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European Journal of Medicinal Chemistry 102 (2015) 375e386

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European Journal of Medicinal Chemistry

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

Mini-review

Recent developments in steroidal and nonsteroidal aromataseinhibitors for the chemoprevention of estrogen-dependent breastcancer

Irshad Ahmad*, Shagufta*

Department of Mathematics and Natural Sciences, School of Arts and Sciences, American University of Ras Al Khaimah, Ras Al Khaimah,United Arab Emirates

a r t i c l e i n f o

Article history:Received 17 April 2015Received in revised form2 August 2015Accepted 4 August 2015Available online 8 August 2015

Keywords:AromataseBreast cancerSteroidal aromatase inhibitorsNonsteroidal aromatase inhibitors

* Corresponding author.E-mail addresses: iahmad@aurak.ac.ae (I. Ahmad)

ae (Shagufta).

http://dx.doi.org/10.1016/j.ejmech.2015.08.0100223-5234/© 2015 Elsevier Masson SAS. All rights re

a b s t r a c t

Aromatase, a cytochrome P450 enzyme complex present in breast tissues, plays a significant role in thebiosynthesis of important endogenous estrogens from androgens. The source of estrogen production inbreast cancer tissues is intra-tumoral aromatase, and inhibition of aromatase may inhibit the growthstimulation effect of estrogens in breast cancer tissues. Consequently, aromatase is considered a usefultherapeutic target in the treatment and prevention of estrogen-dependent breast cancer. Recently,different natural products and synthetic compounds have been rapidly developed, studied, and evaluatedfor aromatase inhibitory activity. Aromatase inhibitors are classified into two categories on the basis oftheir chemical structures, i.e., steroidal and nonsteroidal aromatase inhibitors. This review highlights thesynthetic steroidal and nonsteroidal aromatase inhibitors reported in the literature in the last few yearsand will aid medicinal chemists in the design and synthesis of novel and pharmacologically-potentaromatase inhibitors for the treatment of breast cancer.

© 2015 Elsevier Masson SAS. All rights reserved.

1. Introduction

Breast cancer is the most common form of cancer present inwomen worldwide and is the second leading cause of death afterlung cancer. Reports suggest that one in every eight women de-velops metastatic breast cancer in her lifetime. For 2015, it is esti-mated that over 1.6 million new cancer cases will be diagnosed inthe USA and over 589,000 Americans will die from cancer, i.e., 1620people per day [1]. The presence of high concentrations of estrogenin breast tissue increases the risk of developing breast cancer andthe ability of immature breast tissue cells to strongly bind to car-cinogens, decreasing their DNA repair capacity [2].

In hormone-dependent breast cancer, estrogen plays a signifi-cant role in the stimulation of breast cancer cell proliferation [3].High concentrations of estrogen promote the development ofbreast cancer. To control or block the pathological activity of es-trogens, medicinal chemists have developed two main approaches.The first approach involves the design and synthesis of estrogen

, shagufta.waseem@aurak.ac.

served.

receptor antagonist which provided several anticancer and che-mopreventive drugs, for example, tamoxifen and raloxifene [4].Inhibition of enzyme aromatase is the second approach for thedevelopment of new agents for the breast cancer treatment [5,6].

Aromatase is an enzyme complex formed from two proteins, i.e.,cytochrome P450arom (CYP19) and NADPH-cytochrome P450reductase. CYP19 is a hemoprotein that carries out the conversionof androgens to the estrogens, while NADPH-cytochrome P450reductase is a flavoprotein essential for electron transfer fromNADPH to the P450 enzyme [7].

Aromatase is involved in the final step of the estrogen biosyn-thetic pathway and its selective inhibition will not affect the pro-duction of the other steroids in the pathway [8e10]; therefore,aromatase is considered a useful therapeutic target in the treat-ment and prevention of estrogen-dependent breast cancer [11]. Thecompetitive inhibitors of enzyme aromatase are known as aroma-tase inhibitors (AIs).

AIs, on the basis of their chemical structures, are classified intotwo categories: steroidal and nonsteroidal [12]. Steroidal AIs closelyresemble the shape of androstenedione (1) (Fig. 1), which has af-finity for the aromatase enzyme. These inhibitors interact with thesubstrate-binding site of the enzyme and are identified as Type I

O

O

1

O

O

OH

O

O

CH2

O

O

CH3

O

O

2 3

4 5

Fig. 1. Androstenedione derivatives as steroidal aromatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386376

inhibitors; however, the inhibition may be mechanism-based, inthat the drugs bound to the catalytic site are metabolized to in-termediates, which attach irreversibly to the active site, therebyblocking activity. Type I inhibitors induce a shift in the UV ab-sorption spectrum's Soret band of the heme, from about 420 nm to390 nm. Due to the irreversible nature of the inhibition, the enzymeremains inactive even after the drug is cleared from circulation.Therefore, these inhibitors are marketed as inactivators or as “sui-cide inhibitors.” Nonsteroidal AIs are Type II inhibitors, whichcontain favorably positioned heteroatoms that coordinate to theheme iron of aromatase. Due to this special type of binding, theycause a bathochromic shift in the Soret band. In contrast to Type Iagents, Type II inhibitors are generally reversible and estrogenblockade is dependent on the continuous presence of the drug [6].

Several nonsteroidal AIs are currently in clinical use, namelyanastrozole, letrozole, and exemestane, and some are in thedeveloping stage [13e15]. Due to the development of resistance toAIs and the side effects related to currently utilized compounds, theneed for improved AIs remains [16,17]. Medicinal chemists arecontinuously making efforts to design and synthesize AIs withmore clinical efficacy and minimal side effects. Additionally, re-searchers have also investigated the potential of natural products asAIs. In this review, we summarize the data regarding the steroidaland nonsteroidal inhibitors reported in the literature over the lasttwo decades and focus on synthetic organic compounds that inhibitaromatase.

1.1. Steroidal aromatase inhibitors (AIs)

The literature reveals that the steroidal AIs are mostly built onan androstenedione (1) nucleus by incorporating various chemicalsubstituents at different positions on the steroid. The clinicallyevaluated steroidal AIs are formestane (2), exemestane (3), ata-mestane (4), and MDL-18,962 (5) (Fig. 1). H. J. Brodie et al. reportedthat 4-hydroxyandrostenedione (formestane) was a potent AIcurrently used in the clinic for the treatment of breast cancer. It wasthe most selective and effective AI, but it has the disadvantage ofrequiring administration by intramuscular injection [18]. Exemes-tane is a steroidal inactivator of aromatase, recently approved forclinical use in some countries, and it appears to be more potentthan formestane. An advantage of exemestane is that it can beorally administered [19]. Atamestane showed high affinity foraromatase. Its irreversible inhibition and lack of other endocrineeffects makes it a promising drug for the treatment of estrogen-

dependent disease states [20]. MDL-18,962 was a highly potentirreversible inhibitor of human placental aromatase [21].

Most of the potent steroidal AIs reported in the 1900s were A-and B-modified steroids; therefore, to explore the anti-aromataseactivity of D-modified steroids, Gasi et al. (2001) synthesizedseveral 17a-substituted-17b-hydroxy-16-oximino derivatives of 5-androstene and the corresponding D-seco derivatives. Compound6 (Fig. 2), a D-seco derivative with a 4-en-3-keto system, showedthe highest activity (IC50 ¼ 0.42 mM), compared to compoundsbearing a 5-en-3-hydroxy system or extended linear conjugationin rings A and B [22]. Next, the synthesis and biological activity of16-amino-17-substituted-D-homo steroid derivatives were re-ported; the AI activity of these compounds was much lower thanthe D-seco and 16-oximino derivatives of androstene [23].Considering the anti-aromatase activity of lactones, several ste-roidal D-homo-lactones of 3b-hydroxy-5-androstene series and its4-ene-3-keto,1,4-diene-3-keto,1,4,6-triene-3-keto, and 4,6-diene-3-keto analogs were synthesized. The lactone moiety in the D ringwas favorable for anti-aromatase activity and compound 7 (Fig. 2)was the most potent, with IC50 ¼ 0.25 mM [24]. In view of theseresults, additional D-homo lactones with 1,2-, 3,4-, 5,6- and 6,7-epoxy functions were synthesized and evaluated for anti-aromatase activity; however, these epoxies showed lower activ-ity than formestane [25]. Taking into consideration the importanceof the 4-en-3-one system and systematic conjugation in the A andB rings in steroids and the pyridine moiety in nonsteroidal AIs,several 17-picolyl- and 17-picolinylidene-3b-hydroxy-androst-5-ene derivatives and their 4-en-3-one and 1,4,6-triene-3-one ana-logs were synthesized to study the effects of D-ring-modifiedsteroids on aromatase enzyme. Biological activity data revealedthat 17-picolinylidene-16-one derivatives were more potent AIsthan 17-picolyl and 17-picolinylidene derivatives, and compound 8(Fig. 2) was the most effective inhibitor of aromatase (93.3% inhi-bition) [26]. The presence of a cyano group in the non-steroidal AIsletrozole and anastrozole suggested the introduction of cyanofunctionality in the D ring, and several compounds with a 17-methyl-16,17-seco-16-nitrile-17-one moiety, with 1,4-diene-3-on,4,6-dien-3-on, 1,4,6-trien-3-on, and 4-hydroxy-4-en-3-one sys-tems, were synthesized. Compound 9 (Fig. 2), with a 4,6-dien-3-onsystem, showed promising AI activity with an IC50 of 0.22 mM [27].Later, steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives were synthesized andtested for AI activity. Compound 10 (Fig. 2), bearing a 17-picolynylidene group and a 4-en-3-on system, inhibited aroma-tase activity by 67.9% [28].

Bansal et al. synthesized several new 16E-arylidenosteroidalderivatives and evaluated their aromatase inhibitory activity. Theimidazolyl-substituted steroidal derivatives exhibited good inhibi-tory activity against aromatase and 16-[4-{3-(imidazol-1-yl)pro-poxy}-3-methoxybenzylidene]-5-androstene-3b,17b-diol (11)(Fig. 2) had13 times more activity (IC50 ¼ 2.4 mM) than amino-glutethimide [29]. Next, a new series of 16E-arylidene derivativeswere synthesized by introducing various aryl substituents at the 16position of the steroid skeleton and its impact on aromatase inhi-bition was studied. Among these compounds, 3-keto-4-ene steroid(12) (Fig. 2), with a heteroaromatic ring pyridyl, was the mostpotent (IC50 ¼ 5.2 mM) [30]. Several 16-imidazolyl-substitutedsteroidal derivatives were synthesized and evaluated for anti-aromatase activity. The two compounds, 16b-(imidazol-1-yl)-4-androstene3,17-dione (13) (Fig. 2) and 16b-(imidazole-1-yl)-4-androsta-1,4-diene-3,17-dione (14) (Fig. 2), exhibited strong aro-matase inhibition activity of, i.e., IC50s of ¼ 0.18 mM and 0.16 mM,respectively [31]. Later, a series of imidazolyl-substituted 16E-ary-lindenosteroidal derivatives were prepared and screened for aro-matase inhibition. Themost potent compound of this series was 16-

O

C

CH3

O

6HO

7

O

O

H CH3

O

8

O

H

O

C

CH3

O

9

O

10

H

N

N N

HOH

HO H

H3CO O(CH2)3N

N

11N

12O

O

13O

O

NN

14O

O

NN

15O

O

O(CH2)3 NN

N

Fig. 2. D ring modified steroids as aromatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386 377

[3-{3-(imidazole-1-yl)propoxy}benzylidene]-4-androstene-3,17-dione (15) (Fig. 2), which exhibited an IC50 ¼ 4.4 mM [32].

Yadav et al. designed and synthesized some novel steroidalderivatives by combining the 2,3 positions of ring A of the steroidwith five-membered heterocyclic rings, such as isoxazole andpyrazole. Compound 16 (Fig. 3), with a pyrazole ring fused to the 2,3positions of ring A, showed the most activity (IC50 ¼ 0.51 mM),followed by nitrile derivative 17 (Fig. 3) (IC50 ¼ 1.0 mM) [33].Further, 4-phenylthio function in ring A and the cyano group in ringD were introduced to see the effect of these functionalities onaromatase inhibition; compound 18 (Fig. 3), bearing a 4-phenylthiogroup at the C-4 position and a nitrile group at the 16 position, wasthe most potent (IC50 ¼ 0.17 mM) [34].

Numazawa et al. studied the structureeactivity relationships of2a-substituted androstenedione derivatives for aromatase inhibi-tion activity. Several analogs were synthesized by introducinghalogen, alkyl, hydroxyl, and alkoxy substituents at the 2a position;among these compounds, 2a-fluoro (19) and 2a-ethyl (20) (Fig. 3)steroids were the most potent inhibitors of aromatase, havingIC50s ¼ 0.97 mM and 0.48 mM, respectively [35]. To study thestructureeactivity relationships of the pyridine and other hetero-cyclic derivatives of estrone, a series of pyridine- and other het-erocyclic ring-containing derivatives of 2- and 4-aminoestroneswere synthesized. Compounds 21 and 22 (Fig. 3) were potent AIs,having IC50s ¼ 31 mM and 28 mM, respectively [36]. Considering thearomatase inhibition activity of 6b- and 19-substituted steroids,16b, 19-bridged steroids and epoxy-, cyclo-, epithio-, and methano-androstenediones were synthesized and evaluated for their aro-matase inhibition activity. The 6b,19-epoxy steroid (23) (Fig. 3)showed moderate inhibition (IC50 ¼ 44 mM), whereas other com-pounds were weak to poor competitive inhibitors of aromatase[37].

Considering the importance of the double bond in the C-1

position for increased aromatase inhibition and the non-essentialproperty of the C-3 carbonyl group, Varela et al. synthesized twoseries of steroidal 7a-allylandrostenedione derivatives, i.e., 1-eneand 3-dexo analogs. The biological data explained that theremoval of the C-3 carbonyl group was not favorable for aromataseinhibition, whereas introduction of a double bond in C-1 wasbeneficial for such; compound 24 (Fig. 3) was the most potent AI intheir study (IC50 ¼ 0.47 mM) [38]. To explore the effect of substi-tution of the C-17 carbonyl group in 5-a-androst-3-enes and 3a-4a-epoxy-5a-androstanes, Cep et al. synthesized a series of com-pounds by substituting the original C-17 carbonyl group with hy-droxyl, acetyl, and hydroxylamine groups. Substitution of the C-17carbonyl group reduces the aromatase inhibition of the corre-sponding compounds and the data revealed the importance of theC-17 carbonyl group in the D-ring steroids studied for aromataseinhibition activity [39].

To study the effect of extended linear conjugation in ring(s) Aand/or B, the effect on the position of the epoxide ring, and theeffect of the substituent at the 4-position of a 17-hydroxyiminoandrostane skeleton structure on aromatase inhibition, Pokhrelet al. synthesized 17-hydroxyimino derivatives by adding doublebonds at C-1-C-2, at C-4-C-5, at C-6-C-7, or both positions andsynthesized 17-hydroxyimino derivatives of 1,2- or 4,5-epoxyandrostene and/or -diene and of 4-substituted 4-androstene. The synthesized 17-hydroxyimino steroidal com-pounds showed promising aromatase inhibition activity. Amongthe synthesized compounds, the 4-chloro-3b-hydroxy-4-androsten-17-one oxime (25) (Fig. 3) exhibited the greatest aro-matase inhibition (93.8%) [40]. Ghosh et al. followed the structure-guided approach and designed and synthesized a C-6-substitutedandrosta-1,4-diene-3,17-dionealkynyloxy series and evaluatedthem for aromatase inhibition. A number of C-6-substituted 2-alkynyloxy compounds exhibited promising aromatase inhibition

18

O

16

HO

OH

NNH

17

HO

OH

NC

HO

S

OH

CN

19: R = F20: R = CH2CH3

O

O

R

21

O

HO

HN

O

N

22

O

HO

NHO

N

O

O

O

23

O

O

24

NOH

Cl

HO

25

O

O

O

26

Fig. 3. A and B ring modified steroids as aromatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386378

activity. The most potent compound was a 2-pentynyl oxy deriva-tive (26) (Fig. 3), with an IC50 ¼ 0.012 mM [41].

1.2. Nonsteroidal aromatase inhibitors (AIs)

The discovery of aminoglutethimide (27) (Fig. 4), a potent AI, ledto the design and development of nonsteroidal AIs for the treat-ment of estrogen-dependent breast cancer [42,43].

NH

O O

NH2

N

N

NC

27

30

28

31

N

N

NC

N

CN

NN

Fig. 4. Most representative nonsteroidal aromatase inhibitors. 27, aminoglutethim

The most potent nonsteroidal inhibitors, i.e., those with a highaffinity for the enzyme, are the azole derivatives fadrozole (28),liarozole (29) with an imidazole ring, letrozole (30), anastrozole(31), and vorozole (32), all containing a 1,2,4 triazole ring (Fig. 4).Anastrozole and letrozole are very potent and selective AIs and arewell tolerated by patients. Letrozole is the most active AI among allof the AIs, with 99% aromatase inhibition. The three triazole drugsare the current front-line NSAIs, generally indicated as third-

N

CN

NH

N

N

Cl

29

CN

NN

N

NN

N

32Cl

ide; 28, fadrozole; 29, liarozole; 30, letrozole; 31, anastrozole and 32, vorozol.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386 379

generation compounds [44e47].During the past two decades, the nonsteroidal AIs were recog-

nized as potential therapeutic agents for the treatment of estrogen-related diseases, such as breast cancer. Structure-activity studies ofpotent nonsteroidal AIs revealed that the important structuralfeature is the presence of a heterocyclic nitrogen atom, which in-teracts with the heme iron of the enzyme (P450) while the rest ofthe molecule interacts with the apoprotein moiety of the active site[48e51].

Research groups are engaged in the development of more activeand selective inhibitors of P450. Indeed, selectivity is extremelyimportant because of the widespread occurrence of P450 systemsin the organism, whose concomitant inhibition could cause serioustoxicity [52].

In the 1990s, R.W. Hartmann's group developed a number ofnew and potent nonsteroidal AIs, the most potent of which weretetralone derivatives [53,54]. The first active compound in this se-ries was (E)-2-(4-pyridylmethylene)-1-tetralone (33) (Fig. 5), withan IC50 ¼ 4.6 mM. The potency of 33was increased by replacing the4-pyridyl with a 4-imidazolyl ring. The two isomers of the 4-imidazolyl derivative, i.e., Z isomer 34 and E isomer 35 (Fig. 5),showed IC50s ¼ 0.17 and 0.26 mM, respectively. However, the Eisomer 35 also affected the other steroidogenic P450 enzymes, like17a-hydroxylase-C17,20-lyase (CYP17) and steroid 18-hydroxylase(CYP11B2), and was therefore not selective [55]. The 7-methoxyderivative 36 (Fig. 5) was the most potent AI in the tetralone se-ries (IC50 ¼ 0.041 mM) and it was highly selective for CYP19 [56].Later, the saturated methylene group of 33 was constrained byfusing a cyclopropane ring to positions C1 and C2 of the tetralinnucleus and the 6-methoxy derivative 37 (Fig. 5) was reported as apotent AI. The (þ)-enantiomer of compound 37 showedIC50 ¼ 0.030 mM, whereas its (�)-enantiomer had IC50 ¼ 10 mM;however, compounds 36 and (þ)-37 were unable to show prom-ising in vivo activity in rat models of breast tumors [57].

Further, the tetralinic skeleton was replaced by a quinolinic oneand a series of tetrahydroquinoline derivatives was synthesizedand reported as AIs. Themost interesting compoundwas 38 (Fig. 5).Compound 38was a potent inhibitor of aromatase (IC50 ¼ 0.50 mM)and it was selective with respect to other steroidogenic P450 en-zymes, i.e., CYP17 and CYP11A1 [58].

Le Borgne et al. synthesized several liarozole (29) analogs basedon an indole nucleus bearing the azolylbenzyl fragment at different

O

N

O

NH

NX

N

O

33

37

34: E; X = H35: Z; X = H36: E; X = OCH3

N

N

N

38

Fig. 5. Tetralone and quinoline derivatives as aromatase inhibitors.

positions of the aromatic bicycle. Several modifications wereintroduced, i.e., N-substituent, azole ring, and phenyl substituents;the most effective compounds were 39 (IC50 ¼ 0.10 mM) and 40(IC50 ¼ 0.04 mM) (Fig. 6). In this series, the imidazole nucleus wasmore suitable than the triazole one [59]. Next, Leze et al. modifiedcompound 40 by replacing the ethyl group of the indolic nitrogenwith H and CH3 and changed the phenyl substituent to Cl and Br.The racemic compound 41 (Fig. 6), 5-[(4-chlorophenyl)(1H-imida-zol-1-yl)methyl]-1H-indole, was the most potent AI, withIC50¼ 0.015 mM. The (þ) enantiomer of 41was twice as active as theracemate (IC50¼ 0.009 mM) [60]. Further, the (4-cyano-phenyl)(1H-imidazol-1-yl)methyl chain was introduced at position 6 or 4 of theindole ring and a series of 6- or 4-functionalized indoles weresynthesized as AIs. The most potent compound of this series wasthe racemate 4-[(1H-imidazol-1-yl)(1H-indol-4-yl)(methyl)]ben-zonitrile (42) (Fig. 6), showing a high level of inhibitory activitytoward CYP19with an IC50¼ 0.012 mM [61]. Wang et al. synthesizeda series of indole-imidazole derivatives by moving the phenylgroup present on a central methine in compound 41 to the nitrogenatom in the indole ring. Compound 43 (Fig. 6), with a tri-fluoromethyl (CF3) group at C-40, exhibited the maximum aroma-tase inhibition of the series (IC50 ¼ 0.0049 mM). Study of thestructureeactivity relationship of these compounds revealed that aproton or small electron-withdrawing group at the para position ofthe phenyl ring favored aromatase inhibition, compared to thebulky group [62].

Whomsley et al. reported the [(benzofuran-2-yl) phenylmethyl]imidazoles (44) (Fig. 7) as potent nonsteroidal AIs with IC50s in thenanomolar range [63]. Further, study of the structureeactivityrelationship of this class of molecules revealed that removal of thephenyl ring favored CYP17 inhibition [64], whereas its replacementwith small alkyl groups, together with the introduction of Br or Clatoms on the phenyl moiety of the benzofuran ring, resulted in lowselectivity [65]. Further, a series of compounds were prepared byexchanging the imidazole ring of 44 with a triazole ring. The mostpotent compound was 45 (Fig. 7), with an IC50 ¼ 0.19 mM [66].

Doiron et al. reported the synthesis and aromatase inhibition ofa series of substituted and unsubstituted 1,2,3-triazole, 1,2,4-triazole, and imidazole analogs of letrazole. Structureeactivityrelationship studies of the series revealed that the presence of thenitrogen atom in position 3 or 4 of the 1,2,4 triazole, para cyanogroup and two aryl groups are important for good aromatase in-hibition. The unsubstituted 1,2,3-triazole derivative (46) (Fig. 7)was the most potent compound (IC50 ¼ 0.008 mM) [67].

Pingaew et al. synthesized a series of 1,4-disubstituted-1,2,3-triazole-based sulfonamides bearing 1,2,3,4-tetrahydroisoquinoline(THIQ) and its open-chain derivatives. These compounds were eval-uated for aromatase inhibition and molecular docking studies wereperformed to explore their binding modes. The most potent com-pound of this series was the meta analog of triazole-benzene sul-fonamide, 47 (Fig. 7), containing 6,7-dimethoxy substituents on theisoquinoline ring and coumarinyloxymethyl on the triazole ring(IC50 ¼ 0.2 mM). The molecular docking results of these triazolesrevealed that these compounds comfortably occupy the active site ofaromatase through hydrophobic, pep stacking, and hydrogenbonding. Compound 47 showed hydrogen bonding interactions withMet374 and Ser478, which were suggested to be the amino acid res-idues important for aromatase inhibition [68].

Ferlin et al. chemically modified 2-phenyl-pyrroloquinolinonesand synthesized a small set of pyrrolo[2,3-h]quinolines and pyrrolo[3,2-f]quinolines by introducing either an imidazolylmethyl or atriazolylmethyl group at positions 4 and 9, respectively. The com-pounds were evaluated as nonsteroidal competitive inhibitors ofCYP19, CYP11B1 (steroid-11b-hydroxylase), and CYP17, usingletrozole as a positive control. The results revealed that azolyl

N

N

N

39

N

N

N

F

40

N

N

N

ClH

41

N

N

N

CNH

42

N

43F3C

N

N

Fig. 6. Indole derivatives as nonsteroidal aromatase inhibitors.

O

N

X

YN

44: X = Cl: Y = CH45: X = F; Y = N

N

CN

NN

NC

46

N

O

O SO O

N

NN

OO O

47

N

N

N

NN

48

Fig. 7. Nonsteroidal aromatase inhibitors containing triazole.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386380

methyl derivatives showed promising aromatase inhibition andthat compound 48 (Fig. 7) was highly selective for CYP19(IC50 ¼ 0.13 mM) and had very poor inhibitory activity againstCYP11B1 (IC50 ¼ 14.8 mM) and CYP17 (IC50 � 5000 nM) [69].

Several flavonoid derivatives bearing benzopyranone rings wereevaluated for aromatase inhibitory activity [70]. The flavone chrysin(49) is a potent aromatase inhibitor, with IC50 ¼ 0.50 mM, comparedto several other flavones, such as apigenin (50) with IC50 ¼ 1.2 mM,flavone (51) with IC50 ¼ 8 mM, flavanone (52) with IC50 ¼ 8 mM, andquercetin (53) with IC50 ¼ 12 mM (Fig. 8). The activity of isoflavonestowards aromatase enzyme is considered less than that of flavones.The most potent isoflavone inhibitor is biocchanin A (54) (Fig. 8)with an IC50 of 113 mM.

Recanatini et al. reported a new series of nonsteroidal AIs withchromone and xanthone nuclei. The most interesting AIs of theseries were 55 (IC50 ¼ 0.043 mM) and 56 (IC50 ¼ 0.040 mM) (Fig. 9),which showed good inhibitory activity and selectivity with respectto CYP17 [71]. Brueggemeier et al. introduced a 2-(4-pyridylmethyl)

thio functionality into the isoflavone nucleus, and the analog 3-phenyl-7-(phenylmethoxy)-2-[(40-pyridylmethyl)thio]-4H-1-bezopyran-4-one (57) (Fig. 9) was the most potent inhibitor ofaromatase in their series, with an IC50 ¼ 210 mM [72]. Further seriesof 2-azole and 2-thiazole isoflavones were synthesized usingimidazole, triazole, thioimidazole, and thiotriazole as potent AIs.The result revealed that the aromatase inhibition of isoflavones canbe increased by introducing an appropriate heme-coordinatingnitrogen heterocycle at the 2 position of the isoflavone. Com-pound 58 (Fig. 9), 2-(1H-imidzol-1-yl)-3-phenyl-7-(benzyloxy)-4H-1-benzopyran-4-one showed the maximal aromatase inhibi-tion of that series (IC50 ¼ 0.52 mM) [73].

Bonfield et al. designed and synthesized isoflavanone de-rivatives as potential AIs through structural modification of the Aand B rings of isoflavanones and subsequently evaluated them byfluorescence-based assays utilizing recombinant human aroma-tase. The three compounds, 3-(4-phenoxyphenyl)chroman-4-one(59) (Fig. 9), 6-methoxy-3-phenylchroman-4-one (60), and 3-(pyridine-3-yl)chroman-4-one (61) (Fig. 9), showed promisinginhibitory activity against aromatase, with IC50 values of 2.4 mM,0.26 mM, and 5.8 mM, respectively. The results revealed that iso-flavavnone derivatives bearing methoxy, phenoxy and pyridylfunctional groups are potent AIs, suggesting that the non-planarconfiguration of the isoflavanone structure might be responsiblefor enzyme-ligand binding [74].

Further, Amato et al. synthesized a series of fluorinated andbifunctionalized isoflavanones and evaluated their inhibitory ac-tivity against aromatase using a fluorescence based enzymaticassay. The most potent compounds were 6-methoxy-3-(pyridin-3-yl) chroman-4-one (62) and 6-fluoro-3-(pyridin-3-yl)chroman-4-one (63) (Fig. 9), with IC50 values of 2.5 mM and 0.8 mM, respec-tively [75].

Yahiaoui et al. synthesized 4-imidazolylflavans and 4-triazolylflavans, which were found to significantly inhibit aroma-tase. The most potent compound was 64 (Fig. 10), with anIC50 ¼ 0.041 mM [76]. Later, a benzo ring was introduced at the C-7and C-8 positions on flavanone and several 7,8-benzoflavanoneswere synthesized and evaluated for aromatase inhibition.

The 7,8-benzoflavanones showed promising activity and themost potent compoundwas 65 (Fig.10) with an IC50¼ 0.61 mM [77].

Luqman et al. synthesized several lactone- and lactam-basedneoflavonoids and tetra hydroquinolones and evaluated them forcancer chemopreventive actions using cell and molecular target-

O

O

HO

49

O

O

HO

50OH

O

O

51

O

O

52

O

O

HO

53OH

OH

OH

O

O

54

OH

HO

OH OH

OH O

Fig. 8. Representative flavonoid derivatives as aromatase inhibitors.

O

O X

N

N

55: X = CN56: X = NO2

SO

O

N

O

57

NO

O

ON

58

O

O

N

62

O

O

O

N

63

F

O

O

59

O

O

O

60

O

O

O

61N

Fig. 9. Xanthone and Isoflavone derivatives as aromatase inhibitors.

O

N

N

HO

64

O

O

65

OH

O

O

OH

O

O

O

O

66

Fig. 10. Flavan, flavanone and neoflavanoid derivatives as aromatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386 381

O

67

O

N

N

O O O

N

N

O

F

F

68

Fig. 11. Coumarin derivatives as aromatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386382

based in vitro bioassays. Neoflavonoid compound 66 (Fig. 10)inhibited aromatase with an IC50 value of 12.1 mM [78].

Leonetti et al. designed and synthesized a number of coumarinand fluorene imidazolyl derivatives as potent and selective AIs.The coumarin derivative 67 (Fig. 11), bearing a phenoxy group atC-7 and an imidazolylmethyl substituent at C-4, was the mostpotent compound and exhibited an IC50 ¼ 0.051 mM and a gooddegree of CYP19/CYP17 (17a-hydroxy/17,20-lyase) selectivity [79].To further explore the substituent effects on phenyl andsubstituted phenyl rings on the 4-methylene bridge and differentsubstituents at the C-7 aromatic ring, Stefanachi et al. designedand synthesized several imidazolyl derivatives of 4,7-disubstitutedcoumarins and reported aromatase inhibition and selectivity overP450 17. The most potent CYP19 inhibitor of the series was 7-(3,4-difluorophenoxy)-4-imidazolylmethyl coumarin 68 (Fig. 11),which had an IC50 ¼ 0.047 mM and selectivity over CYP17 (14%inhibition) [80].

A series of resveratrol analogs were designed and synthesizedby Sun et al. and evaluated for their aromatase inhibition. Twocompounds, 69 and 70 (Fig. 12), exhibited maximum aromataseinhibition, with IC50 values of 0.07 mM and 0.036 mM, respectively.The structureeactivity relationship andmolecular modeling resultsrevealed that the para amino group on the trans stilbene benzenering was required for aromatase inhibition and the introduction ofthe imidazole moiety increased the activity [81]. Later, the stilbeneethylenic bridge of resveratrol was replaced with a 1,2,4-thiadiozole heterocycle and modifications were done on two aro-matic rings to afford a 3,5-diaryl-1,2,4-thiadiazole scaffold. Incomparison to trans-resveratrol, the pyridyl derivatives 71 and 72(Fig. 12) had 30-125 times higher aromatase inhibition, with

O2NN

O

O

N

69

H2N

70

SN

N NN

72

Fig. 12. Resveratrol analogs

IC50s ¼ 0.2 mM and 0.8 mM, respectively [82]. Hoshino et al. syn-thesized five resveratrol sulfate metabolites and assessed them foraromatase inhibition. Resveratrol and its sulfates were weak AIs;the most active compound in this series was sulfate metabolite 73(Fig.12), which exhibited 30% inhibition at a concentration of 34 mM[83].

Lv et al. synthesized the tamoxifen metabolites norendoxifen,i.e., (E)-norendoxifen (74), (Z)-norendoxifen (75), and (E,Z)-nor-endoxifen isomers (76) (Fig. 13) and reported aromatase inhibitionand estrogen receptor modulatory activities. The mixed (E,Z)-nor-endofoxin (76) exhibited maximum aromatase inhibition, withIC50 ¼ 0.10 mM, and good binding affinity to both ER-a and ER-b. Foraromatase inhibition, (E)-norendoxifen was the most activecomponent, as it was ten times more active than (Z)-norendoxifen[84]. To further optimize norendoxifen efficacy and improve aro-matase selectivity versus other cytochrome P450 enzymes, severalanalogs were designed, synthesized, and evaluated for aromataseinhibition and estrogen receptor modulatory activities. The com-pound 40-hydroxynorendoxifen (77) (Fig. 13) displayed maximumaromatase inhibition activity (IC50 ¼ 0.045 mM), enhanced affinityfor estrogen receptors, and superior selectivity for aromataseversus other cytochrome P450 enzymes, when compared to nor-endoxifen [85].

Dai et al. reported the synthesis and aromatase inhibition ac-tivity of benzylcarbazole and 12 benzylimidazole derivatives withdifferent substituents on both the phenyl and imidazole rings. Thecompounds bearing carboxyl and ester groups on the phenyl ringor the alkyl group on the imidazole showed good inhibitory ac-tivity. The 2-[2-{(2-ethyl-4-methyl-1H-imidazol-1-yl)methyl}phenyl]acetic acid 78 and benzylcarbazole 79 (Fig. 14) exhibitedthe highest activity, with IC50 values of 6.2 mM and 2.7 mM,respectively [86].

Shen et al. designed and synthesized a library of tetrahydro-b-caboline analogs inspired by callophysin A and tested them by aseries of bioassays related to cancer prevention and treatment. TheS-isomer of callophysin A (80) (Fig. 14) was the most potent againstaromatase, with an IC50 ¼ 10.5 mM [87].

Considering the importance of transition metal complexes asanticancer agents, Prachayasittikul et al. synthesized a series ofmetal (Mn, Cu, and Ni) complexes of 8-hydroxyquinoline (8-HQ)and uracil derivatives and tested their aromatase inhibition. The 8HQ-Cu-uracil complexes (81 and 82) (Fig. 15) showed promising

N

O

O

N

SN

NNN

71

OSO3K

HO

HO

73

as aromatase inhibitors.

NH

N

COOH

OH

*

80 (S)

O OH

N N

N

78 79

Fig. 14. Benzylcarbazole, benzylimidazole and tetrahydro-b-caboline analogs as aromatase inhibitors.

HO ONH2

74

O OH

75

H2N

O OH

76

H2N

77HO O

HO

NH2

Fig. 13. Norendoxifen and its analogs as aromatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386 383

aromatase inhibition with IC50s ¼ 0.30 and 1.7 mM, respectively[88].

Recently, among medicinal chemists, the approach of designingsingle agents that can act against multiple biological targets is ofgreat interest. It is evident that, in addition to inhibition of aro-matase, estrogen production can be reduced by inhibition of theenzyme steroid sulfatase (STS), which converts biologically-inactive estrone sulfate to estrone. Potter and his coworkers pio-neered this approach and designed and synthesized several dualaromatase-steroid sulfatase inhibitors (DASIs). Initially, theletrazole-based compound 83 (Fig. 16), with IC50 values of 3.0 mMfor aromatase and >10 mM for STS, and compound 84 (Fig. 16),which inhibited aromatase and steroid sulfatase with IC50 values of0.003 mM and 2.6 mM, respectively, were reported. Next, severalcompounds were synthesized by increasing the linker length

N

OHCu

HN

NH

O

O

N+O-

O

N OH

Cu

HNNHO

O

HI

8182

Fig. 15. Transition metal complexes as aromatase inhibitors.

between the triazole and the STS pharmacophore. Among thesecompounds, the most potent AI identified was compound 85(Fig. 16), with IC50 value of 0.0001 mM [89].

Further, the core components of the two leading series of DASIs,i.e., 4-((4-bromobenzyl)-[1.2.4]triazole-4-ylamino)benzo-nitrile(86) and biphenyl (87) (Fig. 16), were combined to produce hybridstructures with improved dual activity in the picomolar range.Compound 88 (Fig. 16) had IC50s of 15 pM and 830 pM for aroma-tase and STS, respectively, whereas compound 89 (Fig. 16) had IC50sof 18 pM and130 pM for aromatase and STS, respectively [90].Subsequent synthesis and structure activity relationship studieswere carried out on the derivatives of the dual aromatase-sulfataseinhibitor 4-{[(4-cyanophenyl)(4H-1,2,4-triazol-4-yl)amino]methyl}phenyl sulfamate. The results revealed that parent phenol of DASIswere more potent AIs and replacement of the methylene linkerwith a difluoromethylene group and the para-cyanophenyl ringwith a phenyl, 3,5-difluorophenyl or 2,2-difluorobenzo[d][1,3]dioxol-5-yl ring decreased the aromatase inhibition significantly.The most potent compound was the imidazole derivative 90(Fig.16) with IC50s of: aromatase¼ 0.0002 mMand STS¼ 0.0025 mM[91].

2. Conclusion

This is the first review in the last decade where both steroidaland nonsteroidal AIs reported in the literature have been summa-rized. Aromatase is an important target for breast cancer treatmentand its inhibitors are very beneficial in this respect. AIs haveattracted much attention in medicinal chemistry and providednumerous potent steroidal and nonsteroidal AIs for the chemo-prevention of breast cancer. This review provides researchers

NN

N

H2NO2SO OSO2NH2

83

NN

N

H2NO2SO CN

84

BrN

NN

CN

85

Br

H2NO2SO

NN N

N

R

H2NO2SO

CN

86CN

NN

N

R

OSO2NH2

87

N

N

CN

NN

H2NO2SO

Cl

88

N

N

CN

NN

H2NO2SO

Br

89

N

N

CN

N

H2NO2SO

90

F

Fig. 16. Dual aromatase-steroid sulfatase inhibitors.

I. Ahmad, Shagufta / European Journal of Medicinal Chemistry 102 (2015) 375e386384

working in the field of breast cancer complete knowledge about AIs,which will further help in the design and synthesis of additionalpotent AIs.

Acknowledgment

The authors appreciate the financial support provided by theSchool of Graduate Studies and Research, American University ofRas Al Khaimah through seed grant funded project No. AAS/003/15and AAS/011/15.

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