Bioorganic & Medicinal Chemistry 22 (2014) 2692–2706

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Preliminary investigations into triazole derived androgen receptorantagonists

http://dx.doi.org/10.1016/j.bmc.2014.03.0180968-0896/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +61 3 52272767; fax: +61 3 52271045.E-mail address: luke.henderson@deakin.edu.au (L.C. Henderson).

Jarrad M. Altimari a, Birunthi Niranjan b, Gail P. Risbridger b, Stephanie S. Schweiker c, Anna E. Lohning c,Luke C. Henderson a,d,⇑a Strategic Research Center for Chemistry and Biotechnology, Deakin University, Pigdons Road, Waurn Ponds Campus, Geelong 3216, Victoria, Australiab Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Victoria 3800, Australiac Faculty of Health Sciences and Medicine, Bond University, Gold Coast 4229, Queensland, Australiad Institute for Frontier Materials, Deakin University, Pigdons Road, Waurn Ponds Campus, Geelong 3216, Victoria, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 January 2014Revised 5 March 2014Accepted 13 March 2014Available online 1 April 2014

Keywords:Click chemistryAndrogen receptorTriazoleMolecular modellingProstate cancer

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidalandrogen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amideportion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biologicalactivity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Usingthis methodology a series of 17 N-aryl triazoles were synthesized from commercially available startingmaterials in less than 3 h. After preliminary biological screening at 20 and 40 lM, the most promisingthree compounds were found to display IC50 values of 40–50 lM against androgen dependent (LNCaP)cells and serve as a starting point for further structure–activity investigations. All compounds in thiswork were the focus of an in silico study to dock the compounds into the human androgen receptorligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandro-gens. A comparison of receptor–ligand interactions for the wild type and T877A mutant AR revealedtwo novel polar interactions. One with Q738 of the wild type site and the second with the mutatedA877 residue.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction A common structural feature of non-steroidal AR antagonists is

Prostate cancer (PC) is the most commonly diagnosed cancer inmen and is a leading cause of death in the male population, in 2013prostate cancer was estimated to account for almost 14% of alldiagnosed cancers.1 Antagonistic binding of the steroid-dockingsite within the androgen receptor (AR) is the most common formof small-molecular therapy for PC. The antagonistic binding miti-gates cancer cell proliferation and disease progression by prevent-ing the binding of endogenous testosterone or dihydrotestosterone(5a-DHT) into the AR. The initial inspiration of PC chemotherapywas the chemical modification of naturally occurring steroidal li-gands. Though viable, these compounds were plagued with poorbioavailability, hepatotoxicity and a lack of tissue specific action,leading to their discontinued clinical use.2 As such, non-steroidalAR antagonists, such as Flutamide� 1 (its active metabolite 2),Enzalutamide (MDV3100) and Bicalutamide 3 (Casodex�) are thecurrent state of the art in androgen blockade therapy (Fig. 1).

the extremely electron-deficient N-phenyl amide moiety. The arylportion commonly bears a trifluoromethyl group (CF3) in additionto a strongly deactivating group at the para-position, relative to theamide.3–8 Typical examples of para-substitution are the nitro group(NO2) in the case of 1 and 2, or the nitrile (CN) of 3 and MDV3100.Also, electron deficient N-phenyl amides have found widespreaduse in a variety of fields such as antimicrobials, anti-HIV therapy,Raf inhibitors and have shown potential as antitubercularagents.9–15

Recently, we reported the synthesis of several anti-androgenic(±)-a-lipoic acid derivatives bearing meta- and para- substitutionpatterns of CF3–NO2 and CF3–CN, respectively.8 Though success-fully synthesised, we have found low-to-moderate yields (typically<50%) of the amide formation due to the electron deficient aryl ringdespite extensive optimisation (Scheme 1).

In addition to low yields, another common problem noted by usand others,8,16 is the tedious removal of residual deactivated ani-line present in the crude product from the desired N-phenyl amide.This occurs usually from the inability of the deactivated aniline tobe protonated and washed out during acidic work-up and/or theirtendency to possess poorly defined Rf values (streaking) during

HN

CF3

O2NO

1

4

N

NNR2

R1

HN

CF3

O2NO

2

OH

HN

NCCF3

O

HOS

O

O

F

3R1 = NO2, CF3, CN, etc

R2 = Ph

OH

N

F FF

N N

NH

O

F

S

O

MDV3100

Figure 1. Enzalutamide MDV3100, flutamide 1, hydroxyflutamide 2, bicalutamide 3 and proposed triazoles 4.

H2NPyBrop, CHCl3

100 °C, 1 hr

R1

R2

6 R1 = NO2, R2 = CF35

7 R1 = CN, R2 = CF3

8 R1 = NO2, R2 = F3, 46%

9 R1 = CN, R2 = CF3, 34%

SS

CO2H4

+S

S

4HN

O

R1

R2

Scheme 1. Previously synthesised electron deficient N-arylamides.

Table 1Synthesis of aryl azides

NH2

R2

R1

N3

R2

R1

12 13

R3 R3

1. 1:8, H2O:MeCN then HCl10 minutes, r.t.

2. NaNO2, 1 hr

3. NaN3, 1 hr, 0 °C to r.t.

Entry Compound R1 R2 R3 Yield (%)

1 14 H H H 622 15 CF3 CN H 953 16 H Cl H 674 17 H CN H 955 18 CF3 NO2 H 766 19 CF3 H CF3 687 20 H NO2 H 828 21 CF3 Cl H 95

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2693

column chromatography. To circumvent this problem, Sugai et al.16

used an acetylation technique (derivatising both desired com-pound and residual starting aniline) to allow for chromatographicresolution. Though an effective strategy, the acyl group then hadto be removed from their target compound, adding several syn-thetic steps which is not optimal. Despite these low yields andproblematic purifications, there is no optimised methodology oralternative functional group easily accessible for this type of com-pound. As such we turned our attention to the use of 1,2,3-triazoleswhich have been used extensively as amide bond isosteresthroughout medicinal chemistry for the past decade.17–26 Recently,Antonella and co-workers have synthesised several triazole-derived compounds inspired by Bicalutamide 2.27

As such, the impetus for this study was to replace theN-phenylamide moiety of simple AR antagonists with a 1,4-substi-tuted-1,2,3-triazole and determine if there is any retention ofbiological activity against androgen dependent cells (LNCaP).Therefore, we designed a range of potential triazole derived ARantagonists inspired by anti-androgen Flutamide 1. The syntheticprocedures presented here are rapid, robust and require no purifi-cation techniques. Indeed, in contrast to the electron deficientanilines being a problematic for amide formation, these same deac-tivated anilines, using this approach, are the best performing sub-strates. The isosteric replacement of the amide with a triazole hasan additional benfit in that these compounds will also probe theimportance of the amide carbonyl present in many common ARantagonists. These compounds were then evaluated in vitro fortheir ability to inhibit androgen stimulated cell proliferation usingan androgen dependent (LNCaP) cell line. Following this, com-pounds 23–39 were docked into the human ligand binding domain(hARLBD) of wild type AR and the mutated AR present in LNCaPcells and a comparison of interactions is presented. The latter ofwhich possesses a key point mutation of threonine 877 for analanine (T877A) allowing for correlation to in vitro studies.

2. Results and discussion

2.1. Synthesis of 1,4-substituted-1,2,3-triazoles

We began our investigation into the conversion of anilinicamines into phenyl azides, of general structure 13, via diazo-phenyl

formation in situ facilitated by sodium nitrite in HCl, using modifiedconditions reported by Lear et al. (Table 1).28 Starting with aniline12 (R1–3 = H), an electronically ‘neutral’ substrate, these conditionsfurnished phenyl azide 14 in moderate yield (62%) and in >95%crude purity (Table 1, entry 1). Encouraged by this result we movedon to 3-trifluoromethyl-4-cyanoaniline (R1 = CF3, R2 = CN, R3 = H),an aryl unit commonly used in prostate cancer therapy,8,29–33 whichproceeded much better than the previous example giving the de-sired azide 15 in 95% yield (Table 1, entry 2).

Applying these conditions to a series of substituted anilinesgave the desired azides (16–21) in good to excellent yields (67–95%) and were isolated analytically pure as the crude material. Pre-sumably the azide formation occurs much slower for anilineswhich were obtained in <70% yield (Table 1, entries 1, 3 and 6) thanthe other examples. Nevertheless these compounds were still iso-lated in synthetically usable yields.

With phenyl azides 14–21 in hand, our attention turned to theapplication of these compounds to the copper-alkyne-azide-cyclo-addition (CuAAc) reaction. We chose the CuAAc reaction between21 and phenylacetylene as our model reaction of choice as this pro-vided a simple, yet novel, triazole 22. Our initial reaction condi-tions used a combination of copper(II) sulfate and ascorbic acidin a water/ethanol mixture at room temperature (Table 2, entry1) which only gave a trace (<5%) of the desired compound.

Repeating this reaction at a higher temperature (100 �C) usingmicrowave irradiation, we found that the reaction proceeded in30 min (Table 2, entry 2), giving the desired compound in excellentyield (89%). The drastic reduction of reaction duration usingmicrowave irradiation has been shown several times within ourgroup34–38 and is a phenomena commonly observed in syntheticchemistry.39–43 A copper source which is soluble in organic solvents

Table 3Reaction scope

NN N

R4

R2

R1Alkyne (1.0 eq), CuSO4 (10 mol%),Ascorbic acid (20 mol%), H2O

MW, 100 °C, 30 min

N3

R2

R1 R3

R3

Entry Product R1 R2 R3 Alkyne Yield (%)

1 23 H H H 75

2 24 H NO2 H 52

3 25 CF3 NO2 H 87

4 26 H Cl H 64

5 27 H CN H 96

6 28 CF3 CN H 95

7 29 CF3 H CF3 92

8 30 CF3 Cl H OH 83

9 31 H H H OH 81

10 32 H NO2 H OH 75

11 33 CF3 NO2 H OH 79

12 34 H Cl H OH 80

13 35 H CN H OH 64

14 36 CF3 CN H OH 93

15 37 CF3 H CF3 OH 79

16 38 CF3 NO2 HCO2Me

NHFmoc96

17 39 CF3 CN HNHFmoc

77

2694 J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706

was also used in an effort to increase the product yield within thesame timeframe. Employing copper(I) chloride (Table 2, entry 3) inwater at 100 �C gave a marginally better yield (90%) than that ob-served with copper(II) sulfate, similarly Cu2O (Table 2, entry 4) gavea good yield but required a longer reaction time. As such we consid-ered copper(II) sulfate the optimal catalyst as this can be made as astock solution and is stable on the bench, whereas copper(I) chloriderequires frequent regeneration. We then reduced the reaction timeto 20 and 15 min resulting in slightly reduced yield (Table 2, entries5 and 6, respectively).

Application of our optimal reaction conditions (Table 2, entry 2)to the remaining azides gave the desired triazoles 23–39 in good toexcellent yield (75–96%), with only triazoles 24 and 26, beingisolated in moderate yield (52% and 64%, respectively).

Following the series of phenylacetylene derivatives (Table 3,entries 1–7) we chose 2-methyl-3-butyn-2-ol as the alkyne partneras this group bears significant resemblance to the carbonyl portionof hydroxyflutamide 2, the active androgen receptor antagonistwhich results from in vivo metabolism of Flutamide 1.

Again, under the specified conditions, all the desired triazoleswere obtained in very good yields. Of particular note are 33 and36 which were isolated in excellent yields of 79% and 93%, respec-tively and possess the same aryl substitution pattern present inFlutamide 1 and Bicalutamide 3. Finally, the last alkyne partnerwas Fmoc protected propargyl glycine methyl ester, giving thecorresponding triazoles 38 and 39 (Table 3, entries 16 and 17) inexcellent yield (96% and 77%, respectively) and possess the orthog-onal reactivity of the amino acid group in the scaffold allowing fora myriad of potential synthetic derivatisation.

It is important to note that no purification was required to obtainanalytically pure samples for all compounds presented in Table 3.Examination of the literature showed that triazoles 23, 26 and 27have been made by others,44–47 though in all cases longer reactiontimes were required than those in this work, and ours is the only sys-tem to use an entirely aqueous solvent system.

With a range of the desired triazoles in hand we checked theLipinski parameters of these compounds (refer to ESI) to which tri-azoles 23–39 conformed to Lipinski’s rules well with only 38 and39 having any violations, due to molecular weight. Recently, Guoand co-workers48 synthesised several AR antagonists on thehypothesis that small molecules possessing cLogP values <3.5and polar surface areas (PSA) of >70 Å would demonstrate betterin vivo clearance profiles while possessing sufficient polarity with-

Table 2Investigation into optimal reaction conditions

N3

21

Cl

22

F3C

ClF3C

Ph

"Cu" (10 mol%)

N

NN

Ph

Entry Catalysta Time (h) Tempb (�C) Solvent Yield (%)

1 CuSO4c 24 rt EtOH/H2O Trace

2 CuSO4c 0.5 100 H2O 89

3 CuCl 0.5 100 H2O 904 Cu2O 24 rt CHCl3 835 CuSO4

c 20 min 100 H2O 836 CuSO4

c 15 min 100 H2O 79

a 10 mol % of Cu catalyst was used in all reactions.b Heated using microwave radiation unless at rt.c Carried out with ascorbic acid (20 mol %).

CO2Me

in the AR-LBD to discourage helix-12 closure, thus minimising ARtransactivation. With this in mind the majority of triazoles syn-thesised in this study fulfil these criteria very well and as suchour next focus was investigating the in vitro activity of these com-pounds in LNCaP cells.

2.2. Evaluation of triazoles in vitro

Initially, we undertook a preliminary biological evaluation as ascreening tool to identify compounds for further studies. Thisscreening process involved monitoring cell proliferation of LNCaPcells at two static concentrations, 20 and 40 lM (Table 4). Fromthis data, any compounds showing promising anti-proliferativeproperties would be selected and IC50 values determined.

Evaluation of these compounds for their potential to stopandrogen stimulated growth in LNCaP cells gave a wide varietyof results. While the reduction in LNCaP cell growth was minimalfor the majority of compounds (typically 5–10%), relative to DHT

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2695

control, compounds 32, 34, 36 and 37 displayed moderate to goodlevels of growth inhibition (Table 4, entries 11, 13, 15 and 16).

Compounds 34 and 36 (Table 4, entries 13 and 15), possessingthe aryl substitution pattern for Bicalutamide 3 and Flutamide 1,respectively, also showed very promising levels of LNCaP growthinhibition at both 20 and 40 lM compared to the positive control.Interestingly, 37 (Table 4, entry 16) showed the most promisinginhibitory activity at 40 lM, despite having minimal similarity toexisting AR antagonists. Based on these preliminary results threeof these compounds (32, 36 and 37) were selected for furtherin vitro evaluation to determine IC50 values as these compoundsdisplayed promising activity while also were representative exam-ples of aromatic ring substitution patterns/electronics.50 In theinterest of thoroughness, these three compounds were alsoscreened for IC50 values against PC-3 cells which are androgenindependent and effective inhibition of PC3 cells remains a chal-lenge.49, 51–53

As seen from Figure 2, these compounds possessed moderateactivity for LNCaP growth inhibition, as was expected from the pre-liminary screen, with IC50 values from 40 to 50 lM. Both 32 and 36showed a large increase in IC50 for the PC-3 cell line (relative to

Table 4Preliminary in vitro evaluation of compounds 23–39

Entry # R1 R2 R3 R4

1 3 — — — —

2 23 H H H

3 24 H Cl H

4 25 H CN H

5 26 CF3 CN H

6 27 H NO2 H

7 28 CF3 NO2 H

8 29 CF3 H CF3

9 30 H H H OH

10 31 H Cl H OH

11 32 CF3 Cl H OH

12 33 H CN H OH

13 34 CF3 CN H OH

14 35 H NO2 H OH

15 36 CF3 NO2 H OH

16 37 CF3 H CF3 OH

17 38 CF3 CN H

NHFmoc

O

OMe

18 39 CF3 NO2 H

NHFmoc

O

OMe

a Value given is relative to a positive control using 2 nM of DHT.b Negative values imply agonistic binding to the AR.

LNCaP values), though interestingly 37 possessed an IC50 only mar-ginally increased compared to that of the LNCaP cell line (40 lM vs42 lM). The reason for the similar IC50 displayed by 37 is unknownbut is presumably due to the bis-trifluoromethyl groups on the arylportion. In any case this aryl moiety has been the basis of a newseries of compounds currently under investigation within ourgroup with the aim of developing potent PC-3 inhibitors and anyfindings will be reported in due course. Despite all inhibition val-ues for 23–39 being quite high we were pleased with these levelsof activity considering the rapid synthetic methodology to accessthese compounds and that they are both the first and simplestscaffolds to be accessed. This also suggests that the amide carbonylof existing AR antagonists plays an important role in binding to theAR.

Nevertheless, given the success with which the 1,2,3-triazolescaffold has been used to mimic an amide functionality throughoutmedicinal chemistry we were curious about the low-to-moderateactivities which had been displayed by the majority of these com-pounds. As such we turned to in silico docking of these compoundsto rationalise these results. The LNCaP cell line possesses a keypoint mutation within the ligand binding domain at threonine

LNCaP inhibitiona (20 lM, %) LNCaP inhibitiona (40 lM, %)

28 33

7 6

9 15

4 10

13 6

�3b �11b

�11b �14b

�12b �16b

7 4

5 10

12 21

14 13

20 24

15 16

15 24

15 37

10 11

8 16

32LNCaP IC50 = 49 µMPC-3 IC50 = 69 µM

ClF3C

N

NN

OH

36LNCaP IC50 = 45 µMPC-3 IC50 = 63 µM

NO2

F3C

N

NN

OH

37LNCaP IC50 = 40 µMPC-3 IC50 = 42 µM

F3C

N

NN

OH

CF3

Figure 2. IC50 values for compounds 32, 56 and 37 against LNCaP and PC-3 celllines.

2696 J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706

877, which is replaced with an alanine residue, commonly referredto as T877A. Thus, we carried out molecular docking of these com-pounds into the wild type LBD and T877A mutated LBD, a compar-ison of interactions is provided.

2.3. Molecular modelling

Our initial work in this area was to examine triazoles 23–39 andexamine them from an in silico perspective to map the energeticlandscape within the hARLBD of the T877A mutant and comparewith that of the wild type LBD. Numerous high resolution crystalstructures have been determined bound to either cognate ligands,testosterone and dihydrotestosterone (DHT), or one of a vast arrayof inhibitors, both steroidal and non-steroidal in nature. In thisstudy the compound library was initially docked into the hARstructure, 2AMB.pdb,51 bound to tetrahydrogestrione (THG), a po-tent competitive inhibitor of endogenous androgens. However,since the ligand-based approach of Surflex-Dock uses the volumeand nature of the reference ligand to generate the protomol (i.e.,the conformational space within which the compounds aredocked), a target protein with a larger volume ligand was prefera-ble. For this reason 2PNU.pdb was selected as its bound ligand,EM5744 40, covered a larger space within the ligand binding

Figure 3. A: 2PNU with bound steroidal antagonist, EM5744, 40 (orange), with ribbon bresidues in blue. B: 2OZ7 with bound steroidal antagonist, CPY, 41 (orange). Ribbon backresidues in blue.

domain.54 EM5744 40 was specifically designed to hinder the he-lix-12 (H12) conformational change required for AF-2 transactiva-tion function essential during its transcriptional activity, 40 has anadditional chain at C18 which extends towards the H12 site withinthe hARLBD (Fig. 3). The role of H12 within transactivation is toclose over the LBD after ligand docking (DHT), this reinforces theligand–receptor interaction while also creating a hydrophobic cleftwhich allows the recruitment of co-activators required to initiateAR transcription.55 The crystal structure for the hARLBD withT877A mutation was 2OZ7.pdb with bound cyproterone acetate(CPY, 41).56

Validation for the docking method involved determining theheavy atom root mean square deviation (RMSD) between the origi-nal X-ray structure ligands conformation and its docked poses. AnRMSD of less than 2 Å was considered a good reproduction of thecrystal structure conformation. For 2PNU the RMSD betweenEM5744 40 and its docked pose was found to be 0.482 and0.949 Å for 2OZ7 the RMSD between CYP 41 and its docked pose.Subsequent docking of ligands was thereafter assumed to be rea-sonably well predicted by Surflex-Dock. Surflex-Dock was rankedin the top three docking programs in performance in terms ofdocking accuracy and in ranking known inhibitors in a virtualscreening experiment.57

Surflex-dock’s scoring function ranks ligands in order of highestbinding interaction (total score (pKD), note that binding affinity isnot necessarily predictive of inhibitory potency) and includes con-tributions from (in order of import) hydrophobic complementarity,polar complementarity and entropy with the first two dominatingthe scoring function.55

We initially docked the triazoles 23–39 into the wild-type ARreceptor (2PNU) to gain insight into how these compounds mayinteract with the native AR prior to several key point mutationswhich commonly occur after AR-antagonistic therapy results inthe development of hormone-independent prostate cancer. It hasbeen suggested56 that mutations that lead to increased promiscu-ity within the LBD binding site facilitate AF-2 transactivation viarepositioning H12. While the replacement of T877 with A877 pro-vides space to accommodate more bulky antagonists, a key hydro-gen bonding group is removed. Compounds 23–39 were then

ackbone coloured on secondary structure. Position of H12 shown in cyan, key LBDbone coloured on secondary structure. Position of H12 shown in cyan and key LBD

Table 5Scores for binding to the AR for both wild type (left) and T877A mutant (right)

N

NN

R4R3

R2

R1

Entry # R1 R2 R3 R4 2PNU score (pKD) 2OZ7 scorea (pKD)

1 3 — — — — 6.6 5.6

2 23 H H H 3.8 3.3

3 24 H Cl H 4.0 3.1

4 25 H CN H 3.9 3.0

5 26 CF3 CN H 3.6 3.7

6 27 H NO2 H 3.4 2.9

7 28 CF3 NO2 H 2.4 3.0

8 29 CF3 H CF3 3.9 1.8

9 30 H H H OH 4.9 4.2

10 31 H Cl H OH 4.1 3.3

11 32 CF3 Cl H OH 3.7 3.6

12 33 H CN H OH 5.1 3.7

13 34 CF3 CN H OH 4.8 3.7

14 35 H NO2 H OH 4.6 3.1

15 36 CF3 NO2 H OH 4.5 3.7

16 37 CF3 H CF3 OH 4.5 4.9

17 38R CF3 CN H

NHFmoc

O

OMe 7.1 2.6

18 38S CF3 CN H

NHFmoc

O

OMe 8.6 1.9

19 39R CF3 NO2 H

NHFmoc

O

OMe 5.8 2.1

20 39S CF3 NO2 H

NHFmoc

O

OMe 8.0 1.2

21 40 — — — — 12.4 —22 41 — — — — — 9.923 1 — — — — 6.3 4.424 MVD3100 — — — — 2.7 0.7

a LNCaP cells possess a key point mutation in the AR whereby a threonine is replaced with an alanine (T877A).

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2697

docked into the LBD of the T877A mutant, 2OZ7 to investigate theimpact within the binding site. Additionally, as 38 and 39 are theonly compounds to possess a chiral carbon, both enantiomers wereevaluated in silico, despite only the natural isomer being evaluatedin vitro. Table 5 lists the results of docking into the hARLBD of2PNU.pdb and 2OZ7.pdb. Score (pKD) values are shown with the

higher values corresponding to higher predicted binding affinity,with both crystal structure ligands, EM5744 40 and CYP 41, assum-ing the top position in each run as expected.

The bulky Fmoc compounds (38R/S and 39R/S) generally scoredhighest in the 2PNU site followed by the tertiary alcohols (30–37)and then the aryl compounds (23–29) whereas for the mutated

2698 J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706

2OZ7 site the tertiary alcohols (30–37) scored highest followed bythe aryl compounds (23–29) and then Fmoc compounds (38R/Sand 39R/S) which was surprising as it may be expected that thebulkier groups would be less well accommodated in the smallersite of 2PNU.

The docked poses for the aryl compounds, 23–29, were ob-served to adopt one of two orientations within the 2PNU LBD.The compounds in this series most highly stabilized (23, 24 and29) were oriented to maximize stabilization by two key hydrogenbond interactions between the triazole nitrogen and T877 (Fig. 4).

In this orientation the substituted benzene is directed into thehydrophobic pocket flanked by H12 and W741. The substitutedring of 29 is rotated compared to that of 23 to better accommodatethe two bulky CF3 groups while the chlorine atom of 24 is orientedtowards the polar N738.

The remaining mono and di-substituted compounds (25–28)were docked into the site normally occupied by the steroid nucleusof the cognate ligand achieving a degree of stabilization by hydro-gen bonding the polar substituent to R752 (Fig. 5). The linear CN

Figure 4. 2PNU–aryl compound 23 (atom

Figure 5. 2PNU–compounds 25 (atom colours)

group of 25 scored highest in this position while introducing a sec-ond substituent caused a loss of stability due to steric interference.

Figure 6 depicts all 7 docked aryl substituted compounds,23–29 within the 2OZ7 T877A mutant site. In this case only oneorientation was observed similar to that normally occupied bythe steroid nucleus of the cognate ligand. In all cases the substi-tuted ring is oriented to maximize hydrogen bonding opportunitieswith R752 and N711. Here 26 scores highest facilitating two hydro-gen bonds, one each to R752 and N711, though compounds 23 and24 are small and able to minimize steric issues within the site.

Within the tertiary alcohol series, one of two possible orienta-tions within the 2PNU site were observed depending on positionand length of substituents on the aryl ring (Fig. 7). Those com-pounds that had either no substituent (30), one or two atom singlesubstituents (31 and 33) as well as the meta di-substituted groups(37) were positioned within the small hydrophobic flanked by theH12 and W741.

Stabilization of this position was afforded by hydrogen bondingbetween T877 and both triazole and hydroxyl groups of the

colours), 24 (violet) and 29 (red).

, 26 (cyan), 27 (magenta) and 28 (orange).

Figure 6. 2OZ7–aryl compounds 23 (atom colours), 24 (orange), 25 (cyan), 26 (green), 27 (purple), 28 (beige) and 29 (red).

Figure 7. 2PNU–tertiary alcohols 30 (atom colours), 31 (red), 33 (cyan), 34 (green), 35 (orange), 36 (brown), 37 (violet).

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2699

ligands. Otherwise those ligands with three atom, single substitu-ents (35) and those with ortho disubstituted groups (34 and 36)were oriented to facilitate hydrogen bonding to R752 and betweenthe hydroxyl group of the ligand and T877. Compound 33 had thehighest degree of stabilization in this set due to the additional sta-bilization from the hydrogen bond to N738. The smallest of the set,30, had the second highest total score within the 2PNU LBD. Thenext three (34–36) were next in terms of score and were all stabi-lized by the strong hydrogen bonding interactions with R752 aswell as T877. The most destabilization was seen in 32 (Fig. 8)which was due to the steric hindrance from the bulky CF3 orthoto the chlorine and loss of hydrogen bond to T877.

For the hARLBD T877A mutant site the tertiary alcohols werefound in a different orientation (Fig. 9), having to find alternatearrangements due to the loss of the hydrogen bond to T877. As aresult, the hydroxyl group of compounds 30 and 31 were observedto hydrogen bonding with L704 whereas the longer compounds 33and 35 interacted with N705. By orienting to N705 these

compounds were able to exploit additional hydrogen bonding be-tween their substituted group, CN or NO2 respectively, to Q711and R752.

Di-substitution of the benzene ring results in two possible ori-entations (Fig. 10). Compound 37 had the highest score orientedwith its bulky CF3 group towards Q711. The least bulky 32 adopteda similar orientation within the site and was observed to make ahydrogen bond to N705. Compounds 34 and 36 were flipped in ori-entation with their di-substituted groups closer to L880.

Within the 2PNU binding site both 38S and 38R were observedin an extended conformation stabilized centrally by two hydrogenbonds between the triazole nitrogens and T877 side chain (Fig. 11,top). The substituted benzene ring is positioned in close proximityto the H12 within range for polar interactions with Q738, while theFmoc group was observed to bind in a hydrophobic site normallyoccupied by the steroid nucleus of the cognate ligands. A high de-gree of polar and non-polar stabilization contributes to the rankingof these compounds as first and third in the group.

Figure 8. 2PNU–tertiary alcohol 32.

Figure 9. 2OZ7–tertiary alcohols 30 (atom colours), 31 (red), 33 (cyan), 35 (orange).

2700 J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706

In contrast, a single substitution in the 2OZ7 T887A mutantsite, results in the destabilization of these two compounds intoa folded conformation where the peptide bond acts as a hingeallowing intramolecular interactions between the two ring sys-tems (Fig. 11, bottom). These unfavorable conformations causedan overall destabilizing effect as reflected in the lower scoredespite a high degree of hydrogen bonding. Two hydrogen bondsstabilized 38S between its CN group and both Q711 and R752.Two hydrogen bonds were observed between the carbonyl of38R and both W741 and Q711 with a third between the etheroxygen and Q711.

As for both isomers in 38, 39R also displays two hydrogenbonds between the triazole ring and T877 of the 2PNU site whereasno hydrogen bonding was observed for its stereoisomer (Fig. 12). Inthe more accommodating 2OZ7 site however 39R made threehydrogen bonds, one between the ester O and the Q711 and twobetween the peptide carbonyl and each of W741 and Q711. For39S a unique hydrogen bonding interaction was seen betweenA877 and the CF3 group. In addition one hydrogen bond was pres-ent between the ester carbonyl oxygen and Q711.

A preference for the S stereoisomer was observed within the2PNU site for both 38 and 39 compared to the reverse situation

for the 2OZ7 T877A mutant where the R stereoisomers of 38 and39 had higher scores (Fig. 12). These in silico results have exploredthe conformational space around the hARLBD of both the wild type2PNU and 2OZ7 T887A mutant by docking a set of novel com-pounds into their respective LBDs. While both sites can accommo-date large and flexible compounds, the removal of the threonineside chain in the T877A mutant results in even more space avail-able for large molecules to maneuver. This is consistent with thefindings in binding studies that the T877A mutant shows morepromiscuous ligand binding. The Fmoc group mimics the steroidnucleus of the androgen structure binding into a similar hydropho-bic area. For all ligands at this site, additional hydrogen bondacceptors para- to the triazole ring of 2–3 bonds in length facilitatea stabilizing hydrogen bonding interaction with R752 (Fig. 13).

With similar length, Bicalutamide (3) and Enzalutamide(MVD3100) adopt similar orientations within each site. In bothcases, the di-substituted benzenes with the CN and CF3 groupsare able to hydrogen bond with Q711 and R752. Then, dependingon the conformational space available, the orientation of the otherend of the molecules is either directed towards the H12 helix (for2PNU) or down towards the more hydrophobic pocket around L701(for 2OZ7).

Figure 10. 2OZ7–tertiary alcohols 32 (atom colours), 34 (orange), 36 (violet), 37 (red).

Figure 11. Top, 2PNU 38S (atom colours), 38R (orange), bottom, 2OZ7 T887A mutant 38S (atom colours), 38R (orange).

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2701

Figure 12. Top, 2PNU 39 S (atom colours), 39 R (orange); bottom, 2OZ7 T887A mutant 39 S (atom colours), 39 R (orange).

2702 J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706

The low docking score for MVD3100 can be explained by theflexibility differences where its central thiohydantoin ring limitedits flexibility compared to bicalutamide 3 and this forced the polaramide end into the hydrophobic pocket in the T877A mutant site.

Comparing MVD3100, 3 and 1 to the synthesized triazoles, 26,28, 29, 32, 34, 36 and 37 reveals a prominent hydrogen bondinginteraction between the di-substituted benzene and R752 andQ711. The presence of the two hydrogen bonding groups and thesimilarity in overall molecular length governs the binding modefor these molecules. The smaller triazoles with either no substitu-tion or mono-substitution on the aryl portion showed a variablebinding mode due to the larger conformational space available tothem within the confines of the protomol volume.

Overall, other significant polar interactions that appear to dom-inate the binding are with N705, T877 and pi-stacking with W741.Two novel interactions were identified in this study. Firstly, theinteraction between Q738 side chain NH2 and hydrogen bondacceptors such as the CN group of compound 38 and 39 in the2PNU site. Secondly, a hydrogen bonding interaction between theCF3 group of 39S and the peptide amide of the mutated A877 res-idue. In the 2OZ7 T877A mutant, the removal of one of the key LBDresidues creates a larger space with less hydrogen bonding. All li-gands were observed to bind exclusively into the steroidal bindingsite with the rings of the larger compounds, 38/39, forming a ‘sand-wich’ conformation within the site. It is worth noting here that theS isomer of both 38 and 39 bound with the highest energy of allcompounds within the 2PNU site compared to the 2OZ7 T877A

mutant where the strained ‘sandwich’ conformation resulted inthe least binding affinity of the compounds.

The in silico docking results showed that the compounds thatbound with the highest binding energy within the 2PNU LBD in aconformation that exploited both the steroidal and hydrophobicpocket. These same compounds bound least well within the 2OZ7T877A mutant site and limited to the steroidal binding site. If thepurpose is to specifically target the H12 region of the 2OZ7 T877Amutant to limit transactivation by conformational change, thenthese results indicate that addition of a large hydrophobic moietyonto the alcohol scaffold could promote pi stacking with W741within the hydrophobic site. This could be achieved by ensuring asize greater than that which allowed the ‘sandwich’ conformationin the steroidal site (e.g., 38/39). It is of note that the inhibition as-say results, using the LNCaP cell lines with the T877A mutation,showed 37, a tertiary alcohol, to have the highest percentage inhi-bition which correlated with the highest scoring ligand in this dock-ing experiment despite the lack of hydrogen bond interaction withR752. The meta positioning of the CF3 groups facilitated favourableutilization of the space, which therefore could make a reasonablestarting scaffold for a new series of potential antagonists.

3. Summary

In summary, we have developed a range of novel triazole-de-rived AR antagonists which are easily accessible and have potentialas lead compounds for prostate cancer therapy. In this work we

Figure 13. Top, 2PNU MVD3100 (atom colours), 1 (red), 3 (purple); bottom, 2OZ7 T887A mutant MVD3100 (atom colours), 1 (red), 3 (purple).

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2703

have presented both the in silico and in vivo data which proved tobe variable at of predicting in vitro potency. This is most likely dueto several reasons, such as the mutated AR providing a more pro-miscuous binding site through T877A replacement and the in silicopredicted interactions are not necessarily antagonistic in nature.Nevertheless the docking provided insight into key interactionsand several novel interactions were found via computationalmethods such as the pi-stacking interaction with W741, hydrogenbonds to Q711 and Q738. The standout compounds of this studywere 32, 36 and 37 which will be used for further developmentof novel AR antagonists and computational studies suggest thatthese compounds may still be suitable for wild type AR inhibition.Further exploration of this scaffold to develop more potent ARantagonists is currently underway and will be reported in duecourse.

4. Experimental procedures

4.1. General experimental

All 1H and 13C NMR spectra were recorded on a Jeol JNM-EX270 MHz, Jeol JNM-EX 400 MHz or Bruker AVANCE III 500 MHz

standard bore (solution) as indicated. Samples were dissolved indeuterated chloroform (CDCl3) with the residual solvent peak usedas an internal reference (CDCl3–d H 7.26 ppm). Proton spectra arereported as follows: chemical shift d (ppm), (integral, multiplicity(s = singlet, br s = broad singlet, d = doublet, dd = doublet ofdoublets, t = triplet, q = quartet, m = multiplet), coupling constantJ (Hz), assignment).

Thin Layer Chromatography (TLC) was performed using alumin-ium-backed Merck TLC Silica gel 60 F254 plates, and samples werevisualised using 254 nm ultraviolet (UV) light, and potassium per-manganate/potassium carbonate oxidising dip (1:1:100 KMnO4/K2CO3/H2O w/w). Column chromatography was performed usingsilica gel 60 (70–230 mesh). All solvents used were AR grade. Spe-cialist reagents were obtained from Sigma–Aldrich chemical com-pany and used without further purification. Petroleum spiritsrefers to the fraction boiling between 40–60 �C. HRMS was foundvia a 6210 MSD TOF mass spectrometer under the conditions:gas temperature (350 �C), vaporizer (28 �C), capillary voltage(3.0 kV), cone voltage (40 V), nitrogen flow rate (7.0 L/min), nebul-iser (15 psi). Samples were dissolved in MeOH. Melting pointswere found on a Stuart Scientific Melting Point Apparatus SMP3,v.5 and are uncorrected.

2704 J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706

4.2. General experimental procedure for phenyl azide formation

To a solution of electron withdrawn aniline (20 mmol) in H2O/MeCN (1:8, 36 mL) was added HCl (10 mL, 18 M) and stirred vigor-ously for 10 min at room temperature. Sodium nitrite (40 mmol,2 equiv) was added portion wise and stirred for an additional hourat room temperature, the solution was then cooled to 0 �C and so-dium azide (40 mmol, 2 equiv) was added portion wise. The result-ing solution is then stirred for an additional hour and allowed towarm to room temperature. Water (40 mL) is then added to dis-solve and remaining solid, the solution is then reduced in vaucoto remove residual MeCN. The aqueous phase is then extractedwith CH2Cl2 (4 � 30 mL), the layers combined and washed withbrine (1 � 20 mL), dried (MgSO4 anhydrous) and the solvent re-moved in vacuo to give the desired product.

4.3. General experimental procedure for CuAAC reaction

The azide (1 mmol) and alkyne (1 mmol) are placed in a micro-wave reaction vessel, to this is added a solution of CuSO4 (10 mol %,25 mg/mL aqueous solution) followed by ascorbic acid (20 mol %,20 mg/mL aqueous solution) and the total volume brought to3 mL with water. The reaction is then heated to 100 �C usingmicrowave irradiation for 30 min. The aqueous phase is then ex-tracted with CH2Cl2 (2 � 20 mL), the layers combined and washedwith brine (1 � 20 mL), dried (MgSO4 anhydrous) and the solventremoved in vacuo to give the desired product in >95% crude purityby 1H NMR. Note that despite all compounds being >95% pure by1H NMR spectroscopy when isolated as the crude material, thecompounds were subjected to purification prior to biological eval-uation to ensure no residual metals or other organic impuritieswere present.

4.4. Compound data

4.4.1. 4-(4-Phenyl-1H-1,2,3-triazol-1-yl)-2-(trifluoromethyl)benzonitrile 28

mp 222.8–229.1 �C; 1H NMR (DMSO-d6, 400 MHz): d 9.63 (1H, s,Ar-H) 8.58–8.46 (3H, m, Ar-H) 7.96–7.94 (2H, m, Ar-H) 7.54 (2H, t,Ar-H) 7.44–7.41 (1H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d148.5, 140.2, 138.1, 133.3 (q, J2

C–F = 32 Hz) 130.2, 129.7, 129.2,126.0, 124.2, 122.7 (q, J1

C–F = 272 Hz) 120.7, 118.6, 115.6, 108.4;19F NMR (DMSO-d6, 470 MHz): d �62.2; IR (cm�1): 3329, 1684,1480, 1313, 1034, 653; HRMS, m/z calcd for (C16H10F3N4)315.08576, found 315.08448

4.4.2. 1-(4-Nitro-3-(trifluoromethyl)phenyl)-4-phenyl-1H-1,2,3-triazole 25

mp 156.6–158.2 �C; 1H NMR (DMSO-d6, 400 MHz): d 9.51 (1H, s,Ar-H) 8.43 (1H, m, Ar-H) 8.35–8.32 (1H, m, Ar-H) 8.04 (1H, d,J = 8 Hz, Ar-H) 7.96–7.94 (2H, m, Ar-H) 7.53 (2H, t, J = 8 Hz, Ar-H)7.41 (1H, t, J = 8 Hz, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d148.2, 136.2, 134.0 130.9, 130.5, 129.7, 129.1, 128.5 (q,J2

C–F = 31 Hz) 125.9, 125.7, 124.3 (q, J1C–F = 272 Hz) 120.6, 119.8;

19F NMR (DMSO-d6, 470 MHz): d �63.0; IR (cm�1): 3124, 1494,1309, 1135, 727; HRMS, m/z calcd for (C15H11F3N4O2) 335.0750,found 335.0794.

4.4.3. 1-(3,5-Bis(trifluoromethyl)phenyl)-4-phenyl-1H-1,2,3-triazole 29

mp134.5–136.8 �C; 1H NMR (DMSO-d6, 400 MHz): d 9.63 (1H, s,Ar-H) 8.68 (2H, s, Ar-H) 8.29 (1H, s, Ar-H) 7.95 (2H, t, J = 8 Hz, Ar-H)7.42 (2H, t, J = 8 Hz, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 148.3,138.5, 132.5 (q, J2

C–F = 33 Hz) 130.3, 129.7, 129.1, 125.9, 123.4 (q,J1

C–F = 272 Hz) 122.6 (m), 122.0, 121.1 (m), 120.8; 19F NMR(DMSO-d6, 470 MHz): d �63.0; IR (cm�1): 3098, 1612, 1495,

1362, 1227, 1132, 736; HRMS, m/z calcd for (C16H10F6N3)358.07342, found 358.07849.

4.4.4. 2-(1-(4-Chloro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)propan-2-ol 30

mp 93.7–95.8 �C; 1H NMR (CDCl3, 400 MHz): d 8.05 (1H, s, Ar-H)8.00 (1H, d, J = 2 Hz, Ar-H) 7.83 (1H, dd, J = 8.8 Hz, 2.4 Hz, Ar-H)7.58 (1H, d, J = 8 Hz, Ar-H) 3.69 (1H, br s, OH) 1.64 (6H, s, CH3);13C NMR (DMSO-d6, 100 MHz): d 159.0, 136.4, 133.9, 130.6, 128.5(q, J2

C–F = 31 Hz) 125.6, 122.9 (q, J1C–F = 272 Hz) 120.0, 119.7 (q,

J3C–F = 5 Hz) 67.5, 31.1; 19F NMR (DMSO-d6, 470 MHz): d �61.5;

IR (cm�1): 3353, 2979, 1492, 1310, 1142, 1037, 831736; HRMS,m/z calcd for (C12H11ClF3N3NaO) 328.04404, found 328.04952.

4.4.5. 4-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)benzonitrile 35

mp 108.1–111.2 �C; 1H NMR (DMSO-d6, 400 MHz): d 8.78 (1H, s,Ar-H) 8.17 (1H, d, J = 5.4 Hz, Ar-H) 8.08 (2H, d, J = 5.4 Hz, Ar-H) 3.35(1H, s, OH) 1.54 (6H, s, CH3); 13C NMR (DMSO-d6, 100 MHz): d158.0, 140.3, 134.8, 120.8, 119.7, 118.7, 111.3, 67.5, 31.1; IR(cm�1): 3144, 1614, 1510, 1314, 1137, 1025, 695; HRMS m/z calcdfor (C12H13N4O) 229.10839, found 229.10718.

4.4.6. 4-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)-2-(trifluoromethyl)benzonitrile 36

mp 112.4–114.2 �C; 1H NMR (DMSO-d6, 400 MHz): d 8.23 (1H, s,Ar-H) 8.14–8.11 (2H, m, Ar-H) 8.01 (1H, d, J = 8 Hz, Ar-H) 1.69 (6H,s, CH3) (OH not observed); 13C NMR (DMSO-d6, 100 MHz): d 158.2,140.4, 138.0, 133.3 (q, J2

C–F = 16 Hz) 124.0, 120.16, 116.4 (m),115.6, 108.0, 67.5, 31.0; 19F NMR (DMSO-d6, 470 MHz): d �62.2;IR (cm�1): 3407, 3145, 2981, 1616, 1587, 1365, 1051, 801; HRMS,m/z calcd for (C13H12F3N4O) 297.09577, found 297.09699.

4.4.7. 2-(1-(4-Nitro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)propan-2-ol 33

1H NMR (DMSO-d6, 400 MHz): d 8.94 (1H, s, Ar-H) 8.36 (1H, d,J = 4 Hz, Ar-H) 8.28-8.25 (1H, m, Ar-H) 7.92 (1H, d, J = 8 Hz, Ar-H)1.54 (1H, s, OH); 13C NMR (DMSO-d6, 100 MHz): d 157.9, 136.1,133.8 (m), 130.5, 128.5 (q, J2

C–F = 31 Hz) 125.5, 122.8 (q, J1C–F =

272 Hz) 119.9, 119.5 (m) 67.5, 31.0; 19F NMR (DMSO-d6,470 MHz): d �63.0; IR (cm�1): 3370, 2980, 1492, 1311, 1143,1037, 831; HRMS m/z calcd for (C12H12F3N4O3) 317.08566, found317.08856.

4.4.8. 2-(1-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)propan-2-ol 37

mp 149.8–150.4 �C; 1H NMR (DMSO-d6, 400 MHz): d 9.02 (1H, s,Ar-H) 8.66 (2H, s, Ar-H) 8.22 (1H, s, Ar-H) 5.32 (1H, br s, OH) 1.56(6H, s, CH3); 13C NMR (DMSO-d6, 100 MHz): d 158.0, 144.4, 138.7,132.4 (q, J2

C–F = 34 Hz) 123.4 (q, J1C–F = 272 Hz) 122.1 (m) 121.0 (m)

120.3, 67.5, 31.0, 19F NMR (DMSO-d6, 470 MHz): d �61.5; IR(cm�1): 3290, 3141, 1428, 1275, 1109, 868, 736; HRMS calcd for(C13H12F6N3O)+ 340.08791, found 340.08950.

4.4.9. Methyl-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)propanoate 39

mp 82.5–83.9 �C; 1H NMR (DMSO-d6, 500 MHz): d 8.94 (1H, s,ArH) 8.45–8.38 (3H, m, ArH) 7.95 (2H, d, J = 10 Hz, ArH) 7.86 (2H,d, J = 10 Hz, ArH) 7.63 (2H, t, J = 5 Hz, ArH) 7.38 (2H, t, J = 10 Hz,ArH) 7.27 (2H, dd, J = 5 Hz, 10 Hz, ArH) 4.53–4.48 (1H, m, CH)4.28 (2H, d, J = 10 Hz, CH2) 4.18–4.15 (1H, m, CH) 3.67 (3H, s,CH3); 13C NMR (DMSO-d6, 100 MHz): d 172.2, 156.5, 145.3, 144.3,144.2, 141.3, 140.2, 138.1, 133.2 (q, J2

C–F = 33 Hz) 128.2, 127.6,125.7, 124.1, 122.7, 122.6 (q, J1

C–F = 272 Hz) 120.7, 118.4, 115.6,66.3, 54.1, 52.8, 47.1, 27.7; 19F NMR (DMSO-d6, 470 MHz): d

J. M. Altimari et al. / Bioorg. Med. Chem. 22 (2014) 2692–2706 2705

�61.0; IR (cm�1): 3367, 2954, 1719, 1509, 1467, 1147, 1032, 760;HRMS (ES) calcd for (C29H23F3N5O4)+ 562.16967, found 562.17244.

4.4.10. Methyl-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(4-nitro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)propanoate 38

mp 141.3–142.3 �C; 1H NMR (DMSO-d6, 400 MHz): d 8.79 (1H, s,ArH) 8.29 (1H, d, J = 5 Hz, ArH) 8.22–8.20 (1H, m, ArH) 7.95 (2H, t,J = 10 Hz, ArH) 7.87 (2H, d, J = 5 Hz, ArH) 7.64 (2H, dd, J = 5 Hz,2.5 Hz, ArH) 7.39–7.36 (2H, m, ArH) 7.29, 7.24 (2H, m, ArH)4.51–4.47 (1H, m, CH) 4.29 (2H, d, J = 5 Hz, CH2) 4.18 (1H, t,J = 5 Hz CH) 3.67 (3H, s, CH3); 13C NMR (DMSO-d6, 100 MHz): d172.3, 156.5, 145.0, 144.3, 141.3, 136.1, 133.9, 130.7, 128.5 (q,J2

C–F = 32 Hz) 128.1, 127.6, 125.7, 125.6, 122.8 (q, J1C–F = 272 Hz)

122.4, 120.7, 119.7 (m) 66.3, 54.1, 52.7, 47.1, 27.7; IR (cm�1):3352, 3066, 2852, 1718, 1492, 1289, 1142, 1036, 739, 536; HRMS(ES) calcd for (C28H23F3N5O6)+ 582.1595, found 582.1624.

4.5. Molecular modelling

All modelling work was performed using SYBYL-X version 2.0(SYBYL) unless otherwise stated.57 The Surflex-Dock 2.1 algorithm,based on an incremental construction methodology, was employedfor docking protocols using the ligand-based approach for proto-mol generation. MOLCAD was used to produce surface contours.

4.6. Target preparation

Hydrogens and Gasteiger-Huckel charges were added to targetproteins followed by energy minimisation using the ConjugatedGradient method to a convergence of 0.5 kcal/mol (>4000 itera-tions). Crystal waters and all ligands except for EM7544 and cypro-terone acetate (CPY) were subsequently removed prior to docking.

4.7. Virtual library preparation

Hydrogens and Gasteiger-Huckel charges were added prior toenergy minimisation to a convergence of 0.5 kcal/mol. Additionalstructures were included as positive controls (+ve) known to bindthe androgen binding site (ABS).

4.8. Docking

The protomol was generated using a threshold and bloat valueof 0.5 and 0 respectively. The protomol was analysed to ensure suf-ficient conformation space was included. Consensus scoring (Cscore)was included to identify structures producing a consensus across 4scoring functions. A high Cscore value reflects a high pose bindingscore across all scoring functions.

4.9. Anti-proliferation assays

LNCaP cells were routinely cultured in RPMI media with 10%FCS. For the proliferation assay cells had been treated with phenolred free RPMI medium with 10% charcoal stripped FCS for twodays, in order to steroid starve them. Then 7500 cells per well,were seeded in 96-well culture plates in phenol red free RPMImedium with 10% charcoal stripped FCS. The cells were thengrowth stimulated with 2 nM DHT (dihydrotestosterone). The testcompounds were added along with 2 nM DHT to ascertain theirinhibitory effect at 20 and 40 lM concentrations. Bicalutamidewas used as a positive growth inhibitor control. Culture mediumalong with treatment was refreshed every 3 days. Each treatmentwas carried out in 4 wells. The cells were counted every three daysusing Promega’s Aqueous One solution kit. IC50 measurementswere carried out by GenscriptUSA inc.

Acknowledgements

The authors gratefully acknowledge the CASS foundation andthe Strategic Research Center for Chemistry and Biotechnologyfor the funding to carry out this work. We thank MetabolomicsAustralia for the use of their HRMS service. We would also like tothank the Australian Government for an APA scholarship to J.A.

A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.018.

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