Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis of mycothiol conjugate analogues and evaluation of their antimycobacterial activity Scott W. Riordan, Jessica J. Field, Hilary M. Corkran, Nathaniel Dasyam, Bridget L. Stocker, Mattie S. M. Timmer, Joanne E. Harvey, Paul H. Teesdale-Spittle ⇑ Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand
a r t i c l e
i n f o
Article history: Received 30 January 2015 Revised 2 March 2015 Accepted 24 March 2015 Available online xxxx Keywords: Mycothiol Trichlorovinyl cysteine Antibacterial Antimycobacterial Prodrug
a b s t r a c t Drug-resistant Mycobacterium tuberculosis is a growing health problem. As proof of principle that the bacterial-specific metabolite mycothiol could be used as a delivery agent for antimycobacterial agents, simplified analogues of mycothiol were synthesised containing an S-trichloroethenyl substituted cysteine residue. It was envisaged that uptake of the mycothiol analogue would be followed by release of the known cytotoxin S-trichloroethenyl cysteine by the action of mycothiol S-conjugate amidase or its paralog, mycothiol deacetylase MshB. Promising activity was displayed against model Mycobacteria, although further development will be required to improve selectivity. Ó 2015 Elsevier Ltd. All rights reserved.
Mycobacterial diseases, including tuberculosis, are serious and resurgent global health issues. With the growing prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR) and the recent advent of totally drug resistant (TDR) mycobacteria, there is an imperative to find new antimycobacterial agents.1 There are around 2 billion people infected with Mycobacterium tuberculosis (MTB), leading to up to 2 million deaths annually.2 Of new cases of MTB infection, estimates place approximately 17% as drug resistant to some extent and 3.3% as MDR or worse.2 To overcome the growing trend towards drug resistance in MTB infection, new therapeutics are required. In addition to targeting essential survival pathways in MTB, it is desirable that new agents act through previously unexploited modes of action,2 ideally harnessing metabolic features that are distinct between MTB and a human host. Mycothiol (1, Fig. 1) is a low molecular weight thiol that is unique to actinobacteria, including mycobacteria.3 Consisting of a pseudo-disaccharide motif with pendant cysteine, mycothiol plays an equivalent role in mycobacteria to that of glutathione in eukaryotes. Its roles extend to protection of the bacterium from oxidative stress, antibiotics and electrophiles.4 Following formation of a conjugate (2) with an electrophile, the mycothiol-metabolising enzyme mycothiol S-conjugate amidase (MCA), or the paralogous mycothiol deacetylase MshB, cleave the cysteinyl residue from ⇑ Corresponding author. Tel.: +64 4 463 6094; fax: +64 4 463 5339. E-mail address: [email protected] (P.H. Teesdale-Spittle).
the conjugated mycothiol 2, liberating a mercapturic acid (3) as the final intracellular step in detoxification of electrophiles within the bacterium.3 The N-acetyl group prevents aberrant incorporation into amino acid metabolism. Highly simplified mycothiol variants such as that prepared by Knapp et al. in which the inositol moiety has been replaced with a cyclohexane thiol (4, Fig. 1) have also been shown to be substrates for MCA.5,6 We envisaged that amidase-catalysed cleavage of a substrate lacking an N-acetyl group could be used to liberate an S-chloroalkenyl cysteine. These are known cellular toxins. Their toxicity arises because of aberrant metabolism by pyridoxal phosphate-dependent enzymes which catalyse b-lysis of the cysteine conjugate, liberating powerful electrophiles, including thioacyl halides and thioketenes along with pyruvate and ammonia (Fig. 2).7 This b-lyase enzymatic activity is exhibited by a number of essential amino acid metabolising enzymes that are conserved across species. Conjugation of an S-haloalkenyl cysteine to an MCA substrate could therefore result in selective toxicity towards mycobacteria. As a proof-of-principle, we assessed the ability of Knapp’s simplified MCA substrate to carry a masked form (5) of S-tetrachloroethenyl cysteine (6) (Fig. 3). Release of the electrophile requires two processes that were proposed to be mycobacterial-selective: (1) uptake of a mycothiol analogue and (2) processing by a mycothiol amidase, such as MCA or MshB. In contrast, the b-anomer (7) was not expected to be an amidase substrate, and was synthesized as a control. The N-Boc protected
http://dx.doi.org/10.1016/j.bmcl.2015.03.070 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Riordan, S. W.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.070
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S. W. Riordan et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
OH HO O H O
HO HO O
OH
OH
NH
OH OH
H
OH E-X
HO O
(1)
H
OH
OH
OH HO
S E
HN O
OH
NH
SH
HN
OH
HO O H O
HO
O
O
HO O
(2)
NH
MCA or MshB OH
OH
HO
O (3)
(4)
OH
HO O H O
S E HO
HN
O
OH
H
SH
HN O
H S
OH
H
NH2
OH
Figure 1. Reaction of MSH (1) with an electrophile gives a conjugate (2). Amidases MCA or MshB cleave the conjugate giving a mercapturic acid (3). Knapp0 s analogue (4).
OH HO
O
HO O
MCA or MshB
H R
NH
OH HO
O
HO
Cl
H
X
S
H2 N
X Cl
R NH 2
S
H 2N
OH
O
X
β-lyase
X
OH
O X
X .
S
S X
X HS
X Cl
O
X Cl
NH3
Figure 2. The proposed pathway for generation of reactive electrophiles from a chloroethenyl amidase substrate (X = Cl or H; R = O-inositol or S-cyclohexane).
OH HO HO O H 2N
O
OH
H S
NH
Cl
S (5)
MCA or MshB
OH HO
O
HO Cl
H S
NH 2
HO O
OH
Cl
S
H 2N (6)
Cl
HO O
Cl H 2N
O
H S
NH
Cl
S
Cl
Cl Cl
(7)
Figure 3. Synthetic targets (5) and (7) and proposed route to production of toxic cysteine conjugate (6) in mycobacteria.
precursors of 5 and 7 were also tested. The target compounds lack an N-acetyl group, as amidase cleavage would then liberate a mercapturic acid. Mercapturic acids would be readily excreted from the bacteria. Additionally, the N-acetyl functionality would prevent them undergoing b-lysis, which requires a free amino group on the cysteine conjugate in order to bind to the pyridoxal phosphate cofactor a b-lyase enzyme. Although commercially available, N-Boc-L-cysteine was conveniently prepared through use of di-tert-butyl dicarbonate and a 4-fold excess of cysteine in sodium bicarbonate, providing the product in modest yield (56%) but free from any cystine-containing contaminant and of sufficient purity to obviate the need for further purification. Treatment with tetrachloroethene in the presence of 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) afforded S-trichloroethenyl conjugate 8 (Scheme 1),8 isolated as a pair of rotamers which collapsed to a single S-trichloroethenyl-L-cysteine (6) product on removal of the Boc group. In Knapp’s preparation of compound 4 (Fig. 1), an a-thioglycoside, from b-D-glucosamine pentaacetate, an intermediate
thiazoline set the stereochemistry at the anomeric centre. Opening of the thiazoline with trifluoroacetic acid afforded the thiol group at the anomeric centre, followed by a radical-mediated addition reaction with cyclohexene to generate the a-thioglycoside.5 In our hands the yields from this reaction proved variable, even after canvassing a wide variety of reaction conditions. Prompted, in part, by the work of Xu et al.,9 production of a thioglycoside directly from a-glucosamine pentaacetate was attempted. Xu reports only production of a b-thioglycoside from the Lewis acid-catalysed reaction between b-D-glucosamine pentaacetate and thiophenol, however we proceeded due to our interest in bthioglycoside 10 (Scheme 1) as a precursor to control compound 7 and precedence for anomerisation leading to production of a-glycosides in other reactions of acylated glycosides.10–12 Gratifyingly, treatment of a-D-glucosamine pentaacetate with cyclohexanethiol in the presence of 2 equiv of BF3.OEt2 in dichloroethane at 50 °C produced both a- and b-anomers in a 2:1 ratio. Although the modest combined yield (33%) leaves room for optimisation, we were able to retrieve sufficient material of both desired anomers in a
Please cite this article in press as: Riordan, S. W.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.070
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S. W. Riordan et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
OAc AcO
O O
AcO
H OAc
HN
OH
O
a
SH
Cl
S
HN
Boc
NHAc
OH
Boc
(8)
Cl Cl
OH
b HO OAc AcO
O
H
OAc c
S
AcO
AcO
O
HO O
d
H S
AcO
NHAc
N(Boc)Ac
(9)
(11)
O
H S
NH
Cl
S
RHN
Cl Cl
R = Boc, (Boc- 5)
e
R = H.CF3 CO2H, (5) OAc AcO
O
AcO
H S
OH
OAc c
AcO
O
AcO
NHAc
N(Boc)Ac
( 10 )
( 12)
HO
d
H S
HO O
O
H S
NH
Cl
S
RHN R = Boc, (Boc-7) R = H.CF3CO2 H, (7 )
Cl Cl e
Scheme 1. Reagents and conditions: (a) C2Cl4, DBN, CH3CN (69%); (b) cyclohexanethiol (4 equiv), BF3OEt2 (2 equiv), 1,2-dichloroethane, 0–55 °C, 3 h (9 22%, 10 11%); (c) ditert-butyl dicarbonate (5.4 equiv), 4-(N,N-dimethylamino)pyridine (0.13 equiv), THF, reflux 18 h (11 quant, 12 82%); (d) (i) Na (1 equiv), MeOH, rt, 2 h (ii) HCl, MeOH/water, rt, 18 h, (iii) KOtBu (1 equiv), EDCI (2.5 equiv), 8 (1.5 equiv), DMF, 0 °C–rt, 2 h (Boc-5 26%, Boc-7 35% over 3 steps); (e) TFA, 0 °C–rt, 25 min (5 35%, 7 quant). Synthetic methodology is provided in the Supplementary data.
single step from commercially available starting materials. The anomers were readily separated chromatographically (silica, petroleum ether/ethyl acetate, 1:2) and carried separately through subsequent steps. Although deacetylation can be achieved in a single step by hydrazinolysis at 120 °C,5 we preferred the more mild conditions offered by formation of the N-Boc derivative followed by sequential treatment with sodium methoxide and methanolic HCl at room temperature in a one-pot, two-step global deprotection reaction. Interestingly, the b-anomer of the Boc-protected intermediate could also be recovered as an approximately 1:1 ratio of two rotamers, although these resolved to a single hydrochloride salt upon deprotection. The salts were carried directly into the next step without purification. Coupling of the thioglycoside salts derived from 11 and 12 with 8, mediated by 1-(3-N,N-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), gave protected mycothiol analogues Boc-5 and Boc-7. Removal of the Boc group was achieved using anhydrous trifluoroacetic acid (TFA). Although the TFA salt of the a-anomer proved stable, that of the b-anomer was prone to degradation and was prepared freshly on each occasion for biological testing. The a-anomer was obtained in only modest yield after stringent removal of persistent impurities by chloroform trituration ahead of biological evaluation. Our original hypothesis was that the trichloroethenylcysteinecontaining mycothiol analogue 5 would be selectively taken up into mycobacteria and processed by amidases MCA or MshB to liberate 6, with subsequent b-lyase activity generating reactive electrophiles, such as a halothioketene (Fig. 2). The uptake and activity of the b-anomers of simplified mycothiol analogues, such as 7, have not been previously tested. To our surprise, both anomers were essentially equally active in the model mycobacteria, although the a-anomer was the slightly more active form in the Mycobacterium bovis BCG strain (Table 1), being only 2- to 3-fold
less active than ethambutol which had an MIC of 39 lM in this assay. We propose that either MCA or MshB fail to exhibit the expected substrate selectivity or that an alternative, low-specificity amidase is releasing 6 prior to b-lysis. It is also possible that these compounds induce growth inhibition independently of release of 6, perhaps through inhibition of the mycothiol pathway. As a soil bacterium, Mycobacterium smegmatis has a higher resistance to external stresses than is found in M. bovis BCG or MTB.14 For example, M. smegmatis has a higher relative abundance of mycothiol compared to its oxidized form, mycothione. This renders it more tolerant of stress from oxidants and electrophiles.14 It is therefore no surprise that higher concentrations of 5 and 7 are required to achieve growth inhibition for M. smegmatis than for M. bovis BCG. The promising activity observed for 5 and 7, particularly in the BCG assay, indicates that these compounds are taken up by the bacteria. In contrast their Boc-protected counterparts were inactive, presumably through a failure to undergo catabolism or their inability to inhibit mycothiol-related enzymes. Molecular modelling was undertaken to rationalise the similar activities of 5 and 7. Although there is no crystal structure available for MCA, structures of the paralogous MshB have been determined, including one in which active site acetate and glycerol ligands Table 1 Growth inhibition of model mycobacteria M. smegmatis, M. bovis BCG strain (as minimum inhibitory concentrations, MIC), HL60 and 1A9 cell lines (MTT assay, as half-maximal inhibitory concentrations, IC50) Compound 5 7 Boc-5 Boc-7 a
M. smegmatis (MIC) lM
lM
HL60 (IC50) lM
250–500 250–500 >1000 >1000
62.5–125 125–250 500–1000 P1000
76 ± 1 112 ± 18 60 ± 10 16.5 ± 0.2
BCG (MIC)
a
1A9 (IC50) lM 79 ± 23 121 ± 16 103 ± 4 33 ± 5
IC50 values presented as average ± standard error of the mean.
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a
4
S. W. Riordan et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
analogues could act as selective prodrugs for b-lyase substrates, such as 6. The hypothesis was that both uptake of the prodrug and its catabolic degradation to 6 would occur selectively in mycobacteria. We have shown that highly simplified mycothiol analogues 5 and 7 are taken up by mycobacteria and result in growth inhibition. Further work will be needed to enhance selectivity for mycobacteria over human cells. However, the work presented here successfully demonstrates that compounds of this type inhibit growth of mycobacteria, and so make interesting leads in the search for new antimycobacterial agents. Acknowledgments
Figure 4. Mycothiol analogues 5 (white carbons) and 7 (all grey) are equally accommodated by MshB. The overlaid ligands, discussed binding site residues and protein ribbons are displayed, showing that MshB is able to accommodate both ligands without significant perturbation of the protein structure. MshB coordinates were obtained from the crystal structure of Broadley et al., 4EWL.pdb.13 Modelling methodology and further figures are provided in the Supplementary data.
The authors thank Prof. Greg Cook (University of Otago) for additional testing of compounds against M. bovis BCG. At Victoria University of Wellington, the authors wish to thank Ian Vorster and Jingjing Wang for help with NMR and MS instrumentation, Prof. John Miller for supervision of J.F., Dr. Ronan O’Toole and Dr. Rob Keyzers for supervision of N. D. Supplementary data
define the positioning of the substrate binding site.13 Both 5 and 7 were modelled into the MshB binding site. As shown in Figure 4, both anomers were easily accommodated within the active site cleft. The trichloroethenyl moiety is positioned alongside Tyr142 and the cyclohexyl group in the pocket opening defined by the side chains of Met98, Glu47 and Asn 261. Compounds 5, 7 and their Boc-protected progenitors were tested against HL-60 (human promyelocytic leukemia) and 1A9 (human ovarian carcinoma) cells. Disappointingly, lead compounds 5 and 7 were approximately equipotent towards the human cell lines tested as they were in the BCG assay. The two Boc derivatives were substantially more cytotoxic to the HL-60 and 1A9 cells than they were to mycobacteria. The higher activity of the Boc derivatives, as compared to the target amines, suggests that these compounds act independently of b-lysis. This interpretation is supported by the higher activity of Boc-7 over its alpha-counterpart Boc-5, the reverse of the order of activity for 5 and 7. Given this, Boc-7 may provide a useful lead for a new structural class of anticancer agents. In summary, as part of a search for antibacterial compounds that would be unlikely to be affected by common mechanisms of drug resistance in mycobacteria, we proposed that mycothiol
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 070. References and notes 1. McGrath, M.; Gey van Pittius, N. C.; van Helden, P. D.; Warren, R. M.; Warner, D. F. J. Antimicrob. Chemother. 2014, 69, 292. 2. Field, S. K.; Fisher, D.; Jarand, J. M.; Cowie, R. L. Ther. Adv. Respir. Dis. 2012, 6, 255. 3. Newton, G. L.; Buchmeier, N.; Fahey, R. C. Microbiol. Mol. Biol. Rev. 2008, 72, 471. 4. Fan, F.; Vetting, M. W.; Frantom, P. A.; Blanchard, J. S. Curr. Opin. Chem. Biol. 2009, 13, 451. 5. Knapp, S.; Gonzalez, S.; Myers, D. S.; Eckman, L. L.; Bewley, C. A. Org. Lett. 2002, 4, 4337. 6. Metaferia, B. B.; Fetterolf, B. J.; Shazad-ul-Hussan, S.; Moravec, M.; Smith, J. A.; Ray, S.; Gutierrez-Lugo, M.-T.; Bewley, C. A. J. Med. Chem. 2007, 50, 6326. 7. Anders, M. W. Chem. Res. Toxicol. 2008, 21, 145. 8. Bartels, M. J.; Miner, V. W. J. Labelled Compd. Radiopharm. 1990, 28, 209. 9. Xu, C.; Liu, H.; Li, X. Carbohydr. Res. 2011, 346, 1149. 10. Xue, J. L.; Cecioni, S.; He, L.; Vidal, S.; Praly, J.-P. Carbohydr. Res. 2009, 344, 1646. 11. Pilgrim, W.; Murphy, P. V. J. Org. Chem. 2010, 75, 6747. 12. Malik, S.; Shah, K. J.; Ravindranathan, K. K. P. Carbohydr. Res. 2010, 345, 867. 13. Broadley, S. G.; Gumbart, J. C.; Weber, B. W.; Marakalala, M. J.; Steenkamp, D. J.; Sewell, B. T. Acta Crystallogr., Sect. D. 2012, 68, 1450. 14. Ung, K. S. E.; Av-Gay, Y. FEBS Lett. 2006, 580, 2712.
Please cite this article in press as: Riordan, S. W.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis of mycothiol conjugate analogues and evaluation of their antimycobacterial activity Scott W. Riordan, Jessica J. Field, Hilary M. Corkran, Nathaniel Dasyam, Bridget L. Stocker, Mattie S. M. Timmer, Joanne E. Harvey, Paul H. Teesdale-Spittle ⇑ Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand
a r t i c l e
i n f o
Article history: Received 30 January 2015 Revised 2 March 2015 Accepted 24 March 2015 Available online xxxx Keywords: Mycothiol Trichlorovinyl cysteine Antibacterial Antimycobacterial Prodrug
a b s t r a c t Drug-resistant Mycobacterium tuberculosis is a growing health problem. As proof of principle that the bacterial-specific metabolite mycothiol could be used as a delivery agent for antimycobacterial agents, simplified analogues of mycothiol were synthesised containing an S-trichloroethenyl substituted cysteine residue. It was envisaged that uptake of the mycothiol analogue would be followed by release of the known cytotoxin S-trichloroethenyl cysteine by the action of mycothiol S-conjugate amidase or its paralog, mycothiol deacetylase MshB. Promising activity was displayed against model Mycobacteria, although further development will be required to improve selectivity. Ó 2015 Elsevier Ltd. All rights reserved.
Mycobacterial diseases, including tuberculosis, are serious and resurgent global health issues. With the growing prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR) and the recent advent of totally drug resistant (TDR) mycobacteria, there is an imperative to find new antimycobacterial agents.1 There are around 2 billion people infected with Mycobacterium tuberculosis (MTB), leading to up to 2 million deaths annually.2 Of new cases of MTB infection, estimates place approximately 17% as drug resistant to some extent and 3.3% as MDR or worse.2 To overcome the growing trend towards drug resistance in MTB infection, new therapeutics are required. In addition to targeting essential survival pathways in MTB, it is desirable that new agents act through previously unexploited modes of action,2 ideally harnessing metabolic features that are distinct between MTB and a human host. Mycothiol (1, Fig. 1) is a low molecular weight thiol that is unique to actinobacteria, including mycobacteria.3 Consisting of a pseudo-disaccharide motif with pendant cysteine, mycothiol plays an equivalent role in mycobacteria to that of glutathione in eukaryotes. Its roles extend to protection of the bacterium from oxidative stress, antibiotics and electrophiles.4 Following formation of a conjugate (2) with an electrophile, the mycothiol-metabolising enzyme mycothiol S-conjugate amidase (MCA), or the paralogous mycothiol deacetylase MshB, cleave the cysteinyl residue from ⇑ Corresponding author. Tel.: +64 4 463 6094; fax: +64 4 463 5339. E-mail address: [email protected] (P.H. Teesdale-Spittle).
the conjugated mycothiol 2, liberating a mercapturic acid (3) as the final intracellular step in detoxification of electrophiles within the bacterium.3 The N-acetyl group prevents aberrant incorporation into amino acid metabolism. Highly simplified mycothiol variants such as that prepared by Knapp et al. in which the inositol moiety has been replaced with a cyclohexane thiol (4, Fig. 1) have also been shown to be substrates for MCA.5,6 We envisaged that amidase-catalysed cleavage of a substrate lacking an N-acetyl group could be used to liberate an S-chloroalkenyl cysteine. These are known cellular toxins. Their toxicity arises because of aberrant metabolism by pyridoxal phosphate-dependent enzymes which catalyse b-lysis of the cysteine conjugate, liberating powerful electrophiles, including thioacyl halides and thioketenes along with pyruvate and ammonia (Fig. 2).7 This b-lyase enzymatic activity is exhibited by a number of essential amino acid metabolising enzymes that are conserved across species. Conjugation of an S-haloalkenyl cysteine to an MCA substrate could therefore result in selective toxicity towards mycobacteria. As a proof-of-principle, we assessed the ability of Knapp’s simplified MCA substrate to carry a masked form (5) of S-tetrachloroethenyl cysteine (6) (Fig. 3). Release of the electrophile requires two processes that were proposed to be mycobacterial-selective: (1) uptake of a mycothiol analogue and (2) processing by a mycothiol amidase, such as MCA or MshB. In contrast, the b-anomer (7) was not expected to be an amidase substrate, and was synthesized as a control. The N-Boc protected
http://dx.doi.org/10.1016/j.bmcl.2015.03.070 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Riordan, S. W.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.070
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OH HO O H O
HO HO O
OH
OH
NH
OH OH
H
OH E-X
HO O
(1)
H
OH
OH
OH HO
S E
HN O
OH
NH
SH
HN
OH
HO O H O
HO
O
O
HO O
(2)
NH
MCA or MshB OH
OH
HO
O (3)
(4)
OH
HO O H O
S E HO
HN
O
OH
H
SH
HN O
H S
OH
H
NH2
OH
Figure 1. Reaction of MSH (1) with an electrophile gives a conjugate (2). Amidases MCA or MshB cleave the conjugate giving a mercapturic acid (3). Knapp0 s analogue (4).
OH HO
O
HO O
MCA or MshB
H R
NH
OH HO
O
HO
Cl
H
X
S
H2 N
X Cl
R NH 2
S
H 2N
OH
O
X
β-lyase
X
OH
O X
X .
S
S X
X HS
X Cl
O
X Cl
NH3
Figure 2. The proposed pathway for generation of reactive electrophiles from a chloroethenyl amidase substrate (X = Cl or H; R = O-inositol or S-cyclohexane).
OH HO HO O H 2N
O
OH
H S
NH
Cl
S (5)
MCA or MshB
OH HO
O
HO Cl
H S
NH 2
HO O
OH
Cl
S
H 2N (6)
Cl
HO O
Cl H 2N
O
H S
NH
Cl
S
Cl
Cl Cl
(7)
Figure 3. Synthetic targets (5) and (7) and proposed route to production of toxic cysteine conjugate (6) in mycobacteria.
precursors of 5 and 7 were also tested. The target compounds lack an N-acetyl group, as amidase cleavage would then liberate a mercapturic acid. Mercapturic acids would be readily excreted from the bacteria. Additionally, the N-acetyl functionality would prevent them undergoing b-lysis, which requires a free amino group on the cysteine conjugate in order to bind to the pyridoxal phosphate cofactor a b-lyase enzyme. Although commercially available, N-Boc-L-cysteine was conveniently prepared through use of di-tert-butyl dicarbonate and a 4-fold excess of cysteine in sodium bicarbonate, providing the product in modest yield (56%) but free from any cystine-containing contaminant and of sufficient purity to obviate the need for further purification. Treatment with tetrachloroethene in the presence of 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) afforded S-trichloroethenyl conjugate 8 (Scheme 1),8 isolated as a pair of rotamers which collapsed to a single S-trichloroethenyl-L-cysteine (6) product on removal of the Boc group. In Knapp’s preparation of compound 4 (Fig. 1), an a-thioglycoside, from b-D-glucosamine pentaacetate, an intermediate
thiazoline set the stereochemistry at the anomeric centre. Opening of the thiazoline with trifluoroacetic acid afforded the thiol group at the anomeric centre, followed by a radical-mediated addition reaction with cyclohexene to generate the a-thioglycoside.5 In our hands the yields from this reaction proved variable, even after canvassing a wide variety of reaction conditions. Prompted, in part, by the work of Xu et al.,9 production of a thioglycoside directly from a-glucosamine pentaacetate was attempted. Xu reports only production of a b-thioglycoside from the Lewis acid-catalysed reaction between b-D-glucosamine pentaacetate and thiophenol, however we proceeded due to our interest in bthioglycoside 10 (Scheme 1) as a precursor to control compound 7 and precedence for anomerisation leading to production of a-glycosides in other reactions of acylated glycosides.10–12 Gratifyingly, treatment of a-D-glucosamine pentaacetate with cyclohexanethiol in the presence of 2 equiv of BF3.OEt2 in dichloroethane at 50 °C produced both a- and b-anomers in a 2:1 ratio. Although the modest combined yield (33%) leaves room for optimisation, we were able to retrieve sufficient material of both desired anomers in a
Please cite this article in press as: Riordan, S. W.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.070
3
S. W. Riordan et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
OAc AcO
O O
AcO
H OAc
HN
OH
O
a
SH
Cl
S
HN
Boc
NHAc
OH
Boc
(8)
Cl Cl
OH
b HO OAc AcO
O
H
OAc c
S
AcO
AcO
O
HO O
d
H S
AcO
NHAc
N(Boc)Ac
(9)
(11)
O
H S
NH
Cl
S
RHN
Cl Cl
R = Boc, (Boc- 5)
e
R = H.CF3 CO2H, (5) OAc AcO
O
AcO
H S
OH
OAc c
AcO
O
AcO
NHAc
N(Boc)Ac
( 10 )
( 12)
HO
d
H S
HO O
O
H S
NH
Cl
S
RHN R = Boc, (Boc-7) R = H.CF3CO2 H, (7 )
Cl Cl e
Scheme 1. Reagents and conditions: (a) C2Cl4, DBN, CH3CN (69%); (b) cyclohexanethiol (4 equiv), BF3OEt2 (2 equiv), 1,2-dichloroethane, 0–55 °C, 3 h (9 22%, 10 11%); (c) ditert-butyl dicarbonate (5.4 equiv), 4-(N,N-dimethylamino)pyridine (0.13 equiv), THF, reflux 18 h (11 quant, 12 82%); (d) (i) Na (1 equiv), MeOH, rt, 2 h (ii) HCl, MeOH/water, rt, 18 h, (iii) KOtBu (1 equiv), EDCI (2.5 equiv), 8 (1.5 equiv), DMF, 0 °C–rt, 2 h (Boc-5 26%, Boc-7 35% over 3 steps); (e) TFA, 0 °C–rt, 25 min (5 35%, 7 quant). Synthetic methodology is provided in the Supplementary data.
single step from commercially available starting materials. The anomers were readily separated chromatographically (silica, petroleum ether/ethyl acetate, 1:2) and carried separately through subsequent steps. Although deacetylation can be achieved in a single step by hydrazinolysis at 120 °C,5 we preferred the more mild conditions offered by formation of the N-Boc derivative followed by sequential treatment with sodium methoxide and methanolic HCl at room temperature in a one-pot, two-step global deprotection reaction. Interestingly, the b-anomer of the Boc-protected intermediate could also be recovered as an approximately 1:1 ratio of two rotamers, although these resolved to a single hydrochloride salt upon deprotection. The salts were carried directly into the next step without purification. Coupling of the thioglycoside salts derived from 11 and 12 with 8, mediated by 1-(3-N,N-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), gave protected mycothiol analogues Boc-5 and Boc-7. Removal of the Boc group was achieved using anhydrous trifluoroacetic acid (TFA). Although the TFA salt of the a-anomer proved stable, that of the b-anomer was prone to degradation and was prepared freshly on each occasion for biological testing. The a-anomer was obtained in only modest yield after stringent removal of persistent impurities by chloroform trituration ahead of biological evaluation. Our original hypothesis was that the trichloroethenylcysteinecontaining mycothiol analogue 5 would be selectively taken up into mycobacteria and processed by amidases MCA or MshB to liberate 6, with subsequent b-lyase activity generating reactive electrophiles, such as a halothioketene (Fig. 2). The uptake and activity of the b-anomers of simplified mycothiol analogues, such as 7, have not been previously tested. To our surprise, both anomers were essentially equally active in the model mycobacteria, although the a-anomer was the slightly more active form in the Mycobacterium bovis BCG strain (Table 1), being only 2- to 3-fold
less active than ethambutol which had an MIC of 39 lM in this assay. We propose that either MCA or MshB fail to exhibit the expected substrate selectivity or that an alternative, low-specificity amidase is releasing 6 prior to b-lysis. It is also possible that these compounds induce growth inhibition independently of release of 6, perhaps through inhibition of the mycothiol pathway. As a soil bacterium, Mycobacterium smegmatis has a higher resistance to external stresses than is found in M. bovis BCG or MTB.14 For example, M. smegmatis has a higher relative abundance of mycothiol compared to its oxidized form, mycothione. This renders it more tolerant of stress from oxidants and electrophiles.14 It is therefore no surprise that higher concentrations of 5 and 7 are required to achieve growth inhibition for M. smegmatis than for M. bovis BCG. The promising activity observed for 5 and 7, particularly in the BCG assay, indicates that these compounds are taken up by the bacteria. In contrast their Boc-protected counterparts were inactive, presumably through a failure to undergo catabolism or their inability to inhibit mycothiol-related enzymes. Molecular modelling was undertaken to rationalise the similar activities of 5 and 7. Although there is no crystal structure available for MCA, structures of the paralogous MshB have been determined, including one in which active site acetate and glycerol ligands Table 1 Growth inhibition of model mycobacteria M. smegmatis, M. bovis BCG strain (as minimum inhibitory concentrations, MIC), HL60 and 1A9 cell lines (MTT assay, as half-maximal inhibitory concentrations, IC50) Compound 5 7 Boc-5 Boc-7 a
M. smegmatis (MIC) lM
lM
HL60 (IC50) lM
250–500 250–500 >1000 >1000
62.5–125 125–250 500–1000 P1000
76 ± 1 112 ± 18 60 ± 10 16.5 ± 0.2
BCG (MIC)
a
1A9 (IC50) lM 79 ± 23 121 ± 16 103 ± 4 33 ± 5
IC50 values presented as average ± standard error of the mean.
Please cite this article in press as: Riordan, S. W.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.070
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analogues could act as selective prodrugs for b-lyase substrates, such as 6. The hypothesis was that both uptake of the prodrug and its catabolic degradation to 6 would occur selectively in mycobacteria. We have shown that highly simplified mycothiol analogues 5 and 7 are taken up by mycobacteria and result in growth inhibition. Further work will be needed to enhance selectivity for mycobacteria over human cells. However, the work presented here successfully demonstrates that compounds of this type inhibit growth of mycobacteria, and so make interesting leads in the search for new antimycobacterial agents. Acknowledgments
Figure 4. Mycothiol analogues 5 (white carbons) and 7 (all grey) are equally accommodated by MshB. The overlaid ligands, discussed binding site residues and protein ribbons are displayed, showing that MshB is able to accommodate both ligands without significant perturbation of the protein structure. MshB coordinates were obtained from the crystal structure of Broadley et al., 4EWL.pdb.13 Modelling methodology and further figures are provided in the Supplementary data.
The authors thank Prof. Greg Cook (University of Otago) for additional testing of compounds against M. bovis BCG. At Victoria University of Wellington, the authors wish to thank Ian Vorster and Jingjing Wang for help with NMR and MS instrumentation, Prof. John Miller for supervision of J.F., Dr. Ronan O’Toole and Dr. Rob Keyzers for supervision of N. D. Supplementary data
define the positioning of the substrate binding site.13 Both 5 and 7 were modelled into the MshB binding site. As shown in Figure 4, both anomers were easily accommodated within the active site cleft. The trichloroethenyl moiety is positioned alongside Tyr142 and the cyclohexyl group in the pocket opening defined by the side chains of Met98, Glu47 and Asn 261. Compounds 5, 7 and their Boc-protected progenitors were tested against HL-60 (human promyelocytic leukemia) and 1A9 (human ovarian carcinoma) cells. Disappointingly, lead compounds 5 and 7 were approximately equipotent towards the human cell lines tested as they were in the BCG assay. The two Boc derivatives were substantially more cytotoxic to the HL-60 and 1A9 cells than they were to mycobacteria. The higher activity of the Boc derivatives, as compared to the target amines, suggests that these compounds act independently of b-lysis. This interpretation is supported by the higher activity of Boc-7 over its alpha-counterpart Boc-5, the reverse of the order of activity for 5 and 7. Given this, Boc-7 may provide a useful lead for a new structural class of anticancer agents. In summary, as part of a search for antibacterial compounds that would be unlikely to be affected by common mechanisms of drug resistance in mycobacteria, we proposed that mycothiol
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