Antisulfatase, Osteogenic, and Anticancer Activities of Steroid

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Antisulfatase, osteogenic and anticancer activities of steroid sulfatase inhibitor EO-33 in mice Donald Poirier, Jenny Roy, René Maltais, and Diana Ayan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00382 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Medicinal Chemistry

Journal of Medicinal Chemistry (revised-4)

Antisulfatase, osteogenic and anticancer activities of steroid sulfatase inhibitor EO-33 in mice

Donald Poirier,†,‡,*, Jenny Roy,† René Maltais,† and Diana Ayan†

†

Laboratory of Medicinal Chemistry, Endocrinology and Nephrology Unit, CHU de Québec –

Research Center, Québec, QC, G1V 4G2, Canada ‡

Department of Molecular Medicine, Faculty of Medicine, Université Laval, Québec, QC, G1V

0A6, Canada

(*) Corresponding Author: Donald Poirier Laboratory of Medicinal Chemistry CHU de Québec – Research Center (CHUL, T4-42) 2705 Laurier Boulevard Québec, QC, G1V 4G2, Canada Tel.: 1-418-654-2296; Fax: 1-418-654-2298; E-mail: [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT: Steroid sulfatase (STS) is a key enzyme involved in the biosynthesis of estrogens from inactive sulfated steroids. After we reported EO-33 as a potent in vitro STS inhibitor without undesirable estrogenic activity and with osteogenic properties, we are now interested in validating EO-33’s in vivo potential to inhibit STS, to prevent bone deterioration, and to reduce estrogen-dependent tumor growth. A scale-up synthesis was first elaborated to prepare the multi-gram quantity of EO-33 needed to perform in vivo studies. EO-33 blocked the uterine weight stimulated by estrone sulfate in ovariectomized mice by 69% and the STS activity in liver by 81%. It also produced a selective estrogen receptor modulator effect as assessed by measuring the tibia weight and calcium content. Using human breast cancer (MCF-7 xenograft) model in nude mice, EO-33 blocked 90% of tumor growth induced by estradiol sulfate and no toxic effect was observed by assessing the body and liver weights.

KEYWORDS: Steroid sulfatase, enzyme, inhibitor, selective estrogen receptor modulator, chemical synthesis, cancer, osteoporosis


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INTRODUCTION Steroid sulfatase (STS) represents a key enzyme to target estrogen biosynthesis.1-8 The sulfatases are a group of enzymes that catalyze the conversion of sulfate compounds (R-OSO3H) in corresponding unconjugated compounds (R-OH).9 Nine members of the large family of sulfatases have been isolated from humans, and their corresponding gene identified. Of this group of sulfatases, STS is the only one that catalyzes the hydrolysis of sulfated 3hydroxysteroids (estrone sulfate (E1S), estradiol sulfate (E2S), dehydroepiandrosterone sulfate (DHEAS), and pregnenolone sulfate (PREGS)), inactive on their respective receptor, into corresponding hydroxylated steroids (E1, E2, DHEA, and PREG), assets and/or available for steroidogenesis (Fig. 1).9 There are potentially several advantages in using an inhibitor of STS, since it would prevent the transformation of inactive sulfated steroids into estrogenic hormones in peripheral estrogen-dependent tissues, such as breast, uterus and endometriosis lesions.

O

O

O HO S O O

OH

STS HO E1S

1) Sulfamate compound ( EO-33 ) as STS inhibitor

ER

HO E1

2) Sulfamate compound (EO-33) as ER ligand

Estrogenic (osteogenic) effect in bone

Estrogenic effect in breast cancer and bone tisues

E2 3) Sulfamate compound (EO-33) as ER antagonist

Antiestrogenic effect in breast cancer tissues

Figure 1. Sulfamate derivative EO-33 as a modulator of three different actions: 1) steroid sulfatase (STS) inhibition, 2) estrogen receptor (ER) activation in bone and 3) ER antagonistic effect in breast cancer tissue.



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In the estrogenic pathway, estrogen receptors (ERs) are members of a nuclear receptor superfamily of ligand activated transcription factors.10 Currently, two different ERs (ERα and ERβ) have been described, and they have shown to be critically and differentially involved in the regulation of the normal function of reproductive tissues.11-14 Although the role of ERβ in breast cancer is still being explored, the role of ERα is already well known. Largely expressed (50– 80%) in breast tumors, its presence is the main indicator for therapy using an antiestrogen (AE). Among the family of AE, it is important to mention that selective estrogen receptor modulators (SERMs), contrary to pure AE, interact with ER as agonists or antagonists, depending on the target tissue.15, 16 Typically, an agonist action is wanted in bones, while an antagonist action is targeted in estrogen-dependent tumors. Many efforts have been made to design potent STS inhibitors,4-6 but it is only recently that a molecule (Irosustat) has reached the stage of clinical studies.17,

18

Currently known STS

inhibitors block the estrogen biosynthesis in women, and have demonstrated efficacy in models of breast cancer tumors. However, estrogen blockade resulting from the use of a steroidogenesis inhibitor has an impact on bone calcification, and may promote osteoporosis in treated women, as observed with aromatase inhibitors,19-22 a family of drugs currently used in clinic.23,

24

In

addition to a combination of an AE with an inhibitor of STS or a molecule generating an AE after inhibiting STS,25 obtaining an inhibitor of STS inhibitor with an intrinsic SERM-like behavior (namely ISTS-SERM) would help achieve maximal estrogenic blockade, while avoiding the disadvantages of estrogen deprivation.

The development of ISTS-SERM compounds was initiated in our research group years ago. Using a 1,2,3,4-tetrahydroisoquinolin-7-ol core, we first synthesized libraries of sulfamates and 4 ACS Paragon Plus Environment

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corresponding phenols by parallel solid-phase chemistry. Among the molecules generated, sulfamate (-SO2NH2) derivatives showed interesting inhibitory activity on the STS.26 We then tested the corresponding phenolic derivatives on Saos-2 cells (osteoblasts) to determine their ability to stimulate cell proliferation (in vitro SERM effect). Encouraging results have been obtained, since certain phenols had a proliferative effect that was superior to that of raloxifene, a SERM used in clinic. Unfortunately, our first generation of ISTS-SERM was found to be an estrogenic compound on estrogen-sensitive breast cancer T-47D cells.27 A second round of optimization was undertaken to solve the problem of unwanted estrogenicity on cancer cells, and 3 phenolic compounds, devoid of estrogenic activity and toxicity emerged from this study.28 Sulfamate analogues were then prepared and showed a strong inhibition of STS in transfected HEK-293 cells (IC50 of 3.9, 8.9, and 16.6 nM). Tested at several concentrations on T-47D cells, these phenol and sulfamate compounds showed no undesirable proliferative (estrogenic) activity. To assess their potential as SERMs, the compounds were then tested on Saos-2 bone cells and have demonstrated their ability to significantly stimulate proliferation at 1 μM, and at a level slightly higher than that of the SERM raloxifene.28 These compounds also significantly stimulated the activity of alkaline phosphatase, another marker of the SERM effect in vitro. From these molecules all tested on in vitro assays,26-28 the sulfamate derivative EO-33 (Fig. 2) was the most interesting dual action molecule (inhibiting STS and having a SERM effect); it was then selected for crucial additional in vivo studies in mice.



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RESULTS AND DISCUSSION Chemical synthesis of EO-33 The sulfamate derivative EO-33 was initially obtained in small quantity (a few mg) by solidphase synthesis using an arylsulfamate linker.28 This approach was effective in generating a large number of analogue molecules to rapidly establish structure-activity relationships (SAR), but it was not appropriate to prepare gram quantities of EO-33 for in vivo studies. From commercially available 1,2,3,4-tetrahydro-7-isoquinolinol (1), and by classic solution-phase synthesis, we thus developed a convergent six-step synthesis of EO-33 (Fig. 2). In the first part, we selectively protected the secondary amine of 1 by a fluorenylmethyloxycarbonyl (Fmoc) group to be able to selectively sulfamoylate the phenolic group of 2. The resulting sulfamate 3 was reacted with triphenylmethyl (trityl) chloride and the resulting trityl derivative submitted to Fmoc deprotection using tetrabutylammonium fluoride (TBAF) to afford the free amine 4. In the second part, 2-methylthiophene-5-carboxaldehyde (5) and furfurylamine (6) were condensed in the presence of molecular sieve as dehydrating agent, to provide the secondary amine 7, which reacted with 4-formylbenzoic acid (8) under the reductive amination conditions to provide the tertiary amine 9. In the last part, the secondary amine 4 was coupled with the carboxylic acid moiety 9 to give the amide derivative 10. After a deprotection of the trityl group using hexafluoroisopropanol (HFIP) 30% in dichloromethane (DCM), EO-33 was obtained in high level of purity (97.3% by HPLC) and a satisfactory overall yield of 30% for this short and convergent synthesis. In addition to infrared spectroscopy and mass spectrometry analyses, 1H and

13C

nuclear magnetic resonance (NMR) spectra, including 2D experiments (Supporting

Information), confirmed the right structure of EO-33.


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b

a NH

HO

O H 2N S O O

NFmoc

HO

1

2

NFmoc

3

c, d

Tr

S

N

O H N S O O

g

N O

10

O

O TrHN S O O

S

N HO O

h

9

NH

4

O

O

O

6 5

7

8 9

16

15

O N 10 2 H 2N S O 4 11 1 3 12 O O

EO-33

14

17

13

18

N

f 19

S

22 24 20 21 25 26

O

HOOC

8

23

+ HN

S

e

27 28

S

H

H

O

7

5

H 2N O

6

Figure 2. Chemical synthesis of EO-33, an inhibitor of STS with SERM effect. Reagents and conditions: (a) Fmoc-O-succinimide, NaHCO3, H2O, rt; (b) NH2SOCl, dimethylacetamide (DMA), 0 °C to rt; (c) Trityl chloride, diisopropylethylamine (DIPEA), DCM/DMA, 0 °C to rt; (d) tetrabutylammonium fluoride in dimethylformamide (DMF) (0.05 M), rt; e) 1. Molecular sieve 4 Å, ethanol, rt; 2. NaBH4, ethanol, rt; (f) NaBH(OAc)3, CHCl3, rt; (g) Carboxylic acid 8, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, DIPEA, DMF, rt; (h) HFIP in DCM (30%), rt.

Plasma concentration of EO-33 in mice The sulfamate derivative EO-33 is a hydrophobic molecule (log P = 4.02) whose predictive solubility is relatively low (0.005 mg/mL), which limits the choice of usable vehicle for its 7 ACS Paragon Plus Environment


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administration in mice. Based on a pharmacokinetic study that tested 11 vehicles to administer a hydrophobic steroid derivative,29 we selected and tested 2 mixtures (aqueous 0.4% methyl cellulose (MC):ethanol (EtOH) (92:8) and propylene glycol (PG):dimethylsulfoxide (DMSO) (92:8)) to inject a single dose of EO-33 at 30 mg/kg, subcutaneously (SC) (Table 1). After 7 h, the plasma concentration of EO-33 was higher in MC:EtOH (530 ng/mL) compared to that in PG:DMSO (381 ng/mL). Using the first vehicle, we then performed a preliminary pharmacokinetic study by measuring the plasma concentration of EO-33 at 5 time-points (1, 3, 7, 12, and 24 h) to then estimate the area under curve (AUC)0-24h values for the three doses (2, 10, and 30 mg/kg) of EO-33 injected SC. Except for the shortest time (1 h), a response dose was observed for 3, 7, 12, and 24 h, which is reflected by AUC0-24h values of 1,134 and 5,205 and 13,482 ng.h/mL at doses of 2, 10, and 30 mg/kg, respectively. Table 1. Plasma concentration (PC) of EO-33 following a single SC injection of EO-33 in mice a Mode

Vehicle

Dose

PC at 1 h

PC at 3 h

PC at 7 h

PC at 12 h

PC at 24 h

AUC0-24h

(mg/kg)

(ng/mL)

(ng/mL)

(ng/mL)

(ng/mL)

(ng/mL)

(ng.h/mL)

SC

MC:EtOH

2

681±54

33±2

1±1

1±1

0±0

1134

SC

MC:EtOH

10

2600±158

398±7

15±1

4±1

2±1

5205

SC

MC:EtOH

30

2603±67

2190±87

530±35

64±3

13±1

13482 [269]b

SC

PG:DMSO

30

---

---

381±43

---

---

---

PO

SO:DMSO

30

---

---

49±4

---

---

---

PO

MC:EtOH

30

308±25

19±1

75±2

3±1

0±0

878 [10]b

a

The concentration of EO-33 was determined by liquid chromatography analysis (±SD). The

area under a curve (AUC0-24h) was estimated from the curve (Fig. 3) of the EO-33 concentration


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reported at different time-points (1, 3, 7, 12, and 24 h). b AUC0-24h value for the phenol derivative EO-29 resulting from a single SC injection of sulfamate derivative EO-33.

A gradual disappearance of EO-33 in the blood is observed after the first time-point measured at 1 h. Consequently, a significant concentration of EO-33 was measured at 12 h and 24 h, but only for the highest dose of 30 mg/kg in MC:EtOH given SC (Fig. 3A). When tested orally (by gavage; PO), MC:EtOH was a better vehicle than the mixture of sunflower oil (SO):DMSO in a proportion of 92:8, taking into account the plasma concentration of EO-33 measured at 7 h (75 and 49 ng/mL, respectively). As expected, for the same vehicle (MC:EtOH) and the same dose (30 mg/kg), plasma concentrations of EO-33 were higher for the SC mode than for the PO mode, and results are well represented by the AUC0-24h values (13482 and 878 ng.h/mL, respectively).

Figure 3. Plasma concentration of EO-33 (sulfamate derivative) or EO-29 (phenol derivative) in female mice. A) Plasma concentration of EO-33 following a single SC or PO injection of EO-33 at different doses and in different vehicles. B) Plasma concentration of EO-33 and EO-29



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following a single SC or PO injection of EO-33 at a dose of 30 mg/kg and in MC:EtOH (92:8). Two–three mice per group for each time point. When the error bars (± SEM) are not shown, they are smaller than the symbols.

It is well known that arylsulfamate derivatives are hydrolyzed by STS, thus releasing the corresponding phenolic derivatives and a reactive species inactivating the enzyme.30 In addition to this enzymatic process, a pure chemical hydrolysis is also possible,31 but its importance in a physiological system was not well established. After we identified MC:EtOH (92:8) as the vehicle for in vivo studies in mice, we measured the quantity of both EO-33 (sulfamate derivative; H2NSO3-) and EO-29 (phenol derivative; HO-) following two different modes of administration (SC and PO). As reported in Figure 3B, a very small quantity of phenol was detected in both the SC and PO modes. For the SC and PO modes, phenol EO-29 represents 2.0% and 1.1%, respectively, of the sulfamate EO-33 as estimated using the AUC0-24h values (Table 1). If we suppose a similar stability of EO-33 and EO-29, the results suggest a very low level of the sulfamate hydrolysis and a very good chemical stability of the sulfamate group. Even considering that a phenol derivative can be metabolized by phase-II reactions (glucuronidation, sulfatation, acetylation) with glucuronosyltransferases, which is not the case for a sulfamate derivative, the importance of the phenol EO-29 in blood appears to be very low and the sulfamate EO-33 remains by far the most present in the blood.


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Estrogenic/antiestrogenic activities in mice Estrogenic and antiestrogenic activities of EO-33 were investigated using an ovariectomized (OVX) mouse model,32 by measuring the weight of estrogen-sensitive uterus after 9 days of treatment (Fig. 4). At the end of the protocol, where mice received the vehicle (MC:EtOH (92:8)) only, the mean uterus weight was 51.9 mg and 14.5 mg for intact and OVX mice, respectively. The estrogen deprivation caused by the removal of the ovaries was however reversed by the use of E1S (2 μg/mouse, BID (twice a day), SC); a treatment that increased the mean uterine weight to 106 mg. E1S is not estrogenic as such, but becomes so after its transformation by STS into E1, which will next be reduced by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) to E2,8, 33 the most potent natural estrogen. Compared to the control (CTL) group, an SC injection of EO33 (30 mg/kg, ID (once a day)) did not stimulate the mean uterine weight, suggesting that this inhibitor is not estrogenic (Fig. 4A), as observed by the in vitro cell proliferation results with ER+ cells.28 Using the same mouse model, we also observed that EO-33 (30 mg/kg, ID) was not able to reverse the uterotrophic (estrogenic) effect produced by E1 (0.06 µg/mouse, BID, SC) (Fig. 4B). This result therefore demonstrates that EO-33 does not act as an AE, i.e. an ER antagonist.



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Figure 4. Assessment of estrogenic (A) and antiestrogenic (B) activities of steroid sulfatase (STS) inhibitor EO-33 on the estrogen-sensitive uterine weight of ovariectomized (OVX) mice. Estrone sulfate (E1S) is hydrolyzed by STS into estrone (E1), which is reduced by 17β-HSD1 in potent estrogen estradiol (E2). Five mice per group. Significantly different (** P ˂ 0.01) from OVX/E1S and significantly different (## P ˂ 0.01) from OVX/CTL. INT: intact; CTL: control.

Anti-STS activity in mice After demonstrating that EO-33 did not act as an ER agonist or antagonist in the estrogensensitive mouse uterus, we tested, at two doses, its ability to reverse the uterotrophic effect produced by E1S (Fig. 5A). Since a preliminary protocol with EO-33 (30 mg/kg) injected SC once a day (ID) partly blocked (42%) the stimulation of uterus weight produced by E1S, we used the same dose of EO-33 but injected SC twice daily (BID). EO-33 inhibited the E1S-stimulated mean uterus weight in a dose-dependent manner (49 and 69% at 15 and 30 mg/kg, respectively), demonstrating its ability to inhibit the STS responsible for the activation (hydrolysis) of E1S into E1. At 30 mg/kg, the inhibition of uterine weight (69%) was identical to that obtained using EM1913 (71%), a potent steroidal inhibitor of STS without a SERM effect.34, 35


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Figure 5. Anti-STS activity of inhibitors EO-33 and EM-1913 in ovariectomized (OVX) mice treated 9 days with estrone sulfate (E1S). This later is hydrolyzed by STS into estrone (E1), which is reduced by 17β-HSD1 into potent estrogen estradiol (E2). Five mice per group. A) Effect of EO-33 on estrogen-sensitive mean uterine weight. Significantly different from OVX/CTL (** P ˂ 0.01; * P < 0.05) and from OVX/CTL/E1S (## P < 0.01). B) Effect on the transformation of [3H]-E1S into [3H]-E1 by liver proteins (100 and 500 µg). Significantly different from OVX/E1S (** P ˂ 0.01; * P < 0.05). CTL: control.

The incomplete inhibition of E1S-induced uterine weight stimulation by EO-33 or EM-1913 is explained by the presence of E1 in the E1S batch used for the experiment. Under these conditions, it was therefore not possible to obtain 100% inhibition of the E1S-induced uterine weight stimulation by the STS inhibitor used, as it had been observed previously in another experiment with EM-1913.32 In fact, even considering total inhibition of E1S transformation into E1 by the STS inhibitor used, the E1 contaminating the batch of E1S will be transformed to the potent estrogen estradiol, which will stimulate the estrogen-sensitive uterine weight. However,



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using the liver of mice treated for 9 days with inhibitors, as a source of STS activity, we demonstrated that EO-33 and EM-1913 effectively blocked the transformation of [3H]-E1S into [3H]-E1 by the STS present in 100 and 500 μg of liver protein (Fig. 5B). Thus, the steroidal inhibitor EM-1913 completely blocked (100%) the STS activity, while the non-steroidal inhibitor EO-33 was slightly less effective (71 and 81% inhibition).

Osteogenic activity in mice One of the side effects to the hormonal deprivation-related cancer treatment strategies is bone tissue deterioration (osteoporosis), so the use of an STS inhibitor having a SERM effect by itself would be desirable. In fact, the SERM raloxifene has been approved for the prevention and treatment of osteoporosis in postmenopausal women.36 Based on positive results obtained with EO-33 in Saos-2 cells, a relevant in vitro model used for bone-related research,37 we verify the SERM effect in vivo with OVX young female mice as an animal model. After 70 days of treatment, two parameters related to osteoporosis disease, the tibia weight and the tibia calcium content, were measured to determine the ability of EO-33 to preserve the bone OVX-induced changes. In comparison to sham mice, OVX mice showed a reduction of tibia weight, from 2.44 to 1.93 mg/g of body weight (Fig. 6A) and of the calcium content from 258 to 195 mg/g of tibia weight (Fig. 6B). As expected, the use of E2 at a dose of 5 µg/kg, or the SERM raloxifene at a dose of 1 mg/kg allowed a full recovery (100%) of the tibia weight and calcium content losses resulting from the ablation of the ovaries. EO-33 treatment (30 mg/kg) also produced a full recovery of tibia weight, but only a partial recovery (71%) of calcium content. Estrogen-sensitive uteri were also recovered during necropsy and, as expected, their weights were reduced by OVX and


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stimulated by E2 treatment at a level higher than the sham group. Interestingly, EO-33 treatment produced no estrogenic effect on uterus weight, but a weak stimulation was observed for the group treated with raloxifene (Supporting Information, Fig. S1). Livers were also recovered (Fig. S2) and, except for the OVX group receiving only the vehicle (41.7 mg/g of body weight), their weights did not differ (48.2-50.5 mg/g). In the literature,38,

39

it has been mentioned that

estrogens allow a better vascularization of the liver, which would explain its higher weight for the sham group and the OVX groups treated with E2, raloxifene and EO-33 (due to estrogenic or SERM effects). Finally, the mouse body weights of all groups did not differ significantly, suggesting that EO-33 did not induce apparent toxic effects, after 70 days of treatment (Fig. S3).

Figure 6. Effect of different treatments on two osteoporosis parameters measured on the tibias of ovariectomized (OVX) mice. A) Tibia weight and B) tibia calcium content. Eight mice per group. Significantly different (** P ˂ 0.01) from OVX. Significantly different (# P ˂ 0.01) from Sham. E2: estradiol; Ral: raloxifene.



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Anticancer activity in mice (MCF-7 xenograft) Xenografts of human cancer cells are commonly used as tumor models to determine the in vivo activity of new anti-cancer agents prior to clinical development.40-42 We inoculated human estrogen-sensitive MCF-7 breast cancer cells into female OVX immunodeficient athymic (nude) mice and then injected, three times a week, a dose of E2S (50 μg/mouse/SC) to promote the formation of tumors. After 4 weeks, the tumor rate obtained was 65%, which made it possible to form 3 groups and to start the treatments. After 14 days of treatment, there was a significant difference between the EO-33/E2S-treated group and the group receiving only E2S, and this difference increased further (Fig. 7). At the end of the protocol (28 days of treatment), the size of the tumors in the E2S-stimulated group had increased to 171% of its initial size, while that of the E2S/EO-33 treated group had decreased to 72% of its initial size. The size of the tumors in this group was then not significantly different from that of the OVX group (72% vs 64%, p ˂ 0.01), which clearly demonstrates the ability of the inhibitor EO-33 to block the STS activity present in tumors. In fact, the increase in tumor size results from the stimulation of the proliferation of estrogen-sensitive MCF-7 cells produced from E2S after its transformation by STS into E2.


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Figure 7. Effect of a 28-day treatment with STS inhibitor EO-33 on estradiol sulfate (E2S)induced growth of human MCF-7 breast tumors in OVX nude mice. Tumor sizes are expressed as the percentage of initial tumor area (day 1 of treatment = 100%). EO-33 (30 mg/kg/ID) was SC injected 6 days per week and E2S (50 µg/mouse/ID) was SC injected 3 days per week. Data are expressed as means ± SEM (n = 10–12 tumors and 6-8 mice/group). Significantly different from OVX/E2S (** P < 0.01). Tumor size of OVX and OVX/E2S/EO-33 groups are not significantly different.

No apparent toxic effect was observed by visual analysis of mouse livers and kidneys after necropsy. In addition, there was no significant difference between mean liver weight or body weight of the three group of mice (Supporting Information, Fig. S4 and S5). However, we observed a whitish deposit at the injection site, and the analysis of this deposit by thin-layer chromatography showed the presence of inhibitor EO-33. This observation suggests that MC:EtOH (92:8) is not an optimal injection vehicle for EO-33.

CONCLUSIONS The nonsteroidal STS inhibitor EO-33 was synthesized efficiently in six steps from 1,2,3,4tetrahydroisoquinoline-7-ol in appropriate quantity for in vivo assays. When EO-33 (30 mg/kg) was administered SC in mice using a mixture of MC:EtOH (92:8), its plasma concentration reached 2600 ng/mL at 1 h and generated an AUC0-24h value of 13480 ng.h/mL. At this dose of 30 mg/kg, EO-33 did not induce estrogenic or antiestrogenic effects on the uterus of OVX mice, 17 ACS Paragon Plus Environment


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but it blocked the uterotrophic effect induced by E1S, suggesting its potency to inhibit the STS conversion of E1S into E1. The same inhibitory efficiency was also observed in the liver of mice treated with EO-33. Tested in OVX mice, EO-33 produced a SERM effect as assessed by two in vivo markers, tibia weight and tibia calcium content. In a xenograft tumor model using human breast cancer MCF-7 cells, EO-33 blocked 90% of tumor growth induced by E2S, a precursor of potent estrogen E2. By assessing body weight, liver weight, and liver appearance, no toxic effect was observed in mice. Although the pharmacological profile of EO-33 could be improved, this study reveals it to be a promising nonsteroidal inhibitor of STS having a SERM activity, which opens the door to a more selective approach for treatment of estrogen-related diseases.

EXPERIMENTAL SECTION Chemical synthesis General information. 1,2,3,4-tetrahydroisoquinolin-7-ol and

hexafluoroisopropanol (HFIP)

were purchased from Apollo Scientific (Manchester, UK), Fmoc-O-succinimide from Matrix Innovation (Québec, QC, Canada), tetrabutylammonium fluoride (TBAF) from TCI America (Portland, OR, USA), 2-methylthiophene-5-carboxaldehyde from Acros Organic (Belgium Town, WI, USA), furfurylamine, sodium triacetoxyborohydride and 4-formyl benzoic acid from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol1-yl)uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) from Alfa Aesar (Tewksbury, MA, USA), and sulfamoyl chloride from Matrix Scientific (Columbia, SC, USA). Solvents were obtained from Fisher Scientific (Montréal, QC, Canada) and VWR (Ville


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Mont-Royal, QC, Canada). Flash chromatography was performed on Silicycle 60 230-400-mesh silica gel (Québec, QC, Canada). Thin-layer chromatography was performed on Whatman 0.25mm silica gel 60 F254 plates (Fisher Scientific, Nepean, ON, Canada) and compounds were visualized by exposure to UV light (254 nm) or a solution of ammonium molybdate/sulphuric acid/ethanol (plus heating). Infrared (IR) spectra were recorded on a MB 3000 ABB FTIR spectrometer (Québec, QC, Canada). Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for 1H and 100.6 MHz for

13C

on a Bruker Avance 400 digital spectrometer

(Billerica, MA, USA). The chemical shifts (δ) were expressed in ppm and referenced to chloroform (7.26 and 77.0 ppm), acetone (2.05 and 29.8 ppm) or dimethylsulfoxide (DMSO) (2.50 and 39.5 ppm) for 1H NMR and

13C

NMR, respectively. Low-resolution mass spectra

(LRMS) were recorded on a Shimadzu Prominence apparatus equipped with an atmospheric pressure chemical ionization (APCI) source on positive mode. Purity of tested compounds were found ≥95% as determined by HPLC with a Shimadzu apparatus (Kyoto, Japan) using a Shimadzu SPD-M20A photodiode array detector, an Alltima HP C18 reversed-phase column (250 mm x 4.6 mm, 5 µm) and a solvent gradient of MeOH:water.

Synthesis of 9H-fluoren-9-ylmethyl 7-hydroxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (2). To 1,2,3,4-tetrahydroisoquinolin-7-ol (1) (20.0 g, 0.134 mol) in 1.5 L of tetrahydrofuran (THF)/water (3:1) was added an aqueous solution of NaHCO3 (1.0 M, 402 mL) and Fmoc-Osuccinimide (47.5 g, 0.14 mol). The mixture was stirred overnight at room temperature. The resulting solution was poured into water (2 L) and extracted two times with ethyl acetate (EtOAc). The combined organic layer was washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure to give 49.9 g (99%) of compound 2. The crude



Page 20 of 38

compound was used for the next step without further purification. NMR data agree with those reported in literature.26

Synthesis

of

9H-fluoren-9-ylmethyl

7-(sulfamoyloxy)-3,4-dihydroisoquinoline-2(1H)-

carboxylate (3). Under an argon atmosphere, compound 2 (20.0 g, 0.054 mol) was dissolved in anhydrous dimethylacetamide (DMA) (200 mL) at 0 °C and sulfamoyl chloride (18.5 g, 0.160 mol) was added. The solution was stirred for 15 min at 0 °C and a second portion of sulfamoyl chloride (18.5 g, 0.160 mol) was added. The solution was then allowed to return to room temperature and stirred for an additional 3 h. The resulting solution was poured into water (2 L) and extracted two times with EtOAc. The combined organic layer was washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was triturated with diethyl ether (500 mL), filtered, washed with cold ether and dried under vacuum to give 21.2 g (87%) of compound 3. NMR data agree with those reported in literature.26

Synthesis of 1,2,3,4-tetrahydroisoquinolin-7-yl tritylsulfamate (4). To a solution of compound 3 (21.0 g, 0.047 mol) in a 1.5 L of a solution of anhydrous CH2Cl2/DMA (9:1) at 0 °C was added DIPEA (24.3 mL, 0.140 mol) and trityl chloride (19.5 g, 0.070 mol) under an atmosphere of argon. The solution was stirred for 1 h at room temperature. The resulting solution was poured into water (2 L) and extracted two times with CH2Cl2. The combined organic layers were filtered over a phase-separator (Biotage, Charlotte, NC, USA) and evaporated under reduced pressure to give 43.8 g of crude compound. This crude material was then solubilized in anhydrous dimethylformamide (DMF) (700 mL) and treated with a solution of TBAF in THF (1.0 M, 35.0 mL). The resulting solution was stirred at room temperature for 1 h and then poured


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into water and extracted two times with EtOAc. The combined organic layer was washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was triturated with diethyl ether (500 mL), filtered, washed with cold diethyl ether and dried under vacuum to give 14.3 g (63%, 2 steps) of compound 4. IR (KBr) υ: 3443 and 3279 (NH); 1H NMR (DMSO-d6) δ: 2.69 (t, J = 6.1 Hz, CH2CH2N), 3.01 (t, J = 5.6 Hz, CH2CH2N), 3.11 (broad, NH), 3.85 (s, ArCH2N), 6.51 (s, CH), 6.62 (d, J = 9.1 Hz, CH), 7.01 (d, J = 8.4 Hz, 6-CH), 7.10-7.40 (m, 16H, CH of trityl group and NH); APCI-MS for C28H27O3N2S [M + H]+: 471.20 m/z.

Synthesis of 1-(furan-2-yl)-N-[(5-methylthiophen-2-yl)methyl]methanamine (7). To a mixture of 2-methyl-thiophene-5-carboxaldehyde (15.0 g, 0.119 mol) and furfurylamine (12.4 g, 0.128 mol) in EtOH (150 mL) was added 4 Å molecular sieves (30 g). The resulting suspension was stirred for 10 h at room temperature, then filtered on celite. The filtrate was then ice-cooled and treated with sodium borohydride (1.35 g, 0.36 mol). After 2 h of stirring at room temperature, the mixture was acidified with aqueous HCl 10%. The organic solvent was evaporated under vacuum and the aqueous residue extracted with EtOAc. The aqueous layer was basified with aqueous NaOH 10% and extracted twice with EtOAc. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude oil was purified by flash chromatography with acetone/hexanes (from 5:95 to 10:90) to afford the secondary amine 7 (13.5 g, 54%). 1H NMR (CDCl3) δ: 2.46 (s, CH3), 3.81 (s, CH2NH), 3.89 (s, CH2NH), 6.19 (d, J = 3.2 Hz, CH), 6.32 (dd, J1 = 1.9 Hz, J2 = 3.1 Hz, CH), 6.58 (dd, J1 = 1.0 Hz, J2 = 3.3 Hz, CH), 6.70 (d, J = 3.3 Hz, CH), 7.37 (dd, J1 = 0.7 Hz, J2 = 1.8 Hz, CH).



Page 22 of 38

Synthesis of 4-({(furan-2-ylmethyl)[(5-methylthiophen-2-yl)methyl]amino}methyl)benzoic acid (9). To a solution of amine 7 (13.5 g, 0.065 mol) in chloroform (1.2 L) was added 4-formylbenzoic acid (30.0 g, 0.200 mol) and sodium triacetoxyborohydride (56.3 g, 0.266 mol). The solution was stirred at room temperature for 48 h. The resulting solution was washed with water (1 L), dried over sodium sulfate, filtered and evaporated under reduced pressure. Purification by flash chromatography using EtOAc/hexanes (1:9) gave 22.2 g (97%) of compound 9. IR (KBr) υ: 3150-2200 (OH), 1690 (C=O); 1H NMR (DMSO-d6) δ: 2.34 (s, CH3), 3.55 (s, 2 x CH2N), 3.62 (s, CH2N), 6.24 (d, J = 2.9 Hz, CH), 6.35 (m, CH), 6.56 (d, J = 2.1 Hz, CH), 6.69 (d, J = 3.1 Hz, CH), 7.44 (d, J = 8.1 Hz, 2 x CH), 7.55 (d, J = 0.7 Hz, CH), 7.91 (d, J = 8.1 Hz, 2 x CH), 12.83 (broad s, COOH). APCI-MS for C19H20O3NS [M + H]+: 342.05 m/z.

Synthesis

of

2-{[4-({(furan-2-ylmethyl)[(5-methylthiophen-2-yl)methyl]amino}methyl)

phenyl]carbonyl}-1,2,3,4-tetrahydroisoquinolin-7-yl (tritylamino)methanesulfonate (10). To a solution of carboxylic acid 9 (15.3 g, 0.045 mol) in anhydrous DMF (700 mL) under an argon atmosphere, was added HBTU (17.1 g, 0.045 mol). The solution was stirred for 10 min before the addition of amine 4 (14.2 g, 0.030 mol) and DIPEA (15.7 mL, 0.090 mol) and then stirred for an additional 45 min. The resulting mixture was poured into water (2 L) and extracted two times with EtOAc. The combined organic layer was washed two times with water (1L), washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure. Purification by flash chromatography using a gradient of EtOAc/hexanes from 3:7 to 5:5 gave 20.0 g (84% from 4) of compound 10. IR (KBr) υ: 1620 (N-C=O); 1H NMR (acetone-d6) δ: 2.43 (s, CH3), 2.87 (broad t, CH2CH2N), 3.69 (s, 2 x CH2N), 3.77 (s, CH2N), 3.60-3.90 (broad s, CH2N), 4.65 (broad


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s, CH2N), 6.32 (d, J = 2.5 Hz, CH), 6.40 (m, CH), 6.62 (m, CH), 6.78 (d, J = 2.5 Hz, CH), 7.13 (d, J = 8.3 Hz, CH), 7.28-7.54 (m, 21H, 15 CH of trityl group and 6 x CH). APCI-MS for C47H45O5N3S2 [M + H]+: 795.25 m/z.

Synthesis

of

2-{[4-({(furan-2-ylmethyl)[(5-methylthiophen-2-yl)methyl]amino}methyl)

phenyl]carbonyl}-1,2,3,4-tetrahydroisoquinolin-7-yl sulfamate (EO-33). To a solution of compound 10 (20.0 g, 0.025 mol) in 560 mL of dichloromethane (DCM) were added 240 mL of hexafluoroisopropanol (HFIP). The mixture was stirred overnight and then evaporated to dryness. The resulting crude compound was purified by flash chromatography using a mixture of ethylacetate (EtOAc) and hexanes (1:1) to give 8.4 g (62%) of EO-33. See Figure 2 for the carbon numbering of EO-33. IR (KBr, υ in cm-1): 3356 and 3194 (NH2), 1612 (N-C=O); 1H NMR (CDCl3, δ in ppm): 2.46 (s, 23-CH3), 2.85 (broad m, 8-CH2), 3.65 (s, 17-CH2), 3.67 (s, 24CH2), 3.76 (s, 18-CH2), 3.62 and 3.92 (2 broad s, 9-CH2), 4.60 and 4.82 (2 broad s, 1-CH2), 5.70 (broad s, SONH2), 6.22 (d, J = 3.0 Hz, 26-CH), 6.33 (dd, J1 = 1.9 Hz, J2 = 2.9 Hz, 27-CH), 6.58 (d, J = 2.3 Hz, 21-CH), 6.71 (d, J = 3.2 Hz, 20-CH), 7.14 (m, 3-CH, 5-CH and 6-CH), 7.39 (d, J = 8.0 Hz, 12-CH and 16-CH), 7.40 (s, 28-CH), 7.49 (d, J = 8.0 Hz, 13-CH and 15-CH). 13C NMR (CDCl3) δ: 15.4 (C23), 29.1 (very weak, C8), 44.8 (very weak, C1), 45.2 (very weak, C9), 48.9 (C24), 52.3 (C18), 56.3 (C17), 108.9 (C26), 110.1 (C27), 120.2 (C3), 120.6 (C5), 124.4 (C21), 125.8 (C20), 126.9 (C12 and C16), 128.9 (C13 and C15), 130.1 (C6), 132.7 (C2), 134.0 (C11), 134.5 (C2), 139.4 (C22), 139.9 (C19), 141.8 (C14), 142.0 (C28), 148.6 (C4), 152.1 (C25), 171.3 (C10). APCI-MS for C28H30O5N3S2 [M + H]+: 552.3 m/z. HPLC purity: 97.3%. This purity was determined with a Shimadzu apparatus (Kyoto, Japan) using a Shimadzu SPD-M20A photodiode



Page 24 of 38

array detector, an Alltima HP C18 reversed-phase column (250 mm x 4.6 mm, 5 µm) and a solvent gradient of MeOH:water.

Biological assays

Animals. All experiments were conducted in an animal facility approved by the Canadian Council on Animal Care (CCAC) and the Association for Assessment and Accreditation of Laboratory Animal Care, and protocols were approved by the institutional animal ethics committee (Université Laval, Québec, QC, Canada). All mice were acclimatized to the environmental conditions (temperature, 22 ± 3 °C; humidity, 50 ± 20%; 12-h light/dark cycles, lights on at 07:15 h) for at least 3-5 days before starting the experiments.

Plasma concentration of EO-33 and EO-29 in mice. Female Balb/c mice were obtained from Charles River, Inc. (Saint-Constant, QC, Canada), housed 4 or 5 per cage, and allowed free access to water and food (Teklad Rodent Diet; global 18% protein #2018SX, Envigo, Madison, WI, USA). They received a single dose of EO-33 in 0.1 mL of vehicle fluid by subcutaneous (SC) injection or oral gavage (PO). Depending on the vehicle used, EO-33 was first dissolved in an organic solvent (ethanol (EtOH) or dimethylsulfoxide (DMSO)) and, thereafter, added to the appropriate co-solvent (aqueous 0.4% methylcellulose (MC), propylene glycol (PG) or sunflower oil (SO) to obtain a final 8% concentration of EtOH or DMSO. Mice were fasted with free access to water for 8 h before the injection of EO-33. At the appropriate time point, 7 h for those receiving DMSO as solvent and 5-time points (1, 3, 7, 12, and 24 h) for those receiving EtOH as solvent, mice (2-3 per group) were sacrificed under isoflurane by cardiac puncture followed by cervical dislocation. Blood from each group was pooled and collected into Microvette 24 ACS Paragon Plus Environment

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potassium-EDTA (ethylenediamine tetraacetic acid)-coated tube (Sarstedt, AG & Co., Nümbrecht, Germany) and centrifuged at 3200 rpm for 10 min at 4 °C. The plasma was stored at −80 °C until its analysis. The concentration of EO-29 (phenol) was also determined, but only for plasma samples collected from the experiments using 30 mg of EO-33 (sulfamate) in MC:EtOH (92:8) and injected SC or PO. Concentrations of EO-33 and EO-29 in blood samples were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as described below.

LC-MS/MS measurement of EO-33 and EO-29. The concentration of EO-33 and EO-29 was determined by LC-MS/MS analysis using a procedure developed at the CHU de Québec Research Center (Québec, QC, Canada).43, 44 Briefly, plasma samples (100 µL) were transferred to individual tubes and 1 mM ammonium acetate (600 µL) was added. A methanolic (MeOH) solution (50 µL) containing an internal standard was then added to each tube. Samples were transferred on Strata-X SPE columns (Phenomenex, Torrance, CA, USA), which had been conditioned with MeOH (2 mL) and water (2 mL). Each column was washed with MeOH:water (10:90) (2 mL) and next with a MeOH solution (5 mL) containing 5 mM ammonium acetate. MeOH was evaporated at 45 C under inert atmosphere and the residue dissolved in 100 µL of MeOH:water (85:15). For the analysis, the liquid chromatography (LC) system used a 75 x 4.6mm, 3-μm reversed-phase Luna Phenyl-Hexyl column (Phenomenex, Torrance, CA, USA) at a flow rate of 0.8 mL/min. The inhibitor was detected using an API 4000 mass spectrometer, equipped with TurboIonSpray (Applied Biosystems, Canada). Electrospray ionization in positive ion mode was used.



Page 26 of 38

Uterotrophic/antiuterotrophic/antiSTS activities in mice. Female Balb/c mice weighing 15– 20 g were obtained from Charles River, Inc. (Saint-Constant, QC, Canada), housed 5 per cage, and allowed free access to water and food (Teklad Rodent Diet; global 18% protein #2018SX, Envigo, Madison, WI, USA). Except intact (INT) mice, they were ovariectomized (OVX) under isoflurane-anesthesia via bilateral flank incisions, and randomly assigned to different groups (5 mice/group). Mice in the INT group and OVX control (CTL) group received 0.1 mL of vehicle alone SC (MC:EtOH (92:8)) while the other groups received the same vehicle (0.1 mL) containing the appropriate hormone precursor (E1S [2 µg/mouse/BID/SC] or E1 [0.06 µg/mouse/ID/SC]) with or without the STS inhibitor (EO-33 or EM-1913). The first protocol includes the following groups: Gr-1 (INT), Gr-2 (OVX/CTL), Gr-3 (OVX/E1S), Gr-4 (OVX/EO-33 [30 mg/kg/BID/SC]), Gr-5 (OVX/E1), and Gr-6 (OVX/E1/EO-33 [30 mg/kg/ID/SC]). The second protocol includes the following groups: Gr-1 (OVX/CTL), Gr-2 (OVX/E1S), Gr-3 (OVX/E1S/EO-33 [15 mg/kg/BID/SC]), Gr-4 (OVX/E1S/EO-33 [30 mg/kg/BID/SC]), and Gr-5 (OVX/E1S/EM-1913 [5 mg/kg/ID/SC]). The protocols were performed over a period of 9 days with E1S or E1 injected from day 5 to 10 of the study, while EO-33 or EM-1913 was injected from day 2 to 10. On day 11, the mice were sacrificed by exsanguination followed by cervical dislocation. Uterus and liver from the mice were rapidly dissected, weighed, and kept at -80 °C. Data are expressed as means ± SEM.

AntiSTS activity in liver homogenates. Livers from mice of all groups were resuspended in cold phosphate buffer (pH 7.4) (5 mL/g of tissue) containing protease inhibitors mini-complete (Roche Diagnostics, Laval, QC, Canada). Homogenization was accomplished with a Polytron, using three 10-second bursts separated by a 2-min cooling period in ice. Nuclei and cell debris


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were removed by centrifugation (4 °C) at 2000 g for 30 min, and portions of 1.5 mL of the supernatant were stored at -80 °C. The protein concentration of the supernatants was determined by the Bradford method.45 For the enzymatic assay (transformation of labeled E1S into labeled E1), a mixture of [3H]-E1S (9 nM) (American Radiolabeled Chemicals, Inc) and E1S (Sigma) in PBS buffer (pH 7.4) was added to liver proteins (100 and 500 µg) to obtain a final substrate concentration of 1 µM in a volume of 1 mL. After 2 h of incubation at 37 °C in a shaking incubator, the reaction was stopped by adding 1 mL of xylene. The tubes were stirred and centrifuged at 3000 rpm for 20 min to separate the organic and aqueous phases. The radioactivity was recorded in 500 µL of each phase (organic: [3H]-E1 and aqueous: [3H]-E1S) by liquid scintillation counting with a Wallac 1400 Liquid Scintillation Counter. The results were expressed as the percentage (%) of E1 produced from E1S. All experiments were performed in triplicate and expressed as means ± SEM.

Osteogenic activity in mice (tibia weight and tibia calcium content). Female C57BL/6 mice (63-70 days old; ~19.4 g) were obtained from Charles River (Saint-Constant, QC, Canada), housed 4 or 5 per cage, and allowed free access to water and food (Teklad Rodent Diet; global 18% protein #2018.15, Envigo, Madison, WI, USA). After an acclimation period, the mice were ovariectomized (OVX) to induce osteoporosis, and 8 mice were sham-operated. The control sham group underwent the same procedure, except that the ovaries were identified, but not removed. This procedure was performed under isoflurane-induced anesthesia. On the day of surgery, OVX mice were randomized according to weight, separated into 4 groups of 8 mice, and treated over a period of 70 days. Gr-1 (sham) and Gr-2 (OVX/CTL) were treated with 0.1 mL of vehicle (MC:EtOH (92:8)) alone while the 3 other OVX groups (Gr-3, Gr-4, and Gr-5) received



Page 28 of 38

SC E2 (5 µg/kg), raloxifene (1 mg/kg), and EO-33 (30 mg/kg), respectively, in 0.1 mL of vehicle. At the end of the protocol, the mice were weighed and anesthetized for necropsy. Tibias were immediately excised clean of soft tissues, weighed and fixed with ice-cold formalin 10%. The concentration of calcium was determined with an Agilent 8800 Triple Quad ICP-MS after tibia calcination at 600 C (6 h) and ash digestion with concentrated HNO3 in microwave oven (180 C, 40 min) following a known procedure.46

Anticancer activity in mouse MCF-7 tumor (xenograft). MCF-7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and were routinely grown in suspension in DME-F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with L-glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 µg/mL), fetal bovine serum (FBS, 5% v/v), and estradiol (1 nM), and maintained in a 175 cm2 culture flask at 37 °C under 5% CO2 humidified atmosphere. For the xenograft, homozygous female nu/nu Br athymic mice (28-42 days old, 21-29 g) from Charles River, Inc. (Saint-Constant, QC, Canada) were housed in vinyl cages (4-5 mice/cage) equipped with air lids, kept in laminar airflow hoods, and maintained under pathogen-limiting conditions. Cages, bedding, water, and food (Teklad global 18% protein #2018SX, Envigo, Madison, WI, USA) were autoclaved before use. After the acclimation period, bilateral ovariectomy was performed under isoflurane-induced anesthesia, and mice were treated 3 times per week with estradiol sulfate (E2S) (50 µg/50 µL in MC:EtOH (92/8). Three days after the surgery, 4.1 x 106 MCF-7 cells (passage 20) were inoculated SC in 0.1 mL of DME-F12 medium containing 30% Matrigel in both flanks of each mouse through a 2.5 cm long 22-gauge needle. After 4 weeks of stimulation with E2S, randomization and treatment were started. One day prior to initiation of treatment, all mice bearing tumors were randomly assigned to 3 groups


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(with respect to tumor size): Gr-1 (OVX/CTL, 6 mice, 10 tumors), Gr-2 (OVX/E2S, 7 mice, 10 tumors), and Gr-3 (OVX/E2S/EO-33, 8 mice, 10 tumors). E2S (50 µg/mouse) was given 3 times per week while EO-33 (30 mg/kg) was given to animal 6 days per week by SC injection in 0.1 mL of MC:EtOH (92:8) for 28 days. EO-33 solution was prepared one day prior to initiation of treatment, stored at 4 °C and kept under constant agitation. All mice in the CTL group received only the vehicle. The tumor size was measured two times a week with a caliper. Two perpendicular diameters (L and W) were recorded and tumor area (mm2) was calculated using the formula L/2 x W/2 x π. The area measured on the first day of treatment was taken as 100%. After 28 days of treatment, and 3 h after the last treatment, mice were weighed, anesthetized with isoflurane, and killed by exsanguination (cardiac puncture). The livers were removed and immediately frozen at -80 °C.

Statistical analysis. Statistical significance was determined according to the multiple-range test of Duncan-Kramer.47 P values 0.05 were considered as statistically significant.

ASSOCIATED CONTENT

Supporting Information The supporting information is available free of charge on the ACS Publication website at DOI:………… HPLC chromatogram of final STS inhibitor EO-33, NMR spectra (1H,

13C,

COSY,

NOESY, HSQC, and HMBC) of EO-33, and Figures S1-S5.



Page 30 of 38

Molecular formula strings (CSV)

AUTHOR INFORMATION Corresponding Author Tel: 1-418-654-2296. E-Mail: [email protected] ORCID

Donald Poirier: 0000-0002-7751-3184 René Maltais: 0000-0002-0394-1653 Jenny Roy: 0000-0002-9928-3009 Diana Ayan: 0000-0001-6701-5758 Author Contributions All authors contributed to the manuscript and have given approval to its final version. Funding The authors would like to thank the Canadian Institutes of Health Research (PPP-133374) for providing funding. Notes


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JR and DA declare no competing interests, while DP and RM are owners of a patent on STS inhibitors. ACKNOWLEDGMENTS We would like to thank Dr. Dominic Larivière and Laurence Whitty-Léveillé (Faculty of Sciences and Engineering, Université Laval, Québec, QC, Canada) for determining the calcium content. We also thank Mrs. Micheline Harvey for the careful reading of this manuscript.

ABBREVIATIONS USED AE: antiestrogen; BID: twice a day; CTL: control; DHEAS: dehydroepiandrosterone sulfate; DIPEA: diisopropylethyl amine; E1: estrone; E2; estradiol; E1S: estrone sulfate; E2S: estradiol sulfate;

ER:

estrogen

receptor;

EtOAc:

ethyl

acetate;

ID:

once

a

day;

HFIP:

hexafluoroisopropanol; 17β-HSD1; 17beta-hydroxysteroid dehydrogenase type 1; INT: intact; ISTS-SERM: inhibitor of STS with SERM effect; MC: methyl cellulose; OVX: ovariectomized; PC: plasma concentration; PG: propylene glycol; PREGS: pregnenolone sulfate; RAL: raloxifene; SERM: selective estrogen receptor modulator; SO: sunflower oil; STS: steroid sulfatase.



Page 32 of 38

REFERENCES (1)

Potter, B. V. L. Steroid sulphatase inhibition via aryl sulphamates: clinical progress, mechanism and future prospects. J. Mol. Endocrinol. 2018, 61, T233-T252.

(2)

Rizner, T. L. The important roles of steroid sulfatase and sulfotransferases in gynecological diseases. Front. Pharmacol. 2016, 7, 30.

(3)

Purohit, A.; Foster P. A. Steroid sulfatase inhibitors for estrogen- and androgen-dependent cancers. J. Endocrinol. 2012, 212, 99-110.

(4)

Maltais, R.; Poirier, D. Steroid sulfatase inhibitors: A review covering the promising 20002010 decade. Steroids 2011, 76, 929-948.

(5)

Costa, E. V.; Sousa, E.; Choosang, K.; Singh, S.; Rocha, J.; Lima, R. T.; Pakkong, P.; Ahmed, S.; Vasconcelos, M. H.; Montanari, C. A.; Pinto, M. M. Structure based design, synthesis, and evaluation of potential inhibitors of steroid sulfatase. Curr. Top. Med. Chem. 2014, 14, 1033-1044.

(6)

Mostafa, Y. A.; Taylor, S. D. Steroid derivatives as inhibitors of steroid sulfatase. J. Steroid Biochem. Mol. Biol. 2013, 137, 183-198.

(7)

Subramanian, A.; Salhab, M.; Mokbel, K. Oestrogen producing enzymes and mammary carcinogenesis: a review. Breast Cancer Res. Treat. 2018, 111, 191-202.

(8)

Poirier, D. New cancer drugs targeting the biosynthesis of estrogens and androgens. Drug Dev. Res. 2008, 69, 304-318.

(9)

Hanson, S. R.; Best, M. D.; Wong, C. H. Sulfatases: Structure, mechanism, biological activity, inhibition, and synthetic utility. Angew. Chem. Int. Ed. 2004, 43, 5736-5763.

(10) Hewitt, S. C.; Korach, K. S. Estrogen receptors: New directions in the new millennium. Endocr. Rev. 2018, 39, 664-675. 32 ACS Paragon Plus Environment

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(11) Royuela, M.; de Miguel, M. P.; Bethencourt, F. R.; Sanchez-Chapado, M.; Fraile, B.; Arenas, M. I.; Paniagua, R. Estrogen receptors a and b in the normal, hyperplastic and carcinomatous human prostate. J. Endocrinol. 2001, 168, 447-454. (12) Mosselman, S.; Polman, J.; Dijkema, R. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett. 1996, 392, 49-53. (13) Byers, M.; Kuiper, G. G. J. M.; Gustafsson, J. A.; Park-Sarge, O. K. Estrogen receptor-β mRNA expression in rat ovary: Down-regulation by gonadotropins. Mol. Endocrinol. 1997, 11, 172-182. (14) Paterni, I.; Granchi, C.; Katzenellenbogen, B.; Minutolo, F. Estrogen receptors alpha (ERα) and beta (ERβ): Subtype-selective ligands and clinical potential. Steroids 2014, 90, 13-29. (15) Maximov, P. Y.; Lee, T. M.; Jordan, V. C. The discovery and development of selective estrogen receptor modulators (SERMs) for clinical practice. Curr. Clin. Pharmacol. 2013, 8, 135-155. (16) Jordan, V. C. Antiestrogens and selective estrogen receptor modulators as multi-functional medicines 1. Receptor interactions. J. Med. Chem. 2003, 46, 883-908. (17) Coombes, C.; Cardoso, F.; Isambert, N.; Lesimple, T.; Soulié, P.; Peraire, C.; Fohanno, V.; Kornowski, A.; Ali, T.; Schmid, P. A phase I dose escalation study to determine the optimal biological dose of irosustat, an oral steroid sulfatase inhibitor, in postmenopausal women with estrogen receptor-positive breast cancer. Breast Cancer Res. Treat. 2013, 140, 73-82. (18) Palmieri, C.; Stein, R. C.; Liu, X.; Hudson, E.; Nicholas, H.; Sasano, H.; Guestini, F.; Holcombe, C.; Barrett, S.; Kenny, L.; Reed, S.; Lim, A.; Hayward, L.; Howell, S.;



Page 34 of 38

Coombes, R. C. IRIS trial participants. IRIS study: a phase II study of the steroid sulfatase inhibitor Irosustat when added to an aromatase inhibitor in ER-positive breast cancer patients. Breast Cancer Res. Treat. 2017, 165, 343-353. (19) Alibhai, S. M. H.; Gogov, S.; Allibhai, Z. Long-term side effects of androgen deprivation therapy in men with non-metastasic prostate cancer: A systematic literature review. Crit. Rev. Oncol. Hematol. 2006, 60, 201-215. (20) Angelopoulos, N.; Barbounis, V.; Livadas, S.; Kaltsas, D.; Tolis, G. Effects of estrogen deprivation due to breast cancer treatment. Endocr. Rel. Cancer 2004, 11, 523-535. (21) Rizzoli, R.; Body, A.; De Censi, A.; Reginster, J. Y.; Piscitelli, P.; Brandi, M. L. Guidance for the prevention of bone loss and fractures in postmenopausal women treated with aromatase inhibitors for breast cancer: an ESCEO position paper. Osteoporosis Int. 2012, 23, 2567-2576. (22) Akiyoshi, T.; Shimomura, Y.; Masuda, S.; Okamoto, Y.; Hiura, S.; Kato, H.; Asayama, T.; Ohtani, H. Effect of aromatase inhibitors on bone mineral density in Japanese breast cancer population. Drug Metab. Pharmacokinetic 2013, 26, 446-450. (23) Altundag, K.; Ibrahim, N. K. Aromatase inhibitors in breast cancer: an overview. Oncologist 2006, 11, 553-562. (24) Li J. J. Aromatase inhibitors for breast cancer: exemestane (Aromasin), anastrazole (Arimidex) and letrozole (Femara). 2007. The art of drug synthesis. D.S. Johnson and J.J. Li (Eds), Wiley. 31-38. (25) Rasmussen, L. M.; Zaveri, N. T.; Stenvang, J.; Peters, R. H.; Lykkesfeldt, A. E. A novel dual-target steroid sulfatase inhibitor and antiestrogen: SR 16157, a promising agent for the therapy of breast cancer. Breast Cancer Res. Treat. 2007, 106, 191-203.


Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) Ouellet, É.; Maltais, R.; Ouellet, C.; Poirier, D. Investigation of a tetrahydroisoquinoline scaffold as dual-action steroid sulfatase inhibitors generated by parallel solid-phase synthesis. Med. Chem. Commun. 2013, 4, 681-692. (27) Ouellet, C.; Ouellet, É.; Poirier, D. In vitro evaluation of a tetrahydroisoquinoline derivative as a steroid sulfatase inhibitor and a selective estrogen receptor modulator. Invest. New Drugs 2015, 33, 95-103. (28) Ouellet, C.; Maltais, R.; Ouellet, É.; Barbeau, X.; Lagüe, P.; Poirier, D. Discovery of a sulfamate-based steroid sulfatase inhibitor with intrinsic selective estrogenic receptor modulator properties. Eur. J. Med. Chem. 2016, 119, 169-182. (29) Kenmogne, L. C.; Ayan, D.; Roy, J.; Maltais, R.; Poirier, D. The aminosteroid derivative RM-133 shows in vitro and in vivo antitumor activity in human ovarian and pancreatic cancers. PLoS ONE 2015, 10, e0144890. (30) Woo, L. W. L.; Howart, N. M.; Purohit, A.; Hejaz, H. A. M.; Reed, M. J.; Potter, B. V. L. Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase. J. Med. Chem. 1998, 41, 1068-1083. (31) Spillane, W. J.; Malaubier, J. B. Mechanism of the hydrolysis of the sulfamate EMATE-an irreversible steroid sulfatase inhibitor. Tetrahedron Lett. 2010, 51, 2059-2062. (32) Ciobanu, L. C.; Luu-The, V.; Martel, C.; Labrie, F.; Poirier, D. Inhibition of estrone sulfate-induced uterine growth by potent non-estrogenic steroidal inhibitors of steroid sulfatase. Cancer Res. 2003, 63, 6442-6446. (33) Laplante, Y.; Rancourt, C.; Poirier, D. Relative involvement of three 17β-hydroxysteroid dehydrogenases (types 1, 7 and 12) in the formation of estradiol in various breast cancer cell lines using selective inhibitors. Mol. Cell. Endocrinol. 2009, 301, 146-153.



Page 36 of 38

(34) Ciobanu, L. C.; Boivin, R. P.; Luu-The, V.; Labrie, F.; Poirier, D. Potent inhibition of steroid sulfatase activity by 3-O-sulfamate-17α-benzyl (or 4’-tert-butylbenzyl)estra1,3,5(10)-trienes: combination of two substituents at positions C3 and C17α of estradiol. J. Med. Chem. 1999, 42, 2280-2286. (35) Poirier, D.; Roy, J.; Maltais, R.; Ayan, D. A potent inhibitor of steroid sulfatase (EM-1913) blocks tumor growth in nude mice (MCF-7 xenograft). Curr. Enzyme Inh. 2015, 11, 65-73. (36) Cano, A.; Dapia, S.; Noguera, I.; Pineda, B.; Hermenegildo, C.; del Val, R.; Caeiro, J. R.; Garcia-Pérez, M. A. Comparative effects of 17β-estradiol, raloxifene and genistein on bone 3D microarchitecture and volumetric bone mineral density in the ovariectomized mice. Osteoporos. Int. 2008, 19, 793-800. (37) Prideaux, M.; Wijenayaka, A. R.; Kamarasinghe, D. D.; Ormsby, R. T.; Evdokiou, A.; Findlay, D. M.; Atkins, G. J. SaOS2 osteosarcoma cells as an in vitro model for studying the transition of human osteoblasts to osteocytes. Calcif. Tissue Int. 2014, 95, 183-193. (38) Xu, J.W.; Xiang, Q.L.; Lin, G.P.; Fu, X.D.; Zhou, K.W.; Jiang, P.; Zheng, S.H.; Wang, T.H. Estrogen improved metabolic syndrome through down-regulation of VEGF and HIF1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariextomized female rats. Mol. Biol. Rep. 2012, 39, 8177-8185. (39) Meng, X.; Dai, X.; Liao, T.D.; D’Ambrosio, M.; Wang, F.; Yang, J.J.; Yang, X.P. Dosedependent toxic effects of high-dose estrogen on renal and cardiac injury in surgically postmenopausal mice. Life Sci. 2011, 17, 178-186. (40) Jung, J. Human tumor xenograft models for preclinical assessment of anticancer drug development. Toxicol. Res. 2014, 30, 1-5. (41) Perreault, M.; Maltais, R.; Roy, J.; Picard, S.; Popa, I.; Bertrand, N.; Poirier, D. Induction


Page 37 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of endoplasmic reticulum stress by aminosteroid derivative RM-581 leads to tumor regression

in

PANC-1

xenograft

model.

Invest.

New

Drugs,

in

press,

doi.org/10.1007/s10637-018-0643-4. (42) Ayan, D.; Maltais, R.; Roy, J.; Poirier, D. A new nonestrogenic steroidal inhibitor of 17hydroxysteroid dehydrogenase type I blocks the estrogen-dependent breast cancer tumor growth induced by estrone. Mol. Cancer Ther. 2012, 11, 2096-2104. (43) Labrie, F.; Bélanger, A.; Bélanger, P.; Bérubé, R.; Martel, C.; Cusan, L.; Gomez, J.; Candas, B.; Castiel, I.; Chaussade, V.; Deloche, C.; Leclaire, J. Androgen glucuronides instead of testosterone, as the new markers of androgenic activity in women. J. Steroid Biochem. Mol. Biol. 2006, 99, 182-188. (44) Roy, J.; Lefebvre, J.; Maltais, R.; Poirier, D. Inhibition of dehydroepiandrosterone sulfate in androgen-sensitive tissues by EM-1913, an inhibitor of steroid sulfatase. Mol. Cell. Endocrinol. 2013, 376, 148-155. (45) Bradford, M. M. A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. (46) Lariviere, D.; Packer, A. P.; Marro, L.; Li, C.; Chen, J.; Cornett, R. J. Age dependence of natural uranium and thorium concentrations in bone. Health Physics 2007, 92, 119-126. (47) Kramer, C. Y. Extension of multiple range to group means with unequal numbers of replications. Biometrics 1956, 12, 307-310.



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Table of Contents Graphics


Antisulfatase, Osteogenic, and Anticancer Activities of Steroid

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