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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY

Original article

Synthesis and structure-activity relationship of 1- and 2-substituted- 1,2,3-triazole letrozole-based analogues as aromatase inhibitors

Jérémie Doiron ª,1, Al Haliffa Soultan a,1, Ryan Richarda, Mamadou Mansour Touré ª, Nadia Picota, Rémi Richard ª, Miroslava Čuperlović-Culfb,c, Gilles A. Robichaud ª, Mohamed Touaibia a,*

a Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada

b National Research Council of Canada, Institute for Information Technology, Moncton, NB, Canada

“Department of Chemistry and Biochemistry, Mount Allison University, Sackville, NB, Canada

ARTICLE INFO

Article history: Received 24 February 2011 Received in revised form 30 May 2011

Accepted 31 May 2011

Available online 12 June 2011

Keywords:

Aromatase Triazole Letrozole Structure-activity relationship

ABSTRACT

A series of bis- and mono-benzonitrile or phenyl analogues of letrozole 1, bearing (1,2,3 and 1,2,5)-tri- azole or imidazole, were synthesized and screened for their anti-aromatase activities. The unsubstituted 1,2,3-triazole 10a derivative displayed inhibitory activity comparable with that of the aromatase inhib- itor, letrozole 1. Compound 10a, bearing a 1,2,3-triazole, is also 10000-times more tightly binding than the corresponding analogue 25 bearing a 1,2,5-triazole, which confirms the importance of a nitrogen atom at position 3 or 4 of the 5-membered ring needed for high activity. The effect on human epithelial adrenocortical carcinoma cell line (H295R) proliferation was also evaluated. The compound 10j (IC50 = 4.64 µM), a letrozole 1 analogue bearing para-cyanophenoxymethylene-1,2,3-triazole decreased proliferation rates of H295R cells by 76 and 99% in 24 and 72 h respectively. Computer calculations, using quantum ab initio structures, suggest a possible correlation between anti-aromatase activity and the distance between the nitrogen in position 3 or 4 of triazole nitrogen and the cyano group nitrogen.

1. Introduction

The standard pharmacological treatment for hormone- dependent cancer is to block oestrogen binding to the oestrogen receptor (ER) with tamoxifen [1,2]. Unfortunately, this anti-oes- trogen molecule has many drawbacks. First, as a partial ER agonist in many tissue types [3], its use has been correlated with an increase in the incidences of endometrial cancer [4]. Furthermore, resistance to tamoxifen therapy inevitably results [3]. An alterna- tive approach to tamoxifen treatment is inhibition of oestrogen synthesis. The target for such therapy is aromatase. This key cyto- chrome P450 enzyme encoded by the CYP19 gene catalyzes the rate limiting step in the conversion (aromatization) of androgens to oestrogens [5].

To avoid inhibition of other steroidogenesis enzymes, inhibitor selectivity is crucial. However, non-selective aromatase inhibitors, like non-steroidal aminoglutethimide [6,7], which is a well known and long used therapeutic agent, could affect enzymes controlling

the production of other steroids and induce significant side-effects. New aromatase inhibitors, including letrozole 1 and anastrozole 2 (Fig. 1), were found to be effective challengers of tamoxifen as oestrogen blocker and were highlighted in several recent publica- tions [8-10]. They act through the aromatase pathway, to treat oestrogen-sensitive breast cancers by preventing oestrogen production in the first place. Unlike tamoxifen, that has mixed estrogen agonist and antagonist properties, the aromatase inhibi- tors have no oestrogenic agonist activity. There is now a strong case to be made for selectively inhibiting aromatase in order to achieve a more effective and selective endocrine therapy for hormone- dependent breast cancer.

Many studies have recently been undertaken to evaluate several new families of aromatase inhibitors [11-20]. Le Borgne et al. reported letrozole analogues having an arylindole moiety with imidazole or 1,2,4-triazole heterocycles [21-23]. Structure-activity relationships identified the importance of electron withdrawing groups at the para position of the phenyl with nitrile group being best [24]. This group mimics the carbonyl group of androstenedione as a hydrogen bond acceptor [25,26].

Farag et al. have recently reported new pyrazole-based letrozole and celecoxib analogues with interesting anti-aromatase activities [27,28]. Furthermore, Potter et al. have recently reported on the

* Corresponding author. Tel .: +1 506 858 4493; fax: +1 506 858 4541. E-mail address: mohamed.touaibia@umoncton.ca (M. Touaibia).

1 Both authors contributed equally.

Fig. 1. Letrozole and anastrazole structures.

synthesis and in vitro and in vivo inhibitory activity of novel dual aromatase-sulfatase inhibitors based upon letrozole 1 and anas- trozole 2 [29-32]. Ghosh et al. recently elucidated the crystallo- graphic structure of aromatase with androstenedione [33]. This publically available structure will be essential for establishing the mechanism of the aromatization process, understanding the mode of action of known inhibitors, and is thus indispensable for future drug-discovery efforts for new inhibitors.

In an effort to develop more potent inhibitors of aromatase, letrozole 1 analogues were synthesized. In particular, our struc- ture-activity relationship (SAR) study examines (1) the effect of 1,2,3-triazole instead of the 1,2,4-triazole present in the letrozole 1 structure (Fig. 1), the goal of which was to test various substituted 1,2,3-triazoles and (2) the requirement of one or two cyano or phenyl moieties together with various 1,2,3 -; 1,2,4- and 1,2,5- triazoles. For comparison purposes, the corresponding imidazole analogues were synthesized. The effects of these changes were assayed against aromatase using an in vitro fluorimetric assay [34,35]. The effect on human epithelial adrenocortical carcinoma cell line (H295R) proliferation was also evaluated [36].

2. Results and discussion

2.1. Chemistry

Click chemistry has attracted much attention recently because of its high specificity, quantitative yield and tolerance to various groups [37]. The syntheses of letrozole 1 analogues with a 1,2,3- triazole heterocycle 10a-n and 11a-n are depicted in Scheme 1.

Di(4-cyanophenyl)methane 5, the key intermediate for 10a-n synthesis, was obtained by cold base catalyzed (t-BuOK) conden- sation of p-tolunitrile 3 and p-flurobenzonitrile 4. In order to introduce the azido group, necessary for copper catalyzed 1,3- dipolar cycloaddition, compound 5 was subjected to photochem- ical allylic bromination via N-bromosuccinimide (NBS) in CCl4 under incandescent irradiation (250 W) to give the bromide 6 in good yields. Yields are given in the experimental section. Curiously, benzoyl peroxide catalyzed bromination with NBS failed both thermally and with UV irradiation. The novel azide 8 was thus obtained by conversion of brominated derivative 6 under nucleo- philic substitution at 0 ℃ as all attempts to prepare 8 with heating or at room temperature failed. It should be noted that the synthesis of compounds 5, 6 and 8 were undertaken in an oxygen free atmosphere, as the di(4-cyanophenyl)methyl framework of these compounds is prone to oxidation, producing di(4-cyanophenyl) ketone which is visible as a bright blue spot upon UV illumination of eluted TLC plates of the reaction mixture. As shown in Scheme 1, azide 9 [38] was efficiently synthesized by substitution of commercially available bromide 7 with sodium azide. Treatment of azide 8 or 9 under CuI-catalyzed click reaction conditions with aliphatic as well as substituted propargyl phenol ethers provided the final compounds, with or without para-cyano groups (10a-n or 11a-n respectively), in good yields (Scheme 1). As described in the literature, substituted terminal alkynes used for the synthesis of 10h-n and 11h-n were obtained with treatment of propargyl bromide with desired substituted phenol under basic conditions (K2CO3, DMF) [39].

As shown in Scheme 2, mono-benzonitrile, monophenyl, and bis- benzonitrile letrozole 1 analogues in which the 1,2,4-triazole was substituted with 1,2,3-triazole (17-22, 25) and imidazole (23, 24, 26) were also synthesized. Synthesis began with the nucleophilic substitution of brominated precursors 12 or 13 by the required five member heterocycles in acetone in the presence of potassium carbonate, affording the expected heterocyclic compounds with or without para-cyano groups (17,19,21,23 or 18,20,22,24 respectively) [40,41]. Interestingly, compounds in which a 1H-1,2,3-triazole was to be tied to the aromatic frameworks afforded both the 1,2,3- triazole products (17,18) and the 1,2,5-triazole products (19,20) simultaneously. This phenomenon is most likely due to in situ base- induced tautomerisation of the nitrogenous heterocycle prior to the

iii

3 R1 = CN

4 R1 = CN

5 R1 = CN

6 R1 = CN 7 R1 = H

8 R1 = CN

9 R1 = H

10a R1 = CN; R2 = H

11a R1 = H; R2 = H

10b R1 = CN; R2 = n-Propyl

10c R1 = CN; R2 = n-Hexyl

11b R1 = H; R2 = n-Propyl

10d R1 = CN; R2 = n-Heptyl

11c R1 = H; R2 = n-Hexyl

10e R1 = CN; R2 = n-Decyl 10f R1 = CN; R2 = Cyclohexyl 10g R1 = CN; R2 = Ph

11d R1 = H; R2 = n-Heptyl

11e R1 = H; R2 = n-Decyl 11f R1 = H; R2 = Cyclohexyl 11g R1 = H; R2 = Ph 11h R1 = H; R2 = CH2OPh

10a-n 11a-n

10h R1 = CN; R2 = CH2OPh

10i R1 = CN; R2 = CH2Op-Cl-Ph 10j R1 = CN; R2 = CH2Op-CN-Ph 10k R1 = CN; R2 = CH2Op-NO2-Ph

11i R1 = H; R2 = CH2Op-Cl-Ph 11j R1 = H; R2 = CH2Op-CN-Ph

10l R1 = CN; R2 = CH2Op-CH3-Ph

11k R1 = H; R2 = CH2Op-NO2-Ph

10m R1 = CN; R2 = CH2Op-OCH3-Ph

11l R1 = H; R2 = CH2Op-CH3-Ph

10n R1 = CN; R2 = CH2Op-Ph-Ph

11m R1 = H; R2 = CH2Op-OCH3-Ph 11n R1 = H; R2 = CH2Op-Ph-Ph

Scheme 1. Reagents and conditions: (i) t-BuOK, DMF, -5 ℃, 2 h, DMF; (ii) NBS, CC14, 250 W, 1 h; (iii) NaN3, -5 ℃, DMF, 1 hr for 8; (vi) HC=CR2, NEt3, Cul, rt, THF; 12 h or HC=CH, NEt3, CuI, rt, DMSO, 12 h for 10a and 11a.

Scheme 2. Reagents and conditions: (i) K2CO3, reflux, Acetone, 12 h; (ii) t-BuOK, 4-fluorobenzonitrile, rt, DMF 2.5 h.

Y 11

12 R1 = CN 13 R1 = H

14,15, 16

17 R1 = CN; W = X = N; Y = Z = CH 18 R1 = H; W = X = N; Y = Z = CH 19 R1 = CN; W = Z = N; X = Y = CH 20 R1 = H; W = Z = N; X = Y = CH 21 R1 = CN; W = Y = N; X = Z = CH 22 R1 = H; W = Y = N; X = Z = CH 23 R1 = CN; Y = N; W = X = Z = CH 24 R1 = H; Y = N; W = X = Z = CH

10a R1 = CN; W = X = N; Y = Z = CH 25 R1 = CN; W = Z = N; X = Y = CH 1 R1 = CN; W = Y = N; X = Z = CH 26 R1 = CN; Y = N; W = X = Z = CH

substitution of the bromides [42]. These related compounds were easily separated and isolated by silica gel circular chromatography (see Experimental section). For the 1,2,3-triazoles, the triazole protons were observed as two singlets, each integrating for one proton, at 7.7 and 7.5 ppm. The structure of the 1,2,5-triazole isomers was confirmed by 1H NMR, which gave a singlet, integrating for two protons, at 7.7 ppm for the symmetrical triazole protons. As for the synthesis of letrozole 1 [19] from compound 21, mono-benzonitrile precursors 17, 19 and 23 were subject to base-induced condensation with 4-fluorobenzonitrile 3 to afford the bis-benzonitrile analogues, 10a, 25 and 26 respectively (Scheme 2) [19,43].

Since we were not able to obtain the corresponding bis-phenyl analogues of 18, 20, 22, and 24 under basic condensation with fluorobenzene, commercially available bromide 7, (1,2,4 -; 1,2,3-) triazole, and imidazole were used for the synthesis of bis-phenyl derivatives 11a, 27, 28, and 29 with good yields (Scheme 3) [44]. As for 17, 18, 19, and 20 synthesis (Scheme 2), in addition to the 1,2,3-triazole isomer, the minor 1,2,5-triazole was also obtained. Compounds 11a and 27 were isolated from the same reaction mixture after silica gel chromatography.

Following our interest to explore 1,2,3-triazole derivatives for aromatase inhibition, Scheme 4 describes the preparation of mono-aromatic 1,2,3-triazole bearing letrozole 1 analogues 34b-n and 35b-n. Their synthesis was initiated with the azides 32 [45] and 33 [46] which were prepared by the nucleophilic substitution of the respective benzyl bromide 30 and 31 with sodium azide. As for 10a-n and 11a-n synthesis, Cul-catalysed click reaction with aliphatic alkynes as well as substituted propargyl phenol ethers provided 34b-n and 35b-n in good yield (Scheme 4).

2.2. Biological evaluation

2.2.1. In vitro aromatase inhibitory activity

All compounds were evaluated for aromatase inhibitory activity using the CYP19 high-throughput screening kit (BD Biosciences),

Scheme 3. Reagents and conditions: (i) K2CO3, reflux, Acetone, 12 h.

7 R1 = H

14,15, 16

11a R1 = H; W = X = N; Y = Z = CH 27 R1 = H; W = Z = N; X = Y = CH 28 R1 = H; W = Y = N; X = Z = CH 29 R1 = H; Y = N; W = X = Z = CH

with 7-methoxy-4-trifluoromethyl coumarin (MFC) as the substrate and letrozole 1 as the standard for comparison (Tables 1-3). Letro- zole 1 was determined to have an IC50 value of 0.008 µM.

Given the many reported successes of triazole use in aromatase lead discovery [12,20-23], a systematic series of unsubstituted and substituted 1,2,3-triazole and 1,2,4-triazole analogues of letrozole 1 (Fig. 1) were examined (Scheme 1). Several important trends emerged from the examination of this series (Table 1). As shown in Table 1, the 1,2,3-triazole derivatives 10a-n were all relatively good inhibitors of aromatase (IC50: 0.008-19.32 uM), with optimal activity obtained with the unsubstituted derivative 10a (IC50: 0.008 µM) which is equipotent with letrozole 1. Substitution on carbon 4 of triazole of compound 10a, results in a significant decrease of anti-aromatase activity. In the alkyl series (10b-10f), the more bulky cyclohexyl-substituted 1,2,3-triazole derivative 10f was the least active (10f, IC50: 19.3 uM). Introduction of phenyl (10g, IC50: 10.3 µM) or phenoxymethyl (10h, IC50: 10.9 uM) on carbon 4 of triazole of compound 10a had no impact on the potency. Substituting the para-position of the phenyl ring of 10h with electron withdrawing group, as in 10j (IC50: 4.6 uM) and 10k (IC50: 7.7 µM), enhanced the activity relative to the unsubstituted deriv- ative 10h (IC50: 10.3 uM). Substitution with electron donating groups methyl and methoxy, as in 10l and 10m, had no impact on the activity, while the activity was decreased by the insertion of a chloride group (10i, IC50: 16.1 µM) or a phenyl group (10n, IC50: 13.3 µM).

As indicated by the series of 1,2,3-triazole derivatives 11a-n (Table 1), the presence of the cyano groups appears to be essential for activity. Compound 10a (IC50: 0.008 µM), which bears two para- cyano groups is 500-fold more active than the corresponding cyano-free analogue 11a (IC50: 4.5 uM). The activity was not improved by substitution on carbon 4 of the triazole compound 11a, as alkyl/aryl substituted derivatives (11b-n) were consider- ably less active than letrozole 1, 10a, and 11a (Table 1). The lower IC50 value observed for 10b-n and 11b-n in comparison with 1, 10a, and 11a could be attributed to an impediment of the coordi- nation with the haeme iron of the aromatase by the bulky group (R2).

To investigate the influence of the presence of the two benzo- nitrile or phenyl moieties in 10a and 11a, mono-benzonitrile 17 and monophenyl 18 analogues were synthesized (Scheme 2). Interest- ingly, even when lacking one benzonitrile moiety, 17 (IC50: 0.10 uM) exhibited a good inhibitory potency against aromatase and was 12.5-fold less active than 10a and letrozole 1 (Table 2). In the same vein, 10a analogues bearing asymmetrical (1,2,4-), symmetrical (1,2,5-) triazole and imidazole were investigated (Scheme 2). The presence of a nitrogen atom at position 3 or 4 of the five member ring seems to be crucial for activity. Asymmetrical (1,2,4- and 1,2,3-) triazole and imidazole-containing derivatives (17, IC50: 0.10 uM 21,

Scheme 4. Reagents and conditions: (i) NaN3, DMF, rt; (ii) HC=CR2, NEt3, Cul, rt, THF, 12 h.

NEN

30 R1 = CN 31 R1 = H

32 R1 = CN

34b-n 35b-n

33 R1 = H

34b R1 = CN; R2 = n-Propyl

35b R1 = H; R2 = n-Propyl

34c R1 = CN; R2 = n-Hexyl

35c R1 = H; R2 = n-Hexyl

34d R1 = CN; R2 = n-Heptyl

35d R1 = H; R2 = n-Heptyl

34e R1 = CN; R2 = n-Decyl

35e R1 = H; R2 = n-Decyl

34f R1 = CN; R2 = Cyclohexyl

35f R1 = H; R2 = Cyclohexyl

34g R1 = CN; R2 = Ph

35g R1 = H; R2 = Ph

34h R1 = CN; R2 = CH2OPh

35h R1 = H; R2 = CH2OPh

34i R1 = CN; R2 = CH2Op-CI-Ph

35i R1 = H; R2 = CH2Op-Cl-Ph

34j R1 = CN; R2 = CH2Op-CN-Ph

35j R1 = H; R2 = CH2Op-CN-Ph

34k R1 = CN; R2 = CH2Op-NO2-Ph

35k R1 = H; R2 = CH2Op-NO2-Ph

34l R1 = CN; R2 = CH2Op-CH3-Ph

351 R1 = H; R2 = CH2Op-CH3-Ph

34m R1 = CN; R2 = CH2Op-OCH3-Ph

35m R1 = H; R2 = CH2Op-OCH3-Ph

34n R1 = CN; R2 = CH2Op-Ph-Ph

35n R1 = H; R2 = CH2Op-Ph-Ph

IC50: 0.15 µM; 23, IC50: 0.007 uM) were more active than the symmetrical 1,2,5-triazole-containing derivative (19, IC50: 12.2 µM). Compound 19, bearing 1,2,5-triazole, was 81- and 122- fold less active than the corresponding 1,2,4-(21) and 1,2,3-(17) triazole, respectively. Compound 23, bearing an imidazole five membered ring, was the most active in this series and was equi- potent with letrozole 1 and 10a. The importance of the presence of the nitrogen atom at position 3 or 4 of the five member ring was also confirmed by asymmetrical (1,2,4- and 1,2,3-) triazole and imidazole-containing 11a analogues. Effectively, compounds 18, 22, and 24, bearing asymmetrical triazole (1,2,3- and 1,2,4-) and imidazole, were 1.4-, 4-, and 108-fold more active than the 1,2,5- triazole-containing compound 20, respectively (Table 2). In this fourth series, phenyl para-cyano substitution seems to be crucial for inhibition. Compounds 17, 19, 21, and 23, with a para-cyano substitution, were all more active than the corresponding free para- cyano substitution analogues 18, 20, 22, and 24, respectively (Table 2).

Encouraged by these latest results, symmetrical 1,2,5-triazole and imidazole-containing analogues of 1, 10a, and 11a having the two benzonitrile or phenyl moieties were considered (Schemes 2 and 3). In this series our previous results were confirmed with symmetrical 1,2,5-triazole-containing compounds 25 (IC50: 79.98 µM) being almost 10000-fold less active than 1 and 10a. Imidazole-containing analogue 26 (IC50: 0.004 uM), which is the most potent in this series, was 2- and 20000-fold more active than 1 (and 10a) and 25, respectively (Table 2). For 11a analogues, symmetrical 1,2,5-triazole-containing compound 27 was less active than asymmetrical (1,2,4- and 1,2,3-) triazole and imidazole- containing 28, 11a and 29 respectively (Table 2). The imidazole- containing derivative 29 (IC50: 0.02 uM) was also the most potent

compound in comparison to 27, 11a, and 28 (Table 2). The phenyl para-substitution also seems to be beneficial for inhibition in this series. Except for compound 27 (IC50: 12.01 µM), which was 6-fold more active than the corresponding phenyl para-cyano substitution-free derivative 25 (IC50: 79.98 µM), 10a, 1, and 26 were more active than 11a, 28 and 29 respectively (Table 2). In the last two series, the presence of two phenyl moieties seems to be crucial for inhibition. Except for compound 25 (IC50: 79.98 uM), which was 6-fold less active than 19 (IC50: 12.20 uM); 10a, 1, 26, 11a, 28, and 29 were more active than 17, 21, 23, 18, 22, and 24 respectively (Table 2).

Compound 17, reporting an encouraging inhibitory activity (IC50: 0.1 µM) prompted us to investigate whether introduction of various substituent groups at the triazole, using the classical click chemistry, would further improve the potency of aromatase inhi- bition. Hence, 34b-n were prepared but, as shown in Table 3, the ability of these compounds to inhibit aromatase was low and did not reach the activities of letrozole 1, 10a, and 17 or other 1,2,3- triazoles (Table 3). Given IC50 values of 2-3 uM, the heptyl (34d, IC50: 3.07 µM), phenyl (34g, IC50: 3.38 uM), 4-chlorophenyl (34j, IC50: 3.91 µM), and 4-methoxyphenyl (34m, IC50: 2.34 uM) substituted compounds were the most potent. As shown with compounds 34b-n (Table 3), the introduction of various substit- uent groups at the 1,2,3-triazole of compound 18 (IC50: 11.88 uM) lead to an increase of activity in general. The para-cyanophenoxy- methylene-containing analogue 35j (IC50: 1.36 uM), which is the most potent in this series, was 8-fold more active than 18 (Table 3). By comparison between Tables 1 and 3, compounds 34n-b and 35b-n are more active than their counterparts with two para- cyanophenyl (10b-n) or phenyl (11b-n), respectively. Unlike other series, the presence of the cyano group does not seem to influence

Table 1 IC50 data for analogues 10a-n and 11a-n using CYP19 high-throughput screening kit [47].
Compound	IC50 (uM)	Compound	IC50 (uM)	Compound	IC50 (µM)	Compound	IC50 (µM)
10a	0.008	10h	10.97	11a	4.58	11h	31.26
10b	4.70	10i	16.11	11b	10.96	11i	13.37
10c	5.07	10j	4.64	11c	14.83	11j	16.41
10d	10.60	10k	7.73	11d	19.36	11k	11.61
10e	16.02	10l	10.95	11e	13.54	11l	26.77
10f	19.32	10m	12.16	11f	13.72	11m	15.21
10g	10.33	10n	13.35	11g	12.94	11n	15.48

Table 2 IC50 data for analogues 17-29, 1, 10a, and 11a using CYP19 high-throughput screening kit [47].
Compound	IC50 (UM)	Compound	IC50 (UM)	Compound	IC50 (AM)	Compound	IC50 (AM)
17	0.10	18	11.88	10a	0.008	11a	4.58
19	12.20	20	16.31	25	79.98	27	12.01
21	0.15	22	4.11	1	0.008	28	1.10
23	0.007	24	0.15	26	0.004	29	0.02

Table 3 IC50 data for analogues 34b-n, 35b-n, 19, and 20 using CYP19 high-throughput screening kit [47].
Compound	IC50 (μM)	Compound	IC50 (μM)	Compound	IC50 (AM)	Compound	IC50 (uM)
19	0.10	34h	6.09	20	11.88	35h	10.60
34b	4.37	34i	3.91	35b	12.63	35i	5.04
34c	5.26	34j	9.16	35c	7.47	35j	1.36
34d	3.07	34k	5.62	35d	9.53	35k	2.78
34e	7.88	34l	4.17	35e	6.40	35l	2.27
34f	10.70	34m	2.34	35f	7.98	35m	17.34
34g	3.38	34n	11.57	35g	9.77	35n	10.70

activity. Paradoxically, the best compound (35j, IC50: 1.36 uM) in this series has a para-cyanophenoxymethylene at position 4 of the 1,2,3-triazole.

2.2.2. Antiproliferative activity

Some of the compounds from each series that showed the best biological activities in our first test, the triazoles (10a-c, 10j, 35j-1) and the imidazoles (23, 26, 29), were evaluated for their effects on the human epithelial adrenocortical carcinoma cell line (H295R) proliferation (see Fig. 2). The H295R cell line is a commonly used model for the study of aromatase activity [48-51]. The results showed that most of our compounds dis- played an antiproliferative activity against H295R cells compa- rable with that of letrozole 1. Furthermore, compound 10j with a para-cyanophenoxymethylene-1,2,3-triazole moiety exhibits the most potent antiproliferative activity in comparison to letrozole

1(Fig. 2), suggesting other effects in addition to aromatase inhi- bition. Previous studies have demonstrated that at a concentra- tion of 10 nM, letrozole 1 inhibits nearly 10% of H295R proliferation [52]. While at the same concentration, 10j inhibited 76% of cell proliferation in 24 h and 99% in 72 h. Compound 35j, cyano-free group analogue, did not exhibit significant activity. Analogue 26 in which the 1,2,4-triazole was substituted with an imidazole inhibited 40% of H295R proliferation.

2.3. Computer modelling

Comparison of the IC50 data for 10a and 25 (0.008 uM vs 79.98 µM) clearly shows the importance of carbon substitution at position 3 or 4 of the five member ring by nitrogen (Table 2)). At the same time, experimental analysis of the interaction between aro- matase cytochrome P450 and its substrate androstenedione shows the importance of hydrogen bonding in positioning and stabiliza- tion of the substrate [33]. Hydrogen bond acceptors in androste- nedione are the two oxygen atoms of carbonyl functions at 10.25 Å separation [33]. Finally, virtual screening and analysis of known, strong, aromatase inhibitors resulted in recommendations for the necessary requirements for a pharmacophore including the need for two hydrogen bond acceptors [54]. Similarly the work of Neves et al. [55] points also to the importance of hydrogen bonding in aromatase inhibitor binding. Geometric differences of the studied compounds particularly related to hydrogen bonding potential have been explored by using quantum chemical structure and electronic structure simulations. From theoretically obtained

Fig. 2. Relative growth rates of the human epithelial adrenocortical carcinoma cell line (H295R) treated with different test compounds (10 nM) [53].

1.2

Fluorescence relative to control

0,8

0,6

0,4

0,2

Control

10a

10b

10c

10j

35j

35k

351

Control

10a

10b

10c

10]

35]

35k

351

24 h

Time of exposure (h)

72h

structures for these compounds (Fig. 2), the geometric differences in the orientation of the asymmetrical 1,2,3-triazole and the symmetrical 1,2,5-triazole rings are apparent (Figure S1: see supplementary material). Theoretical calculations revealed a correlation between the inter-nitrogen distances, as shown for 10a and 25 (Table S1: see supplementary material), and biological activity. As a cyano group in the para position of the phenyl ring is an essential structural requirement for inhibitory activity with azole-type aromatase inhibitors [24,56], the presence of another nitrogen on the heterocyclic ring, at a particular position and distance, seems to be also crucial for inhibition with such compounds [26,55]. Nitrogens in the studied compounds are expected to be hydrogen bond acceptors as was confirmed by the HOMO and LUMO analysis (data not shown). The distance between the nitrogens in computationally analyzed structures of androste- nedione, 1, 10a, and 25 are provided in Table S1 (see supplementary material) [57]. It can be observed that in compound 10a and letrozole 1 distances between the nitrogen of the cyano group and the nitrogen at position 3 or 4 of the triazole are similar to the distance between oxygens in the natural substrate (Figure S1: see supplementary material). At the same time, nitrogens at position 2 of the triazole and that of the second cyano group in compounds 1, 10a and 25 could possibly provide an additional hydrogen bond with another binding partner in the target protein which might explain the observed difference in the IC50 values.

3. Conclusions

In summary, we have described the synthesis of a systematic series of unsubstituted and substituted 1,2,3-triazole, 1,2,4-triazole and imidazole analogues of letrozole 1. The key step of the synthesis of 1,2,3-triazole analogues is a copper(I)-catalyzed Huis- gen 1,3-dipolar cycloaddition (click) onto azides (8, 9, 32, or 33) with selected aliphatic alkynes or substituted propargyl phenol ethers. Mono-benzonitrile and monophenyl letrozole 1 analogues in which the 1,2,4-triazole was substituted with (1,2,3 or 1,2,5)- triazole and imidazole were also synthesized by nucleophilic substitution onto brominated precursors by the required five member heterocycles. Base-induced condensation with 4- fluorobenzonitrile onto mono-benzonitrile precursors afforded the bis-benzonitrile analogues.

From the structure-activity point of view, the synthesized and tested molecules in this study allowed us to conclude that the nitrogen atom in position 3 or 4 of 1,2,4-triazole is crucial for inhibition of aromatase. Analogues with a 1,2,3-triazole or imid- azole are more active than the corresponding 1,2,5-triazole analogue. The presence of a para-cyano group and two aryl groups also seems important for good activity. Moreover, among our analogues with 1,2,3-triazole, synthesized via click chemistry, compounds bearing a para-cyanophenoxymethylene (10j and 35j) could serve as potential lead compounds. With IC50’s of 4.6 and 1.36 µM, respectively (10j and 35j) and with excellent inhibition of cell proliferation for 10j, these compounds will be the starting point of another series of molecules in which the cyano group position, the nature, as well as the length of the linker between the phenyl and the triazole can be investigated.

4. Experimental section

4.1. Chemistry

All chemicals used were purchased from Aldrich (CA). Purifica- tion of compounds was carried out by silica gel circular chroma- tography (chromatotron®, model 7924, Harrison Research). TLC was run on silica gel coated aluminium sheets (SiliaPlate TLC,

Silicycle®) with detection by UV light (254 nm, UVS-11, Miner- alight® shortwave UV lamp). Melting points were obtained using a MEL-TEMP® (model 1001D) melting point apparatus. FTIR spectra were recorded on a Nicolet® Impact 400 spectrometer. NMR spectra were recorded on a Bruker® Avance III 400 MHz spec- trometer. High resolution mass measurements were performed on an Agilent® LC-MSD-TOF instrument (model 6210) in positive electrospray. Protonated ions (M + nH)n+ were used for empirical formula confirmation.

4.1.1. 4,4’-Dicyanodiphenylmethane (5)

At -5 ℃ and under inert atmosphere, 7.68 g (69 mmol) of t-BuOK are suspended in 20 mL anhydrous DMF with vigourous stirring. Over a period of 1 h, 2.34 g (19.9 mmol) of 4-methylbenzonitrile dissolved in 5 mL DMF are added dropwise to the reaction mixture, followed by dropwise addition of 3.66 g (30.3 mmol) of 4- fluorobenzonitrile dissolved in 5 mL DMF over 30 min. After 30 min the solution is quenched with 120 mL water, which is then extracted with 4 x 40 mL AcOEt. The organic fraction is dried over MgSO4, filtered and concentrated under vacuum to give a yellow oil which slowly crystallizes. The resulting solid is recrystallized in 20 mL tertbutyl ether to give 2.25 g (5.64 mmol, yield = 52%) of 4,4’- dicycanodiphenylmethane 5 as fluffy beige crystals. Mp = 166-167 ℃ (lit. 168 ℃), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.62 (d, 4H, J = 8.2 Hz, N=CC(CH)), 7.29 (d, 4H, J = 8.2 Hz, N=CC(CHCH)), 4.11 (s, 2H, Ar-CH2-Ar), 13C NMR (101 MHz, CDCI3, 25 °℃), ô (ppm) = 144.82, 132.58, 129.72, 118.66, 110.78, 41.90; HRMS m/z calc. for C15H10N2 + (H+): 219.0917; found: 219.0913.

4.1.2. 4,4’-Dicyanodiphenylbromomethane (6)

Under nitrogen and with stirring, 2.027 g (9.29 mmol) of 4,4’- dicyanodiphenylmethane 5 and 1.689 g (9.29 mmol) of NBS are dissolved in 10 mL CCl4. A 250 W incandescent lamp is then placed 10-15 cm from the reaction vessel, which quickly comes to reflux. After 1hr, TLC analysis (30% AcOEt/Hex) shows disappearance of the starting material. The resulting mixture is cooled, filtered and the residual solid washed with CH2Cl2 until no more solid dissolves to give an orange solution which is then evaporated under vacuum. The resulting oil is diluted with 40 mL CH2Cl2, washed twice with water, twice with brine, dried over MgSO4, filtered and concen- trated. The oily residue is then recrystallized from 60% AcOEt/Hex to give 1.739 g (5.85 mmol, yield = 63%) of the desired bromide 6 as a white solid. Mp = 118-119 ℃, Rf = 0.55 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.68 (d, 4H, J = 8.3 Hz, N=CC(CH)), 7.55 (d, 4H, J = 8.3 Hz, N=CC(CHCH)), 6.25 (s, 1H, CHBr), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) =144.65, 132.69, 129.15, 118.07, 112.59, 51.61; HRMS m/z calc. for C15H9BrN2 + (H+): 297.0022; found: 297.0021.

4.1.3. 4,4’-Dicyanodiphenylazidomethane (8)

To a stirred solution of 1.404 g (21.6 mmol) NaN3 in anhydrous DMF at -5 ℃ and under N2 was added 1.727 g (5.81 mmol) of 4,4’- dicyanodiphenylbromomethane 6. After 1 h the reaction is quenched with 75 mL H2O and extracted with 3 x 25 mL diethyl ether. The combined organic fractions were then washed with water, brine, dried over MgSO4 and concentrated. The resulting translucent oil is purified by silica gel circular chromatography using 10% AcOEt/Hex to afford 1.450 g (5.60 mmol, yield = 96%) of the desired azido derivative 8 as a white solid. Mp = 63-64 ℃, Rf = 0.67 (100% CH2Cl2), I.R. 2232 cm-1, 2117 cm-1, 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (d, 4H, J = 8.3 Hz, N=CC(CH)), 7.45 (d, 4H, J = 8.3 Hz, N=CC(CHCH)), 5.82 (s, 1H, CHN3), 13C NMR (101 MHz, CDCl3, 25℃), ô (ppm) = 143.44, 132.87, 128.03, 118.11, 112.73, 62.23; HRMS m/z calc. for C15H9N5 + (H+): 260.0931; found: 260.0930.

4.1.4. 1-(4,4’-Dicyanodiphenylmethyl)-1H-1,2,3-triazole (10a)

To a stirred solution of 180 mg (0.7 mmol) 4,4’-dicyanodiphe- nylazidomethane 8 in 6 mL DMSO was added 13 mg (0.07 mmol) CuI. The reaction vessel was then purged with acetylene gas, after which 85 mg (0.84 mmol) triethylamine were added. The reaction mixture was left under acetylene atmosphere (balloon pressure) with stirring overnight before being quenched with 125 mL satu- rated sodium chloride solution. The resulting mixture was extrac- ted with 4 x 25 mL AcOEt after which the organic phases were combined and washed with 2 x 25 mL concentrated NH4Cl and 2 × 25 mL brine, dried over MgSO4 and concentrated to yield a yellow oil which, after silica gel circular chromatography (0-40% AcOEt/Hex) afforded the desired triazole as a white powder, yield = 29% Mp = 210 ℃ (dec.), Rf = 0.49 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.83 (s, 1H, =CHN), 7.73 (d, 4H, J = 8.3 Hz, HAr), 7.52 (s, 1H, =CHN), 7.27 (d, 4H, J = 8.3 Hz, HAr), 7.16 (s, 1H, Ph-CH-Ph); 13C NMR (101 MHz, CDC13, 25 ℃), ô (ppm): 141.79, 134.37, 133.02, 128.87, 123.70, 117.81, 113.41, 66.91; HRMS m/ z calc. for C17H11N5 + (H+): 286.1087; found: 286.1084.

4.2. General procedure for the preparation of 1,2,3-triazoles (10b-10n, 11b-11n)

To a stirred solution of the azido precursor 8 (0.5 mmol) in 4 mL THF at room temperature are sequentially added 1.5 eq of the desired alkyne (0.75 mmol), 0.1 eq. of copper (I) iodide (0.05 mmol) and 1.2 eq. of triethylamine (0.6 mmol). The solution is stirred for 12 h then diluted with 30 mL ethyl acetate, washed twice with a saturated solution of NH4Cl, twice with brine, dried over MgSO4, filtered and evaporated. The residue is purified by silica gel circular chromatography (eluent: EtOAc/Hex or MeOH/CH2Cl2).

4.2.1. 1-(4,4’-Dicyanodiphenylmethyl)-4-propyl-1H-1,2,3- triazole (10b)

From azide 8 and 1-pentyne. Dark brown oil after silica gel circular chromatography (0-40% AcOEt/Hex), yield = 58%. Rf = 0.19 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (d, 4H, J = 8.4 Hz, N=CC(CH)), 7.26 (d, 4H, J = 8.4 Hz, N=CC(CHCH)), 7.20 (s, 1H, =CHN), 7.09 (s, 1H, Ph-CH-Ph), 2.73-2.70 (t, 2H, J = 7.6 Hz, CH2CH2CH3), 1.75-1.70 (m, 2H, CH2CH2CH3), 0.99-0.95 (t, 3H, J = 7.4 Hz, CH3), 13C NMR (101 MHZ, CDCl3, 25 ℃), ô (ppm) = 148.97, 142.07, 132.96, 128.87, 120.73, 117.89, 113.23, 66.84, 27.67, 22.56, 13.79; HRMS m/z calc. for C20H17N5 + (H+): 328.1557; found: 328.1556.

4.2.2. 1-(4,4’-Dicyanodiphenylmethyl)-4-hexyl-1H-1,2,3-triazole (10c)

From azide 8 and 1-octyne. Translucent oil after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 80%. Rf = 0.41 (35% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.72 (d, 4H, J= 8.5 Hz, N=CC(CH)), 7.22 (d, 4H, J = 8.5 Hz, N=CC(CHCH)), 7.19(s, 1H, =CHN), 7.08 (s, 1H, Ph-CH-Ph), 2.73 (t, 2H, J = 7.8 Hz, CH2CH2(CH2)3CH3), 1.67 (m, 2H, CH2CH2(CH2)3CH3), 1.41-1.25 (m, 6H, CH2CH2(CH2)3CH3), 0.89 (t, 3H, J = 6.9 Hz, CH3). 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm) = 149.24, 142.06, 132.95, 128.87, 120.60, 117.87, 113.25, 66.85, 31.47, 29.25, 28.91, 25.71, 22.52, 14.02; HRMS m/z calc. for C23H23N5 + (H+): 370.2020; found: 370.2019.

4.2.3. 1-(4,4’-Dicyanodiphenylmethyl)-4-heptyl-1H-1,2, 3-triazole (10d)

From azide 8 and 1-nonyne. Translucent oil after silica gel circular chromatography (0-30% AcOEt/Hex), yield = 69%. Rf = 0.38 (35% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (d, 4H, J = 8.5 Hz, N=CC(CH)), 7.26 (d, 4H, J = 8.5 Hz, N=CC(CHCH)), 7.19 (s, 1H, =CHN), 7.08 (s, 1H, Ph-CH-Ph), 2.73 (t, 2H, J = 7.8 Hz, CH2CH2(CH2)4CH3), 1.67 (m, 2H, CH2CH2(CH2)4CH3),

1.34-1.22 (m, 8H, CH2CH2(CH2)4CH3), 0.88 (t, 3H, J = 6.9 Hz, CH3), 13C NMR (101 MHz, CDC13, 25 ℃), ô (ppm) = 149.23, 142.08, 132.95, 128.88, 120.62, 117.88, 113.23, 66.84, 31.70, 29.29, 29.21, 28.94, 25.71, 22.59, 14.07; HRMS m/z calc. for C24H25N5 + (H+): 384.2180; found: 384.2180.

4.2.4. 1-(4,4’-Dicyanodiphenylmethyl)-4-decyl-1H-1,2,3- triazole (10e)

From azide 8 and 1-dodecyne. Translucent oil after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 79%. Rf = 0.43 (35% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (d, 4H, J = 8.3 Hz, N=CC(CH)), 7.26 (d, 4H, J = 8.3 Hz, N=CC(CHCH)), 7.19 (s, 1H, =CHN), 7.08 (s, 1H, Ph-CH-Ph), 2.73 (t, 2H, J = 7.8 Hz, CH2CH2(CH2)7CH3), 1.67 (m, 2H, CH2CH2(CH2)7CH3), 1.40-1.23 (m, 14H, CH2CH2(CH2)7CH3), 0.89 (t, 3H, J = 6.9 Hz, CH3), 13C NMR (101 MHz, CDC13, 25 ℃), ô (ppm) = 149.25, 142.07, 132.95, 128.87, 120.60, 117.87, 113.25, 66.85, 31.89, 29.56, 29.53, 29.30, 25.72, 22.67, 14.11; HRMS m/z calc. for C27H31N5 + (H+): 426.2652; found: 426.2648.

4.2.5. 1-(4,4’-Dicyanodiphenylmethyl)-4-cyclohexyl-1H-1,2,3- triazole (10f)

From azide 8 and ethynylcyclohexane. White solid after silica gel circular chromatography (0-35% AcOEt/Hex), yield = 63%. Mp = 150-152 ℃, Rf = 0.34 (35% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (d, 4H, J = 8.1 Hz, N=CC(CH)), 7.25 (d, 4H, J = 8.1 Hz, N=CC(CHCH)), 7.15 (s, 1H, =CHN), 7.07 (s, 1H, Ph-CH-Ph), 2.80-2.75 (m, 1H, cyclohexyl), 2.11-2.00 (m, 2H, cyclohexyl), 1.84-1.65 (m, 3H, cyclohexyl), 1.48-1.33 (m, 4H, cyclohexyl), 1.33-1.19 (m, 1H, cyclohexyl), 13C NMR (101 MHZ, CDCl3, 25 ℃), ô (ppm) = 154.42, 142.12, 132.94, 128.89, 119.32, 117.89, 113.21, 66.89, 35.31, 32.91, 26.04, 25.93; HRMS m/z calc. for C23H21N5 + (H+): 368.1870; found: 368.1865.

4.2.6. 1-(4,4’-Dicyanodiphenylmethyl)-4-phenyl-1H-1,2,3- triazole (10g)

From azide 8 and 1-ethynylbenzene. White solid after silica gel circular chromatography (0-0.3% MeOH/CH2Cl2), yield = 91%. Mp = 82-85 ℃, Rf = 0.52 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.83 (m, 2H, HAr), 7.74 (d, 4H, J = 8.4 Hz, N=CC(CH)), 7.68 (s, 1H, =CHN), 7.46-7.42 (m, 2H, HAr), 7.40-7.35 (m, 1H, HAr), 7.32 (d, 4H, J = 8.4 Hz, N=CC(CHCH)), 7.18 (s, 1H, Ph-CH-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 148.38, 141.76, 133.06, 129.75, 128.97, 128.93, 128.73, 125.78, 119.50, 117.83, 113.42, 67.10; HRMS m/z calc. for C23H15N5 + (H+): 362.1400; found: 362.1393.

4.2.7. 1-(4,4’-Dicyanodiphenylmethyl)-4-(phenoxymethyl)-1H- 1,2,3-triazole (10h)

From azide 8 and 1-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-0.3% MeOH/CH2Cl2), yield = 71%. Mp = 55-59 ℃, Rf= 0.27 (1.5% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.72 (d, 4H, J = 8.5 Hz, N=CC (CH)), 7.56 (s, 1H, =CHN), 7.33-7.29 (m, 2H, HAr), 7.26 (d, 4H, J = 8.5 Hz, N=CC(CHCH)), 7.12 (s, 1H, Ph-CH-Ph), 7.03-6.99 (m, 3H, HAr), 5.25 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm) = 157.96, 145.09, 141.64, 133.03, 129.62, 128.89, 122.81, 121.55, 117.82, 114.80, 113.41, 67.14, 62.00; HRMS m/z calc. for C24H17N50 + (H+): 392.1506; found: 392.1503.

4.2.8. 1-(4,4’-Dicyanodiphenylmethyl)-4-((4-chlorophenoxy) methyl)-1H-1,2,3-triazole (10i)

From azide 8 and 1-chloro-4-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-0.3% MeOH/ CH2Cl2), yield = 60%. Mp = 59-63 ℃, Rf = 0.27 (1.5% MeOH/

CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.72 (d, 4H, J = 8.4 Hz, N=CC(CH)), 7.55 (s, 1H, =CHN), 7.27-7.24 (m, 6H, N=CC(CHCH) + CIC(CH)), 7.12 (s, 1H, Ph-CH-Ph), 6.91 (m, 2H, CIC(CHCH)), 5.20 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 156.58, 144.57, 141.56, 133.05, 129.49, 128.87, 126.48, 122.90, 117.79, 116.14, 113.47, 67.18, 62.25; HRMS m/z calc. for C24H16CIN50 + (H+): 426.1116; found: 426.1111.

4.2.9. 1-(4,4’-Dicyanodiphenylmethyl)-4-((4-cyanophenoxy) methyl)-1H-1,2,3-triazole (10j)

From azide 8 and 4-(prop-2-ynyloxy)benzonitrile. White solid after silica gel circular chromatography (0-0.4% MeOH/CH2Cl2), yield = 97%. Mp = 72-75 ℃, Rf = 0.32 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.73 (d, 4H, J = 8.5 Hz, N=CC(CH)), 7.63-7.59 (m, 3H, HAr + =CHN), 7.28 (d, 4H, J = 8.5 Hz, N=CC(CHCH)), 7.13 (s, 1H, Ph-CH-Ph), 7.07 (m, 2H, HAr), 5.27 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm) = 161.24, 143.68, 141.48, 134.11, 133.08, 128.89, 123.18, 118.91, 117.75, 115.46, 113.53, 104.89, 67.26, 62.00; HRMS m/z calc. for C25H16N60 + (H+): 417.1458; found: 417.1449.

4.2.10. 1-(4,4’-Dicyanodiphenylmethyl)-4-((4-nitrophenoxy) methyl)-1H-1,2,3-triazole (10k)

From azide 8 and 1-nitro-4-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-0.4% MeOH/ CH2Cl2), yield = 71%. Mp = 72-76 °℃, Rf = 0.21 (1% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 8.22 (dt, 2H, J = 7.0 Hz, 2.8 Hz, NO2C(CH)), 7.73 (d, 4H,J = 8.5 Hz, N=CC(CH)), 7.63 (s,1H,= CHN), 7.29 (d, 4H, J = 8.5 Hz, N=CC(CHCH)), 7.13 (s, 1H, Ph-CH-Ph), 7.08 (dt, 2H, J = 7.0 Hz, 2.8 Hz, NO2C(CHCH)), 5.32 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 162.90, 143.48, 142.06, 141.44, 133.09, 128.88, 125.98, 123.24, 117.74, 114.77, 113.56, 67.30, 62.33; HRMS m/z calc. for C24H16NGO3 + (H+): 437.1357; found: 437.1351.

4.2.11. 1-(4,4’-Dicyanodiphenylmethyl)-4-((p-tolyloxy)methyl)-1H- 1,2,3-triazole (10l)

From azide 8 and 1-methyl-4-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-0.4% MeOH/ CH2Cl2), yield = 77%. Mp = 55-64 ℃, Rf = 0.47 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (d, 4H, J = 8.4 Hz, N=CC(CH)), 7.56 (s, 1H, =CHN), 7.26 (d, 4H, J = 8.4 Hz, N=CC(CHCH)), 7.12-7.09 (m, 3H, HAr + Ph-CH-Ph), 6.87 (m, 2H, HAr), 5.20 (s, 2H, CH2-0-Ph), 2.31 (s, 3H, CH3), 13C NMR (101 MHZ, CDCl3, 25 ℃), ô (ppm) = 155.90, 145.23, 141.68, 133.02, 130.86, 130.04, 128.90, 122.82, 117.84, 114.67, 113.37, 67.12, 62.19, 20.50; HRMS m/z calc. for C15H19N50 + (H+): 416.1662; found: 406.1659.

4.2.12. 1-(4,4’-Dicyanodiphenylmethyl)-4-((4-methoxyphenoxy) methyl)-1H-1,2,3-triazole (10m)

From azide 8 and 1-methoxy-4-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-0.4% MeOH/ CH2Cl2), yield = 70%. Mp = 55-63 ℃, 1H NMR (400 MHZ, CDCI3, 25 ℃), ô (ppm) = 7.72 (d, 4H, J = 8.4 Hz, N=CC(CH)), 7.53 (s, 1H, = CHN), 7.26 (d, 4H, J = 8.4 Hz, N=CC(CHCH)), 7.11 (s, 1H, Ph-CH-Ph), 6.92-6.89 (m, 2H, HAr), 6.87-6.83 (m, 2H, HAr), 5.19 (s, 2H, CH2-0-Ph), 3.79 (s, 3H, OCH3), 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm) = 154.41, 152.06, 145.26, 141.64, 133.03, 128.88, 122.75, 117.81, 115.97, 114.70, 113.43, 67.13, 62.82, 55.70; HRMS m/z calc. for C25H19N502 + (H+): 422.1612; found: 422.1603.

4.2.13. 1-(4,4’-Dicyanodiphenylmethyl)-4-((4-phenylphenoxy) methyl)-1H-1,2,3-triazole (10n)

From azide 8 and 1-phenyl-4-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-0.25% MeOH/

CH2Cl2), yield = 80%. Mp = 66-70 ℃, Rf = 0.28 (1% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.72 (d, 4H, J = 8.3 Hz, N=CC(CH)), 7.58-7.53 (m, 5H, HAr + =CHN), 7.47-7.42 (m, 2H, HAr), 7.36-7.32 (m, 1H, HAr), 7.27 (d, 4H, J = 8.3 Hz, N=CC(CHCH)), 7.12 (s, 1H, Ph-CH-Ph), 7.08-7.04 (m, 2H, HAr), 5.29 (s, 2H, CH2O), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 157.53, 145.00, 141.62, 140.44, 134.64, 133.04, 128.89, 128.83, 128.26, 126.94, 126.71, 122.86, 117.80, 115.11, 113.44, 67.18, 62.15; HRMS m/z calc. for C30H21N50 + (H+): 468.1819; found: 468.1813.

4.2.14. 1-(Diphenylmethyl)- 1H-1,2,3-triazole (11a)

Compound 11a was prepared from azide 9 [32] and acetylene using the general procedure for the synthesis of 10a. Purification by chromatography (0-20% AcOEt/Hex) gave 11a as a white solid; yield = 92%. Mp = 119-120 ℃, Rf = 0.27 (40% AcOEt/hexane), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.75 (s, 1H, =CHN=), 7.45 (s, 1H, =CHN-Ph), 7.40-7.37, (m, 6H, HAr), 7.17 (s, 1H, CH-Ph), 7.14-7.12 (m, 4H, HAr), 13C NMR (101 MHz, CDC13, 25 ℃), ô (ppm): 138.19, 133.65, 128.94, 128.59, 128.08, 123.56, 67.94; HRMS m/z calc. for C15H13N3 + (H+): 236.1182; found: 236.1178.

4.2.15. 1-(Diphenylmethyl)-4-propyl-1H-1,2,3-triazole (11b)

From azide 9 and 1-pentyne and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chro- matography (0-20% AcOEt/Hex), yield = 40%. Mp = 89-90 ℃, Rf = 0.25 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.40-7.34 (m, 6H, HAr), 7.15 (s, 1H, =CHN), 7.14-7.11 (m, 4H, HAr), 7.10 (s, 1H, Ph-CH-Ph), 2.70 (t, 2H, J = 7.4 Hz, CH2CH2CH3), 1.69 (sext., 2H, J = 7.4 Hz, CH2CH2CH3), 0.96 (t, 3H, J = 7.4 Hz, CH3), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 148.10, 138.44, 128.87, 128.45, 128.09, 120.66, 67.88, 27.81, 22.70, 13.80. HRMS m/z calc. for C18H19N3 + (H+): 278.1652; found: 278.1645.

4.2.16. 1-(Diphenylmethyl)-4-hexyl-1H-1,2,3-triazole (11c)

From azide 9 and 1-octyne and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chro- matography (0-13% AcOEt/Hex), yield = 68%. Mp = 70-72 ℃, Rf = 0.11 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.40-7.35 (m, 6H, HAr), 7.162-7.109 (m, 5H, HAr + =CHN), 7.10 (s, 1H, Ph-CH-Ph), 2.71 (t, 2H, J = 7.7 Hz, CH2CH2(CH2)3CH3), 1.66 (quint., 2H, J = 7.7 Hz, CH2CH2(CH2)3CH3), 1.39-1.27 (m, 6H, CH2CH2(CH2)3CH3), 0.88 (m, 3H, CH3), 13C NMR (101 MHz, CDCl3, 25 °C ô (ppm): 148.35, 138.45, 128.86, 128.44, 128.09, 120.56, 67.87, 31.51, 29.39, 28.92, 25.83, 22.54, 14.03. HRMS m/z calc. for C21H25N3 + (H+): 320.2121; found 320.2116.

4.2.17. 1-(Diphenylmethyl)-4-heptyl-1H-1,2,3-triazole (11d)

From azide 9 and 1-nonyne and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 66%. Mp = 70-71 ℃, Rf = 0.29 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDC13, 25 ℃) ô (ppm): 7.40-7.34 (m, 6H, HAr), 7.16-7.11 (m, 5H, HAr + =CHN), 7.10 (s, 1H, Ph-CH-Ph), 2.71 (t, 2H, J = 7.6 Hz, CH2CH2(CH2)4CH3), 1.66 (quit., 2H, J = 7.6 Hz, CH2CH2(CH2)4 CH3), 1.36-1.27 (m, 8H, CH2CH2(CH2)4CH3), 0.88 (m, 3H, CH3). 13C NMR (101 MHz, CDC13, 25 ℃) ô (ppm): 148.35, 138.46, 128.86, 128.44, 128.09, 120.56, 67.88, 31.74, 29.43, 29.22, 28.97, 25.82, 22.59, 14.08. HRMS m/z calc. for C22H27N3 + (H+): 334.2278; found: 334.2270.

4.2.18. 1-(Diphenylmethyl)-4-decyl-1H-1,2,3-triazole (11e)

From azide 9 and 1-dodecyne and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 88%. Mp = 83-84 ℃, Rf = 0.52 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCl3, 25 ℃)

ô (ppm): 7.41-7.35 (m, 6H, HAr), 7.15-7.11 (m, 5H, HAr + =CHN), 7.09 (s, 1H, Ph-CH-Ph), 2.71 (t, 2H, J = 7.6 Hz, CH2CH2(CH2)7CH3), 1.64 (quit., 2H, J = 7.6 Hz, CH2CH2(CH2)7CH3), 1.33-1.29 (m, 14H, CH2CH2(CH2)7CH3), 0.89 (m, 3H, CH3). 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 148.36, 138.46, 128.85, 128.43, 128.09, 120.55, 67.88, 31.90, 29.56, 29.44, 29.32, 25.83, 22.68, 14.11. HRMS m/z calc. for C25H33N3 + (H+): 376.2747; found: 375.2740.

4.2.19. 1-(Diphenylmethyl)-4-cyclohexyl-1H-1,2,3-triazole (11f)

From azide 9 and ethynylcyclohexane and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 51%. Mp = 160-162 ℃, Rf = 0.31 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.40-7.33 (m, 6H, HAr), 7.15-7.10 (m, 5H, HAr + =CHN), 7.09 (s, 1H, Ph-CH-Ph), 2.80-2.74 (m, 1H, cyclo- hexyl), 2.07-2.05 (m, 2H, cyclohexyl), 1.81-1.69 (m, 3H, cyclo- hexyl), 1.44-1.29 (m, 4H, cyclohexyl), 1.29-1.23 (m, 1H, cyclohexyl). 13C NMR (101 MHz, CDCl3, 25 C) ô (ppm): 153.60, 138.50, 128.85, 128.42, 128.11, 119.20, 67.90, 35.42, 33.02, 26.15, 26.06. HRMS m/z calc. for C21H23N3 + (H+): 318.1965; found: 318.1958.

4.2.20. 1-(Diphenylmethyl)-4-phenyl-1H-1,2,3-triazole (11g)

From azide 9 and 1-ethynylbenzene and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 78%. Mp = 176-178 ℃, Rf = 0.35 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.84-7.82 (m, 2H, HAr), 7.63 (s, 1H, =CHN), 7.43-7.38 (m, 8H, HAr), 7.37-7.31 (m, 1H, HAr), 7.20-7.19 (m, 4H, HAr), 7.18 (s, 1H, Ph-CH-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 147.58, 138.15, 130.57, 128.97, 128.79, 128.62, 128.16, 128.13, 125.73, 119.58, 68.16. HRMS m/z calc. for C21H17N3 + (H+): 312.1495; found: 312.1489.

4.2.21. 1-(Diphenylmethyl)-4-phenoxymethyl-1H-1,2,3-triazole (11h)

From azide 9 and 1-(prop-2-ynyloxy)benzene and using the general procedure for the synthesis of 10b-n. Pale yellow solid after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 72%. Mp = 153-154 ℃, Rf = 0.28 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.50 (s, 1H, =CHN), 7.41-7.36 (m, 5H, HAr), 7.32-7.28 (m, 3H, HAr), 7.14-7.12 (m, 5H, HAr + Ph-CH-Ph), 7.01-6.97 (m, 3H, HAr), 5.22 (s, 2H, CH2-0-Ph), 13C NMR, (101 MHz, CDCl3, 25 ℃) ô (ppm): 158.21, 144.08, 138.02, 129.51, 128.97, 128.63, 128.10, 122.79, 121.30, 114.90, 68.23, 62.23. HRMS m/z calc. for C22H19N30 + (H+): 342.1601; found: 342.1597.

4.2.22. 1-(Diphenylmethyl)-4-chloro-phenoxymethyl-1H-1,2,3- triazole (11i)

From azide 9 and 1-chloro-4-(prop-2-ynyloxy)benzene and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 70%. Mp = 137-138 ℃, Rf = 0.47 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.48 (s, 1H, =CHN), 7.42-7.36 (m, 6H, HAr), 7.26-7.22 (m, 2H, HAr), 7.17-7.10 (m, 5H, HAr + Ph-CH-Ph), 6.94-6.90 (m, 2H, HAr), 5.18 (s, 2H, CH2-O-Ph), 13C NMR (101 MHz, CDC13, 25 ℃) ô (ppm): 156.78, 143.57, 137.95, 129.39, 128.99, 128.68, 128.07, 126.22, 122.91, 116.27, 68.25, 62.48. HRMS m/z calc. for C22H18N3OCI + (H+): 376.1211; found: 376.1207.

4.2.23. 1-(Diphenylmethyl)-4-cyano-phenoxymethyl-1H-1,2,3- triazole (11j)

From azide 9 and 4-(prop-2-ynyloxy)benzonitrile and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 78%. Mp = 116-117 ℃, Rf = 0.37 (30% AcOEt/Hex). 1H NMR (400 MHZ,

CDC13, 25 ℃) ô (ppm): 7.61-7.52 (m, 2H, HAr), 7.51 (s, 1H, =CHN), 7.42-7.37 (m, 6H, HAr), 7.15-7.11 (m, 5H, HAr + Ph-CH-Ph), 7.07-7.04 (m, 2H, HAr), 5.25 (s, 2H, CH2-O-Ph), 13C NMR (101 MHZ, CDC13, 25 ℃) ô (ppm): 161.42, 142.73, 137.84, 134.03, 129.03, 128.75, 128.06, 123.15, 119.06, 115.60, 104.61, 68.33, 62.23. HRMS m/z calc. for C23H19N40 + (H+): 367.1553, found: 367.1547.

4.2.24. 1-(Diphenylmethyl)-4-nitro-phenoxymethyl-1H-1,2,3- triazole (11k)

From azide 9 and 1-nitro-4-(prop-2-ynyloxy)benzene and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-13% AcOEt/Hex), yield = 88%. Mp = 134-135 ℃, Rf = 0.17 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCI3, 25 ℃) ô (ppm): 8.24-8.20 (m, 2H, HAr), 7.53 (1H, s, =CHN), 7.41-7.37 (6H, m, HAr), 7.16-7.12 (5H, m, HAr + Ph-CH-Ph), 7.10-7.06 (2H, m, HAr), 5.30 (2H, s, CH2-0-Ph), 13C NMR (101 MHZ, CDCI3, 25 ℃) ô (ppm): 163.12, 142.53, 141.91, 137.82, 129.04, 128.77, 128.06, 125.93, 123.22, 114.89, 68.36, 62.57. HRMS m/z calc. for C22H18N403 + (H+): 387.1452; found: 387.1442.

4.2.25. 1-(Diphenylmethyl)-4-methyl-phenoxymethyl-1H-1,2,3- triazole (11l)

From azide 9 and 1-methyl-4-(prop-2-ynyloxy)benzene and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 99%. Mp = 121-122 °℃, Rf = 0.58 (30% AcOEt/Hex). 1H NMR (400 MHz, CDCl3, 25 ℃) ô (ppm): 7.49 (s, 1H, =CHN), 7.41-7.36 (m, 6H, HAr), 7.16-7.09 (m, 7H, HAr + Ph-CH-Ph), 6.90-6.87 (m, 2H, HAr), 5.19 (s, 2H, CH2-0-Ph), 2.31 (s, 3H, CH3), 13C NMR (101 MHZ, CDCl3, 25 ℃) ô (ppm): 156.14, 144.24, 138.04, 130.57, 129.95, 128.96, 128.62, 128.11, 122.77, 114.80, 68.21, 62.46, 20.50. HRMS m/z calc. for C23H21N30 + (H+): 356.1757; found: 356.1749.

4.2.26. 1-(Diphenylmethyl)-4-methoxy-phenoxymethyl-1H-1,2,3- triazole (11m)

From azide 9 and 1-methoxy-4-(prop-2-ynyloxy)benzene and using the general procedure for the synthesis of 10b-n. White solid after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 40%. Mp = 122-123 ℃, Rf = 0.53 (30% AcOEt/Hex). 1H NMR (400 MHz, CDCl3, 25 ℃) ô (ppm): 7.48 (s, 1H, =CHN), 7.41-7.28 (m, 6H, HAr), 7.14-7.11 (m, 5H, HAr + Ph-CH-Ph), 6.94-6.90 (m, 2H, HAr), 6.86-6.82 (m, 2H, HAr) 5.17 (s, 2H, CH2-O-Ph), 3.73 (s, 3H, CH3), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 154.25, 152.33, 144.23, 138.03, 128.96, 128.62, 128.10, 122.79, 116.09, 114.63, 68.20, 63.06, 55.69. HRMS m/z calc. for C23H21N3O2 + (H+): 372.1707; found: 372.1698.

4.2.27. 1-(Diphenylmethyl)-4-phenyl-phenoxymethyl-1H-1,2,3- triazole (11n)

From azide 9 and 1-phenyl-4-(prop-2-ynyloxy)benzene and using the general procedure for the synthesis of 10b-n. Grey solid after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 80%. Mp = 180-182 ℃, Rf = 0.51 (30% AcOEt/Hex). 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.58-7.53 (m, 5H, HAr + =CHN), 7.46-7.28 (m, 9H, HAr), 7.16-7.13 (m, 5H, HAr et Ph-CH-Ph), 7.08-7.06 (m, 2H, HAr), 5.27 (s, 2H, CH2-0-Ph), 13C NMR (101 MHZ, CDC13, 25 ℃) ô (ppm): 157.79, 143.98, 140.66, 138.01, 134.37, 128.99, 128.75, 128.66, 128.19, 128.11, 126.78, 126.76, 122.88, 115.21, 68.26, 62.38. HRMS m/z calc. for C28H23N3O + (H+): 418.1914; found: 418.1903.

4.3. General procedure for the preparation of compounds 17-24

To a stirred solution of 500 mg of the appropriate bromide 12 or 13 in 25 mL anhydrous acetone, under N2 at room temperature, is

added 1.5 eq. of the desired nitrogenous heterocycle followed by 1.5 eq. of K2CO3 and 0.05 eq. of KI. The reaction mixture is left to react overnight at reflux temperature. The resulting solution is diluted with 100 ml of water and extracted with 3 x 30 mL EtOAc. The organic fractions are combined, washed twice with 30 mL KOH 1 M, twice with brine, dried over MgSO4, filtered and concentrated. The residue is purified by silica gel circular chromatography (eluent: EtOAc/Hex or MeOH/CH2Cl2) to yield the pure heterocyclic compounds. 1-substituted-1H-1,2,3-triazoles and 2-substituted- 2H-1,2,3-triazoles (17/18, 19/20) were obtained simultaneously. These isomers were separated by silica gel circular chromatography.

4.3.1. 1-(4-Cyanophenylmethyl)-1H-1,2,3-triazole (17) and 2-(4- cyanophenylmethyl)-2H-1,2,3-triazole (19)

From bromide 12 and 1H-1,2,3-triazole 14, white solid after silica gel circular chromatography (0-80% AcOEt/Hex), yield (17) = 52%. Mp = 82-83 ℃, Rf = 0.40 (2% MeOH/CH2Cl2). (17): 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.77 (s, 1H, =CHN=), 7.70 (d, 2H, J = 8.1 Hz, HAr), 7.62 (s, 1H, =CHN-Ph), 7.35 (d, 2H, J= 8.1 Hz, HAr), 5.66 (s, 2H, CH2); 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 199.75, 139.98, 134.59, 132.87, 128.32, 123.71, 118.14, 112.72, 53.18; HRMS m/z calc. for C10H8N4 + (H+): 185.0822; found: 185.0823. Yield (19) = 35%. Mp = 80-81 ℃, Rf = 0.83 (2% MeOH/CH2Cl2). (19): 1H NMR (400 MHz, CDCl3, 25 ℃), ô (ppm) = 7.70 (s, 2H, NCHCHN), 7.66 (d, 2H, J = 8.1 Hz, HAr), 7.38 (d, 2H, J = 8.1 Hz, HAr), 5.69 (s, 2H, CH2); 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm) = 140.36, 135.04, 132.61, 128.49, 118.38, 112.34, 57.79; HRMS m/z calc. for C10HgN4 + (H+): 185.0822; found: 185.0819.

4.3.2. 1-(Phenylmethyl)-1H-1,2,3-triazole (18) and 2- (phenylmethyl)-2H-1,2,3-triazoles (20)

From bromide 13 and 1H-1,2,3-triazole 14, white solid after silica gel circular chromatography (0-50% AcOEt/Hex), yield (18) = 75%. Mp = 56-57 ℃, Rf = 0.17 (40% AcOEt/hexane). (18): 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.75 (s, 1H, =CH-N=), 7.49 (s, 1H, =CH-N-Ph), 7.42-7.35, (m, 3H, HAr), 7.29-7.27 (m, 2H, HAr), 5.59 (s, 2H, CH2-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm): 134.70, 134.27, 129.12, 128.75, 128.02, 123.30, 53.98. HRMS m/z calc. for C9HgN3 + (H+): 160.0869; found: 160.0868. Yield (20) = 23%. Mp = 39-41 ℃, Rf = 0.63 (40% AcOEt/hexane). (20) 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.65 (s, 2H, =CHCH=), 7.39-7.32, (5H, m, HAr), 5.63 (s, 2H, CH2-Ph), 13C NMR (101 MHZ, CDC13, 25 ℃), ô (ppm): 135.25, 134.53, 128.80, 128.33, 128.01, 58.59; HRMS m/z calc. for C9HgN3 + (H+): 160.0869; found: 160.0868.

4.3.3. 1-(4-Cyanophenylmethyl)-1H-1,2,4-triazole (21)

From bromide 12 and 1H-1,2,4-triazole 15, white solid after silica gel circular chromatography (0-80% AcOEt/Hex), yield = 56%. Mp = 66-68 ℃, Rf = 0.14 (70% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 8.17 (s, 1H, NCHN), 8.02 (s, 1H, NCHN), 7.68 (d, 2H, J = 8.1 Hz, HAr), 7.35 (d, 2H, J = 8.1 Hz, HAr), 5.44 (s, 2H, CH2), 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm): 152.70, 143.46, 139.91, 132.84, 128.31, 116.16, 112.67, 52.74; HRMS m/z calc. for C10H18N4 + (H+): 185.0822; found: 185.0821.

4.3.4. 1-(Phenylmethyl)-1H-1,2,4-triazole (22)

From bromide 13 and 1H-1,2,4-triazole 15, yellow solid after silica gel circular chromatography (0-50% AcOEt/Hex), yield = 63%. Mp = 90 ℃, Rf = 0.45 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCI3, 25 °℃), ô (ppm) = 8.05 (s, 1H, NCHN), 7.95 (s, 1H, NCHN), 7.45-7.23 (m, 5H, HAr), 5.34 (s, 2H, CH2). 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 153.10, 144.00, 135.50, 130.04, 129.64, 128.98, 54.57; HRMS m/z calc. for C9H9N3 + (H+): 160.0869; found: 160.0870.

4.3.5. 1-(4-Cyanophenylmethyl)-1H-imidazole (23)

From bromide 12 and imidazole 16, white solid after silica gel circular chromatography (0-1% MeOH/CH2Cl2), yield = 50%. Mp = 89-90 °C, Rf = 0.10 (1% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71-7.65 (d, 2H,J = 8.1 Hz, HAr), 7.58 (s, 1H, NCH=N), 7.27-7.21 (d, 2H, J = 8.1 Hz, HAr), 7.18 (s, 1H, NCHCHN), 6.95 (s, 1H, NCHCHN), 5.24 (s, 1H, CH2-Ph); 13C NMR (101 MHZ, CDC13, 25 ℃), ô (ppm): 141.63, 137.55, 132.78, 130.40, 127.59, 119.28, 118.25, 112.22, 50.11; HRMS m/z calc. for C11H9N3 + (H+): 184.0869; found: 184.0867.

4.3.6. 1-(Phenylmethyl)-1H-imidazole (24)

From bromide 13 and imidazole 16, yellow solid after silica gel circular chromatography (0-50% AcOEt/Hex), yield = 67%. Mp = 68-70 ℃, Rf = 0.21 (50% AcOEt/Hex), 1H NMR (200 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.52 (s, 1H, NCHN), 7.45-7.00 (m, 5H, HAr), 6.97 (s, 1H, NCHCHN), 6.89 (s, 1H, NCHCHN), 5.00 (s, 2H, CH2), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 137.45, 136.21, 129.82, 128.98, 128.25, 127.27, 119.29, 50.78; HRMS m/z calc. for C10H10N2 + (H+): 159.0917; found: 159.0914.

4.4. General procedure for the preparation of compounds 10a, 25, and 26

At -5 ℃ and under inert atmosphere, 2.0 mmol of t-BuOK are suspended in 3.5 mL anhydrous DMF with vigourous stirring. Over a period of 1 h, 0.75 mmol of 17 (19, or 23) dissolved in 1 mL DMF is added dropwise to the reaction mixture, followed by dropwise addition of 133 mg (1.09 mmol) of 4-fluorobenzonitrile over 30 min. After 1hr the mixture is quenched with 3 M HCl until acidic (pH < 5), neutralized with NaHCO3, diluted with 100 mL water then extracted with 4 x 25 mL EtOAc. The organic fraction is washed with brine, dried over MgSO4, filtered and concentrated. The residue is purified by silica gel circular chromatography (eluent: EtOAc/Hex) to yield the pure compounds.

4.4.1. 1-(4,4’-Dicyanodiphenylmethyl)-1H-1,2,3-triazole (10a)

From 17 and 4-fluorobenzonitrile, white powder after silica gel circular chromatography (0-40% AcOEt/Hex), yield = 26%. Mp = 210 ℃ (dec.), Rf= 0.49 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.83 (s, 1H, =CHN), 7.73 (d, 4H, J = 8.3 Hz, HAr), 7.52 (s, 1H, =CHN), 7.27 (d, 4H, J = 8.3 Hz, HAr), 7.16 (s, 1H, Ph-CH-Ph); 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm): 141.79, 134.37, 133.02, 128.87, 123.70, 117.81, 113.41, 66.91; HRMS m/z calc. for C17H11N5 + (H+): 286.1087; found: 286.1084.

4.4.2. 2-(4,4’-Dicyanodiphenylmethyl)-2H-1,2,3-triazoles (25)

From 19 and 4-fluorobenzonitrile, yellow crystals after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 58%. Mp = 154-156 ℃, Rf = 0.82 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.76 (s, 2H, NCHCHN), 7.69 (d, 4H, J= 8.3 Hz, HAr), 7.33 (d, 4H, J = 8.3 Hz, HAr), 7.15 (s, 1H, Ph-CH-Ph); 13C NMR (101 MHz, CDC13, 25 ℃), ô (ppm): 142.38, 135.37, 132.66, 129.03, 118.11, 112.88, 70.91; HRMS m/z calc. for C17H11N5 + (H+): 286.1087; found: 286.1083.

4.4.3. 1-(4,4’-Dicyanodiphenylmethyl)-1H-imidazole (26)

From 23 and 4-fluorobenzonitrile, yellow syrup after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 66%. Rf = 0.30 (2% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25℃), ô (ppm) =7.72 (d, 4H, J = 8.1 Hz, HAr), 7.49 (s, 1H, NCH=N), 7.22 (d, 4H, J = 8.1 Hz, HAr), 7.16 (s, 1H, Ph-CH-Ph), 6.89 (s, 1H, NCHCHN), 6.72 (s, 1H, NCHCHN); 13C NMR (101 MHz, CDC13, 25 ℃), ô (ppm): 142.87, 137.15, 132.97, 130.33, 128.81, 118.88, 117.93, 113.01, 63.83; HRMS m/z calc. for C18H12N4 + (H+): 285.1135; found: 285.1133.

4.4.4. 2-(Diphenylmethyl)-2H-1,2,3-triazoles (27) and 1- (diphenylmethyl)-1H-1,2,3-triazole (11a)

Compound 27 and 11a were simultaneously prepared from bromide 7 (400 mg, 1.6 mmol) and 1H-1,2,3-triazole 14 (223.5 mg, 3.23 mmol) using the general procedure for the synthesis of 17-24. Purification by chromatography (0-20% AcOEt/Hex) gave 27 as a white solid; yield = 22%. Mp = 89-91 ℃, Rf = 0.67 (40% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.71 (s, 2H, =CHCH=), 7.40-7.34, (m, 6H, HAr), 7.25-7.23 (m, 4H, HAr), 7.11 (s, 1H, Ph-CH-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm): 138.67, 134.49, 128.59, 128.27, 128.22, 72.26; HRMS m/z calc. for C15H13N3 + (H+): 236.1182; found: 236.1178. Purification by chromatography (0-20% AcOEt/ Hex) gave 11a as a white solid; yield = 71%. Mp = 119-120 ℃, Rf = 0.27 (40% AcOEt/hexane), 1H NMR (400 MHZ, CDCI3, 25 ℃), ô (ppm) = 7.75 (s, 1H, =CHN=), 7.45 (s, 1H, CHN-Ph), 7.40-7.37, (m, 6H, HAr), 7.17 (s, 1H, CH-Ph), 7.14-7.12 (m, 4H, HAr), 13C NMR (101 MHz, CDCI3, 25 ℃), ô (ppm): 138.19, 133.65, 128.94, 128.59, 128.08, 123.56, 67.94; HRMS m/z calc. for C15H13N3 + (H+): 236.1182; found: 236.1178.

4.4.5. 1-(Diphenylmethyl)-1H-1,2,4-triazole (28)

Compound 28 was prepared from bromide 7 (500 mg, 2.03 mmol) and 1H-1,2,4-triazole 15 (209 mg, 3.04 mmol) using the general procedure for the synthesis of 17-24. Purification by chromatography (0-20% AcOEt/Hex) gave 27 as a white solid; yield = 51%. Mp = 89-91 ℃, Rf = 0.71 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 8.02 (s, 1H, NCHN), 7.91 (s, 1H, NCHN), 7.50-7.00 (m, 10H, HAr), 6.72 (s, 1H, Ph-CH-Ph), 13C NMR (101 MHz, CDCl3, 25 °℃), ô (ppm) = 152.30, 143.56, 137.95, 128.97, 128.63, 128.15, 67.89; HRMS m/z calc. for C15H13N3 + (H+): 236.1182; found: 236.1182.

4.4.6. 1-(Diphenylmethyl)-1H-imidazole (29)

Compound 29 was prepared from bromide 7 (500 mg, 2.03 mmol) and imidazole 16 (206 mg, 3.03 mmol) using the general procedure for the synthesis of 17-24. Purification by chromatography (0-20% AcOEt/Hex) gave 29 as a yellow solid; yield = 55%. Mp = 83-84 ℃, Rf = 0.47 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃), ô (ppm) = 7.40 (s, 1H, NCHN), 7.38-7.12 (m, 11H, NCHCHN + HAr), 6.89 (s, 1H, NCHCHN), 6.54 (s, 1H, Ph-CH-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃), ô (ppm) = 139.15, 137.44, 129.39, 128.88, 128.38, 128.08, 119.39, 65.04; HRMS m/z calc. for C16H14N2 + (H+): 235.1230; found: 236.1228.

4.5. General CuAAC procedure for the preparation of 1,2,3-triazoles (34b-n, 35b-n)

To a stirred solution of the appropriate azido precursor 32 [39], or 33 [40] (0.5 mmol) in 4 mL THF at room temperature are sequentially added 1.5 eq of the desired alkyne (0.75 mmol), 0.1 eq. of copper (I) iodide (0.05 mmol) and 1.2 eq. of triethylamine (0.6 mmol). The solution is stirred for 12 h then diluted with 30 mL ethyl acetate, washed twice with a saturated solution of NH4Cl, twice with brine, dried over MgSO4, filtered and evaporated. The residue is purified by silica gel circular chromatography (eluent: EtOAc/Hex or MeOH/CH2Cl2).

4.5.1. 1-(4-Cyanophenylmethyl)-4-propyl-1H-1,2,3-triazole (34b)

From azide 32 and pent-1-yne. Yellow crystals after silica gel circular chromatography (0-30% AcOEt/Hex), yield = 40%. Mp = 80-82 ℃, Rf = 0.57 (50% AcOEt/Hex), 1H NMR (200 MHZ, CDC13, 25 ℃) ô (ppm): 7.67 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.45-7.35 (m, 3H, =CHN + N=CC(CHCH)), 5.60 (s, 2H, CH2-Ph), 2.72 (m, 2H, -CH2CH2CH3), 1.70 (m, 2H, -CH2CH2CH3), 0.95 (m, 3H, CH3). 13C

NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 149.12, 140.34, 132.79, 128.26, 120.92, 118.21, 112.51, 53.15, 27.64, 22.57, 13.74. HRMS m/z calc. for C13H14N4 + (H+): 227.1291; found: 227.1290.

4.5.2. 1-(4-Cyanophenylmethyl)-4-hexyl-1H-1,2,3-triazole (34c)

From azide 32 and oct-1-yne. White powder after silica gel circular chromatography (0-30% AcOEt/Hex), yield = 76%. Mp = 84-86 ℃, Rf = 0.74 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.68 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.32 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 7.28 (s, 1H, =CHN), 5.60 (s, 2H, CH2-Ph), 2.75 (m, 2H, -CH2CH2(CH2)3CH3), 1.82-1.60 (m, 2H, -CH2CH2(CH2)3CH3), 1.55-1.20 (m, 6H, -CH2CH2(CH2)3CH3), 0,89 (m, 3H, CH3), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 149.47, 140.30, 132.83, 128.25, 120.73, 118.18, 112.63, 53.20, 31.52, 29.31, 28.89, 25.68, 22.53, 14.04. HRMS m/z calc. for C16H20N4 + (H+): 269.1761; found: 269.1758.

4.5.3. 1-(4-Cyanophenylmethyl)-4-heptyl-1H-1,2,3-triazole (34d)

From azide 32 and non-1-yne. White solid after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 71%. Mp = 70-72 ℃, Rf = 0.18 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.68 (d, 2H, J = 8.4, N=CC(CH)), 7.35 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 7.25 (s, 1H, =CHN), 5.56 (s, 2H, CH2-Ph), 2.72 (m, 2H, CH2CH2(CH2)4CH3), 1.80-1.58 (m, 2H, CH2CH2(CH2)4CH3), 1.55-1.10 (m, 8H, CH2CH2(CH2)4CH3), 0.86 (m, 3H, CH3).13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 149.43, 140.31, 132.80, 128.25, 120.74, 118.17, 112.59, 53.18, 31.703, 29.33, 29.17, 28.97, 25.67, 22.59, 14.05. HRMS m/z calc. for C17H22N4 + (H+): 283.1917; found: 283.1914.

4.5.4. 1-(4-Cyanophenylmethyl)-4-decyl-1H-1,2,3-triazole (34e)

From azide 32 and dodec-1-yne. White solid after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 58%. Mp = 98-100 ℃, Rf = 0.19 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.68 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.35 (d, 2H, J = 8.4, N=CC(CHCH)), 7.30 (s, 1H, =CHN), 5.60 (s, 2H, CH2-Ph), 2.72 (m, 2H, CH2CH2(CH2)7CH3), 1.80-1.60 (m, 2H, CH2CH2(CH2)7CH3), 1.60-1.15 (m, 14H, CH2CH2(CH2)7CH3), 0.90 (m, 3H, CH3). 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 149.46, 140.30, 132.81, 128.24, 120.73, 118.18, 112.60, 53.18, 31.88, 29.56, 29.52, 29.33, 29.24, 25.68, 22.66, 14.10. HRMS m/z calc. for C20H28N4 + (H+): 325.2387; found: 325.2381.

4.5.5. 1-(4-Cyanophenylmethyl)-4-cyclohexyl-1H-1,2,3-triazole (34f)

From azide 32 and ethynylcyclohexane. White powder after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 86%. Mp = 159-160 ℃, Rf = 0.17 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.68 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.34 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 7.22 (s, 1H, =CHN), 5.57 (s, 2H, CH2-Ph), 2.78-2.75 (m, 1H, cyclohexyl), 2.07-2.04 (m, 2H, cyclo- hexyl), 1.82-1.67 (m, 4H, cyclohexyl), 1.45-1.34 (m, 2H, cyclohexyl), 1.29-1.22 (m, 2H, cyclohexyl). 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 154.72, 140.27, 132.85, 128.29, 119.41, 118.21, 112.63, 53.23, 35.30, 32.96, 26.09, 25.99. HRMS m/z calc. for C16H18N4 + (H+): 267.1604; found: 267.1597.

4.5.6. 1-(4-Cyanophenylmethyl)-4-phenyl-1H-1,2,3-triazole (34g)

From azide 32 and 1-ethynylbenzene. White solid after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 63%. Mp = 120-124 ℃, Rf = 0.68 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.95-7.62 (5H, m, HAr + =CHN), 7.60-7.36 (5H, m, HAr), 5.65 (2H, s, CH2-Ph). 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 148.70, 139.90, 132.94, 130.12, 128.92, 128.49, 128.36, 125.75, 119.64, 118.13, 112.85, 53.48. HRMS m/z calc. for C16H12N4 + (H+): 261.1135; found: 261.1132.

4.5.7. 1-(4-Cyanophenylmethyl)-4-phenoxymethyl-1H-1,2,3- triazole (34h)

From azide 32 and 1-(prop-2-ynyloxy)benzene. White solid after silica gel circular chromatography (0-30% AcOEt/Hex), yield = 77%. Mp = 78-80 ℃, Rf = 0.20 (50% AcOEt/Hex), 1H NMR (400 MHz, CDCl3, 25 ℃) ô (ppm): 7.75-7.65 (m, 3H, HAr + =CHN), 7.45-7.30 (m, 4H, HAr), 7.05-6.98 (m, 3H, HAr), 5.62 (s, 2H, CH2-Ph), 5.25 (s, 2H, CH2-0-Ph). 13C NMR (101 MHz, CDC13, 25 ℃) ô (ppm): 158.07, 145.27, 139.69, 132.91, 129.58, 128.40, 122.80, 121.40, 114.76, 112.84, 61.97, 53.45. HRMS m/z calc. for C17H14N40 + (H+): 291.1240; found: 291.1236.

4.5.8. 1-(4-Cyanophenylmethyl)-4-((4-chlorophenoxy)methyl)-1H- 1,2,3-triazole (34i)

From azide 32 and 1-chloro-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-40% AcOEt/Hex), yield = 78%. Mp = 124-128 ℃, Rf = 0.44 (50% AcOEt/Hex), 1H NMR (400 MHZ, CDC13, 25 ℃) ô (ppm): 7.61 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.60 (s, 1H, =CHN), 7.38 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 7.30-7.42 (m, 2H, HAr), 7.00-6.90 (m, 2H, HAr), 5.61 (s, 2H, CH2-Ph), 5.20 (s, 2H, CH2-0-Ph). 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 156.67, 144.76, 139.59, 132.93, 129.45, 128.43, 126.32, 122.88, 118.08, 116.12, 112.91, 62.24, 53.49. HRMS m/z calc. for C17H13CIN40 + (H+): 325.0851; found: 325.0844.

4.5.9. 1-(4-Cyanophenylmethyl)-4-((4-cyanophenoxy)methyl)-1H- 1,2,3-triazole (34j)

From azide 32 and 4-(prop-2-ynyloxy)benzonitrile. White solid after silica gel circular chromatography (0-2% MeOH/CH2Cl2), yield = 98%. Mp = 142-144 ℃, Rf = 0.60 (5% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.80-7.55 (d, 5H, HAr + = CHN), 7.48-7.40 (m, 2H, HAr), 7.15-7.05 (m, 2H, HAr), 5.62 (s, 2H, CH2-Ph), 5.27 (s, 2H, CH2-0-Ph). 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 161.34, 143.82, 139.51, 134.06, 132.93, 128.50, 123.22, 119.00, 118.06, 115.52, 112.91, 104.64, 62.02, 53.54. HRMS m/z calc. for C18H13N50 + (H+): 316.1193; found: 316.1189.

4.5.10. 1-(4-Cyanophenylmethyl)-4-((4-nitrophenoxy)methyl)-1H- 1,2,3-triazole (34k)

From azide 32 and 1-nitro-4-(prop-2-ynyloxy)benzene. White flakes after silica gel circular chromatography (0-1% MeOH/ CH2Cl2), yield = 76%. Mp = 108-110 ℃, Rf = 0.20 (5% MeOH/ CH2Cl2), 1H NMR (400 MHz, DMSO-d6, 25 ℃) ô (ppm): 8.42 (s, 1H, = CHN), 8.22 (d, 2H, J = 8.6 Hz, NO2C(CH)), 7.88 (d, 2H, J = 8.4 Hz, N=CC(CH)) 7.55 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 7.29 (d, 2H, J = 8.6 Hz, NO2C(CHCH)), 5.75 (s, 2H, CH2-Ph), 5.36 (s, 2H, CH2-0-Ph). 13C NMR (101 MHz, DMSO-d6, 25 ℃) ô (ppm): 163.28, 142.25, 141.39, 141.12, 132.80, 128.79, 125.89, 125.41, 118.54, 115.372, 111.05, 61.93, 52.31. HRMS m/z calc. for C17H13N503 + (H+): 336.1091; found: 336.1086.

4.5.11. 1-(4-Cyanophenylmethyl)-4-((4-methylphenoxy)methyl)- 1H-1,2,3-triazole (34l)

From azide 32 and 1-methyl-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-30% AcOEt/ Hex), yield = 71%. Mp = 112-114 ℃, Rf = 0.20 (50% AcOEt/Hex), 1H NMR (400 MHz, CDCl3, 25 ℃) ô (ppm): 7.69 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.60 (s, 1H, =CHN), 7.36 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 7.10 (d, 2H, J = 8.3 Hz, CH3C(CH)), 6.88 (d, 2H, J = 8.3 Hz, CH3C(CHCH)), 5.62 (s, 2H, CH2-Ph), 5.21 (s, 2H, CH2-0-Ph), 2.31 (s, 3H, CH3). 13C NMR (101 MHz, CDC13, 25 ℃) ô (ppm): 155.97, 145.48, 139.69, 132.92, 130.70, 130.01, 128.40, 122.72, 118.11, 114.62, 112.87, 62.17, 53.46, 20.48. HRMS m/z calc. for C18H16N40 + (H+): 305.1397; found: 305.1392.

4.5.12. 1-(4-Cyanophenylmethyl)-4-((4-methoxyphenoxy)methyl)- 1H-1,2,3-triazole (34m)

From azide 32 and 1-methoxy-4-(prop-2-ynyloxy)benzene. Beige powder after silica gel circular chromatography (0-2% MeOH/CH2Cl2), yield = 95%. Mp = 168-170℃, Rf= 0.77 (5% MeOH/ CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.67 (d, 2H, J = 8.4 Hz, N=CC(CH)), 7.58 (s, 1H, =CHN), 7.32 (d, 2H, J = 8.4 Hz, N=CC(CHCH)), 6.97-6.80 (m, 4H, HAr), 5.60 (s, 2H, CH2-Ph), 5.15 (s, 2H, CH2-0-Ph), 3.76 (s, 3H, OCH3). 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 154.30, 152.19, 145.45, 139.71, 132.90, 128.40, 122.77, 115.88, 114.70, 62.76, 55.70, 53.44. HRMS m/z calc. for C18H16N4O2 + (H+): 321.1346; found: 321.1341.

4.5.13. 1-(4-Cyanophenylmethyl)-4-((4-phenylphenoxy)methyl)- 1H-1,2,3-triazole (34n)

From azide 32 and 1-phenyl-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-50% AcOEt/ Hex), yield = 88%. Mp = 154-156 ℃, Rf = 0.48 (5% MeOH/CH2Cl2), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.75-7.29 (m, 12H, = CHN + HAr), 7.12-7.02 (m, 2H, HAr), 5.63 (s, 2H, CH2-Ph), 5.25 (s, 2H, CH2-0-Ph). 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 157.62, 145.20, 139.64, 134.50, 132.94, 128.79, 128.43, 128.25, 126.86, 126.73, 122.83, 118.10, 115.07, 112.89, 62.12, 53.49. HRMS m/z calc. for C23H18N40 + (H+): 367.1553; found: 367.1546.

4.5.14. 1-(Phenylmethyl)-4-propyl-1H-1,2,3-triazole (35b)

From azide 33 and pent-1-yne. Yellow oil after silica gel circular chromatography (0-13% AcOEt/Hex), yield = 77%. Rf = 0.11 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.41-7.35 (m, 3H, HAr), 7.28-7.25 (m, 2H, HAr), 7.20 (s, 1H, =CHN), 5.50 (s, 2H, CH2-Ph), 2.68 (t, 2H, J = 7.4 Hz, -CH2CH2CH3), 1.68 (sext., 2H, J = 7.4 Hz, -CH2CH2CH3), 0.95 (t, 3H, J = 7.4, CH3), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 148.76, 135.03, 129.05, 128.59, 127.95, 120.54, 53.96, 27.73, 22.66, 13.78. HRMS m/z calc. for C12H15N3 + (H+): 202.1339; found: 202.1336.

4.5.15. 1-(Phenylmethyl)-4-hexyl-1H-1,2,3-triazole (35c)

From azide 33 and oct-1-yne. White powder after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 82%. Mp = 51-53 ℃, Rf = 0.36 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCI3, 25 ℃) ô (ppm): 7.38-7.36 (m, 3H, HAr), 7.27-7.26 (m, 2H, HAr), 7.25 (s, 1H, =CHN), 5.50 (s, 2H, CH2-Ph), 2.69 (m, 2H, -CH2CH2(CH2)3CH3), 1.66-1.62 (m, 2H, -CH2CH2(CH2)3CH3), 1.34-1.27 (m, 6H, -CH2CH2(CH2)3CH3), 0.88 (m, 3H, CH3). 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 149.00, 135.05, 129.04, 128.57, 127.94, 120.45, 53.96, 31.54, 29.37, 28.91, 25.74, 14.04. HRMS m/z calc. for C15H21N3 + (H+): 244.1808; found: 244.1801.

4.5.16. 1-(Phenylmethyl)-4-heptyl-1H-1,2,3-triazole (35d)

From azide 33 and non-1-yne. White powder after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 84%. Mp = 61-62 ℃, Rf = 0.30 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.36-7.34 (m, 3H, HAr), 7.29-7.25 (m, 2H, HAr), 7.19 (s, 1H, =CHN), 5.51 (s, 2H, CH2-Ph), 2.69 (m, 2H, -CH2CH2(CH2)4CH3), 1.72-1.63 (m, 2H, -CH2CH2(CH2)4CH3), 1.35-1.27 (m, 8H, -CH2CH2(CH2)4CH3), 0.88 (t, 3H, J = 7.0 Hz, CH3). 13C NMR (101 MHz, CDC13, 25 ℃) ô (ppm): 149.01, 135.04, 129.04, 128.58, 127.95, 120.44, 53.97, 31.73, 29.41, 29.20, 29.00, 25.74, 22.61, 14.08. HRMS m/z calc. for C16H23N3 + (H+): 258.1965; found: 258.1960.

4.5.17. 1-(Phenylmethyl)-4-decyl-1H-1,2,3-triazole (35e)

From azide 33 and dodec-1-yne. White powder after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 29%. Mp = 78-79 ℃, Rf = 0.32 (30% AcOEt/Hex), 1H NMR (400 MHZ,

CDCl3, 25 ℃) ô (ppm): 7.40-7.35 (m, 3H, HAr), 7.28-7.25 (m, 2H, HAr), 7.19 (s, 1H, =CHN), 5.51 (s, 2H, CH2-Ph), 2.69 (m, 2H, -CH2CH2(CH2)7CH3), 1.66-1.61 (m, 2H, -CH2CH2(CH2)7CH3), 1.31-1.26 (m, 14H, -CH2CH2(CH2)7CH3), 0.89 (m, 3H, CH3), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 149.02, 135.05, 129.04, 128.58, 127.95, 120.44, 53.97, 31.89, 29.57, 29.54, 29.42, 29.35, 29.32, 29.27, 25.75, 22.68, 14.12. HRMS m/z calc. for C19H29N3 + (H+): 300.2434; found: 300.2427.

4.5.18. 1-(Phenylmethyl)-4-cyclohexyl-1H-1,2,3-triazole (35f)

From azide 33 and ethynylcyclohexane. Grey powder after silica gel circular chromatography (0-20% AcOEt/Hex), yield = 94%. Mp = 105-108 ℃, Rf = 0.32 (30% AcOEt/Hex), 1H NMR (400 MHz, CDCl3, 25 ℃) ô (ppm): 7.41-7.35 (m, 3H, HAr), 7.29-7.26 (m, 2H, HAr), 7.16 (s, 1H, =CHN), 5.50 (s, 2H, CH2-Ph), 2.76-2.73 (m, 1H, cyclohexyl), 2.05-2.02 (m, 2H, cyclohexyl), 1.81-1.69 (m, 2H, cyclohexyl), 1.43-1.35 (m, 4H, cyclohexyl), 1.28-1.22 (m, 2H, cyclohexyl), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 154.23, 135.04, 129.04, 128.57, 128.00, 119.14, 53.98, 35.35, 33.00, 26.14, 26.03. HRMS m/z calc. for C15H19N3 + (H+): 242.1652; found: 242.1647.

4.5.19. 1-(Phenylmethyl)-4-phenyl-1H-1,2,3-triazole (35g)

From azide 33 and 1-ethynylbenzene. Yellow powder after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 64%. Mp = 128-130 ℃, Rf = 0.20 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.83-7.81 (m, 2H, HAr), 7.68 (s, 1H, =CHN), 7.43-7.36 (m, 5H, HAr), 7.35-7.31 (m, 3H, HAr), 5.59 (s, 2H, CH2-Ph), 13C NMR (101 MHz, CDC13, 25 ℃) ô (ppm): 148.25, 134.70, 130.55, 129.17, 128.81, 128.17, 128.07, 125.71, 119.48, 54.24. HRMS m/z calc. for C15H13N3 + (H+): 236.1182; found: 236.1177.

4.5.20. 1-(Phenylmethyl)-4-phenoxymethyl-1H-1,2,3-triazole (35h)

From azide 33 and 1-(prop-2-ynyloxy)benzene. Grey powder after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 87%. Mp = 119-120 ℃, Rf = 0.40 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.55 (s, 1H, =CHN), 7.41-7.37 (m, 3H, HAr), 7.32-7.28 (m, 4H, HAr), 7.00-6.96 (m, 3H, HAr), 5.55 (s, 2H, CH2-Ph), 5.21 (s, 2H, CH2-O-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 158.20, 144.73, 134.73, 129.53, 129.16, 128.82, 128.13, 122.55, 121.25, 114.17, 62.07, 54.26. HRMS m/z calc. for C16H15N30 + (H+): 266.1288; found: 266.1283.

4.5.21. 1-(Phenylmethyl)-4-chloro-phenoxymethyl-1H-1,2,3- triazole (35i)

From azide 33 and 1-chloro-4-(prop-2-ynyloxy)benzene. Grey powder after silica gel circular chromatography (0-15% AcOEt/ Hex), yield = 81%. Mp = 98-99 ℃, Rf = 0.20 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.53 (s, 1H, =CHN), 7.39 (s, 3H, HAr), 7.29-7.23 (m, 4H, HAr), 6.92-6.90 (m, 2H, HAr), 5.55 (s, 2H, CH2-Ph), 5.17 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 156.77, 144.24, 134.38, 129.40, 129.18, 128.87, 128.13, 126.18, 122.63, 116.13, 62.33, 54.29. HRMS m/z calc. for C16H14N30CI + (H+): 300.0898; found: 300.0893.

4.5.22. 1-(Phenylmethyl)-4-cyano-phenoxymethyl-1H-1,2,3- triazole (35j)

From azide 33 and 4-(prop-2-ynyloxy)benzonitrile. White powder after silica gel circular chromatography (0-15% AcOEt/ Hex), yield = 88%. Mp = 109-110 ℃, Rf = 0.12 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDC13, 25) ô (ppm): 7.60-7.57 (m, 2H, HAr), 7.55 (s, 1H, =CHN), 7.42-7.38 (m, 3H, HAr), 7.31-7.28 (m, 2H, HAr), 7.07-7.04 (m, 2H, HAr), 5.56 (s, 2H, CH2-Ph), 5.24 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 161.40, 143.39, 134.25, 134.04, 129.22, 128.96, 128.17, 122.85, 119.05, 115.54,

104.59, 62.14, 54.37. HRMS m/z calc. for C17H14N40 + (H+): 291.1240; found: 291.1232.

4.5.23. 1-(Phenylmethyl)-4-nitro-phenoxymethyl-1H-1,2,3-triazole (35k)

From azide 33 and 1-nitro-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-10% AcOEt/ Hex), yield = 99%. Mp = 100-101 ℃, Rf = 0.15 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 8.23-8.19 (m, 2H, HAr), 7.57 (s, 1H, =CHN), 7.42-7.39, (m, 3H, HAr), 7.31-7.28 (m, 2H, HAr), 7.09-7.05 (m, 2H, HAr), 5.57 (s, 2H, CH2-Ph), 5.29 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 163.10, 143.19, 141.89, 134.22, 129.24, 128.98, 128.19, 125.93, 122.92, 114.85, 62.48, 54.39. HRMS m/z calc. for C16H14N403 + (H+): 311.1139; found: 311.1131.

4.5.24. 1-(Phenylmethyl)-4-methyl-phenoxymethyl-1H-1,2,3- triazole (35l)

From azide 33 and 1-methyl-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-25% AcOEt/Hex), yield = 91%. Mp = 107-108 ℃, Rf = 0.38 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCI3, 25 ℃) ô (ppm): 7.53 (s, 1H, =CHN), 7.42-7.35, (m, 3H, HAr), 7.32-7.28 (m, 2H, HAr), 7.10-7.08 (m, 2H, HAr), 6.90-6.86 (m, 2H, HAr), 5.54 (s, 2H, CH2-Ph), 5.18 (s, 2H, CH2-O-Ph), 2.30 (s, 3H, CH3), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 156.11, 144.91, 134.50, 130.53, 129.96, 129.15, 128.81, 128.13, 122.50, 114.66, 62.27, 54.24, 20.48. HRMS m/z calc. for C17H17N30 + (H+): 280.1444; found: 280.1439.

4.5.25. 1-(Phenylmethyl)-4-methoxy-phenoxymethyl-1H-1,2,3- triazole (35m)

From azide 33 and 1-methoxy-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-15% AcOEt/Hex), yield = 96%. Mp = 91-92 ℃, Rf = 0.12 (30% AcOEt/ Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.53 (s, 1H, =CHN), 7.42-7.37, (m, 3H, HAr), 7.30-7.27 (m, 2H, HAr), 6.94-7.90 (m, 2H, HAr), 6.85-6.81 (m, 2H, HAr), 5.55 (s, 2H, CH2-Ph), 5.15 (s, 2H, CH2-0-Ph), 3.78 (s, 3H, CH3-O-Ph), 13C NMR (101 MHz, CDCI3, 25 ℃) ô (ppm): 154.20, 152.33, 144.90, 134.49, 129.15, 128.81, 128.12, 122.53, 115.88, 114.65, 62.85, 55.70, 54.24. HRMS m/z calc. for C17H17N3O2 + (H+): 296.1394; found: 296.1386.

4.5.26. 1-(Phenylmethyl)-4-phenyl-phenoxymethyl-1H-1,2,3- triazole (35n)

From azide 33 and 1-phenyl-4-(prop-2-ynyloxy)benzene. White powder after silica gel circular chromatography (0-20% AcOEt/ Hex), yield = 49%. Mp = 194-196 ℃, Rf = 0.22 (30% AcOEt/Hex), 1H NMR (400 MHZ, CDCl3, 25 ℃) ô (ppm): 7.57 (s, 1H, =CHN), 7.55-7.52 (m, 4H, HAr), 7.45-7.38 (m, 5H, HAr), 7.35-7.28 (m, 3H, HAr), 7.08-7.04 (m, 2H, HAr), 5.56 (s, 2H, CH2-Ph), 5.25 (s, 2H, CH2-0-Ph), 13C NMR (101 MHz, CDCl3, 25 ℃) ô (ppm): 157.76, 144.65, 140.65, 134.46, 134.35, 129.18, 128.85, 128.74, 128.21, 128.15, 126.77, 126.75, 122.60, 115.08, 62.22, 54.29 HRMS m/z calc. for C22H19N30 + (H+): 342.1601; found: 342.1592.

4.6. Aromatase inhibition assay

Inhibition of aromatase (CYP19) by synthesized compounds (IC50) was determined using the P450 Inhibition Kit CYP19/MFC (BD Biosciences, Two Oak Park Bedford, MA, USA) according to the manufacturer’s instructions. Briefly, 100 ul of each compound was diluted in NADPH-cofactor mix for every concentration tested (10000, 3333, 1111, 370, 123, 41, 13, 4 and 0 nM) and placed in duplicate on a 96-well plate. The plate was then incubated at 37 ℃

for 10 min. After incubation, 100 ul of the enzyme/substrate mix was added to the treated conditions and the plate was then incu- bated at 37 ℃ for 30 min. After incubation, 75 ul of Stop Reagent was added to the entirety of the plate and 100 ul of the enzyme/ substrate mix was added in the blank columns. Thereafter, the plate was subjected to a colorimetric analysis and read on a fluorescence microplate reader (FLUOstar Optima, BMG Labtechnologies, Nexc = 410 nm/2em = 520 nm). IC50 values were determined using the colorimetric data analyzed through normalized non-linear regres- sion using the GraphPad Prism 5 software. The less active mole- cules (IC50 > 10 µM) were assayed by two independent experiments (n = 2), each being run in technical triplicate. The most active compounds (IC50 ≤ 10 uM) were assayed by three independent experiments (n = 3), each being run in technical triplicate.

4.7. Cell proliferation assay

Human epithelial adrenocortical carcinoma cell line (NCI- H295R) was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DMEM/F12 media supplemented with 10% Nu-Serum Serum Replacements, 1% ITS+ Universal Culture Supplement Premix, 2 L equivalent (BD Biosci- ences), L-glutamine (2 mM), penicillin G (100 U mL-1), and strep- tomycin (100 mg mL-1) (Invitrogen). During experiments, cells were either left untreated, treated with DMSO (0.01%) or were incubated with 10 nM of synthesized compounds (reconstituted in DMSO) in DMEM/F12 phenol red free media supplemented with 1.5% Nu-Serum (minimum concentration for cells to proliferate), 1% ITS, L-glutamine (2 mM), penicillin G (100 U mL-1), and strepto- mycin (100 mg mL-1) for 24 and 72 h prior to analysis of cell proliferation. Cell proliferation assays were performed on seeded cells (2 x 104 cells/well) in 96-well plates and analyzed for cellular viability using a multiplexed assay of CellTiter Blue (Promega, Madison, WI) kit according to the manufacturer’s instructions. Briefly, 20 ul of the CellTiter Blue substrate was added to 100 ul of cell suspension and incubated for 1 h at 37 ℃. Thereafter, the plate was subjected to a colorimetric analysis and read on a fluorescence microplate reader (FLUOstar Optima, BMG Labtechnologies, Àexc = 560 nm/2em = 590 nm).

Acknowledgement

This research was financially supported by Université de Mon- cton, the New Brunswick Innovation Foundation (NBIF), the Medical Research Fund of New Brunswick, and the Canada Foun- dation for Innovation (CFI). M.T. thanks Dr. C. Pepin (Université d’Avignon) for graciously recommending A.S. Special thanks to Dr. A. Culf and Dr. J. Jean-Francois for their excellent comments and suggestions during the preparation of this manuscript.

Appendix. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.ejmech.2011.05.074.

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