = 4.8 Hz, 2H, SO2-NH-CH2-CH2), 4.95 (t, J = 4.8 Hz, 1H, SO2-NH), 6.21 (t, J = 4.8 Hz, 1H, SO2-NH-CH2-CH2-NH), 7.61 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 10.04 (bs, 1H, NH-CO-Ar) ppm. 13C NMR (100 MHz, CDCl3): δ 22.10, 23.05, 24.01, 24.06, 28.35, 40.24, 43.44, 79.99, 12
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94.88, 114.59, 129.62, 131.57, 132.12, 132.61, 132.90, 134.42, 136.12, 136.57, 145.83, 156.34, 161.00 ppm. ESI HRMS calcd. for [M-(H)]- 571.0643, found 571.0644. IR (KBr) νmax: 3414, 3077, 2974, 2227, 1683, 1637, 1616, 1579, 1481, 1447, 1400, 1383, 1369, 1347, 1327, 1286, 1259, 1167, 1105, 1078 cm-1. 5-[(2-aminoethyl)sulfamoyl]-2,4-dichloro-N-(3-cyano-4,5,6,7-tetrahydro-1-
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4.3.3.
benzothiophen-2-yl)benzamide hydrochloride (4). To a solution of 3 (0.095 g, 0.166 mmol) in DCM (10 mL), CF3COOH (0.6 mL, 8.0 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure and the product
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was dissolved in 1 M HCl in EtOAc (3 mL). EtOAc was removed and the product was dried in a desiccator overnight.
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Yield = 100%; pale brown crystals, mp = 204–206 °C; 1H NMR (400 MHz, MeOD): δ 1.85– 1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.62 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 2.72 (t, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 3.10 (t, J = 6.0 Hz, 2H, SO2-NH-CH2), 3.22 (t, J = 6.0 Hz, 2H, SO2-NH-CH2-CH2), 7.91 (s, 1H, Ar-H), 8.33 (s, 1H, Ar-H) ppm, NH3+ and CONH are exchanged. 13C NMR (100 MHz, MeOD): δ 23.31, 24.23, 24.91, 25.09, 40.77, 41.13, 97.21, 114.61, 131.01, 133.05, 133.15, 134.25, 134.82, 135.66, 137.55, 137.99, 146.59, 164.12 ppm. ESI HRMS calcd. for [M+(H)]+ 473.0276, found 473.0268. IR (KBr) νmax: 3437, 2928, 2361, 220 nm, 100% at 254 nm).
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2228, 1671, 1582, 1448, 1334, 1295, 1174, 1084, 1034 cm-1. HPLC tR = 11.977 min (100% at
4.3.4. General procedure for synthesis of compounds 7 and 8.
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To a solution of 5-hydroxybenzoic acid (5) (17.4 mmol) in pyridine (10 mL), acetic anhydride was added dropwise (6.6 mL, 70.0 mmol). The reaction mixture was stirred at room temperature for 1 h and was subsequently poured into water (50 mL). The water phase was
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acidified to pH 2 and extracted with EtOAc (3 × 40 mL). The combined organic phases were washed with 0.1 M HCl (1 × 100 mL), brine (1 × 100 mL), and dried with Na2SO4. The solvent was evaporated under reduced pressure and the crude product was used without further purification.
4.3.4.1. 3-acetoxybenzoic acid (7). Yield = 89%; white crystals, mp = 127–129 °C (lit. [31] 128.7–131.3 °C); 1H NMR (400 MHz, DMSO-d6): δ 2.28 (s, 3H, CO-CH3), 7.39 (ddd, J1 = 8.1, J2 = 2.4, J3 = 1.1 Hz, 1H, Ar-H), 7.52–7.56 (m, 1H, Ar-H), 7.65–7.69 (m, 1H, Ar-H), 7.80–7.86 (m, 1H, Ar-H), 13.18 (bs, 1H, COOH) ppm.
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4.3.5. General procedure for synthesis of 9 and 10. To a solution of 7 (14.0 mmol) in anhydrous DCM (30 mL), DMF (catalytic amount) and (COCl)2 (3.60 mL, 42 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at room temperature. The solvent was evaporated to dryness and the residue was dissolved in
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DCM (20 mL). A mixture of 2-aminothiophene (2.245 g, 12.6 mmol) and pyridine (3.30 mL, 42 mmol) in DCM (30 mL) was added drop-wise, and the reaction was stirred overnight at room temperature. The reaction mixture was washed with 1 M HCl (40 mL), saturated aqueous NaHCO3 (40 mL) and brine (40 mL), and dried (Na2SO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography
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and recrystallized from ethanol.
4.3.5.1. 3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenyl acetate (9).
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Yield = 65%; white crystals, mp = 161–162 °C; 1H NMR (400 MHz, CDCl3): δ 1.82–1.88 (m, 4H, CH2-CH2-CH2-CH2), 2.34 (s, 3H, OCO-CH3), 2.59–2.71 (m, 4H, CH2-CH2-CH2-CH2), 7.35 (ddd, J1 = 8.1, J2 = 2.3, J3 = 1,0 Hz, 1H, Ar-H), 7.54 (t, J = 8.0 Hz, 1H, Ar-H), 7.65 (t, J = 2.0 Hz, 1H, Ar-H), 7.78 (ddd, J1 = 7.8, J2 = 1.7, J3 = 1.0 Hz, 1H, Ar-H), 8.99 (s, 1H, CONH) ppm. 13C NMR (100 MHz, CDCl3): δ 21.11, 22.12, 23.09, 23.98, 94.26, 114.54, 121.12, 124.93, 126.26, 128.94, 130.07, 131.25, 133.41, 146.53, 150.95, 162.87, 169.30 ppm. ESI
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HRMS calcd. for [M+(H)]+ 341.0960, found 341.0966. IR (KBr) νmax: 3237, 3202, 3074, 2936, 2842, 2365, 2345, 2223, 2212, 1765, 1671, 1572, 1545, 1458, 1369, 1325, 1291, 1272, 1206, 1146, 1094,1010 cm-1. Anal. Calcd. for C18H16N2O3S × 0.1 H2O: C, 63.18; H, 4.77; N, 8.19. Found C, 62.90; H, 4.45; N, 8.05.
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4.3.6. General procedure for synthesis of 11 and 12. To a suspension of 9 (3.0 g, 8.0 mmol) in methanol (20 mL), K2CO3 (1.69 g, 12.0 mmol) was
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added. The reaction mixture was stirred at room temperature for 1 h, and the solvent was evaporated under reduced pressure. The residue was dissolved in 1 M HCl (40 mL) and extracted with EtOAc (3 × 40 mL). The combined organic phases were washed with brine, dried with Na2SO4, and the solvent was removed under reduced pressure. The product was used without further purification. 4.3.6.1.
N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-hydroxybenzamide
(11).
1
Yield = 87%; small white crystals, mp = 249–250°C; H NMR (400 MHz, acetone-d6): δ 1.80–1.90 (m, 4H, CH2-CH2-CH2-CH2), 2.53–2.71 (m, 4H, CH2-CH2-CH2-CH2), 7.07–7.13 (m, 1H, Ar–H), 7.35–7.42 (m, 1H, Ar–H), 7.45–7,54 (m, 2H, 2 × Ar–H), 8.85 (s, 1H, CO–
14
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NH), 10.52 (s, 1H, Ar–OH) ppm.
13
C NMR (100 MHz, acetone-d6): δ 22.92, 23.84, 24.46,
24.67, 96.20, 114.52, 115.62, 119.87, 120.44, 129.47, 130.70, 132.17, 134.96, 147.33, 158.47, 165,45 ppm. ESI HRMS calcd. for [M+(H)]+ 299.0854, found 299.0856. IR (KBr) νmax: 3380, 3244, 3077, 2992, 2943, 2922, 2840, 2345, 2222, 1660, 1616, 1587, 1576, 1559, 1490, 1458,
C, 64.41; H, 4.73; N, 9.39. Found C, 64.19; H,4.56; N, 9.27. 4.3.7. General procedure for synthesis of 14-29, 20a-b and 21a-b.
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1399, 1327, 1306, 1283, 1260, 1216, 1147, 1078 , 1029 cm-1. Anal. Calcd. for C16H14N2O2S:
Method A: To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-
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hydroxybenzamide (12) (0.270 g, 0.90 mmol) in DMF (5 mL), K2CO3 or Cs2CO3 (1.35 mmol) was added. After 15 min, the corresponding bromide or chloride (1.35 mmol) was added and the reaction mixture was stirred for 16 h at 50 °C. The solvent was evaporated under reduced
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pressure and the residue was dissolved in EtOAc (40 mL). The organic phase was washed with water (2 × 30 mL), 1 M HCl (1 × 30 mL), brine (1 × 30 mL), and dried with Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography. 4.3.7.1.
3-((4-chlorobenzyl)oxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)benzamide (14). Yield = 44%; white crystals, mp = 195–196 °C; 1H NMR (400 MHz,
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CDCl3): δ 1.79–1.90 (m, 4H, CH2-CH2-CH2-CH2), 2.58–2.70 (m, 4H, CH2-CH2-CH2-CH2), 5.11 (s, 2H, O-CH2-Ar), 7.17–7.21 (m, 1H, Ar-H), 7.34–7.42 (m, 4H, 4 × Ar-H), 7.42–7.48 (m, 2H, 2 × Ar-H), 7.51–7.55 (m, 1H, Ar-H), 8.85 (s, 1H, CO-NH) ppm.
13
C NMR (100
MHz, CDCl3): δ 22.12, 23.10, 24.00, 69.46, 94.08, 113.64, 114.49, 119.42, 120.07, 128.87,
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128.92, 130.26, 131.12, 133.29, 134.06, 134.74, 146.57, 159.04, 163.27 ppm. ESI HRMS calcd. for [M+(H)]+ 429.0934, found 423.0939. IR (KBr) νmax: 3446, 3252, 3200, 3076, 2932,
AC C
2855, 2366, 2217, 1889, 1670, 1602, 1595, 1573, 1558, 1490, 1470, 1456, 1411, 1399, 1378, 1327, 1296, 1279, 1229, 1164, 1153, 1116, 1095, 1082, 1054, 1032, 1014 cm-1. Anal. Calcd. for C23H19ClN2O2S × 0.3 H2O: C, 64.49; H, 4.61; N, 6.54. Found C, 64.29; H, 4.32; N, 6.44. Method B: To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3hydroxybenzamide (12) (0.250 g, 0.838 mmol) in anhydrous THF (5 mL), the corresponding alcohol (0.838 mmol) and PPh3 (0.330 g, 1.25 mmol) were added. The reaction mixture was cooled on ice bath and DIAD (0.25 mL, 1.25 mmol) was added. The reaction was stirred overnight at room temperature. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography.
15
ACCEPTED MANUSCRIPT
4.3.7.2.
3-((1H-pyrazol-1-yl)methoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)benzamide (16). Yield = 26%; pale yellow crystals, mp = 86–88 °C; 1H NMR (400 MHz, CDCl3): δ 1.69–1.79 (m, 4H, CH2-CH2-CH2-CH2), 2.44 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2CH2), 2.56 (t, J = 4.8 Hz, 2H, CH2-CH2-CH2-CH2), 6.09 (s, 2H, CH2-O), 6.37 (t, J = 2.4 Hz,
RI PT
1H, CH-CH-CH-pyrazol), 6.84 (ddd, J1 = 8.0, J2 = 2.8, J3 = 0.8 Hz, 1H, Ar-H), 6.92 (dt, J1 = 7.6, J2 = 1.6 Hz, 1H, Ar-H), 6.98 (dd, J1 = 2.4, J2 = 1.6 Hz, 1H, Ar-H), 7.10 (t, J = 8.0 Hz, 1H, Ar-H), 7.54 (dd, J1 = 1.6, J2 = 0.4 Hz, 1H, CHA-CH-CHB-pyrazole), 7.62 (bs, 1H, CO-NH), 7.84 (d, J = 2.4 Hz, 1H, CHA-CH-CHB-pyrazole) ppm. 13C NMR (100 MHz, CDCl3): δ 21.62, 22.62, 24.12, 24.62, 64.25, 107.30, 110.01, 112.66, 115.25, 118.54, 119.74, 129.46, 131.28,
SC
134.42, 134.79, 136.96, 140.30, 148.79, 156.04, 170.75 ppm. ESI HRMS calcd. for [M+(H)]+ 379.1229, found 379.1220. IR (KBr) νmax: 3418, 2937, 2225, 1668, 1599, 1449, 1376, 1291,
M AN U
1248, 1194, 1128, 1086, 1054 cm-1. HPLC tR = 12.696 min (100% at 220 nm, 100% at 254 nm).
4.3.8. General procedure for synthesis of 22a-b and 23a-b.
To a solution of Boc-protected amine (20a-b and 21a-b) (0.1 mmol) in DCM, CF3COOH (3– 5 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. Solvent was evaporated and some products were converted into hydrochloride salts with HCl
4.3.8.1.
TE D
in EtOAc.
3-(2-aminoethoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide
(22a). Yield = 76%; white crystals, mp = 158–160 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.73–1.84 (m, 4H, CH2-CH2-CH2-CH2), 2.54–2.67 (m, 4H, CH2-CH2-CH2-CH2), 3.25–3.31
EP
(m, 2H, NH-CH2-CH2-O), 4.22–4.30 (m, 2H, NH-CH2-CH2-O), 7.25–7.31 (m, 1H, Ar-H), 7.50-7.57 (m, 2H, 2 × Ar–H), 7.58–7.64 (m, 1H, Ar-H), 7.93–8.14 (bs, 3H, NH3+) ppm.
13
C
AC C
NMR (100 MHz, DMSO-d6): δ 21.67, 22.57, 23.45, 23.59, 38.32, 64.63, 96.29, 114.18, 114.30, 118.81, 121.07, 128.84, 129.82, 131.45, 133.80, 146.16, 157.74, 164.79 ppm. ESI HRMS calcd. for [M+(H)]+ 342.1276, found 342.1277. IR (KBr) νmax: 3434, 3235, 3195, 3080, 2936, 2220, 1669, 1576, 1559, 1511, 467, 1450, 1326, 1297, 1282, 1230, 1203, 1183, 1128, 1076, 1025 cm-1. Anal. Calcd. for C18H19N3O2S × CF3COOH: C, 52.74; H, 4.43; N, 9.23. Found C, 52.56; H, 4.22; N, 9.13. 4.3.9. General procedure for synthesis of compounds 24a, 25a-b, 40a-e and 41. To a solution of amine (22a, 23a-b, 35, 38a-e or 39c) (0.150 mmol) in anhydrous DMF (5 mL), di-Boc-S-methylisothiourea (0.150 mmol), Et3N (0.300 mmol) and HgCl2 (0.150 mmol)
16
ACCEPTED MANUSCRIPT
were added. The reaction mixture was stirred for 2 h at room temperature. The formed precipitate was filtered off and the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (30 mL), washed with water (3 × 30 mL), saturated NaHCO3 (30 mL), brine (30 mL), and dried with Na2SO4. The crude product was purified by flash 4.3.9.1.
RI PT
chromatography. Tert-butyl(tert-butoxycarbonylamino)(2-(3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]
thiophen-2-ylcarbamoyl)phenoxy)ethylamino)methylencarbamate (24a). Yield = 51%; white crystals, mp = 165–166 °C; 1H NMR (400 MHz, CDCl3): δ 1.53 (dd, J1 = 6.8, J2 = 1.2 Hz, 16H, 2 × COO-(CH3)3), 1.81–1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.61–2.74 (m, 4H, CH2-CH2-
SC
CH2-CH2), 3.90 (q, J = 5.3 Hz, 2H, NH-CH2-CH2-O), 4.20 (t, J = 5.2 Hz, 2H, NH-CH2-CH2O), 7.18–7.27 (m, 1H, Ar-H), 7.41–7.50 (m, 2H, 2 × Ar-H), 7.50–7.56 (m, 1H, Ar-H), 8.78 (t, 13
C
M AN U
J = 5.3 Hz, 1H, C-NH-CH2), 8.90 (s, 1H, CO-NH-C), 11.20 (s, 1H, C-NH-Boc) ppm.
NMR (100 MHz, acetone-d6): δ 14.37, 22.92, 23.30, 23.83, 24.46, 24.68, 28.14, 28.47, 32.32, 40.70, 67.21, 79.02, 83.84, 96.29, 114.50, 114.93, 119.77, 121.46, 129.56, 130.78, 132.27, 135.00, 159.79 ppm. ESI HRMS calcd. for [M+(H)]+ 584.2543, found 584.2545. IR (KBr) νmax: 3341, 3285, 3080, 2976, 2934, 2362, 2344, 2219, 1741, 1656, 1623, 1576, 1546, 1493, 1459, 1434, 1415, 1397, 1362, 1330, 1300, 1276, 1223, 1146, 1086, 1062, 1046, 1027 cm-1. Anal. Calcd. for C29H37N5O6S × 0.9 H2O: C, 58.06; H, 6.52; N, 11.67. Found C, 58.33; H,
TE D
6.44; N, 11.27.
4.3.10. General procedure for synthesis of compounds 26a, 27a-b, 42a-e and 43. To a solution of Boc-protected guanidine (24a, 25a-b, 40a-e and 41) (0.095 mmol) in DCM
EP
(5 mL), CF3COOH (0.210 mL, 2.84 mmol) was added, and the mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and 1 M HCl in EtOAc (3 ml) was added to the residue. The formed precipitate was collected by filtration. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2-guanidinoethoxy)
AC C
4.3.10.1.
benzamide (26a). Yield = 86%; white crystals, mp = 179–181 °C; 1H NMR (400 MHz, acetone-d6): δ 1.84–1.89 (m, 4H, CH2-CH2-CH2-CH2), 2.57–2.61 (m, 2H, CH2-CH2-CH2CH2), 2.68–2.72 (m, 2H, CH2-CH2-CH2-CH2), 3.78–3.83 (m, 2H, NH-CH2-CH2-O), 4.33 (t, J = 5.2 Hz, 2H, NH-CH2-CH2-O), 7.25–7,29 (m, 1H, Ar-H), 7.48–7.53 (m, 1H, Ar-H), 7.62– 7.69 (m, 2H, 2 × Ar-H), 7.79 (bs, 3H, C–NH3+), 8.68 (bs, 1H, NH), 10.72 (bs, 1H, NH) ppm. 13
C NMR (100 MHz, MeOD): δ 22.91, 23.82, 24.47, 24.68, 41.68, 41.79, 67.71, 96.50,
114.50, 114.94, 119.87, 121.62, 129.64, 130.74, 132.31, 135.02, 147.20, 159.52, 165.34 ppm. ESI HRMS calcd. for [M+(H)]+ 384.1494, found 384.1493. IR (KBr) νmax: 3425, 3366, 3179,
17
ACCEPTED MANUSCRIPT
2936, 2855, 2363, 2344, 2225, 1674, 1579, 1558, 1454, 1397, 1326, 1304, 1272, 1206, 1186, 1134, 1063, 1028 cm-1. HPLC tR = 11,836 min, (98.2% at 220 nm, 100% at 254 nm). 4.3.11. Methyl 2-chloro-5-hydroxybenzoate (28). To a cooled (0°C) solution of 2-chloro-5hydroxybenzoic acid (6) (4 g, 23.6 mmol) in methanol (50 mL), thionyl chloride (2.5 mL, 35
RI PT
mmol) was added drop-wise. The reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (100 ml). The organic phase was washed with saturated NaHCO3 (3 × 50 mL), water (50 mL) and brine (50 mL), dried with Na2SO4 and evaporated under reduced pressure.
SC
Yield = 99%; white crystals, mp = 96–97 °C; 1H NMR (400 MHz, CDCl3): δ 3.91 (s, 3H, OCH3), 6.91 (dd, J1 = 8.7, J2 = 3.1 Hz, 1H, Ar-H), 7.27 (d, J = 8.7 Hz, 1H, Ar-H), 7.31 (d, J = 3.1 Hz, 1H, Ar-H).
13
C NMR (100 MHz, CDCl3): δ 52.79, 118.15, 120.33, 124.67, 130.25,
M AN U
132.14, 154.51, 166.77 ppm. ESI HRMS calcd. for [M+(H)]+ 187.0162, found 187.0155. HPLC tR = 10.618 min (100% at 220 nm, 100% at 254 nm).
4.3.12. Methyl 5-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-chlorobenzoate (29). To a solution of 28 (1.00 g, 5.36 mmol) in DMF (15 mL), K2CO3 (1.11 g, 8.04 mmol) was added. After 10 min at room temperature, tert-butyl 2-bromoethylcarbamate (1.56 g, 6.97 mmol) was added
TE D
and the reaction mixture was stirred at 80 °C for 16 h. DMF was evaporated, the crude residue was dissolved in EtOAc (50 mL), washed with water (2 × 30 mL), 1 M NaOH (2 × 30 mL), water (1 × 50 mL), brine (10 mL), and dried with Na2SO4. The crude residue was purified by flash chromatography.
EP
Yield = 73%; colourless oil; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, 3 × CH3), 3.56 (q, J = 5.6 Hz, 2H, O-CH2-CH2-NH), 3.95 (s, 3H, O-CH3), 4.05 (t, J = 4.8 Hz, 2H, O-CH2-CH2-
AC C
NH), 4.98 (bs, 1H, NH-CO), 6.98 (dd, J1 = 8.8, J2 = 2.8 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H), 7.37 (d, J = 6.0 Hz, 1H, Ar-H) ppm.
13
C NMR (100 MHz, CDCl3): δ 28.38, 39.95, 52.53,
67.71, 79.72, 116.72, 119.22, 125.39, 130.56, 131.95, 155.83, 156.88, 165.87 ppm. ESI HRMS calcd. for [M+(H)]+ 330.1108, found 330.1103. 4.3.13. 5-(2-(tert-butoxycarbonylamino)ethoxy)-2-chlorobenzoic acid (30). To a solution of 29 (1.00 g, 3.03 mmol) in a mixture of dioxane and water (1/1, 30 mL), NaOH (0.24 g, 6.00 mmol) was added. The reaction mixture was stirred at room temperature for 4 h. Dioxane was evaporated under reduced pressure and the remaining water was acidified with 1 M HCl to pH 3. The water phase was extracted with EtOAc (3 × 30 mL). The combined organic phases
18
ACCEPTED MANUSCRIPT
were washed with brine (1 × 50 mL), dried with Na2SO4 and evaporated under reduced pressure. Yield = 98%; white crystals, mp = 117–119 °C; 1H NMR (400 MHz, CDCl3): δ 1.48 (s, 9H, 3 × CH3), 3.58 (q, J = 5.6 Hz, 2H, O-CH2-CH2-NH), 4.07 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH),
RI PT
5.03 (bs, 1H, NH-CO), 7.03 (dd, J1 = 8.0, J2 = 2.4 Hz, 1H, Ar-H), 7.40 (d, J = 8.8 Hz, 1H, ArH), 7.52 (d, J = 2.8 Hz, 1H, Ar-H), 7.64 (bs, 1H, COOH) ppm. 13C NMR (100 MHz, acetoned6): δ 28.63, 40.51, 68.35, 78.98, 117.93, 119.83, 124.93, 132.34, 132.64, 156.77, 158.37, 166.56 ppm. ESI HRMS m/z calcd. for [M-(H)]+ 314.0795, found 314.0791. IR (KBr) νmax:
SC
3403, 2981, 2576, 1717, 1669, 1605, 1572, 1527, 1478, 1464, 1430, 1394, 1366, 1275, 1252, 1207, 1162, 1110, 1077, 1053, 1037 cm-1.
M AN U
4.3.14. 2-(3-carboxy-4-chlorophenoxy)ethan aminium 2,2,2-trifluoroacetate (31). To a solution of 30 (1.00 g, 3.17 mmol) in DCM, CF3COOH (7.0 mL, 91.4 mmol) was added and stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and the crude product was used without further purification.
Yield = 100%; mp = 147–149 °C; 1H NMR (400 MHz, acetone-d6): δ 4.29 (t, J = 5.6 Hz, 2H, O-CH2-CH2-NH2), 4.51 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH2), 7.17 (dd, J1 = 8.8, J2 = 3.2 Hz,
TE D
1H, Ar-H), 7.42 (d, J = 3.2 Hz, 1H, Ar-H), 7.43 (d , J = 8.8 Hz, 1H, Ar-H) ppm. 4.3.15. 2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzoic acid (32). To a suspension of 31 (1.00 g, 4.64 mmol) in anhydrous DCM (15 mL), anhydrous pyridine (1.12 mL, 13.9 mmol) was added and the reaction mixture was cooled to 0°C. (CF3CO)2O (0.72 mL, 5.10
EP
mmol) was added drop-wise and the mixture was stirred for 2 h at 0 °C then allowed to warm to room temperature. The reaction mixture was diluted with DCM (30 mL), washed with 1 M
AC C
HCl (2 × 30 mL), water (30 mL), and saturated NaHCO3 (3 × 30 ml). The combined alkaline water phases were acidified with concentrated HCl to pH = 1 and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with water (50 mL), brine (50 mL) and dried with Na2SO4. The solvent was evaporated under reduced pressure. Yield = 76%; white crystals, mp = 141–143°C; 1H NMR (400 MHz, acetone-d6): δ 3.79 (q, J = 5.6 Hz, 2H, O-CH2-CH2-NH), 4.28 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 7.15 (dd, J1 = 8.8, J2 = 3.2 Hz, 1H, Ar-H), 7.43 (d, J = 2.8 Hz, 1H, Ar-H), 7.45 (d, J = 8.4 Hz, 1H, Ar-H), 8.79 (bs, 1H, NH-CO) ppm. 13C NMR (100 MHz, acetone-d6): δ 39.98 (d, 4JF,C = 12 Hz, CH2-NH), 67.01, 117.08 (q, 1JF,C = 285.8 Hz, CF3), 117.89, 119.86, 125.21, 132.46, 132.70, 157.93 (qd,
19
ACCEPTED MANUSCRIPT
2
JF,C = 36.5 Hz, 2JF,C = 8 Hz, CO-CF3), 158.07, 166.52 ppm. ESI HRMS m/z calcd. for [M-
(H)]+ 310.0094, found 310.0088. IR (KBr) νmax: 3414, 3316, 3110, 2927, 2633, 1710, 1597, 1560, 1486, 1469, 1432, 1414, 1384, 1374, 1349, 1321, 1277, 1260, 1235, 1206, 1182, 1116,
4.3.16. General procedure for synthesis of compounds 33a and 33b.
RI PT
1061, 1052, 1039 cm-1. HPLC tR = 10.996 min (100% at 220 nm, 100% at 254 nm).
To a solution of 32 (2.15 g, 6.88 mmol) in anhydrous DCM (30 mL), DMF (catalytic amount) and (COCl)2 (1.77 mL, 20.6 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at room temperature. The solvent was evaporated to dryness and the residue was dissolved
SC
in DCM (20 mL). A mixture of the corresponding 2-aminothiophene (1.73 g, 6.19 mmol) and pyridine (1.63 mL, 20.6 mmol) in DCM (20 mL) was added drop-wise, and the reaction was stirred overnight at room temperature. The reaction mixture was washed with 1 M HCl (30
M AN U
mL), saturated aqueous NaHCO3 (30 mL) and brine (30 mL), and dried (Na2SO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography.
4.3.16.1. Tert-butyl 2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano4,5-dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (33a). Yield = 79%; orange crystals, mp = 89–91 °C; 1H NMR (400 MHz, CDCl3): δ 1.46 (s, 9H, 3 × CH3), 2.67 (t, J = 5.6 Hz,
TE D
2H,CH2-CH2-N), 3.68 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.78 (q, J = 5.4 Hz, 2H, O-CH2-CH2NH), 4.13 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 4.51 (s, 2H, CH2-N-CO), 6.98 (bs, 1H, NHCO-CF3), 7.01 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.37 (d, J = 8.7 Hz, 1H, Ar-H), 7.48 (d, J = 3.1 Hz, 1H, Ar-H), 9.84 (s, 1H, NH-CO-thiophene) ppm.
13
C NMR (100 MHz, CDCl3): δ
EP
24.07, 28.39, 31.45, 36.56, 39.18, 66.27, 80.65, 94.18, 113.65, 114.32, 116.95, 117.18, 119.95, 122.99, 131.64, 131.98, 147.04, 154.54, 157.21, 157.57 (q, 2JC,F = 37.4 Hz), 161.82, 162.62 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 573.1186, found 573.1201. IR (KBr) νmax
AC C
3261, 2982, 2935, 2217, 1666, 1545, 1459, 1416, 1367, 1287, 1237, 1210, 1153, 1118, 1063, 1038, 1005 cm-1. HPLC tR = 16.299 min (100% at 220 nm, 100% at 254 nm). 4.3.17. 2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium trifluoroactetate (34). To a solution of 33a (2.13 g, 3.72 mmol) in DCM (30 mL), CF3COOH (8.60 mL, 112 mmol) was added and stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and the residue was dried in a desiccator overnight.
20
ACCEPTED MANUSCRIPT
Yield = 96%; orange crystals, mp = 90–92 °C; 1H NMR (400 MHz, CDCl3): δ 3.07 (t, J = 6.2 Hz, 2H, CH2-CH2-N), 3.60 (t, J = 6.2 Hz, 2H, CH2-CH2-N), 3.81 (q, J = 5.1 Hz, 2H, O-CH2CH2-NH), 4.16 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 4.39 (s, 2H, CH2-NH2+), 6.71 (t, J = 5.7 Hz, 1H, NH-CO-CF3), 7.07 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.44 (d, J = 8.8 Hz, 1H, Ar13
C NMR (100
RI PT
H), 7.56 (d, J = 3.1 Hz, 1H, Ar-H), 10.00 (s, 1H, NH-CO-thiophene) ppm.
MHz, MeOD): δ 22.26, 27.78, 40.29, 42.35, 42.76, 67.46, 95.33, 113.83, 116.08, 116.57, 118.94, 119.78, 121.38, 124.03, 130.43, 132.32, 135.79, 149.83, 158.83, 161.78 (q, J = 36.8 Hz), 166.53 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 473.0662, found 473.0668. IR (KBr)
= 9.914 min (96.6% at 220 nm, 97.1% at 254 nm).
SC
νmax 2992, 2842, 2221, 1711, 1667, 1547, 1462, 1419, 1292, 1135, 1067, 1039 cm-1. HPLC tR
4.3.18. General procedure for synthesis of compounds 35, 36 and 38a-f.
M AN U
To a solution of 33b, 34 or 37a-f (0.17 mmol) in methanol (2 mL), 1 M NaOH (2 mL) was added. The reaction mixture was stirred overnight. The methanol was evaporated and the residue was diluted with water (20 mL). The water phase was extracted with EtOAc (3 × 20 mL) and the combined organic phases were washed with water (30 mL), brine (30 mL) and dried with Na2SO4. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography.
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-tetrahydro
TE D
4.3.18.1.
benzo[b]thiophen-2-yl)benzamide (35). Yield = 100%; white crystals, mp = 162–164 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.81–1.90 (m, 1H, CH-CHAHB-CH2), 1.97–2.02 (m, 1H, CHCHAHB-CH2), 2.61–2.68 (m, 3H, CH2-CH-CH2-CH2), 2.80–2.92 (m, 2H, CH2-CH), 3.22 (t, J
EP
= 4.8 Hz, 2H, O-CH2-CH2-NH2), 4.22 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH2), 6.72 (d, J = 8.4 Hz, 2H, Ar-H), 7.09–7.13 (m, , 3H, 3 × Ar-H), 7.24 (d, J = 3.2 Hz, 1H, Ar-H), 7.45 (d, J = 8.8 Hz, 1H, Ar-H), 9.25 (bs, 1H, NH-CO) ppm.
13
C NMR (100 MHz, pyridine-d6): δ 26.76,
AC C
32.06, 34.27, 41.92, 42.18, 69.31, 97.49, 116.97, 117.96, 118.38, 120.29, 125.17, 130.44, 130.72, 133.03, 133.68, 138.26, 138.39, 149.53, 159.40, 159.57, 167.05 ppm. ESI HRMS calcd. for [M+(H)]+ 468.1149, found 468.1143. IR (KBr) νmax: 3549, 3475, 3413, 3237, 3084, 3012, 2922, 2224, 2032, 1677, 1656, 1638, 1617, 1574, 1556, 1514, 1460, 1408, 1384, 1317, 1296, 1234, 1201, 1178, 1156, 1139, 1074, 1017 cm-1. HPLC tR = 11.363 min (100% at 220 nm, 100% at 254 nm). 4.3.19. General procedure for synthesis of compounds 37a-f. Method A: To a solution of 34 (0.30 g, 0.511 mmol) in DMF (3 mL), Et3N (1.5 mmol) was added. After 10 min of stirring at room temperature, the corresponding benzyl bromide (0.766 21
ACCEPTED MANUSCRIPT
mmol) was added. The reaction mixture was stirred overnight at room temperature. DMF was then evaporated, the crude residue was dissolved in EtOAc (20 mL) and washed with water (3 × 10 mL), brine (10 mL) and dried with Na2SO4. The crude product was purified by flash chromatography.
RI PT
Method B: To a solution of 34 (0.50 g, 0.852 mmol) in THF (5 mL), the corresponding benzaldehyde (1.28 mmol) and Na(OAc)3BH (0.45 g, 2.13 mmol) were added, and the reaction mixture was stirred overnight at room temperature. To quench the reaction, saturated aqueous NaHCO3 (10 mL) was used, and the water phase was extracted with EtOAc (3 × 20 The crude residue was purified by flash chromatography. 4.3.19.1.
SC
mL). The combined organic phases were washed with brine (30 mL) and dried with Na2SO4.
N-(6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2-chloro-5-(2-
M AN U
(2,2,2-trifluoroacetamido)ethoxy)benzamide (37a). Yield = 35%; pale brown crystals, mp = 71–73 °C; 1H NMR (400 MHz, acetone-d6): δ 2.68 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 2.85 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.76–3.79 (m, 4H, N-CH2-Ar+ O-CH2-CH2NH ), 4.26 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 7.11 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.25–7.29 (m, 2H, 2 × Ar-H), 7.33–7.37 (m, 2H, 2 × Ar-H), 7.39-7.44 (m, 3H, 3 × Ar-H), 8.79 (bs, 1H, NH-CO-CF3), 11.01 (s, 1H, NH-CO-thiophene) ppm.
13
C NMR (100 MHz,
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acetone-d6): δ 24.98, 40.04, 50.15, 51.41, 62.04, 67.18, 95.27, 114.08, 115.69, 116.38, 118.54, 119.28, 132.30, 127.55, 128.05, 129.21, 129.77, 131.14, 131.85, 135.97, 139.53, 147.26, 158.29, 164.51 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 563.1132, found 563.1137. IR νmax 3319, 3081, 2923, 2807, 2214, 1714, 1667, 1545, 1456, 1405, 1366, 1288, 1209, 1154,
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1057, 1036, 1007 cm-1. HPLC tR = 11.907 min (100% at 220 nm, 100% at 254 nm).
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4.4. Molecular docking studies
A docking study was performed using the crystal structure of MurF enzyme from S. pneumoniae (PDB code: 3zm5) and OEDocking software (Release 3.0.1, OpenEye Scientific Software, Inc.).
4.4.1. Ligand preparation The molecules were built with ChemBioDraw Ultra 12.0 (CambridgeSoft). The ligand geometries were optimized with ChemBio 3D Ultra 12.0 (CambridgeSoft) using MM2 force field until a minimum 0.100 root mean square (RMS) gradient was reached. The optimized structure was refined with GAMESS interface using the semi-empirical AM1 method, QA 22
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optimization algorithm and Gasteiger Hückel charges for all atoms for 100 steps. Amines and guanidines were kept in their ionized state, corresponding to pH 7.4. FRED requires a set of input conformers for each ligand, which were generated with OMEGA 2.4.6, with maximum number of conformations set to 1000, and using the RMS cut-off value (Root Mean Square
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(RMS) cartesian distance below which two conformers are duplicates) of 0.3 Å. All the other options were left as default values. 4.4.2. Receptor preparation & docking protocol
The crystal structure of MurF enzyme from S. pneumoniae in complex with the ligand (PDB
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code: 3zm5) was taken as a starting-point. The ligand (2,4-dichloro-N-[3-cyano-6-[(4hydroxyphenyl)methyl]-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium-2-yl]-5-
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morpholinosulfonyl-benzamide) was taken as a reference structure and a grid box including all active-site atoms (including hydrogens) with a volume of 32562 Å, dimensions of 36.22 Å × 36.33 Å × 24.67 Å, and outer contour of 2953 Å, was created around it as a “docking receptor” using Make Receptor 3.0.1.
The docking software FRED (OEDocking 3.0.1) was used for docking studies with the default settings, except for dock resolution, which was set to “high”, and number of poses, which was
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set to 50. The proposed 10 binding modes with the highest rank of the docked inhibitors were evaluated using final score and root mean square deviation (RMSD) as a tool to explore relative structural differences between proposed binding modes. The graphical representations of the proposed binding positions of all the molecules were obtained using Accelrys
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Discovery Studio 2.5 (Accelrys Software Inc.). 4.4.3. Validation of the docking protocol
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The docking procedure should be able to correctly predict the binding poses of the molecules in the PDB database i.e., the crystal structures of ligands in complex with proteins. We have attempted to reproduce the pose of the ligand as seen in its X-ray complex with the target MurF enzyme from S. pneumoniae (PDB code: 3zm5). The top 10 docking poses of the docked ligand were within 1.5 Å RMSD of the ligand crystal structure. The predicted top score pose was the one that best fitted the crystal structure of the ligand, overlapping it almost completely, which offers the proof of the protocol validation (Fig. 3).
ACKNOWLEDGEMENTS
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We thank OpenEye Scientific Software, Inc. for free academic licenses of their software, and the Ministry of Higher Education, Science and Technology of the Republic of Slovenia for financial support. This study was partially supported by a Young Researcher grant to MH, and
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an L1-4039 grant to SG, both from the Slovenian Research Agency.
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REFERENCES [1] A.J. Alanis, Resistance to antibiotics: are we in the post-antibiotic era?, Arch. Med. Res. 36 (2005) 697–705.
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[2] P.C. Appelbaum, 2012 and beyond: potential for the start of a second pre-antibiotic era?, J. Antimicrob. Chemoth. 67 (2012) 2062–2068.
[3] I. Chopra, The 2012 Garrod Lecture: Discovery of antibacterial drugs in the 21st century, J. Antimicrob. Chemoth., (2012).
[4] T.D.H. Bugg, D. Braddick, C.G. Dowson, D.I. Roper, Bacterial cell wall assembly: still an
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attractive antibacterial target, Trends Biotechnol. 29 (2011) 167–173.
[5] A. Gautam, R. Vyas, R. Tewari, Peptidoglycan biosynthesis machinery: A rich source of
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drug targets, Cr. Rev. in Biotechn. 31 (2011) 295–336.
[6] D.W. Green, The bacterial cell wall as a source of antibacterial targets, Expert Opin. Ther. Tar. 6 (2002) 1–20.
[7] H. Barreteau, A. Kovač, A. Boniface, M. Sova, S. Gobec, D. Blanot, Cytoplasmic steps of peptidoglycan biosynthesis, FEMS Microbiol. Rev. 32 (2008) 168–207. [8] A. El Zoeiby, F. Sanschagrin, R.C. Levesque, Structure and function of the Mur enzymes:
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development of novel inhibitors, Mol. Microbiol. 47 (2003) 1–12. [9] C.A. Smith, Structure, function and dynamics in the mur family of bacterial cell wall ligases, J. Mol. Biol. 362 (2006) 640–655. [10] D. J. Miller, S. M. Hammond, D. Anderluzzi, T. D. H. Bugg, Aminoalkylphosphinate
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inhibitors of D-Ala-D-Ala adding enzyme, J. Chem. Soc., Perk. T. 1. (1998) 131–142. [11] Y.G. Gu, A.S. Florjancic, R.F. Clark, T. Zhang, C.S. Cooper, D.D. Anderson, C.G. Lerner, J.O. McCall, Y. Cai, C.L. Black-Schaefer, G.F. Stamper, P.J. Hajduk, B.A.
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Beutel, Structure–activity relationships of novel potent MurF inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 267–270. [12] G.F. Stamper, K.L. Longenecker, E.H. Fry, C.G. Jakob, A.S. Florjancic, Y.G. Gu, D.D. Anderson, C.S. Cooper, T. Zhang, R.F. Clark, Y. Cia, C.L. Black-Schaefer, J. Owen McCall, C.G. Lerner, P.J. Hajduk, B.A. Beutel, V.S. Stoll, Structure-based optimization of MurF inhibitors, Chem. Biol. Drug Des. 67 (2006) 58–65. [13] E.Z. Baum, S.M. Crespo-Carbone, D. Abbanat, B. Foleno, A. Maden, R. Goldschmidt, K. Bush, Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF, Antimicrob. Agents Ch. 50 (2006) 230–236.
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[14] E.Z. Baum, S.M. Crespo-Carbone, A. Klinger, B.D. Foleno, I. Turchi, M. Macielag, K. Bush, A MurF inhibitor that disrupts cell wall biosynthesis in Escherichia coli, Antimicrob. Agents Ch. 51 (2007) 4420–4426. [15] E.Z. Baum, S.M. Crespo-Carbone, B.D. Foleno, L.D. Simon, J. Guillemont, M.
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Macielag, K. Bush, MurF inhibitors with antibacterial activity: Effect on muropeptide levels, Antimicrob. Agents Ch. 53 (2009) 3240–3247.
[16] S. Turk, A. Kovač, A. Boniface, J.M. Bostock, I. Chopra, D. Blanot, S. Gobec, Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening, Bioorg. Med. Chem. 17 (2009) 1884–
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[17] S. Turk, M. Hrast, I. Sosič, H. Barreteau, D. Mengin-Lecreulx, D. Blanot, S. Gobec,
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Biochemical characterization of MurF from Streptococcus pneumoniae and the identification of a new MurF inhibitor through ligand-based virtual screening, Acta Chim. Slov. 60 (2013) 294–299.
[18] M. Hrast, S. Turk, I. Sosič, D. Knez, C.P. Randall, H. Barreteau, C. Contreras-Martel, A. Dessen, A.J. O’Neill, D. Mengin-Lecreulx, D. Blanot, S. Gobec, Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF, Eur. J. Med. Chem. 66 (2013) 32–45.
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[19] P.D. Leeson, A.M. Davis, Time-related differences in the physical property profiles of oral drugs. J. Med. Chem. 47 (2004) 6338–6348. [20] R. O’Shea, H.E. Moser, Physicochemical Properties of Antibacterial Compounds: Implication for Drug Discovery, J. Med. Chem. 51 (2008) 2871–2878.
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[21] I. Dvoretzky, G.H. Richter, Formaldehyde condensation in the pyrazole series, J. Org. Chem. 15 (1950) 1285–1288.
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[22] P.A. Lanzetta, L.J. Alvarez, P.S. Reinach, O.A. Candia, An improved assay for nanomole amounts of inorganic phosphate, Anal. Biochem. 100 (1979) 95–97. [23] S.L. McGovern, B.T. Helfand, B. Feng, B.K. Shoichet, A specific mechanism of nonspecific inhibition, J. Med. Chem. 46 (2003) 4265–4272. [24] J.K. Hobbs, K. Miller, A.J. O’Neill et al. Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus. J. Antimicrob. Chemother. 62 (2008) 1003–1008. [25] S. Dementin, A. Bouhss, G. Auger, C. Parquet, D. Mengin-Lecreulx, O. Dideberg, J. van Heijenoort, D. Blanot, Evidence of a functional requirement for a carbamoylated lysine
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residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments, Eur. J. Biochem. 268 (2001) 5800–5807. [26] A.J. O'Neill, Staphylococcus aureus SH1000 and 8325-4: comparative genome sequences of key laboratory strains in staphylococcal research. Lett. Appl. Microbiol. 51
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(2010) 358–361. [27] A.J. O'Neill, J.M. Bostock, A.M. Moita, I. Chopra, Antimicrobial activity and mechanisms of resistance to cephalosporin P1, an antibiotic related to fusidic acid. J. Antimicrob. Chemother. 50 (2002) 839–848.
[28] E. Shapiro, F. Baneyx, Stress-based identification and classification of antibacterial
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agents: second-generation Escherichia coli reporter strains and optimization of detection, Antimicrob. Agents Chemother. 46 (2002) 2490–2497.
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[29] P.A. Wayne, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-Ninth Edition. Clinical and Laboratory Standards Institute (2012) CLSI Document M07–A9.
[30] R.A. Dixon, I. Chopra, Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide, Antimicrob. Agents Chemother. 29 (1986) 781–788. [31] E.R. Marshall, J.A. Kuck, R.C. Elderfield, Studies on lactones related to the cardiac aglycones. X. Synthesis of simple hydroxylated β-substituted ∆α,β-butenolides, J. Org.
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Chem. 7 (1942) 444–456.
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List of table and figure captions:
Table 1. Inhibitory activities of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a and 27b
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against MurF from S. pneumoniae and E. coli.
Table 2. Inhibitory activities of compounds 33-38f, 39c, 42a-e and 43 against MurF from S. pneumoniae and E. coli.
and 43 against S. aureus and E. coli bacterial strains.
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Table 3. Antibacterial activities of compounds 23a-b, 26a, 27a-b, 33a, 34-38f, 39c, 42a-e
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Figure 1. Two-step design of new cyanothiophene MurF inhibitors. Overlapping scaffold essential for good inhibitory potency is shown in red.
Figure 2. New cyanothiophene MurF inhibitors (sticks rendering, coloured by atom) docked to MurFSp (PDB code: 3zm5) with a co-crystallized inhibitor (sticks rendering, in green). The best ranked predicted binding mode can be seen for a) amine, and b) guanidine of truncated
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analogue of II; guanidine is predicted to fit into the “morpholine” pocket (binds morpholine moiety from the co-crystallized inhibitor) by forming 4 H-bonds with Asn134, Asn137 and Asn326 (in green, demonstrated by the distances between guanidine protons and aminoacid carbonyl oxygens), while the rest of the molecule overlaps the structure of a co-crystallized
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cyanothiophene scaffold. Virtually the same binding mode is predicted for c) amine with benzyl substituent attached to position 6 of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine (c), while its guanidine analogue (d) moves towards the interior of the binding site to form close contact
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with Asp323 (in green, ionic bond and two H-bonds).
Figure
3.
Crystal
structure
of
the
ligand
(2,4-dichloro-N-[3-cyano-6-[(4-
hydroxyphenyl)methyl]-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium-2-yl]-5-morpholino sulfonyl-benzamide, shown as green sticks) and top docked pose of the ligand, as predicted by FRED (shown as stick colored by atom type). The top docked ligand pose had an RMSD of 1.15 Å.
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Scheme 1: Reagents and conditions: (a) tert-butyl (2-aminoethyl)carbamate, Et3N, DCM, 0 °C to rt, 16 h; (b) 1. (COCl)2, DMF, DCM, 0 °C to rt, 0.5 h; 2. 2-aminothiophene, pyridine, DCM, 0 °C to rt, 16 h; (c) 1. CF3COOH, DCM, rt, 2 h; 2. 1 M HCl in EtOAc.
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Scheme 2: Reagents and conditions: (a) Ac2O, pyridine, rt, 1 h; (b) 1. (COCl)2, DMF, DCM, 0 °C to rt, 0.5 h; 2. amine, pyridine, DCM, 0 °C to rt, 16 h; (c) K2CO3, MeOH, rt, 1 h; (d) A: K2CO3 or Cs2CO3, corresponding halogenide, 50 °C, 16 h; B: (1H-pyrazol-1-yl)methanol (13) or cyclopentylmethanol, PPh3, DIAD, THF, 0 °C to rt, 16 h; (e) CF3COOH, DCM, rt, 2 h, 1 M HCl in EtOAc; (f) di-Boc-methyl-thiourea, HgCl2, Et3N, DMF, rt, 4 h; (g) 1. CF3COOH,
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DCM, rt, 2 h; 2. 1 M HCl in EtOAc.
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Scheme 3: Reagents and conditions: (a) SOCl2, MeOH, rt, 3 h; (b) K2CO3, DMF, 80 °C, 16 h; (c) NaOH, dioxane/water, rt, 16 h; (d) CF3COOH, DCM, rt, 2 h; (e) (CF3CO)2O, pyridine, DCM, 0 °C to rt, 2 h; (f) 1. (COCl)2, DMF, DCM, 0 °C to rt, 1 h; 2. corresponding 2aminothiophene, pyridine, DCM, 0 °C to rt, 16 h; (g) CF3COOH, DCM, rt, 2 h; (h) 1 M NaOH, MeOH, rt, 16 h; (i) A: K2CO3, corresponding benzyl bromide, DMF, rt, 16 h; B: corresponding benzaldehyde, Na(OAc)3BH, AcOH, THF, rt, 16 h; (j) di-Boc-methyl-thiourea,
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HgCl2, Et3N, DMF, rt, 4 h; (k) 1. CF3COOH, DCM, rt, 2 h; 2. 1 M HCl in EtOAc.
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RAa (%) or IC50b (µM) Compound
R1
X
MurFEc
-H
15
-H
16
-H
17
-Cl
18
-Cl
19
-Cl
22a
102%
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14
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MurFSp
97%
99%
89%
85%
81%
82%
102%
97%
87% c
74%c
-H
98%
93%
22b
-H
53%c
75%c
23a
-Cl
170 µM
208 µM
23b
-Cl
128 µM
272 µM
26a
-H
167 µM
258 µM
27a
-Cl
75 µM
56 µM
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81%
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27b
a
78 µM
-Cl
102 µM
Residual activity (%) of the enzyme at 100 µM of the tested compound. Data are means of
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two independent experiments. Standard deviations were within ±10% of the means; b
Concentration of the inhibitor for which the residual activity of the enzyme is 50%; cResidual
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activity (%) of the enzyme at 250 µM of the tested compound.
N
33b
C
34
N
35
C
-H2+CF3COO-
-NH2
N N
37b
N
37c
N
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37a
-NH2
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R
RAa (%) or IC50b (µM) MurFSp
MurFEc
96%
102%
98%
101%
99%
91%
58 µM
81 µM
85%
65%
172 µM
46%
154 µM
81%
55%
255 µM
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R
3
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-H
37d
N
71%
56%
37e
N
55%
45%
37f
N
103 µM
152 µM
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N
-NH2
51 µM
84 µM
38b
N
-NH2
125 µM
73 µM
38c
N
-NH2
663 µM
336 µM
38d
N
-NH2
119 µM
150 µM
38e
N
-NH2
107 µM
76 µM
38f
N
-NH2
454 µM
83 µM
39c
N
-NH2
109 µM
88 µM
42a
N
55 µM
57 µM
42b
N
61 µM
78 µM
42c
N
37 µM
80 µM
42d
N
117 µM
143 µM
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38a
42e
N
61 µM
66 µM
43
C
20 µM
25 µM
Residual activity (%) of the enzyme at 250 µM of the tested compound. Data are means of
two independent experiments. Standard deviations were within ±10% of the means; b
Concentration of the inhibitor for which the residual activity of the enzyme is 50%.
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MIC (µg/ml) S. aureus
Compound SH1000
E. coli 1411a
1411+
AB734+
PMBNd AB734a
PMBNd
>256
128
>256
128
128
64
23a
128
128
32
128
32
32
32
23b
128
64
32
64
32
32
32
26a
64
64
64
64
>256
16
16
27a
64
16
8
16
8
8
8
27b
>256
32
32
32
32
16
8
33a
>256
>256
>256
>256
>256
>256
>256
34
>256
>256
>256
>256
>256
>256
>256
35
>256
>256
16
>256
16
32
8
36
>256
>256
>256
>256
>256
>256
>256
37a
>256
>256
>256
>256
>256
>256
>256
37b
>256
>256
>256
>256
>256
>256
>256
37c
>256
>256
>256
>256
>256
>256
>256
37d
>256
>256
>256
>256
>256
>256
>256
37e
>256
>256
>256
>256
>256
>256
>256
37f
>256
>256
>256
>256
>256
>256
64
38a
64
64
32
64
32
32
16
38b
128
128
64
128
64
64
32
38c
>256
>256
>256
>256
>256
>256
>256
38d
>256
128
64
128
64
64
64
38e
128
256
>256
>256
>256
>256
>256
39c
128
>256
64
>256
64
64
32
42a
8
32
16
32
16
32
8
42b
32
32
32
32
32
32
8
42c
8
64
32
64
32
16
8
42d
32
128
64
128
64
64
32
42e
16
128
32
256
32
16
16
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>256
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4
SM1411b ES100c
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43 a
4
64
16
64
16
16
4
E. coli 1411 and AB734 are parental strains of SM1411 and ES100, respectively; bSM1411
is 1411 deleted for acrAB; cES100 is AB734 deleted for tolC; dPMBN, polymyxin B
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nonapeptide.
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O
O OH
HO
O
HO
a X
S
O O
X 5 (X = H) 6 (X = Cl)
O
N H
b
7 (X = H) 8 (X = Cl)
O
X
CN
9 (X = H) 10 (X = Cl) c
O R1
O
N H
S d
11 (X = H) 12 (X = Cl) O N H
O
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NHBoc
n
S
e
NH3+Cl-
n
X CN 22a (X = H, n = 1) 22b (X = H, n = 2) 23a (X = Cl, n = 1) 23b (X = Cl, n = 2)
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CN
X
CN
O N H
OH
N H
X CN 14-19, 20a,b, 21a,b
S
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O
S
f
O
NH
S
O
N H CN
n
N H
X
HCl
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26a (X = H, n = 1) 27a (X = Cl, n = 1) 27b (X = Cl, n = 2)
NH2
NBoc
O
g
S
N H
O
X CN 24a (X = H, n = 1) 25a (X = Cl, n = 1) 25b (X = Cl, n = 2)
n
N H
NHBoc
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Highlights
Design, synthesis and evaluation of new series of cyanothiophene-based inhibitors of MurF. Low micromolar inhibitors of MurF from Escherichia coli and Streptococcus pneumoniae
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were obtained.
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The inhibitors show antibacterial activity against strains of S. aureus and E. coli.
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SUPPORTING INFORMATION ON: Manuscript Title: Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold
Barreteau3, Alex J. O’Neill2, Didier Blanot3 and Stanislav Gobec1*
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Authors: Martina Hrast1, Marko Anderluh1, Damijan Knez1, Christopher P. Randall2, Hélène
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
2
School of Molecular and Cellular Biology and Antimicrobial Research Centre, University of
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1
Leeds, Leeds LS 9JT, U.K.
Univ Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS,
91405 Orsay, France
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3
*Corresponding author: Stanislav Gobec University of Ljubljana
1000 Ljubljana, Slovenia Tel: +386-1-4769500 Fax: +386-1-4258031.
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Faculty of Pharmacy, Aškerčeva 7
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Table of Contents
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E-mail: [email protected]
Compound Characterization.………………......................................................….………...S2 References………………………………………………………………………………......S17 1
H NMR spectra for compounds 23a-b, 27b, 33, 35, 36, 37a-c, 37e-f, 38a-f, 39c, 40c, 42a,
42c-e and 43…………………………………………………………… …………………..S18 13
C NMR spectra for compounds 23b, 27b, 33, 35, 36, 37a-c, 37e-f, 38a, 38c, 38e-f, 39c, 40c,
42a-c, 42e and 43…………………………………………………………………………...S31 Table S1: ClogP values of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a, 27b, 33-38f, 39c, 42a-e and 43………………………………………………………………………..……....S42
S1
ACCEPTED MANUSCRIPT
5-acetoxy-2-chlorobenzoic acid (8). Yield = 95%; white crystals, mp = 117–120 °C, 1H NMR (400 MHz, CDCl3): δ 2.35 (s, 3H, CO-CH3), 7.28 (dd, J1 = 8.4, J2 = 2.8 Hz, 1H, Ar-H), 7.52 (d, J = 8.7 Hz, 1H, Ar-H), 7.79 (d, J = 2.3 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, CDCl3):
RI PT
δ 21.01, 125.62, 127.06, 129.12, 131.81, 132.39, 148.85, 168.91, 169.20 ppm. ESI HRMS calcd. for [M+(H)]+ 212.9955, found 212.9950. IR (KBr) νmax: 3070, 1762, 1709, 1602, 1576, 1474, 1412, 1374, 1308, 1262, 1217, 1112, 1049, 1014, 941, 907, 871, 833 cm-1.
4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenyl
acetate
SC
(10). Yield = 60%; pale yellow crystals, mp = 169–176 °C; 1H NMR (400 MHz, CDCl3): δ 1.81–1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.35 (s, 3H, O-CH3), 2.62–2.74 (m, 4H, CH2-CH2CH2-CH2), 7.27 (dd, J1 = 8.7, J2 = 2.8 Hz, 1H, Ar-H), 7.53 (d, J = 8.7 Hz, 1H, Ar-H), 7.70 (d, 13
C NMR (100 MHz, CDCl3): δ 20.99,
M AN U
J = 2.8 Hz, 1H, Ar-H), 9.82 (s, 1H, CO-NH) ppm.
22.10, 23.07, 24.00, 24.03, 94.81, 114.13, 125.07, 126.50, 127.78, 129.32, 131.38, 131.85, 132.15, 145.77, 149.69, 160.91, 168.77 ppm. ESI HRMS calcd. for [M+(H)]+ 375.0570, found 375.0558. IR (KBr) νmax: 3504, 3248, 3193, 3081, 3000, 2953, 2228, 1762, 1683, 1575, 1560, 1464, 1438, 1409, 1371, 1348, 1329, 1292, 1269, 1238, 1206, 1150, 1116, 1051, 1014,
TE D
969, 951, 914, 876, 842, 820 cm-1.
2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-hydroxybenzamide
(12).
Yield = 100%; pale brown powder, mp = 175–180 °C; 1H NMR (400 MHz, CDCl3): δ 1.82– 1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.61–2.74 (m, 4H, CH2-CH2-CH2-CH2), 6.80 (s, 1H, ArOH), 7.02 (dd, J1 = 8.7, J2 = 3.0 Hz, 1H, Ar-H), 7.35 (d, J = 8.8 Hz, 1H, Ar-H), 7.62 (d, J =
EP
3.0 Hz, 1H, Ar-H), 9.86 (s, 1H, CO-NH) ppm. 13C NMR (100 MHz, CDCl3): δ 22.07, 23.06, 24.01, 24.07, 94.99, 114.12, 118.56, 121.00, 121.89, 129.50, 131.13, 131.50, 132.22, 145.75,
AC C
155.48, 161.95 ppm. ESI HRMS calcd. for [M+(H)]+ 333.0465, found 333.0460. IR (KBr) νmax: 3430, 3321, 2936, 2840, 2209, 1647, 1602, 1573, 1557, 1484, 1466, 1423, 1310, 1259, 1217, 1118, 1036, 876, 822, 811 cm-1. 1H-pyrazol-1-ylmethanol (13).To a solution of 1H-pyrazol (0.750 g, 11.0 mmol) in diethyl ether (5 ml), 37% formaldehyde (0.9 mL, 11.0 mmol) was added. The reaction mixture was stirred over night at room temperature. The solvent was evaporated under reduced pressure and crude product was used without further purification [1].
S2
ACCEPTED MANUSCRIPT
Yield = 99%; white cubical crystals, mp = 84–86°C; 1H NMR (400 MHz, DMSO-d6): δ 5.37 (d, J = 7.6 Hz, 2H, CH2), 6.28 (t, J = 2.0 Hz, 1H, Ar-H), 6.72 (t, J = 7.6 Hz, 1H, OH), 7.48 (dd, J1 = 2.0, J2 = 0.8 Hz, 1H, Ar-H), 8.79 (dd, J1 = 2.0, J2 = 0.8 Hz, 1H, Ar-H) ppm. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(cyclopentylmethoxy)benzamide (15).
RI PT
Yield = 32%; yellow crystals, mp = 128–130 °C; 1H NMR (400 MHz, CDCl3): δ 1.35–1.40 (m, 2H), 1.59–1.69 (m, 4H), 1.79–1.86 (m, 6H), 2.38–2.45 (m, 1H), 2.54-2.58 (m, 4H), 4.27 (d, J = 7.1 Hz, 2H, O-CH2-CH), 6.96 (dd, J1 = 8.0, J2 = 2.6 Hz, 1H, Ar-H), 7.00–7.06 (m, 2H, 2 × Ar-H), 7.26 (d, J = 8.0 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, CDCl3): δ 21.07, 22.11,
SC
23.23, 23.49, 24.35, 28.47, 37.73, 71.82, 99.35, 114.31, 114.82, 117.30, 119.31, 119.92, 128.74, 131.05, 131.95, 154.98, 157.19, 161.43 ppm. ESI HRMS calcd. for [M+(H)]+ 381.1637, found 381.1634. IR (KBr) νmax: 3340, 3026, 2943, 2867, 2479, 2365, 2217, 2164,
M AN U
1942, 1869, 1610, 1586, 1488, 1458, 1402, 1385, 1352, 1301, 1262, 1242, 1214, 1172, 1100, 1082, 1026 cm-1. Anal. Calcd. for C22H24N2O2S × 0.7 H2O: C, 67.22; H, 6.51; N, 7.13. Found C, 67.15; H, 6.37; N, 7.12.
5-(benzyloxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide (17). Yield = 6%; white needles, mp = 162–164 °C; 1H NMR (400 MHz, CDCl3): δ 1.83–1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.66–2.72 (m, 4H, CH2-CH2-CH2-CH2), 5.14 (s, 2H, CH2-O), 7.12
TE D
(dd, J1= 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.33-7.49 (m, 6H, 6 × Ar-H), 7.65 (dd, J1 = 3.2, J2 = 1.5 Hz, 1H, Ar-H), 9.68 (s, 1H, CO-NH) ppm.
13
C NMR (100 MHz, CDCl3): δ 21.67, 22.61,
24.09, 24.48, 53.99, 70.44, 109.71, 113.12, 118.68, 121.87, 127.53, 128.16, 128.20, 128.65, 129.05, 130.49, 134.05, 135.54, 135.90, 136.19 , 148.55, 156.98, 167.95 ppm. ESI HRMS
EP
calcd. for [M+(H)]+ 423.0934, found 423.0937. IR (KBr) νmax: 3436, 3341, 2946, 2206, 1661, 1550, 1458, 1312, 1272, 1236, 1024, 879, 824 cm-1.
AC C
2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-((4-cyanobenzyl)oxy) benzamide (18). Yield = 30%; yellow powder, mp = 239–243 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.70–1.78 (m, 4H, CH2-CH2-CH2-CH2), 2.36–2.53 (m, 4H, CH2-CH2-CH2-CH2), 5.24 (s, 2H, CH2-O), 6.92 (dd, J1 = 8.7, J2 = 3.1 Hz,1 H, Ar-H), 7.20 (d, J = 3.0 Hz, 1H, ArH), 7.25 (d, J = 8.8, 1H, Ar-H), 7.67 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 7.87 (d, J = 8.2 Hz, 2H, 2 × Ar-H) ppm.
13
C NMR (100 MHz, DMSO-d6): δ 22.49, 23.35, 23.80, 24.17, 68.41, 90.28,
110.40, 115.04, 115.71, 118.76, 118.94, 121.43, 122.51, 128.06, 128.23, 130.21, 132.40, 142.44, 142.81, 156.05, 168.13 ppm. ESI HRMS calcd. for [M+(H)]+ 448.0887, found
S3
ACCEPTED MANUSCRIPT
448.0877. IR (KBr) νmax: 3431, 2929, 2845, 2228, 2197, 1592, 1533, 1470, 1392, 1360, 1304, 1257, 1228, 1153, 1130, 1106, 1049, 1026, 985, 889, 871, 817 cm-1. 5-(2-amino-2-oxoethoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2yl)benzamide (19). Yield = 57%; pale orange crystals, mp = 217–218 °C; 1H NMR (400 MHz,
RI PT
DMSO-d6): δ 1.75–1.81 (m, 4H, CH2-CH2-CH2-CH2), 2.53 (t, J = 6.4 Hz, 2H, CH2-CH2-CH2CH2), 2.64 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 4.51 (s, 2H, O-CH2-CO-NH2), 7.13 (dd, J1 = 8.8, J2 = 2.4 Hz, 1H, Ar-H), 7.21 (d, J = 3.2 Hz, 1H, Ar-H), 7.45 (bs, 1H, CO-NHAHB), 7.50 (d, J = 8.8 Hz, 1H, Ar-H), 7.61 (bs, 1H, CO-NHAHB), 12.32 (bs, 1H, NH-CO) ppm. 13C
SC
NMR (100 MHz, DMSO-d6): δ 21.68, 22.57, 23.39, 23.54, 66.98, 94.51, 113.94, 115.70, 117.93, 121.86, 128.20, 130.48, 131.30, 135.28, 145.55, 156.33, 164.02, 169.36 ppm. ESI HRMS calcd. for [M+(H)]+ 390.0679, found 390.0680. IR (KBr) νmax: 3413, 2927, 2205,
M AN U
1668, 1640, 1616, 1564, 1549, 1458, 1404, 1320, 1287, 1271, 1237, 1125, 1061, 1040 cm-1. HPLC tR = 13.462 min (96.4% at 220 nm, 98.9% at 254 nm). Tert-butyl
(2-(3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenoxy)
ethyl)carbamate (20a). Yield = 14%; white crystals, mp = 181–182 °C; 1H NMR (400 MHz, CDCl3): δ 1.48 (s, 9H, COO-(CH3)3), 1.83–1.93 (m, 4H, CH2-CH2-CH2-CH2), 2.62–2.73 (m, 4H, CH2-CH2-CH2-CH2), 3.59 (q, J = 5.1 Hz, 2H, NH-CH2-CH2-O), 4.12 (t, J = 5.1 Hz, 2H,
TE D
NH-CH2-CH2-O), 5.03 (s, 1H, CO-NH-CH2), 7.13–7.18 (m, 1H, Ar-H), 7.44–7.50 (m, 3H, 3 × Ar-H), 8.86 (s, 1H, CO-NH) ppm.
13
C NMR (100 MHz, CDCl3): δ 22.12, 23.10, 23.99,
28.40, 39.97, 67.49, 79.67, 94.14, 113.68, 114.54, 119.20, 119.46, 128.84, 130.17, 131.13, 133.30, 146.64, 155.89, 159.09, 163.43 ppm. ESI HRMS calcd. for [M+(H)]+ 442.1801,
EP
found 442.1812. IR (KBr) νmax: 3358, 3245, 3077, 2979, 2939, 2880, 2223, 1684, 1670, 1576, 1539, 1465, 1395, 1365, 1330, 1295, 1280, 1268, 1254, 1228, 1172, 1135, 1111, 1051, 1001
9.42.
AC C
cm-1. Anal. Calcd. for C23H27N3O4S: C, 62.56; H, 6.16; N, 9.52. Found C, 62.34; H, 6.23; N,
Tert-butyl
(3-(3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenoxy)
propyl)carbamate (20b). Yield = 69%; white crystals, mp = 164–166 °C; 1H NMR (400 MHz, CDCl3): δ 1.44 (s, 9H, COO-(CH3)3), 1.81–1.89 (m, 4H, CH2-CH2-CH2-CH2), 1.98–2.03 (m, 2H, O-CH2-CH2-CH2), 2.61 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 2.67 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 3.35 (q, J = 6.4 Hz, 2H, CH2-NH-CO), 4.09 (t, J = 6.0 Hz, 2H, O-CH2CH2), 4.83 (t, J = 5.6 Hz, 1H, CH2-NH-COO), 7.13 (ddd, J1 = 8.4, J2 = 2.4, J3 = 1.2 Hz, 1H, Ar-H), 7.41 (t, J = 8.0 Hz, 1H, Ar-H), 7.44–7.48 (m, 2H, 2 × Ar-H), 9.09 (bs, 1H, NH-CO) S4
ACCEPTED MANUSCRIPT
ppm.
13
C NMR (100 MHz, acetone-d6): δ 22.93, 23.84, 24.48, 24.69, 28.66, (peak for
CH2CH2CH2 under solvent peak), 38.15, 66.64, 78.54, 96.34, 114.51, 114.77, 119.64, 120.95, 129.57, 130.62, 132.25, 134.88, 147.25, 156.75, 160.17, 165.39 ppm. ESI HRMS calcd. for [M+(H)]+ 456.1957, found 456.1960. IR (KBr) νmax: 3545, 3417, 3234, 2965, 2928, 2222,
RI PT
1616, 1544, 1441, 1384, 1287, 1268, 1224, 1170, 1099, 1046 cm-1. Anal. Calcd. for C24H29N3O4S: C, 63.27; H, 6.42; N, 9.22. Found C, 63.28; H, 6.34; N, 9.18. Tert-butyl
(2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)
phenoxy)ethyl)carbamate (21a). This compound was used for further reaction without
Tert-butyl
SC
purification.
(2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)
M AN U
phenoxy)propyl)carbamate (21a). This compound was used for further reaction without purification.
3-(3-aminopropoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide
(22b).
Yield = 73%; pale brown crystals, mp = 194–196 °C; 1H NMR (400 MHz, MeOD): δ 1.86– 1.93 (m, 4H, CH2-CH2-CH2CH2), 2.21 (p, J = 6.8 Hz, 2H, CH2-CH2-CH2), 2.64 (t, J = 4.8 Hz, 2H, CH2-CH2-CH2-CH2), 2.72 (t, J = 5.2 Hz, 2H, CH2-CH2-CH2-CH2), 3.22 (p, J = 7.6
TE D
Hz, 2H, CH2-NH2), 4.23 (t, J = 5.6 Hz, 2H, CH2-CH2-O), 7.25 (ddd, J1 = 8.4, J2 = 2.8, J3 = 1.2 Hz, 1H, Ar-H), 7.50 (t, J = 8.4 Hz, 1H, Ar-H), 7.55 (dd, J1 = 2.4, J2 = 1.6 Hz, 1H, Ar-H), 7.64 (ddd, J1 = 7.6, J2 = 1.6, J3 = 1.2 Hz, 1H, Ar-H) ppm.
13
C NMR (100 MHz, pyridine-d5): δ
24.26, 25.20, 26.03, 26.22, 29.77, 39.19, 67.74, 99.12, 117.02, 117.25, 121.03, 123.34,
EP
131.25, 131.87, 133.98, 136.89, 149.51, 160.97, 168.03 ppm. ESI HRMS calcd. for [M+(H)]+ 356.1433, found 356.1424. IR (KBr) νmax: 3549, 3472, 3412, 3233, 2925, 2215, 2028, 1616, nm).
AC C
1544, 1464, 1383, 1271, 1148 cm-1. HPLC tR = 11.563 min (97.1% at 220 nm, 96.9% at 254
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) benzamide (23a). Yield = 12%; pale yellow crystals, mp = 198–202 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.75–1.80 (m, 4H, CH2-CH2-CH2-CH2), 2.50–2.55 (m, 2H, CH2-CH2-CH2-CH2, under solvent peak), 2.63 (t, 2H, J = 4.8 Hz, H, CH2-CH2-CH2-CH2), 3.24 (t, J = 5.2 Hz, 2H, H2NCH2-CH2-O), 4.22 (t, J = 7.0 Hz, 2H, H2N-CH2-CH2-O), 7.16 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.21 (d, J = 3.1 Hz, 1H, Ar-H), 7.51 (d, J = 8.7, 1H, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 22.31, 23.18, 23.74, 24.00, 55.99, 66.27, 91.15, 115.34, 115.86, 116.60, 117.91, 122.46, 122.89, 128.86, 130.32, 140.95, 156.26, 167.26 ppm. ESI HRMS calcd. for [M+(H)]+ S5
ACCEPTED MANUSCRIPT
376.0887, found 376.0875. IR (KBr) νmax: 3371, 2934, 2363, 2216, 1719, 1660, 1578, 1462, 1398, 1326, 1289, 1262, 1229, 1121, 1025, 976, 896, 826 cm-1. HPLC tr = 11.650 min (94.7% at 220 nm, 95.0% at 25 4 nm). 5-(3-aminopropoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)
RI PT
benzamide (23b). Yield = 16%; yellow crystals, mp = 87–90°C; 1H NMR (400 MHz, CDCl3): δ 1.59–2.44 (bs, 2H, -NH2), 1.83–1.90 (m, 4H, CH2-CH2-CH2-CH2), 1.94 (q, J = 6.5 Hz, 2H, H2N-CH2-CH2-CH2-O), 2.62–2.73 (m, 4H, CH2-CH2-CH2-CH2), 2.85 (t, J = 6.8 Hz, 2H, H2NCH2-CH2-CH2-O), 4.08 (t, J = 6.1 Hz, 2H, H2N-CH2-CH2-CH2-O), 7.02 (dd, J1 = 8.8, J2 = 3.1
SC
Hz, 1H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.46 (d, J = 3.1 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 22.40, 23.26, 23.77, 24.09, 27.30, 36.54, 64.95, 90.65, 114.98, 115.46, 118.45, 122.12, 128.50, 130.14, 134.34, 141.74, 156.43, 167.81, 173.26 ppm. ESI
M AN U
HRMS calcd. for [M+(H)]+ 390.1043, found 390,1047. IR (KBr) νmax: = 3436, 2930, 2196, 1508, 1466, 1370, 1289, 1223, 1025, 815 cm-1. HPLC tr = 12.038 min (93.9% at 220 nm, 93.5% at 25 4nm).
Tert-butyl(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-4,5,6,7-tetrahydrobenzo [b]thiophen-2-yl)carbamoyl)phenoxy)ethylamino)methylencarbamate (25a). Yield = 96%; pale yellow crystals; mp = 103–106 °C; 1H NMR (400 MHz, CDCl3): δ 1.52 (s, 9H, COO-
TE D
(CH3)3), 1.54 (s, 9H, COO-(CH3)3), 1.80–1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.65 (d, J = 5.7 Hz, 2H, CH2-CH2-CH2-CH2), 2.70 (d, J = 5.6 Hz, 2H, CH2-CH2-CH2-CH2), 3.89 (q, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 4.17 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 7.13 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.42 (d, J = 8.8 Hz, 1H, Ar-H), 7.56 (d, J = 3.1 Hz, 1H, Ar-H), 8.82 (s,
EP
1H, NH-CH2-CH2-O), 9.72 (s, 1H, NH-CO), 11.48 (s, 1H, NH-Boc) ppm. Tert-butyl(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-4,5,6,7-tetrahydrobenzo
AC C
[b]thiophen-2-yl)carbamoyl)phenoxy)propylamino)methylencarbamate (25b). Yield = 71%, white powder; mp = 109–113 °C; 1H NMR (400 MHz, CDCl3): δ 1.52 (s, 18H, 2 × COO(CH3)3), 1.81–1.94 (m, 4H, CH2-CH2-CH2-CH2), 2.82 (q, J = 6.3 Hz, 2H, O-CH2-CH2-CH2NH), 2.61–2.75 (m, 4H, CH2-CH2-CH2-CH2), 3.68 (q, J = 6.4 Hz, 2H, O-CH2-CH2-CH2-NH), 4.11–4.16 (m, 2H, O-CH2-CH2-CH2-NH), 7.14 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.40 (d, J = 8.9 Hz, 1H, Ar-H), 7.59 (d, J = 3.1 Hz, 1H, Ar-H), 8.58–8.77 (m, 1H, NH-CH2-CH2-CH2O), 9.70 (s, 1H, NH-CO), 11.53 (s, 1H, NH-Boc) ppm.
13
C NMR (100 MHz, DMSO-d6): δ
21.69, 22.58, 23.38, 23.53, 27.55, 27.94, 31.28, 53.60, 55.88, 67.20, 78.08, 82.85, 94.31,
S6
ACCEPTED MANUSCRIPT
113.19, 115.47, 117.31, 121.32, 128.07, 130.42, 131.19, 133.93, 135.38, 152.00, 155.13, 156.95, 163.02 ppm Amino((2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl) phenoxy)ethyl)amino)methaniminium chloride (27a). Yield = 93%; yellow crystals, mp = 99–
RI PT
103 °C; 1H NMR (400 MHz, MeOD): δ 1.80–1.96 (m, 4H, CH2-CH2-CH2-CH2), 2.58–2.75 (m, 4H, CH2-CH2-CH2-CH2), 3.63–3.69 (m, 2H, O-CH2-CH2-NH), 4.20 (t, J = 5.0 Hz, 2H, OCH2-CH2-NH), 7.15 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.23 (d, J = 3.0 Hz, 1H, Ar-H), 7.48 (d, J = 8.9 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 21.67, 22.56, 23.38,
SC
23.53, 66.70, 94.49, 113.95, 115.26, 118.07, 121.80, 128.21, 130.26, 131.29, 135.41, 145.56, 156.60, 156.98, 163.98 ppm. (1 C covered by the signal of the solvent). ESI HRMS calcd. for [M+(H)]+ 418.1104, found 418.1108. IR (KBr) νmax: 3403, 2934, 2214, 1654, 1550, 1458,
M AN U
1290, 1218, 1065, 819 cm-1. HPLC tR = 12.342 min (95.3% at 220 nm, 97.7% at 254 nm). Amino((2-(4-chloro-3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl) phenoxy)propyl)amino)methaniminium chloride (27b). Yield = 99%; yellow solid, mp = 94– 97 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.72–1.83 (m, 4H, CH2-CH2-CH2-CH2), 1.94 (p, J = 6.4 Hz, 2H, O-CH2-CH2-CH2-NH), 2.50–2.55 (m, 2H, CH2-CH2-CH2-CH2, under solvent peak), 2.59–2.67 (m, 2H, CH2-CH2-CH2-CH2), 3.28 (q, J = 6.5 Hz, 2H, O-CH2-CH2-CH2-
TE D
NH), 4.08 (t, J = 6.1 Hz, 2H, O-CH2-CH2-CH2-NH), 7.13 (dd, J1 = 8.8, J2 = 3.0 Hz, 1H, ArH), 7.19 (d, J = 3.0 Hz, 1H, Ar-H), 7.49 (d, J = 8.8 Hz, 1H, Ar-H), 7.74 (t, J = 5.7 Hz, 1H, NH-Boc), 12.30 (s, 1H, NH-CO) ppm.
13
C NMR (100 MHz, DMSO-d6): δ 21.67, 22.56,
23.38, 23.53, 28.00, 37.73, 65.49, 94.48, 113.95, 115.18, 117.87, 121.41, 128.18, 130.52,
EP
131.28, 135.38, 145.56, 156.82, 156.92, 164.07 ppm. HPLC tR = 12.821 min (95.2% at 220 nm, 96.8% at 254 nm).
AC C
4-(2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5,6,7tetrahydrobenzo[b]thiophen-6-yl)phenyl acetate (33b). Yield = 70%; yellow crystals, mp = 194–196 °C; 1H NMR (400 MHz, CDCl3): δ 1.99 (qd, J1 = 11.2, J2 = 5.6 Hz, 1H, CHCHAHB-CH2), 2.18–2.24 (m, 1H, CH-CHAHB-CH2), 2.33 (s, 3H, CO-CH3), 2.72–2.89 (m, 3H, CH2CH-CH2-CH2), 2.98–3.12 (m, 2H, CH2-CH), 3.85 (q, = 5.6 Hz, 2H, O-CH2-CH2-NH), 4.19 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 6.77 (bs, 1H, NH-CO-CF3), 7.07 (dd, J1 = 8.8, J2 = 3.2 Hz, 1H, Ar-H), 7.08 (d, J = 8.8 Hz, 2H, Ar-H), 7.30 (d, J = 8.0 Hz, 2H, Ar-H), 7.45 (d, J = 8.8 Hz, 1H, Ar-H), 7.58 (d, J = 3.2 Hz, 1H, Ar-H), 9.77 (bs, 1H, NH-CO) ppm. 13C NMR (100 MHz, CDCl3): δ 21.18, 24.28, 29.38, 31.83, 39.18, 40.22, 66.27, 94.41, 114.12, 115.73 S7
ACCEPTED MANUSCRIPT
(q, 1JC,F =285.8 Hz, CF3), 116.95, 120.01, 121.70, 123.02, 127.85, 128.63, 131.10, 131.68, 132.06, 142.53, 146.41, 149.26, 157.20, 157.52 (q, 2JC,F = 37.1 Hz, CO-CF3), 161.57, 169.75 ppm. ESI HRMS calcd. for [M+(H)]+ 606.1077, found 606.1081. IR (KBr) νmax: 3414, 3198, 3078, 2927, 2222, 1730, 1685, 1560, 1507, 1461, 1384, 1297, 1242, 1204, 1186, 1155, 1039,
RI PT
1020 cm-1. HPLC tR = 16.721 min (96.3% at 220 nm, 100% at 254 nm). 5-(2-aminoethoxy)-2-chloro-N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)
benzamide (36). Yield = 76%; pale brown solid, mp = 128–130 °C; 1H NMR (400 MHz, MeOD): δ 2.79 (t, J = 5.8 Hz, 2H, CH2-CH2-N), 3.30 (t, J = 5.8 Hz, 2H, CH2-CH2-N), 3.35 (t,
SC
J = 5.0 Hz, 2H, O-CH2-CH2-NH2), 4.09 (s, 2H, CH2-N), 4.26 (t, J = 5.0 Hz, 2H, O-CH2-CH2NH2), 7.15 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.27 (d, J = 3.1 Hz, 1H, Ar-H), 7.46 (d, J = 8.9 Hz, 1H, Ar-H) ppm.
13
C NMR (100 MHz, MeOD): δ 23.11, 40.33, 43.03, 43.73, 66.43,
M AN U
96.06, 114.73, 116.77, 119.43, 124.81, 125.42, 131.14, 132.42, 137.16, 150.59, 158.36 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 377.0839, found 377.0833. IR (KBr) νmax 3068, 2949, 2718, 2547, 2202, 1642, 1549, 1508, 1463, 1401, 1366, 1327, 1295, 1261, 1217, 1180, 1153, 1123, 1083, 1020, 1003 cm-1. HPLC tR = 6.518 min (100% at 220 nm, 100% at 254 nm). 2-chloro-N-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2(2,2,2-trifluoroacetamido)ethoxy)benzamide (37b). Yield = 53%; white crystals, mp = 88–90
TE D
°C; 1H NMR (400 MHz, CDCl3): δ 2.72 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 2.81 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 3.55 (s, 2H, CH2-N), 3.65 (s, 2H, N-CH2-Ar), 3.77–3.81 (m, 5H, O-CH3 + O-CH2-CH2-NH), 4.13 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 6.75 (bs, 1H, NH-CO-CF3), 6.86 (d, J = 8.6 Hz, 2H, Ar-H), 7.02 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.25 (d, J = 8.6 Hz, 2H,
EP
Ar-H), 7.39 (d, J = 8.8 Hz, 1H, Ar-H), 7.51 (d, J = 3.1 Hz, 1H, Ar-H), 9.73 (s, 1H, NH-COthiophen) ppm. 13C NMR (100 MHz, acetone-d6): δ 24.99, 40.04, 50.02, 51.29, 55.53, 61.47,
AC C
67.18, 95.26, 114.09, 114.54, 115.70, 116.38, 118.55, 119.28, 123.30, 127.62, 130.97, 131.16, 131.24, 131.85, 135.96, 147.21, 158.29, 159.99, 164.48 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 593.1237, found 593.1251. IR (KBr) νmax 3326, 2214, 1715, 1668, 1546, 1511, 1459, 1366, 1290, 1242, 1210, 1158, 1059, 1034, 1007 cm-1. HPLC tR = 12.107 min (98.9% at 220 nm, 100% at 254 nm). Methyl
4-((2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5-
dihydrothieno[2,3-c]pyridin-6(7H)-yl)methyl)benzoate (37c). Yield = 59%; yellow crystals, mp = 109–111 °C; 1H NMR (400 MHz, acetone-d6): δ 2.70 (t, J = 5.7 Hz, 2H, CH2-CH2-N), 2.87 (t, J = 5.7 Hz, 2H, CH2-CH2-N), 3.65 (s, 2H, CH2-N), 3.77 (q, 2H, J = 5.4 Hz, 2H, OS8
ACCEPTED MANUSCRIPT
CH2-CH2-NH-CO), 3.85 (s, 2H, N-CH2-Ar), 3.88 (s, 3H, COO-CH3), 4.26 (t, J = 5.4 Hz, 2H, O-CH2-CH2-NH-CO), 7.11 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.28 (d, J = 3.0 Hz, 1H, ArH), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 7.56 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 7.99 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 8.77 (bs, 1H, NH-CO-CF3), 11.02 (s, 1H, NH-CO-thiophene) ppm. 13C NMR
RI PT
(100 MHz, acetone-d6): δ 24.93, 40.04, 50.23, 51.45, 52.34, 61.50, 67.19, 95.24, 114.06, 115.69, 116.38, 118.55, 119.28, 123.30, 127.37, 129.74, 130.11, 130.33, 131.11, 131.85, 135.96, 145.27, 147.33, 158.29, 164.52, 167.19 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 621.1186, found 621.1190. IR (KBr) νmax 3308, 3088, 2957, 2911, 2754, 2214, 1733, 1697, 1666, 1611, 1577, 1459, 1434, 1416, 1372, 1330, 1283, 1220, 1183, 1140, 1112, 1057, 1037,
SC
1011 cm-1. HPLC tR = 12.004 min (100% at 220 nm, 100% at 254 nm).
2-chloro-N-(3-cyano-6-(3-hydroxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2-
M AN U
(2,2,2-trifluoroacetamido)ethoxy)benzamide (37d). Yield = 60%; pale yellow crystals, mp = 228–230 °C; 1H NMR (400 MHz, acetone-d6): δ 2.68 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 2.85 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.69 (s, 2H, N-CH2-Ar), 3.78 (q, J = 5.4 Hz, 2H, O-CH2-CH2-NH), 4.26 (t, J = 5.4 Hz, 2H, O-CH2-CH2-NH), 6.68-6.87 (m, 3H, 2 × Ar-H + NH-CO-CF3), 6.92 (s, 1H, Ar-H), 7.10–7.18 (m, 2H, 2 × Ar-H), 7.28 (d, J = 3.0 Hz, 1H, Ar-H), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 8.78 (s, 1H, NH-CO-thiophen), 11.01 (s, 1H, OH)
TE D
ppm. ESI HRMS m/z calcd. for [M+(H)]+ 579.1081, found 579.1076. IR (KBr) νmax 3323, 2216, 1772, 1730, 1656, 1581, 1552, 1481, 1457, 1400, 1345, 1310, 1262, 1211, 1182. 1137, 1121, 1084, 1062, 1043 cm-1. HPLC tR = 11.233 min (100% at 220 nm, 100% at 254 nm). 2-chloro-N-(3-cyano-6-(4-cyanobenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2-
EP
(2,2,2-trifluoroacetamido)ethoxy)benzamide (37e). Yield = 74%; yellow crystals, mp = 170– 172 °C; 1H NMR (400 MHz, CDCl3): δ 2.77 (t, J = 5.0 Hz, 2H, CH2-CH2-N), 3.85 (t, J = 5.0
AC C
Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2-Ar), 3.82 (q, J = 4.8 Hz, 2H, O-CH2-CH2-NH-CO), 4.16 (t, J = 4.8 Hz, 2H, O-CH2-CH2-NH-CO), 6.75 (bs, 1H, NH-COCF3), 7.0 (d, J = 8.9 Hz, 1H, Ar-H), 7.43 (d, J = 8.9 Hz, 1H, Ar-H), 7.49–7.55 (m, 3H, 3 × ArH), 7.65 (d, J = 7.3 Hz, 1H, Ar-H), 9.79 (s, 1H, NH-CO-thiophene) ppm.
13
C NMR (100
MHz, CDCl3): δ 24.03, 39.37, 49.70, 51.08, 61.25, 66.52, 94.41, 111.51, 114.00, 117.24, 117.33, 119.04, 120.42, 123.27, 126.61, 129.57, 130.14, 131.60, 132.39, 132.57, 143.91, 146.90, 157.41, 157.82, 161.64 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 588.1084, found 588.1081. IR νmax 3315, 3053, 2835, 2219, 1703, 1654, 1585, 1554, 1481, 1467, 1455, 1434, 1394, 1339, 1319, 1299, 1276, 1230, 1202, 1178, 1161, 1112, 1065, 1050, 1034, 1010 cm-1. HPLC tR = 11.750 min (97.4% at 220 nm, 100% at 254 nm). S9
ACCEPTED MANUSCRIPT
N-(6-(4-(1H-tetrazol-5-yl)benzyl)-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamide (37f). Yield = 45%; pale yellow crystals, mp = 137–139 °C; 1H NMR (400 MHz, MeOD): δ 2.89 (t, J = 5.5 Hz, 2H, CH2-CH2N), 3.29 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 3.69 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 4.03 (s,
RI PT
2H, CH2-N), 4.15 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 4.19 (s, 2H, N-CH2-Ar), 7.10 (d, J = 8.9 Hz, 1H, Ar-H), 7.19 (d, J = 3.0 Hz, 1H, Ar-H), 7.42 (d, J = 8.9 Hz, 1H, Ar-H), 7.62 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 8.08 (d, J = 8.4 Hz, 2H, 2 × Ar-H) ppm. 2 × NH exchanged. 13C NMR (400 MHz, MeOD): δ 24.11, 40.39, 50.61, 51.30, 61.46, 67.54, 95.65, 114.21, 116.18, 116.63, 119.75, 124.14, 124.54, 128.59, 129.03, 131.16, 132.21, 132.38, 136.11, 136.97, 149.19,
SC
158.93, 159.26, 159.63, 166.49 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 631.1254, found 631.1249. IR (KBr) νmax 2942, 2218, 1712, 1673, 1546, 1460, 1429, 1368, 1288, 1209, 1184,
M AN U
1154, 1062, 1036, 1012 cm-1. HPLC tR = 10.862 min (100% at 220 nm, 100% at 254 nm). 5-(2-aminoethoxy)-N-(6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2chlorobenzamide (38a). Yield = 71%; yellow crystals, mp = 122–124 °C; 1H NMR (400 MHz, MeOD): δ 2.72 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 2.88 (t, J = 5.6 Hz, 2H, CH2-CH2-N), 3.13 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.59 (s, 2H, CH2-N), 3.76 (s, 2H, N-CH2-Ar), 4.12 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 7.07 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.22 (d, J = 13
C NMR (100 MHz, DMSO-d6):
TE D
3.1 Hz, 1H, Ar-H), 7.28–7.41 (m, 6H, 6 × Ar-H) ppm.
24.01, 35.80, 49.46, 51.09, 60.95, 65.33, 90.72, 115.66, 115.98, 117.56, 121.18, 122.64, 127.03, 127.68, 128.27, 128.46, 128.80, 129.04, 130.43, 138.46, 140.54, 156.17, 162.31, 167.19 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 467.1309, found 467.1298. IR (KBr) νmax
EP
2926, 2880, 2804, 2762, 2199, 1657, 1574, 1512, 1463, 1406, 1365, 1318, 1275, 1224, 1165, 1132, 1080, 1053 cm-1. HPLC tR = 8.527 min (100% at 220 nm, 100% at 254 nm). [2,3-
AC C
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-tetrahydrothieno
c]pyridin-2-yl)benzamide (38b). Yield = 75%; yellow crystals, mp = 123–125 °C; 1H NMR (400 MHz, MeOD): δ 2.71 (t, J = 5.8 Hz, 2H, CH2-CH2-N), 2.86 (t, J = 5.4 Hz, 2H, CH2-CH2N), 3.12 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH2), 3.56 (s, 2H, CH2-N), 3.69 (s, 2H, N-CH2-Ar), 3.79 (s, 3H, O-CH3), 4.11 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH2), 6.91 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.06 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.21 (d, J = 3.0 Hz, 1H, Ar-H), 7.30 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.38 (d, J = 8.9 Hz, 1H, Ar-H) ppm. ESI HRMS m/z calcd. for [M+(H)]+ 497.1414, found 497.1409. IR νmax 3354, 3298, 2820, 2363, 2217, 1736, 1662, 1586, 1512, 1458, 1418, 1365, 1312, 1276, 1244, 1226, 1169, 1132, 1070, 1049, 1029 cm-1. HPLC tR = 8.794 min (100% at 220 nm, 100% at 254 nm). IR νmax 3354, 3298, 2920, 2820, S10
ACCEPTED MANUSCRIPT
2363, 2217, 1736, 1662, 1586, 1512, 1458, 1418, 1365, 1312, 1276, 1244, 1226, 1169, 1132, 1070, 1049, 1029 cm-1. HPLC tR = 8.794 min (100% at 220 nm, 100% at 254 nm). 4-((2-(5-(2-aminoethoxy)-2-chlorobenzamido)-3-cyano-4,5-dihydrothieno[2,3-c]pyridin6(7H)-yl)methyl)benzoic acid (38c). Yield = 79 %; pale yellow crystals, mp = 175–177 °C; 1H
RI PT
NMR (400 MHz, DMSO-d6): δ 2.53 (t, J = 5.2 Hz, 2H, CH2-CH2-N), 2.73 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 3.19 (t, 2H, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.43 (s, 2H, CH2-N), 3.73 (s, 2H, N-CH2-Ar), 4.15 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 6.95 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, Ar-H), 7.23 (d, J = 3.1 Hz, 1H, Ar-H), 7.33(d, J = 8.8 Hz, 1H, Ar-H), 7.46 (d, J = 8.3 Hz,
SC
2H, 2 × Ar-H), 7.991 (d, J = 8.3 Hz, 2H, 2 × Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 23.99, 38.56, 49.56, 51.16, 60.50, 65.39, 90.61, 115.60, 115.99, 117.66, 120.95, 122.64, 127.59, 128.65, 129.33, 130.36, 130.42, 140.70, 143.46, 145.45, 156.18, 167.31, 167.51 ppm.
M AN U
ESI HRMS m/z calcd. for [M+(H)]+ 511.1207, found 511.1203. IR (KBr) νmax 2927, 2804, 2215, 2198, 1640, 1584, 1568, 1516, 1462, 1366, 1306, 1275, 1224, 1164, 1131, 1078, 1053, 1022 cm-1. HPLC tR = 7.718 min (100% at 220 nm, 100% at 254 nm). 5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(3-hydroxybenzyl)-4,5,6,7-tetrahydrothieno
[2,3-
c]pyridin-2-yl)benzamide (38d). Yield = 89%; pale yellow crystals, mp = 113–115 °C; 1H NMR (400 MHz, MeOD): δ 2.72 (t, J = 5.2 Hz, 2H, CH2-CH2-N), 2.87 (t, J = 5.2 Hz, 2H,
TE D
CH2-CH2-N), 3.12 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.59 (s, 2H, CH2-N), 3.68 (s, 2H, NCH2-Ar), 4.11 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 6.72 (d, J = 8.0 Hz, 1H, Ar-H), 6.83– 6.86 (m, 2H, 2 × Ar-H), 7.06 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.15–7.22 (m, 2H, 2 × ArH), 7.39 (d, J = 8.9 Hz, 1H, Ar-H) ppm. 13C NMR (100 MHz, MeOD): δ 25.16, 41.34, 50.75,
EP
54.96, 62.79, 69.42, 95.69, 115.70, 116.68, 117.52, 118.89, 121.91, 124.39, 126.74, 129.08, 130.62, 131.12, 131.60, 132.17, 138.17, 139.91, 158.84, 167.99 ppm. ESI HRMS m/z calcd.
AC C
for [M+(H)]+ 483.1258, found 483.1245. IR νmax 3063, 2925, 2809, 2210, 1663, 1584, 1545, 1457, 1405, 1365, 1278, 1225, 1154, 1120, 1054, 1008 cm-1. HPLC tR = 7.930 min (99.2% at 220 nm, 99.5% at 254 nm). 5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-cyanobenzyl)-4,5,6,7-tetrahydrothieno[2,3c]pyridin-2-yl)benzamide (38e). Yield = 83%; yellow solid, mp = 125–127 °C; 1H NMR (400 MHz, acetone-d6): δ 2.69 (t, J = 5.7 Hz, 2H, CH2-CH2-N), 3.87 (t, J = 5.7 Hz, 2H, CH2-CH2N), 3.56 (t, J = 5.8 Hz, 2H, O-CH2-CH2-NH2), 3.66 (s, 2H, CH2-N), 3.86 (s, 2H, N-CH2-Ar), 4.25 (t, J = 5.8 Hz, 2H, O-CH2-CH2-NH2), 7.09 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.27 (d, J = 3.0 Hz, 1H, Ar-H), 7.40 (d, J = 8.9 Hz, 1H, Ar-H), 7.64 (d, J = 8.3 Hz, 2H, 2 × Ar-H), S11
ACCEPTED MANUSCRIPT
7.76 (d, J = 8.3 Hz, 2H, 2 × Ar-H) ppm.
13
C NMR (100 MHz, acetone-d6): δ 24.87, 50.22,
51.19, 51.48, 61.27, 70.02, 95.08, 111.73, 114.26, 116.43, 119.02, 119.50, 122.76, 126.97, 130.49, 130.97, 131.71, 133.08, 136.05, 145.66, 147.98, 158.83, 164.77 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 492.1261, found 492.1271. IR (KBr) νmax 2926, 1823, 2215, 1668, 1553,
RI PT
1458, 1413, 1363, 1293, 1218, 1167, 1134, 1116, 1067, 1012 cm-1. HPLC tR = 8.375 min (94.5% at 220 nm, 95.6% at 254 nm).
N-(6-(4-(1H-tetrazol-5-yl)benzyl)-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-5-(2aminoethoxy)-2-chlorobenzamide (38f). Yield = 64%; yellow crystals, mp = 221–223 °C; 1H
SC
NMR (400 MHz, DMSO-d6): δ 2.61 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 2.78 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 3.25 (t, 2H, J = 4.9 Hz, 2H, O-CH2-CH2-NH2), 3.53 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2-Ar), 4.21 (t, J = 4.9 Hz, 2H, O-CH2-CH2-NH2), 7.10 (dd, J1 = 8.8, J2 = 3.0 Hz,
M AN U
1H, Ar-H), 7.24 (d, J = 3.0 Hz, 1H, Ar-H), 7.37 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 7.46 (d, J = 8.9 Hz, 1H, Ar-H), 7.95 (d, J = 8.2 Hz, 2H, 2 × Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 23.78, 38.36, 49.00, 50.42, 60.47, 65.14, 93.22, 115.61, 117.66, 122.24, 125.90, 128.99, 129.54, 130.37, 130.61, 137.33, 156.34, 159.82, 164.88 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 535.1431, found 535.1428. IR νmax 3633, 2837, 2217, 2116, 2022, 1646, 1566, 1583, 1476, 1451, 1403, 1365, 1312, 1289, 1233, 1166, 1122, 1056, 1023, 1011 cm-1. HPLC
Methyl
TE D
tR = 7.689 min (97.1% at 220 nm, 100% at 254 nm).
4-((2-(5-(2-aminoethoxy)-2-chlorobenzamido)-3-cyano-4,5-dihydrothieno[2,3-
c]pyridin-6(7H)-yl)methyl)benzoate (39c). Yield = 71%; pale yellow crystals, mp = 136–138 °C; 1H NMR (400 MHz, MeOD): δ 2.73 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 2 .87 (t, J = 5.4 Hz,
EP
2H, CH2-CH2-N), 3.33 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 3.62 (s, 2H, CH2-N), 3.83 (s, 2H, N-CH2-Ar), 3.91 (s, 3H, COO-CH3), 4.24 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH2), 7.15 (dd,
AC C
J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.26 (d, J = 3.1 Hz, 1H, Ar-H), 7.46 (d, J = 8.9 Hz, 1H, ArH), 7.52 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 8.01 (d, J = 8.9 Hz, 2H, 2 × Ar-H) ppm.
13
C NMR
(100 MHz, MeOD): δ 25.20, 40.43, 50.70, 51.90, 52.77, 62.20, 66.72, 96.16, 114.74, 116.73, 119.56, 124.74, 127.70, 130.64, 130.85, 131.65, 132.44, 136.81, 144.69, 148.83, 158.43, 166.54, 168.52, 178.99 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 525.1363, found 525.1354. IR νmax 2950, 2911, 2797, 2211, 1718, 1608, 1574, 1530, 1459, 1434, 1401, 1311, 1275, 1194, 1175, 1105, 1017 cm-1. HPLC tR = 8.752 min (96.9% at 220 nm, 95.2% at 254 nm). Tert-butyl
(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(benzyl)-4,5,6,7-tetra-
hydrothieno[2,3-c]pyridine-2-ylcarbamoyl)phenoxy)ethylamino)methylenecarbamate
(40a). S12
ACCEPTED MANUSCRIPT
Yield = 56%; yellow solid, mp = 92–94 °C; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, O(CH3)3), 1.49 (s, 9H, O-(CH3)3), 2.77 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 2 .88 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.58 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2-Ar), 3.88 (q, J = 5.3 Hz, 2H, O-CH2CH2-NH), 4.16 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 7.13 (dd, J1 = 8.8, J2 = 3.1 Hz, 1H, ArCH2-NH), 9.76 (s, 1H, NH-Boc), 11.46 (s, NH-CO-thiophene) ppm.
RI PT
H), 7.27–7.38 (m, 6H, 6 × Ar-H), 7.56 (d, J = 3.1 Hz, 1 H, Ar-H), 8.74 (t, J = 5.3 Hz, O-CH213
C NMR (100 MHz,
CDCl3): δ 24.24, 28.25, 28.47, 40.08, 49.57, 50.87, 61.85, 67.13, 79.69, 83.51, 94.21, 114.07, 117.28, 120.61, 122.69, 126.74, 127.74, 128.72, 129.34, 130.06, 131.56, 132.09, 137.62, 146.96, 153.25, 156.54, 157.86, 161.86, 163.55 ppm. ESI HRMS m/z calcd. for [M+(H)]+
SC
709.2575, found 709.2565. IR νmax 3334, 2977, 2221, 1720, 1682, 1638, 1617, 1547, 1478, 1452, 1412, 1361, 1322, 1285, 1253, 1229, 1137, 1048, 1023 cm-1.
(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-
M AN U
Tert-butyl
tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylene-carbamate (40b). Yield = 74%; pale yellow crystals, mp = 107–109 °C; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, O-(CH3)3), 1.48 (s, 9H, O-(CH3)3), 2.73 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 2 .84 (t, J = 5.4 Hz, 2H, CH2-CH2-N), 3.57 (s, 2H, CH2-N), 3.67 (s, 2H, N-CH2-Ar), 3.79 (s, 3H, OCH3), 3.83 (q, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 4.11 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH),
TE D
6.86 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.08 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.26 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.37 (d, J = 8.9 Hz, 2H, Ar-H), 7.51 (d, J = 3.0 Hz, 1 H, Ar-H),8.69 (t, J = 5.2 Hz, O-CH2-CH2-NH), 9.75 (s, 1H, NH-Boc), 11.46 (s, NH-CO-thiophene) ppm. 13C NMR (100 MHz, CDCl3): δ 24.26, 28.26, 28.48, 40.09, 49.44, 50.77, 55.49, 61.23, 67.15, 79.68,
EP
83.52, 94.24, 114.06, 117.33, 020.66, 122.70,126.21, 126.84, 129.67, 130.11, 130.56, 131.54, 132.13, 146.94, 153.28, 156.56, 157.91, 159.24, 161.84, 163.60 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 739.2654, found 739.2672. IR νmax 3332, 2976, 2933, 2216, 1721, 1638, 1613,
AC C
1546, 1512, 1457, 1411, 1364, 1320, 1284, 1244, 1134, 1048, 1024 cm-1. Methyl
4-((2-(5-(2-(2,3-bis(tert-butoxycarbonyl)guanidino)ethoxy)-2-chlorobenzamido)-3-
cyano-4,5-dihydrothieno[2,3-c]pyridin-6(7H)-yl)methyl)benzoate (40c). Yield = 54%; yellow crystals, mp = 106–108 °C; 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H, O-(CH3)3), 1.49 (s, 9H, O-(CH3)3), 2.75 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 2 .87 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.61 (s, 2H, CH2-N), 3.79 (s, 2H, N-CH2-Ar), 3.84 (q, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 3.90 (s, 3H, COO-CH3), 4.12 (t, J = 5.3 Hz, 2H, O-CH2-CH2-NH), 7.09 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.37 (d, J = 8.9 Hz, 1H, Ar-H), 7.44 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 7.52 (d, J = 3.1 Hz, 1 H, Ar-H), 8.01 (d, J = 8.3 Hz, 2H, 2 × Ar-H),8.72 (t, J = 5.3 Hz, O-CH2-CH2-NH), 9.77 S13
ACCEPTED MANUSCRIPT
(s, 1H, NH-Boc), 11.45 (s, NH-CO-thiophene) ppm.
13
C NMR (100 MHz, CDCl3): δ 24.23,
28.27, 28.49, 40.15, 49.69, 51.01, 52.25, 61.44, 67.15, 79.87, 83.60, 94.24, 114.04, 117.38, 120.72, 122.73, 126.51, 129.15, 129.67, 130.07, 131.48, 132.16, 143.12, 147.07, 153.28, 156.60, 157.91, 161.87, 163.47, 167.11 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 767.2630,
RI PT
found 767.2632. Tert-butyl (tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(3-hydroxybenzyl)-4,5,6,7 tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylene-carbamate
(40d).Yield = 36%; yellow solid, mp = 117–119 °C; 1H NMR (400 MHz, CDCl3): δ 1.45 (s,
SC
9H, O-(CH3)3), 1.47 (s, 9H, O-(CH3)3), 2.63 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 2.78 (t, J = 5.5 Hz, 2H, CH2-CH2-N), 3.47 (s, 2H, CH2-N), 3.59 (s, 2H, N-CH2-Ar), 3.71 (q, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 3.90 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 6.80–6.83 (m, 3H, 3 × Ar-H),
M AN U
6.93 (dd, J1 = 8.9, J2 = 3.1 Hz, 1H, Ar-H), 7.13-7.15 (m, 2H, 2 × Ar-H), 7.28 (d, J = 8.9 Hz, 1H, Ar-H), 8.71 (t, J = 5.5Hz, 1H, O-CH2-CH2-NH), 10.00 (bs, 1H, NH-Boc), 11.46 (bs, NHCO-thiophene). ESI HRMS m/z calcd. for [M+(H)]+ 725.2524, found 725.2503. Tert-butyl
(tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(4-cyanobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylenecarbamate
Tert-butyl
TE D
(40e). Yield = 23%.This compound was used for further reaction without purification. (tert-butoxycarbonylamino)(2-(4-chloro-3-(3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-ylcarbamoyl)phenoxy)ethylamino)methylene
carbamate
(41). Yield = 63%; yellow crystals, mp = 133–135 °C; 1H NMR (400 MHz, MeOD): δ 1.49
EP
(s, 9H, O-(CH3)3), 1.53 (s, 9H, O-(CH3)3), 1.97 (qd, J1 = 10.8, J2 = 5.6 Hz, 1H, CH-CHAHBCH2), 2.10–2.16 (m, 1H, CH-CHAHB-CH2), 2.67–2.80 (m, 3H, CH2-CH-CH2-CH2), 2.91–3.03 (m, 2H, CH2-CH), 3.79 (t, J = 5.2 Hz, 2H, O-CH2-CH2-NH), 4.20 (t, J = 5.2 Hz, 2H, O-CH2-
AC C
CH2-NH2), 6.76 (d, J = 8.8 Hz, 2H, Ar-H), 7.14 (d, J = 8.4 Hz, 2H, Ar-H), 7.17 (dd, J1 = 8.8, J2 = 3.2 Hz, 1H, Ar-H), 7.24 (d, J = 2.8 Hz, 1H, Ar-H), 7.44 (d, J = 8.8 Hz, 1H, Ar-H) ppm. 13
C NMR (100 MHz, acetone-d6): δ 25.14, 28.15, 28.49, 30.61, 31.63, 32.61, 40.83, 67.56,
79.09, 83.87, 95.40, 114.31, 116.16, 116.55, 119.26, 123.24, 128.69, 129.33, 131.83, 132.02, 136.01, 137.21, 147.09, 153.72, 156.85, 157.15, 158.34, 164.46, 164.53 ppm. ESI HRMS calcd. for [M+(H)]+ 710.2415, found 710.2419. IR (KBr) νmax: 3549, 3412, 2976, 2930, 2213, 1786, 1724, 1636, 1617, 1575, 1549, 1516, 1456, 1411, 1384, 1368, 1324, 1285, 1230, 1140, 1051, 1024 cm-1. Anal. Calcd. for C35H40ClN5O7S × 0.4 H2O: C, 58.59; H, 5.73; N, 9.76. Found C, 58.29; H, 5.32; N, 10.11. S14
ACCEPTED MANUSCRIPT
1-(2-(3-((6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)carbamoyl)-4chlorophenoxy)ethyl)guanidinium chloride (42a). Yield = 63%; white solid, mp = 150– 152°C; 1H NMR (400 MHz, MeOD): δ 3.09 (t, J = 5.1 Hz, 2H, CH2-CH2-N), 3.63 (t, J = 4.7 Hz, 1H, O-CH2-CH2-NH), 3.78 (t, J = 4.7 Hz, 1H, O-CH2-CH2-NH), 4.18 (t, J = 4.8 Hz, 1H,
RI PT
O-CH2-CH2-NH), 4.25 (t, J = 4.8 Hz, 1H, O-CH2-CH2-NH), 4.45 (s, 2H, CH2-N), 4.66 (s, 2H, N-CH2-Ar), 7.16 (dt, J1 = 8.8, J2 = 3.0 Hz, 1H, Ar-H), 7.23 (t, J = 3.3 Hz, 1H, Ar-H), 7.47 (dd, J1 = 9.0, J2 = 2.1 Hz, 1H, Ar-H), 7.53–7.59 (m, 3H, 3 × Ar-H), 7.60-7.62 (m, 2H, 2 × ArH) ppm. (1 × CH2 under solvent peak). 13C NMR (100 MHz, MeOD): δ 22.85, 28.27, 42.19, 50.46, 60.95, 68.21, 95.27, 113.79, 116.66, 116.76, 119.85, 119.89, 120.90, 124.51, 124.59,
SC
130.30, 130.57, 130.74, 131.69, 132.53, 132.58, 136.04, 150.34, 158.75, 166.58 ppm. IR νmax 3117, 2944, 2677, 2553, 2221, 1738, 1670, 1591, 1543, 1456, 1398, 1375, 1275, 1248, 1212,
M AN U
1150, 1067, 1038 cm-1. HPLC tR = 8.957 min (98.9% at 220 nm, 98.9% at 254 nm). 1-(2-(4-chloro-3-(3-cyano-6-(4-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2ylcarbamoyl)phenoxy)ethyl)guanidinium chloride (42b).Yield = 87%; pale yellow crystals, mp =75–77 °C; 1H NMR (400 MHz, MeOD): δ 3.08 (t, J = 4.7 Hz, 2H, CH2-CH2-N), 3.78 (t, J = 4.7 Hz, 2H, CH2-CH2-N), 3.85 (s, 3H, Ar-O-CH3), 4.25 (t, J = 5.1 Hz, 2H, O-CH2-CH2NH), 4.39 (s, 2H, CH2-N), 4.42 (s, 2H, N-CH2-Ar), 4.49 (t, 2H, J = 4.9 Hz, O-CH2-CH2-NH),
TE D
7.07 (d, J = 8.5 Hz, 2H, 2 × Ar-H), 7.16 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.22 (t, J = 3.0 Hz, 1H, Ar-H), 7.47 (d, J = 8.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.5 Hz, 2H, 2 × Ar-H) ppm. 13C NMR (100 MHz, MeOD): δ 22.87, 28.27, 42.37, 50.27, 56.08, 60.57, 67.72, 95.26, 113.80, 115.98, 116.76, 116.66, 119.89, 120.99, 121.91, 124.59, 130.60, 132.53, 134.09, 136.07,
EP
150.31, 158.60, 162.92, 166.53 ppm. HRMS m/z calcd. for [M+(H)]+ 539.1632, found 539.1630. IR νmax 3146, 2939, 2838, 2576, 2361, 2218, 1735, 1673, 1612, 1585, 1545, 1515, 1458, 1398, 1370, 1249, 1180, 1147, 1065, 1028 cm-1. HPLC tR = 9.256 min (98.9% at 220
AC C
nm, 99.2% at 254 nm).
1-(2-(4-chloro-3-((3-cyano-6-(4-(methoxycarbonyl)benzyl)-4,5,6,7-tetrahydrothieno[2,3c]pyridin-2-yl)carbamoyl)phenoxy)ethyl)guanidinium chloride (42c). Yield = 85%; pale yellow solid, mp = 112–114 °C; 1H NMR (400 MHz, MeOD): δ 3.10 (t, J = 4.9 Hz, 2H, CH2CH2-N), 3.63 (t, J = 4.8 Hz, 2H, CH2-CH2-N), 3.83 (s, 3H, COO-CH3), 4.18 (t, J = 5.1 Hz, 2H, O-CH2-CH2-NH), 4.45 (s, 2H, CH2-N), 4.65 (s, 2H, N-CH2-Ar), 7.15 (dd, J1 = 8.8, J2 = 2.9 Hz, 1H, Ar-H), 7.22 (d, J = 2.9 Hz, 1H, Ar-H), 7.46 (d, J = 8.8 Hz, 1H, Ar-H), 7.75 (d, J = 8.1 Hz, 2H, 2 × Ar-H), 8.16 (d, J = 8.1 Hz, 2H, 2 × Ar-H) ppm. 1 × CH2 under solvent peak. 13
C NMR (100 MHz, MeOD): δ 22.86, 42.16, 50.73, 50.75, 53.09, 60.22, 68.20, 95.24, S15
ACCEPTED MANUSCRIPT
113.79, 116.65, 119.85, 120.80, 124.40, 130.53, 131.58, 132.50, 132.85, 133.35, 135.19, 136.03, 150.38, 158.76, 159.17, 166.58, 167.76 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 567.1581, found 567.1592. IR νmax 3156, 2950, 2568, 2218, 1721, 1675, 1586, 1545, 1457, 1436, 1371, 1281, 1248, 1187, 1148, 1111, 1064, 1039, 1022 cm-1. HPLC tR = 9.219 min
RI PT
(100% at 220 nm, 100% at 254 nm). 1-(2-(4-chloro-3-((3-cyano-6-(3-hydroxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-
yl)carbamoyl)phenoxy)ethyl)guanidinium chloride (42d). Yield = 76%; white solid, mp = 159–161 °C; 1H NMR (400 MHz, MeOD): δ 3.07 (bs, 2H, CH2-CH2-N), 3.63 (bs, 2H, O-
SC
CH2-CH2-NH), 4.17 (t, J = 4.7 Hz, 2H, CH2-CH2-N), 4.42 (s, 2H, N-CH2-Ar), 4.46 (s, 2H, OCH2-CH2-NH), 6.93–7.03 (m, 3H, 3 × Ar-H), 7.14–7.21 (m, 2H, 2 × Ar-H), 7.33 (t, J = 7.4 Hz, 1H, Ar-H), 7.46 (d, J = 8.6 Hz, 1H, Ar-H) ppm. (1 × CH2 under solvent peak, NH, OH
M AN U
exchanged). 13C NMR (100 MHz, MeOD): δ 22.82, 42.16, 50.52, 60.91, 65.28, 68.20, 95.16, 113.70, 116.55, 118.38, 119.02, 119.73, 120.80, 123.06, 124.29, 130.43, 131.41, 131.72, 132.39, 135.92, 150.21, 158.64, 159.07, 159.63, 166.47 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 525.1476, found 525.1468. IR (KBr) νmax 3319, 3161, 2720, 2582, 2219, 1737, 1664, 1590, 1546, 1481, 1457, 1421, 1371, 1284, 1243, 1149, 1065, 1039, 1023 cm-1. HPLC tR = 8.355 min (99.1% at 220 nm, 99.2% at 254 nm).
TE D
1-(2-(4-chloro-3-((3-cyano-6-(4-cyanobenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2yl)carbamoyl)phenoxy)ethyl)guanidinium chloride (42e). Yield = 82%; white solid, mp = 169–171 °C; 1H NMR (400 MHz, MeOD): δ 3.12 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.65 (t, J = 5.3 Hz, 2H, CH2-CH2-N), 3.74 (s, 2H, O-CH2-CH2-NH), 4.20 (t, J = 5.0 Hz, 2H, O-CH2-CH2-
EP
NH), 4.45 (s, 2H, CH2-N), 4.66 (s, 2H, N-CH2-Ar), 7.17 (dd, J1 = 8.9, J2 = 3.0 Hz, 1H, Ar-H), 7.24 (d, J = 3.0 Hz, 1H, Ar-H), 7.48 (d, J = 8.9 Hz, 1H, Ar-H), 7.83 (d, J = 8.2 Hz, 2H, 2 ×
AC C
Ar-H), 7.92 (d, J = 8.3 Hz, 2H, 2 × Ar-H) ppm.13C NMR (100 MHz, MeOD): δ 22.92, 28.26, 42.16, 50.76, 60.03, 68.20, 95.27, 113.80, 115.44, 116.65, 119.13, 119.85, 120.96, 124.42, 130.55, 132.51, 133.53, 134.38, 135.74, 136.04, 150.33, 158.76, 159.18, 166.59 ppm. ESI HRMS m/z calcd. for [M+(H)]+ 534.1479, found 534.1475. IR (KBr) νmax 3329, 3154, 2545, 2221, 1736, 1661, 1587, 1546, 1458, 1419, 1370, 1290, 1200, 1173, 1124, 1065, 1039 cm-1. HPLC tR = 8.796 min (98.4% at 220 nm, 98.8% at 254 nm). Amino((2-(4-chloro-3-((3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2yl)carbamoyl)phenoxy)ethyl)amino)methaniminium chloride (43). Yield = 79%; pale brown crystals, mp = 205–206 °C; 1H NMR (400 MHz, MeOD): δ 1.98 (m, 1H, CH-CHAHB-CH2), S16
ACCEPTED MANUSCRIPT
2.11–2.17 (m, 1H, CH-CHAHB-CH2), 2.69–2.82 (m, 3H, CH-CH2-CH2), 2.93–3.02 (m, 2H, CH2-CH-CH2-CH2), 3.65 (t, J = 4.8 Hz, 2H, CH2-NH-C), 4.20 (t, J = 4.8 Hz, 2H, CH2-CH2O), 6.77 (d, J = 8.8 Hz, 2H, 2 × Ar-H), 7.14 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.16 (dd, J1 = 5.6, J2 = 3.2 Hz, 1H, Ar-H), 7.24 (d, J = 3.2 Hz, 1H, Ar-H), 7.48 (d, J = 8.8 Hz, 1H, Ar-H) ppm. 13
RI PT
C NMR (100 MHz, MeOD): δ 25.59, 31.06, 33.06, 41.43, 42.07, 68.10, 96.56, 114.89,
116.34, 116.52, 119.62, 124.29, 128.88, 130.55, 132.35, 132.77, 136.33, 137.65, 147.35, 156.99, 158.60, 159.05, 166.15 ppm. ESI HRMS calcd. for [M+(H)]+ 510.1367, found 510.1361. IR (KBr) νmax: 3414, 2224, 1652, 1616, 1575, 1511, 1458, 1387, 1305, 1239, 1123,
SC
1061, 1040 cm-1. HPLC tR = 11.881 min (98.1% at 220 nm, 97.2% at 254 nm). REFERENCES:
M AN U
[1] K. Sundaravel, E. Suresh, M. Palaniandavar, Iron(III) complexes of tridentate N3 ligands as models for catechol dioxygenases: Stereoelectronic effects of pyrazole coordination,
AC C
EP
TE D
Inorg. Chim. Acta. 363 (2010) 2768–2777.
S17
ppm
7
6,5
% 50 100150
23b (1D 1H) CDCl3 400MHz
6
5,5
5
%
2,21
1,04 1,00
1,00
0
10
20
30
4,5
4,5
4
4
3,5
3,5
3
3
4,16
2,08 1,58
2,39
5,88
M AN U
5
2,866 2,849 2,832 2,712 2,701 2,686 2,660 2,649 2,646 2,635 1,965 1,949 1,932 1,916 1,907 1,900 1,893 1,878 1,870 1,863 1,852 1,848 1,842 1,832
5,5
4,093 4,077 4,062
TE D
ppm 7,465 7,458 7,398 7,376 7,285 7,039 7,031 7,017 7,009
100
6
7,48
EP
80
6,5
2,41 2,12
60
AC C
40
7
2,14
20 7,5
2,15
0 ppm
1,00
1,00 1,00
50
SC
40
70
2,5
2,5
1,79 1,78 1,77 1,76 1,75
2,63 2,54 2,51 2,50 2,50 2,50 2,49
3,24
3,37
4,22
ppm 7,52 7,50 7,22 7,21 7,17 7,16 7,15 7,14
80
% 50 100150
RI PT
60
1
%
ACCEPTED MANUSCRIPT
H NMR spectra for compounds 23a-b, 27b, 33, 35, 36, 37a-c, 37e-f , 38a-f, 39c, 40c, 42a,
42c-e, 43
23a (1D 1H) DMSO 400MHz
2 1,5
2
S18
ppm
0,92
0
5
9
8
7
6
5
4
3 3
1,463
1,725
4
9,05
2,674
5
2,00
6
2,00 2,01
7
4,144 4,131 4,118 3,786 3,772 3,682
8
1,97
9
33 (1D 1H) CDCl3 400MHz
%
0,96
0,77
0
1,98 3,16
1,89
1,98
2,00
1,00 1,07
1,06
M AN U
% 50 100150
10
4,505
7,483 7,475 7,385 7,363 7,240 7,021 7,014 6,999 6,992
ppm 9,841
25
11
2,00
20
TE D
15
12
1,94
EP
10 ppm
0,99 0,99
AC C
%
SC
10
30
40
RI PT
20
50
60
4,081 3,670 3,306 3,290 3,275 3,258 2,651 2,639 2,625 2,516 2,512 2,507 2,502 2,498 2,090 1,972 1,956 1,940 1,924 1,913 1,908 1,779
7,754 7,740 7,726 7,496 7,474 7,188 7,181 7,146 7,139 7,124 7,117
12,295
ppm
% 50 100150
ACCEPTED MANUSCRIPT
27b (1D 1H) DMSO 400MHz
2
2
S19
AC C EP TE D
ppm 9 8 7 6
2,00
2,05
0,88 3,03
0,88
0,82
0
5
4
3
0,93 0,95
3,37
2,27
2,06
M AN U
%
4
SC
2
8
10
12
RI PT
6
14
16
7,485 7,463 7,233 7,225 7,130 7,115 7,106 7,094 6,720 6,699 4,224 4,212 4,199 3,344 3,321 3,229 3,216 3,216 3,204 2,914 2,886 2,884 2,865 2,830 2,829 2,817 2,683 2,681 2,678 2,677 2,674 2,669 2,664 2,656 2,643 2,635 2,623 2,509 2,505 2,500 2,495 2,491 2,010 2,008 2,004 2,002 1,997 1,995 1,976 1,974 1,973 1,907 1,891 1,883 1,882 1,877 1,873 1,871 1,869 1,861 1,860 1,852 1,849 1,844 1,841 1,840 1,836 1,830 1,811
9,251
ppm
% 50 100150
ACCEPTED MANUSCRIPT
35 (1D 1H) DMSO 400MHz
2
S20
%
1,0
10
20
ppm
10
9,5
9
8,5
8
7,5
7
6,5
6
5
5,5
5
4
4,5
4
3,5
2,1 2,0
6
2,829 2,814 2,800 2,732 2,719
37b (1D 1H) CDCl3 400MHz
%
2,00
4,01
4,07 2,02
2,06
1,05
3,02 2,13 2,15
0,68
0,64
0
M AN U
% 50 100150
7
5,3 2,0 2,0
4,142 4,129 4,116 4,106 4,088 3,812 3,796 3,785 3,772 3,650 3,548
7,517 7,403 7,381 7,265 7,260 7,249 7,244 7,240 7,031 7,023 7,009 7,001 6,875 6,870 6,858 6,853
9,733
ppm
8
2,8
70
9
2,1 1,0
60
TE D
50
10
1,0
40
EP
30 ppm
1,0 1,0
AC C
0
SC
20
60
80
RI PT
40
100
4,274 4,261 4,247 3,794 3,781 3,767 3,759 3,618 3,614 3,609 2,863 2,854 2,848 2,834 2,696 2,692 2,682 2,678 2,673 2,663 2,061 2,055 2,050 2,044 2,039
8,779 7,442 7,442 7,420 7,416 7,415 7,398 7,398 7,368 7,365 7,365 7,361 7,347 7,344 7,343 7,332 7,328 7,297 7,297 7,294 7,290 7,283 7,275 7,261 7,257 7,254 7,127 7,119 7,105 7,097
ppm 11,011
% 50 100150
ACCEPTED MANUSCRIPT
37a (1D 1H) Acetone 400MHz
3 2
3
2,5
S21
ppm
0,96
0
50
10
9,5
9
8,5
8
7,5
7
6,5
6
5,5
5
4,5
4
1,98 2,01
4
3,5
2,854 2,842 2,765
3,829 3,817 3,788 3,608
5
4,162
6
2,01
7
37e (1D 1H) CDCl3 400MHz
%
2,01 0,96 1,01 1,00
2,04
0,70
0,69
0
1,98
3,78
1,95
3,04 2,13
2,05
M AN U
7,661 7,642 7,548 7,518 7,499 7,470 7,439 7,417 7,264 7,066 7,044 6,755 6,752
9,786
% 50 100150
8
1,99 1,89
400 ppm
9
2,05
350
10
0,93
300
TE D
250
11
1,00
200 ppm
2,31 3,08 1,06
150
EP
100
AC C
%
SC
20
60
80
RI PT
40
4,275 4,262 4,248 3,883 3,850 3,781 3,768 3,654 3,650 3,646 2,886 2,871 2,856 2,710 2,700 2,696 2,692 2,061 2,055 2,050 2,044 2,039
8,004 7,999 7,988 7,983 7,566 7,562 7,545 7,443 7,421 7,284 7,277 7,129 7,121 7,106 7,099
8,776
11,023
ppm
100
% 50 100150
ACCEPTED MANUSCRIPT
37c (1D 1H) Acetone 400MHz
3 2
3
2,5
S22
ppm
6,09
0
7
6,5
6
5,5
5
4,5
4
4
3,5
3,5
1,91
4,5
1,87
5
3,763 3,593 3,334 3,329 3,318 3,314 3,310 3,306 3,302 3,146 3,133 3,125 3,120 2,890 2,875 2,868 2,861 2,737 2,723 2,712 2,710
5,5
4,131 4,119 4,106
4,888
6
38a (1D 1H) MeOD 400MHz
%
2,01 2,13
0,94 0,98
1,03
2,07
2,05
0
2,00
6,69
2,09
1,96
M AN U
% 50 100150
6,5
1,89
10 ppm 7,413 7,409 7,404 7,392 7,389 7,381 7,376 7,372 7,359 7,355 7,340 7,319 7,315 7,311 7,305 7,298 7,291 7,284 7,281 7,277 7,220 7,212 7,087 7,080 7,065 7,058 7
2,12
8
TE D
6
7,5
2,00
EP
4
8
2,14
1,00
2 ppm
1,03
AC C
%
SC
20
60
80
RI PT
40
100
3,700 3,687 3,674 3,318 3,314 3,310 3,306 3,302 3,288 3,273 2,905 2,891 2,877
4,190 4,168 4,154 4,141 4,025
4,909 4,903
ppm 8,091 8,070 7,641 7,635 7,620 7,614 7,435 7,430 7,413 7,407 7,196 7,188 7,121 7,116 7,108 7,094 7,086
% 50 100150
ACCEPTED MANUSCRIPT
37f (1D 1H) MeOD 400MHz
3 2,5
3
S23
%
ppm
8
7,5
7
50
7,470 7,449 7,338 7,316 7,231 7,223 6,969 6,961 6,947 6,939
7,919 7,902 7,898
ppm
6,5
6
5,5
5
4,5
4
3,5
2,50
2,01
3,207 3,194 3,181
4
2,741 2,727 2,714 2,545 2,534 2,534 2,509 2,504 2,500 2,495 2,491
3,426
4,5
2,60
5
3,731
5,5
4,165 4,152 4,139
6
38c (1D 1H) DMSO 400MHz
%
2,11
2,18
0,99
1,01
1,01 2,01
0
3,5
3
2,00
2,01
1,89
2,03
2,08
3,13
M AN U
% 50 100150
TE D
40
6,5
2,57
2,00
EP
30
7
0,99
0,99 0,91
20
AC C
10 ppm
1,97
2,00
0
4
SC
2
8
10
12
14
RI PT
6
16
18
3,798 3,696 3,568 3,318 3,314 3,310 3,306 3,302 3,137 3,129 3,117 3,104 2,874 2,859 2,845 2,729 2,724 2,715 2,703 2,701
4,120 4,107 4,094
4,888
ppm 7,393 7,371 7,314 7,293 7,216 7,208 7,074 7,066 7,052 7,044 6,924 6,920 6,903 6,895
% 50 100150
ACCEPTED MANUSCRIPT
38b (1D 1H) MeOD 400MHz
3 2,5
2,5
S24
AC C EP TE D
ppm 7,5 7 6,5 6 5,5
2,09
0,99
2,21
1,06
2,13
0,95
0
5
4,5
4
3,5 3
2,00
3,30
2,03
2,01
2,63
M AN U
%
SC
5
15
20
RI PT
10
25
30 % 50 100150
3,684 3,585 3,318 3,314 3,310 3,306 3,302 3,137 3,124 3,111 2,886 2,879 2,867 2,850 2,733 2,720
4,124 4,111 4,098
4,892
ppm 7,398 7,376 7,218 7,211 7,185 7,164 7,145 7,079 7,071 7,057 7,049 6,859 6,842 6,839 6,735 6,731 6,717 6,713 6,710
ACCEPTED MANUSCRIPT
38d (1D 1H) MeOD 400MHz
2,5
S25
ppm
2,09
0
8
7,5
7
4,220 4,208
6,5
6
5,5
5
4,5
4
3
3,5
3
2,00
3,5
3,260 3,248 3,235 2,798 2,784 2,770 2,620 2,608 2,509 2,505 2,500 2,496 2,492
4
3,530
4,5
2,24
5
2,00
5,5
2,14
6
38f (1D 1H) DMSO 400MHz
%
2,01
0,95
0,96
0,94
2,00
2,04
0
2,05
2,11
2,20
2,17
2,73
M AN U
% 50 100150
6,5
3,705
TE D
7,473 7,451 7,384 7,363 7,242 7,234 7,113 7,106 7,091 7,084
7,956 7,935
ppm
100
7
2,26
2,03
0,99
80
7,5
0,98
60 ppm
1,00 1,95
40
EP
20
AC C
%
SC
20
60
RI PT
40
80
2,885 2,870 2,856 2,703 2,693 2,689 2,685 2,674 2,586 2,568 2,551 2,533
3,869 3,659 3,655 3,650 3,577 3,563 3,548
4,262 4,247 4,233
ppm 7,771 7,766 7,755 7,750 7,654 7,649 7,633 7,412 7,390 7,274 7,267 7,108 7,100 7,086 7,078
% 50 100150
ACCEPTED MANUSCRIPT
38e (1D 1H) Acetone 400MHz
2,5
2,5
S26
%
ppm
11
10
9
8
7
6
5
4
3
10,02 9,00
3,5
1,487 1,480 1,468
4
2,26 2,01
4,5
2,933 2,881 2,867 2,856 2,764 2,751
5
3,06 2,09 1,93 1,94
4,118 4,104 4,086 3,899 3,843 3,829 3,789 3,617
5,5
40c (1D 1H) CDCl3 400MHz
%
2,01
1,03
0,88
2,03
2,04
0
2,00
2,00
1,92
1,94
3,19 2,12
M AN U
% 50 100150
6
2,78
8,016 7,995 7,523 7,515 7,452 7,431 7,385 7,363 7,240 7,102 7,094 7,080 7,072
8,730 8,716 8,704
9,769
ppm 11,452
6,5
0,96
60
TE D
50
7
0,97 2,05 1,03
40
EP
30
7,5
2,20
20
8
0,94
10
AC C
0 ppm
0,89
0,97
SC
20
60
80
RI PT
40
3,620 3,344 3,331 3,318 3,314 3,310 3,306 3,302 2,888 2,873 2,860 2,740 2,726 2,713
3,907 3,829
4,257 4,245 4,232
4,903
7,535 7,515 7,468 7,446 7,261 7,254 7,169 7,161 7,147
ppm 8,021 8,001
100
% 50 100150
ACCEPTED MANUSCRIPT
39c (1D 1H) MeOD 400MHz
3 2,5
2
S27
ppm
2,00
0
10
8
7,5
50 % 50 100150
7 6
6,5 5,5
42c (1D 1H) MeOD 400MHz
%
2,00
1,06 1,03
2,27 3,25 1,13
0
6
5,5
4,078 4,065 4,053 3,832
3,534 3,522 3,511
1,65
3,00
2,07
1,55
4,5
3,5
3
4,5
4
4
3,208 3,204 3,200 3,196 3,192 2,992
4,344
5
1,58
5
2,13
1,34
1,33
1,11 1,12
2,02
M AN U
4,780
7,364 7,342 7,112 7,105 7,058 7,051 7,036 7,029
7,646 7,626
ppm 8,061 8,041
6,5
4,538
TE D
40
7
1,92
EP
30
7,5
0,73 0,83
20 ppm
0,91
1,99
AC C
%
SC
5
15
RI PT
10
20
3,090
3,318 3,314 3,310 3,306 3,302
3,793 3,782 3,771 3,645 3,634 3,622
4,437 4,260 4,249 4,238 4,189 4,177 4,165
4,567
4,887
ppm 7,622 7,615 7,606 7,598 7,558 7,548 7,548 7,542 7,534 7,481 7,476 7,459 7,454 7,235 7,226 7,218 7,177 7,170 7,163 7,155 7,148 7,141
% 50 100150
ACCEPTED MANUSCRIPT
42a (1D 1H) MeOD 400MHz
3,5 3
S28
ppm
2,05
0
5
10
8
7,5
7
6,5
6
5,5
5
4,5
4,5
3,738 3,665 3,652 3,640 3,338 3,334 3,330 3,326 3,321 3,131 3,117 3,103
4,208 4,196 4,183
5
4,447
4,660
5,5
4
4
1,91
42e (1D 1H) MeOD 400MHz
%
2,09 1,64
3,06
2,12
1,07
1,22
0
2,00
2,64
2,01
M AN U
4,907
7,496 7,474 7,243 7,236 7,189 7,181 7,167 7,159
7,912 7,841 7,820
% 50 100150
6
1,83 1,93
40 ppm
6,5
2,05
35
TE D
30
7
2,00
EP
25
7,5
2,02
0,98 1,00
20
8
1,00
15 ppm
2,00
AC C
%
SC
10
30
40
RI PT
20
50
60
3,5
3,089
3,326
3,641
4,184 4,116 4,098
4,476 4,435 4,431
4,912
7,483 7,461 7,348 7,225 7,174 7,153 7,041 7,018 6,963 6,943
ppm
% 50 100150
ACCEPTED MANUSCRIPT
42d (1D 1H) MeOD 400MHz
3,5 3
3
S29
AC C EP TE D
ppm 8 7,5 7 6,5 6 5,5
2,01
2,00
0,99 3,02
1,00
0
5
4,5
4
3,5 3 2,5
0,99
1,01
2,85
2,06
2,06
M AN U
%
2
SC
1
4
5
6
RI PT
3
7
8
7,493 7,471 7,241 7,234 7,168 7,161 7,151 7,146 7,139 7,135 7,130 6,774 6,753 4,904 4,209 4,197 4,184 3,662 3,650 3,638 3,336 3,332 3,328 3,324 3,319 3,060 3,018 3,016 3,011 3,002 2,986 2,985 2,970 2,938 2,925 2,813 2,783 2,777 2,776 2,771 2,764 2,755 2,749 2,742 2,739 2,733 2,731 2,727 2,725 2,724 2,720 2,719 2,165 2,164 2,163 2,160 2,158 2,155 2,151 2,145 2,131 2,127 2,124 2,000 1,999 1,987 1,985 1,973 1,970 1,965 1,958
ppm
% 50 100150
ACCEPTED MANUSCRIPT
43 (1D 1H) MeOD 400MHz
2
S30
%
0
2
6
AC C
4
10
EP
8
ppm
160
150
27b (1D 13C) DMSO 400MHz
140
TE D
130
120
110
100 90
90 80
80 70
70 60
60 50
50
40
40
30
28,023 23,554 23,404 22,577 21,687
100
40,107 39,899 39,692 39,480 39,272 39,065 38,857 37,749
%
0
1
2
M AN U
110
65,512
120
94,503
130
117,895 115,205 113,973
140
121,431
150
131,306 130,544 128,200
160
135,403
% 50 100150 ppm
145,592
156,940 156,871
164,096
ppm
SC
3
5
40,111 39,903 39,692 39,484 39,276 39,068 38,857 38,591 36,564 27,312 24,104 23,783 23,280 22,416
64,968
90,675
122,109 118,478 115,752 115,726 115,485 115,008
130,169 128,521
141,765
156,459
162,434
167,836
ppm
% 50 100150
RI PT
4
ACCEPTED MANUSCRIPT
13
C NMR spectra for compounds 23b, 27b, 33, 35, 36, 37a-c, 37e-f, 38a, 38c, 38e-f, 39c, 40c,
42a-c, 42e,43
23b (1D 13C) DMSO 400MHz
30 20
20
S31
ppm
0
2
4
AC C
%
8
10
EP
6
14
16
18
TE D
12 ppm
170 150
160 140
150 130
140 120
130
120 110 100
35 (1D 13C) Pyr 400MHz
%
110
100
90 70
80
60
70
60
50 40
50
40
26,766
80
34,279 32,067
90
42,197 41,931
69,315
M AN U
97,500
160
152,122 151,852 151,582 149,537 138,397 138,270 137,785 137,537 137,293 133,684 133,035 130,731 130,443 125,762 125,515 125,267 120,298 118,384 117,969 116,981
159,580 159,413
% 50 100150 ppm
167,049
20
10
SC
0
30
RI PT
20
40
30
24,143
31,448 28,390 28,047
36,559
39,180
66,265
80,646 77,379 77,062 76,745
94,181
122,993 119,953 117,179 116,949 114,321 113,647
131,983 131,640
147,038
ppm 162,622 161,820 157,759 157,383 157,212 154,536
% 50 100150
ACCEPTED MANUSCRIPT
33 (1D 13C) CDCl3 400MHz
30 20
20
S32
ppm
160
150
140
130
120
110
100
90
80
70
5
10
AC C
0
EP
15
25
62,039
67,183
95,266
139,528 135,970 131,851 131,144 129,773 129,211 129,047 129,011 128,049 127,553 123,302 119,278 119,161 116,376 114,080
147,260
158,294
164,506
50
40
30,496 30,303 30,110 29,920 29,727 29,534 29,340 24,981
37a (1D 13C) Acetone 400MHz
M AN U
30 % 50 100150 ppm
TE D
20
40,039
60
51,409 50,148
%
SC
RI PT
ACCEPTED MANUSCRIPT
30
20
S33
ppm
0
2
AC C
%
6
EP
4
10
12
14
TE D
8
160
150
160 140
140 120
130
120 100
37c (1D 13C) Acetone 400MHz
110
%
100
90
80
70
60
50
40
30,496 30,303 30,110 29,920 29,727 29,534 29,340 24,926
60
40,043
80
52,342 51,453 50,228
61,503
67,186
95,244
M AN U
135,955 131,854 131,107 130,331 130,112 129,744 127,374 123,302 119,282 118,545 116,387 116,384 115,687 114,058
% 50 100150 ppm
147,329 145,266
158,294
ppm 167,189 164,517
2
4
SC
0
8
10
12
14
RI PT
6
16 ppm
30,496 30,303 30,110 29,920 29,727 29,534 29,340 24,992
40,036
51,285 50,020
55,532
61,470
67,179
95,259
135,959 131,851 131,238 131,235 131,162 131,158 130,972 127,622 127,618 123,302 119,282 118,549 116,376 115,695 114,535 114,087
147,212
159,985 158,287
164,480
18
% 50 100150
ACCEPTED MANUSCRIPT
37b (1D 13C) Acetone 400MHz
40 20
30
20
S34
AC C EP TE D
ppm 160 140 120 100
M AN U
%
5
15
20
25
RI PT
10
SC
0
30
80
60
40
0,211
24,303
39,370
51,082 49,704
61,253
66,524
77,547 77,230 76,913
94,410
132,566 132,387 131,596 130,138 129,570 126,613 123,267 120,424 119,038 117,329 117,241 114,000 111,514
143,907
146,903
161,638 157,821 157,413
ppm
35
% 50 100150
ACCEPTED MANUSCRIPT
37e (1D 13C) CDCl3 400MHz
20 0
S35
ppm
0
2
AC C
%
6
EP
4
160
140
10 ppm
120
100
80
60
40
23,999
51,164 49,560 40,133 39,926 39,718 39,510 39,299 39,091 38,883 38,664 38,562
60,503
65,385
90,607
% 50 100150
38c (1D 13C) DMSO 400MHz
M AN U
130,417 130,363 129,331 128,657 128,653 127,585 122,638 120,954 117,659 115,993 115,599
145,454 143,460 140,697
156,179
167,508 167,312
TE D
8
SC
RI PT
ACCEPTED MANUSCRIPT
20
S36
ppm
0
2
AC C
%
6
EP
4
10
12
TE D
8
14 % 50 100150
170
160
150
140 120
130 110
120 100
38f (1D 13C) DMSO 400MHz
110
%
100
90
80
60
70
50
60
40
50
30
40
30
23,780
70
50,417 48,999 40,133 39,926 39,718 39,510 39,299 39,091 38,883 38,358
80
60,474
90
65,144
M AN U
93,220
130
117,655 115,606
140
130,610 130,370 129,535 128,985 125,901 122,244
150
137,329 137,325
156,339
160
159,820
ppm
164,884
ppm
2
SC
0
6
8
10
RI PT
4
12
14 % 50 100150
11,867
30,470 30,276 30,087 29,894 29,700 29,507 29,318 24,845
51,448 51,168 50,198 46,986
61,243
69,992
95,054
136,020 133,053 131,686 131,682 130,946 130,465 126,940 122,736 119,470 118,996 116,405 116,401 114,236 111,698
145,636
158,803
ppm 164,745
ACCEPTED MANUSCRIPT
38e (1D 13C) Acetone 400MHz
20 10
20
S37
ppm
0
5
AC C
%
15
20
EP
10
30
35
40
160
150
140
140
130
120
120
40c (1D 13C) CDCl3 400MHz
%
0
110
100
90
80
70
60
50
40
28,579 28,492 28,372 28,270 28,189 24,231
60
40,146
52,351 51,013 49,690
80
61,435
100
67,151
83,595 79,866 77,879 77,551 77,438 77,230 76,913
94,235
M AN U
114,044
160
117,376
132,161 131,476 130,073 129,668 129,154 126,515 122,727 120,722
143,134
% 50 100150
180
147,067
153,279
157,905 156,603
163,471 161,867
167,109
ppm
ppm
TE D
25
SC
2
6
8
RI PT
4
10
25,204
62,197 52,773 51,902 50,703 49,792 49,577 49,365 49,150 48,939 48,727 48,512 40,434
66,720
96,164
136,806 132,446 132,442 131,651 130,853 130,714 130,638 127,700 124,736 119,560 116,738 116,734 114,744
144,694
148,828
158,426
168,524 166,544
178,993
ppm
% 50 100150
ACCEPTED MANUSCRIPT
39c (1D 13C) MeOD 400MHz
40 20
30
S38
ppm
0
1
AC C
%
3
EP
2
5
6
TE D
4 ppm
160 140
140 130 120
120 110
42b (1D 13C) MeOD 400MHz
%
100 80
80
70
60
60 50 40
40
22,871
90
28,270
67,719 60,574 56,076 50,645 50,422 50,368 50,265 50,145 49,788 49,577 49,365 49,150 48,939 48,723 48,512 42,370
100
86,176
95,263
M AN U
124,594 121,911 120,992 119,888 116,756 116,658 115,976 113,796
150
136,069 134,086 132,526 130,598
160
150,312
158,597
% 50 100150 ppm
162,917
166,530
7
2
SC
0
6
RI PT
4
8
10
22,853
68,211 60,946 50,710 50,670 50,553 50,382 50,313 50,240 50,204 50,167 50,094 50,021 49,792 49,577 49,365 49,150 48,939 48,727 48,512 42,169 28,273
86,176
95,267
136,044 132,581 132,526 131,691 130,736 130,565 130,299 124,586 120,897 119,891 119,851 116,760 116,658 113,793
150,341
158,754
166,581
ppm
% 50 100150
ACCEPTED MANUSCRIPT
42a (1D 13C) MeOD 400MHz
30 20
20
S39
ppm
0
1
AC C
%
3
EP
2
5
6
160
150
% 50 100150
160 140
140
120
130
120
100
42e (1D 13C) MeOD 400MHz
110
%
0
100
90
80
70
60
40
50
40
30
22,922
60
28,262
50,761 49,792 49,577 49,365 49,150 48,939 48,727 48,512 42,155
80
60,028
68,200
95,267
M AN U
136,040 135,741 134,564 134,378 133,718 133,532 132,508 130,550 124,590 124,415 120,956 119,848 119,129 116,749 116,647 115,437 113,804
150,333
159,217 159,177 158,761
166,592
ppm
ppm
TE D
4
SC
1
3
4
5
RI PT
2
6
22,856
60,217 53,091 50,754 50,736 49,788 49,573 49,361 49,150 48,935 48,723 48,508 42,158
68,204
95,238
136,029 135,187 133,353 132,854 132,497 131,575 130,528 124,404 120,799 119,851 116,651 113,789
150,381
159,170 158,758
167,762 166,577
ppm
% 50 100150
ACCEPTED MANUSCRIPT
42c (1D 13C) MeOD 400MHz
20
20
S40
AC C EP TE D
ppm 160 140 120
0
M AN U
%
SC
5
15
20
RI PT
10
25
30 % 50 100150
100
80
60
40
25,595
33,067 31,066
49,716 49,501 49,289 49,078 48,863 48,651 48,436 42,086 41,444
68,110
96,573
119,637 116,531 116,349 114,898
124,303
137,663 136,343 132,778 132,363 130,555 128,885
147,363
159,054 158,609 157,001
166,158
ppm
ACCEPTED MANUSCRIPT
43 (1D 13C) MeOD 400MHz
20
S41
ACCEPTED MANUSCRIPT
SC
Structure
M AN U
Compound
RI PT
Table S1: ClogP values of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a, 27b, 33-38f, 39c, 42a-e and 43.
I
TE D
II
2.83
3.40
2.42
AC C
EP
4
ClogP
5.87
15
5.48
16
3.16
14
S42 17
5.07
ACCEPTED MANUSCRIPT
4.50
RI PT
18
1.80
19
2.59
M AN U
SC
22a
22b
TE D
23a
AC C
26a
EP
23b
2.99
2.50
2.90
1.96
27a
1.88
27b
2.23
S43
ACCEPTED MANUSCRIPT
3.52
RI PT
33
4.26
SC
33b
M AN U
34
35
EP
AC C
37a
TE D
36
2.04
3.24
0.38
3.82
37b
3.74
37c
3.79
S44
ACCEPTED MANUSCRIPT
3.15
RI PT
37d
3,25
SC
37e
M AN U
37f
38a
TE D
38b
2.82
2.74
0.29
AC C
EP
38c
3.31
38d
2.15
38e
2.25
S45
ACCEPTED MANUSCRIPT
2.31
RI PT
38f
2.79
39c
M AN U
SC
42a
42b
TE D
42c
AC C
42e
EP
42d
43
2.19
2.11
2.16
1.53
1.63
2.62
S46
