Estrone Sulfatase Inhibitors as New Anticancer Agents

Enzyme steroid sulfatase (STS) is considered as a promising therapeutic target for the treatment of hormone-dependent oncological diseases such as breast, endometrial, prostate cancers, and endometriosis. The discovery of potent and irreversible STS inhibitors stimulated huge efforts of preclinical and clinical work. Various STS inhibitors such as steroid sulfamate, steroid nonsulfamate, nonsteroidal sulfamate, and nonsteroidal nonsulfamate-based inhibitors have been developed. In the review known STS inhibitors from the point of view of their safety, side-effects and perspectives for clinical application are considered. Among STS inhibitors several dual (multitargeted) compounds have huge potential being nonestrogenic and acting in nanomolar levels on the targets. The dual aromatase-sulfatase inhibitors (DASI) approach has a great potential when a synergy between STS and aromatase inhibition is expected and, thus it could address acquired resistance mechanisms. Among STS inhibitors based on steroid skeleton 17 α -benzyl-, 17 β -arylsulfonamides, 17-diisopropylcarbamoyl-3-O-sulfamates exhibit the best properties, especially as dual anticancer potential drugs. The same modifications result in the increased activity against STS in 2-OMe-3-O-sulfamates as well as 2-OMe-3, 17 β -bissulfamates, which are also active against triple negative breast cancer. 8 α -Steroid estrogen analogs without estrogenic properties also possess high STS-inhibitory activity and block breast cancer cells growth with the activity comparable to tamoxifen.


Introduction
Breast cancer (BC) is the most common malignant tumor in women (12%) worldwide and is the second leading cause of cancer mortality after lung cancer (26%) [1].
Approximately 95-97% of tumors are estrogen-dependent in the early stages of their development [2, 3] and more than 70% express very high levels of estrogen receptor alpha (ERα) [4]. The fundamental difference of extragonadal estrogen synthesis is its autocrine nature-that an organ producing estrogens is a target organ at the same time. Thus, local concentration of estrogens in such organs may be markedly elevated. Peripheral estrogens formation is increased after menopause, and compensates estrogens deficiency in different organs and tissues [5]. Extragonadal estrogens' production may rise with the aging. Moreover, it was continually emphasized in the literature that the increased level of estrogens in the body is considered as a risk of the BC development [6,7].
Free hormones are formed from sulfates of estrogen and androgens under action of steroid sulfatase. At high concentrations, androgens compete for binding with ERs. The activation of ERα under the action of androstenediol and DHEA in BC cells has been detected. It is confirmed by the inhibition of cell growth in the presence of antiestrogens. The evaluation of E1S level during diagnostic of various oncological diseases (for example, prostate cancer) is of high importance [30].

Approaches for the manipulation of estrogen level in tumors 2.1 Endocrine therapy
Hormonal (endocrine) therapy is effectively used for the treatment of HDBC. Most types of BCs are estrogen-dependent, with approximately 55% in premenopausal women and 75% in postmenopausal women [31][32][33][34].
Selective estrogen receptor modulators (SERMs) or down-regulators (SERD), such as tamoxifen, raloxifene, ospemifine, and fulvestrant are compounds that are currently used in clinical practice to treat BC [9,35]. In breast tissues, SERMs effectively block the activation of ER(α) by endogenous ligands, preventing the transcription of genes mediated by estrogen response elements [36,37]. SERMs have tissue-specific effects on ERα that results in antagonist activity in breast and uterus tissues as well as agonist activity in bone. Although tamoxifen and raloxifene possess the desired SERM activity, they also increase the risk of venous thromboembolism [38] and exhibit toxicity [22]. Given that resistance (de novo or acquired resistance) is a major limiting factor in the use of endocrine therapy, additional endocrine therapies with other mechanisms of action are needed [39,40].

Inhibitors of enzymes responsible for the estrogen formation in tumors
The aromatase enzyme is responsible for the conversion of testosterone and androstenedione to E2 and E1, respectively. Thus, inhibition of the aromatase enzyme is one of the approaches for the development of new drugs to treat BC [41][42][43].
Nonsteroidal third-generation aromatase inhibitors (AIs), such as anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin), are often used for postmenopausal hormone-dependent BC treatment in clinical practice. Despite the success of AIs in the clinic, numerous BC patients still progress after AI therapy due to the development of resistance to AIs and side-effects such as osteoporosis caused by whole-body deprivation of estrogen [44,45]. Mechanisms of AI resistance include ligand-independent activation of the ER and signaling via other growth factor receptors; new insights about resistance are published recently [45].
The overall response rates for AIs (40-50%) suggest the presence of alternative sources of estrogens. The production of E1, DHEA and androstenediol is an important mechanism of resistance to AI treatment [46].
It was demonstrated that AIs used sequentially with tamoxifen had higher efficacy compared to tamoxifen alone, with an improvement in overall survival [47].
There are other factors involved in tumor growth [48]. The enzymes STS and 17β-HSD1 have been identified as essential parts in E2 production and subsequent promotion of cancer growth. Recently it was shown that 17β-HSD7 also plays a key role in increasing the E2/E1 ratio in BC tumors [49]. Very recently, some evaluations of the "sulfatase pathways" in tumor stroma have been carried out [50]. The STS is also responsible for the hydrolysis of DHEAS to DHEA, which is an immediate precursor of androstenediol, a potent estrogenic steroid [51], whose formation is not influenced by AIs. DHEAS stimulates proliferation of MCF-7 cells from BC, which could be blocked by an antiestrogen or STS inhibitor but by an AI. E1S and DHEAS are particularly abundant in blood circulation and could act as a reservoir of steroid precursors, specifically in BC [29,52]. The formation of DHEA through the STS pathway accounts for the production of 90% of the androgen androstenediol [52], which possesses estrogenic properties, that are 100-times weaker than estradiol [13,53]. Androstenediol is present at 100-fold higher concentrations than estradiol in the circulation, and may have estrogenic properties that are equal to estradiol [54]. Thus, inhibition of STS has the dual property of reducing local androstenediol biosynthesis [55,56].

Steroid sulfatase enzyme (STS)
The STS enzyme (EC 3.1.6.2, aryl sulfatase C, steryl-sulfatase) is widely distributed throughout the body and plays critical role in steroidogenesis [54]. Publications in recent years indicate the role of STS activity in gynecological diseases [57], mentioning diminished endometriosis in vivo under the action of STS inhibitors [58,59]. However, a phase II trial with STS inhibitors in endometrium cancer patients with advanced disease revealed no superior effects as compared to progestin megestrol acetate, and further studies are ongoing [60]. STS inhibitors are also useful for the treatment of ovary cancers and prostate cancer [16,61].
According to the in vitro studies, STS is the main enzyme responsible for estrogen production in hormone-dependent breast tumors, and has several hundred times higher activity in liver and normal/malignant breast tissues than aromatase [13, 53,62]. STS mRNA expression (74%) in ERα-positive breast tumors is an independent prognostic indicator in predicting relapse-free survival, with higher levels of expression being associated with a poor prognosis [63]. Like aromatase inhibitors, sulfatase inhibition can only be used in postmenopausal women. Probably, the greatest benefit with sulfatase inhibition is in those cases where DHEAS levels are high. To date, STS inhibitors are still in an early stage of development [53,64,65].
The human STS is a protein, integrated in microsomal membrane. Its threedimensional structure has been determined (PDBcode 1P49) [66]. However, knowledge about regulation of its expression as well as activity is limited. The topology of the active site of the steroid sulfatase and the arylsulfatases A and B is similar [66].
Most of the STS inhibitors discovered to date, act as irreversible active-sitedirected inhibitors. An aryl sulfamate group (ArOSO 2 NH 2 ) is considered as the pharmacophore for irreversible inhibition of the enzyme. One of the first time-, pH-, and concentration-dependent irreversible active-site directed-steroidal inhibitor is estrone-3-O-sulfamate (EMATE), which inhibit STS in MCF-7 cells from BC by 99% at 0.1 μM and has an IC 50 value of 65 pM (IC 50 = 80 nM in placental microsomes). EMATE was evaluated in clinical trials [67]. The highest effectiveness of EMATE has been demonstrated in rats (subcutaneous and oral administration). STS activity was also inhibited when EMATE was administered to humans in dose 0.5 mg/kg [68]. Despite the exceptional potency of the EMATE [67,68], it is not used in clinical practice to treat hormone-dependent BC because metabolic conversion of EMATE by STS releases estrone, which act via estrogen receptors, and can directly promote tumor growth [69]. Nevertheless, EMATE is now the prototypical inhibitor, and used as standard during evaluation of other potential STS inhibitors [19].

Mechanisms of inactivation of steroid sulfatase
Several research groups made attempts to establish the mechanism(s) of sulfatase inactivation. However, the precise mechanism of inhibition is still uncertain. In 2010, Spillane and Malaubier have established that the hydrolysis of EMATE occurs by two different mechanisms: an SN2 mechanism below pH 9.5 and E1cB mechanisms involving N-sulfonylamines at higher pHs [70]. Detailed presumable mechanisms have been discussed in recent reviews [71][72][73].
Based on the mechanisms, the result of the hydrolysis is free estrone. Moreover, under per os administration, the activity of EMATE is several times higher than the activity of estrone, due slow metabolism of EMATE in liver [68]. EMATE is not subjected to metabolic inactivation in red blood cells. Thus, consideration of hormonal activity and side-effects of steroids with free phenolic group is important in the modeling of sulfatase inhibitors for therapeutic use [54,74,75].
The knowledge of the crystal structure opens the rational drug design of molecules for the inactivation of steroid sulfatase.

Nonsteroidal STS inhibitors
Many investigations have been carried out to develop nonsteroidal STS inhibitors, because nonsteroidal drugs and their metabolites may have less undesirable effects.
A search of an orally active, nonestrogenic, nonsteroidal STS inhibitors among tricyclic compounds based around the coumarin core resulted in the discovery of Irosustat (667-coumate, STX64, BN83495) [77], which is the first-in-class irreversible time-and concentration-dependent STS inhibitor for the treatment of hormone-dependent BC in postmenopausal women that has been clinically evaluated in breast, endometrial, and prostate cancers [77] and there is potential for innovative dual-targeting approaches [78,79], with an IC 50  Efficiency of Irosustat has also been demonstrated in a phase II study in (ER+) endometrial cancer in women with advanced or recurrent disease [82]. The high bioavailability of Irosustat is explained by the prevention of degradation by sequestration inside red blood cells where it, similarly to EMATE, binds to (and inhibits) carbonic anhydrase II (IC 50 = 22 nM) [83]. The inactivation mechanism suggests that a sulfamate group is transferred to the gem-diol form of formylglycine 75 of steroid sulfatase due to a facile E1cB elimination of sulfamate anion to give the corresponding coumarin, which has a long half-life in blood [84]. However, the further development of Irosustat in monotherapy was stopped in the phase I/II clinical studies, because Irosustat does not possess superior properties to the current standard of care megesterol acetate, and its relative bioavailability decreases with increasing dose. The study of its combination with other hormonal therapies (for example, with the aromatase inhibitor anastrozole) is underway [85]. Metabolism of Irosustat has been investigated [86]. Irosustat also inhibits skin and liver STS [86]. Poirier with colleagues, among tetrahydroisoquinoline-N-substituted derivatives [90], found second-generation dual-action compounds that inhibit STS and act as a SERM. These compounds are devoid of estrogenic activity and toxicity. Their sulfamate derivatives possess high inhibitory activity toward STS (IC 50 of 3.9, 8.9, and 16.6 nM). Both phenolic and their sulfamate derivatives show no estrogenic activity and moderate antiestrogenic properties. All compounds significantly stimulate osteoblast-like Saos-2 cell proliferation, thus suggesting a SERM activity. The results of molecular docking experiments suggest that the most active compounds 6 and 7 bind in a competitive manner with E2 [91].

Dual aromatase/STS inhibitors
Another approach for the treatment of hormone-dependent BC is the development of DASIs, which may have an additive or synergistic antitumor effect. The potential advantages of a single chemical agent with the ability to interact with Estrone Sulfatase Inhibitors as New Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.85850 multiple biological targets were highlighted previously [92]. In the case of DASIs, this goal is being pursued by the introduction of the critical sulfamate unit in structures with known aromatase-inhibiting properties [93,94]. All DASIs are still in preclinical investigations [95].
One of the most potent dual inhibitor is compound 10 with 98 and 85% inhibition of STS and aromatase, respectively, at 10 μM [97]. A series of DASIs have been investigated [98,99]. Compound 11 (STX681, IC 50 = 0.82 nM for aromatase and IC 50 = 39 nM for STS) and similar analog 12 also exhibit an excellent profile against aromatase (IC 50 = 0.13 nM) and STS (IC 50 = 3.5 nM) and are not estrogenic [100]. Bissulfamate 13 at a single oral dose of 10 mg/kg inhibits aromatase and rat liver STS by 60 and 88%, respectively. The anastrazole inspired compound 12 is also potent dual inhibitor in vivo [101,102].
Potter with coauthors published the successful realization of the strategy when the core components of the two leading DASIs resulted in the hybrid structures that exhibit a very high level of dual inhibition against aromatase and STS in vitro (IC 50 = 0.015-0.75 nM). Most active compound is analog 17 (IC 50 for aromatase = 0.0002 μM, for STS = 0.0025 μM) [104].
The latest achievements in the field of nonsteroidal AIs are presented in recent reviews [105,106].

Steroid-based STS inhibitors with sulfamate group
The estrogenicity of EMATE and estradiol-3-O-sulfamate (26, E2MATE, PGL2001, J995) is the serious restriction for their development as anticancer agents. E2MATE effectively inhibits STS activity in endometrial tissue in vitro and in vivo (in doses 1.0 and 0.5 mg/kg) without affecting systemic E2 levels [58,59,115,116], and is introduced into Phase IIa of clinical trials [117]. E2MATE has been also clinically investigated as a pro-drug for hormone-replacement therapy and some limited clinical data are available. EMATE and E2MATE are bound to carbonic anhydrase (for EMATE IC 50 = 23 nM) within red blood cells, being dual inhibitors of carbonic anhydrases and STS [118].
The sulfamate 27 (NOMATE) was evaluated as an STS inhibitor. This steroid without the 17-carbonyl group possesses ablated estrogenicity as well as reduced STS activity compared to EMATE. NOMATE was shown to exhibit antitumor activity against a range of tumor cell lines [119].
D-ring lactone 28 has been developed as an orally available STS inhibitor [114]. The latest together with related lactam 29 were independently developed by Imperial College and University of Bath [120]. These compounds are potent STS inhibitors (98 and 91% inhibition of STS activity in MCF-7 cells at 0.1 μM, respectively; oral dose of 2 mg/kg/day) without estrogenic effects.
Simple modifications of the D-ring have led to dramatic variations in estrogenicity. Thus, the conversion of EMATE to the oxime results in a super-estrogen. From the other hand, D-ring heterocyclic derivatives exhibit reduced estrogenicity [121,122].
The replacement of ring D with N-substituted piperidinedione moiety results in the loss of estrogenic properties and greater STS inhibitory activity in vivo compared to STX64, as it was shown by the compounds STX213 (31) [123] and STX1938 (30) [124]. The STX1938 (30) and STX213 (31) inhibit STS with IC 50 of 1 nM and 35 pM correspondingly (90-and 18-fold more potent than EMATE, respectively) [125]. STX213 and STX1938 possess superior properties in comparison with STX64 in vivo models with once weekly oral dose 1 mg/kg [125,126].  [127].
Cyclic sulfamate 36 is an effective STS inhibitor (IC 50 = 9.3 nM) in vivo with dose regime 1 mg/mouse/day for 5 weeks [128]. The derivatives of oxathiazine 36 are claimed as estrogen-ablative agents; however, no data on their activity have been published [129]. Cyclic sulfamates with six-membered ring are time-dependent inactivators [130]. Acyclic mono-alkylated sulfamates are not time-dependent inactivators of sulfatases. Probably, imino compound 36 hydrolyzes to the orthoformyl sulfamate in situ [53]. The five-membered ring compounds such as 37 are not time-dependent inactivators of STS [131].

Dual 17β-HSD1/STS inhibitors
17βHSD converts E1 to E2 and DHEA to androstanediol [132]. Several inhibitors based on steroidal skeleton have been successfully developed [133,134]. Few dual inhibitors of 17β-HSD and STS for the treatment of steroid hormone-dependent diseases are patented [135]. The example of such inhibitors is represented by the compound 38.
A-ring-modified steroidal sulfamates, for example, series of 2-OMe-estradiol sulfamates and analogs have been investigated as nonestrogenic STS inhibitors [136,137]. 2-MeO-EMATE 39 demonstrates the excellent inhibitory properties in the relation to STS in vitro (IC 50 = 30 nM) and in vivo and is not estrogenic [138]. It strongly shows the antiproliferative effects toward BC cells by inducing apoptosis and cell cycle arresting in the G2/M phase [139].
2-Ethyl-EMATE 40 was identified as a promising multitargeted anticancer agent with strong ability to arrest the cell cycle, inhibit angiogenesis, as well as inhibit tumor growth in a xenograft model [140]. It was found that 2-ethylestrone (desulfamoylated compound 40) belongs to series of potent superoxide dismutase inhibitors [141].
It is known that 2-methoxyestradiol, a metabolite of E2, possesses antiangiogenic properties and prevents tumor growth through disrupting tubulin polymerization by binding at the colchicine-binding site [142,143]. 2-Methoxyestradiol is considered as the perspective compound for the treatment of endometriosis [144].
The anticancer effects of the 2-substituted sulfamate estrogen derivatives arise from disruption of tubulin polymerization, and the compounds also binding at the colchicine site [145]. 3,17β-Bissulfamates of estrogens are other representatives of multitargeted antitumor agents, acting as STS inhibitors with antiproliferative activity (IC 50 = 18-250 nM) [146]. Such bissulfamates compete with colchicine for tubulin binding and disrupt microtubules resulting into cell cycle arrest just by apoptosis in vitro and in vivo [147,148] and inhibit angiogenesis in vitro and in vivo [149]. The STS inhibitory activity of bissulfamate 41 is comparable to EMATE activity [150]. Bissulfamoylated derivatives with 2-MeO (42, STX140) and 2-Et (44, STX243) substituents in steroidal skeleton exhibit high STS inhibitory activity (IC 50 = 39 and 1000 nM, respectively) [151].
Coordination of the 17-sulfamate residue to the zinc in active site of the complex of STX140 with human carbonic anhydrase II is revealed [155].
STX140 depolarizes mitochondrial bioenergetics, activates caspase 3/7 causing apoptosis through the intrinsic mitochondrial pathway, and downregulates DOI: http://dx.doi.org/10.5772/intechopen.85850 the expression of caspase inhibitors [156]. The activity of such compounds is also explained by their ability to disrupt the tubulin-microtubule equilibrium in cells as being central to their antitumor activity. STX140 and STX243 bind with the colchicines binding site of tubulin. 2-( 11 C)Methoxy-3,17β-OO-bis(sulfamoyl)estradiol has been proposed as a new potential PET agent for imaging of steroid sulfatase in cancers [157].
Among a series of C17-ketone and amide-modified estrone-derived sulfamates, compound KW-2581 (49, 17-diisopropylcarbamoyl-1,3,5(10),16-estratetraen-3-ylsulfamate) is the most promising, not estrogenic, orally active anticancer agent for the treatment of hormone-dependent BC and endometrial cancer [160]. KW-2581 as STS inhibitor is five times more potent compared to STX-64 (IC 50 = 4 nM) [161]. It was also demonstrated that the compound inhibits the ability of androstanediol-S to stimulate the in vivo growth of MCF-7 cells from BC overexpressing STS. However, KW-2581 is practically insoluble in water (approx. 0.1 ng/mL). The attempts to increase its oral bioavailability showed that the milled powder exhibited poorer properties than the intact sample, including a lower level of crystallinity, higher water content, and increased decomposition rate [162].
Compound EM-1913 (50) is nonestrogenic steroidal STS inhibitor with IC 50 = 0.05 nM [165], which also inhibits dehydroepiandosterone sulfate action in androgen-sensitive tissues, being therefore considered as a potential drug for the treatment of prostate cancer [166].
17α-Benzyl substituent yields reversible STS inhibitors in the absence of a sulfamate group, and incorporation of an aryl sulfamate onto the A-ring results in a potent time-dependent irreversible inhibitor. The IC 50 of the tert-butylbenzyl derivative 51 is low (8.3 nM); however, steroid 51 is estrogenic. A-ring substitution leads to the reduced estrogenicity. 2-Methoxyderivative 52 has an IC 50 = 0.04 nM. The compound without the tert-butyl group is nonestrogenic and effective STS inhibitor in vivo [167].

Dual STS/SERM inhibitors
Maximum estrogen blockade in the treatment of (ER+) BC may be achieved using dual ERα antagonists and STS inhibitors, which might cause osteoporosis as a side effect [169]. In this case, the compound possessing SERM properties is needed.
Desulfamoylation of SR16157 (54) results in SR16137 (55), which is a tissueselective antiestrogen with beneficial effects on bone and cardiovascular system [171]. SR16157 is 10 times more potent as a growth inhibitor of MCF-7 cells than either the antiestrogens tamoxifen or SR1613. Additionally, SR16137 has a 10-fold higher affinity for ERα as compared to tamoxifen. SR16157 was shown to possess minimal genotoxic activity [172]. SR16157 has been recommended in initial phase I of clinical trials with the starting dose of 1.3 mg/kg/day administered as a single dose in humans.
We demonstrated that 8-alpha-analogs of steroid estrogens effectively inhibit the growth of BC cells, including triple negative BC [173,174].

Conclusions
Manipulation of hormone biosynthesis in tumors by enzymes inhibitors is a very attractive approach for the treatment of hormone-dependent tumors such as breast, prostate cancer, and endometriosis.
The importance of STS in human body has been underlined by many investigations. Thus, STS-catalyzed hydrolysis of pregnolone-3-sulfate and dehydroepiandro-sterone-3-sulfate in the brain regulates neurosteroid synthesis and influences memory. STS inhibition for the potentiation of memory in sufferers of neurological diseases such as Alzheimer's disease and dementia has been postulated [175]. The role of STS inhibitors as agents to reveal beneficial endogenous glucocorticoid effects was also claimed. The use of STS inhibitors in combination with the immunosuppressive ascomycin for the treatment of acne, seborrhoea, androgenetic alopecia, and hirsutism is patented. The administration of an estrogen (including norgestimate and norelgestromin), in combination with a progestogen in hormonereplacement therapy act by inhibiting STS, thus reduce estrogen production and protect the endometrium and breast from hormone-dependent cancers [176]. STS inhibitors prevent ovarian cycle disturbance, prolonged unopposed secretion of estrogens, and ovarian follicular cyst formation in premenopausal women, as well as prevent premature uterine contractions, particularly for preterm labor [177].
The importance of STS inhibition in endometriosis, prostate cancer, as well as latest discussions about mechanism of inhibition is well considered in the review of Prof. Potter [178]. The significance of steroid sulfatase and sulfotransferases in gynecological diseases are summarized in the review [57]. As far as estrogenic compounds may stimulate tumor cells growth, the main requirement for STS inhibitors and their metabolites is the absence of estrogenicity. Among nonsteroidal STS inhibitors only one nonestrogenic compound-Irosustate was evaluated in clinical trials with excellent properties, however its further development was stopped. Currently, the action of Irosustate in the combination with AIs is investigated.
The discovery of dual (multitargeted) inhibitors is the most promising nowadays. For example, several DASIs based on anastrazole, letrozole, and vorozole templates inhibit both STS and aromatase in nanomolar concentrations, being nonestrogenic; and have a chance to be introduced in clinical trials.
Among STS inhibitors based on steroid skeleton 17α-benzyl-derivatives, 17β-arylsulfonamides, and 17-diisopro-pylcarbomoyl-3-O-sulfamates exhibit the best properties, especially as multitargeted (dual) anticancer potential drugs. The same modifications result in the increased activity against STS in the case of 2-OMe-3-O-sulfamates as well as 2-OMe-3,17β-bissulfamates. The latter also possess activity against most aggressive form-triple negative BC.
Additionally, 8α-steroid estrogen analogs without estrogenic properties possess high STS activity and block BC cells growth with the activity comparable to standard of care for BC treatment tamoxifen.
The authors confirm that this article content has no conflicts of interest.