Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part I

Bioorganic & Medicinal Chemistry 23 (2015) 2568–2578

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Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part I Katsuji Aikawa a,⇑, Toshio Miyawaki a, Takenori Hitaka a, Yumi N. Imai a, Takahito Hara a, Junichi Miyazaki b, Masuo Yamaoka a, Masami Kusaka c, Naoyuki Kanzaki a, Akihiro Tasaka d, Mitsuru Shiraishi e, Satoshi Yamamoto a a

Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1 Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-85555, Japan Pharmaceutical Production Division, Takeda Pharmaceutical Company Ltd, 17-85 Jusohommachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan CMC Center, Takeda Pharmaceutical Company Ltd, 26-1 Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-85555, Japan d Environment & Safety Department, Takeda Pharmaceutical Company Ltd, 17-85 Jusohommachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan e Laboratories of Medicinal & Organic Chemistry Pharmaceutical Sciences, Himeji Dokkyo University, 7-2-1, Kamiohno, Himeji-shi, Hyogo 670-8524, Japan b c

a r t i c l e

i n f o

Article history: Received 1 February 2015 Revised 6 March 2015 Accepted 8 March 2015 Available online 19 March 2015 Keywords: Androgen receptor AR Selective androgen receptor modulators (SARMs) Testosterone

a b s t r a c t To develop effective drugs for hypogonadism, sarcopenia, and cachexia, we designed, synthesized, and evaluated selective androgen receptor modulators (SARMs) that exhibit not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also neutral or antagonistic effects on the prostate. Based on the information obtained from a docking model with androgen receptor (AR), we modified a hit compound A identified through high-throughput screening. Among the prepared compounds, 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j had highly potent AR agonistic activities in vitro and good tissue selectivity in vivo. These derivatives increased the weight of the levator ani muscle without influencing the prostate and seminal vesicle. In addition, these compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Steroid hormones comprise a group of important chemical mediators that exert various physiological effects.1 Androgen is a general term for C19 steroid hormones.2 Androgens elicit diverse biological responses through the androgen receptor (AR), including anabolic, osteoblastic, and hematopoietic effects as well as maintenance of libido. Testosterone is one of the major androgens in the body and is synthesized mainly from cholesterol in the testis.3 In the prostate, 5a-reductase converts testosterone to dihydrotestosterone (DHT), which shows the most potent AR agonistic activity among all naturally occurring androgens.4 Testicular dysfunction with aging or morbidity causes a decline in serum testosterone levels.5 This decline is believed to be related to various symptoms such as muscle weight loss, osteoporosis, depression, and decreased libido.6 This syndrome is called lateonset hypogonadism (LOH). Several recent clinical studies have shown that testosterone supplementation in androgen replacement therapy (ART) can be effective for increasing lean body mass ⇑ Corresponding author. Tel.: +81 466 32 1157. E-mail address: [email protected] (K. Aikawa). http://dx.doi.org/10.1016/j.bmc.2015.03.032 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

and improving muscle strength.7 However, ART can potentially induce side effects such as exacerbation of benign prostatic hypertrophy (BPH) or progression of latent prostate cancer. In addition, testosterone and its esters cannot be administered orally because of rapid hepatic elimination and hepatotoxicity. Consequently, the compounds are administered by inconvenient intramuscular injection, surgical implantation, or transdermal delivery using patches or gels.8 Although androgenic anabolic steroids are available as oral formulations, hepatotoxicity limits extensive use of such analogs in chronic therapy.9,10 Under these circumstances, there has been growing interest in nonsteroidal tissue-selective androgen receptor modulators (SARMs).11 The concept of SARMs evolved from selective estrogen receptor modulators (SERMs),12 which have been clinically used for over 2 decades to replenish the diminishing circulating estrogens in postmenopausal conditions. SARMs are expected to show not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also antagonistic or neutral effects on the prostate. SARMs could provide advantages over conventional ART for the treatment of LOH. Several nonsteroidal SARMs such as Ostarine (GTx/Merck),13,14 BMS564929 (Bristol–Myers Squibb),15 and LGD2941 (Ligand)16

K. Aikawa et al. / Bioorg. Med. Chem. 23 (2015) 2568–2578

have been reported to be in the clinical stage. Particularly, Ostarine has been proven to increase lean body mass and improve some muscle functions of patients with cancer cachexia in a Phase II study, although further development appears to have been halted for undisclosed reasons (Fig. 1). At the beginning of our research for novel SARMs, a promising lead compound, 1-(4-nitro-1-naphthyl)pyrrolidine (compound A), was found through high-throughput screening (HTS). Compound A showed strong AR binding affinity (IC50 = 2.4 nM) and moderate AR agonistic activity (EC50 = 41 nM). The binding modes of compound A to the ligand binding domain (LBD) of AR were explored using program Gold (ver.1.1, Cambridge Crystallographic Data Center, Cambridge, UK) in a docking study. We used in-house protein crystal structure in docking study, not derived from the Protein Data Bank. The study findings suggested that there may be some interactions with amino acids in the LBD (Fig. 2). First, the nitro group at the 4-position of the naphthalene ring forms a hydrogen bond with Arg752. Second, the naphthalene ring interacts with Leu707, Leu873, Met745, Met749, Met787, and Phe764 through hydrophobic contacts. Third, there are 2 extra spaces around the pyrrolidine ring, a hydrophobic pocket around the 2position constructed with Trp741, Met742, and Met745, and a

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hydrophilic pocket around the 3-position surrounded by Asn705 and Thr877. This information prompted us to design agonists more potent than compound A using the following strategies: (1) replacement of the 4-nitro group with a hydrogen bond-forming substituent exhibiting low toxicity (X), (2) installation of a hydrophobic group (R1) at the 2-position of the pyrrolidine ring to accommodate the hydrophobic pocket, and (3) introduction of a polar substituent (R2) to form hydrogen bonds with Asn705 and Thr877. In this paper, we describe the design, synthesis, and in vitro and in vivo characterizations of 1-(4-substituted-1-naphthyl)pyrrolidine derivatives. 2. Chemistry Synthesis of the requisite pyrrolidines 3, 8a, and 8b,c is depicted in Schemes 1–3. Preparation of 2-(S)-vinylpyrrolidine 2 was performed by oxidation of commercially available 2-(S)-hydroxymethyl pyrrolidine 1, followed by Wittig reaction with methyltriphenylphosphonium bromide. Subsequent hydrogenation of compound 2 afforded 2-(S)-ethyl pyrrolidine 3 in 72% yield (Scheme 1). Boc-protected L-alanine 4 was condensed using Meldrum’s acid, and the following cyclization was accomplished

Figure 1. Known nonsteroidal SARMs.

Figure 2. Docking study of compound A with the LBD of AR and the design of more potent agonists.

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Scheme 1. Reagents: (a) (1) SO3-Py, Et3N, DMSO, (2) Ph3PMeBr, NaH, DMSO; (b) H2, Pd–C, MeOH.

Scheme 2. Reagents: (a) (1) Meldrum’s acid, CDI, CH2Cl2, (2) AcOEt, reflux; (b) NaBH4, AcOH, CH2Cl2; (c) (1) 4 M HCl–AcOEt, (2) Amberlyst A-21, MeOH, (3) recryst. from IPE; (d) Red-Al, THF.

Scheme 3. Reagents: (a) K2CO3, acetone; (b) 12, TsOH, toluene, reflux; (c) H2, PtO2, MeOH; (d) MeMgBr, THF, Et2O, 78 °C; (e) LAH, THF; (f) H2, Pd–C, MeOH; (g) (CO2H)2, MeOH.

by refluxing the mixture in EtOAc to give compound 5 in 53% yield. Treatment of 5 with sodium tetrahydroborate–AcOH caused cis-selective reduction to provide (2S,3S)-pyrrolidone 6 in 65% yield. Deprotection of the Boc group by adding 4 M HCl in EtOAc and subsequent reduction of the amide group by adding Red-Al yielded (2S,3S)-pyrrolidine 8a quantitatively (Scheme 2). Synthesis of pyrrolidine 8b,c was initiated by installation of a cyclopropyl group into methyl acetoacetate 9 using 1,2-dibromoethane to afford 11 in 49% yield (Scheme 3). Successive coupling of 11 with an (S)-benzylamine derivative 12 gave dihydropyrrole 13. Optically pure (2S,3S)-pyrrolidine 14 was prepared by diastereo-selective hydrogenation of 13 using platinum (IV) oxide under hydrogen atmosphere driven by the optically pure (S)-benzyl group. Compound 14 was converted into compound 15a,b by treatment with methylmagnesium bromide or lithium aluminum hydride, and subsequent cleavage of the benzyl group by hydrogenation using palladium on carbon under hydrogen atmosphere yielded optically active pyrrolidine 8b,c. A coupling reaction of pyrrolidine 3 or 8 with 4-fluoronaphthonitrile 16 was performed using potassium carbonate or lithium carbonate as a base in dimethyl sulfoxide under 100 °C to yield 4-pyrrolidinonaphthonitrile derivatives 17a–k. For Boc- or benzyl-protected pyrrolidines, protecting groups (R) were removed by treatment with acid or hydrogenation before the coupling reaction. We used commercially purchased materials 8b–d,f,g,k for the reactions (Scheme 4). Further modifications of 4-pyrrolidinonaphthonitrile derivatives are illustrated in Schemes 5–7. The cyano group of 17a was hydrolyzed by potassium hydroxide under reflux to afford

Compd.

R1

R1'

R2

R

Methods

17a

H

H

H

H

e

91

17b

Me

H

H

Boc

a+e

49

17c

H

Me

H

H

e+f

45

17d

Me

Me

H

Bn

c+e

3

17e

Et

H

H

Boc

b+e+f

18

17f

i-Pr

H

H

Bn

d+e

28

17g

CONH2

H

H

H

e

37

17h

Me

H

OH

H

e

65

17i

Me

H

CH2OH

H

e

30

17j

Me

H

C(Me)2OH

H*

e**

73

17k

Me

H

CO2Me

Bn

c+e

22

Yield (%)

*1/2 (CO2H) salt, **Li2CO3 was used instead of K2CO3.

Scheme 4. Methods: (a) TFA, toluene; (b) HCl, EtOAc; (c) H2, Pd–C, MeOH, HCl; (d) H2, Pd–C, MeOH, AcOH; (e) K2CO3, DMSO; (f) Chiral HPLC.

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Scheme 5. Reagents: (a) KOH, EtOH, H2O, reflux.

Scheme 6. Reagents: (a) TFAA, Et3N, CH2Cl2.

Scheme 7. Reagents: (a) (1) 3,4-dimethoxybenzoic acid, Ph3P, DEAD, THF, toluene, (2) NaOHaq, THF, MeOH, H2O; (b) DMSO, (COCl)2, Et3N, CH2Cl2.

carboxylic acid derivative 18 in 1% yield (Scheme 5), and the carbamoyl group of 17g was converted to a cyano group using trifluoroacetic anhydride to provide 17l in 76% yield (Scheme 6). Preparation of (2S,3R)-pyrrolidine 17m was achieved by Mitsunobu reaction of (2S,3S)-pyrrolidone derivative 17h followed by hydrolysis, and subsequent Swern oxidation afforded 3-oxo pyrrolidine 17n in 53% yield (Scheme 7).

Table 1 In vitro activities of 18, 17a, and A

N

X

3. Results and discussion 3.1. AR binding and reporter gene assay AR binding affinities were evaluated by competitive displacement of a radiolabeled [3H]mibolerone from AR, and the data were reported as IC50 values. Functional activities were determined by luciferase activities and described as EC50 values. First, we tried to replace the nitro group of lead compound A with hydrogen bond-forming substituents X, as shown in Table 1. Conversion to a carboxyl group (18) resulted in significant loss of the binding affinity and agonistic activity. In contrast, introduction of a cyano group (17a) retained the binding affinity and showed a 15-fold stronger agonistic activity (EC50 = 2.5 nM) than that of compound A. Based on this finding, we fixed a cyano group as substituent X and focused on introducing substituents to the pyrrolidine ring. Initially, the substituent effect of R1 at the 2-position of the pyrrolidine ring was evaluated (Table 2). Introduction of an (S)methyl group (17b) led to a 10-fold increase in the agonistic activity (EC50 = 0.19 nM), whereas (R)-methyl pyrrolidine derivative 17c markedly reduced the agonistic activity. These results suggested that AR clearly recognized the (S)-methyl group. Incorporation of an (S)-ethyl group was also effective for increasing the agonistic activity (17e) comparable to that of the (S)-methyl derivative 17b. In contrast, dimethyl (17d) and isopropyl (17f) groups caused decreases in the agonistic activity, which indicated that the space around the 2-position on the pyrrolidine ring is limited. Next, we examined the influence of polar substituents such as carbamoyl (17g) and cyano (17l) groups. However, these derivatives resulted in decreased agonistic activity, which suggested that the space around the 2-position is hydrophobic. Then, we evaluated the effects of substituents at the 3-position 0 (R2, R2 ) on the pyrrolidine ring. Both (S)-OH (17h) and (R)-OH (17m) groups maintained highly potent agonistic activity. On the other hand, 3-oxopyrrolidine derivative 17n was less potent than 17b. Based on these results, it was believed that a hydrogen donator at the 3-position on the pyrrolidine ring could be responsible

a b

Compd

X

AR bindinga IC50b (nM)

AR reportera EC50b (nM)

18 17a A

CO2H CN NO2

>10,000 2.5 2.5

— 2.5 41

Human AR was used. IC50 and EC50 values shown are the mean values of duplicate measurements.

Table 2 Effect of substitutions on the pyrrolidine ring R 2' R2 N

R 1' R1

CN

a b

0

Compd

R1

R1

17b 17c 17d 17e 17f 17g 17l 17h 17m 17n 17i 17j 17k 17a

Me H Me Et i-Pr CONH2 CN Me Me Me Me Me Me H

H Me Me H H H H H H H H H H H

0

R2

R2

AR bindinga IC50b (nM)

AR reportera EC50b (nM)

H H H H H H H OH H

H H H H H H H H OH

@O (S)-CH2OH (S)-C(Me)2OH (S)-CO2Me H

H H H H

0.84 20 7 1.1 1.4 34 1 2.2 2.4 37 4.1 1.4 7.5 2.5

0.19 >1000 21 0.24 2 21 160 0.25 0.32 2.2 1.2 45 >1000 2.5

Human AR was used. IC50 and EC50 values shown are the mean values of duplicate measurements.

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for the agonistic activity. Replacement of the OH group with CH2OH (17i) resulted in a 5-fold decrease in the agonistic activity relative to that of 17h. Furthermore, introduction of a dimethyl group (17j) caused a 38-fold decrease in the agonistic activity, which indicated that distance and steric hindrance should also be important for agonistic activity together with the hydrogen-donating ability of the OH group. As additional examinations, incorporation of more polar substituents such as a methoxycarbonyl (17k) group resulted in marked decreases in the agonistic activity. 3.2. Tissue selectivity in vivo (3-week-old immature rats, 0.25 mg/kg, bid, 4-day treatment) Tissue selectivity was investigated using the Hershberger assay.17 In this assay, the weight of the levator ani muscle was used as an indicator of anabolic effects, and the weights of the prostate and seminal vesicle were used as indicators of androgenic activity. We selected strong agonists 17h (0.15, 0.5, 1.5 mg/kg, bid, po) and

17m and a weak agonist 17j (0.15, 0.5, 1.5, 2.5 mg/kg, bid, po) in this in vivo study. These compounds were administered to immature castrated male SD rats (3 week old) for 4 days. To extrapolate this rat assay to a human study, testosterone propionate (TP, 0.5 mg/kg/day, sc, qd.) was administered along with the test compounds to complement adrenal testosterone, which is not produced in rats. In this type of study, TP was reported to have equivalent potency and efficacy for the prostate and levator ani muscle when dosed subcutaneously. Strong agonists 17h (EC50 = 0.25 nM) and 17m (EC50 = 0.32 nM) demonstrated tissue-selective in vivo pharmacological activity. They increased the weight of the levator ani muscle markedly at the minimal dose (0.15 mg/kg, bid). The effects were observed in a dose-dependent manner, and at a dose of 1.5 mg/kg (bid), they stimulated growth of the levator ani muscle up to 150% relative to that in TP-treated vehicle rats. On the other hand, these 2 compounds did not influence the weights of the prostate and seminal vesicle at the highest dose. A weak agonist 17j (EC50 = 45 nM) also

Figure 3. Tissue selectivity in vivo ⁄p 6 0.05 (Student’s t-test) when compared with the TP (0.5 mg/kg)-treated control group.

K. Aikawa et al. / Bioorg. Med. Chem. 23 (2015) 2568–2578

showed anabolic effects in a dose-dependent manner, however, it did not increase the levator ani muscle significantly at the dose of 0.15/mg/kg (bid). This result was consistent with the weak in vitro profiles. Compound 17j did not stimulate the prostate and seminal vesicle in the same manner as 17h and 17m. These results showed in vivo proof-of-concept for this series as tissue SARMs. 3.3. Sexual behavior induction assay To confirm the agonistic activity on the CNS, a sexual behavior induction assay was performed (Table 3). Selected fertile male rats were castrated to eliminate sexual behavior. After treatment with the compounds for 3 weeks, sexual behavior induction was confirmed by the pseudopregnancy rate in female rats. Compounds 17h and 17m induced sexual behavior at a dosage of 1.5 mg/kg (bid, po). Compound 17j also induced sexual behavior at a dosage of 2.5 mg/kg (bid, po). These results proved that these SARM compounds could act as androgen agonists on the CNS.

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the Hershberger assay was not because of low concentration in the prostate. 4. Conclusion With the aim of developing SARMs, we modified hit compound A on the basis of information obtained from a docking model study using AR. 1-(4-Cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j were discovered and showed highly potent AR agonistic activities. As a result of further investigation, they showed good tissue selectivity in the Hershberger assay, increasing levator ani muscle dose dependently, whereas showing no influence on the prostate and seminal vesicle. These compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS. Further optimization studies using this series of compounds are under way, and the results will be reported in due course. 5. Experimental section

3.4. Tissue distribution of 17j

5.1. Chemistry

To determine the reason for tissue selectivity in vivo, we checked the tissue distribution of compound 17j (male SD rats, 5 mg/kg, po, Fig. 4). The data showed that the order of concentration of the compound was brain > plasma = prostate > levator ani muscle and did not change throughout the experiment. The data demonstrated that the reason for no influence on the prostate in

Melting points were determined on a Büchi melting point apparatus and were not corrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Gemini-200 (200 MHz), a Varian Mercury-300 (300 MHz), or a Bruker DPX300 (300 MHz) instrument. Chemical shifts are reported as d values (ppm) downfield from internal tetramethylsilane of the indicated solution. Peak multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; qui, quintet; dd, doublet of doublet; ddd, doublet of doublet of doublets; dt, doublet of triplet; br s, broad singlet; m, multiplet. Coupling constants (J values) are given in Hertz (Hz). Elemental analyses were performed by Takeda Analytical Laboratories. Reaction progress was determined by thin layer chromatography (TLC) analysis on silica gel 60 F254 plates (Merck) or NH TLC plates (Fuji Silysia Chemical Ltd). Chromatographic purification was performed on silica gel columns 60 (0.063–0.200 mm or 0.040–0.063 mm, Merck), basic silica gel (ChromatorexNH, 100–200 mesh, Fuji Silysia Chemical Ltd), or Purif-Pack (SI 60 lM or NH 60 lM, Fuji Silysia, Ltd). Commercial reagents and solvents were used without additional purification. Mass spectra (MS) were acquired using an Agilent LC/MS system (Agilent 1200SL/Agilent 6130MS), Shimadzu LC/MS system (LC-10ADvp high-pressure gradient system/LCMS-2010A), or Shimadzu UFLC/MS (Prominence UFLC high-pressure gradient system/LCMS-2020) operating in the electron spray ionization mode (ESI+). The column used was an L-column 2 ODS (3.0  50-mm I.D., 3 lm, CERI, Japan) with a temperature of 40 °C and a flow rate of 1.2 or 1.5 mL/min. Mobile phase A was 0.05% TFA in ultrapure water. Mobile phase B was 0.05% TFA in acetonitrile, which was increased linearly from 5% to 90% over 2 min and maintained at 90% over the next 1.5 min, after which the column was equilibrated to 5% for 0.5 min. Compounds 17a–k, 17h0 , 17m0 , and 18 were confirmed to be >95% pure by either LC/MS or elemental analysis. The yields were not optimized. The following abbreviations are used. DMSO = dimethyl sulfoxide, Et2O = diethyl ether, EtOAc = ethyl acetate, MeOH = methanol, IPE = diisopropyl ether, THF = tetrahydrofuran, TFA = trifluoroacetic acid.

Table 3 Sexual behavior induction assay Compd

17h 17m 17j *

Dose (mg/kg, bid, po for 21 days) 0

0.5

1.5

2.5

3.5

5

0 0 0

44* 38 —

70* 67* —

— — 57*

— — 57*

80* 67* 50*

p 6 0.05 (Steel test) when compared with the vehicle-treated control group.

Figure 4. Tissue distribution of 17j.

5.1.1. tert-Butyl (2R)-2-vinylpyrrolidine-1-carboxylate (2) To a mixture of tert-butyl (2R)-2-(hydroxymethyl)pyrrolidine1-carboxylate 1 (5.10 g, 25.3 mmol) in DMSO (35 mL) were added triethylamine (12.1 mL, 86.8 mmol) and sulfur trioxide pyridine complex (13.8 g, 86.7 mmol) at 10 °C. After stirring for 2.5 h, the mixture was poured into ice water and extracted with CH2Cl2.

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The separated organic layer was washed with 50% aqueous citric acid, saturated aqueous NaHCO3, and brine; dried over MgSO4; and concentrated in vacuo to give the crude product as a colorless oil (2.30 g, 11.5 mmol, 46%). A mixture of NaH (60%, oil dispersion) (402 mg, 10.1 mmol) in DMSO (10 mL) was stirred at 55 °C for 1 h, followed by addition of methyl triphenylphosphonium bromide (3.59 g, 10.5 mmol) in DMSO (15 mL). After stirring for 45 min, the mixture was cooled to room temperature. Then, the mixture was added to the crude product (2.00 g, 10.0 mmol) in DMSO (30 mL). The mixture was stirred at room temperature for 15 h. The mixture was poured into water and extracted with CH2Cl2. The organic layer was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 2 (191 mg, 0.968 mmol, 9.6%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 1.44 (9H, s), 1.64–2.10 (4H, m), 3.34–3.45 (2H, m), 4.20–4.38 (1H, m), 5.03– 5.06 (2H, m), 5.66–5.80 (1H, m). 5.1.2. tert-Butyl (2S)-2-ethylpyrrolidine-1-carboxylate (3) The mixture of compound 2 (110 mg, 0.558 mmol) and 10% Pd/C (50% wet, 119 mg) in MeOH (3.0 mL) was stirred at room temperature under hydrogen atmosphere for 20 h. The mixture was filtered through a pad of Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 3 (80.0 mg, 0.401 mmol, 72%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 0.86 (3H, t, J = 7.5 Hz), 1.25–1.37 (1H, m), 1.46 (9H, s), 1.59–1.99 (5H, m), 3.26–3.74 (3H, m). 5.1.3. tert-Butyl (2S)-3-hydroxy-2-methyl-5-oxo-2,5-dihydro-1Hpyrrole-1-carboxylate (5) To a solution of N-(tert-butoxycarbonyl)-L-alanine 4 (1170 g, 6.17 mol) in CH2Cl2, 2,2-dimethyl-1,3-dioxane-4,6-dione (934 g, 6.48 mol) was added portionwise at 0 °C for 20 min. Then, 1,10 -carbonyldiimidazole (1200 g, 7.40 mol) was added portionwise to the mixture at 0 °C for 30 min, and the mixture was stirred at room temperature for 46 h. Thereafter, 5% aqueous KHSO4 (50 L) was added to the mixture at 0 °C, and the mixture was stirred at 0 °C for 10 min. The organic layer was separated, dried over MgSO4, and concentrated in vacuo. The residue was dissolved in EtOAc (15 L) and refluxed for 2 h. After cooling to 0 °C, 5% aqueous NaHCO3 (30 L) was added to the mixture. The aqueous layer was separated and acidified with citric acid (2000 g, 10.4 mol) (pH = 3–4). The mixture was extracted with EtOAc (10 L) 4 times. The organic layer was combined, washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was recrystallized from EtOAc to give 5 (699 g, 3.28 mol, 53%) as colorless solid. [a]D = +85.3° (c 0.521, MeOH). 1H NMR (300 MHz, CDCl3) d 1.51 (3H, d, J = 6.9 Hz), 1.57 (9H, s), 3.15–3.31 (2H, m), 4.42 (1H, q, J = 6.9 Hz). 5.1.4. tert-Butyl (2S,3S)-3-hydroxy-2-methyl-5-oxopyrrolidine1-carboxylate (6) To a solution of 5 (698 g, 3.27 mol) in CH2Cl2 (16 L), acetic acid (1630 mL, 28.4 mol) was added dropwise at 30 °C to 25 °C for 30 min. Then, sodium tetrahydroborate (9.85 g, 0.260 mol) was added portionwise to the mixture at 35 °C to 30 °C for 40 min, and the mixture was stirred at 0 °C for 18 h. Ice water (10 L) was added to the mixture, which was stirred for 10 min. The organic layer was separated, and NaCl (1000 g) was added to the aqueous layer, followed by extraction with EtOAc (10 L) 3 times. The organic layer was combined, dried over MgSO4, and concentrated in vacuo. To a solution of the residue in CH2Cl2 (16 L), acetic acid (1630 mL, 28.4 mol) was added dropwise at 30 °C to 25 °C for 30 min. Then, sodium tetrahydroborate (9.85 g, 0.260 mol) was added portionwise to the mixture at 30 °C to 30 °C for 40 min, and the mixture was stirred at 0 °C for 18 h. Ice water (10 L) was added

to the mixture, which was stirred for 10 min. The organic layer was separated, and NaCl (1000 g) was added to the aqueous layer, followed by extraction with EtOAc (10 L) 3 times. The organic layer was combined, dried over MgSO4, and concentrated in vacuo. The residue (700 g, 3.25 mol, 99%) was recrystallized from IPE to give 6 (460 g, 2.14 mol, 65%) as colorless solid. 1H NMR (300 MHz, CDCl3) d 1.33 (3H, d, J = 6.6 Hz), 1.53 (9H, s), 2.58 (1H, dd, J = 17.1, 8.7 Hz), 2.71 (1H, dd, J = 17.1, 8.7 Hz), 4.21–4.30 (1H, m), 4.46–4.54 (1H, m). 5.1.5. (4S,5S)-4-Hydroxy-5-methylpyrrolidin-2-one (7) To a solution of 6 (459 g, 2.13 mol) in EtOAc (4.6 L) was added 4 M HCl–EtOAc solution (1600 mL, 6.40 mol). After stirring at room temperature for 1 h, the mixture was concentrated in vacuo. The residue was dissolved in MeOH (800 mL), and Amberlyst A-21 [1070 g, previously washed with MeOH (3 L) 3 times to remove water] was added. After stirring for 2 h at room temperature, Amberlyst A-21 was filtered off and washed with MeOH (1.5 L) 4 times. The filtrate was combined and concentrated in vacuo. The residue was purified using basic silica gel column chromatography (EtOAc/MeOH = 4:1) and recrystallized from IPE to give 7 (207 g, 1.80 mol, 84%) as colorless crystals. Mp 139–140 °C. 1H NMR (300 MHz, DMSO-d6) d 1.03 (3H, d, J = 6.6 Hz), 1.95 (1H, dd, J = 16.5, 3.3 Hz), 3.52–3.61 (1H, m), 4.10–4.17 (1H, m), 4.93 (1H, d, J = 5.1 Hz), 7.50 (1H, br s). Anal. Calcd for C5H9NO2: C, 52.16; H, 7.88; N, 12.17. Found: C, 52.18; H, 7.83; N, 12.10. 5.1.6. (2S,3S)-2-Methylpyrrolidin-3-ol (8a) To a solution of 7 (40.0 g, 0.347 mol) in THF (1.6 L), sodium bis(2-methoxyethoxy)aluminum dihydride (70% toluene solution, 388 g, 1.34 mol) was added dropwise at room temperature for 20 min, and the mixture was stirred at 85 °C for 3 h. After cooling to 0 °C, sodium carbonate decahydrate (159 g, 0.556 mol) was added portionwise for 20 min, and the mixture was stirred at room temperature overnight. Insoluble portions were filtered off through a pad of Celite and washed with THF. The filtrate was concentrated in vacuo to give 8a (35.0 g, 0.346 mol, 100%) as a brown oil. 1H NMR (300 MHz, DMSO-d6) d 0.99 (3H, d, J = 6.6 Hz), 1.50–1.60 (1H, m), 1.78–1.89 (1H, m), 2.49–2.66 (2H, m), 2.89–2.97 (1H, m), 3.83–3.87 (1H, m). 5.1.7. Methyl 1-acetylcyclopropanecarboxylate (11) A mixture of methyl 3-oxobutanoate (1650 g, 14.2 mol) and potassium carbonate (5880 g, 42.6 mol) in acetone (13.7 L) was refluxed for 30 min. Then, 1,2-dibromoethane (4000 g, 21.3 mol) was added dropwise to the mixture for 1 h at room temperature, and the mixture was refluxed for 60 h. After cooling to room temperature, insoluble portions were filtered off through a pad of Celite and washed with Et2O. The filtrate was concentrated in vacuo, and the residue was distilled to give 11 (983 g, 6.92 mol, 49%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 1.47 (4H, s), 2.46 (3H, s), 3.74 (3H, s). 5.1.8. Methyl (2S,3S)-2-methyl-1-((1S)-1-phenylethyl)pyrrolidine3-carboxylate (14) The mixture of 11 (983 g, 6.91 mol), (1S)-1-phenylethanamine (838 g, 6.91 mol), and p-toluenesulfonic acid monohydrate (65.8 g, 0.346 mol) in toluene (12.3 L) was refluxed for 40 h with a Dean–Stark trap. The mixture was cooled to room temperature, and the insoluble portions were filtered off through a pad of Celite. The filtrate was concentrated in vacuo to give 13. PtO2 (6.39 g, 28.1 mol) was added to a solution of 13 in MeOH (7.0 L), and the mixture was stirred at room temperature under hydrogen atmosphere for 60 h. The insoluble portions were filtered off through a pad of Celite, and the filtrate was concentrated in vacuo. The residue was purified using basic silica gel column

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chromatography (Hexane/EtOAc = 9:1) and washed with hexane to give 14 (643 g, 2.60 mol, 38%) as a colorless solid. 1H NMR (300 MHz, CDCl3) d 0.79 (3H, d, J = 6.3 Hz), 1.35 (3H, d, J = 6.9 Hz), 1.82–1.93 (1H, m), 2.11–2.24 (1H, m), 2.54 (1H, t, J = 7.2 Hz), 2.70 (1H, td, J = 9.3 and 3.9 Hz), 3.02–3.15 (1H, m), 3.44–3.62 (2H, m), 3.67 (3H, s), 7.21–7.36 (5H, m). 5.1.9. 2-((2S,3S)-2-Methyl-1-((1S)-1-phenylethyl)pyrrolidin-3-yl) propan-2-ol (15a) To a solution of 14 (643 g, 2.60 mol) in dry THF (9.19 L), 3 M methylmagnesium bromide diethyl ether solution (2570 mL, 7.72 mol) was added dropwise at 78 °C under argon atmosphere. After stirring for 30 min, the mixture was warmed to room temperature and stirred for 3 h. Saturated aqueous NH4Cl (15 L) was added to the mixture and extracted with EtOAc (18 L). The organic layer was washed with brine (15 L), dried over MgSO4, and concentrated in vacuo to give 15a (643 g, 2.60 mol, 100%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 1.03 (3H, d, J = 6.6 Hz), 1.22 (3H, s), 1.32 (3H, s), 1.34 (3H, d, J = 6.3 Hz), 1.70–1.89 (2H, m), 2.11–2.19 (1H, m), 2.44–2.58 (2H, m), 3.38 (1H, qui, J = 6.3 Hz), 3.60 (1H, q, J = 6.6 Hz), 7.19–7.35 (5H, m). 5.1.10. 2-((2S,3S)-2-Methylpyrrolidin-3-yl)propan-2-ol 0.5 oxalic acid salt (8b) To a solution of 15a (710 g, 2.87 mol) in MeOH (10.2 L) was added 10% Pd/C (50% wet, 56.0 g), and the mixture was stirred at room temperature under hydrogen atmosphere for 40 h. The insoluble portions were filtered off through a pad of Celite, and the filtrate was concentrated in vacuo. The residue was dissolved in MeOH (5.0 L), and oxalic acid (164 g, 1.82 mol) was added. The mixture was concentrated in vacuo, and the residue was recrystallized in Et2O to give 8b (433 g, 2.30 mol, 80%) as a colorless solid. Mp 120–122 °C. 1H NMR (300 MHz, DMSO-d6) d 1.12–1.13 (6H, m), 1.17 (3H, s), 1.74–2.08 (3H, m), 3.01–3.19 (2H, m), 3.56–3.65 (1H, m). Anal. Calcd for C8H17NO20.5C2H4O20.5H2O: C, 54.80; H, 9.71; N, 7.10. Found: C, 54.43; H, 9.44; N, 6.76. 5.1.11. ((2S,3S)-2-Methyl-1-((1S)-1-phenylethyl)pyrrolidin-3-yl) methanol (15b) To a mixture of 14 (740 mg, 2.99 mmol) in THF (8.0 mL) was added lithium aluminum hydride (114 mg, 3.00 mmol) at 0 °C, and the mixture was stirred at 0 °C for 3 h. Then, water (0.11 mL), 25% aqueous potassium hydroxide (0.11 mL), and water (0.33 mL) were added to the mixture at 0 °C, and the mixture was stirred at room temperature for 15 h. The insoluble portions were filtered off through a pad of Celite, and the filtrate was concentrated in vacuo to give 15b (656 mg, 2.99 mmol, 100%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 1.14 (3H, d, J = 6.3 Hz), 1.29 (3H, d, J = 6.9 Hz), 1.67–1.79 (1H, m), 1.85–1.97 (1H, m), 2.04–2.09 (1H, m), 2.36–2.46 (1H, m), 2.62 (1H, td, J = 9.9, 3.6 Hz), 2.87–2.95 (1H, m), 3.47 (1H, dd, J = 9.9, 3.3 Hz), 3.86– 3.99 (2H, m), 7.22–7.33 (5H, m). 5.1.12. ((2S,3S)-2-Methylpyrrolidin-3-yl)methanol (8c) A mixture of 15b (581 mg, 2.65 mmol) and 10% Pd/C (50% wet, 564 mg) in MeOH (9.0 mL) was stirred at room temperature under hydrogen atmosphere for 20 h. The insoluble portions were filtered off through a pad of Celite, and the filtrate was concentrated in vacuo to give 8c (260 mg, 2.26 mmol, 85%) as a pale yellow oil. 1 H NMR (300 MHz, CDCl3) d 1.23 (3H, d, J = 6.6 Hz), 1.76–1.87 (1H, m), 1.96–2.10 (2H, m), 2.82–2.90 (1H, m), 3.06–3.23 (2H, m), 3.56 (1H, dd, J = 10.2, 4.2 Hz), 3.81 (1H, dd, J = 10.2, 4.2 Hz). 5.1.13. 4-(Pyrrolidin-1-yl)-1-naphthonitrile (17a) A mixture of pyrrolidine (0.127 mL, 1.52 mmol), 4-fluoro-1naphthonitrile 16 (200 mg, 1.17 mmol), and potassium carbonate

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(484 mg, 3.51 mmol) in DMSO (10 mL) was stirred at 100 °C for 20 h. The mixture was cooled to room temperature, poured into water, and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography and recrystallized from EtOAc–hexane to give 17a (187 mg, 0.840 mmol, 72%) as pale yellow crystals. Mp 109–110 °C. 1H NMR (200 MHz, CDCl3) d 2.01–2.08 (4H, m), 3.59–3.66 (4H, m), 6.69 (1H, d, J = 8.4 Hz), 7.39–7.48 (1H, m), 7.55–7.62 (1H, m), 7.72 (1H, d, J = 8.0 Hz), 8.13–8.17 (1H, m), 8.26 (1H, d, J = 8.2 Hz). IR (KBr) 2203, 1563, 1518 cm1. Anal. Calcd for C15H14N2: C, 81.05; H, 6.35; N, 12.60. Found: C, 80.99; H, 6.33; N, 12.47. 5.1.14. 4-((2S)-2-Methylpyrrolidin-1-yl)-1-naphthonitrile (17b) To a mixture of tert-butyl (2S)-2-methylpyrrolidine-1-carboxylate (1.25 g, 6.75 mmol) in toluene (2.0 mL) was added TFA (4.0 mL) and the mixture was stirred at room temperature for 5 h. The mixture was concentrated in vacuo, and the residue was dissolved in DMSO (10 mL). 4-Fluoro-1-naphthonitrile 16 (0.855 g, 5.00 mmol) and potassium carbonate (2.80 g, 20.2 mmol) were added to the solution, and the mixture was stirred at 100 °C for 5 h. The mixture was cooled to room temperature, poured into water, and extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography and washed with hexane to give 17b (0.780 g, 3.30 mmol, 66%) as a pale yellow solid. Mp 73–74 °C. [a]D = 251.5° (c 0.470, MeOH). 1 H NMR (200 MHz, CDCl3) d 1.18 (3H, d, J = 5.8 Hz), 1.60–2.15 (3H, m), 2.20–2.40 (1H, m), 3.25–3.40 (1H, m), 3.90–4.15 (2H, m), 6.82 (1H, d, J = 8.0 Hz), 7.40–7.70 (2H, m) 7.76 (1H, d, J = 8.0 Hz), 8.15–8.25 (2H, m). IR (KBr) 2209, 1565, 1514, 1327, 763 cm1. Anal. Calcd for C16H16N2: C, 81.32; H, 6.82; N, 11.85. Found: C, 81.35; H, 6.87; N, 11.84. 5.1.15. 4-((2R)-2-Methylpyrrolidin-1-yl)-1-naphthonitrile (17c) The mixture of 2-mehtylpyrrolidine (3.00 g, 35.2 mmol), 16 (5.00 g, 29.2 mmol), and potassium carbonate (5.00 g, 36.2 mmol) in DMSO (30 mL) was stirred at 100 °C for 5 h. The mixture was cooled to room temperature, poured into water, and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 4-(2-methylpyrrolidin-1-yl)-1naphthonitrile (4.76 g, 20.1 mmol, 69%). 4-(2-Methylpyrrolidin-1yl)-1-naphthonitrile (1.08 g, 4.57 mmol) was resolved using HPLC to afford optically pure 17b (804 mg, 3.40 mmol, 48%) and 17c (805 mg, 3.41 mmol, 48%). [Column: CHIRALPAK AS (50 mm  500 mm); column temperature, 25 °C; mobile phase, hexane/EtOH = 97:3; flow rate, 60 mL/min; UV detection at 254 nm]. 17c [a]D = +257.7° (c 0.410, MeOH). Analytical HPLC showed 99.5% purity. 5.1.16. 4-(2,2-Dimethylpyrrolidin-1-yl)-1-naphthonitrile (17d) To a mixture of 1-benzyl-2,2-dimethylpyrrolidine (0.750 g, 3.96 mmol) in 1 M aqueous HCl (4.00 mL, 4.00 mmol) and MeOH (40 mL), 10% Pd/C (50% wet, 0.40 g) was added, and the mixture was stirred at room temperature under hydrogen atmosphere for 18 h. The insoluble portions were filtered off through a pad of Celite, and the filtrate was concentrated in vacuo. The residue was dissolved in DMSO (6.0 mL) and 16 (0.260 g, 1.52 mmol), and potassium carbonate (0.830 g, 6.01 mmol) was added. After stirring at 100 °C for 20 h, the mixture was cooled to room temperature, poured into water, and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 17d (46.0 mg, 0.184 mmol, 12%) as a yellow oil. 1H NMR (200 MHz, CDCl3) d 1.17 (6H, s), 1.90–2.15 (4H, m),

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7.34 (1H, d, J = 8.0 Hz), 7.48–7.70 (2H, m), 7.83 (1H, d, J = 8.0 Hz), 8.14–8.22 (1H, m), 8.42–8.49 (1H, m). IR (KBr) 2218, 1570 cm1. Analytical HPLC showed 98.0% purity. 5.1.17. 4-((2S)-2-Ethylpyrrolidin-1-yl)-1-naphthonitrile (17e) A solution of 3 (70.0 mg, 0.351 mmol) in 4 M HCl–EtOAc solution (1.00 mL, 4.00 mmol) was stirred at room temperature for 1.5 h. The mixture was concentrated in vacuo, and the residue was washed with hexane to give a colorless solid. The mixture of solid 16 (50.0 mg, 0.292 mmol) and potassium carbonate (104 mg, 0.753 mmol) in DMSO (1.0 mL) was stirred at 100 °C for 3 h. The mixture was cooled to room temperature, poured into water, and extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 17e (33.0 mg, 0.132 mmol, 38%, 24.4% ee), which was resolved using HPLC to afford optically pure 17e (16.0 mg, 63.9 lmol, 18%, 99.1% ee) as a yellow oil. [Column: CHIRALPAK AS (50 mm  500 mm); column temperature, 25 °C; mobile phase, hexane/EtOH = 95:5; flow rate, 60 mL/min; UV detection at 254 nm]. [a]D = 294.6° (c 0.330, MeOH). 1H NMR (300 MHz, CDCl3) d 0.90 (3H, t, J = 7.8 Hz), 1.30–1.45 (1H, m), 1.68–1.86 (3H, m), 1.95–2.05 (1H, m), 2.26–2.34 (1H, m), 3.32–3.38 (1H, m), 3.83–3.92 (1H, m), 3.95–4.03 (1H, m), 6.79 (1H, d, J = 8.1 Hz), 7.45 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.59 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.73 (1H, d, J = 8.1 Hz), 8.13–8.18 (2H, m). IR (KBr) 2963, 2209, 1564 cm1. Analytical HPLC showed 99.4% purity. 5.1.18. 4-((2R)-2-Isopropylpyrrolidin-1-yl)-1-naphthonitrile (17f) A method similar to that described for 17d using acetic acid instead of aqueous HCl was used to prepare this compound in 28% yield as a pale yellow oil. [a]D = 337.2° (c 0.776, MeOH). 1H NMR (200 MHz, CDCl3) d 0.81 (3H, d, J = 7.0 Hz), 0.94 (3H, d, J = 7.0 Hz), 1.60–2.20 (5H, m), 3.30–3.46 (1H, m), 3.88–4.08 (2H, m), 6.85 (1H, d, J = 8.8 Hz), 7.40–7.68 (2H, m), 7.73 (1H, d, J = 8.8 Hz), 8.10–8.22 (2H, m). IR (KBr) 2210, 1566 cm1. Analytical HPLC showed 98.9% purity. 5.1.19. 1-(4-Cyano-1-naphthyl)-D-prolinamide (17g) A method similar to that described for 17a was used to prepare this compound in 37% yield as colorless crystals. Mp 176–177 °C. [a]D = 194.6° (c 0.380, MeOH). 1H NMR (200 MHz, CDCl3) d 1.82–2.30 (3H, m), 2.50–2.72 (1H, m), 3.30–3.42 (1H, m), 4.10– 4.48 (2H, m), 5.29 (1H, br s), 6.38 (1H, br s), 6.97 (1H, d, J = 8.0 Hz), 7.50–7.75 (2H, m), 7.78 (1H, d, J = 8.0 Hz), 8.20–8.32 (2H, m). IR (KBr) 2210, 1690, 1568 cm1. Analytical HPLC showed 96.9% purity.

[a]D = 144.0° (c 0.270, MeOH). 1H NMR (DMSO-d6) d 1.10 (3H, d, J = 6.3 Hz), 1.86–1.93 (2H, m), 3.25–3.31 (1H, m), 3.96–4.04 (1H, m), 4.21–4.32 (2H, m), 6.89 (1H, d, J = 8.4 Hz), 7.55 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.70 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.89 (1H, d, J = 8.4 Hz), 7.97–8.00 (1H, m), 8.23–8.26 (1H, m). IR (KBr) 2228, 1223 cm1. Anal. Calcd for C16H16N2OH2SO41.2 H2O: C, 51.66; H, 5.53; N, 7.53. Found: C, 51.60; H, 5.52; N, 7.62. 5.1.22. 4-((2S,3S)-3-(Hydroxymethyl)-2-methylpyrrolidin-1-yl)1-naphthonitrile (17i) A method similar to that described for 17a was used to prepare this compound in 30% yield as a pale yellow solid. Mp 158–159 °C. [a]D = 258.9° (c 0.320, MeOH). 1H NMR (300 MHz, CDCl3) d 1.44 (3H, d, J = 6.3 Hz), 1.99–2.20 (3H, m), 2.55–2.66 (1H, m), 3.01–3.09 (1H, m), 3.79–4.02 (3H, m), 4.11 (1H, qui, J = 6.3 Hz), 6.96 (1H, d, J = 8.4 Hz), 7.54 (1H, ddd, J = 8.4, 6.9, 1.5 Hz), 7.65 (1H, ddd, J = 8.4, 6.9, 1.5 Hz), 7.82 (1H, d, J = 8.4 Hz), 8.14–8.21 (2H, m). IR (KBr) 2211, 1568, 1323 cm1. Anal. Calcd for C17H18N2O0.2H2O: C, 75.64; H, 6.87; N, 10.38. Found: C, 75.77; H, 6.83; N, 10.46. 5.1.23. 4-((2S,3S)-3-(2-Hydroxypropan-2-yl)-2-methylpyrrolidin1-yl)-1-naphthonitrile (17j) A method similar to that described for 17a using lithium carbonate instead of potassium carbonate was used to prepare this compound in 73% yield as a colorless solid. Mp 67 °C. [a]D = 229.0° (c 1.000, MeOH). 1H NMR (300 MHz, CDCl3) d 0.98 (3H, d, J = 6.6 Hz), 1.27 (1H, s), 1.34 (3H, s), 1.39 (3H, s), 2.13– 2.21 (2H, m), 2.49–2.58 (1H, m), 3.26–3.34 (1H, m), 3.76–3.84 (1H, m), 4.39–4.48 (1H, m), 6.89 (1H, d, J = 8.4 Hz), 7.49–7.54 (1H, m), 7.60–7.65 (1H, m), 7.77 (1H, d, J = 8.4 Hz), 8.10–8.13 (1H, m), 8.16–8.19 (1H, m). IR (KBr) 2211, 1569 cm1. Anal. Calcd for C19H22N2O: C, 77.52; H, 7.53; N, 9.52. Found: C, 77.50; H, 7.71; N, 9.52. 5.1.24. Methyl (2S,3S)-1-(4-cyano-1-naphthyl)-2methylpyrrolidine-3-carboxylate (17k) A method similar to that described for 17d was used to prepare this compound in 22% yield as a colorless oil. [a]D = 236.3° (c 0.408, MeOH). 1H NMR (200 MHz, CDCl3) d 1.03 (3H, d, J = 6.4 Hz), 2.00–2.52 (2H, m), 3.10–3.48 (2H, m), 3.76 (3H, s), 4.05–4.45 (2H, m), 6.93 (1H, d, J = 8.6 Hz), 7.50–7.70 (2H, m), 7.80 (1H, d, J = 8.0 Hz), 8.14–8.28 (2H, m). IR (KBr) 2213, 1737, 1570 cm1. Analytical HPLC showed 95.9% purity.

5.1.20. 4-((2S,3S)-3-Hydroxy-2-methylpyrrolidin-1-yl)-1naphthonitrile (17h) A method similar to that described for 17a was used to prepare this compound in 65% yield as a yellow oil. [a]D = 270.2° (c 0.618, MeOH). 1H NMR (200 MHz, CDCl3) d 1.25 (3H, d, J = 6.2 Hz), 1.93 (1H, d), 2.00–2.20 (2H, m), 3.18–4.02 (1H, m), 4.20–4.40 (1H, m), 4.40–4.58 (1H, m), 6.86 (1H, d, J = 8.0 Hz), 7.46–7.70 (2H, m), 7.78 (1H, d, J = 8.0 Hz), 8.12–8.28 (2H, m). IR (KBr) 2211, 1565, 1515 cm1. Anal. Calcd for C16H16N2O: C, 76.16; H, 6.39; N, 11.10. Found: C, 76.06; H, 6.41; N, 11.00.

5.1.25. 4-(Pyrrolidin-1-yl)-1-naphthoic acid (18) A mixture of 4-(pyrrolidin-1-yl)-1-naphthonitrile 17a (1.76 g, 7.92 mmol) and 2 M aqueous potassium hydroxide (2.70 mL, 5.40 mmol) in EtOH (2.7 mL) was stirred at 100 °C for 2 days. The insoluble portions were filtered off through a pad of Celite, and the filtrate was acidified with 1 M aqueous HCl. The mixture was extracted with EtOAc, washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was washed with Et2O to give 18 (17.0 mg, 70.5 lmol, 0.89%) as a brown solid. Mp 194 °C. 1H NMR (200 MHz, DMSO-d6) d 1.94–2.00 (4H, m), 3.48–3.54 (4H, m), 6.82 (1H, d, J = 8.4 Hz), 7.38–7.47 (1H, m), 7.50–7.58 (1H, m), 8.09 (1H, d, J = 8.4 Hz), 8.22–8.26 (1H, m), 9.05–9.09 (1H, m), 12.27 (1H, br s). Analytical HPLC showed 99.7% purity.

5.1.21. 4-((2S,3S)-3-Hydroxy-2-methylpyrrolidin-1-yl)-1naphthonitrile sulfate (17h0 ) To a solution of 17h (20.0 mg, 79.2 lmol) in EtOAc (1.0 mL), sulfuric acid (0.500 mL, 9.38 mmol) in EtOAc (0.3 mL) was added at room temperature. After stirring for 1 h, the mixture was concentrated in vacuo. The residue was recrystallized from EtOAc to give 17h0 (18 mg, 51.4 lmol, 65%) as a colorless solid. Mp 111–112 °C.

5.1.26. (2R)-1-(4-Cyano-1-naphthyl)pyrrolidine-2-carbonitrile (17l) To a solution of 17g (160 mg, 0.603 mmol) in CH2Cl2, trifluoroacetic anhydride (0.250 mL, 180 mmol) and triethylamine (0.560 mL, 4.02 mmol) were added, and the mixture was stirred at room temperature for 0.5 h. The mixture was basified with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer

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was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 17l (113 mg, 0.457 mmol, 76%) as a yellow powder. [a]D = +92.3° (c 0.39, MeOH). 1H NMR (200 MHz, CDCl3) d 2.00–2.72 (4H, m), 3.35–3.50 (1H, m), 3.65–3.82 (1H, m), 4.70– 4.80 (1H, m), 7.18 (1H, d, J = 8.0 Hz), 7.55–7.80 (2H, m), 7.88 (1H, d, J = 8.0 Hz), 8.04–8.15 (1H, m), 8.20–8.32 (1H, m). IR (KBr) 2216, 1573 cm1. Analytical HPLC showed 98.4% purity.

[a]D = 253.9° (c 0.270, MeOH). 1H NMR (300 MHz, CDCl3) d 1.17 (3H, d, J = 6.6 Hz), 2.69 (1H, ddd, J = 18.0, 7.5, 4.2 Hz), 2.79 (1H, dt, J = 18.0, 7.8 Hz), 3.15 (1H, ddd, J = 9.9, 7.8, 7.5 Hz), 3.89 (1H, q, J = 6.6 Hz), 4.07 (1H, ddd, J = 9.9, 7.8, 4.2 Hz), 7.09 (1H, d, J = 8.1 Hz), 7.63 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.71 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.89 (1H, d, J = 8.1 Hz), 8.24–8.28 (2H, m). IR (KBr) 2216, 1759, 1574 cm1. Anal. Calcd for C16H14N2O: C, 76.78; H, 5.64; N, 11.19. Found: C, 76.52; H, 5.63; N, 11.30.

5.1.27. 4-((2S,3R)-3-Hydroxy-2-methylpyrrolidin-1-yl)-1-naphthonitrile (17m) To a solution of 17h (1.00 g, 3.96 mmol), 3,4-dimethoxybenzoic acid (1.08 g, 5.95 mmol), triphenylphosphine (3.28 g, 12.5 mmol) in toluene (30 mL), and diethyl azodicarbonate (40% toluene solution, 5.17 g, 11.9 mmol) were added dropwise at room temperature under nitrogen atmosphere. After stirring at room temperature for 16 h, the insoluble portions were filtered off through a pad of Celite and washed with toluene. The filtrate was concentrated in vacuo and purified using silica gel column chromatography. The residue was washed with MeOH to give (2S,3R)-1-(4-cyano-1-naphthyl)-2-methylpyrrolidin-3-yl 3,4dimethoxybenzoate (975 mg, 2.34 mmol, 59%) as a colorless solid. To a solution of (2S,3R)-1-(4-cyano-1-naphthyl)-2-methylpyrrolidin-3-yl 3,4-dimethoxybenzoate (950 mg, 2.28 mmol) in MeOH (35 mL) and THF (9 mL), 1 M aqueous sodium hydroxide (6.84 mL, 6.84 mmol) was added, and the mixture was stirred at room temperature for 1 h. The mixture was poured into brine and extracted with IPE twice. The organic layer was combined, washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 17m (549 mg, 2.18 mmol, 95%) as a pale yellow oil. [a]D = 268.6° (c 0.515, MeOH). 1H NMR (200 MHz, CDCl3) d 1.15(3H, d, J = 6.2 Hz), 1.80–2.20 (2H, m), 2.0–2.50 (1H, m), 3.20– 3.38 (1H, m), 3.77–4.00 (2H, m), 4.10–4.30 (1H, m), 6.89 (1H, d, J = 8.0 Hz), 7.46–7.68 (2H, m), 7.19 (1H, d, J = 8.0 Hz), 8.14–8.26 (2H, m). IR (KBr) 2211, 1567, 1514 cm1. Analytical HPLC showed 99.0% purity.

5.2. Biology

5.1.28. 4-((2S,3R)-3-Hydroxy-2-methylpyrrolidin-1-yl)-1-naphthonitrile methanesulfonate (17m0 ) To a solution of 17m (280 mg, 1.11 mmol) in THF (1.0 mL), methanesulfonic acid (72.0 lL, 1.11 mmol) was added at room temperature. Et2O was added to the mixture, and the residual solid was collected by filtration to give 17m0 (205 mg, 0.588 mmol, 53%) as a colorless solid. Mp 107–108 °C. [a]D = 174.5° (c 0.350, MeOH). 1H NMR (300 MHz, CDCl3) d 1.44 (3H, d, J = 6.3 Hz), 2.40–2.50 (1H, m), 2.80–2.90 (1H, m), 2.89 (3H, s), 3.98–4.36 (3H, m), 4.58–4.64 (1H, m), 7.83–7.94 (3H, m), 8.02 (1H, d, J = 7.8 Hz), 8.37–8.40 (1H, m), 8.68–8.71 (1H, m). IR (KBr) 3320, 2228, 1194 cm1. Anal. Calcd for C16H16N2OCH3SO3H0.1H2O: C, 58.30; H, 5.81; N, 8.00. Found: C, 58.13; H, 5.77; N, 7.97. 5.1.29. 4-((2S)-2-Methyl-3-oxopyrrolidin-1-yl)-1-naphthonitrile (17n) To a mixture of DMSO (0.220 mL, 3.10 mmol) and CH2Cl2 (4.0 mL), oxalyl chloride (0.140 mL, 1.61 mmol) was added at 78 °C under argon atmosphere. After stirring for 10 min, a solution of 17m (210 mg, 0.832 mmol) in CH2Cl2 (2.0 mL) was added dropwise to the mixture. The mixture was stirred for 15 min, followed by addition of triethylamine (0.570 mL, 4.09 mmol). After stirring at 78 °C for 10 min, the mixture was warmed to room temperature. The mixture was poured into water and extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified using silica gel column chromatography to give 17n (150 mg, 0.599 mmol, 73%) as a colorless solid. Mp 113–114 °C.

5.2.1. AR-binding inhibitory test To a solution containing a wild-type AR, radiolabeled mibolerone (3 nM) and a compound were added, and the mixture was incubated at 4 °C for 3 h. Bound (B) and free (F) compounds were separated using the dextran/charcoal (DCC) method. The label count of B was measured, and the inhibitory rate of the compound was calculated. 5.2.2. Compound evaluation in the AR reporter assay Cos-7 cells (at a density of 5  106) were sown in a 150-cm2 flask and grown in a culture medium [DMEM medium containing 10% DCC–fetal bovine serum (FBS) and 2 mM glutamine] for 24 h. Vector DNA containing AR genes and vector DNA containing the luciferase gene bound at the downstream of an androgenresponsive promoter derived from mouse mammary tumor virus (MMTV) were co-transfected using a liposome method. After culturing for 4 h, the cells were harvested, and 10,000 cells were plated in a 96-well plate and cultured for 3 h. In the agonistic assay, DHT or a compound was added, and the cells were further cultured for 24 h, after which the luciferase activity was measured. In the antagonistic assay, DHT (0.1 lM) and a compound were added, and the cells were further cultured for 24 h, after which the luciferase activity was measured. The rate of inhibition by the compound was calculated by setting the luciferase activity induced by the addition of DHT (0.1 lM) as 100. 5.2.3. Animals Male CD(SD)IGS rats at the age of 4–8 weeks were purchased from Charles River Japan. Female Wistar Imamichi rats at the age of 8 weeks were purchased from CLEA Japan, Inc. These animals were maintained on a 12-h/12-h light/dark cycle (lights on at 0800 h) with constant temperature (25 °C). Food and water were available ad libitum. Vaginal smears of female rats were examined every morning, and animals that had 4-day regular menstrual cycles were selected for sexual behavior experiments. Bilateral orchidectomies were performed under ether anesthesia. 5.2.4. Reagents Chemicals and solvents were of reagent grade. The compounds were suspended in 0.5% methylcellulose (Metlose, Shin-Etsu Chemical Co., Ltd, Japan) aqueous solution for oral administration or dissolved in corn oil (Nippon Shokuhin Kako, Ltd, Japan) containing 20% benzyl benzoate (Wako Pure Chemical Industries, Ltd, Japan) for subcutaneous administration. 5.2.5. Hershberger assays with immature rats17 Male CD(SD)IGS rats at 4 weeks of age were used for assessing the tissue-specific action of compounds. The rats were castrated 5 days before treatment with the compounds started. The compounds were administered twice a day for 4 days consecutively by the oral route. All rats were also treated with 0.5 mg/kg/day of testosterone. On the next day after the last administration, the animals were sacrificed by collecting blood from the heart under ether anesthesia, and ventral and dorsal prostates, seminal vesicles with their fluid (both sides), and levator ani muscles were

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extirpated for weighing. 17h0 and 17m0 were used on behalf of 17h and 17m, respectively. The amounts of 17h0 and 17m0 were calculated for the dose shown in Figure 3 as the free form, 17h and 17m. 5.2.6. Sexual behavior tests Male CD(SD)IGS rats were castrated at the age of 9 weeks, and subcutaneous implantation of a DHEA pellet (1.5 mg, 21-day release; Innovative Research of America, Sarasota, FL) was performed under ether anesthesia at the age of 12 weeks. On the next day after the pellet transplantation, treatment with the compounds was started. Tests for sexual behavior were started 18–21 days after first administration of the compounds. The following testing procedure was used. Each male was placed in a plastic cage. Five minutes later, a proestrus female rat at 16 weeks of age that had regular 4-day menstrual cycles was introduced, and the pair was housed in the cage together overnight. Vaginal smears were examined daily for at least 9 days after mating to determine the pseudopregnancy rate. Acknowledgments We thank Dr. Tomoyasu Ishikawa for helpful discussions; Takashi Santou for the AR binding assay and reporter gene assay; and Yumiko Akinaga, Tsuneo Masaki, Hiromi Shinohara, Megumi Morimoto, Hideo Araki, and Kazuyo Nakamura for performing the biological assays.

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