Synthesis, pharmacological evaluation, and σ1 receptor interaction analysis of hydroxyethyl substituted piperazines.

Subscriber access provided by Universitaetsbibliothek | Erlangen-Nuernberg

Article

Synthesis, pharmacological evaluation and #1 receptor interaction analysis of hydroxyethyl substituted piperazines Frauke Weber, Steffi Brune, Katharina Korpis, Patrick J Bednarski, Erik Laurini, Valentina Dal Col, Sabrina Pricl, Dirk Schepmann, and Bernhard Wünsch J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401707t • Publication Date (Web): 11 Mar 2014 Downloaded from http://pubs.acs.org on March 18, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Medicinal Chemistry

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Synthesis, pharmacological evaluation and σ1 receptor interaction analysis of hydroxyethyl substituted piperazines Frauke Weber,a Stefanie Brune,a Katharina Korpis,b Patrick J. Bednarski,b Erik Laurini,c Valentina Dal Col,c Sabrina Pricl,c,d Dirk Schepmann,a Bernhard Wünscha,*

a

Institute of Pharmaceutical and Medicinal Chemistry, University of Münster,

Corrensstr. 48, D-48149 Münster, Germany Tel.: +49-251-83-33311; Fax: +49-251-83-32144; E-mail: [email protected] b

Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry,

University of Greifswald, F.-L.-Jahn-Straße 17, 17487 Greifswald, Germany c

Molecular Simulations Engineering (MOSE) Laboratory, Department of Engineering

and Architecture (DEA), University of Trieste, Via Valerio 6, 34127 Trieste, Italy d

National Interuniversity Consortium for Material Science and Technology (INSTM),

Research Unit MOSE-DEA, University of Trieste, Via Valerio 6, 32127 Trieste, Italy

Abstract Starting from (S)- or (R)-aspartate three synthetic strategies were explored to prepare hydroxyethyl substituted piperazines with different substituents at the N-atoms. σ receptor affinity was recorded using receptor material from both animal and human origin. σ1 affinities determined with guinea pig brain and human RPMI 8226 tumor cell

lines

differed

slightly

but

showed

the

same

tendency.

(S)-2-[4-

(Cyclohexylmethyl)-1-(naphthalene-2-ylmethyl)piperazin-2-yl]ethanol (7c) revealed the highest affinity at human σ1 receptors (Ki = 6.8 nM). The potent σ1 receptor ligand 7c was able to inhibit selectively the growth of three human tumor cell lines with IC50 values in the low micromolar range. The reduced growth of the RPMI-8226 cell line ACS Paragon Plus Environment


2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was caused by apoptosis. The interaction of 7c with the σ1 receptor was analyzed in detail using the 3D homology model of the σ1 receptor. The calculated free binding energies of all hydroxyethylpiperazines nicely correlate with their recorded affinities towards the human σ1 receptor.

Keywords hydroxyethyl piperazines, σ1 ligands, structure affinity relationships (SARs), cytotoxic activity; apoptosis; 3D homology model; docking

Introduction σ Receptors became generally recognized as a unique receptor class when it was unequivocally demonstrated that their pharmacological profile differs from that of opioid receptors, which they were formerly confused with.1,2 Two subtypes of σ receptors have been identified so far, classified as σ1 and σ2 receptors. Both subtypes differ in their distribution and in their ligand binding profile.3,4 The σ1 receptor has been cloned from different tissues like guinea pig liver5 and human 6

placental choriocarcinoma cells. Much less is known about the σ2 receptor, but new findings hypothesize an association of the σ2 receptor with the PGRMC-1 protein.7 This protein was found in liver microsomes first and could be cloned from human and mammalian tissues. Both protein types show related intracellular distribution, similar molecular weight and the same high affinity for [3H]-progesterone. Knockout of the PGRMC-1 gene led to decreased affinity of σ2 ligands.7,8 σ1 Receptors were shown to be involved in the modulation of neurotransmitter systems and in the regulation of ion channels.9-11 In addition, both subtypes are reported to regulate cancer cell growth. Various tumor cell lines express large numbers of σ receptors, and, σ2 ACS Paragon Plus Environment

Page 2 of 36

Page 3 of 36


3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

receptors are overexpressed especially in proliferating tumor cells.12 Several studies reported on the cytotoxic effects of σ1 antagonists and σ2 agonists.13,14 However, their impact on the cell cycle or mechanism of cell death is not clearly understood. Compounds I – III bearing a hydroxyalkyl substituted piperazine ring represent well established σ1 receptor ligands. For example, recent studies have shown the high σ receptor binding of 2-hydroxyethyl substituted piperazines II in contrast to the homologous hydroxypropyl derivatives III.15 With respect to selectivity towards the σ2 subtype, the hydroxyethyl derivatives II are superior to the hydroxymethyl ligands I showing the same σ1 affinity.

Scheme 1. Piperazine derivatives I - III with different lengths of the hydroxyalkyl side chain. Herein we present different synthetic strategies to afford hydroxyethylpiperazines of general formula II with varying substitution patterns. The σ1 and σ2 affinities of all synthesized compounds were evaluated using receptor material from animal and human origin. The interaction of the piperazine derivatives with the σ1 receptor was analyzed in detail performing molecular dynamics simulations of the compounds in complex with our recently validated σ1 3D homology model. The cytotoxic activity of the most potent σ1 ligand was investigated in a panel of human tumor cell lines. The ability of this compound to induce apoptosis was also studied.

ACS Paragon Plus Environment


Page 4 of 36

4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Synthesis The chloroacetamide 3 was prepared from (S)-aspartic acid (1) in four steps. This reaction sequence includes esterification of diacid 1 to obtain the diester HCl 2.HCl,16 followed by reductive alkylation with benzaldehyde and subsequent acylation with chloroacetyl chloride. The resulting chloroacetamide 3 was reacted with different primary amines to obtain the dioxopiperazines 4a-e in a domino reaction, consisting of a SN2 reaction followed by intramolecular aminolysis. Reduction with LiAlH4 in refluxing THF led to the piperazine ethanols 5a-e (Scheme 2). The (R)-enantiomer of 5e was synthesized in the same manner using (R)-aspartic acid as starting material. All derivatives shown are single enantiomers with (S)-configuration, unless otherwise stated. The enantiomers are characterized with the prefix ent. The high σ1 and σ2 affinity of the cyclohexylmethyl derivatives 5e and ent-5e prompted us to investigate the σ affinity of piperazines 7 with different residues at N-1. For this purpose, the benzyl group of 5e was cleaved off by transfer hydrogenolysis and the secondary amine

6

was

reductively

alkylated

with

bulky

biphenyl-4-carbaldehyde

or

naphthalene-1-carbaldehyde in presence of NaBH(OAc)3 to obtain 7a and 7b in 7 – 8 % overall yields (Scheme 2).


Page 5 of 36


5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

R1 CH3 CH2CH2CH2CH3 CH2CH2CH(CH3)2 (CH2)2C6H5 CH2C6H11

4, 5 a b c d e

7 a b

R2 4-Ph-Ph 1-Naph

Scheme 2. Reagents and reaction conditions: (a) (H3C)3SiCl, H3COH, rt, 16 h; (b) 1. PH-CH=O, NEt3, CH2Cl2, rt, 16 h; 2. NaBH4, H3COH, 0 °C, 40 min; 3. ClCH2COCl, NEt3, CH2Cl2, rt, 2.5 h; (c) R1-NH2, NEt3, CH3CN, rt, 16 h – 3 d; (d) LiAlH4, THF, reflux, 16 h. The enantiomer ent-5e was prepared in the same manner starting with (R)-aspartic acid ent-1. (e) HCO2NH4, Pd/C, H3COH, 3.5 h, reflux; (f) R-CHO, NaBH(OAc)3, CH3CN. Since the bulky N-1-residues appeared to be the preferred substituents for high σ1 receptor binding, derivatives of 5a and 5b were synthesized by combining methyl and butyl residues at N-4 with naphthylmethyl- and biphenylylmethyl residues at N-1. In order to obtain piperazines 11 with different substituents on both N-atoms via a shorter route, the diester.HCl 2.HCl was acylated with chloroacetamide to afford the secondary amide 8 (Scheme 3). Ring closure of 8 with methylamine and butylamine led to the dioxopiperazines 9a and 9c, which were reduced by LiAlH4 to give the N-1 unsubstituted piperazines 10a and 10c, respectively. The substituted piperazines



Page 6 of 36

6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11a-d were prepared by reductive alkylation of 10a and 10c with naphthalene-1carbaldehyde, biphenyl-4-carbaldehyde, 4-methoxybenzaldehyde and NaBH(OAc)3.

9-11 a b c d

R1 CH3 CH3 CH2CH2CH2CH3 CH2CH2CH2CH3

R2 1-Naph 4-Ph-Ph 4-Ph-Ph 4-MeO-Ph

Scheme 3. Reagents and reaction conditions: (a) ClCH2COCl, NaHCO3, CH2Cl2, 16 h, rt; (16) (b) R1-NH2, NEt3, CH3CN, 16 h, rt; (c) LiAlH4, THF, reflux, 16 h; R2-CHO, NaBH(OAc)3, CH3CN, 7 d. The reductive alkylation of N-1 unsubstituted piperazines 6 and 10 generated low yields of products 7 and 11, respectively. This may be due to the hydroxyethyl side chain that sterically shields the adjacent secondary amine. We assume that the aldehydes could react with the γ-aminoalcohols 6 and 10 to yield 1,3-oxazinanes (N/O-acetals), which are less reactive leading to undesired side products and reduced yields of 7 and 11. Furthermore, it was not possible to perform the ring closure of chloroacetamide 8 with cyclohexylmethylamine to yield piperazines with the preferred N-4-residue for σ1 affinity. We assume that the chloroacetamide 8 without a substituent on N-1 adopt another conformation which is disfavored for the cyclization with large amines like cyclohexylmethylamine.


Page 7 of 36


7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

.

2 HCl

CO2CH3

(a) R2

CO2CH3

N

CO2CH3

(b) R2

H

15a-d

Cl

CO2CH3

(c) O

CO2CH3

N

(d)

N

O

H O

R

N

H

OH

CO2CH3

N R2

17a-d

N

H

16a-d

2

(e)

CO2CH3

N H

7, 15-18 a b c d

H

18a-d

R2 4-Ph-Ph 1-Naph 2-Naph 6-MeO-2-Naph

R2

7a-d Scheme 4. Reagents and reaction conditions: (a) R2-CH=O, NEt3, CH2Cl2, rt, 16 h; (b) NaBH4, H3COH, 0 °C, 40 min; (c) ClCH2COCl, NEt3, CH2Cl2, rt, 2.5 h; (d) C6H11CH2-NH2, NEt3, CH3CN, rt, 16 h; (e) LiAlH4, THF, reflux, 16 h. The enantiomers ent-7a and ent-7b were prepared in the same manner starting with ent-2.HCl. Therefore,

an

alternate

synthetic

route

was

explored

to

generate

N-4-

cyclohexylmethyl substituted derivatives 5e, ent-5e, 7a, 7b, 7c and 7d with high σ1 affinity. The large hydrophobic aryl residue at N-1 was introduced at the second step to avoid the reductive alkylation on the stage of piperazine ethanol 6 or 10. The diester 2.HCl was reacted with different aldehydes and the resulting imines 15 were reduced with NaBH4 to provide the secondary amines 16 (Scheme 4). Acetylation of 16 with chloroacetyl chloride led to the chloroacetamides 17, which were reacted with cyclohexylmethylamine to obtain the piperazinediones 18. Reduction of 18 led to the piperazinylethanols 7a-d in high yields. Thus, compounds 7a-d could be more efficiently prepared according to the route outlined in Scheme 4.



8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Pharmacological evaluation Receptor binding studies The σ1 and σ2 affinity of the test compounds was determined in competition experiments with radioligands. All compounds were tested at σ1 and σ2 receptors of animal origin prepared from guinea pig brain and rat liver, respectively. To investigate the correlation of receptor binding, potent ligands were also assayed with human σ1 and σ2 receptors obtained from human tumor cell lines RPMI 8226 and RT-4, respectively. Membrane preparations containing the membrane bound animal and human σ1 and σ2 receptors were obtained by homogenization, centrifugation and washing of the respective tissue or cell line. The implementation of the receptor binding assays using human tumor cell line membrane preparations was previously described.17-20 Both σ1 assays were performed with [3H]-(+)-pentazocine as radioligand. [3H]-DTG was used as radioligand in the σ2 assays. Compounds with high affinity were tested three times. For compounds with low σ affinity, only the inhibition (in %) of the radioligand binding at a concentration of 1 µM is given. The σ receptor affinities of the different piperazines are presented in Table 1. Whereas the compounds 5a, 11a and 11b with a small methyl residue on N-4 do not show strong affinity to the guinea pig σ1 receptor, the introduction of larger substituents leads to increased σ1 affinity in the order methyl < butyl < isopentyl < cyclohexylmethyl. The cyclohexylmethyl derivatives 5e, ent-5e, 7a, 7b, ent-7a, ent-7b and 7c have Ki values in the low nanomolar range but show low selectivity against the σ2 receptor. The introduction of a methoxy group into the hydrophobic aryl residue of 7c leads to a decrease in both σ1 and σ2 receptor affinity as it is demonstrated by the pairs 5b/11d and 7c/7d. The absolute configuration has a


Page 8 of 36

Page 9 of 36


9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

negligible effect on the receptor affinity, since (R)- and (S)-configured enantiomers bind with similar IC50 values (e.g. 5e and ent-5e). Table 1 σ1 and σ2 receptor affinity of hydroxyethyl substituted piperazines σ1 σ2 σ1 (hum)c) σ2 a) b) d) (gp) (rat) (hum) K [nM] i R2 R1 Ki [nM] Ki [nM] ± SEM Ki [nM] ± SEM ± SEM ± SEM 5a Me Ph 28 %e) 27 %e) n.d. n.d. f) e) 5b Bu Ph 279 10 % n.d. n.d. 5c CH2CH2C(CH3)2 Ph 46 ± 1 877f) 107 ± 13 n.d. 5d Ph 31 ± 3 779f) 41 ± 13 n.d. (CH2)2C6H5 5e CH2C6H11 Ph 4.2 ± 1.1 116f) 21 ± 4 168f) ent-5e CH2C6H11 Ph 1.9 ± 0.4 60 ± 11 23 ± 8 251f) 7a 4-Ph-Ph 3.5 ± 0.5 73 ± 45 34 ± 8 43 ± 13 CH2C6H11 ent-7a CH2C6H11 4-Ph-Ph 7.8 ± 2.4 38 ± 1 29 ± 7 39 ± 11 7b CH2C6H11 1-Naph 1.9 ± 0.6 26 ± 12 29 ± 10 16 ± 4 ent-7b 1-Naph 0.9 ± 0.2 14 ± 4 7.5 ± 2.7 37 ± 21 CH2C6H11 7c CH2C6H11 2-Naph 4.7 ± 1.8 69 ± 24 6.8 ± 2.3 31 ± 13 7d 6-MeO-2-Naph CH2C6H11 43 ± 10 253e) 27 ± 4 n.d. e) e) 11a 0% n.d. n.d. Me 1-Naph 3% e) e) f) 11b Me 4-Ph-Ph 17 % 3% 711 n.d. 11c 230f) n.d. n.d. Bu 4-Ph-Ph 465f) f) e) 11d Bu 4-MeO-Ph 491 16 % n.d. n.d. (+)pentazocine 5.4 ± 0.5 36 ± 5.2 haloperidol 6.6 ± 0.9 78 ± 2.3 40 ± 5.3 200 ± 33 di-o-tolylguanidine 71 ± 8 58 ± 18 208 ± 26 20 ± 5.8 a) guinea pig brain, b) rat liver c) RPMI 8226 cells, d) RT-4 cells, e) Inhibition of radioligand binding at 1µM concentration of test compound, f) result from one measurement, n.d.: not determined

The affinity of the synthesized compounds towards human σ1 receptors (RPMI 8226 cell line) is reduced compared to the affinity recorded in the guinea pig assay. The same trend was observed for the reference compounds (+)-pentazocine, haloperidol and di-o-tolylguanidine. However, the data show the same tendency indicating that the results obtained with receptor material from guinea pig brain and human tumor cell line RPMI 8226 are well comparable. We assume that the higher Ki values



10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

obtained in the human tumor cell line based σ1 assay originate from different test materials and assay conditions. For the discussion of the σ1/σ2 selectivity, the data recorded in the traditional guinea pig brain (σ1) and rat liver (σ2) assays are used. These data indicate more than tenfold selectivity for most of the piperazine derivatives for the σ1 over the σ2 subtype. Due to the higher Ki(σ1)-values obtained with human tumor RPMI 8226 cell line the σ1/σ2 selectivity is considerably reduced, when these values are compared with the Ki(σ2) values obtained with rat liver membrane preparations. Moreover, the Ki(σ2) values recorded with rat liver and human RT-4 cell line preparations are very similar indicating reduced σ1/σ2 selectivity when compared with the Ki(σ1)-values obtained with human RPMI 8226 cell line preparations. The very potent σ1 ligands 7a, ent-7a and 7b interacting in the low nanomolar range with the guinea pig σ1 receptors (Ki(σ1) = 1.9-3.5 nM) reveal high selectivity over the rat σ2 receptor (14 to 31-fold), but can be regarded as non-selective, when considering the Ki(σ1) values obtained with the human tumor cell line RPMI 8226. These variations of the Ki values are attributed to species differences and different preparations of receptor material. In this work these species differences were observed for the first time and should be considered in further σ1/σ2 selectivity discussions. Within this series of compounds, the cyclohexylmethyl derivative 7c represents the most potent σ1 ligand with the lowest Ki value in the human σ1 assay (Ki = 6.8 nM) and a promising σ1/σ2 selectivity for both the animal (15-fold) and the human tumor cell based assays (5-fold). Therefore 7c was selected for further characterization in the cytotoxicity assay with the aim to understand better the pharmacological properties of potent σ1 ligands.


Page 10 of 36

Page 11 of 36


11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cytotoxicity of 7c in human cancer cell lines The antiproliferative effect of 7c was investigated in seven human tumor cell lines, namely 5637 and RT-4 (bladder cancer), A427 (small cell lung cancer), LCLC-103H (large cell lung cancer), MCF-7 (breast cancer), DAN-G (pancreas cancer) and RPMI 8226 (multiple myeloma). In case of the suspension-cell line RPMI 8226, 7c was tested in a MTT assay.21 The optical density of the resulting formazane product was used to determine the cytotoxic activity of the compound. For the adherent cell lines, crystal violet assay21 was used. The results are given as part of adherent cells in relation to an untreated control. Five serially diluted stock solutions were used to determine the IC50 values. Table 2 Average IC50 values (± SD) for the growth inhibition of 7c employing different tumor cell lines. Results are from at least 3 independent experiments. cell line 5637 RT-4 A-427 LCLC-103H DAN-G MCF-7 RPMI 8226

IC50 [µM] 2.1 ± 1.9 ˃20 2.5 ± 2.6 ˃20 ˃20 15 ± 2 7.3 ± 3.9

7c inhibited the growth of the cell lines 5637, A-427 and RPMI 8226, with IC50 values in the low micromolar range, whereas lower activity was observed for the MCF-7 cell line. The growth inhibition of LCLC-103H, RT-4, and DAN-G cell lines was not reduced at a concentration of 20 µM of 7c. These results indicate a selective mechanism of cytotoxicity for 7c. Induction of apoptosis in RPMI-8226 cell lines For these studies, RPMI-8226 cells were used because of their high density of σ1 receptors.18 After incubation of RPMI-8226 cells with 7c at the IC50- and IC90-



12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

concentrations for 24 or 48 h, the cells were harvested and double-stained with annexin-V and propidium iodide (PI). The relative numbers of early (i.e. those stained with annexin-V-FITC) and late apoptotic cells (i.e. those stained with both annexin-VFITC and PI) were quantified by flow cytometry.

Figure 1. Population of annexin-V positive, PI negative RPMI-8226 cells after 24 h and 48 h treatment with 7c.

Table 3 Apoptotic activity in untreated controls and in 7c treated RPMI-8226 cells 24 h incubation period 48 h incubation period Concentration early-apoptotic late-apoptotic early-apoptotic late-apoptotic (%) cells (%)a cells (%)b cells (%)a cells (%)b 0 12.8 ± 2.59 8.11 ± 1.44 14.6 ± 3.09 6.43 ± 0.77 7.3 8.60 ± 1.50 9.71 ± 2.41 22.55 ± 5.46 10.59 ± 4.16 13.2 9.94 ± 0.76 15.67 ± 7.55 25.82 ± 5.89 17.74 ± 6.86 a) annexin-V positive, PI negative; b) annexin-V positive, PI positive The results presented in Figure 1 and Table 3 show a significant increase in the amount of early-apoptotic cells (annexin-V positive, PI negative cells) in relation to the untreated control after 48 h. Shorter incubations (i.e. 24 h) did not result in an increased number of apoptotic cells, neither at the IC50 nor at the IC90 concentration.


Page 12 of 36

Page 13 of 36


13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Further studies will be necessary to elucidate the mechanism of apoptosis; e.g., whether it is a caspase dependent or independent event.

Molecular Modeling To support the SAR analysis developed above with a molecular-based rationale for the binding mode of these series of new, potent piperazine σ1 ligands, the model structure of the best derivative 7c was docked into the putative binding site of the 3D

σ1 receptor homology model22 and the drug/protein free energy of binding (∆Gbind) was estimated using a validated molecular dynamics (MD) procedure23 based on MM/PBSA calculations24 (see Supporting Information for details). The analysis of the corresponding MD trajectories reveals that four major types of interactions are involved in the binding mode of 7c to the σ1 receptor, as highlighted in Figure 2: 1) π interactions between residues Arg119 (π-cation) and Tyr120 (T-stacking π−π), respectively, with the naphthalene ring of 7c; 2) a hydrogen bond (HB) between the side chain –OH donor of Thr181 and the acceptor oxygen atom of the hydroxyethyl chain of 7c; 3) a salt bridge between the –NH+ of the N-alkyl moiety of 7c and the protein Asp126 –COO- and 4) a robust network of stabilizing van der Waals interactions between the high hydrophobic cavity leaned by the side chains of Ile128, Phe133, Tyr173, and Leu186 and the cyclohexylmethyl portion of 7c.



Page 14 of 36

14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a)

b) 173Tyr

133Phe

T181

Ile128

D126

L186

186Leu

I128 OH

R119

Hydrophobic interaction

F133 Y120

126Asp

Y173

HO salt bridge N

OH

nd bo H-

O N

Thr181

OH H

interaction OH

NH

H2N HN

Tyr120

Arg119

Figure 2. a) 2D schematic representation of the identified interactions between the 3D homology model σ1 receptor and 7c. The lines/arrows indicate key interactions between the receptor and its ligand. b) Equilibrated MD snapshot of the complex of the σ1 receptor with 7c showing the key interactions depicted in part a. The main protein residues involved in these interactions are Arg119 and Tyr120 (π-interactions, cyan), Asp126 (salt bridge, red), Ile128, Phe133, Tyr173 and Leu186 (hydrophobic interaction, magenta) and Thr181 (hydrogen bond, green). Compound 7c is shown in atom-colored sticks-and-balls (C, gray; N, blue; and O, red). H atoms are omitted, but the salt bridge and the H-bond are indicated as black lines or black dotted lines, respectively. Na+ and Cl- ions are represented as dark gray and light gray sphere, respectively. Water molecules are not shown for clarity. The 3D model of the σ1 receptor was obtained 22 by homology modeling techniques starting from the following model templates: 3ClA.pdb, 1l24.pdb, 2Z2Z.pdb, and 2Q8l.pdb.

All the interactions described above are quantified by a calculated ∆Gbind of 7c for σ1 equal to -10.85 ± 0.36 kcal/mol, corresponding to an estimated affinity σ1Ki,calcd value of 11.2 nM (Table 4), in stringent agreement with the experimental quantity σ1Ki,exp of 6.8 nM (Table 1). ACS Paragon Plus Environment

Page 15 of 36


15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 4 Major components and total binding free energy (kcal/mol) for compound 7c in complex with the σ1 receptor. The calculated σ1Ki (σ1Ki,calcd, nM) is also reported for comparison with the corresponding experimental value Components

7c

∆EVDW

-54.40 ± 0.05

∆EELE

-90.84 ± 0.09

∆EMM

-145.24 ± 0.10

∆GPB

126.11 ± 0.14

∆GNP

-4.31 ± 0.03

∆GSOL

121.80 ± 0.14

∆Hbind

-23.44 ± 0.18

-T∆Sbind

12.59 ± 0.31

∆Gbind

-10.85 ± 0.36

σ1Ki,calcda

11.2

a

The σ1Ki,calc values were obtained from the corresponding ∆Gbind values using the relationship ∆Gbind = -RT ln(1/σ1Ki,calcd). Similarly to other σ1/ligand complexes,22,24,25 the nonbonded mechanical energy components of ∆Gbind, ∆EVDW and ∆EELE, afford a substantial, favorable contribution to binding. On the other hand, the desolvation penalty paid by 7c upon binding (∆GPB) is also quite substantial, so that the net, resulting electrostatic contribution to the affinity of 7c for the σ1 receptor is markedly unfavorable. Specifically, the mean value of the electrostatic energy (∆EELE + ∆GPB) is 35.27 kcal/mol, whilst the corresponding value of the van der Waals and hydrophobic interaction energies (∆EVDW + ∆GNP) is -58.71 kcal/mol. Accordingly, it follows that the association between the σ1 receptor and 7c is mainly driven by more favorable nonpolar



16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

interactions in the complex than in the solution, in harmony with a proposed general scheme for noncovalent association.

To investigate in details the binding mode of 7c to the σ1 receptor, a deconvolution of the enthalpic component (∆Hbind) of the binding free energy into contributions from each protein residue was carried out, as illustrated in Figure 3.

Figure 3. Per residue binding enthalpy decomposition for the σ1/7c complex. Only σ1 amino acids from position 100 to 200 are shown, as for all remaining residues the contribution to ligand binding is irrelevant. As seen in Figure 4, substantial van der Waals and electrostatic interactions are contributed by residues Arg119 (-0.75 kcal/mol) and Y120 (-1.64 kcal/mol) - through the aforementioned π-cation and T-stacking π−π interaction, respectively - and by the residues belonging to the hydrophobic pocket Ile128 (-1.12 kcal/mol), Phe133 (-0.97 kcal/mol), Tyr173 (-0.88 kcal/mol) and Leu186 (-0.83 kcal/mol) (Figure 2) The stable salt bridge (average dynamic length (ADL) = 3.91 ± 0.07 Å) involving Asp126 and the permanent hydrogen bond (ADL = 1.99 ± 0.05 Å) featured by Thr181are responsible for stabilizing contributions of -2.67 kcal/mol and -2.33 kcal/mol, respectively. All other receptor residues were characterized by negligible interaction enthalpy values (|∆Hbind| < 0.30 kcal/mol).


Page 16 of 36

Page 17 of 36


17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

To corroborate the experimental structure-activity relationship formulated for this series of σ1 ligands, the same computational recipe was applied to all remaining 15 compounds. Accordingly, the corresponding receptor/ligand complexes were built and optimized by a combined molecular mechanics/molecular dynamics procedure, and the relevant values of ∆Gbind/Ki,calcd evaluated applying the MM-PBSA computational ansatz. These results are listed in Table 5. Table 5 Binding free energies ∆Gbind (kcal/mol) and σ1Ki,calcd values (nM) for the entire set of the 12 bioactive ligands in complex with the σ1 receptor. Errors are given in parenthesis as standard errors of the mean. The experimental σ1Ki values (nM) and the calculated σ1Ki (nM), as estimated from the corresponding ∆Gbind values (∆Gbind = -RT ln(1/σ1Ki,calcd)), are also reported for comparison. Compound 5a 5b 5c 5d 5e ent-5e 7a ent-7a 7b ent-7b 7c 7d 11a 11b 11c

-∆H (kcal/mol) -17.44 (0.15) -20.13 (0.18) -22.02 (0.19) -22.43 (0.17) -22.19 (0.16) -22.25 (0.16) -23.31 (0.18) -23.27 (0.19) -23.34 (0.17) -23.11 (0.17) -23.44 (0.18) -23.26 (0.19) -18.87 (0.19) -18.73 (0.16) -20.80

-T∆S (kcal/mol) -10.40 (0.31) -11.62 (0.32) -12.22 (0.29) -12.11 (0.29) -12.01 (0.28) -12.00 (0.30) -13.02 (0.29) -12.91 (0.31) -12.62 (0.27) -12.41 (0.30) -12.59 (0.31) -12.78 (0.31) -11.72 (0.29) -11.63 (0.28) -11.74

∆Gbind (kcal/mol) -7.04 (0.34) -8.51 (0.37) -9.80 (0.35) -10.32 (0.35) -10.18 (0.32) -10.25 (0.34) -10.29 (0.34) -10.36 (0.36) -10.72 (0.32) -10.70 (0.34) -10.85 (0.36) -10.48 (0.36) -7.15 (0.35) -7.10 (0.32) -9.06


σ1Ki,calcd [nM]

σ1Ki [nM] ± SEM

6900

28%

580

279

66

107 ± 13

27

41 ± 13

35

21 ± 4

31

23 ± 8

29

34 ± 8

26

29 ± 7

14

29 ± 10

15

7.5 ± 2.7

11

6.8 ± 2.3

21

27 ± 4

5800

3%

6300

17%

230

465


Page 18 of 36

18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11d

(0.15) -20.02 (0.18)

(0.30) -11.53 (0.30)

(0.34) -8.49 (0.35)

600

491

It is interesting to observe how, notwithstanding all approximation involved, the applied methodology was able to rank correctly the entire set of compounds with respect to their affinity towards the human σ1 receptor protein. Specifically, only those compounds carrying a bulky substituent on N-1 (5c-e and 7a-d) are endowed with the most favorable free energy of binding (∆Gbind < 10 kcal/mol). The presence of a butyl chain (5b and 11c-d) reflects in a weak but still appreciable ligand binding capacity while the introduction of the small methyl moiety on N-1 (5a and 11a-b) leads to a dramatic loss of protein/ligand affinity (3-4 kcal/mol, Table 5). On the other hand, an aromatic substituent in R2 and, even more intriguingly, the absolute configuration of the enantiomeric compounds appear to play a negligible role in receptor binding (e.g., 5e and ent-5e, 7a and ent-7a, and 7b and ent-7b, Table 5). Lastly, the dissection of the binding enthalpy into per residue contribution applied to 7c has been extended to the entire molecular series of σ1 ligands. To facilitate a direct comparison with the lead compound 7c, the values of ∆Hbind for the critical σ1 residues were clustered according to a specific underlying interaction type, as follow: Arg119 and Tyr120 were considered for π interactions (π), Asp126 for salt bridge (SB), Thr181 for hydrogen bond (HB) and Ile128, Phe133, Tyr173, and Leu186 for hydrophobic interactions (HY). Moreover, compounds 5a, 7a, and 11c were selected for a meaningful comparison with 7c. These results are reported in Figure 4.


Page 19 of 36


19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Comparison of per residue binding enthalpy decomposition for compounds 5a, 7a, 7c, and 11c in complex with the σ1. Critical receptor residues are clustered according to the specific underlying interactions: π (π interactions); SB (salt bridge); HB (hydrogen bond); HY (hydrophobic interactions) (see legend and main text for more details).

Figure 4 clearly shows that the lead compound 7c and the derivative 7a exhibit a similar interaction profile except for negligible quantitative differences. Indeed, the naphthalene ring (7c) and the biphenyl moiety (7a) are both able to establish the best interactions with residues Arg119 and Tyr120; furthermore, the similar scaffold of these two compounds allows for the adoption of an optimal conformational fit of the molecule within the entire binding pocket. In contrast, the absence of a large, hydrophobic substituent on N-1 as in compounds 5a and 11c leads to a drastic loss not only in HY stabilization (∆H(HY,5a) = -0.75 kcal/mol, ∆H(HY,11c) = -1.57 kcal/mol vs. ∆H(HY,7c) = -3.80 kcal/mol) but also in all other main interactions, by virtue of a global rearrangement of the receptor/complex binding site. Accordingly, the resultant affinity for the σ1 protein of 7a is similar to that of 7c, whilst the binding of 5a and 11a to the same receptor is substantially weaker.



20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Conclusion Hydroxyethylpiperazines with a small methyl group at an N-atom do not reveal high σ1 receptor affinity, whereas analogs with larger lipophilic residues at both N-atoms show high σ1 affinity. The cyclohexylmethyl group proved to be the preferred substituent at the N-4 position, as demonstrated for the compounds 5e, ent-5e, 7a-d and ent-7a,b. The configuration in 2-position of the piperazine ring does not exert a strong impact on σ1 receptor affinity. The Ki values recorded with the human tumor cell line RPMI 8226 are slightly higher, but show the same tendency as the Ki values obtained with receptor material from guinea pig brain. Since the cyclohexylmethyl substituted derivative 7c showed the highest affinity towards the human σ1 receptor, the growth inhibition of seven tumor cell lines was investigated. It was shown that 7c inhibited selectively the growth of three tumor cell lines indicating a relationship between σ1 affinity and growth inhibition of these tumor cell lines. The growth inhibition by 7c is mediated by inducing apoptosis. The interaction of 7c with the human σ1 receptor was analyzed in detail by docking studies using the recently reported 3D homology model of the σ1 receptor. Four types of interactions were found to be crucial for high σ1 affinity: π interactions between Arg119/Tyr120 and the naphthalene ring, a hydrogen bond between Thr181 and the O-atom of the hydroxyethyl moiety, an ionic interaction between Asp126 and a protonated piperazine N-atom, and finally lipophilic van der Walls interactions between the side chains Ile128, Phe133, Tyr173, and Leu186 with the cyclohexylmethyl substituent. The contribution of the single amino acids to the free energy resulting from binding of 7c with the human σ1 receptor was analyzed.


Page 20 of 36

Page 21 of 36


21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The interaction of the whole set of hydroxyethylpiperazines towards the human σ1 receptor was analyzed in detail and it was found that the calculated free binding energies correlate nicely with the recorded Ki values. It should be emphasized that this is the first study using the human tumor cell line RPMI 8226 for recording σ1 affinity of a set on novel ligands. Therefore this is the first study, which allows the correlation of the calculated free binding energy with recorded Ki values.

Experimental Part Chemistry, general Thin layer chromatography: Silica gel 60 F254 plates (Merck). Flash chromatography (fc): Silica gel 60, 40–43 µm (Merck); parentheses include: diameter of the column, eluent, Rf value. Melting point: Melting point apparatus SMP 3 (Stuart Scientific), uncorrected. 1H NMR (400 MHz),

13

C NMR (100 MHz): Unity Mercury Plus AS 400

NMR spectrometer (Varian); δ in ppm related to tetramethylsilane; coupling constants are given with 0.5 Hz resolution; the assignments of

13

C and 1H NMR signals were

supported by 2D NMR techniques. The purity of all compounds was determined by HPLC analysis. HPLC (method ACN): Merck Hitachi Equipment; UV detector: L7400; autosampler:L-7200; pump: L-7100; degasser: L-7614; column: LiChrospher® 60 RP-select B (5 µm); LiCroCART® 250-4 mm cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 µL; detection at λ = 210 nm; solvent A: demineralized water with 0.05% (v/v) trifluoroacetic acid; solvent B: acetonitrile with 0.05% (v/v) trifluoroacetic acid: gradient elution (% A): 0-4 min: 90.0 %; 4-29 min: gradient from 90 % to 0 %; 29-31 min: 0 %; 31-31.5 min: gradient from 0 % to 90.0 %; 31.5-40 min: 90 %. According to HPLC analysis the purity of all test compounds is > 95 %.



22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

General Procedure A: Under N2, 1 equivalent of the monocyclic piperazinedione was dissolved in THF abs. and the mixture was cooled down to 0 °C. At this temperature, lithium aluminium hydride (6 equivalents) was added. The reaction mixture was stirred at 0 °C for 10 min and then heated to reflux for 16 h. Finally water was added under ice-cooling until H2 liberation was finished. The mixture was stirred at 0 °C for 10 min and then heated to reflux for 30 min. After cooling to room temperature, the mixture was filtered and the solvent was removed in vacuo. The residue was purified by fc. Dimethyl (S)-2-aminobutanedioate hydrochloride (2.HCl)16 Under ice cooling, chlorotrimethylsilane (33.2 mL, 263.0 mmol) was added dropwise to a suspension of (S)-aspartate (1) (10 g, 75.1 mmol) in methanol abs. (150 mL) and the reaction mixture was stirred for 16 h at room temperature. The solvent and volatile byproducts were evaporated in vacuo. The residue was suspended in methanol (1 x 30 mL) and the solvent was removed in vacuo. Then diethyl ether was added (30 mL) and the solvent was removed under reduced pressure. This procedure was repeated three times. The product was dried in high-vacuum. Colorless solid, mp 112 – 115 °C, yield 15 g (99.9 %). C6H12ClNO4, Mr = 197.6. 1H NMR (CD3OD): δ [ppm] = 3.07 (d, J = 5.3 Hz, 2H, CHCH2CO2CH3), 3.75 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.40 (t, J = 5.4 Hz, 1H, CHCH2CO2CH3). The signal for the protons of the NH3+-group is not seen. Dimethyl (S)-2-[2-chloro-N-(naphthalen-2-ylmethyl)acetylamino]butanedioate (17c) 2.HCl (1 g, 5.06 mmol) was dissolved in CH2Cl2 abs. (15 mL) and triethylamine (0.74 mL, 5.06 mmol) and 2-naphthaldehyde (0.79 g, 5.06 mmol) were added slowly. Then Na2SO4 was added and the mixture was stirred for 16 h at room temperature. For ACS Paragon Plus Environment

Page 22 of 36

Page 23 of 36


23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

work-up, the suspension was filtered, the precipitate was washed with CH2Cl2 and the filtrate was concentrated in vacuo. Diethyl ether was added to the resulting residue and the mixture was filtered again. Evaporation of the filtrate gave the imine 15c (1.24 g, 4.14 mmol) as a pale yellow solid, which was dissolved in methanol (15 mL) and converted directly by addition of NaBH4 (260.4 mg, 7.0 mmol) over 40 min under ice cooling. The mixture was stirred at room temperature for another 5 min, before the solvent was evaporated in vacuo. Water (15 mL) was added to the residue and the aqueous layer was extracted with diethyl ether (5 x 10 mL). The combined organic layers were dried (K2CO3), filtered and the solvent was removed under reduced pressure. The oily residue (16c, 1.03 g, 3.42 mmol) was dissolved in CH2Cl2 abs. (15 mL). Triethylamine (0.38 mL, 2.73 mmol) and chloroacetyl chloride (0.44 mL, 5.47 mmol) were added dropwise under N2-atmosphere and ice cooling. The mixture was stirred for 2.5 h at room temperature. The solvent was removed in vacuo and diethyl ether (50 mL) was added to the resulting residue. The suspension was filtered, washed and the filtrate was concentrated in vacuo. Purification of the residue was performed by fc (∅ 3.5 cm, h = 16 cm, v = 30 mL, cyclohexane/ethyl acetate = 4/1, Rf = 0.12). Pale yellow oil, yield 291.6 mg (15 % over 3 steps). C19H20ClNO5, Mr = 377.8.

1

H NMR (CDCl3): δ [ppm] = 2.69 (dd, J = 17.4 / 7.9 Hz, 0.2H,

CHCH2CO2CH3NR), 2.80 (dd, J = 17.1 / 6.9 Hz, 0.8H, CHCH2CO2CH3HR), 3.01 (dd, J = 17.3 / 6.4 Hz, 0.2H, CHCH2CO2CH3NR), 3.30 (dd, J = 17.1 / 6.3 Hz, 0.8H, CHCH2CO2CH3HR), 3.40 (s, 3 x 0.2H, OCH3NR), 3.57 (s, 3 x 0.2H, OCH3NR), 3.60 (3 x 0.8H, OCH3HR), 3.68 (s, 3 x 0.8H, OCH3HR), 4.11 (d, J = 12.7 Hz, 0.8H, O=CCH2ClHR), 4.17 (d, J = 12.6 Hz, 0.8H, O=CCH2ClHR), 4.37 (d, J = 15.8 Hz, 0.2H, NCH2ArNR), 4.51 (s, broad, 2 x 0.2H, O=CCH2ClNR), 4.59 (t, J = 6.6 Hz, 0.8H, CHCH2CO2CH3HR), 4.84 (d, J = 16.7 Hz, 0.8H, NCH2ArHR), 4.91 (d, J = 16.8 Hz,



24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.8H, NCH2ArHR), 5.05 – 5.15 (m, 2 x 0.2H, NCH2ArNR + CHCH2CO2CH3NR), 7.33 (d, J = 8.5 Hz, 0.2H, Ar-HNR), 7.43 – 7.45 (m, 0.2H, Ar-HNR + 0.8H, Ar-HHR), 7.50 – 7.54 (m, 2 x 0.8H, Ar-HHR), 7.59 (s, broad, 0.2H, NCH2ArNR), 7.77 (d, J = 8.6 Hz, 3 x 0.2H, NCH2ArNR), 7.78 – 7.88 (m, 0.2H, Ar-HNR + 4 x 0.8H, Ar-HHR). Ratio of rotamers is 80:20. (S)-Methyl 2-[4-(cyclohexylmethyl)-1-(naphthalen-2-ylmethyl)-3,6-dioxopiperazin-2-yl]acetate (18c) 17c (200 mg, 0.53 mmol) was dissolved in dry acetonitrile (molecular sieves 3 Å, 10 mL) and triethylamine (0.1 mL, 0.64 mmol) and cyclohexylmethylamine (0.1 mL, 0.69 mmol) were added slowly. The reaction mixture was stirred at room temperature for 16 h. For workup, the solvent was evaporated in vacuo and the residue was dissolved in ethyl acetate (10 mL). The organic layer was washed with 0.5 M HCl (2 x 4 mL), 0.5 M NaOH (1 x 4 mL) and brine (1 x 4 mL), dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by fc (∅ 2 cm, h = 20 cm, v = 10 mL, cyclohexane/ethyl acetate = 4/1, Rf = 0.12). Colorless solid, mp 119 - 123 °C, yield 173 mg (77 %). C25H30N2O4, Mr = 422.5. 1H NMR (CDCl3): δ [ppm] = 0.91 – 1.00 (m, 2H, NCH2C6H11), 1.13 – 1.28 (m, 3H, NCH2C6H11), 1.65 – 1.74 (m, 6H, NCH2C6H11), 2.82 (dd, J = 17.2 / 5.1 Hz, 1H, CHCH2CO2CH3), 3.02 (dd, J = 17.2 / 3.7 Hz, 1H, CHCH2CO2CH3), 3.18 – 3.30 (m, 2H, NCH2C6H11), 3.54 (s, 3H, OCH3), 3.97 (d, J = 17.2 Hz, 1H, O=CH2N), 4.13 (t, J = 4.1 Hz, 1H, CHCH2CO2CH3), 4.38 (d, J = 17.5 Hz, 1H, O=CH2N), 4.43 (d, J = 15.3 Hz, 1H, NCH2Ar), 5.20 (d, J = 15.1 Hz, 1H, NCH2Ar), 7.35 (dd, J = 8.4 / 1.8 Hz, 1H, Ar-H), 7.46 – 7.51 (m, 2H, Ar-H), 7.69 (s, 1H, Ar-H1-H), 7.77 – 7.83 (m, 3H, Ar-H).


Page 24 of 36

Page 25 of 36


25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(S)-2-[4-(Cyclohexylmethyl)-1-(naphthalen-2-ylmethyl)piperazin-2-yl]ethanol (7c) 7c was synthesized according to General Procedure A: Reaction of 18c (150 mg, 0.36 mmol) with LiAlH4 solution (1 M in THF, 2.1 mL, 2.1 mmol) was performed in THF abs. (12 mL). Fc (∅ 2 cm, h = 18 cm, v = 10 mL, cyclohexane/ethyl acetate = 3/2, Rf = 0.13). Colorless solid, mp 55 – 59 °C, yield 75.2 mg (57 %). C24H34N2O, Mr = 366.5. 1H NMR (CDCl3): δ [ppm] = 0.81 – 0.90 (m, 2H, NCH2C6H11), 1.13 – 1.23 (m, 2H, NCH2C6H11), 1.45 – 1.52 (m, 1H, NCH2C6H11), 1.63 – 1.79 (m, 5H, NCH2C6H11, CHCH2CH2OH), 1.89 – 1.91 (m, 2H, NCH2C6H11, CHCH2CH2OH), 2.04 – 2.07 (m, 1H, NCH2C6H11 or CHCH2CH2OH), 2.11 (d, J = 7.2 Hz, 2H, NCH2C6H11), 2.32 – 2.42 (m, 4H, NCH2), 2.57 – 2.60 (m, 1H, NCH2), 2.86 – 2.89 (m, 1H, NCH), 2.92 – 2.98 (m, 1H, NCH2), 3.57 (d, J = 12.9 Hz, 1H, NCH2Ar), 3.77 – 3.83 (m, 1H, CHCH2CH2OH), 3.87 – 3.92 (m, 1H, CHCH2CH2OH), 4.28 (d, J = 12.2 Hz, 1H, NCH2Ar), 7.42 – 1.51 (m, 3H, Ar-H), 7.72 (s, 1H, Ar-H1-H), 7.79 – 7.83 (m, 3H, Ar-H). The signal for the proton of the OH group is not seen. Molecular modeling The model structures of compounds 5a-e, 7a-d, and 11a-d were sketched and geometrically optimized using Discovery Studio (DS, version 2.5, Accelrys, San Diego, CA). A conformational search was then carried out using a well-validated, ad hoc developed combined molecular mechanics/molecular dynamics simulated annealing (MDSA) protocol22 - 24,

26 - 32

using Amber 1233 and the ff03 force field.34

Accordingly, the relaxed structures were subjected to five repeated temperature cycles (from 300 to 1000 K and back) using constant-volume/constant-temperature (NVT) molecular dynamics (MD) conditions. At the end of each annealing cycle, the



26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

structures were again energy minimized to converge below 10-4 kcal/mol Å, and only the structures corresponding to the minimum energy were used for further modeling. The atomic partial charges for the geometrically optimized compounds were obtained using the RESP procedure,35-37 and the electrostatic potentials were produced by single-point quantum mechanical calculations at the Hartree – Fock level with a 631G * basis set, using the Merz − Singh − Kollman van der Waals parameters. Eventual ff03 missing force field parameter of Amber 12 were generated using the general Amber force field (GAFF).38 The compound optimized structures were then docked into the σ1 putative binding pocket by applying a consolidated procedure performed with Autodock 4.3/Autodock Tools 1.4.639 on a win64 platform. The resulting structures were visualized and subjected to cluster analysis with a 1 Å tolerance for an all-atom root-mean-square (rms) deviation from a lower energy structure representing each cluster family. Then, for each compound only the molecular conformation satisfying the combined criteria of having the lowest (i.e., more favorable) Autodock energy and belonging to a highly populated cluster was selected to carry for further modeling. Each ligand/receptor complex obtained from the docking procedure was further refined in Amber 12 using the quenched molecular dynamics (QMD) method.40–49 According to QMD, 1 ns MD simulations at 300 K were employed to sample the conformational space of each ligand/receptor complex in the GB/SA continuum solvation environment.50,51 The integration step was equal to 1 fs. After each picosecond, each system was cooled to 0 K, and the structure was extensively minimized and stored. To prevent global conformational changes of the protein, the backbone atoms of the protein binding site were constrained by a harmonic force constant of 100 kcal/Å, whereas the amino acid side chains and the ligands were ACS Paragon Plus Environment

Page 26 of 36

Page 27 of 36


27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

allowed to move without any constraint. The best energy configuration of each complex resulting from the previous step was subsequently solvated by a cubic box of TIP3P52 water molecules extending at least 10 Å in each direction from the solute. Each system was then neutralized and, furthermore, the solution ionic strength was adjusted to the physiological value of 0.15 M by adding the required amounts of Na+ and Cl- ions. Each solvated system was relaxed by 500 steps of steepest descent followed by 500 other conjugate-gradient minimization steps and then gradually heated to a temperature of 300 K in intervals of 50 ps of NVT MD, using a Verlet integration time step of 1.0 fs. The Langevin thermostat was used to control temperature, with a collision frequency of 2.0 ps-1. The SHAKE method53 was used to constrain all of the covalently bound hydrogen atoms, while long-range nonbonded van der Waals interactions were truncated by using dual cutoffs of 6 and 12 Å. The particle mesh Ewald (PME) method54 was applied to treat long-range electrostatic interactions. The protein was restrained with a force constant of 2.0 kcal/(mol Å), and all simulations were carried out with periodic boundary conditions. The density of the system was subsequently equilibrated via MD runs in the isothermal − isobaric (NPT) ensemble, with isotropic position scaling and a pressure relaxation time of 1.0 ps, for 50 ps with a time step of 1 fs. Each system was further equilibrated using NPT MD runs at 300 K, with a pressure relaxation time of 2.0 ps. Five equilibration steps were performed, each 2 ns long and with a time step of 2.0 fs. To check the system stability, the fluctuations of the rmsd of the simulated position of the backbone atoms of the σ1 receptor with respect to those of the initial protein were monitored. All chemico-physical parameters and rmsd values showed very low fluctuations at the end of the equilibration process, indicating that the systems reached a true equilibrium condition. ACS Paragon Plus Environment


Page 28 of 36

28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The equilibration phase was followed by a data production run consisting of 20 ns of MD simulations in the canonical (NVT) ensemble. Only the last 10 ns of each equilibrated MD trajectory were considered for statistical data collections. All simulations were carried out using the Sander and Pmemd modules of Amber 12, running

on

the

EURORA-CPU/GPU

calculation

cluster

of

the

CINECA

supercomputer facility (Bologna, Italy). The entire MD simulation and data analysis procedure was optimized by integrating Amber 12 in modeFRONTIER, a multidisciplinary and multiobjective optimization and design environment.55 Supporting Information Available Supporting

Information

is

available

free

of

charge

via

the

Internet

at

http://pubs.acs.org. and includes physical, spectroscopic and purity data of all compounds, synthetic methods and description of the σ receptor binding assays, cytotoxicity assay and of the molecular modeling methods.

Corresponding author Bernhard Wünsch* Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, D-48149 Münster, Germany Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: [email protected]

Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the

Cells-in-motion Cluster of Excellence (EXC, 1003-CiM), University of Münster, Germany.


Page 29 of 36


29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abbreviations APCI, atmospheric pressure chemical ionization; DTG, di-o-tolylguanidine; EM, exact mass; MM/PBSA, molecular mechanics/Poisson Boltzmann Surface Area; MTT ,

3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PGRMC-1, progesterone receptor membrane component 1; PI, propidium iodide; SEM, standard error of the mean.



30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References (1)

Tam, S. W. Naloxone-inaccessible σ receptor in rat central nervous system.

Proc. Natl. Acad. Sci. USA 1983, 80, 6703-6707. (2)

Vaupel, D.B. Naltrexone fails to antagonize the σ effects of PCP and SKF 10,047 in the dog. Eur. J. Pharmacol. 1983, 92, 269-274.

(3)

Hellewell, S. B.; Bruce, A.; Feinstein, G.; Orringer, J.; Williams, W.; Bowen, W. D. Rat liver and kidney contain high densities of sigma 1 and sigma 2 receptors: characterization by ligand binding and photoaffinity labeling. Eur. J. Pharmacol. 1994, 268, 9-18.

(4)

Quirion, R.; Bowen, W. D.; Itzhak, Y.; Junien, J. L.; Musacchio, J. M.; Rothman, R. B.; Su, T. P.; Tam, S. W.; Taylor, D. P. A proposal for the classification of sigma binding sites. Trends Pharmacol. Sci. 1992, 13, 85-86.

(5)

Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H. G.; Striessnig, J.; Kemper, E.; Glossmann, H. Purification, molecular cloning, and expression of mammalian sigma1-binding site. Proc. Natl. Acad. Sci. USA 1996, 93, 80728077.

(6)

Kekuda, R.; Prasad, P.D.; Fei, Y. J.; Leibach, F. H.; Ganapathy, V. Cloning and functional expression of the human type 1 sigma receptor (hSigmaR1).

Biochem. Biophys. Res. Comm. 1996, 229, 553-558. (7)

Xu, J.; Zeng, C.; Chu, W.; Pan, F.; Rothfuss, J. M.; Zhang, F.; Tu, Z.; Zhou, D.; Zeng, D.; Vangveravong, S.; Johnston, F.; Spitzer, D.; Chang, K. C.; Hotchkiss, R. S.; Hawkins, W. G.; Wheeler, K. T.; Mach, R. H. Identification of the PGRMC1 protein complex as putative sigma-2 receptor binding site. Nature

Comm. 2011, 1. (8)

Rohe, H. J.; Ahmed, I. S.; Twist, K. E.; Craven, R. J. PGRMC1 (progesterone receptor membrane component 1): A targetable protein with multiple functions in steroid signaling, P450 activation and drug binding. Pharmacol. The. 2009,

121, 14-19. (9)

Lupardus, P. D.; Wilke, R. A.; Aydar, E.; Palmer, C. P.; Chen, Y.; Ruoho, A. E.; Jackson, M. B. Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-Protein nor ATP.

J. Physiol. 2000, 526, 527-539. (10) Hong, W.; Werling, L. L. Evidence that the σ1 receptor is not directly coupled to G proteins. Eur. J. Pharmacol. 2000, 408, 117-125. ACS Paragon Plus Environment

Page 30 of 36

Page 31 of 36


31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Vilner, B. J.; Bowen, W.D. Modulation of cellular calcium by sigma-2 receptors: release from intracellular stores in human SK-N-SH neuroblastoma cells. J.

Pharm. Exp. Ther. 2000, 292, 900-911. (12) Vilner, B. J.; John, C. S.; Bowen, W. D. Sigma-1 and sigma-2 receptors are expressed in a wide variety of human and rodent tumor cell lines. Cancer

Research 1995, 55, 408-413. (13) Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.; Abate, C.; Perrone, R.; Tortorella, V. Antiproliferative and cytotoxic effects of some σ2 agonists and σ1 antagonists in tumor cell lines. Naunyn Schmiedebergs Arch. Pharmacol. 2004,

370, 106-113. (14) Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.; Niso, M.; Simone, G. M.; Perrone, R.; Paradiso, A. Cyclohexylpiperazine derivative PB28, a sigma2 agonist and sigma1 antagonist receptor, inhibits cell growth, modulates pglycoprotein, and synergizes with antracyclines in breast cancer. Mol. Cancer

Therap. 2006, 5, 1807-1816. (15) Holl, R.; Schepmann, D.; Wünsch, B. Homologous piperazine-alcanols: chiral pool synthesis and pharmacological evaluation. Med. Chem. Commun. 2012, 3, 673-679. (16) Holl, R.; Dykstra, M.; Schneiders, M.; Fröhlich, R.; Kitamura, M.; Würthwein, E. U.; Wünsch, B. Synthesis of 2,5-diazabicylo[2.2.2]octanes by Dieckmann analogous cyclization. Aust. J. Chem. 2008, 61, 914-919. (17) De Haven Hudkins, D. L.; Fleissner, L. C.; Ford-Rice, F. Y. Characterization of the binding of [3H]-(+)-pentazocine to σ recognition sites in guinea pig brain.

Eur. J. Pharmacol.: Mol. Pharmacol. Sect. 1992, 227, 371-378. (18) Brune, S.; Schepmann, D.; Lehmkuhl, K.; Frehland, B.; Wünsch, B. Characterization of ligand binding to the σ1 receptor in a human tumor cell line (RPMI 8226) and establishment of a competitive receptor binding assay. Assay

Drug Dev. Technol. 2012, 10, 365-374. (19) Schepmann, D.; Lehmkuhl, K.; Brune, S.; Wünsch, B. Expression of sigma receptors of human urinary bladder tumor cells (RT-4 cells) and development of a competitive receptor binding assay for the determination of ligand affinity to human sigma2 receptors. J. Pharm. Biomed. Anal. 2011, 55, 1136-1141. (20) Mach, R. H.; Smith, C. R.; Childers, S. R. Ibogaine possesses a selective affinity for σ2 receptors. Life Sci. 1995, 57, 57-62. ACS Paragon Plus Environment


Page 32 of 36

32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(21) Bracht, K.; Boubakari; Grünert, R.; Bednarski, P. J. Correlations between the activities of 19 antitumor agents and the intracellular GSH concentrations in a panel of 14 human cancer cell lines. Comparisons with the NCI data. Anticancer

drugs 2006, 17, 41-51. (22) Laurini, E.; Dal Col, V.; Mamolo, M. G.; Zampieri, D.; Posocco, P.; Fermeglia, M.; Vio, V.; Pricl, S. Homology model and docking-based virtual screening for ligands of the sigma 1 receptor. ACS Med. Chem. Lett. 2011, 2, 834-839. (23) Laurini, E.; Marson, D.; Dal Col, V.; Fermeglia, M.; Mamolo, M. G.; Zampieri, D.; Vio, L.; Pricl, S. Another brick in the wall. Validation of the σ1 receptor 3D model by computer-assisted design, synthesis, and activity of new σ1 ligands. Mol.

Pharm. 2012, 9, 3107-3126. (24) Meyer, C.; Schepmann, D.; Yanagisawa, S.; Yamaguchi, J.; Dal Col, V.; Laurini, E.; Itami, K.; Pricl, S.; Wünsch, B. Pd-catalyzed

direct

C-H

bond

functionalization of spirocyclic sigma-1 ligands: generation of a pharmacophore model and analysis of reverse binding mode by docking into a 3D homology model of the sigma-1 receptor. J. Med. Chem. 2012, 55, 8047-8065. (25) Rossi, D.; Pedrali, A.; Gaggeri, R.; Marra, A.; Pignataro, L.; Laurini, E.; Dal Col, V.; Fermeglia, M.; Pricl, S.; Schepmann, D.; Wünsch, B.; Peviani, M.; Curti, D.; Collina, S. Chemical, pharmacological, and in vitro metabolic stability studies on enantiomerically pure RC-33 compounds: promising neuroprotective agents acting as σ1 receptor agonists. Chem. Med. Chem. 2013, 8, 1514-27. (26) Laurini, E.; Zampieri, D.; Mamolo, M. G.; Vio, L.; Zanette, C.; Florio, C.; Posocco, P.; Fermeglia, M.; Pricl, S. A 3D-pharmacophore model for sigma-2 receptors

based on a series

of

substituted benzo[d]oxazol-2(3H)-one

derivatives. Bioorg. Med. Chem. Lett. 2010, 20, 2954-2957. (27) Mazzei, M.; Nieddu, E.; Miele, M.; Balbi, A.; Ferrone, M.; Fermeglia, M.; Mazzei, M. T.; Pricl, S.; La Colla, P.; Marongiu, F.; Ibba, C.; Loddo, R. Activity of Mannich bases of 7-hydroxycoumarin against flaviviridae. Bioorg. Med. Chem. 2008, 16, 2591-2605. (28) Zampieri, D.; Mamolo, M. G.; Vio, L.; Banfi, E.; Scialino, G.; Fermeglia, M.; Ferrone, M.; Pricl, S. Synthesis, antifungal and antimycobacterial activities of new bis-imidazole derivatives, and prediction of their binding to P450(14DM) by molecular docking and MM/PBSA method. Bioorg. Med. Chem. 2007, 15, 74447458. ACS Paragon Plus Environment

Page 33 of 36


33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(29) Carta, A.; Loriga, M.; Paglietti, G.; Ferrone, M.; Fermeglia, M.; Pricl, S.; Sanna, T.; Ibba, C.; La Colla, P.; Loddo, R. Design, synthesis, and preliminary in vitro and in silico antiviral activity of [4,7]phenanthrolines and 1-oxo-1,4-dihydro[4,7]phenantrolines against single-stranded positive-sense RNA genome viruses. Bioorg. Med. Chem. 2007, 15, 1914-1927. (30) Carta, A.; Loriga, G.; Piras, S.; Paglietti, G.; Ferrone, M.; Fermeglia, M.; Pricl, S.; La Colla, P.; Secci, B.; Collu, G.; Loddo, R. Synthesis and in vitro evaluation of

the

anti-viral

activity

of

N-[4-(1H(2H)-benzotriazol-1(2)-

yl)phenyl]alkylcarboxamides. Med. Chem. 2006, 2, 577-589. (31) Frecer, V.; Kabeláč, M.; De Nardi, P.; Pricl, S.; Miertuš, S. Structure-based design of inhibitors of NS3 serine protease of hepatitis C virus. J. Mol. Graphics

Modell. 2004, 22, 209-220. (32) Felluga, F.; Pitacco, G.; Valentin, E.; Coslanich, A.; Fermeglia, M.; Ferrone, M.; Pricl, S. Studying enzyme enantioselectivity using combined ab initio and free energy calculations: alfa-chymotrypsin and methyl cis- and trans-5-oxo-2pentylpirrolidine-3-carboxylates. Tetrahedron: Asymmetry 2003, 14, 3385-3399. (33) Case, D. A.; Darden, T.; III Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Kolossvary, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M-J., Cui, G.; Roe, D. R.; Mathews, D. H.; Seetin, M. G.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER 11; University of California: San Francisco, CA, 2010. (34) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 2003,

24, 1999-2012. (35) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 1993, 97, 10269-10280. (36) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Kollman, P. A. Application of the RESP charges to calculate conformational energies, hydrogen bond energies, and free energies of solvatation. J. Am. Chem. Soc. 1993, 115, 9620-9631. ACS Paragon Plus Environment


34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(37) Cieplak, P.; Cornell, W.; Bayly, C.; Kollman, P. Application of the multimolecule and multiconformational RESP methodology to biopolymers: charge derication for DNA; RNA, and proteins. J. Comput. Chem. 1995, 116, 1357-1376. (38) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 2004, 25, 11571174. (39) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785-2791. (40) Woolf, T. B.; Roux, B. Structure, energetics, and dynamics of lipid-protein interactions: A molecular dynamics study of the gramicidin A channel in a DMPC bilayer. Proteins 1996, 24, 92-114. (41) Jo, S.; Kim, T.; Im, W. Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PLoS One. 2007, 2, 880. (42) Jo, S.; Lim, J. B.; Klauda, J.B.; Im, W. CHARMM-GUI membrane builder for mixed bilayers and its application to yeast membranes. Biophys. J. 2009, 97, 50-8. (43) Skjevik, Å. A.; Madej, B .D.; Walker, R. C.; Teigen, K.LIPID11: A Modular framework for lipid simulations using Amber. J. Phys. Chem. B. 2012, 116, 11124-36. (44) Carta, A.; Briguglio, I.; Piras, S.; Boatto, G.; La Colla, P.; Loddo, R.; Tolomeo, M.; Grimaudo, S.; Di Cristina, A.; Pipitone, R. M., Laurini, E.; Paneni, M. S.; Posocco,

P.;

Fermeglia,

M.;

Pricl,

S.

3-Aryl-2-[1H-benzotriazol-1-

yl]acrylonitriles: a novel class of potent tubulin inhibitors. Eur. J. Med. Chem. 2011, 46, 4151-4167. (45) Liu, X.; Chen, H.; Laurini, E. Wang, Y.; Dal Col, V.; Posocco, P.; Ziarelli, F. Fermeglia, M.; Zhang, C. C.;. Pricl, S.; Peng, L. 2-Difluoromethylene-4methylenepentanoic acid, A paradoxical probe able To mimic the signaling role of 2-oxoglutaric acid in cyanobacteria. Org. Lett. 2011, 13, 2924-2927. (46) Giliberti, G.; Ibba, C.; Marongiu, E.; Loddo, R.; Tonelli, M.; Boido, V.; Laurini, E.; Posocco, P.; Fermeglia, M.; Pricl, S. Synergistic experimental/computational studies on arylazoenamine derivatives that target the bovine viral diarrhea virus RNA-dependent RNA polymerase. Bioorg. Med. Chem. 2010, 18, 6055-6068.


Page 34 of 36

Page 35 of 36


35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47) Tonelli, M.; Boido, V.; La Colla, P.; Loddo, R.; Posocco, P.; Paneni, M. S.; Fermeglia, M.; Pricl, S. Pharmacophore modeling, resistant mutant isolation, docking, and MM-PBSA analysis: Combined experimental/computer-assisted approaches to identify new inhibitors of the bovine viral diarrhea virus (BVDV).

Bioorg. Med. Chem. 2010, 18, 2304-2316. (48) Carta, A.; Pricl, S.; Piras, S.; Fermeglia, M.; La Colla, P.; Loddo, R. Activity and molecular modeling of a new small molecule active against NNRTI-resistant HIV-1 mutants. Eur. J. Med. Chem. 2009, 44, 5117-5122. (49) Tonelli, M.; Vazzana, I.; Tasso, B.; Boido, V.; Sparatore, F.; Fermeglia, M.; Paneni, M. S.; Posocco, P.; Pricl, S.; La Colla, P.; Ibba, C.; Secci, B.; Collu, G.; Loddo, R. Antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives. Bioorg. Med. Chem. 2009, 17, 4425-4440 and references therein. (50) Onufriev, A.; Bashford, D.; Case, D.A. Modification of the generalized born model suitable for macromolecules. J. Phys. Chem. B 2000, 104, 3712-3720. (51) Feig, M.; Onufriev, A.; Lee, M. S.; Im, W.; Case, D. A.; Brooks III, C. L. Performance comparison of generalized born and poisson methods in the calculation of electrostatic solvation energies for protein structures. J. Comput.

Chem. 2004, 25, 265-284. (52) Jorgensen, W. L.;. Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem.

Phys. 1983, 79, 926-935. (53) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327-341. (54) Toukmaji, A.; Sagui, C.; Board, J.; Darden, T. Efficient particle-mesh Ewald based approach to fixed and induced dipolar interactions. J. Chem. Phys. 2000,

113, 10913-10927. (55) http://www.esteco.com/home/mode_frontier/mode_frontier.html.



Page 36 of 36

36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic

R1 COOH H2N

N

COOH H

T181 N R2

H

D126

L186

OH

7c R1 = CH2C6H11 2 R = 2-naphthylmethyl σ1 (guinea pig): Ki = 4.7 nM σ2 (rat): Ki = 69 nM σ1 (human): Ki = 6.8 nM σ2 (human): Ki = 31 nM

I128 R119


F133 Y120 Y17

Molecular design and synthesis of 1,4-disubstituted piperazines as α(1)-adrenergic receptor blockers.

Synthesis and quantitative structure-activity relationships of antibacterial 1-(substituted benzhydryl)-4-(5-nitro-2-furfurylideneamino) piperazines.

De Novo Assembly of Highly Substituted Morpholines and Piperazines.

Pharmacological evaluation of the beta-adrenoceptor agonist and thromboxane receptor blocking properties of 1-benzyl substituted trimetoquinol analogues.

Antidepressant and anticonvulsant activity of 1-(5-phenyl-4-oxazolin-2-yl)-4-substituted piperazines.

Synthesis and pharmacological evaluation of some 3-substituted octahydropyrido(2,1-c)(1,4)oxazines.

Palladium-Catalyzed Modular Synthesis of Substituted Piperazines and Related Nitrogen Heterocycles.

Synthesis of N-substituted C-normorphinans and their pharmacological properties.

kappa opioid receptor dual selectivity.

Synthesis and pharmacological evaluation of carvacrol propionate.

4-aroylpiperidines and 4-(α-hydroxyphenyl)piperidines as selective sigma-1 receptor ligands: synthesis, preliminary pharmacological evaluation and computational studies.

Synthesis and evaluation of high affinity, aryl-substituted [18F]fluoropropylbenzamides for dopamine D-2 receptor studies.

Synthesis and evaluation of N⁶-substituted apioadenosines as potential adenosine A₃ receptor modulators.

Synthesis and evaluation of antidepressant-like activity of some 4-substituted 1-(2-methoxyphenyl)piperazine derivatives.

Synthesis and biological evaluation of some novel 1-substituted fentanyl analogs in Swiss albino mice.

Synthesis and pharmacological evaluation of amino, azido, and nitrogen mustard analogues of 10-substituted cannabidiol and 11- or 12-substituted delta 8-tetrahydrocannabinol.

Synthesis and pharmacological evaluation of M4 muscarinic receptor positive allosteric modulators derived from VU10004.

Synthesis, pharmacological evaluation and molecular modeling studies of triazole containing dopamine D3 receptor ligands.

Correction: Synthesis, dynamic NMR characterization and XRD studies of novel N,N'-substituted piperazines for bioorthogonal labeling.

Synthesis and initial biological evaluation of substituted 1-phenylamino-2-thio-4,5-dimethyl-1H-imidazole derivatives.

Synthesis and pharmacological characterization of 1-benzyl-4-aminoindole-based thyroid hormone receptor β agonists.

Design, synthesis, and pharmacological evaluation of novel 2-(4-substituted piperazin-1-yl)1, 8 naphthyridine 3-carboxylic acids as 5-HT3 receptor antagonists for the management of depression.

Synthesis and pharmacological evaluation of mono-arylimidamides as antileishmanial agents.

Ester Prodrugs of Ketoprofen: Synthesis, Hydrolysis Kinetics and Pharmacological Evaluation.