Steroid-triazole conjugates: A brief overview of synthesis and their application as anticancer agents

Steroids are biomolecules that play pivotal roles in various physiological and drug discovery processes. Abundant research has been fuelled towards steroid-heterocycles conjugates over the last few decades as potential therapeutic agents against various diseases especially as anticancer agents. In this context various steroid-triazole conjugates have been synthesized and studied for their anticancer potential against various cancer cell lines. A thorough search of the literatures revealed that a concise review pertaining the present topic is not compiled. Therefore, in thus review we summarize the synthesis, anticancer activity against various cancer cell lines and structure activity relationship (SAR) of various steroid-triazole conjugates. This review can lay down the path towards the development of various steroid-heterocycles conjugates with lesser side effects and profound efficacy.


Introduction
Steroids are naturally occurring biomolecules that play vital roles in biological systems. Due to their pivotal pharmacological properties and enthralling rigid framework, they are one of the most thrilling molecules in drug discovery [1][2][3]. Steroids possess a rigid tetracyclic core with a highly specific perhydrocyclopentano[α]phenanthrene orientation possessing varied functional groups on it, which provides diversity in their biological actions, making them versatile substrates for different targets. In general, the core structure of a steroid consists of 17 carbon atoms connected in 4 fused rings, among which three are cyclohexanes (A, B, and C) and the fourth one is a cyclopentane (D ring) ring. The numbering in perhydrocyclopentano[α]phenanthrene system is carried out in ascending order starting from ring A, continuing to ring B, C and ending in ring D (Fig. 1) [4,5].
In particular, a variety of steroid conjugates have been explored as anticancer agents in the past few decades. It has been understood that steroid conjugates exert their anticancer activity either via enzyme inhibition (steroid sulfatase inhibitors, aromatase inhibitors, hydroxysteroid dehydrogenase inhibitors) or by binding to receptors (antiestrogens, antiprogestins) [1,[46][47][48][49][50][51][52][53][54]. As a result, steroids have served as backbone in bioconjugation approach to improve the physicochemical (increased lipid solubility) and biological properties (target specificity or receptor selectivity) properties of bioactive nonsteroidal pharmacophores. In addition, steroids uniquely bind to the nuclear and membrane receptors and penetrate the cell membranes. Furthermore, a slight change in the steroidal framework induces a significant response in the associated biological systems.
Thus, extensive research has been fuelled towards the rational modification of steroid molecules, especially via conjugation chemistry to obtain anticancer lead compounds that are less toxic, less vulnerable to multi-drug resistance (MDR) and highly bioavailable. Among the studied steroid heterocyclic conjugates, steroid triazoles have been extensively designed and studied towards the development of novel anticancer agents. In addition, various steroid conjugated bioactive molecules are also reported against various activities and are under clinal trials to improve the properties of bioactive molecules [55][56][57][58][59][60][61].
In view to its synthetic analogues many semi-synthetic triazole analogues of natural products are also reported to possess various biological activities especially as anticancer agents [139][140][141][142][143]. For instance, Chandrashekhar et. al. reported 1,2,3-triazole hybrids of myrrhanone C and studied their invitro cytoxicity against human lung cancer cell line (A549), human cervical cancer cell line (Hela), human breast cancer cell line (MCF-7), human prostate cancer cell line (DU-145) and human liver cancer cell line (HepG2) [144]. Same group in 2019, reported side chain analogues of myrrhanones A & B with triazoles as anti-inflammatory and cytotoxic agents against human prostate cancer cell line (DU-145), human colon cancer cell line (HT-29),  human breast cancer cell line (MCF-7), human cervical cancer cell line (Hela), human glioblastoma cancer cell line (U87MG) and human normal lung cell line (L132) [145]. In continuation to their efforts the same group in 2020, reported synthesis of 1,2,3-triazole hybrids of myrrhanone B and studied their antiproliferative activity against human lung cancer cell line (A549), human prostate cancer cell line (DU-145), human breast cancer cell line (MDAMB-231), human cervical cancer cell line (SiHa), human glioblastoma cancer cell line (U87MG), human prostate cancer cell line (PC3), human colon cancer cell line (HT-29) and human normal lung cell line (L132) [146].
In the above context, synthesis of triazoles have attracted considerable attention over the past decades. The most common reaction for the synthesis of 1,2,3-triazole is the thermal cycloaddition of azide and terminal alkyne [147]. However, this method suffers with several disadvantages such as regioselectivity, low yield and high temperatures [148]. To overcome all these drawbacks in 2001, sharpless reported the Cu-catalyzed azide-alkyne cycloaddition reaction (CuAAC) for the synthesis of 1,2,3-triazole in good yields and high regioselectivity ( Fig. 3) [149]. Further, many other metal-catalyzed [150], and metalfree strategies [151][152][153][154][155][156] have been employed for the synthesis of triazole scaffold with diverse functionality either by multistep or multicomponent approach [157] in recent years.
The present review summarizes the synthesis, anticancer activity results and the conclusive SAR studies on steroid-triazole conjugates. We believe this review will act as a resource on anticancer properties of steroid-triazole conjugates, and thus should stimulate more research around steroid heterocycles conjugates against various clinical indications, especially against different types of cancer (Fig. 5).

Synthesis and anticancer activity of steroid-triazoles conjugates
It has been well understood that the presence of different functional groups around the rigid tetracyclic steroidal framework leads to diversity in their biological actions, and thus, such functionalized steroids have served as substrates for different targets. As a result, tuning the target binding properties of steroid-based biologically important molecules has become a major research challenge in recent years, and exhaustive efforts have been devoted to the generation of novel steroidal analogues via chemical conjugation. Most of the steroid-based pharmaceuticals are semi-synthetic compounds prepared by connecting a special functionality to the core structure of a steroid [5]. In this realm, heterocyclic moieties that exhibit hydrophobicity and potent receptor binding properties are often used to derivatize/functionalize steroid frameworks. The synthesis of such modified steroidal-heterocyclic conjugates have exhibited valuable pharmacological activities [6][7][8], overcoming pertinent drug development issues, such as relatively high risk of toxicity, bacterial resistance and/or pharmacokinetic deficiencies [9].
In particular, the basicity and hydrophilicity of an azole moiety have altered the biological function of a steroid, [11,12] and the resultant steroidal azoles have also been found to be potent inhibitors of 17-hydroxylase-C17,20-lyase, the enzyme that catalyses the conversion of progesterone and pregnenolone into the androgens. Since androgens are implicated in the etiology of a number of androgen dependant diseases (e.g. prostate cancer); the inhibitors of such enzymes are useful for the  treatment of these diseases [11,12]. Steroidal azoles have also been found to have strong inhibitory effect on 5-reductases that makes them promising lead compounds against various types of tumours. In recent years, considerable interest has been focused on steroidal-triazole in view of the broad spectrum of their biological activities. Several novel steroid-triazole conjugates have been synthesized and described as potent inhibitors of 17-hydroxylase-C17,20-lyase (P45017) that can block androgen synthesis at an early stage and may therefore be useful in the treatment of prostatic carcinoma [1][2][3].
Below is an account on the synthesis and anticancer evaluation of several steroid-triazole conjugates.
Among all the derivatives synthesized, compound 41a was found to be the most potent with a growth inhibition of 96.9% against HeLa, while 41a was found to possess growth inhibition of 83.7% and 88.5% against MCF7 and A2780, respectively. The pyridyl derivative (41b) showed growth inhibition of 93.2%, 63.8% and 43.3% against HeLa, MCF7 and A2780, respectively. No clear structure-activity relationship could be established, but it was found that aromatic moiety with metaand para-substitution exhibited better anticancer activity as compared to the ortho-substitution, with the exception of m-amino substitution. In addition, heteroaromatic substituted derivatives were found to be more potent as compared to aromatic or alkyl substituted derivatives (Scheme 3) [177].
Among all the analogues synthesized (45), only few showed activities below the concentration of 30 µM. Compounds 45a, 45b, 45c, 45d and 45e exhibited 32.1, 40.9, 32.3, 29.7 and 43.5% growth inhibition at 30 µM against HeLa as compared to the cisplatin (99.9%), respectively. The pyridyl group containing analogue (45e) was found to exhibit the best activity with 43.5 % of growth inhibition at 30 µM. Compounds with phenyl substitution (45a) exhibited a modest activity, except for p-OMe substitution on phenyl ring. Aryl or heteroaryl substituted derivatives were found to be more potent as compared to cycloalkyl substituted derivatives (Scheme 4) [180].
In 2012, Eva Frank and co-workers reported the synthesis of 15βtriazolyl-5α-estrone (50) and 15β-triazolyl-5α-androstane derivatives (53) and evaluated their antiproliferative activity against three different human cancer cell lines, including cervix cancer cell line (HeLa), skin epidermoid cancer cell line (A431) and breast cancer cell line (MCF7). The azido group was introduced stereoselectively at the 15β-position of sterane skeleton (46) by the 1,4-Michael addition with NaN 3 using acetic acid in THF to produce corresponding azido derivative (47) [181], which followed by reductive acylation with KBH 4 /Ac 2 O in MeOH/pyridine afforded 48. Finally, 48 on reaction with different terminal alkynes (27) using CuI, Ph 3 P and DIPEA in toluene at 111 • C yielded 15β-triazolyl-5α-estrone derivatives (49), which finally on oxidation using Jones reagent in acetone furnished 15β-triazolyl-5α-estrone (50). A similar approach was used for the synthesis of 15β-triazolyl-5α-androstane derivatives (53) (Scheme 5) [182]. Among all the derivatives (49, 50, 52 and 53) screened for their antiproliferative activity, compound 49a was found to possess an IC 50 value of 1.70 µM against A431 as compared to cisplatin (IC 50 = 2.84 µM). Compounds 52a, 52b and 52c were found to possess an IC 50 value of 7.70, 9.40 and 6.52 µM against HeLa as compared to cisplatin (IC 50 = 12.43 µM). Compounds 53a, 53b, 53c and 53d were found to be active against MCF-7 with IC 50 value in the range of 1. 69-8.40 µM as compared to cisplatin (IC 50 = 9.63 µM). Compound 53a was found to be most active compound with an IC 50 value of 1.69 µM against MCF-7. Further, cell cycle analysis and apoptotic study were carried out to study the possible mechanism of action. Cell cycle analysis revealed that the number cells increase in the subG1 phase, thus suggesting that the compounds exert their action by making the cells prone for apoptosis. More mechanistic investigation using cell morphology and cell integrity revealed a concentration dependent increase in the nuclear condensation and cell membrane permeability, thus making the cells to undergo apoptosis (Scheme 5) [182].
Among all the compounds (84 & 89) screened for their cytotoxicity, 89c and 84 were found to be most active with an IC 50 value of 0.7 µM and 0.8 µM against K562. In addition, 89a-d and 84 were also found to be active against OVCAR-3 and WM36 with an IC 50 value in the range of 0.9-2.2 µM and 1.9-3.8 µM. With respect to tubulin polymerase inhibition activity 84 was found to be most active with an IC 50 value of 2.2 µM. SAR showed that flouro-substitution was found to be beneficial for cytotoxicity as well as tubulin polymerase inhibition activity, as it enhances the lipophilicity. The same concept was found to be true for methoxy-substituted derivative, but it was found to be inactive against tubulin polymerase (Scheme 9) [197].
All the compounds (94) screened showed varied growth inhibitory effect against various cancer cell lines, so a clear SAR could not be established. 94a showed % inhibition of 53.60, 43.93 and 43.30 at 10 -5 M concentration against CNS, renal and prostate cancer cell line, respectively. Flow cytometry was carried out in prostate cancer cell line. Cell cycle results revealed that cell percentage increased in the S phase and G2-M phase from 27.87% to 31.76% and 13.12% to 14.75%, respectively as compared to untreated cells. While the cells population in the Go-G1 phase was reduced to 53.94% as compared to 59.01% in untreated cells. 94a also showed tolerated drug ability to varied physicochemical parameters (Scheme 10) [199].
In The 16-olefin (95) was synthetised from 13α-estrone 3-benzyl ether as reported in the literature [200]. The epoxidation of 16-olefin (95) was carried using magnesium monoperoxyphthalate in DCM:MeOH (1:1) at rt to give 16β,17β -(96) and 16α,17α-epoxide (99) in the ratio of 3:1 [200]. The epoxide ring was opened by reacting with NaN 3 in DMSO: Among all the derivatives (98 & 101), only 98a with 16α,17β configuration was found to be active against HeLa with an IC 50 value of 2.0 µM. C-16β, C-17 α configuration derivatives (101a-e) were found to possess anticancer activity comparable to reference cisplatin. The results of the growth inhibitory effects clearly displayed the signification of configuration at C-16 and C-17. The activity was also greatly affected by the substitution on the phenyl ring of the acetylenes. The parasubstituted triazolyl derivatives were found to be more potent as compared to meta-and unsubstituted triazolyl derivatives. More precisely, halogen substituted triazolyl derivatives were found to be less potent than their corresponding alkyl-substituted triazolyl derivatives. Cell cycle analysis of against HeLa showed a significant increase in the number of cells in the S and G2/ M phase as compared to the G1 phase after 24 h of exposure. While after an exposure of 48 h, the number of cells increased in the subdiploid (subG1) and G2/M phase as compared to the G1 phase. Which indicated the arrest of the cells in the G2/M phase of the cell cycle. Apoptotic study revealed an increase in the number of cells in the early apoptosis and secondary necrosis. Enzyme study revealed activated caspase-3 and caspase-9 thus inducing apoptosis via intrinsic pathway (Scheme 11) [201].   Among all the D-ring-substituted 16α-triazolylestrone derivatives (105 & 106), among all the 105 derivatives, 105c was found to be most active with an IC 50 value of 8.07 µM against MCF-7 as compared to cisplatin (IC 50 = 9.63 µM). 106a was found active against HeLa, MCF-7 and A431 with an IC 50 value of 5.08 µM, 7.88 µM and 6.77 µM, respectively. Further, flowcytometry analysis revealed that an increase in the hypodiploid population (subG1) and a cell cycle arrest in the G2/ M phase in a concentration-and exposure-dependent manner. Fluorescent microscopy analysis revealed disturbance in the cell membrane integrity and nuclear condensation. Caspase study showed increased activity of caspase-3 and caspase-9 without the activation of the caspase-8, thus causing apoptosis in the treated cell via intrinsic pathway. RT-PCR analysis revealed that the levels of cell cycle regulators (CDK1, cyclin B1/B2 and cdc25B) decreased while the ratio of Bax/Bcl-2 increased. Western blotting showed that the derivatives increased the level of phosphorylation of stathmin (Scheme 12) [203].
The synthesized triazolyl 13α-estrone-nucleoside conjugates (1 1 3) were evaluated for their antiproliferative property using MTT assay. 113b was found to be most potent possessing an IC 50 value of 9.0 µM, 9.0 µM and 10.4 µM against A2780, HeLa and MCF-7, respectively. However, 113a was also found to be active against A2780 and HeLa with an IC 50 value of 10.9 µM and 16.3 µM, respectively. However, the mechanism of action of the conjugates could not be explained but authors hypothesised that the lower activity of the conjugates as compared to the reference steroid, could be due to the polar property and steric effect in the conjugates due to presence of nucleosides. This hypothesis was also supported by the fact that the cytostatic activity of the protected 13α-estrone-nucleoside conjugates (113a-b) was found to be more profound than that of unprotected 13α-estrone-nucleoside conjugates (113c-d). This fact also highlighted the importance of size and polar characteristics of the group at C-3 triazolyl moiety of 13α-estrone. Based on the above fact the authors also suggested that passive transport of the unprotected 13α-estrone-nucleoside conjugates (113c-d) through the cell membrane could not take place which contributed to the lower concentration of the conjugates inside the cell, thus displaying weak cytostatic activity (Scheme 13) [206].
Among all the derivatives synthesized (69, 126, 128, 129 & 130), the reduced diastereomers (129 & 130) were found to be most active. For instance, 128a was found to be active against A431, MCF-7, A2780 and HeLa with an IC 50 value of 0.3 µM, 0.6 µM, 0.5 µM and 0.9 µM, respectively as compared to cisplatin. 130a was also found to be active against A431, MCF-7, A2780 and HeLa with an IC 50 value of 0.5 µM, 0.5 µM, 0.6 µM and 1.6 µM, respectively. In addition, 129a was also found active with an IC 50 value of 0.8 µM, 1.0 µM, 1.2 µM, and 2.5 µM against A431, MCF-7, A2780 and HeLa, respectively as compared to the standard cisplatin. Based on the results authors postulated that presence of 17-keto or 17-hydroxy functionality in the triazole derivatives is crucial for the activity. It was established that introduction of triazolyl moiety at C-3 position is beneficial for the activity as compared to the presence of ether functionality at C-3 (Scheme 15) [205].
Among all the derivatives (1 3 3) synthesized, 133b was found to be active against human breast cancer cell line (HBL-100) with an IC 50 value of 5.77 µM. Among other derivatives, 139a and 139e were found to be active with an IC 50 value of 5.54 and 5.16, 8.20 µM against A549 and HT-29, HCT-116, respectively. The varied activity of different derivatives revealed that not only the position of the functional group but the presence of different functional group also plays a critical role in the activity of the derivatives (Scheme 16) [208].
Among all the derivatives (1 5 7), 157d was found to be more active against HepG-2 with an IC 50 value of 9.10 µM as compared to 5-FU (IC 50 = 10.59 µM). In addition, 157d was also found active against HCT116 and MCF-7 with IC 50 value of 31.04 and 9.18 µM, respectively. 157c was found to be active against A549, HeLa, BEL7402, HCT116, and MCF-7 with an IC 50 value of 17.46,15.11,15.96,11.86 and 14.93 µM,respectively (Scheme 20). Flow cytometry was carried on 157d to study the cell cycle arrest in HepG-2. The study revealed that, a significant decrease in the cells in mitotic G2 phase (1.47% (control) to 11.14%  synthesized analogues, SAR was established which suggested that presence of electron-withdrawing group on the triazole ring significantly enhances the activity as compared to the presence of electrondonating group on the triazole ring. All the 2 and 3-substituted derivatives were found to be less potent than 4-substituted derivatives, except for 3-substituted flouro derivative. Surprisingly, 3,4-disubsituted Cl derivative was also found to be potent [217].
The synthesized conjugates (1 7 6) were evaluated for activity against MCF-7 and 4 T1, 176a was found to be most potent with an IC 50 value of 2.61 ± 0.70 µM against MCF-7. While, 176c was found active against both MCF-7 and 4 T1 cells with an IC 50 value of 5.71 ± 1.00 µM and 8.76 ± 1.29 µM, respectively (Scheme 24). 176a, 176b and 176c were also found to be non-toxic against HEK 293 with cell viability greater than 86%. Apoptotic study of 176c in MCF-7 revealed that, it induces higher apoptosis of 46.09% as compared to 176a which induces only 33.89% apoptosis. While in case of 4 T1 cells, 176a and 176c showed similar apoptotic potential. Pharmacokinetic study of 176c revealed mean residence time (MRT) of 8.47 h and a half-life of 5.63 h. SAR studies revealed that ester derivatives of triazolyl aryl ketones appended cholic acid derivatives were found to be more active as compared to their than their corresponding amide derivatives. The same was also found true in case of deoxycholic acid derivatives. The unsubstituted aryl ketone derivatives found to be more active as compared to the substituted derivatives. Presence of electron withdrawing group on the aryl ketone was found to beneficial for the activity as compared to the presence of electron donating group [224].

Conclusions
The present review concisely and systematically describes the updates on the synthesis and anticancer properties of the steroid-triazole conjugates. Its lays a very important emphasis on the synthesis and anticancer activity of different steroids-triazoles conjugates against Scheme 24. Synthesis and anticancer activity of cholic acid/deoxycholic acid appended triazolyl aryl ketones (1 7 6). D.S. Agarwal et al. various human cancer cell lines. In addition, the reviews also shed some light on the SAR and MOA of these conjugates which could lead to better understanding and lay the founding stone for the targeted based drug discovery. Thus, it is believed that this review would be very useful for the future development of therapeutics against various types of cancers in humans.

Author contributions
DS Agarwal organised the literature and drafted the manuscript. DS Agarwal, R Sakhuja, RM Beteck and LJ Legoabe carefully revised and finalized the manuscript for publication.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.