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European Journal of Medicinal Chemistry 133 (2017) 139e151

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European Journal of Medicinal Chemistry

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

Mini-review

Quest for steroidomimetics: Amino acids derived steroidal andnonsteroidal architectures

Shagufta a, **, Irshad Ahmad a, Gautam Panda b, *

a Department of Mathematics and Natural Sciences, School of Arts and Sciences, American University of Ras Al Khaimah, Ras Al Khaimah, United ArabEmiratesb Medicinal and Process Chemistry Division, Sector 10, Jankipuram Extension, Sitapur Road, CSIR-Central Drug Research Institute, Lucknow, 226 031, UP,India

a r t i c l e i n f o

Article history:Received 24 December 2016Received in revised form21 March 2017Accepted 24 March 2017Available online 27 March 2017

Keywords:SteroidomimeticsChiral amino acidSteroidalNonsteroidalSeco-steroids

* Corresponding author.** Corresponding author.

E-mail addresses: shagufta.waseem@aurak.ac.aecdri.res.in (G. Panda).

http://dx.doi.org/10.1016/j.ejmech.2017.03.0540223-5234/© 2017 Elsevier Masson SAS. All rights res

a b s t r a c t

The chiral pool amino acids have been utilized for the construction of steroidal and non-steroidal ar-chitectures in the quest for steroidomimetics. Chirality derived from amino acid-based architecturesprovides new and easy to incorporate chiral chemical space, which is otherwise very difficult to intro-duce and comprised of several synthetic steps for asymmetric steroids. The different and exciting ligand-receptor interactions may arise from the use of each amino acid enantiomer that was introduced into thechiral steroidal backbone. The A and D rings of steroidal architectures can be mimicked by the phenylgroup of the amino acid tyrosine. The Mitsunobu reaction, nucleophilic substitution and elimination, etc.were utilized for constructing diverse tri- and tetracyclic steroidal skeletons as well as benzofused seco-steroids from amino acids. These benzofused, amino acid-derived steroidal and nonsteroidal moleculeshad promising biological activity in hormonal related disorders.

© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Steroids belong to a class of natural and synthetic organiccompounds characterized by a rigid framework of 17 carbon atomsformed from four fused rings with varying levels of functionaliza-tion. The rings characteristically are three six-membered rings andone five membered ring arranged in 6-6-6-5 manner and the ringsare identified by capital letters and the numbering of carbon atomsare shown in Fig. 1 [1]. Steroids are widely distributed in nature,having diversity in the structures and possess a broad biologicalactivity profile due to their ability to penetrate cell membranes andbind to nuclear and membrane receptors. The most significantsteroidal compounds are a) sex hormones [2e4], includingandrostane, pregnane and estrane series, which exhibit varioushormonal activities; b) bile acids [5] responsible for lipid digestionand absorption; c) corticosteroids [6] involved in several physio-logical processes such as regulation of inflammation, carbohydratemetabolism, protein catabolism and blood electrolyte level; d)

(Shagufta), gautam_panda@

erved.

cardiac glycosides [7] used for heart failure treatment; e) sterols [8]as important constituents of cell membranes having a significantrole in their stability, cell growth, proliferation as well as precursorof bile acids and hormonal steroids (Fig. 1). The diverse action andwide spectrum of biological activities of steroids have not onlyinspired biochemists and endocrinologists, but also encouragedorganic chemist to develop synthetic strategies for partial and totalsynthesis of steroids. In recent years the structurally modifiedsteroids have attracted much attention owing to the increasingchallenges in the development of new therapeutic agents withminimum side effects and for the specific, selective physiologicalactivity.

Hydrophobic steroids have wide occurrence, rigid frameworkwith diverse functionalization, extensive biological activity profileand ability to penetrate cell membranes and bind to specific hor-monal receptors and thus steroids are considered as promisingscaffold for further modifications either through alteration of ringor changes at the periphery [9]. The steroidal skeletons are usuallyaltered by partial cleavage followed by reconstruction throughsimultaneous incorporation of heteroatoms or other structuralmoieties. Based on the type of alteration performed on the steroidalskeleton, the modified steroids can be divided into different cate-gories: a) heterosteroids [10e12] as the replacement of one ormore

Fig. 1. Steroid skeleton and the structures of most significant steroids.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151140

carbon atoms of a steroid molecule with heteroatoms, affecting thechemical properties of a steroid and having examples like azaste-roids, oxasteroids and thiasteroids; b) homosteroids [13,14] withexpansion of one or more carbon atoms to the ring skeleton in anyof the four rings; c) steroidal hybrids/conjugates [15] by integrationand/or linkage of steroids with other biomolecules such as aminoacids, polyamines and carbohydrates, drugs and other functionalmolecules; d) steroidal architectures containing new ring fused toring A, ring D or bridging rings A and B [16]. Most of these steroidalskeletons have proven to be promising candidates in pharmaceu-ticals; several of them are currently in the market as anti-inflammatory, immuno depressant and anti-cancer agents. How-ever, in the therapeutic application steroids are associated withseveral deficiencies due to their poor bioavailability and side effectsdue to hormonal imbalance. Additionally, the chemical structuresof steroids are very complex which makes it difficult to synthesizeand thus being expensive. Therefore the medicinal chemist'sthought was to develop more potential therapeutic agents for thehormonal related disorders with minimum side effect as well ascost effective. This led to the discovery of highly effective and se-lective non-steroidal molecules, deprived of steroidal frameworkand with minimum side effects.

A number of tailored steroid molecules have been synthesizedas potent inhibitors of specific enzymes such as aromatase, sulfa-tase and P450 etc. for the treatment of hormone dependent cancer[17e20]. During the past few years for the treatment of hormone-resistant cancers, the development of steroidal derivatives, whichinhibit the angiogenesis, tubulin polymerization and the up regu-lation of apoptotic pathways through complex signal transductionmechanisms, has attained much attention [21]. The new steroidalscaffolds with reduced undesirable hormonal activity were devel-oped through various synthetic ways, such as: elimination or

modification of functional groups responsible for binding to thehormone receptor, reducing the substrate-receptor interactions byintroducing the chemical substituents near the original group toincrease the steric hindrance, varying the number of ring membersor primary stereo structure or designing the heterocyclic de-rivatives not recognized by the receptor protein due to the differ-ence with the natural hormone in specific structure or geometry[22e26].

While going through the vast literature of steroidomimeticsleading to steroidal and nonsteroidal molecules, it appears to usamino acids as chiron were not utilized to mimic the skeleton ofsteroids to evaluate their possible properties. The importance ofchirally pure a-amino acids in asymmetric synthesis of structurallycomplex or simple drug or drug-like molecules is well documented[27]. The easy availability of amino acids in enantiomericallyenriched form and possibility of functional group transformationmade it an important scaffold for the pharma industries, and thisleads to the synthesis of several molecules for various therapeuticareas such as antimicrobials, infectious diseases, cardiovascular andnervous system disorders, genital tract diseases, estrogen-relateddisorders and bone remodeling. The molecules derived fromamino acids are very much like natural ligands with high level ofbiocompatibility, water solubility and enhanced resistance to pro-teolysis. The promising biological activity profile and scope ofstructural modification of the steroid and amino acids scaffoldencouraged us to utilize the amino acids as a chiral pool to developnovel steroidomimetics with potent therapeutic activity. Wedesigned initially asymmetric heterosteroids with synthetic possi-bility from chiral amino acids and later we shifted our attentiontowards the synthesis of amino acid derived non-steroidal struc-tures having structural similarities of steroidal framework. In thiscase study, we have compiled the amino acid derived steroidal and

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non-steroidal architectures in the last decade from our group. Thefocus of this article is to make the reader acquainted with thesynthetic methodologies towards steroidomimetics along withtheir biological significance from amino acids. The study is dividedinto two sections: the first section deals with the chiral amino acidderived steroidal architectures and the later section involves theamino acid derived non-steroidal architectures.

2. Amino acids derived steroidal architectures

2.1. Amino acids derived tetracyclic structures

The heterocyclic azasteroids are steroidal systems bearing singlenitrogen or several nitrogen atoms at different positions in thecyclopentanophenanthrene skeleton. The D-ring modified steroidderivatives obtained by ring expansion or by replacement of one ormore carbon atoms by heteroatom affect the chemical properties ofsteroids and thus play an important role towards new activepharmaceuticals [28e31]. The D-ring modified aza steroids arereported not only as non-estrogenic, steroidal sulfatase inhibitorsfor the treatment of hormone dependent breast cancer, but also actas antifungal, anti-inflammatory and antiallergic agents [32e36].Additionally the steroidal D-lactones are known to have anti-aro-matase and anti-hypercholesterolemic activities [37,38].

Considering the significance of D-ringmodified steroids, in 2006we designed and synthesized a new type of tetracyclic hetero-steroid through incorporation of chiral amino acids into the D-ringof steroidal skeleton [39]. Until 2006 only one example of 9-membered D-ring on steroid nucleus was reported and thus itwas anticipated that modification of D-ring of steroidal frameworkinto constrained 9 membered D-ring with hetero atoms will pro-vide highly efficient synthetic route to novel heterosteroid. For thesynthesis of target molecule 1, the benzylated estriol 2, synthesizedfrom benzylated estrone was used as starting material. The treat-ment of vicinal hydroxyl groups of 2 with NaIO4 afforded the dia-ldehyde which on further reduction with LAH provided the diol 3.The C-16 hydroxy group of diol 3 was protected selectively withTBDMS to afford D-secoestrone alcohol 4. The oxidation of the C-17hydroxyl group provided the required D-secoestrone aldehyde 5.The aldehyde 5 was reacted with amino acid methyl ester hydro-chloride to give imine which on further reduction afforded theamines 6. The conversion of secondary amine of 6 to tertiary amine,deprotection of the C-16 hydroxy group followed by hydrolysis ofmethyl ester provided the hydroxy acid 7. The macrolactonizationreaction using Yamaguchi coupling of hydroxy acid 7 provided thecorresponding lactones 8, which could be converted to targetmolecules 1 through deprotection of benzyl group (Scheme 1). Thecompounds were evaluated for anti-breast cancer activity usingER þ ve and -ve cell lines; but they showed moderate activity.However, the successful incorporation of amino acids into thesteroidal skeleton encouraged us to work further in this direction.

Keeping in view the importance of azasteroids [40,41] in thefield of enzyme inhibition [42e45] as they block the biosynthesis ofphysiologically undesirable steroids, we were encouraged todevelop efficient and convergent methodologies towards theasymmetric synthesis of azasteroids using amino acids as chiron.We noticed that there are no reports on the asymmetric synthesisof steroidal systemswith nitrogen at 14-position. Therefore, we haddecided to use proline and its derivatives as chiral synthon for theintroduction of nitrogen at the desired 14-position and to inducespecific stereo orientation in the steroidal system. Thus, in 2013, wedesigned and synthesized nitrogenous steroidal core 9 bearing ni-trogen at 14-position and unsaturation at D9(11) position [46].

The starting materials used in the synthesis of azasteroid 9 i.ebromo derivative 10 and aldehyde 11 was obtained in good yield

using the literature procedure [47]. The lithiated derivative of thesubstrate 10 was prepared using n-BuLi and was reacted withaldehyde 11 to afford the intermediate carbinols 12a and 12a'asdiastereomeric mixture, and which were used in the next stepwithout any separation as newly generated stereo center is lost atthe cyclization stage. Finally the NHBoc group deprotection incarbinol 12a and 12a’ followed by 6-endo trig cyclization using Et3Nas base provided the separable mixture of unsaturated D9(11) 14-azasteroid 9a and 9a’ in 1.2: 1 ratio (Scheme 2). The cyclizationworked through intramolecular SN20 reaction involving the attackof proline derived pyrrolidine nitrogen atom onto a tetralonederived allylic alcohol. It's our believe that the library of azasteroidscan be easily synthesized from readily available D and L amino acidsand by exploring the double bond at D9(11) of 14-azasteroids. Theproline derived novel azasteroids were also evaluated in humanbreast cancer cells MCF-7 and MDA-MB-231, and human prostatecancer cell lines PC-3 and DU-145. These compounds showed sig-nificant activity and specificity towards breast cancer and caused anotable decrease in progesterone, its metabolite 5-a preganene andsteroid 5-a reductase in MCF-7 cells. The binding modes of theseazasteroids were studiedwith the homologymodel of 5a-reductaseand the results revealed that p-systems of the azasteroid scaffoldand the aromatic residues are involved in the formation of non-covalent interactions such as cation-p and p-p leading to tightbinding. The series of azasteroids showed promising results and thelead compound could serve as a base for the development of newgroups of effective breast cancer therapeutics. We are in process tocompile the detail biological activity as well as binding studies ofthese synthesized azasteroids [48].

2.2. Amino acids derived tri and tetracyclic miscellaneousstructures

The chroman core is abundant in numerous natural productsand also present in several pharmaceutically important moleculeswith a wide range of biological activities especially as selectiveestrogen receptor modulators (SERMs), NF-kB inhibitors and antiHIV agents. Furthermore synthetic and natural products bearingoxazepinone skeleton are well known to exhibit anti HIV andantimalarial properties. Thus encouraged by the pharmaceuticalapplications of chroman and oxazepinone core, we thought todesign and synthesize a new type of steroidal scaffold 13 bycombining the chroman ring with chiral oxazepinone ring havingcontiguous quaternary and tertiary stereocenters from the aminoacid L-proline. We believed that tetracyclic scaffold 13 is a newexample of steroidomimetics and the synthesis could be easilyaccomplished from chiral amino acid and chroman moiety [49].

The starting material tetrahydropyrrolo[1,2-c]oxazol-3(1H)-onederivative 15 was prepared in five steps from L-Proline and bromochromanone derivative 14 was accessed following the knownprocedure [50e52]. The bromo chromanone derivative 14 wasreacted with n-BuLi to generate anions which on reaction with 15afforded the conjugated product 16 through oxazolone ring open-ing. Finally the treatment of 16 with sodium hydroxide andhydrogen peroxide in ethanol provided the desired product 13 inlow yields via epoxide formation, followed by intramolecularnucleophilic substitution (Scheme 3). We developed the route forthe one pot synthesis of a new class of chroman fused S-prolinederived chiral oxazepinones from the amino acid L-proline andchroman which worked through efficient cascade reactions;installing contiguous chiral quaternary and tertiary stereo centers.We anticipated that several natural products and pharmaceuticallyimportant molecules having tetracyclic skeleton or resemblancewith steroidal framework could be further synthesized using thedeveloped synthetic procedures.

Scheme 1. Reagents and Conditions: (a) NaI4, Methanol, H2O, 0 �C; (b) LAH, THF, 0 �C; (c) TBDMSCl, Imidazole, DCM, 0 �C-rt. (d) TPAP, NMMO, MS 4A, DCM, rt; (e) amino acid methylester hydrochloride, TEA, Methanol:THF (4:1), reflux-rt; (f) NaBH4, Methanol, 0e5 �C; (g) MeI, K2CO3, Acetone, rt; (h) AcOH:H2O:THF (3:1:1), 60 �C; (i) 1N NaOH, Dioxane, 60 �C; (j)2,4,6-Cl3C6H2COCl, Et3N, THF, rt, then DMAP, PhMe, reflux.

Scheme 2. Reagents and conditions: (a) i) n-BuLi, dry THF, �78 �C; ii) 11, �78 �C to rt (b) 4M HCl in dioxane, 0 �C-rt; (c) Et3N, rt.

Scheme 3. Reagents and Conditions: (a) i) n-BuLi, THF, �78 �C; ii) 15, �78 �C to rt; (b) H2O2, NaOH, EtOH, 0 �C.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151142

Next our focus was on biologically important nitrogen-containing benzofused steroidal tricycles i.e. pyrazino[1,2-a]qui-noxalines (17) which may be elaborated into typical steroidal tet-racyclic skeletons. Prior to our synthesis of pyrazino[1,2-a]quinoxalines from amino acids [53], only two synthetic approacheswere reported [54,55]. We started the synthesis of 17 by reacting 1-fluoro-2-nitrobenzene 18 with S-amino acid 19. Both the reactantswere reacted in the presence of K2CO3 and then treated with SOCl2and MeOH to produce methyl esters 20. The hydroxyl group pro-tection using TBDMSCl followed by ester group hydrolysis and thenreaction with different N-tosyl protected amino acid methyl estersunder Mitsunobu condition furnished 21 in good yields. Next, thedesilylation, reduction of aromatic nitro group followed by itsprotection with tosyl group provided derivative 22 which underMitsunobu condition furnished the enantiomerically pure

substituted 1,2,3,4-tetrahydroquinoxalines 23. The reduction ofester group followed by 6-exo-tet cyclization using PPh3/I2/imid-azole afforded the final product pyrazino[1,2-a]quinoxalines(Scheme 4).

Following the similar approach of synthesizing other tetracycliccompounds using amino acid, we further targeted the synthesis oftetrahydro-5H-isoquinolino[2,3-a]quinoxaline (24), a tetracyclicsteroidal compound [56]. The compound 25 was used as startingmaterial for the synthesis of the target molecule, and it was pre-pared from S-Amino acid and 1-fluoro-2-nitrobenzene by followingthe same steps as described previously for the synthesis of 20. Thereduction of ester group and nitro group of 25, followed by bocprotection furnished 26. Further Swern oxidation of alcohol 26 toaldehyde followed by Wittig reaction afforded 27. Finally to obtainthe required target compound, the 27 were subjected to

Scheme 4. Reagents and Conditions: (a) anhy. K2CO3, DMF, 80 �C; (b) SOCl2. MeOH, 0 �C-rt; (c) TBDMSOTf, 2,6-lutidine, DCM, 78 �C (d) LiBH4 (2M), THF, 0 �C-rt; (e) PPh3, DEAD, N-tosyl protected amino acid methyl esters, 0 �C-rt; (f) Acetic acid: THF: water (3:1:1), 120 �C; (g) Pd-C (10%), H2 50 psi, rt; (h) Py, TsCl, 0 �C-rt; (i) PPh3, DEAD, 0 �C; (j) LiBH4 (2M), dryTHF, 0 �C-rt; (k) PPh3, I2, imidazole, 40 �C.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151 143

deprotection followed by reaction with substituted benzaldehydeand it was anticipated that through intermediate 28 followed byelectron demand inverse Diels-Alder reaction tetrahydro-5H-iso-quinolino[2,3-a]quinoxaline (24) will be produced. However,instead of target compound 24 we obtained dihy-drobenzodiazepine 29 and its structure was confirmed throughdifferent NMR analytical techniques (Scheme 5).

3. Amino acids derived non-steroidal benzofused seco-steroid like architectures

The rate of recognition and binding of steroids to their relatedreceptors or metabolizing enzymes depends on its structure andconformation and thus the compounds which mimic steroidalconformation i.e steroidomimetics can easily bind to these cellulartargets [57]. Steroidomimetics deprived of the steroidal cyclo-pentanoperhydrophenanthrene nucleus are characterized as non-steroidal compounds. These non-steroidal compounds can in-crease or decrease the steroidal activity and therefore provide thebasis of various pharmaceuticals to treat hormone related diseases.The main biological targets for non-steroidal compounds are

Scheme 5. Reagents and Conditions: (a) LiBH4 (2M), anhyd. THF, 0 �C-rt; (b) H2, Pd-C, MeOHPh3PþCH3I�, KHMDS, dry THF, �78 �C; (f) i) TFA, dry DCM, 0 �C-rt; ii) Substituted benzalde

steroid receptors or enzymes, which are involved in steroidbiosynthesis and metabolism. The non-steroidal compounds act aspotent inhibitors of several enzymes such as human 17a-hydrox-ylase-17,20-lyase (CYP 17), 17b-hydroxysteroid dehydrogenase typeI and II (17b-HSD1 and 17b-HSD1), 5a-reductase type I and II, andoxidosqualene cyclase and are mainly used for the treatment ofosteoporosis, cancer, benign prostatic hyperplasia (BPH) and car-diovascular diseases [58e65]. The advantages of using non-steroidal drugs over steroidal drugs are due to enhanced receptorselectivity, improved pharmacokinetic properties, such as oralbioavailability and hepatic metabolism. Due to these propertiesnonsteroidal compounds are considered as attractive targets fornovel drug discovery. The non-steroidal molecules developed tomimic steroids and used clinically in treating different diseases[66e69] are shown in Fig. 2. Tamoxifen is a nonsteroidal drug,widely used in breast cancer treatment and is currently the world'slargest selling breast cancer drug. Tamoxifen was synthesized anddeveloped by the former ICI (now AstraZeneca) [70] and is soldunder the brand names Nolvadex, Istubal, and Valodex. Tamoxifenworks as an antagonist in breast tissue, is agonist on bone in postmenopausal women but displays some estrogen like effects in the

, 50 Psi, rt; (c) Boc2O, NaHCO3, EtOH, RT; (d) (COCl)2, DMSO, Et3N, dry DCM, �78 �C; (e)hyde, rt.

Fig. 2. Non-steroidal drugs available in the market.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151144

uterus [71]. Raloxifene synthesized by Eli Lilly Pharmaceuticals andused for the treatment of osteoporosis and is sold under the brandname Evista. It acts as an antagonist in both breast and uterinetissue, but is an agonist in bone [72]. Centchroman was the firstnon-steroidal oral contraceptive that is developed by the CentralDrug Research Institute in India [73]. It is the first nonsteroidal oralcontraceptive, which shows the antagonist profile in breast anduterus, but is an agonist in bone. Clomiphene is the first triaryl-ethylene to enter the drug market. It is used as a profertility drugfor the induction of ovulation [74]. Letrozole containing 1,2,4 tri-azole ring is very potent and selective aromatase inhibitors having99% aromatase inhibition and are well tolerated by patients [75].667-COUMATE is a tricyclic coumarin based most potent sulfataseinhibitor which is non-estrogenic, orally active [76].

From the last one decade the research of our group is mainlyfocused on two major areas: a) to develop new pharmacophores asanti-breast cancer agents and in this respect we have reported thephenanthrene based compounds containing amino alkyl chainswith promising activity; b) to develop new and efficient strategiesfor the syntheses of amino acid based molecules in quest for ster-oidomimetics [77e83]. The pharmacological importance of ben-zoxazepines in medicinal chemistry encouraged us to work on thisscaffold and thus we designed and synthesized benzoxazepines 30from building blocks, substituted benzene derivatives 31 and S-Amino acid derived N-tosyl amino esters 32 and towards accessingnonsteroidal architectures (Scheme 6) [84].

Scheme 6. Reagents and Conditions: (a) DEAD, PPh3, THF, 0 �C to rt, N2; (b) LAH, THF

For the synthesis of benzoxazepines, Mitsunobu reaction of S-amino acid derivative 32 with benzene derivative 31 provided thecompound 33 which on reduction with LAH followed by deben-zylation afforded 34 bearing the free alcoholic and phenolic hy-droxyl groups. The compound 34 was subjected to Mitsunobureaction conditions to provide the desired enantiomerically purebenzoxazepine derivatives 30. The synthesized compounds wereevaluated for anti-tumor activity in human breast cancer cells andxenograft model. The biological result of the S-amino acid derivedbenzoxazepines showed the compound bearing benzene ring inside chain exhibiting better activity and the compound derivedfrom tyrosine was the most potent.

It is well known that the compounds with promising anti-estrogenic activity in breast cancer cells mostly bear at least one ofthe following characteristic feature: a) strategically placed hydroxyor methoxy groups b) alkyl amino ethyl chain [85]. Based on theabove mentioned reports, we believed that our synthesized ben-zoxazepines derived from the amino acid tyrosine are nonsteroidalsteroidomimetic where benzene/substituted benzene of benzox-azepine core is mimicking the A and B rings of steroids respectivelyand benzene ring acquired from amino acid tyrosine, is resemblingthe D ring of steroidal skeleton. To further study the effect of alkylamino ethyl chain on the anti-tumor activity of tyrosine-basedbenzoxazepine 35, we incorporated the various amino alkylchains on tyrosine benzene and prepared 36 (Scheme 7) [86].

The new benzoxazepines particularly works to inhibit growth of

, 0 �C; (c) H2, 10% Pd/C, MeOH, rt, 50 psi, RT; (d) DEAD, PPh3, THF, 0 �C to rt, N2.

Scheme 7. Reagents and Conditions: (a) anhyd K2CO3, R1Cl, dry acetone, reflux.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151 145

breast cancer cell lines, MCF-7 and MDA-MB-231, but lack cyto-toxicity to normal HEK-293 cells. The most active compound of theseries was 36a and 36b bearing piperidine ethyl and azepane ethylgroup, respectively and inhibit growth of MCF-7 and MDA-MB-231 cells at IC50 in the micromolar range (Table 1).

The inhibition of cell growth induced by the active compoundswas because of cell cycle arrest at G0/G1 phase. The efficacy of 36aand 36b for inducing apoptosis rather than necrosis was estab-lished by assessing cellular processes associated with apoptosissuch as: phosphatidylserine exposure (Annexin V-PI; Flow cytom-etry), DNA fragmentation and PRP Cleavage. 36a at the concentra-tion of 5 mM and 10 mM considerably increases Annexin-V positiveand PI negative cell population indicating induction of apoptosis

Table 1IC50 (mM) value of benzoxazepine derivatives (36a-b and 37a-c) in breast cancer cell line

Compound No Structure

36a

36b

37a

37b

37c

Results were calculated from three independent experiments with triplicate of each obs

compared with untreated control (Fig. 3). The compound 36bconcentration dependently increased phosphatidylserine expo-sure, induced PARP cleavage and DNA fragmentation (Fig. 4).Moreover, 36b activated the apoptosis through activation ofcaspase-8 and -9, mitochondrial membrane depolarization andincrease in Bax/Bc12 ratio. Through the use of selective caspaseinhibitors, it was proved that the major contributor to 36b inducedapoptosis is caspase-8-dependent pathway. 36a and 36b causedsignificant reduction in tumor volume of MCF-7 xenograft tumor innude mice model. 36a at the dose of 16 mg kg�1 day�1 displayedsignificantly higher relative tumor volume regression than thecontrol group and its activity was comparable to that of tamoxifencitrate at 30 days of treatment (Fig. 5). Similarly the treatment of

s using MTT assay after 24 h treatment.

MCF- 7 MDA-MB-231

9.62 10.2

6.78 7.2

10.34 13.21

14.5 18.6

12.45 15.67

ervation.

Fig. 4. 36b induces Apoptosis in MCF-7 cells A)- 36b increases phosphatidylserine exposure- MCF-7 cells were treated either with vehicle (V) or indicated concentrations of 36b for24 h and phosphatidylserine exposure was measured by flow cytometry using FITC labelled Annexin V & PI staining. Representative dot plots and relative apoptotic cells are shown.B) Effect of 36b treatment on PARP Cleavage e36b treated MCF-7 cell lysates were immunoblotted against PARP, Densitometry data of cleaved PARP is plotted. C) Effect of 36btreatment on DNA Fragmentation - cells were treated with 36b and 4 OHT as indicated and DNA Fragmentation after 24 h of treatment was quantified. Graph represents relativeDNA fragmentation taking þ ve control (PC) as 100% (#). Data represent mean ± SEM from three independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to V.Reprinted with permission from Dwivedi et al. [86].

Fig. 3. Negative cell population at 5 mM and 10 mM of 36a; After 24 h, the cells were harvested, stained with Annexin-V and propidium iodide, and analyzed by flow cytometry. Dataare expressed as % of total cell Count. Reprinted with permission from Samanta et al. [84].

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151146

36b (16 mg kg�1 day�1) to these mice for 21 days resulted in nearly50% reduction in relative tumor volume (Fig. 6A). However, whencompared with vehicle-treated controls no significant differencesin the initial and final body weights were observed (Fig. 6B).

Further biological activity results of tyrosine-based benzox-azepine derivatives bearing various amino alkyl chains on benzeneof the amino acid tyrosine and literature reports of the 7asubstituted estradiol derivatives, especially ICI 164 182, 384 780,and EM-139 exhibiting antiestrogenic activity on breast cancer cells[87e93] encouraged us to design and synthesize a new type oftyrosine based benzoxazepine derivatives 37 bearing alkyl aminoethyl chains on benzene ring, which is present on nitrogen atom ofbenzoxazepine skeleton [86]. We anticipated that benzene ringbearing alkyl amino ethyl chain substituent on nitrogen might actas 7a substituent as in compoundsmentioned in Fig. 7 and thus willexert antiestrogenic effect on breast cancer cells.

The synthesis of benzoxazepines derivatives 37 was done in

several steps. The bromo derivative 39 obtained from benzylalcohol 38 was reacted with tyrosine derivative 40 to afford amine41. The Boc protection of secondary amine, reduction of methylester followed by debenzylation reaction provided alcohol 42. Un-der the Mitsunobu reaction condition compound 42 after cycliza-tion afforded benzoxazepane derivative 43. The boc groupdeprotection followed by reaction with (4-(bromomethyl)phenox-y)(tert-butyl)dimethylsilane (44) produced compound 45. Finallydifferent kinds of amino alkyl chain were incorporated on hydroxylgroup of 45 to produce compound 37 (Scheme 8). The newly syn-thesized compounds were evaluated for anti-proliferative activityin both MCF-7 (ER þ ve) and MDA-MB-231 (ER-ve) cells using MTTassay. Several compounds of this series (37a, 37b, 37c) were foundto inhibit growth of MCF-7 and MDA-MB-231 cells at IC50 of lessthan 20.0 mM (Table 1).

Later detailed biological studies were conducted to find outin vitro and in vivo effects of a tyrosine based benzoxazepine, (4-[4-

Fig. 6. 36b inhibits growth of MCF-7 cancer cell xenograft in athymic nude mice. (A) Increase in relative tumor volume and (B) Average body weight over time in control and 36btreatment groups indicated. N ¼ 10 mice/group; data represent mean ± SEM. *P < 0.05 and **P < 0.01 compared to control. Reprinted with permission from Dwivedi et al. [86].

Fig. 5. Regression in mean relative tumor volume in human breast cancer cell (MCF-7) xenograft model. Animals were treated with 36a at the dose of 8 mgkg�1 day�1 and16 mg kg�1 day�1 after tumor induction for 30 days. The data presented are average of relative tumor volume (n ¼ 10 animals/group). Reprinted with permission from Samanta et al.[84].

Fig. 7. 7a-substituted antiestrogens.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151 147

(toluene-4-sulfonyl)-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-3-ylmethyl]-phenol) [THBP, 35a] in human breast cancer cells, withan objective of examining its target [94]. Treatment of MCF-7 and

MDA-MB231 with 35a for 24 h gave concentration-dependent lossof viability with IC50 of ~20 mM and ~30 mM, respectively. In mo-lecular docking studies, THBP positioned in the ATP binding pocket

Scheme 8. Reagents and conditions: (a) PPh3, CBr4, DCM, 0 �C-rt; (b) K2CO3, DMF, RT; (c) NaHCO3, Boc2O, EtOH, RT; (d) LiBH4, THF, RT; (e) H2, 10% Pd-C, 50 psi, 0 �C; (f) PPh3, DEAD,0 �C-rt (g) 6N HCl, MeOH, 70 �C; (h) K2CO3, DMF, 44, RT; (i) R2Cl, K2CO3, acetone, 60 �C.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151148

of IGF-1 receptor (IGF-1R) and selectively abrogated increasedviability of MCF-7 cells by insulin-like growth factor 1 (IGF-1). InMCF-7 cells, THBP inhibited IGF-1-induced phosphorylation of IGF-1R and insulin receptor substrate-1 (IRS-1) without inhibiting in-sulin signaling. In athymic nude mice, compared with vehicle, thegrowth of MCF-7 xenograft tumors was decreased significantlywith treatment of 35a because of inhibition of cancer cell prolif-eration as well as promotion of cell death that correlated withreduced phospho-IGF-1R levels. Thus THBP 35a is a an orally effi-cacious suppressor of ER-positive human breast cancers by selec-tive abrogation of IGF-1R signaling. Further efforts are going on tostudy the THBP effect on drug-resistant tumors, its distribution andpossible host toxicity. We are anticipating that the result of ourcurrent study will support to the design of a new and efficient classof IGF-1R inhibitors for future preclinical and clinical trials.

4. Miscellaneous section of benzofused secosteroids

Following the same strategy of utilizing the amino acid for thesynthesis of heterocyclic compounds mimicking the steroidalskeleton we synthesized several benzo fused derivatives andevaluated them for biological activities related to estrogen relateddisorders. All these molecules were designed as C-seco steroids andwe believed that benzene ring mimic the A ring of steroid, B ring isin the modified form and if R substituent is CH2Ph, then Phenylgroup mimics the D-ring of the steroid skeleton. The moleculeswere synthesized and evaluated for biological activity. The bio-logical profile of the synthesized benzo fused seco-steroids/C-secosteroids were encouraging and thus the synthetic strategies forthese compounds supports the goal i.e. development of newchemical entities for estrogen related activities. The benzo fusedsecosteroids derivatives synthesized by our group using aminoacids are (Fig. 8) [95e101]: a) chiral 3-substituted-1,4-benzodiazepin-2-ones (46) synthesized by coupling of 2-nitrobenzyl bromide with a series of amino acids followed by dia-zepine ring formation with Fe/AcOH; b) bicyclic amino acid-annulated seven and eight membered ring heterocycles i.e. benzo[1,4]oxazepines (47), benzo[1,4]diazepines (48), benzo[1,4]thiaze-pine (49), benzo[1,4]diazocines (50) and benzo[1,4]oxazocines (51)synthesized in chiral form derived from naturally abundant S-amino acids; c) 3,4-dihydro-2H-benzo[b][1,4]thiazines (52) syn-thesized via-a copper-catalyzed intramolecular N-aryl amination

reaction on substituted 2-(2-bromophenylthio)-ethanamines, pre-pared by the nucleophilic substitution reaction of 2-bromobenzenethiol with Boc-protected amino alcohol de-rivatives. d) Enantiomerically enriched indolines (53) and tetrahy-droisoquinolines (54) were synthesized from easily accessible (S)-amino acids derived chiral carbocations. Here Friedel-Crafts reac-tion promoted by Lewis acid (AlCl3) offered trans-diaster-eoselectivity. The rate of the reaction and diastereoselectivity of theproduct is significantly influenced by steric hindrance of sub-stituents of amino acids and the nature of the aryl groups. e) Thesubstituted 1,2,3,4-tetrahydroquinoxaline (55), benzo-annulatedunsymmetrical chiral [9]-N3 peraza and [12]-N4 peraza-macrocycles (56 and 57) were prepared using inter- and intra-molecular Mitsunobu reaction from S-amino acids.

5. Conclusion

We have established that chiral amino acids could be efficientlyused in the development of steroidal and nonsteroidal ster-oidomimetics. The amino acid-derived tetracyclic and tricyclicstructures, non-steroidal benzofused seco-steroids, and miscella-neous heterocycles were prepared from chiral amino acids.Chirality derived from amino acid-based architectures gives aconvenient way to incorporate chiral chemical space within thesteroidal skeleton, which is otherwise very difficult to introducewith multiple synthetic steps. The different and interesting ligand-receptor interactions may originate with the use of each amino acidenantiomer that has been used to construct an asymmetric ste-roidal backbone. Also, it has been revealed that a few of thesecompounds have significant biological activity against breast can-cer. Considering the importance of new chemical entities in drugindustry, this strategy will be of interest to organic and medicinalchemists. Our amino acid-derived steroidomimetics strategy pro-vides a scope for the development of amino acid-derived scaffoldsfor other therapeutic targets; future research in this area couldprovide encouraging results.

Moreover, the advantages of using non-steroidal drugs oversteroidal drugs are due to their enhanced receptor selectivity, whiletheir improved pharmacokinetic properties give a platform to thedesign and synthesis of amino acid-derived nonsteroidal com-pounds as attractive targets for novel drug discovery. The A and Drings of steroidal architectures can be mimicked with the phenyl

Fig. 8. Miscellaneous benzofused seco-steroids derived from amino acids.

Shagufta et al. / European Journal of Medicinal Chemistry 133 (2017) 139e151 149

group of the amino acid tyrosine. Strategically placed hydroxy ormethoxy groups from tyrosine and an alkyl amino ethyl chain willprovide a range of new amino acid-derived nonsteroidals with aprobable anti-estrogenic profile. Among all the compounds syn-thesized and evaluated, non-steroidal benzoxazepines derivedfrom tyrosine gave promising results. We are expecting that thisarticle will update researchers with the synthetic strategiesinvolved in the synthesis of amino acid-derived steroidomimeticsand thus encourage them to design and synthesize promisingsteroidomimetics and other organic scaffolds with significantmedical properties using chiral amino acids.

Acknowledgements

The authors, Dr. Shagufta and Dr. Irshad Ahmad are thankful tothe School of Graduate Studies and Research, American Universityof Ras Al Khaimah for their support. Gautam Panda thanksDepartment of Science & Technology (DST SB/S1/OC-93/2013) forthe continued support for his research. This has CDRI Communi-cation no 9472.

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