Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine

Article pubs.acs.org/jmc

Discovery of a 1‑Methyl-3,4-dihydronaphthalene-Based Sphingosine1-Phosphate (S1P) Receptor Agonist Ceralifimod (ONO-4641). A S1P1 and S1P5 Selective Agonist for the Treatment of Autoimmune Diseases Haruto Kurata,*,† Kensuke Kusumi,† Kazuhiro Otsuki,† Ryo Suzuki,† Masakuni Kurono,† Takaki Komiya,‡ Hiroshi Hagiya,‡ Hirotaka Mizuno,‡ Hiroki Shioya,‡ Takeji Ono,‡ Yuka Takada,‡ Tatsuo Maeda,‡ Norikazu Matsunaga,§ Tetsu Kondo,§ Sachiko Tominaga,§ Ken-ici Nunoya,§ Hidekazu Kiyoshi,∥ Masaharu Komeno,∥ Shinji Nakade,‡ and Hiromu Habashita† †

Medicinal Chemistry Research Laboratories, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan ‡ Exploratory Research Laboratories and §Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., 17-2 Wadai, Tsukuba, Ibaragi 300-4247, Japan ∥ Safety Research Laboratories, Ono Pharmaceutical Co., Ltd., 50-10 Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan S Supporting Information *

ABSTRACT: The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, is described. While it has been revealed that the modulation of the S1P1 receptor is an effective way to treat autoimmune diseases such as relapsing−remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P3 receptor is implicated in some undesirable effects. We carried out a structure−activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P1 over S1P3 and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing).

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INTRODUCTION Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that mediates a wide variety of biological responses through the S1P1−5 receptors.1 During the course of the clinical development of 1 (fingolimod, FTY720)2 (Figure 1), which is now on the market as the first orally active medicine for the treatment of relapsing−remitting multiple sclerosis (RRMS), it was reported that its phosphorylated metabolite 2 is a S1P1,3,4,5 pan agonist2c and the S1P1 receptor plays a critical role in sequestering peripheral lymphocytes in the lymph nodes and thymus to prevent lymphocytes from entering into inflammatory tissues.3 Transient bradycardia was reported4 as one of the © 2017 American Chemical Society

adverse events in the clinical studies of 1, and the S1P3 receptor was thought to be implicated in the heart rate reduction observed in rodents.5 On the basis of these observations, many S1P3-sparing S1P1 receptor agonists have been reported6 and several have entered into clinical trials7 including 3 (siponimod, BAF312),8 4 (ponesimod, ACT-128800),9 2-amino-2-[2-[2chloro-4-[[3-(phenylmethoxy)phenyl]thio]phenyl]ethyl]-1,3propanediol hydrochloride (KRP-203),10 1-{5-[(3R)-3-amino4-hydroxy-3-methylbutyl]-1-methyl-1H-pyrrol-2-yl}-4-(4-methReceived: May 29, 2017 Published: November 9, 2017 9508

DOI: 10.1021/acs.jmedchem.7b00785 J. Med. Chem. 2017, 60, 9508−9530

Journal of Medicinal Chemistry

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Figure 1. Selected examples of S1P1 agonists in clinical trials and on the market (excerpt from the literature7).

Figure 2. Our early work to identify a central dihydronaphthalene core.

ylphenyl)-1-butanone (CS-0777),11 5 (ozanimod, RPC1063),12 and (R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)acetic acid (etrasimod, APD334)13 (Figure 1). Among them, we discovered 1({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641),14 a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, which showed potential for the treatment of RRMS in phase 2 proof of concept studies. It was reported that S1P3-sparing S1P1 receptor agonists including 13n can induce transient bradycardia in humans.7,14c Nevertheless, S1P3-sparing S1P1 agonists should be a safer option than a nonselective S1P receptor agonist for the treatment of autoimmune diseases such as RRMS because the activation of the S1P3 receptor has been reported to be implicated in undesirable effects such as bronchoconstriction and blood pressure increase.15 Herein, we describe the discovery, synthesis, and detailed structure− activity relationship (SAR) study of 13n. We first identified hit compound 6 by screening our proprietary library.16 Compound 6 with a β-alanine moiety in the hydrophilic head region and a 5-phenylpentyl moiety in the lipophilic tail region showed 26-fold selectivity for S1P1 over S1P3 in a Ca2+ influx assay and had an ED50 of 26 mg/kg (4 h after oral dosing) in a peripheral lymphocyte lowering (PLL) test in mice. Subsequently, the central region and hydrophilic

amino acid region of hit compound 6 were constrained by a double bond and ring closures to investigate conformational preferences for the S1P1 receptor and the S1P3/S1P1 selectivity, which led to a selective and potent lead compound with a central dihydronaphthalene core in our early work.17 Further, a SAR study of the lipophilic tail region revealed that incorporation of both a fluorine at the 4-position on the terminal phenyl ring in the 3-phenylpropyloxy moiety linked to the 6-position of the dihydronaphthalene core and a methyl group at the 2-position on the propylene linker improved the potency toward the S1P1 receptor, the ED50 result from the PLL test, and the selectivity for S1P1 over S1P3. As a result, compound 7 was identified with a preferred (S)-methyl substituent at the above-mentioned 2-position and exhibited 26 000-fold selectivity for S1P1 over S1P3 and was potent in the PLL test in mice (ED50 = 0.22 mg/kg, 24 h after oral dosing)18 (Figure 2). This outcome encouraged us to further optimize the lipophilic tail region in the dihydronaphthalene series leading to the discovery of a clinical candidate that was more potent than 7 in the PLL test while maintaining or elevating the S1P3/S1P1 selectivity of 7.

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RESULTS AND DISCUSSION Chemistry. In general, amino acid derivatives with the general structure 13 were prepared by using 6-hydroxyl-1methyldihydronaphthalene-2-aldehyde derivative 8 as the key

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Scheme 1. General Synthetic Route for Amino Acid Derivatives 13

Scheme 2. Synthesis of Benzyl Alcohols 9f−k with Isobutyl Substituents at the 4-Position on the Benzylic Phenyl Ringa

Reagents and conditions: (a) BH3·THF complex, THF, 0 °C to rt, overnight, quantitative yield; (b) Tf2O, pyridine, DCM, 0 °C, 20 min to 1 h, 97% to quantitative yield; (c) i-BuMgBr/Et2O, cat. Fe(acac)3, THF, NMP, 0 °C to rt or rt, 0.5−3.5 h, 50% to quantitative yield; (d) t-BuLi, TMEDA, nhexane, −78 to 0 °C, 4 h and then DMF, THF, 0 °C, 4 h, 31%; (e) NaBH4, MeOH, 0 °C, 1.5 h, quantitative yield; (f) LiAlH4, THF, 0 °C, 10 min to 1 h, 46−75% in two steps; (g) SOCl2, MeOH, 0 °C to rt, 2.5 days, quantitative yield. a

reagent in the presence of a catalytic amount of Fe(acac)3.19 The resulting 1-methoxy-3-isobutylbenzene 17 was orthoformylated using t-BuLi/TMEDA in n-hexane followed by the addition of DMF to give 18 in 31% yield, followed by quantitative conversion to alcohol 9g after reaction with NaBH 4 . Alcohols 9h and 9i were derived from the corresponding methyl 4-hydroxybenzoates 19h and 19i by reaction with Tf2O to yield the aryl triflate derivatives 20h and 20i followed by the reaction with the isobutyl Grinard reagents to yield the methyl 4-isobutylbenzoate derivatives 21h and 21i and then reaction with LiAlH4. Alcohols 9j and 9k were synthesized using the same reaction conditions employed to prepare the alcohols 9h and 9i using methyl and ethyl 4chlorobenzoates 23j and 23k, respectively, instead of the aryltriflates as substrates.

intermediate (Scheme 1). The intermediate 8 was converted to 6-alkoxy-1-methyldihydronaphthalene-2-aldehyde derivative 11 by the reaction with alcohol 9 (route A) or alkyl halide 10 derived from alcohol 9 (route B) under Mitsunobu and basic conditions, respectively. The aldehyde 11 was subsequently converted to amino acid derivative with the general structure 13 by reductive alkylation with 3-azetidinecarboxylic acid (route C) or reductive alkylation with methyl 3-azetidine carboxylate followed by saponification of the resulting esters (routes D and E). Benzyl alcohol derivatives 9f−k with the isobutyl groups at the 4-position on the benzylic phenyl ring were prepared as shown in Scheme 2. Alcohol 9f was readily available from the reduction of the corresponding carboxylic acid 14 using a BH3· THF complex. The isobutyl group was introduced to aryl triflate 16 under Fürstner conditions using an isobutyl Grignard 9510



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Scheme 3. Synthesis of Benzyl Alcohols 9l−p with Substituents Other than an Isobutyl Group at the 4-Position on the Benzylic Phenyl Ringa

Reagents and conditions: (a) BH3·THF complex, THF, 0 °C to rt, 5 h, 92%; (b) (i) MOMCl, K2CO3, DMF, 50 °C, overnight for 26m, MOMCl, iPr2NEt, THF, 0−100 °C, 15 h for 26n, (ii) t-BuLi, n-hexane, 0 °C, 1 h, and then DMF, 0 °C, 15 min, 23% in two steps for 27m or CO2 gas, 81% in two steps for 27n; (c) (i) HCl/1,4-dioxane, rt, overnight, (ii) MeI, K2CO3, DMF, rt, 3.5−15 h, (iii) NaBH4, MeOH, 0 °C to rt, 1 h, 74% in three steps for 9m or LiAlH4, THF, 0 °C, 1 h, 75% in three steps for 9n; (d) (i) Tf2O, pyridine, CH2Cl2/THF, 0 °C to rt, 6 h, (ii) n-BuMgBr/THF, cat. Fe(acac)3, THF, NMP, 0 °C to rt, 1 h; (e) LiAlH4, THF, 0 °C, 1 h, 22% in three steps for 9o and 17% in two steps for 9p; (f) n-BuLi, i-Pr2NH, THF, DMPU, −78 °C, 2 h and then acetone, −78 to 0 °C, 1.5 h. a

Scheme 4. Synthesis of Pyridine Methanols 9q and 9ra

Reagents and conditions: (a) H2SO4, MeOH, PhMe, 110 °C, 15−18 h, 78% for 33q and 51% for 33r; (b) (i) n-PrMgBr/Et2O, cat. Fe(acac)3, THF, NMP, rt, 2 h for 33q or 0 °C to rt, 2 h for 33r, (ii) NaOMe, MeOH, rt, 18 h to 50 °C, 6 h for 34q or 0−50 °C, 2 h for 34r; (c) LiAlH4, THF, 0 °C, 20 min, 10% in three steps for 9q or rt, 20 min, 19% in three steps for 9r.

a

with methyl iodide, and reduction of the formyl group by NaBH4. Alcohol 9n with a n-propyl substituent at the 4position was prepared using similar procedures as above in which the phenol moiety of 3-n-propylphenol 26n was protected by the methoxymethyl group, and then the less hindered ortho-position to the methoxymethyl group was carboxylated using CO2 gas instead of DMF to yield carboxylic acid derivative 27n. This was converted to alcohol 9n in 75% yield by the deprotection of the methoxymethyl group under acidic conditions, concomitant methylation of both the phenol and the carboxylate with methyl iodide, and reduction of the ester by LiAlH4. Alcohol 9o was synthesized by similar procedures to yield 9h and 9i in 22% yield over three steps using an n-butyl Grignard reagent instead of the isobutyl Grignard reagents used to synthesize 9h and 9i. Alcohol 9p

Benzyl alcohol derivatives 9l−p with alkyl substituents other than an isobutyl group at the 4-position on the benzylic phenyl ring were prepared as shown in Scheme 3. Alcohol 9l was obtained from the reduction of carboxylic acid 25 using a BH3· THF complex in 92% yield. Alcohol 9m with an ethyl substituent at the 4-position was prepared by using an ortholithiation method. The phenol moiety of 3-ethylphenol 26m was protected by a methoxymethyl group using methoxymethyl chloride and potassium carbonate and the resulting methoxymethyl ether was ortho-formylated at the less hindered orthoposition to the methoxymethoxy group in the presence of tBuLi followed by DMF to yield the aldehyde derivative 27m in 23% yield. Compound 27m was converted to alcohol 9m in 74% yield by the deprotection of the methoxymethyl group under acidic conditions, methylation of the resulting phenol 9511



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Scheme 5. Synthesis of Dihydronaphthalene Intermediates 8x and 8ya

Reagents and conditions: (a) (i) BH3·THF complex, THF, 0 °C to rt, 3 h for 35x or LiAlH4, THF, 0 °C to rt, 30 min for 35y, (ii) PPh3, CCl4, 50− 70 °C, 14−16 h, 82−98% in two steps; (b) (i) CH2CHCO2Et, Pd(OAc)2, n-Bu3N, 95 °C, 14 h for 36x or 130 °C, 20 min under microwave irradiation for 36y, (ii) H2, 10% Pd−C, MeOH or EtOH, rt, 3 h, (iii) 2 mol/L, NaOH-aq, MeOH, THF, rt, 16 h, 30% in three steps for 37x or EtOH, THF, rt, 2 h, 68% in three steps for 37y; (c) (COCl)2, DMF, CH2Cl2, 0 °C to rt, 20−30 min and then SnCl4, PhMe, 0 °C to rt, 20−30 min, 67% to quantitative yield; (d) (i) MeMgCl/THF, THF, 0 °C, 1.5−2 h, (ii) 1 mol/L, HCl-aq, EtOAc, rt, 100 min, 86% for 39x or 5 mol/L, HCl-aq, THF, rt, 80 min, 96% for 39y; (e) (i) POCl3, DMF, 0 °C to rt, 2−2.5 h, 69%−72%, (ii) BBr3, CH2Cl2, −78 to 0 °C, 4 h, 54%−91%. a

with a methyl group at the 7-position on the core was also prepared by the same procedure as 8x. Amino acid derivatives 13a−u were prepared according to Scheme 6. 6-Substituted-1-methyl-3,4-dihydro-2-naphthalenecarbaldehydes 11b−f, 11h−k, 11m, and 11o,p were synthesized from the reaction of the dihydronaphthalene intermediate 8w18 with the corresponding alcohols under Mitsunobu conditions (alcohols 9b−e were commercially available, and the preparation of 9f, 9h−k, 9m, and 9o,p are described in Schemes 2−4) (route A). Aldehydes 11a, 11g, 11l, 11n, and 11q,r were synthesized from the reaction of the dihydronaphthalene 8w with the corresponding alkyl bromide under basic conditions (route B). The alkyl bromide for 11a was commercially available, while the alkyl bromides for 11g, 11l, 11n, and 11q,r were generated from the corresponding alcohols 9g, 9l, 9n, and 9q,r from the reaction with CBr4 and PPh3 in CH2Cl2 or PBr3 in Et2O. All the in-house-synthesized alkyl bromides were used without purification. The resulting aldehydes 11a−r were converted to the amino acid derivatives 13a−r using two procedures. One involved the reductive alkylation22 of the aldehydes 11a−f, 11h−m, and 11q,r with 3azetidinecarboxylic acid to directly yield the desired amino acids 13a−f, 13h−m, and 13q,r (route C). The other employed the reductive alkylation of the aldehydes 11g and 11n−p with methyl 3-azetidinecarboxylate hydrochloride to the esters 12g and 12n−p followed by the saponification of the esters to the desired amino acid derivatives 13g and 13n−p (routes D and E). Amino acid derivatives 13s−u were obtained using the same procedures as mentioned above starting from dihydronaphthalene 8x or 8y via routes B and C. Biology. In vitro potency was evaluated by measuring the intracellular Ca2+ concentration in CHO-K1 cells stably expressing the human S1P1 (hS1P1) or S1P3 (hS1P3) receptor as a primary assay to examine SAR. In vivo potency toward PLL was evaluated by determining the number of lymphocytes 4 or 24 h after oral dosing of the test compounds in mice. Structure−Activity Relationship Study. With compound 7 in hand, which exhibited 26 000-fold selectivity for S1P1 over S1P3 and an ED50 of 0.22 mg/kg at 24 h after oral dosing in the

with a tertiary alcohol was prepared via intermediate 31 obtained by the reaction of acetone with a benzyl anion species generated in situ from methyl 2-methoxy-4-methylbenzoate 30 using lithium diisopropylamide. Pyridine methanol derivatives 9q and 9r were prepared as shown in Scheme 4. 2,6-Dichloronicotinic acid 32q and 4,6dichloronicotinic acid 32r were converted to the aryl ester derivatives 33q and 33r using a catalytic amount of H2SO4 in MeOH in 78% and 51% yield, respectively. Then they were subjected to the above-mentioned Fe(acac)3-catalyzed coupling reactions with the n-propyl Grignard reagent followed by SNAr reactions with sodium methoxide and reduction by LiAlH4 to yield the pyridine methanol derivatives 9q and 9r. Dihydronaphthalene intermediates 8x and 8y with methyl substituents at the 5- and 7-positions, respectively, were prepared as described in Scheme 5. 3-Methoxy-2-methylbenzoic acid 35x was converted to the corresponding benzyl chloride 36x by reaction with a BH3·THF complex followed by CCl4 in the presence of PPh3 in 98% yield. The C-3 homologation reaction of the benzyl chloride 36x was carried out by using ethyl acrylate in the presence of n-Bu3N and a catalytic amount of Pd(OAc)2.20 The resulting E/Z mixture of the unsaturated ester derivatives was hydrogenated to the corresponding saturated ester that was saponified to 4arylbutanoic acid 37x in 30% yield over three steps. The carboxylic acid 37x was converted to an acid chloride in situ by reaction with oxalyl chloride in the presence of a catalytic amount of DMF followed by subjection to a Friedel−Crafts type cyclization reaction to quantitatively give tetralone 38x. The tetralone 38x was converted to the dihydronaphthalene derivative 39x by the addition of a methyl Grignard reagent to the ketone followed by dehydration under acidic conditions. Introduction of a formyl group at the 2-position on the dihydronaphthalene 39x using the Vilsmeier reaction21 and deprotection of the methyl group from the methoxy group at the 6-position on the dihydronaphthalene core by BBr3 in CH2Cl2 afforded dihydronaphthalene intermediate 8x in 33% yield from 39x. The other dihydronaphthalene intermediate 8y 9512



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Scheme 6. Synthesis of Amino Acid Derivatives 13a−ua

a

Reagents and conditions: (route A) R-OH, TMAD, PPh3, THF, rt, 2.5 h to overnight; (route B) R-Br (commercially available or derived from reaction of R-OH with CBr4, PPh3, CH2Cl2, 0 °C to rt, 30 min to 1 h or with PBr3, Et2O, 0 °C, 1.5 h), K2CO3, DMF, 0 °C to rt, 8 h to overnight; (route C) 3-azetidinecarboxylic acid, NaOH (powder), HC(OMe)3, MeOH/THF, 0 °C, 3−4 h or 0 °C to rt, 5−6 h, and then NaBH4, 0 °C, 0.5−3 h; (route D) methyl 3-azetidinecarboxylate hydrochloride, NaBH(OAc)3, AcOH, DMF, 0 °C to rt, 2 h to overnight; (route E) NaOH-aq, MeOH or MeOH/THF, 0 °C to rt, 0.5−4.5 h.

with compound 13v. While 13c and 13e bearing indane rings were tolerated in terms of their potency toward the S1P1 receptor, they were less potent than compound 13v in the PLL test in mice. Next, we turned our attention to derivatives with benzyl moieties in the lipophilic tail region because they provided the opportunity for thorough investigation of the effects of varying the substituents and substitution patterns on the benzylic aryl ring. A representative of this series from our library was compound 13f having an isobutyl substituent at the 4-position

PLL test in mice, we aimed to discover compounds more potent than 7 using the PLL test by undertaking a SAR study on the dihydronaphthalene series. Starting from compound 13v with a naked phenyl ring at the end of the propylene linker, a structure discovered during the course of identification of compound 7 from our earlier work,18 we first investigated the effects of conformational restrictions in the lipophilic tail region (Table 1). Amino acid derivatives 13a, 13b, and 13d with naphthalene and tetrahydronaphthalene moieties decreased the potency toward the S1P1 receptor by 6- to 32-fold compared 9513



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Table 1. Structure−Activity Relationship Study of the Effects of Conformational Restrictions in the Lipophilic Tail Region

a EC50 and ED50 values are shown as the mean of at least two and five data points, respectively (one experiment with five mice in the peripheral lymphocyte lowering test). N.T.: not tested.

be tolerated in terms of both S1P1 receptor potency and the PLL test, indicating that a specific hydrophilic functionality is tolerated in the lipophilic tail region (ClogP of 4.97 for 13g and 2.91 for 13p). Among them, compound 13n with the most promising S1P3/S1P1 selectivity (>30 000) and potency in the PLL test in mice (ED50 of 0.018 and 0.029 mg/kg 4 and 24 h after oral dosing, respectively) was selected for a further SAR study. Following identification of compound 13n, the introduction of a nitrogen atom in the benzylic aryl ring was investigated (Table 4). While compound 13r with a 5-pyridine ring (Y = N) decreased the potency toward the S1P1 receptor by 57-fold compared with 13n, compound 13q with a 3-pyridine ring (X = N) had comparable potencies to 13n toward the S1P1 receptor and the PLL test 4 h after oral dosing (ED50 = 0.011 mg/kg). As the 3-pyridine ring in 13q contributed to reducing the ClogP (4.57 for 13n vs 3.97 for 13q), the S1P3/S1P1 selectivity was reduced compared with 13n. Finally, a SAR study of the substituents on the dihydronaphthalene core was undertaken. While compound 13t with a methyl group at the 7-position on the core resulted in a 540fold decrease in potency toward the S1P1 receptor, compound 13s with a methyl group at the 5-position on the core was welltolerated in terms of both S1P1 receptor potency and the PLL test (ED50 = 0.032 mg/kg 4 h after oral dosing) while maintaining S1P3/S1P1 selectivity (>25 000) (Table 4). A comparison between 13q and 13u bearing a 3-pyridine ring in the benzylic part revealed that the methyl group at the 5-

on the benzylic phenyl ring and less than 50% of maximal activity toward the S1P3 receptor at 30 μM (Table 2). To develop a SAR around 13f, the influence of an additional substituent on the benzylic aryl ring was investigated using the methoxy group as a probe. Compound 13g with a 2-methoxy group improved the potency toward the S1P1 receptor and the PLL test 4 h after oral dosing (ED50 = 0.0092 mg/kg) by 5-fold and 42-fold, respectively, compared with 13f. While compound 13h with a 3-methoxy group also improved the potency toward the S1P1 receptor and the PLL test 4 h after oral dosing (ED50 = 0.034 mg/kg), it was less selective for S1P1 over S1P3 (S1P3/ S1P1 = 1300) and less potent than 13g. Further investigation of the substituents at the 2-position revealed that compound 13g with the methoxy group was more potent than compounds 13i, 13j, and 13k with a chlorine atom, a methyl group, and a fluorine atom, respectively, in terms of both its in vitro S1P1 and in vivo PLL potencies. This encouraging result for 13g prompted us to optimize the substituents at the 4-position on the benzylic aryl ring. The optimization of the alkyl chain at the 4-position revealed that methyl (13l), ethyl (13m), and n-propyl (13n) groups were the optimal length for S1P1 receptor potency and selectivity for S1P1 over S1P3 at concentrations up to 30 μM (Table 3). Removing the terminal methyl group from the isobutyl group of 13g contributed to attenuating the activation of the S1P3 receptor without affecting the potency toward the S1P1 receptor (13n). Interestingly, introduction of a tertiary alcohol to the isobutyl group of 13g to give 13p was found to 9514



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Table 2. SAR Study of the Effects of Substituents on the Benzylic Aryl Ring

EC50 and ED50 values are shown as the mean of at least two and five data points, respectively (one experiment with five mice in the peripheral lymphocyte lowering test). a

The potential efficacy of 13n to treat autoimmune diseases was evaluated using a collagen-induced arthritis (CIA) model24 in female Lewis rats (Figure 3). The clinical scores of the 13n 0.1 mg/kg group were significantly lower than those of the control group and were considered to be consistent with the PLL potency of 13n in the female Lewis rats where the maximum effect was induced at a dose of 0.1 mg/kg.14a Subsequently the effects of 13n on heart rate and peripheral lymphocyte counts were examined in monkeys. When compound 13n was orally administered in a single dose to unrestrained conscious monkeys at dosages of 1, 10, and 100 mg/kg, a significant decrease in heart rate was observed at 4− 24 h after administration at a dosage of 100 mg/kg and the maximum decrease of heart rate to the pretreatment value was 19% at 4 h after administration (Table SI-1 in Supporting Information). The plasma concentrations of 13n were increased with increasing dosages (320, 3570, and 13 500 ng/ mL at 4 h after dosing at 1, 10, and 100 mg/kg, respectively), and the plasma concentration at 100 mg/kg at 24 h after dosing was maintained at 11 100 ng/mL. On the other hand, a significant decrease in peripheral lymphocyte counts was observed at 4−24 h at dosages of 0.01 and 0.03 mg/kg after oral administration of 13n (Table SI-2). Our SAR study was driven by the compound’s ability to activate the human S1P1 receptor and its effect in the mouse PLL test. The amino acid sequences lacking the N-terminal

position on the core was effective to attenuate the activation toward the S1P3 receptor without impairing its potencies toward the S1P1 receptor and in the PLL test. Compounds 13n, 13s, and 13u were subjected to further evaluation. Further Evaluation of Selected Compounds. We evaluated three representative compounds, 13n, 13s, and 13u, identified from the SAR studies. As summarized in Table 5, compound 13n was more potent than 13s and 13u, both in the PLL test in mice 24 h after oral dosing by 2.5- and 1.7-fold, respectively, and in a cAMP accumulation assay in cells expressing the S1P5 receptor by 3.0- and 2.8-fold, respectively. The activation of the S1P5 receptor is believed to contribute to positive effects in neurodegenerative disorders.13,23 On the basis of these results, compound 13n was selected for further evaluation. The pharmacokinetic properties of 13n were investigated after single intravenous injection and oral administration in male Crl:CD(SD) rats and monkeys. The total clearance (CLtot), oral half-life (T1/2), and bioavailability (BA) of 13n were 338 mL h−1 kg−1, 17 h, and 75.9% and 63.6 mL h−1 kg−1, 29 h, and 66.5% in rats and monkeys, respectively, at 0.1 mg/kg as shown in Table 6. In liver microsomes, metabolism of [3H]13n was slow and similar across species as shown in Table 7. Additionally, the serum protein binding of [3H]-13n at concentrations of 50−5000 ng/mL showed no significant interspecies differences as shown in Table 7. 9515



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Table 3. SAR Study of the Effects of Alkyl Substituents at the 4-Position on the Benzylic Aryl Ring

a EC50 and ED50 values are shown as the mean of at least two and five data points, respectively (one experiment with five mice in peripheral lymphocyte lowering test). N.T.: not tested.

Table 4. SAR Study of the Effects of Introducing a Nitrogen Atom on the Benzylic Phenyl Ring and Varying the Methyl Substituents on the Dihydronaphthalene Ring in 13n

a EC50 and ED50 values are shown as the mean of at least two and five data points, respectively (one experiment with five mice in the peripheral lymphocyte lowering test). N.T.: not tested.

extracellular and C-terminal intracellular domains containing a ligand binding pocket (Ser49 in TM1 (transmembrane domain) through Thr314 in TM7) are 99.6% identical between the mouse and rat. Furthermore, the homology between the

mouse S1P1 receptor and the human homolog is 97.4%. Therefore, we considered that any interspecies differences in the potency of 13n toward the S1P1 receptor would be negligible. In fact, 13n inhibited the binding of [33P]-S1P to the 9516



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Table 5. In Vitro and in Vivo Profiles of Three Representative Compounds 13n, 13s, and 13u

EC50 and ED50 values are shown as the mean of at least two and five data points, respectively (one experiment with five mice in the peripheral lymphocyte lowering test). N.T.: not tested. a

Table 6. Pharmacokinetic Properties of 13n species (dose) a

rat (0.1 mg/kg) monkeya (0.1 mg/kg) a

route

AUCinf (ng·h/mL)

iv po iv po

302 (48) 229 (14) 1640 (390) 1090 (60)

Cmax (ng/mL)

CLtot (mL h−1 kg−1) 338 (54)

7.47 (0.54) 63.6 (16.8) 28.4 (2.1)

T1/2 (h) 16 17 30 29

(1) (4) (2) (1)

Vss (mL/kg)

BA (%)

7150 (540)

75.9

2660 (480)

66.5

Values are the mean from three animals. Standard deviation is given in parentheses.

Table 7. In Vitro DMPK Properties of 13n species mouse SD rat Lewis rat cynomolgus monkey human

unchanged form of 13n after 2 h incubation in the liver microsomes (%)a

serum protein binding (%)b

93.3 92.6 not applicable 94.1

96.2−97.1 97.7−98.2 97.2−97.7 97.1−97.3

95.2

96.8−97.1

a

Values represent the mean of two metabolism experiments. bValues represent the mean of four protein binding experiments. Concentrations of compound 13n were 50, 500, and 5,000 ng/mL.

hS1P1 and rS1P1 receptors with similar Ki values of 0.626 nM and 0.772 nM, respectively, and in the cAMP accumulation assay, 13n activated hS1P1 and rS1P1 to the same extent with EC50 values of 0.0273 nM and 0.0286 nM, respectively.14a The peripheral lymphocyte count has been used as a pharmacodynamic biomarker to gauge efficacy across species.8,14b Therefore, we used the PLL potency in mice as the primary indicator to triage compounds, and thus, the most potent compound 13n was evaluated in the rat CIA model. On the basis of reports that the egress of lymphocytes from the lymph nodes requires the S1P1 receptor3 and the PLL effect by S1P1 agonists are induced by the downregulation of the S1P1 receptor,2d,12 i.e., by blocking the S1P1 signal as functional antagonists, we performed PK/PD analysis of 13n tested in the CIA model. In the female Lewis rats used for the model, the maximum concentrations (Cmax) obtained by single oral administration of 13n were 5.76 nM and 21.5 nM at 0.03 mg/kg and 0.1 mg/kg, respectively. From the expression analysis of the human S1P1

Figure 3. Clinical Scores of 13n in the CIA model. Arthritis was induced by immunizing with bovine type II collagen emulsified in incomplete Freund’s adjuvant on days 0 and 7 to the female Lewis rats. Arthritis was not induced in the normal control group. Arthritis efficacy was evaluated by using the arthritis score characterized by edema and/or erythema in the paws. 13n (0.03 or 0.1 mg/kg) or 0.5% methylcellulose was administered orally once daily from day 0 to day 27. The arthritis scores were expressed as the mean value ± standard error for the normal control (open circle, n = 5), control (filled circle, n = 10), 13n 0.03 mg/kg (square, n = 9), 13n 0.1 mg/kg (triangle, n = 9). The steel test was used to compare the scores between the control and 13n groups, with a p-value of less than 5%: (∗∗) p < 0.01; (∗∗∗) p < 0.001.

receptor on the plasma membrane of genetically engineered CHO cells, the concentration of 13n required to induce the downregulation of the S1P1 receptor by approximately 90% (25 nM)14a was consistent with the Cmax at the efficacious dose of 9517



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with the assigned structures. Proton nuclear magnetic resonance spectra (1H NMR) were taken on a Varian Mercury 300 or VNMRS 600 spectrometer using deuterated chloroform (CDCl3) or deuterated methanol (CD3OD) or deuterated dimethylsulfoxide (DMSO-d6) or mixture of those as the solvent. The chemical shift values were reported in parts per million (δ) and coupling constants (J) in hertz (Hz). Electron spray ionization (ESI) was determined on a Waters ZQ2000 spectrometer. Fast atom bombardment mass spectra (FABMS, HRMS) and electron ionization (EI) were obtained on a JEOL JMS-700 spectrometer. Atmospheric pressure chemical ionization (APCI) was determined on a HITACHI M-1200H spectrometer. Elemental analysis (EA) was determined on a PE2400 series CHNS/O analyzer. Melting point (mp) was determined on a Yanagimoto MP500D. Microwave irradiation was carried out by CEM Discover. Column chromatography was carried out on silica gel [Merck silica gel 60 (0.063−0.200 mm), Wako gel C200, or Fuji Silysia FL60D] or Biotage horizon (QUAD, HORIZON). TLC was performed on silica gel (Merck TLC or HPTLC plates, silica gel 60 F254). All final compounds were assessed for purity by ultrahigh performance liquid chromatography (UHPLC) on Waters Acquity UPLC I-Class system with photodiode array (PDA) detector, MS, and ELSD via the following conditions. Column: YMC Triart C18 2.0 mm × 30 mm, 1.9 μm. Mobile phase A: 0.10% TFA in water (v/v). Mobile phase B: 0.10% TFA in MeCN (v/v). Gradient: 95.0% water/5.0% MeCN linear to 5% water/95% MeCN in 1.2 min, hold at 5% water/95% MeCN to 1.5 min. Flow: 1.0 mL/min. According to these LC−MS analyses, all final compounds showed a purity of ≥95% by ELSD. 6-Hydroxy-1,5-dimethyl-3,4-dihydro-2-naphthalenecarbaldehyde (8x). To POCl3 (0.32 mL, 3.6 mmol) was added dry DMF (2 mL) at ambient temperature. The reaction mixture was stirred for 30 min at ambient temperature. To the mixture was added a solution of 39x (304 mg, 1.6 mmol) at ambient temperature. The reaction mixture was poured into ice/water (1.1 L) and extracted with (hexane/EtOAc, 1/3) and (hexane/EtOAc, 1/1) which was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 6-methoxy-1,5-dimethyl-3,4-dihydronaphthalene-2-carbaldehyde (250 mg, 72% yield) as a pale yellow powder. TLC Rf = 0.35 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.31 (s, 1 H), 7.42 (d, J = 8.5 Hz, 1 H), 6.77 (d, J = 8.5 Hz, 1 H), 3.87 (s, 3 H), 2.74−2.68 (m, 2 H), 2.52−2.47 (m, 5 H), 2.19 (s, 3 H). To a solution of 6-methoxy-1,5-dimethyl-3,4-dihydronaphthalene-2carbaldehyde (139 mg, 0.64 mmol) in dry CH2Cl2 (3 mL) was added a solution of BBr3 in dry CH2Cl2 (1.0 mol/L, 3.3 mL, 3.3 mmol), −78 °C. After stirring for 4 h at 0 °C, the reaction mixture was poured into ice−water. The mixture was extracted with EtOAc twice. The combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure. The residue was triturated in MTBE and the precipitate was collected by filtration to yield 8x (70 mg, 54% yield) as a greenish powder. TLC Rf = 0.31 (hexane/EtOAc, 2/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.34 (d, J = 8.4 Hz, 1 H), 6.72 (d, J = 8.4 Hz, 1 H), 5.19 (s, 1 H), 2.65−2.76 (m, 2 H), 2.42−2.55 (m, 5 H), 2.22 (s, 3 H); MS (EI, Pos) m/z 202 (M)+ (base peak), 187, 173, 159, 158. 6-Hydroxy-1,7-dimethyl-3,4-dihydro-2-naphthalenecarbaldehyde (8y). The same procedure as 8x was performed using 3,5-dimethyl-7,8-dihydronaphthalen-2-yl methyl ether 39y (1.42 g, 7.5 mmol), POCl3 (1.5 mL, 16.6 mmol), and dry DMF (10 mL) to yield 6-methoxy-1,7-dimethyl-3,4-dihydronaphthalene-2-carbaldehyde (1.12 g, 69% yield) as a yellow powder. TLC Rf = 0.45 (hexane/EtOAc, 3/ 1); 1H NMR (300 MHz, CDCl3) δ 10.29 (s, 1 H), 7.31 (s, 1 H), 6.67 (s, 1 H), 3.87 (s, 3 H), 2.74−2.69 (m, 2 H), 2.53−2.48 (m, 5 H), 2.23 (s, 3 H). The same procedure as 8x was performed using 6-methoxy-1,7dimethyl-3,4-dihydronaphthalene-2-carbaldehyde (1.0 g, 4.6 mmol), BBr3 (2.2 mL, 23 mmol), and dry CH2Cl2 (13 mL) to yield 8y (855 mg, 91% yield) as a brown powder. TLC Rf = 0.19 (hexane/EtOAc, 3/ 1); 1H NMR (300 MHz, CDCl3) δ 10.21 (s, 1 H), 9.82 (s, 1 H), 7.36 (s, 1 H), 6.64 (s, 1 H), 2.52−2.59 (m, 2 H), 2.44 (s, 3 H), 2.26−2.34 (m, 2 H), 2.12 (s, 3 H); MS (ESI, Pos 20 V) m/z 203 (M + H)+.

0.1 mg/kg in the model. We concluded this analysis supports the hypothesis that the S1P1 agonists induce the PLL effect via the downregulation of the S1P1 receptor resulting in improvement of the clinical scores in the animal model. Given the selectivity of 13n for S1P1 as shown by the magnitude of its inhibition of the specific binding of [33P]-S1P to the human S1P1, S1P2, S1P3, S1P4, and S1P5 receptors, with Ki values of 0.626 nM, >5450 nM, >5630 nM, 28.8 nM, and 0.574 nM, respectively,14a we considered compound 13n to be a S1P1 and S1P5 dual agonist. Suitable profile including the data on 13n discussed above, its efficacy in an EAE model in mice,14a and prediction of a favorable human response14b encouraged us to advance 13n into clinical trials.

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CONCLUSIONS Starting from the hit compound 6 with a simple amino acid structure, 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n, a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, was discovered as a clinical candidate through a detailed SAR study. Facilitated by the convergent synthetic strategy using the intermediates 8w−y with a central dihydronaphthalene core, the tolerability of conformational restrictions in the lipophilic tail region was investigated. The subsequent SAR study focused on the benzyl motif revealed that the methoxy group at the 2-position on the benzylic aryl ring improved potency in the PLL test in mice by 42-fold (13f vs 13g). Optimization of the alkyl substituents at the 4-position on the benzylic aryl ring led to the discovery of 13n which exhibited >30 000-fold selectivity for S1P1 over S1P3 in the Ca2+ influx assay and was highly potent in the PLL test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing). The benzyl series allowed us to investigate various substituents and substitution patterns in the lipophilic tail region compared with the phenylpropyl series as exemplified by 7 previously identified in our earlier work. A further SAR study revealed hydrophilic groups such as a tertiary alcohol (13p) and 3pyridine (13q) in the lipophilic tail region were tolerated in terms of their potencies toward the S1P1 receptor and the PLL test while a methyl substituent at the 5-position on the central dihydronaphthalene core attenuated the activity toward the S1P3 receptor (13q vs 13u). Compound 13n was potent in the cAMP accumulation assay toward not only S1P1 but also S1P5, properties which are expected to lead to additional preferred effects for the treatment of RRMS. Suitable profile including pharmacokinetic properties for a once-daily administration in rat and monkey, significant efficacies in autoimmune disease models such as the rat CIA model, little interspecies difference in in vitro DMPK properties, significant difference in the dose levels to induce lymphopenia and bradycardia in monkeys, and prediction of human response encouraged us to advance 13n into clinical trials. The direct-acting S1P1 agonist 13n may allow more accurate control of its pharmacological activities in vivo compared with prodrugs that require conversion to phosphorylated active metabolites in vivo.

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EXPERIMENTAL SECTION

It should be noted that final compounds 13a−u described herein can induce biological effects at very low dose in human (see ref 14c). Chemistry. General. Unless noted otherwise, all reagents and solvents were used as purchased without further purification. Analytical samples were homogeneous as confirmed by thin layer chromatography (TLC) and afforded spectroscopic results consistent 9518



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(4-Isobutyphenyl)methanol (9f). To a stirred solution of 4isobutylbenzoic acid 14 (1.0 g, 5.6 mmol) in THF (5 mL) was added a solution of BH3·THF complex (1.0 mol/L, 8.4 mL, 8.4 mmol) at 0 °C. The reaction mixture was stirred overnight at ambient temperature. After MeOH was added to the reaction mixture, the mixture was concentrated under reduced pressure and residue was extracted with CHCl3 from water. The organic layer was washed with brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 9f (1.09 g, quantitative yield) as a colorless oil. (4-Isobutyl-2-methoxyphenyl)methanol (9g). To a stirred solution of 18 (171 mg, 0.89 mmol) in MeOH (10 mL) was added NaBH4 (100 mg, 2.6 mmol) at 0 °C. After the reaction mixture was stirred for 1.5 h at 0 °C, the reaction mixture was concentrated under reduced pressure. To the residue was added NH4Cl-aq, and the mixture was extracted with MTBE twice. The combined organic layer was washed with NH4Cl-aq, NaHCO3-aq, water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield 9g (175 mg, quantitative yield) as a colorless oil. TLC Rf = 0.44 (hexane/ EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.15 (d, J = 7.5 Hz, 1 H), 6.72 (dd, J = 7.5, 1.4 Hz, 1 H), 6.67 (d, J = 1.4 Hz, 1 H), 4.65 (s, 2 H), 3.86 (s, 3 H), 2.47 (d, J = 7.1 Hz, 2 H), 2.27 (s, 1 H), 1.79−1.94 (m, 1 H), 0.91 (d, J = 6.6 Hz, 6 H); MS (EI, Pos) m/z 194 (M+) (base peak), 151, 137, 123, 91. (4-Isobutyl-3-methoxyphenyl)methanol (9h). To a suspension of LiAlH4 (227 mg, 6.0 mmol) in dry THF (11 mL) was added a solution of methyl 4-isobutyl-3-methoxybenzoate 21h (1.104 g, 5.0 mmol) at 0 °C. After the reaction mixture was stirred for 10 min at 0 °C, saturated Na2SO4-aq and MTBE were added to the mixture. After the reaction mixture was stirred for 40 min at ambient temperature, MgSO4 was added to the reaction mixture. The insoluble was removed through Celite and the filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel (hexane/EtOAc) to yield 9h (869 mg, 90% yield) as a colorless oil. TLC Rf = 0.17 (hexane/EtOAc, 5/1); 1H NMR (300 MHz, CDCl3) δ 7.05 (d, J = 7.5 Hz, 1 H), 6.87 (s, 1 H), 6.84 (d, J = 7.5 Hz, 1 H), 4.66 (d, J = 6.0 Hz, 2 H), 3.82 (s, 3 H), 2.47 (d, J = 7.0 Hz, 2 H), 1.82−1.97 (m, 1 H), 1.60 (t, J = 6.0 Hz, 1 H), 0.89 (d, J = 7.0 Hz, 6 H); MS (EI, Pos) m/z 194 (M)+, 151, 91 (base peak). (2-Chloro-4-isobutylphenyl)methanol (9i). The same procedure as 9h was performed using 21i (300 mg, 1.3 mmol), LiAlH4 (59 mg, 1.6 mmol), and dry THF (6 mL) to yield 9i (crude 209 mg, 79% yield) as a colorless oil. TLC Rf = 0.41 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.36 (d, J = 8.0 Hz, 1 H), 7.16 (d, J = 1.5 Hz, 1 H), 7.05 (dd, J = 8.0, 1.5 Hz, 1 H), 4.75 (s, 2 H), 2.45 (d, J = 7.0 Hz, 2 H), 1.79−1.94 (m, 2 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (EI, Pos) m/z 198 (M)+, 138 (base peak). (4-Isobutyl-2-methylphenyl)methanol (9j). The same procedure as 9h was performed using crude 24j (590 mg, 2.8 mmol), LiAlH4 (128 mg, 3.4 mmol), and dry THF (10 mL) to yield 9j (220 mg, 46% yield in 2 steps) as a colorless oil. TLC Rf = 0.36 (hexane/ EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.24 (d, J = 8.5 Hz, 1 H), 6.96−7.00 (m, 2 H), 4.67 (s, 2 H), 2.43 (d, J = 7.5 Hz, 2 H), 2.35 (s, 3 H), 1.78−1.93 (m, 1 H), 1.55 (s, 1 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (EI, Pos) m/z 178 (M)+, 160, 135 (base peak). (2-Fluoro-4-isobutylphenyl)methanol (9k). The same procedure as 9h was performed using 24k (1.0 g (content 810 mg), 3.6 mmol), LiAlH4 (165 mg, 4.3 mmol), and dry THF (20 mL) to yield 9k (492 mg, 75% yield in 2 steps) as a colorless oil. TLC Rf = 0.35 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.29 (t, J = 8.0 Hz, 1 H), 6.92 (dd, J = 8.0, 1.5 Hz, 1 H), 6.85 (dd, J = 11.0, 1.5 Hz, 1 H), 4.73 (d, J = 6.0 Hz, 2 H), 2.46 (d, J = 7.5 Hz, 2 H), 1.80−1.92 (m, 1 H), 1.72 (t, J = 6.0 Hz, 1 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (EI, Pos) m/z 182 (M)+, 139, 122 (base peak). (2-Methoxy-4-methylphenyl)methanol (9l). The same procedure as 9f was performed using 2-methoxy-4-methylbenzoic acid 25 (1.70 g, 10.0 mmol), BH3·THF complex (1.0 mol/L, 15.0 mL, 15.0 mmol), THF (10 mL) to yield 9l (1.44 g, 92% yield) as a colorless oil. TLC Rf = 0.44 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 7.14 (d, J = 7.5 Hz, 1 H), 6.75 (d, J = 7.5 Hz, 1 H), 6.71 (s, 1 H), 4.64

(s, 2 H), 3.85 (s, 3 H), 2.35 (s, 3 H), 2.08−2.29 (m, 1 H); MS (EI, Pos) m/z 152 (M)+. (4-Ethyl-2-methoxyphenyl)methanol (9m). To a solution of 27m (282 mg, 1.6 mmol) in 1,4-dioxane (3 mL) was added a solution of HCl in 1,4-dioxane (4 mol/L, 0.80 mL, 3.2 mmol) at ambient temperature. After the reaction mixture was stirred for 16 h at ambient temperature, the reaction mixture was concentrated under reduced pressure to yield 4-ethyl-2-hydroxybenzaldehyde (247 mg, quantitative yield) as a reddish brown oil. It was used for the next reaction without purification. TLC Rf = 0.53 (hexane/EtOAc, 5/1). To a stirred solution of 4-ethyl-2-hydroxybenzaldehyde (238 mg, 1.6 mmol) and methyl iodide (0.15 mL, 2.4 mmol) in DMF (2 mL) was added K2CO3 (439 mg, 3.2 mmol) at ambient temperature. After stirring for 4 h at ambient temperature, cold water was added to the mixture which was extracted with (hexane/EtOAc, 1/1) twice. Combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 4-ethyl-2-methoxybenzaldehyde (262 mg, quantitative yield) as a yellow oil. It was used for the next reaction without purification. TLC Rf = 0.44 (hexane/EtOAc, 5/1). The same procedure as 9g was performed using 4-ethyl-2methoxybenzaldehyde (260 mg, 1.6 mmol), NaBH4 (90 mg, 2.4 mmol), and MeOH (3 mL) to yield 9m (195 mg, 74% in 3 steps) as a colorless oil. TLC Rf = 0.24 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.17 (d, J = 8.0 Hz, 1 H), 6.78 (dd, J = 8.0, 1.0 Hz, 1 H), 6.73 (s, 1 H), 4.65 (s, 2 H), 3.87 (s, 3 H), 2.65 (q, J = 7.5 Hz, 2 H), 2.24−2.20 (m, 1 H), 1.24 (t, J = 7.5 Hz, 3 H). (2-Methoxy-4-propylphenyl)methanol (9n). The same procedure as 9m was performed using 27n (3.80 g, 17.0 mmol), a solution of HCl in 1,4-dioxane (4 mol/L, 17.0 mL, 68 mmol), and 1,4-dioxane (5 mL) to yield 2-hydroxy-4-propylbenzoic acid (2.35 g, 77% yield) as a pale yellow powder after the concentrated residue from the reaction mixture was triturated with hexane. TLC Rf = 0.30 (CHCl3/MeOH/ H2O, 100/10/1); 1H NMR (300 MHz, CDCl3) δ 10.38 (s, 1 H), 7.81 (d, J = 8.0 Hz, 1 H), 6.83 (d, J = 1.5 Hz, 1 H), 6.76 (dd, J = 8.0, 1.5 Hz, 1 H), 2.60 (t, J = 7.5 Hz, 2 H), 1.59−1.73 (m, 2 H), 0.95 (t, J = 7.5 Hz, 3 H); MS (EI, Pos) m/z 180 (M)+, 162, 134, 105 (base peak). The same procedure as 9m was performed using 2-hydroxy-4propylbenzoic acid (700 mg, 3.9 mmol), MeI (0.73 mL, 11.7 mmol), K2CO3 (1.77 g, 12.8 mmol), and DMF (15 mL) to yield methyl 2methoxy-4-propylbenzoate (783 mg, 97% yield) as a yellow oil. It was used for the next reaction without purification. TLC Rf = 0.50 (hexane/EtOAc, 2/1); 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J = 8.0 Hz, 1 H), 6.77−6.82 (m, 2 H), 3.90 (s, 3 H), 3.87 (s, 3 H), 2.61 (t, J = 7.5 Hz, 2 H), 1.59−1.73 (m, 2 H), 0.95 (t, J = 7.5 Hz, 3 H); MS (EI, Pos) m/z 208 (M)+, 177, 105, 90, 77 (base peak). The same procedure as 9h was performed using methyl 2-methoxy4-propylbenzoate (1.57 g, 7.5 mmol), LiAlH4 (330 mg, 8.6 mmol), and THF (32.5 mL) to yield 9n (1.41 g, quantitative yield) as a colorless oil. It was used for the next reaction without purification. TLC Rf = 0.43 (hexane/EtOAc, 2/1); 1H NMR (300 MHz, CDCl3) δ 7.16 (d, J = 7.5 Hz, 1 H), 6.76 (dd, J = 7.5, 1.5 Hz, 1 H), 6.71 (d, J = 1.5 Hz, 1 H), 4.65 (s, 2 H), 3.86 (s, 3 H), 2.58 (t, J = 7.5 Hz, 2 H), 2.20 (s, 1 H), 1.58−1.72 (m, 2 H), 0.95 (t, J = 7.5 Hz, 3 H); MS (EI, Pos) m/z 180 (M)+, 137 (base peak), 91. (4-Butyl-2-methoxyphenyl)methanol (9o). The same procedure as 9h was performed using methyl 4-butyl-2-methoxybenzoate 29 (56 mg, 0.25 mmol), LiAlH4 (9.5 mg, 0.25 mmol), and THF (1 mL) to yield 9o (48 mg, quantitative yield) as an oil. The residue after workup was used for next reaction without purification. TLC Rf = 0.50 (hexane/EtOAc, 3/1). 1-[4-(Hydroxymethyl)-3-methoxyphenyl]-2-methyl-2-propanol (9p). The same procedure as 9h was performed using crude 31 (300 mg, 1.3 mmol), LiAlH4 (57 mg, 1.5 mmol), and THF (10 mL) to yield 9p (216 mg, 17% yield in 2 steps) as a white powder. TLC Rf = 0.32 (hexane/EtOAc, 1/2); 1H NMR (300 MHz, CDCl3) δ 7.19 (d, J = 7.5 Hz, 1 H), 6.79 (d, J = 7.5 Hz, 1 H), 6.76 (s, 1 H), 4.67 (s, 2 H), 3.87 (s, 3 H), 2.76 (s, 2 H), 2.26 (s, 1 H), 1.37 (s, 1 H), 1.24 (s, 6 H); MS (EI, Pos) m/z 210 (M)+, 165, 152, 134 (base peak). 9519



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(2-Methoxy-6-propyl-3-pyridinyl)methanol (9q). The same procedure as 9h was performed using methyl 2-methoxy-6propylnicotinate 34q (267 mg, 1.3 mmol), LiAlH4 (73 mg, 1.9 mmol), and dry THF (2 mL) to yield 9q (210 mg, 91% yield) as a colorless oil. TLC Rf = 0.70 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 7.43 (d, J = 7.3 Hz, 1 H), 6.69 (d, J = 7.3 Hz, 1 H), 4.60 (s, 2 H), 3.98 (s, 3 H), 2.55−2.73 (m, 2 H), 2.16−2.37 (m, 1 H), 1.66−1.84 (m, 2 H), 0.95 (t, J = 7.4 Hz, 3 H); MS (EI, Pos) m/z 181 (M)+, 166, 153 (base peak), 136. (4-Methoxy-6-propyl-3-pyridinyl)methanol (9r). The same procedure as 9h was performed using methyl 4-methoxy-6propylnicotinate 34r (159 mg, 0.76 mmol), LiAlH4 (43 mg, 1.1 mmol), and dry THF (1 mL) to yield 9r (130 mg, 94% yield) as a yellow solid. TLC Rf = 0.45 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1 H), 6.64 (s, 1 H), 4.65 (s, 2 H), 3.90 (s, 3 H), 2.65−2.79 (m, 2 H), 2.17−2.62 (m, 1 H), 1.65−1.84 (m, 2 H), 0.97 (t, J = 7.3 Hz, 3 H); MS (FAB, Pos) m/z 182 (M + H)+. 1-Methyl-6-(2-naphthylmethoxy)-3,4-dihydro-2naphthalenecarbaldehyde (11a). Typical Procedure for the Aldehyde Derivative 11 (Route B). To a stirred solution of 6hydroxy-1-methyl-3,4-dihydro-2-naphthalenecarbaldehyde 8w (100 mg, 0.53 mmol) and 2-naphthylmetyl bromide (141 mg, 0.64 mmol) in DMF (2 mL) was added K2CO3 (147 mg, 1.06 mmol) at ambient temperature. After stirring for 14 h at ambient temperature, cold water was added to the mixture. Resulting precipitates were collected by filtration and purified by chromatography on silica gel using (hexane/EtOAc) as an eluent. Collected sample was triturated with MTBE to yield 11a (108 mg, 62% yield) as a white powder. TLC Rf = 0.39 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.82−7.91 (m, 4 H), 7.47−7.56 (m, 4 H), 6.92 (dd, J = 8.5, 2.5 Hz, 1 H), 6.88 (d, J = 2.5 Hz, 1 H), 5.28 (s, 2 H), 2.70−2.77 (m, 2 H), 2.48−2.55 (m, 5 H); MS (EI, Pos) m/z 328 (M+), 141 (base peak), 115. 1-Methyl-6-(1,2,3,4-tetrahydro-2-naphthalenylmethoxy)3,4-dihydro-2-naphthalenecarbaldehyde (11b). Typical Procedure for the Aldehyde Derivative 11 (Route A). To a solution of 8w (100 mg, 0.53 mmol) and 1,2,3,4-tetrahydro-2-naphthalenemethanol 9b (103 mg, 0.64 mmol) in dry THF (1.5 mL) were added PPh3 (181 mg, 0.69 mmol) and TMAD (119 mg, 0.69 mmol) at ambient temperature. After the reaction mixture was stirred for 15 h, the mixture was purified by chromatography on silica gel using (hexane/EtOAc) as an eluent to yield 11b (170 mg, 96% yield) as a white powder. TLC Rf = 0.47 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.49 (d, J = 8.5 Hz, 1 H), 7.10−7.13 (m, 4 H), 6.83 (dd, J = 8.5, 2.5 Hz, 1 H), 6.78 (d, J = 2.5 Hz, 1 H), 3.98 (d, J = 6.5 Hz, 2 H), 2.96−3.05 (m, 1 H), 2.85−2.92 (m, 2 H), 2.59−2.77 (m, 3 H), 2.47−2.55 (m, 5 H), 2.24−2.40 (m, 1 H), 2.04− 2.19 (m, 1 H), 1.56−1.68 (m, 1 H); MS (EI, Pos) m/z 332 (M+) (base peak), 145. 6-(2,3-Dihydro-1H-inden-2-ylmethoxy)-1-methyl-3,4-dihydro-2-naphthalenecarbaldehyde (11c). The same procedure as 11b was performed using 8w (100 mg, 0.53 mmol), (2,3-dihydro-1Hinden-2-yl)methanol (87 mg, 0.59 mmol), PPh3 (181 mg, 0.69 mmol), TMAD (119 mg, 0.69 mmol), and THF (1.5 mL) to yield 11c (130 mg, 77% yield) as a white powder. TLC Rf = 0.33 (hexane/EtOAc, 5/ 1); 1H NMR (300 MHz, CDCl3) δ 10.31 (s, 1 H), 7.47 (d, J = 8.5 Hz, 1 H), 7.19−7.25 (m, 2 H), 7.13−7.19 (m, 2 H), 6.81 (dd, J = 8.5, 2.5 Hz, 1 H), 6.76 (d, J = 2.5 Hz, 1 H), 4.00 (d, J = 7.0 Hz, 2 H), 3.18 (dd, J = 15.5, 7.0 Hz, 2 H), 2.91−3.08 (m, 1 H), 2.86 (dd, J = 15.5, 5.5 Hz, 2 H), 2.67−2.76 (m, 2 H), 2.46−2.55 (m, 5 H); MS (EI, Pos) m/z 318 (M+) (base peak), 131. 1-Methyl-6-[2-(1,2,3,4-tetrahydro-1-naphthalenyl)ethoxy]3,4-dihydro-2-naphthalenecarbaldehyde (11d). The same procedure as 11b was performed using 8w (100 mg, 0.53 mmol), 1,2,3,4tetrahydro-1-naphthaleneethanol (123 mg, 0.69 mmol), PPh3 (181 mg, 0.69 mmol), TMAD (119 mg, 0.69 mmol), and THF (2 mL) to yield 11d (180 mg, 92% yield) as a white solid. TLC Rf = 0.63 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.49 (d, J = 8.6 Hz, 1 H), 7.04−7.23 (m, 4 H), 6.82 (dd, J = 8.6, 2.7 Hz, 1 H), 6.77 (d, J = 2.7 Hz, 1 H), 4.11 (t, J = 6.4 Hz, 2 H), 3.01−

3.18 (m, 1 H), 2.67−2.87 (m, 4 H), 2.51 (s, 3 H), 2.44−2.58 (m, 2 H), 2.12−2.33 (m, 1 H), 1.97−2.11 (m, 1 H), 1.82−1.97 (m, 2 H), 1.68− 1.82 (m, 2 H); MS (EI, Pos) m/z 346 (M)+ (base peak), 188, 173, 159, 131, 117, 91. 6-[2-(2,3-Dihydro-1H-inden-1-yl)ethoxy]-1-methyl-3,4-dihydro-2-naphthalenecarbaldehyde (11e). The same procedure as 11b was performed using 8w (100 mg, 0.53 mmol), 2-(2,3-dihydro1H-inden-1-yl)ethan-1-ol (112 mg, 0.69 mmol), PPh3 (181 mg, 0.69 mmol), TMAD (119 mg, 0.69 mmol), and THF (2 mL) to yield 11e (120 mg, 68% yield) as a white solid. TLC Rf = 0.60 (hexane/EtOAc, 3/1). 6-[(4-Isobutylbenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenecarbaldehyde (11f). The same procedure as 11b was performed using 8w (60 mg, 0.32 mmol), 9f (52 mg, 0.32 mmol), PPh3 (100 mg, 0.38 mmol), TMAD (82 mg, 0.48 mmol), and THF (3 mL) to yield 11f (77 mg, 73% yield) as a yellow oil. TLC Rf = 0.66 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.31 (s, 1 H), 7.48 (d, J = 8.60 Hz, 1 H), 7.33 (d, J = 8.1 Hz, 2 H), 7.17 (d, J = 8.1 Hz, 2 H), 6.87 (dd, J = 8.6, 2.4 Hz, 1 H), 6.83 (d, J = 2.4 Hz, 1 H), 5.06 (s, 2 H), 2.67−2.77 (m, 2 H), 2.50 (s, 3 H), 2.43−2.55 (m, 4 H), 1.79−1.95 (m, 1 H), 0.91 (d, J = 6.6 Hz, 6 H); MS (ESI, Pos 20 V) m/ z 335 (M + H)+. 6-[(4-Isobutyl-2-methoxybenzyl)oxy]-1-methyl-3,4-dihydro2-naphthalenecarbaldehyde (11g). Typical Procedure for the Aldehyde Derivative 11 by the Reaction with the Alkyl Bromide 10 Derived from the Alcohol 9. To a stirred solution of 9g (200 mg, 1.0 mmol) in CH2Cl2 (10 mL) were added PPh3 (405 mg, 1.5 mmol) and CBr4 (511 mg, 1.5 mmol) at 0 °C. After the reaction mixture was stirred for 1 h at ambient temperature, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DMF (10 mL) and K2CO3 (213 mg, 1.5 mmol), and 8w (233 mg, 1.2 mmol) was added to the solution. After the reaction mixture was stirred for 4 h at ambient temperature, water was added to the reaction mixture. The mixture was extracted with EtOAc twice. The combined organic layer was washed with NH4Cl-aq, water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield 11g (278 mg, 77% yield) as a white powder. TLC Rf = 0.54 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.48 (d, J = 8.6 Hz, 1 H), 7.32 (d, J = 7.7 Hz, 1 H), 6.84−6.93 (m, 2 H), 6.74−6.79 (m, 1 H), 6.69−6.72 (m, 1 H), 5.11 (s, 2 H), 3.86 (s, 3 H), 2.67−2.78 (m, 2 H), 2.50 (s, 3 H), 2.46−2.55 (m, 2 H), 2.48 (d, J = 7.1 Hz, 2 H), 1.80−1.97 (m, 1 H), 0.92 (d, J = 6.6 Hz, 6 H); MS (ESI, Pos 20 V) m/z 365 (M + H)+, 177. 6-[(4-Isobutyl-3-methoxybenzyl)oxy]-1-methyl-3,4-dihydro2-naphthalenecarbaldehyde (11h). The same procedure as 11b was performed using 8w (150 mg, 0.80 mmol), 9h (178 mg, 0.92 mmol), PPh3 (251 mg, 0.96 mmol), TMAD (165 mg, 0.96 mmol), and THF (1.5 mL) to yield 11h (204 mg, 70% yield) as a yellow powder. TLC Rf = 0.28 (hexane/EtOAc, 4/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.49 (d, J = 8.5 Hz, 1 H), 7.10 (d, J = 7.5 Hz, 1 H), 6.87−6.95 (m, 3 H), 6.85 (d, J = 2.5 Hz, 1 H), 5.06 (s, 2 H), 3.83 (s, 3 H), 2.69−2.77 (m, 2 H), 2.46−2.55 (m, 7 H), 1.84−1.98 (m, 1 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (ESI, Pos 20 V) m/z 365 (M + H)+. 6-[(2-Chloro-4-isobutylbenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenecarbaldehyde (11i). The same procedure as 11b was performed using 9i (200 mg, 1.0 mmol), 8w (171 mg, 0.91 mmol), PPh3 (286 mg, 1.1 mmol), TMAD (188 mg, 1.1 mmol), and THF (1.7 mL) to yield 11i (127 mg, 38% yield) as a white powder. TLC Rf = 0.43 (hexane/EtOAc, 4/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.50 (d, J = 8.5 Hz, 1 H), 7.42 (d, J = 8.0 Hz, 1 H), 7.21 (d, J = 1.5 Hz, 1 H), 7.07 (dd, J = 8.0, 1.5 Hz, 1 H), 6.88 (dd, J = 8.5, 2.5 Hz, 1 H), 6.85 (d, J = 2.5 Hz, 1 H), 5.17 (s, 2 H), 2.70−2.78 (m, 2 H), 2.44−2.55 (m, 7 H), 1.80−1.93 (m, 1 H), 0.91 (d, J = 6.5 Hz, 6 H); MS (ESI, Pos 20 V) m/z 371, 369 (M + H)+. 6-[(4-Isobutyl-2-methylbenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenecarbaldehyde (11j). The same procedure as 11b was performed using 9j (218 mg, 1.2 mmol), 8w (200 mg, 1.1 mmol), PPh3 (335 mg, 1.3 mmol), TMAD (220 mg, 1.3 mmol), and THF (2 mL) to yield 11j (240 mg, 65% yield) as a pale yellow powder. TLC Rf = 0.52 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 9520



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6-{[4-(2-Hydroxy-2-methylpropyl)-2-methoxybenzyl]oxy}-1methyl-3,4-dihydro-2-naphthalenecarbaldehyde (11p). The same procedure as 11b was performed using 9p (84 mg, 0.40 mmol), 8w (75 mg, 0.40 mmol), PPh3 (125 mg, 0.48 mmol), TMAD (82 mg, 0.48 mmol), and THF (2 mL) to yield 11p (32 mg, 21% yield) as a white powder. TLC Rf = 0.36 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 10.30 (s, 1 H), 7.47 (d, J = 8.5 Hz, 1 H), 7.36 (d, J = 7.5 Hz, 1 H), 6.88 (dd, J = 8.5, 2.5 Hz, 1 H), 6.80−6.86 (m, 2 H), 6.78 (d, J = 1.0 Hz, 1 H), 5.12 (s, 2 H), 3.86 (s, 3 H), 2.77 (s, 2 H), 2.69−2.76 (m, 2 H), 2.47−2.54 (m, 5 H), 1.25 (s, 6 H); MS (ESI, Pos 20 V) m/z 381 (M + H)+. 6-[(2-Methoxy-6-propyl-3-pyridinyl)methoxy]-1-methyl-3,4dihydro-2-naphthalenecarbaldehyde (11q). The same procedure as 11g was performed using 9q (90 mg, 0.50 mmol), CBr4 (200 mg, 0.60 mmol), PPh3 (157 mg, 0.60 mmol), and CH2Cl2 (1 mL) in the bromination reaction and 8w (78 mg, 0.40 mmol), K2CO3 (138 mg, 1.0 mmol), and DMF (1 mL) in the alkylation reaction to yield 11q (121 mg, 86% yield) as a yellow powder. TLC Rf = 0.55 (hexane/ EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.30 (s, 1 H), 7.57 (d, J = 7.5 Hz, 1 H), 7.47 (d, J = 8.6 Hz, 1 H), 6.80−6.90 (m, 2 H), 6.72 (d, J = 7.5 Hz, 1 H), 5.05 (s, 2 H), 3.98 (s, 3 H), 2.60−2.78 (m, 4 H), 2.44−2.56 (m, 5 H), 1.65−1.86 (m, 2 H), 0.97 (t, J = 7.4 Hz, 3 H); MS (EI, Pos) m/z 351 (M)+, 164 (base peak). 6-[(4-Methoxy-6-propyl-3-pyridinyl)methoxy]-1-methyl-3,4dihydro-2-naphthalenecarbaldehyde (11r). The same procedure as 11g was performed using 9r (130 mg, 0.72 mmol), CBr4 (381 mg, 1.15 mmol), PPh3 (302 mg, 1.15 mmol), and CH2Cl2 (2 mL) in the bromination reaction and 8w (95 mg, 0.50 mmol), K2CO3 (207 mg, 1.5 mmol), and DMF (2 mL) in the alkylation reaction to yield 11r (87 mg, 50% yield) as a pale yellow solid. TLC Rf = 0.43 (hexane/ EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 8.43 (s, 1 H), 7.49 (d, J = 8.6 Hz, 1 H), 6.79−6.95 (m, 2 H), 6.70 (s, 1 H), 5.08 (s, 2 H), 3.91 (s, 3 H), 2.67−2.83 (m, 4 H), 2.43−2.59 (m, 5 H), 1.70−1.85 (m, 2 H), 0.99 (t, J = 7.3 Hz, 3 H); MS (FAB, Pos) m/z 352 (M + H)+, 164. 6-[(2-Methoxy-4-propylbenzyl)oxy]-1,5-dimethyl-3,4-dihydro-2-naphthalenecarbaldehyde (11s). The same procedure as 11g was performed using 9n (61 mg, 0.34 mmol), CBr4 (177 mg, 0.53 mmol), PPh3 (132 mg, 0.53 mmol), and CH2Cl2 (3 mL) in bromination reaction and 8x (68 mg, 0.50 mmol), K2CO3 (93 mg, 0.67 mmol), and DMF (3 mL) in alkylation reaction to yield 11s (0.10 g, 81% yield) as a pale yellow powder. TLC Rf = 0.31 (hexane/EtOAc, 5/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.41 (d, J = 8.5 Hz, 1 H), 7.36 (d, J = 7.5 Hz, 1 H), 6.88 (d, J = 8.5 Hz, 1 H), 6.81 (dd, J = 7.5, 1.5 Hz, 1 H), 6.74 (d, J = 1.5 Hz, 1 H), 5.13 (s, 2 H), 3.87 (s, 3 H), 2.69−2.77 (m, 2 H), 2.56−2.64 (m, 2 H), 2.46−2.53 (m, 5 H), 2.26 (s, 3 H), 1.59−1.71 (m, 2 H), 0.96 (t, J = 7.50 Hz, 3 H); MS (ESI, Pos 20 V) m/z 365 (M + H)+. 6-[(2-Methoxy-4-propylbenzyl)oxy]-1,7-dimethyl-3,4-dihydro-2-naphthalenecarbaldehyde (11t). The same procedure as 11g was performed using 9n (89 mg, 0.50 mmol), CBr4 (246 mg, 0.74 mmol), PPh3 (195 mg, 0.74 mmol), and CH2Cl2 (4 mL) in bromination reaction and 8y (100 mg, 0.50 mmol), K2CO3 (137 mg, 1.07 mmol), and DMF (4 mL) in alkylation reaction to yield 11t (93 mg, 52% yield) as a yellow powder. TLC Rf = 0.41 (hexane/ EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.29 (s, 1 H), 7.36 (d, J = 7.5 Hz, 1 H), 7.33 (s, 1 H), 6.78−6.82 (m, 2 H), 6.73 (s, 1 H), 5.12 (s, 2 H), 3.86 (s, 3 H), 2.66−2.74 (m, 2 H), 2.60 (t, J = 7.5 Hz, 2 H), 2.46−2.54 (m, 5 H), 2.29 (s, 3 H), 1.59−1.73 (m, 2 H), 0.97 (t, J = 7.5 Hz, 3 H); MS (ESI, Pos 20 V) m/z 365 (M + H)+. 6-[(2-Methoxy-6-propyl-3-pyridinyl)methoxy]-1,5-dimethyl3,4-dihydro-2-naphthalenecarbaldehyde (11u). The same procedure as 11g was performed using 9q (124 mg, 0.68 mmol), CBr4 (305 mg, 0.92 mmol), PPh3 (241 mg, 0.92 mmol), and CH2Cl2 (1 mL) in bromination reaction and 8x (81 mg, 0.40 mmol), K2CO3 (111 mg, 0.80 mmol), and DMF (2 mL) in alkylation reaction to yield 11u (72 mg, 50% yield) as a pale yellow powder. TLC Rf = 0.52 (hexane/ EtOAc, 5/1); 1H NMR (300 MHz, CDCl3) δ 10.33 (s, 1 H), 7.62 (d, J = 7.3 Hz, 1 H), 7.41 (d, J = 8.8 Hz, 1 H), 6.85 (d, J = 8.8 Hz, 1 H), 6.74 (d, J = 7.3 Hz, 1 H), 5.08 (s, 2 H), 3.99 (s, 3 H), 2.61−2.81 (m, 4

(s, 1 H), 7.50 (d, J = 8.5 Hz, 1 H), 7.29 (d, J = 7.5 Hz, 1 H), 6.98−7.03 (m, 2 H), 6.90 (dd, J = 8.5, 2.5 Hz, 1 H), 6.85 (d, J = 2.5 Hz, 1 H), 5.04 (s, 2 H), 2.69−2.79 (m, 2 H), 2.48−2.57 (m, 5 H), 2.45 (d, J = 7.0 Hz, 2 H), 2.36 (s, 3 H), 1.80−1.93 (m, 1 H), 0.91 (d, J = 6.5 Hz, 6 H); MS (ESI, Pos 20 V) m/z 349 (M + H)+. 6-[(2-Fluoro-4-isobutylbenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenecarbaldehyde (11k). The same procedure as 11b was performed using 9k (139 mg, 0.77 mmol), 8w (120 mg, 0.64 mmol), PPh3 (201 mg, 0.77 mmol), TMAD (132 mg, 0.77 mmol), and THF (1.5 mL) to yield 11k (151 mg, 67% yield) as a pale yellow powder. TLC Rf = 0.45 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.49 (d, J = 8.5 Hz, 1 H), 7.37 (t, J = 8.0 Hz, 1 H), 6.95 (dd, J = 8.0, 1.5 Hz, 1 H), 6.84−6.92 (m, 3 H), 5.13 (s, 2 H), 2.69− 2.77 (m, 2 H), 2.46−2.55 (m, 7 H), 1.81−1.93 (m, 1 H), 0.91 (d, J = 6.5 Hz, 6 H); MS (EI, Pos) m/z 352 (M)+, 165 (base peak). 6-[(2-Methoxy-4-methylbenzyl)oxy]-1-methyl-3,4-dihydro2-naphthalenecarbaldehyde (11l). The same procedure as 11g was performed using 9l (114 mg, 0.75 mmol), CBr4 (400 mg, 1.2 mmol), PPh3 (314 mg, 1.2 mmol), and CH2Cl2 (1 mL) in bromination reaction and 8w (94 mg, 0.50 mmol), K2CO3 (138 mg, 1.0 mmol), and DMF (1 mL) in alkylation reaction to yield 11l (117 mg, 72% yield) as a pale yellow powder. TLC Rf = 0.37 (hexane/ EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.30 (s, 1 H), 7.46 (d, J = 8.4 Hz, 1 H), 7.28 (d, J = 7.7 Hz, 1 H), 6.82−6.92 (m, 2 H), 6.78 (d, J = 7.7 Hz, 1 H), 6.73 (s, 1 H), 5.10 (s, 2 H), 3.85 (s, 3 H), 2.66−2.78 (m, 2 H), 2.44−2.55 (m, 5 H), 2.36 (s, 3 H); MS (EI, Pos) m/z 322 (M)+, 199, 136, 135, 105 (base peak). 6-[(4-Ethyl-2-methoxybenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenecarbaldehyde (11m). The same procedure as 11b was performed using 9m (110 mg, 0.66 mmol), 8w (124 mg, 0.66 mmol), PPh3 (174 mg, 0.66 mmol), TMAD (114 mg, 0.66 mmol), and THF (2 mL) to yield 11m (53 mg, 24% yield) as a pale yellow powder. TLC Rf = 0.40 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.33 (d, J = 7.5 Hz, 1 H), 6.90 (dd, J = 8.5, 2.5 Hz, 1 H), 6.85 (d, J = 2.5 Hz, 1 H), 6.82 (dd, J = 7.5, 1.5 Hz, 1 H), 6.76 (d, J = 1.5 Hz, 1 H), 5.11 (s, 2 H), 3.87 (s, 3 H), 2.62− 2.77 (m, 4 H), 2.47−2.55 (m, 5 H), 1.25 (t, J = 7.5 Hz, 3 H); MS (ESI, Pos 20 V) m/z 337 (M + H)+. 6-[(2-Methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenecarbaldehyde (11n). To a solution of 9n (14.6 g, 81.4 mmol) in Et2O (160 mL) was added PBr3 (4.3 mL, 40.7 mmol) at 0 °C. After the reaction mixture was stirred for 1.5 h at 0 °C, the reaction mixture was added dropwise to NaHCO3-aq. The mixture was extracted with MTBE twice, and the combined organic layer was washed with brine and dried over Na2SO4. The filtrate was concentrated under reduced pressure, and the residue (23.5 g) was used for next reaction without purification. The same procedure as 11a was performed using the residue above (23.5 g), 8w (13.0 g, 69.1 mmol), K2CO3 (12.4 g, 89.5 mmol), and DMF (150 mL) to yield 11n (21.3 g, 88% yield) as a light gray powder. TLC Rf = 0.25 (hexane/EtOAc, 6/1); 1H NMR (300 MHz, CDCl3) δ 10.30 (s, 1 H), 7.46 (d, J = 8.5 Hz, 1 H), 7.31 (d, J = 7.5 Hz, 1 H), 6.89 (dd, J = 8.5, 2.5 Hz, 1 H), 6.84 (d, J = 2.5 Hz, 1 H), 6.79 (dd, J = 7.5, 1.5 Hz, 1 H), 6.73 (d, J = 1.5 Hz, 1 H), 5.10 (s, 2 H), 3.86 (s, 3 H), 2.68−2.76 (m, 2 H), 2.56−2.63 (m, 2 H), 2.47−2.54 (m, 5 H), 1.58−1.73 (m, 2 H), 0.96 (t, J = 7.5 Hz, 3 H); MS (ESI, Pos 20 V) m/z 351 (M + H)+, 163. HRMS (FAB, Pos) calcd for C23H26O3: 351.1960. Found: 351.1962 (M + H)+. 6-[(4-Butyl-2-methoxybenzyl)oxy]-1-methyl-3,4-dihydronaphthalene-2-carbaldehyde (11o). The same procedure as 11b was performed using 9o (48 mg, 0.25 mmol), 8w (69 mg, 0.37 mmol), PPh3 (97 mg, 0.37 mmol), TMAD (63 mg, 0.37 mmol), and THF (1 mL) to yield 11o (37 mg, 41% yield) as a pale yellow oil. TLC Rf = 0.37 (hexane/EtOAc, 6/1); 1H NMR (300 MHz, CDCl3) δ 10.31 (s, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.32 (d, J = 8.0 Hz, 1 H), 6.89 (dd, J = 8.5, 2.5 Hz, 1 H), 6.85 (d, J = 2.5 Hz, 1 H), 6.80 (dd, J = 8.0, 1.5 Hz, 1 H), 6.74 (d, J = 1.5 Hz, 1 H), 5.10 (s, 2 H), 3.86 (s, 3 H), 2.75−2.69 (m, 2 H), 2.62 (t, J = 7.5 Hz, 2 H), 2.53−2.48 (m, 5 H), 1.65−1.55 (m, 2 H), 1.43−1.31 (m, 2 H), 0.94 (t, J = 7.0 Hz, 3 H). 9521



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(Route C). To a solution of 3-azetidinecarboxylic acid (48 mg, 0.47 mmol), NaOH (powder 19 mg, 0.47 mmol), and HC(OMe)3 (0.052 mL, 0.47 mmol) in MeOH (5 mL) was added a solution of 11a (103 mg, 0.31 mmol) in MeOH (1.5 mL) and THF (6.5 mL) at 0 °C. After stirring for 4 h at 0 °C, NaBH4 (18 mg, 0.47 mmol) was added to the mixture at 0 °C. After stirring for 30 min at 0 °C, a solution of 4 mol/L HCl in EtOAc was added to the mixture to pH 4−6. The mixture was concentrated under reduced pressure and the residue was purified by chromatography on silica gel using (CHCl3/MeOH/NH4OH) as an eluent to yield free form of 13a (43 mg, 33% yield). To a suspension of free form of 13a (43 mg, 0.10 mmol) in THF (3 mL) was added a solution of HCl-aq (1 mol/L, 0.16 mL, 0.16 mmol) at 0 °C. THF (20 mL) and water (1 mL) were added to the mixture. To the resulting solution was added Et2O, and precipitates were collected to yield a hydrochloric acid salt form of 13a (25 mg, 56% yield) as a white powder. TLC Rf = 0.32 (CHCl3/MeOH/NH4OH, 80/20/4); 1H NMR (300 MHz, CD3OD) δ 7.81−7.91 (m, 4 H), 7.54 (dd, J = 8.5, 1.5 Hz, 1 H), 7.44−7.51 (m, 2 H), 7.35 (d, J = 8.5 Hz, 1 H), 6.91 (dd, J = 8.5, 2.5 Hz, 1 H), 6.87 (d, J = 2.5 Hz, 1 H), 5.26 (s, 2 H), 4.19− 4.45 (m, 4 H), 4.16 (s, 2 H), 3.64−3.77 (m, 1 H), 2.74 (t, J = 8.0 Hz, 2 H), 2.20−2.29 (m, 5 H); MS (ESI, Pos 20 V) m/z 827 (2M + H)+, 414 (M + H)+, 313, 141. HRMS (FAB, Pos) calcd for C27H27NO3: 414.2069. Found: 414.2059 (M + H)+. 1-{[1-Methyl-6-(1,2,3,4-tetrahydro-2-naphthalenylmethoxy)-3,4-dihydro-2-naphthalenyl]methyl}-3-azetidinecarboxylic Acid (13b). The same procedure as 13a was performed using 11b (170 mg, 0.51 mmol), 3-azetidinecarboxylic acid (77 mg, 0.76 mmol), NaOH (powder, 31 mg, 0.78 mmol), HC(OMe)3 (0.084 mL, 0.77 mmol), THF (4 mL), MeOH (10 mL), and NaBH4 (29 mg, 0.77 mmol) to yield 13b (99 mg, 46% yield) as a white powder. TLC Rf = 0.31 (CHCl3/MeOH/NH4OH, 80/20/4); 1H NMR (300 MHz, CD3OD) δ 7.33 (d, J = 8.5 Hz, 1 H), 7.04−7.06 (m, 4 H), 6.81 (dd, J = 8.5, 2.5 Hz, 1 H), 6.77 (d, J = 2.5 Hz, 1 H), 4.11−4.24 (m, 4 H), 4.08 (s, 2 H), 3.96 (d, J = 6.5 Hz, 2 H), 3.35−3.47 (m, 1 H), 2.96 (dd, J = 16.0, 5.0 Hz, 1 H), 2.80−2.88 (m, 2 H), 2.70−2.77 (m, 2 H), 2.61 (dd, J = 16.0, 10.5 Hz, 1 H), 2.19−2.28 (m, 6 H), 2.06−2.14 (m, 1 H), 1.53−1.64 (m, 1 H); MS (ESI, Pos 20 V) m/z 835 (2M + H)+, 418 (M + H)+, 317. HRMS (FAB, Pos) calcd for C27H31NO3: 418.2382. Found: 418.2404 (M + H)+. 1-{[6-(2,3-Dihydro-1H-inden-2-ylmethoxy)-1-methyl-3,4-dihydro-2-naphthalenyl]methyl}-3-azetidinecarboxylic Acid Hydrochloride (13c). The same procedure as 13a was performed using 11c (126 mg, 0.40 mmol), 3-azetidinecarboxylic acid (61 mg, 0.60 mmol), NaOH (powder, 24 mg, 0.60 mmol), HC(OMe)3 (0.066 mL, 0.60 mmol), THF (3 mL), MeOH (5 mL), and NaBH4 (23 mg, 0.61 mmol) to yield a free form of 13c (95 mg, 59% yield), which was treated with 1 mol/L HCl-aq in THF at ambient temperature for 30 min. Et2O was added to the mixture and resulting precipitates were collected to yield a hydrochloric acid salt form of 13c (90 mg, 87% yield) as a pale yellow powder. TLC Rf = 0.20 (CHCl3/MeOH/ NH4OH, 80/20/4); 1H NMR (300 MHz, CD3OD) δ 7.33 (d, J = 8.5 Hz, 1 H), 7.15−7.21 (m, 2 H), 7.07−7.13 (m, 2 H), 6.79 (dd, J = 8.5, 2.5 Hz, 1 H), 6.74 (d, J = 2.5 Hz, 1 H), 4.21−4.41 (m, 4 H), 4.15 (s, 2 H), 3.97 (d, J = 7.0 Hz, 2 H), 3.63−3.76 (m, 1 H), 3.06−3.18 (m, 2 H), 2.78−2.99 (m, 3 H), 2.69−2.77 (m, 2 H), 2.19−2.29 (m, 5 H); MS (ESI, Pos 20 V) m/z 807 (2M + H)+, 404 (M + H)+, 303. HRMS (FAB, Pos) calcd for C26H29NO3: 404.2226. Found: 404.2227 (M + H)+. 1-({1-Methyl-6-[2-(1,2,3,4-tetrahydro-1-naphthalenyl)ethoxy]-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13d). The same procedure as 13a was performed using 11d (180 mg, 0.51 mmol), 3-azetidinecarboxylic acid (77 mg, 0.76 mmol), NaOH (powder, 30 mg, 0.76 mmol), HC(OMe)3 (0.086 mL, 0.76 mmol), THF (1.5 mL), MeOH (1.5 mL), and NaBH4 (58 mg, 1.53 mmol) to yield 13d (113 mg, 51% yield) as a pale yellow amorphous powder. TLC Rf = 0.37 (CHCl3/MeOH/NH4OH, 80/20/ 4); 1H NMR (300 MHz, CD3OD) δ 7.33 (d, J = 8.5 Hz, 1 H), 7.09− 7.15 (m, 1 H), 7.01−7.08 (m, 3 H), 6.79 (dd, J = 8.5, 2.5 Hz, 1 H), 6.74 (d, J = 2.5 Hz, 1 H), 4.03−4.25 (m, 8 H), 3.34−3.49 (m, 1 H), 3.01−3.10 (m, 1 H), 2.69−2.80 (m, 4 H), 2.09−2.29 (m, − H), 1.70−

H), 2.42−2.56 (m, 5 H), 2.26 (s, 3 H), 1.66−1.84 (m, 2 H), 0.97 (t, J = 7.3 Hz, 3 H); MS (FAB, Pos) m/z 366 (M + H)+, 164. Methyl 1-({6-[(4-Isobutyl-2-methoxybenzyl)oxy]-1-methyl3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylate (12g). Typical Procedure for the Amino Acid Ester Derivative 12 (Route D). Methyl 3-Azetidinecarboxylate Hydrochloride. To MeOH (70 mL) was added SOCl2 (23.4 mL, 321 mmol) at 0 °C with stirring. After 15 min, 3-azetidinecarboxylic acid (25 g, 247 mmol) was added to the mixture in five portions at 0 °C. After the reaction mixture was stirred for 2 h at ambient temperature, the reaction mixture was concentrated under reduced pressure under 30 °C. The residue was washed with Et2O twice to yield methyl 3azetidinecarboxylate hydrochloride (35.8 g, 95% yield) as an orange powder. TLC Rf = 0.68 (CHCl3/MeOH/NH4OH, 80/20/4); 1H NMR (300 MHz, CD3OD) δ 4.18−4.33 (m, 4 H), 3.72−3.81 (m, 4 H); MS (ESI, Pos 20 V) m/z 231 (2M + H)+, 116 (M + H)+. To a stirred solution of 11g (800 mg, 2.2 mmol), methyl 3azetidinecaboxylate hydrochloride (400 mg, 2.6 mmol), and AcOH (0.50 mL) in DMF (10 mL) was added NaBH(OAc)3 (558 mg, 2.6 mmol) at 0 °C. After reaction mixture was stirred overnight at ambient temperature, it was concentrated under reduced pressure and NaHCO3-aq was added to the mixture. Reaction mixture was extracted with EtOAc (5 times), and the combined organic layer was washed with NaHCO3-aq, water, brine and dried over Na2SO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel (hexane/EtOAc) to yield 12g (856 mg, 84% yield) as a colorless oil. TLC Rf = 0.46 (hexane/EtOAc, 1/5). 1H NMR (300 MHz, CDCl3) δ 7.33 (d, J = 7.7 Hz, 1 H), 7.19 (d, J = 8.2 Hz, 1 H), 6.78−6.85 (m, 2 H), 6.75 (dd, J = 7.7, 1.5 Hz, 1 H), 6.69 (d, J = 1.5 Hz, 1 H), 5.06 (s, 2 H), 3.85 (s, 3 H), 3.70 (s, 3 H), 3.49−3.59 (m, 2 H), 3.23−3.41 (m, 5 H), 2.63−2.73 (m, 2 H), 2.47 (d, J = 7.1 Hz, 2 H), 2.21−2.31 (m, 2 H), 2.09 (s, 3 H), 1.81−1.94 (m, 1 H), 0.92 (d, J = 6.6 Hz, 6 H); MS (ESI, Pos 20 V) m/z 927 (2M + H)+, 464 (M + H)+, 349, 177. Methyl 1-({6-[(2-Methoxy-4-propylbenzyl)oxy]-1-methyl3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylate (12n). To a stirred solution of 11n (22.0 g, 62.8 mmol) and AcOH (9.5 mL) in DMF (190 mL) was added methyl 3-azetidinecaboxylate hydrochloride (12.4 g, 81.7 mmol) at 0 °C. After the reaction mixture was stirred for 10 min, NaBH(OAc)3 (17.3 g, 81.7 mmol) was added to the reaction mixture at 0 °C in several portions. After the reaction mixture was stirred for 8 h at ambient temperature, the reaction mixture was poured into (ice (∼500 mL)/saturated NaHCO3-aq (1.9 L)) and extracted with MTBE twice. Combined organic layer was washed with brine and dried over Na2SO4. The filtrate was concentrated under reduced pressure to yield 12n (31.0 g, quantitative yield) as a brown oil. The residue after workup was used for next reaction without purification. TLC Rf = 0.23 (CHCl3/MeOH, 20/1). Methyl 1-({6-[(4-Butyl-2-methoxybenzyl)oxy]-1-methyl-3,4dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylate (12o). The same procedure as 12g was performed using 11o (34 mg, 0.093 mmol), methyl 3-azetidinecaboxylate hydrochloride (21 mg, 0.14 mmol), NaBH(OAc)3 (29 mg, 0.14 mmol), AcOH (0.025 mL), and DMF (0.50 mL) to yield 12o (29 mg, 67% yield) as a yellow oil. TLC Rf = 0.29 (hexane/EtOAc, 1/2); 1H NMR (300 MHz, CDCl3) δ 7.32 (d, J = 7.5 Hz, 1 H), 7.18 (d, J = 8.5 Hz, 1 H), 6.82−6.75 (m, 3 H), 6.71 (s, 1 H), 5.05 (s, 2 H), 3.84 (s, 3 H), 3.70 (s, 3 H), 3.56−3.52 (m, 2 H), 3.39−3.24 (m, 5 H), 2.68 (t, J = 7.5 Hz, 2 H), 2.61−2.55 (m, 2 H), 2.28−2.24 (m, 2 H), 2.08 (s, 3 H), 1.70−1.59 (m, 4 H), 0.95 (t, J = 7.0 Hz, 3 H). Methyl 1-[(6-{[4-(2-Hydroxy-2-methylpropyl)-2methoxybenzyl]oxy}-1-methyl-3,4-dihydronaphthalen-2-yl)methyl]azetidine-3-carboxylate (12p). The same procedure as 12g was performed using 11p (29 mg, 0.076 mmol), methyl 3azetidinecaboxylate hydrochloride (17 mg, 0.11 mmol), NaBH(OAc)3 (24 mg, 0.11 mmol), AcOH (0.10 mL), and DMF (1 mL) to yield crude 12p (22 mg) as an oil. TLC Rf = 0.23 (EtOAc). MS (ESI, Pos 20 V) m/z 959 (2M + H)+, 480 (M + H)+, 365, 193. 1-{[1-Methyl-6-(2-naphthylmethoxy)-3,4-dihydro-2naphthalenyl]methyl}-3-azetidinecarboxylic Acid Hydrochloride (13a). Typical Procedure for the Amino Acid Derivative 13 9522



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2.03 (m, 5 H); MS (ESI, Pos 20 V) m/z 863 (2M + H)+, 432 (M + H)+, 331. HRMS (FAB, Pos) calcd for C28H33NO3: 432.2539. Found: 432.2531 (M + H)+. 1-({6-[2-(2,3-Dihydro-1H-inden-1-yl)ethoxy]-1-methyl-3,4dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13e). The same procedure as 13a was performed using 11e (120 mg, 0.36 mmol), 3-azetidinecarboxylic acid (77 mg, 0.76 mmol), NaOH (powder, 30 mg, 0.76 mmol), HC(OMe)3 (0.086 mL, 0.76 mmol), THF (1.5 mL), MeOH (1.5 mL), and NaBH4 (58 mg, 1.53 mmol) to yield 13e (65 mg, 43% yield) as a white powder. TLC Rf = 0.32 (CHCl3/MeOH/NH4OH, 80/20/4); 1H NMR (300 MHz, CD3OD) δ 7.33 (d, J = 8.5 Hz, 1 H), 7.16−7.22 (m, 2 H), 7.08−7.13 (m, 2 H), 6.79 (dd, J = 8.5, 2.5 Hz, 1 H), 6.75 (d, J = 2.5 Hz, 1 H), 4.14−4.25 (m, 4 H), 4.07−4.14 (m, 4 H), 3.35−3.48 (m, 1 H), 3.28− 3.32 (m, 1 H), 2.69−3.01 (m, 4 H), 2.19−2.39 (m, 7 H), 1.71−1.91 (m, 2 H); MS (ESI, Pos 20 V) m/z 835 (2M + H)+, 418 (M + H)+, 317. HRMS (FAB, Pos) calcd for C27H31NO3: 418.2382. Found: 418.2400 (M + H)+. 1-({6-[(4-Isobutylbenzyl)oxy]-1-methyl-3,4-dihydro-2naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13f). The same procedure as 13a was performed using 11f (75 mg, 0.22 mmol), 3-azetidinecarboxylic acid (23 mg, 0.22 mmol), NaOH (powder, 9.0 mg, 0.22 mmol), HC(OMe)3 (0.027 mL, 0.24 mmol), MeOH (2 mL), and NaBH4 (13 mg, 0.33 mmol) to yield 13f (40 mg, 39% yield) as a white powder. TLC Rf = 0.20 (n-BuOH/AcOH/H2O, 20/4/1); 1H NMR (300 MHz, CD3OD) δ 7.24−7.43 (m, 3 H), 7.15 (d, J = 8.1 Hz, 2 H), 6.73−6.94 (m, 2 H), 5.04 (s, 2 H), 4.16 (s, 2 H), 4.06−4.47 (m, 4 H), 3.60−3.82 (m, 1 H), 2.73 (t, J = 8.4 Hz, 2 H), 2.48 (d, J = 7.1 Hz, 2 H), 2.13−2.31 (m, 5 H), 1.69−1.98 (m, 1 H), 0.90 (d, J = 6.6 Hz, 6 H); MS (APCI, Neg 20 V) m/z 418 (M − H)−. HRMS (FAB, Pos) calcd for C27H33NO3: 420.2539. Found: 420.2520 (M + H)+. 1-({6-[(4-Isobutyl-2-methoxybenzyl)oxy]-1-methyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13g). Typical Procedure for the Amino Acid Derivative 13 (Route E). To a stirred solution of 12g (829 mg, 1.8 mmol) in MeOH (15 mL) was added 1 mol/L NaOH-aq (5 mL, 5 mmol) at 0 °C. After the reaction mixture was stirred for 3 h at ambient temperature, 1 mol/L HCl-aq (5 mL, 5 mmol) was added to the mixture to neutralize. The reaction mixture was concentrated under reduced pressure, and the residue was triturated with MeOH/water. Collected powder was purified by recrystallization from THF (2.5 mL)/water (4.5 mL) to yield 13g (721 mg, 90% yield) as a white solid. TLC Rf = 0.23 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.32 (d, J = 8.6 Hz, 1 H), 7.26 (d, J = 7.7 Hz, 1 H), 6.83 (dd, J = 8.6, 2.7 Hz, 1 H), 6.77−6.80 (m, 2 H), 6.71−6.75 (m, 1 H), 5.04 (s, 2 H), 4.11−4.27 (m, 4 H), 4.09 (s, 2 H), 3.84 (s, 3 H), 3.34−3.48 (m, 1 H), 2.67−2.77 (m, 2 H), 2.48 (d, J = 7.3 Hz, 2 H), 2.20 (s, 3 H), 2.18− 2.28 (m, 2 H), 1.81−1.95 (m, 1 H), 0.91 (d, J = 6.8 Hz, 6 H); MS (ESI, Pos 20 V) m/z 899 (2M + H)+, 450 (M + H)+, 349, 177. HRMS (FAB, Neg) calcd for C28H35NO4: 448.2488. Found: 448.2493 (M − H)−. 1-({6-[(4-Isobutyl-3-methoxybenzyl)oxy]-1-methyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13h). The same procedure as 13a was performed using 11h (118 mg, 0.32 mmol) and 3-azetidinecarboxylic acid (49 mg, 0.49 mmol), NaOH (powder, 19 mg, 0.49 mmol), HC(OMe)3 (0.053 mL, 0.49 mmol), THF (3 mL), MeOH (5 mL), and NaBH4 (18 mg, 0.49 mmol) to yield 13h (76 mg, 52% yield) as a white powder. TLC Rf = 0.17 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.32 (d, J = 8.5 Hz, 1 H), 7.05 (d, J = 7.5 Hz, 1 H), 6.98 (d, J = 1.5 Hz, 1 H), 6.90 (dd, J = 7.5, 1.5 Hz, 1 H), 6.85 (dd, J = 8.5, 2.5 Hz, 1 H), 6.81 (d, J = 2.5 Hz, 1 H), 5.04 (s, 2 H), 4.12−4.26 (m, 4 H), 4.08 (s, 2 H), 3.80 (s, 3 H), 3.34−3.47 (m, 1 H), 2.69−2.77 (m, 2 H), 2.46 (d, J = 7.0 Hz, 2 H), 2.19−2.29 (m, 5 H), 1.81−1.95 (m, 1 H), 0.86 (d, J = 7.0 Hz, 6 H); MS (ESI, Pos 20 V) m/z 899 (2M + H)+, 450 (M + H)+, 349. HRMS (FAB, Neg) calcd for C28H35NO4: 448.2488. Found: 448.2493 (M − H)−. 1-({6-[(2-Chloro-4-isobutylbenzyl)oxy]-1-methyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13i).

The same procedure as 13a was performed using 11i (125 mg, 0.34 mmol) and 3-azetidinecarboxylic acid (51 mg, 0.51 mmol), NaOH (powder, 20 mg, 0.51 mmol), HC(OMe)3 (0.056 mL, 0.51 mmol), THF (1 mL), MeOH (5 mL), and NaBH4 (19 mg, 0.51 mmol) to yield 13i (88 mg, 57% yield) as a white powder. TLC Rf = 0.23 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.43 (d, J = 8.0 Hz, 1 H), 7.34 (d, J = 8.5 Hz, 1 H), 7.23 (d, J = 1.5 Hz, 1 H), 7.11 (dd, J = 8.0, 1.5 Hz, 1 H), 6.85 (dd, J = 8.5, 2.5 Hz, 1 H), 6.81 (d, J = 2.5 Hz, 1 H), 5.13 (s, 2 H), 4.10−4.24 (m, 4 H), 4.08 (s, 2 H), 3.36−3.47 (m, 1 H), 2.70−2.78 (m, 2 H), 2.48 (d, J = 7.0 Hz, 2 H), 2.19−2.28 (m, 5 H), 1.81−1.92 (m, 1 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (ESI, Pos 20 V) m/z 909, 907 (2M + H)+, 456, 454 (M + H)+, 355, 353. HRMS (FAB, Neg) calcd for C27H32ClNO3: 452.1992. Found: 452.2007 (M − H)−. 1-({6-[(4-Isobutyl-2-methylbenzyl)oxy]-1-methyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13j). The same procedure as 13a was performed using 11j (143 mg, 0.41 mmol) and 3-azetidinecarboxylic acid (62 mg, 0.62 mmol), NaOH (powder, 25 mg, 0.62 mmol), HC(OMe)3 (0.068 mL, 0.62 mmol), THF (0.5 mL), MeOH (5 mL), and NaBH4 (23 mg, 0.62 mmol) to yield 13j (67 mg, 38% yield) as a white powder. TLC Rf = 0.21 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.31 (d, J = 8.5 Hz, 1 H), 7.25 (d, J = 7.5 Hz, 1 H), 6.99 (s, 1 H), 6.95 (d, J = 7.5 Hz, 1 H), 6.85 (dd, J = 8.5, 2.5 Hz, 1 H), 6.80 (d, J = 2.5 Hz, 1 H), 5.02 (s, 2 H), 4.01−4.18 (m, 4 H), 3.99 (s, 2 H), 3.34−3.45 (m, 1 H), 2.69−2.77 (m, 2 H), 2.44 (d, J = 7.0 Hz, 2 H), 2.33 (s, 3 H), 2.18−2.29 (m, 5 H), 1.77−1.93 (m, 1 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (ESI, Pos 20 V) m/z 867 (2M + H)+, 434 (M + H)+, 333. HRMS (FAB, Neg) calcd for C28H35NO3: 432.2539. Found: 432.2539 (M − H)−. 1-({6-[(2-Fluoro-4-isobutylbenzyl)oxy]-1-methyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13k). The same procedure as 13a was performed using 11k (150 mg, 0.43 mmol) and 3-azetidinecarboxylic acid (65 mg, 0.64 mmol), NaOH (powder, 26 mg, 0.64 mmol), HC(OMe)3 (0.070 mL, 0.64 mmol), THF (1 mL), MeOH (6 mL), and NaBH4 (24 mg, 0.64 mmol) to yield 13k (78 mg, 42% yield) as a white powder. TLC Rf = 0.31 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.31−7.42 (m, 2 H), 6.90−7.01 (m, 2 H), 6.86 (dd, J = 8.5, 2.5 Hz, 1 H), 6.81 (d, J = 2.5 Hz, 1 H), 5.09 (s, 2 H), 4.11−4.24 (m, 4 H), 4.08 (s, 2 H), 3.35−3.47 (m, 1 H), 2.69−2.77 (m, 2 H), 2.49 (d, J = 7.0 Hz, 2 H), 2.19−2.28 (m, 5 H), 1.79−1.94 (m, 1 H), 0.90 (d, J = 6.5 Hz, 6 H); MS (ESI, Pos 20 V) m/z 875 (2M + H)+, 438 (M + H)+, 337. HRMS (FAB, Neg) calcd for C27H32FNO3: 436.2288. Found: 436.2289 (M − H)−. 1-({6-[(2-Methoxy-4-methylbenzyl)oxy]-1-methyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13l). The same procedure as 13a was performed using 11l (117 mg, 0.36 mmol) and 3-azetidinecarboxylic acid (61 mg, 0.60 mmol), NaOH (powder, 24 mg, 0.60 mmol), HC(OMe)3 (0.066 mL, 0.60 mmol), THF (1 mL), MeOH (1.5 mL), and NaBH4 (20 mg, 0.52 mmol) to yield 13l (87 mg, 59% yield) as a white powder. TLC Rf = 0.19 (CHCl3/MeOH/NH4OH, 20/5/1) ; 1H NMR (300 MHz, CD3OD) δ 7.31 (d, J = 8.4 Hz, 1 H) 7.23 (d, J = 7.5 Hz, 1 H) 6.69−6.88 (m, 4 H) 5.03 (s, 2 H) 4.10−4.26 (m, 4 H) 4.07 (s, 2 H) 3.84 (s, 3 H) 3.33− 3.49 (m, 1 H) 2.64−2.79 (m, 2 H) 2.33 (s, 3 H) 2.15−2.29 (m, 5 H); MS (ESI, Pos 20 V) m/z 815 (2M + H)+, 408 (M + H)+, 307, 135. HRMS (FAB, Neg) calcd for C25H29NO4: 406.2018. Found: 406.2013 (M − H)−. 1-({6-[(4-Ethyl-2-methoxybenzyl)oxy]-1-methyl-3,4-dihydro2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13m). The same procedure as 13a was performed using 11m (50 mg, 0.15 mmol), 3-azetidinecarboxylic acid (22 mg, 0.22 mmol), NaOH (powder, 9 mg, 0.22 mmol), HC(OMe)3 (0.024 mL, 0.22 mmol), THF (0.7 mL), MeOH (2.5 mL), and NaBH4 (8 mg, 0.22 mmol) to yield 13m (21 mg, 33% yield) as a white powder. TLC Rf = 0.28 (CHCl3/MeOH/ NH4OH, 80/20/4); 1H NMR (300 MHz, CD3OD) δ 7.30 (d, J = 8.5 Hz, 1 H), 7.25 (d, J = 7.5 Hz, 1 H), 6.74−6.85 (m, 4 H), 5.04 (s, 2 H), 4.08−4.24 (m, 4 H), 4.06 (s, 2 H), 3.85 (s, 3 H), 3.35−3.46 (m, 1 H), 2.60−2.76 (m, 4 H), 2.18−2.28 (m, 5 H), 1.24 (t, J = 7.5 Hz, 3 H); 9523



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MS (ESI, Pos 20 V) m/z 843 (2M + H)+, 422 (M + H)+, 321, 149. HRMS (FAB, Neg) calcd for C26H31NO4: 420.2175. Found: 420.2176 (M − H)−. 1-({6-[(2-Methoxy-4-propylbenzyl)oxy]-1-methyl-3,4dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic Acid (13n). To a stirred solution of 12n (27.8 g, 61.9 mmol) in THF (37 mL) and MeOH (37 mL) was added 5 mol/L NaOH-aq (18.5 mL, 93.0 mmol) at 0 °C. After the reaction mixture was stirred for 3 h at ambient temperature, 5 mol/L HCl-aq (18.5 mL, 93.0 mmol) was added to the mixture to neutralize. The reaction mixture was concentrated under reduced pressure, and the mixture of PhMe (333 mL) and EtOH (167 mL) was added to the residue, and inorganic salts were removed through Celite. The filtrate was concentrated under reduced pressure and the residue was recrystallized three times from MeOH, (MeOH/H2O) and (MeOH/Et2O) to yield 13n (10.9 g, 42% yield in 2 steps) as a white powder. TLC Rf = 0.20 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (600 MHz, CD3OD) δ 7.31 (d, J = 8.5 Hz, 1 H), 7.25 (d, J = 8.0 Hz, 1 H), 6.83−6.81 (m, 2 H), 6.78 (d, J = 2.5 Hz, 1 H), 6.76 (dd, J = 8.0, 1.5 Hz, 1 H), 5.04 (s, 2 H), 4.24−4.14 (m, 4 H), 4.09 (s, 2 H), 3.84 (s, 3 H), 3.41 (quintet, J = 8.0 Hz, 1 H), 2.72 (t, J = 7.5 Hz, 2 H), 2.59 (t, J = 7.5 Hz, 2 H), 2.24 (t, J = 7.5 Hz, 2 H), 2.20 (s, 3 H), 1.68−1.61 (m, 2 H), 0.94 (t, J = 7.5 Hz, 3 H); 13C NMR (150 MHz, CD3OD) δ 177.1, 160.2, 158.6, 145.5, 139.2, 136.7, 129.9, 129.6, 126.7, 123.8, 122.5, 121.5, 115.1, 113.4, 111.9, 66.1, 58.5, 58.1, 55.9, 39.2, 36.4, 29.5, 28.1, 25.7, 15.1, 14.1; MS (ESI, Pos 20 V) m/z 871 (2M + H)+, 436 (M + H)+, 335, 163. HRMS (FAB, Neg) calcd for C27H33NO4: 434.2331. Found: 434.2335 (M − H)−. Elemental analysis (average of three experiments) calcd for C27H33NO4: C, 74.45%; H, 7.64%; N, 3.22%. Found: C, 74.47%; H, 7.73%; N, 3.22%. Melting point: 156− 159 °C. 1-({6-[(4-Butyl-2-methoxybenzyl)oxy]-1-methyl-3,4-dihydro2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13o). The same procedure as 13g was performed using 12o (24 mg, 0.052 mmol), 1 mol/L NaOH-aq (1.0 mL, 1.0 mmol), MeOH (1 mL), and THF (1 mL) to yield 13o (19 mg, 82% yield) as a white powder. TLC Rf = 0.36 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.31 (d, J = 8.6 Hz, 1 H), 7.25 (d, J = 7.5 Hz, 1 H), 6.72 v 6.86 (m, 4 H), 5.04 (s, 2 H), 4.12−4.29 (m, 4 H), 4.10 (s, 2 H), 3.84 (s, 3 H), 3.34−3.49 (m, 1 H), 2.72 (t, J = 6.8 Hz, 2 H), 2.61 (t, J = 7.7 Hz, 2 H), 2.15−2.31 (m, 5 H), 1.54−1.67 (m, 2 H), 1.30−1.44 (m, 2 H), 0.94 (t, J = 7.3 Hz, 3 H); MS (ESI, Pos 20 V) m/z 899 (2M + H)+, 450 (M + H)+, 349, 177. HRMS (FAB, Neg) calcd for C28H35NO4: 448.2488. Found: 448.2493 (M − H)−. 1-[(6-{[4-(2-Hydroxy-2-methylpropyl)-2-methoxybenzyl]oxy}-1-methyl-3,4-dihydro-2-naphthalenyl)methyl]-3azetidinecarboxylic Acid (13p). The same procedure as 13g was performed using 12p (22 mg, 0.046 mmol), 1 mol/L NaOH-aq (0.40 mL, 0.40 mmol), MeOH (0.4 mL), and THF (0.4 mL) to yield 13p (20 mg, 57% yield in 2 steps) as a white powder. TLC Rf = 0.25 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.31 (d, J = 8.5 Hz, 1 H), 7.27 (d, J = 7.5 Hz, 1 H), 6.89 (d, J = 1.5 Hz, 1 H), 6.77−6.85 (m, 3 H), 5.06 (s, 2 H), 4.11−4.25 (m, 4 H), 4.08 (s, 2 H), 3.86 (s, 3 H), 3.35−3.46 (m, 1 H), 2.68−2.77 (m, 4 H), 2.19 v 2.28 (m, 5 H), 1.18 (s, 6 H); MS (ESI, Pos 20 V) m/z 931 (2M + H)+, 466 (M + H)+, 365, 193. HRMS (FAB, Neg) calcd for C28H35NO4: 464.2437. Found: 464.2430 (M − H)−. 1-({6-[(2-Methoxy-6-propyl-3-pyridinyl)methoxy]-1-methyl3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13q). The same procedure as 13a was performed using 11q (121 mg, 0.34 mmol), 3-azetidinecarboxylic acid (61 mg, 0.60 mmol), NaOH (powder, 24 mg, 0.60 mmol), HC(OMe)3 (0.066 mL, 0.60 mmol), THF (1 mL), MeOH (1.5 mL), and NaBH4 (20 mg, 0.52 mmol) to yield 13q (66 mg, 44% yield) as a white powder. TLC Rf = 0.19 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.61 (d, J = 7.5 Hz, 1 H), 7.32 (d, J = 8.6 Hz, 1 H), 6.73−6.87 (m, 3 H), 5.01 (s, 2 H), 4.10−4.29 (m, 4 H), 4.08 (s, 2 H), 3.96 (s, 3 H), 3.34−3.49 (m, 1 H), 2.57−2.81 (m, 4 H), 2.15−2.31 (m, 5 H), 1.67−1.82 (m, 2 H), 0.95 (t, J = 7.4 Hz, 3 H); MS (ESI, Pos

20 V) m/z 873 (2M + H)+, 437 (M + H)+, 336, 168. HRMS (FAB, Neg) calcd for C26H32N2O4: 435.2284. Found: 435.2292 (M − H)−. 1-({6-[(4-Methoxy-6-propyl-3-pyridinyl)methoxy]-1-methyl3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13r). The same procedure as 13a was performed using 11r (87 mg, 0.25 mmol), 3-azetidinecarboxylic acid (38 mg, 0.38 mmol), NaOH (powder, 15 mg, 0.38 mmol), HC(OMe)3 (0.042 mL, 0.38 mmol), THF (0.5 mL), MeOH (2 mL), and NaBH4 (12 mg, 0.33 mmol) to yield 13r (39 mg, 36% yield) as a white powder. TLC Rf = 0.27 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 8.28 (s, 1 H), 7.33 (d, J = 8.6 Hz, 1 H), 6.96 (s, 1 H), 6.77−6.88 (m, 2 H), 5.06 (s, 2 H), 4.09−4.28 (m, 4 H), 4.07 (s, 2 H), 3.94 (s, 3 H), 3.34−3.48 (m, 1 H), 2.67−2.78 (m, 4 H), 2.15−2.32 (m, 5 H), 1.65−1.83 (m, 2 H), 0.97 (t, J = 7.3 Hz, 3 H); MS (FAB, Neg) m/z 435 (M − H)−. HRMS (FAB, Neg) calcd for C26H32N2O4: 435.2284. Found: 435.2290 (M − H)−. 1-({6-[(2-Methoxy-4-propylbenzyl)oxy]-1,5-dimethyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13s). The same procedure as 13a was performed using 11s (60 mg, 0.16 mmol), 3-azetidinecarboxylic acid (25 mg, 0.25 mmol), NaOH (powder, 10 mg, 0.25 mmol), HC(OMe)3 (0.027 mL, 0.25 mmol), THF (1.5 mL), MeOH (3 mL), and NaBH4 (9 mg, 0.25 mmol) to yield 13s (24 mg, 32% yield) as a white powder. TLC Rf = 0.19 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.27 (d, J = 7.5 Hz, 1 H), 7.21 (d, J = 8.5 Hz, 1 H), 6.80−6.85 (m, 2 H), 6.76 (dd, J = 7.5, 1.0 Hz, 1 H), 5.05 (s, 2 H), 4.10−4.25 (m, 4 H), 4.07 (s, 2 H), 3.85 (s, 3 H), 3.35−3.46 (m, 1 H), 2.70−2.77 (m, 2 H), 2.56−2.63 (m, 2 H), 2.17−2.27 (m, 8 H), 1.59− 1.72 (m, 2 H), 0.95 (t, J = 7.5 Hz, 3 H); MS (ESI, Pos 20 V) m/z 899 (2M + H)+, 450 (M + H)+, 349, 163. HRMS (FAB, Neg) calcd for C28H35NO4: 448.2488. Found: 448.2486 (M − H)−. 1-({6-[(2-Methoxy-4-propylbenzyl)oxy]-1,7-dimethyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13t). The same procedure as 13a was performed using 11t (70 mg, 0.19 mmol), 3-azetidinecarboxylic acid (30 mg, 0.30 mmol), NaOH (powder, 12 mg, 0.30 mmol), HC(OMe)3 (0.031 mL, 0.30 mmol), THF (1.2 mL), MeOH (3 mL), and NaBH4 (11 mg, 0.30 mmol) to yield 13t (32 mg, 37% yield) as a white powder. TLC Rf = 0.20 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.28 (d, J = 7.5 Hz, 1 H), 7.18 (s, 1 H), 6.82 (d, J = 1.5 Hz, 1 H), 6.75−6.79 (m, 2 H), 5.06 (s, 2 H), 4.10−4.25 (m, 4 H), 4.07 (s, 2 H), 3.85 (s, 3 H), 3.35−3.47 (m, 1 H), 2.66−2.73 (m, 2 H), 2.59 (t, J = 7.5 Hz, 2 H), 2.18−2.26 (m, 8 H), 1.59−1.72 (m, 2 H), 0.95 (t, J = 7.5 Hz, 3 H); MS (ESI, Pos 20 V) m/z 899 (2M + H)+, 450 (M + H)+, 349, 163. HRMS (FAB, Neg) calcd for C28H35NO4: 448.2488. Found: 448.2493 (M − H)−. 1-({6-[(2-Methoxy-6-propyl-3-pyridinyl)methoxy]-1,5-dimethyl-3,4-dihydro-2-naphthalenyl}methyl)-3-azetidinecarboxylic Acid (13u). The same procedure as 13a was performed using 11u (73 mg, 0.20 mmol), 3-azetidinecarboxylic acid (38 mg, 0.38 mmol), NaOH (powder, 15 mg, 0.38 mmol), HC(OMe)3 (0.042 mL, 0.38 mmol), THF (0.5 mL), MeOH (2 mL), and NaBH4 (12 mg, 0.33 mmol) to yield 13u (29 mg, 32% yield) as a white powder. TLC Rf = 0.40 (CHCl3/MeOH/NH4OH, 20/5/1); 1H NMR (300 MHz, CD3OD) δ 7.62 (d, J = 7.3 Hz, 1 H), 7.22 (d, J = 8.8 Hz, 1 H), 6.82 (d, J = 8.8 Hz, 1 H), 6.77 (d, J = 7.3 Hz, 1 H), 5.00 (s, 2 H), 4.08−4.24 (m, 4 H), 4.05 (s, 2 H), 3.96 (s, 3 H), 3.34−3.48 (m, 1 H), 2.69−2.77 (m, 2 H), 2.61−2.69 (m, 2 H), 2.14−2.29 (m, 8 H), 1.64− 1.85 (m, 2 H), 0.95 (t, J = 7.4 Hz, 3 H); MS (FAB, Neg) m/z 449 (M − H)−. HRMS (FAB, Neg) calcd for C27H34N2O4: 449.2440. Found: 449.2445 (M − H)−. 3-Methoxyphenyl Trifluoromethanesulfonate (16). To a solution of 3-methoxyphenol 15 (5.0 g, 40.3 mmol) in CH2Cl2 (30 mL) were added pyridine (15.6 mL, 193 mmol) and Tf2O (8.1 mL, 48 mmol) at 0 °C. After the reaction mixture was stirred for 20 min at 0 °C, the reaction mixture was poured into saturated NaHCO3-aq. The mixture was extracted with CH2Cl2 and organic layer was washed with saturated NaHCO3-aq, water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure, and the residue was dissolved in MTBE, and organic layer was washed with 1 mol/L HClaq, saturated NaHCO3-aq, water, brine and dried over MgSO4. The 9524



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filtrate was concentrated under reduced pressure to yield 16 (10.0 g, 97% yield) as an orange oil. TLC Rf = 0.59 (hexane/EtOAc, 6/1); 1H NMR (300 MHz, CDCl3) δ 7.35 (t, J = 8.3 Hz, 1 H), 6.93 (dd, J = 8.3, 2.38 Hz, 1 H), 6.87 (dd, J = 8.3, 2.4 Hz, 1 H), 6.81 (t, J = 2.4 Hz, 1 H), 3.83 (s, 3 H); MS (EI, Pos) m/z 256 (M+), 123. 1-Methoxy-3-(2-methylpropyl)benzene (17). To a stirred solution of 16 (2.0 g, 7.8 mmol) in THF (35 mL) and NMP (3.8 mL) were added Fe(acac)3 (276 mg, 0.78 mmol) and isobutylmagnesium bromide (2 mol/L in Et2O, 4.7 mL, 9.4 mmol) at 0 °C. After the reaction mixture was stirred for 3 h at ambient temperature, NH4Cl-aq was added to the reaction mixture. The mixture was extracted with MTBE twice. The combined organic layer was washed with NH4Cl-aq, water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield 17 (923 mg, 72% yield) as a colorless oil. TLC Rf = 0.48 (hexane/EtOAc, 20/1); 1H NMR (300 MHz, CDCl3) δ 7.17 (t, J = 7.8 Hz, 1 H), 6.67−6.75 (m, 3 H), 3.79 (s, 3 H), 2.44 (d, J = 7.3 Hz, 2 H), 1.78−1.94 (m, 1 H), 0.90 (d, J = 6.6 Hz, 6 H); MS (EI, Pos) m/z 164 (M+), 121 (base peak), 107. 4-Isobutyl-2-methoxybenzaldehyde (18). To a stirred solution of TMEDA (389 mg, 3.4 mmol) in n-hexane (9 mL) were added tBuLi (1.6 mol/L in n-pentane, 2.1 mL, 3.4 mmol) and 17 (500 mg, 3.0 mmol) in n-hexane (6 mL) at −78 °C. After the reaction mixture was stirred for 4 h at 0 °C, the reaction mixture was added to a solution of DMF (5 mL) and THF (30 mL) at 0 °C. After the reaction mixture was stirred for 4 h at 0 °C, the reaction mixture was poured into NH4Cl-aq. The mixture was extracted with EtOAc twice. The combined organic layer was washed with NH4Cl-aq, water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield 18 (184 mg, 31% yield) as a pale yellow oil. TLC Rf = 0.66 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.39 (s, 1 H), 7.73 (d, J = 7.9 Hz, 1 H), 6.81 (d, J = 7.9 Hz, 1 H), 6.73 (d, J = 0.9 Hz, 1 H), 3.91 (s, 3 H), 2.51 (d, J = 7.3 Hz, 2 H), 1.83−1.98 (m, 1 H), 0.92 (d, J = 6.6 Hz, 6 H); MS (EI, Pos) m/z 192 (M+), 174, 150, 149, 121 (base peak), 91. Methyl 3-Methoxy-4-{[(trifluoromethyl)sulfonyl]oxy}benzoate (20h). The same procedure as 16 was performed using methyl 4-hydroxy-3-methoxybenzoate 19h (3.0 g, 16.5 mmol), Tf2O (3.3 mL, 19.6 mmol), pyridine (2.7 mL, 33.4 mmol), and CH2Cl2 (30 mL) to yield 20h (5.7 g, quantitative yield) as a yellow oil. TLC Rf = 0.32 (hexane/EtOAc, 5/1); 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J = 2.5 Hz, 1 H), 7.67 (dd, J = 8.5, 2.5 Hz, 1 H), 7.27 (d, J = 8.5 Hz, 1 H), 3.98 (s, 3 H), 3.94 (s, 3 H); MS (EI, Pos) m/z 314 (M)+, 283, 181 (base peak). Methyl 2-Chloro-4-{[(trifluoromethyl)sulfonyl]oxy}benzoate (20i). The same procedure as 16 was performed using methyl 2chloro-4-hydroxybenzoate 19i (700 mg, 3.7 mmol), Tf2O (0.95 mL, 5.6 mmol), pyridine (0.76 mL, 9.4 mmol), and CH2Cl2 (7 mL) to yield 20i (1.15 g, 97%) as a yellow oil. TLC Rf = 0.21 (hexane/EtOAc, 10/1); 1H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 8.5 Hz, 1 H), 7.40 (d, J = 2.5 Hz, 1 H), 7.26 (dd, J = 8.5, 2.5 Hz, 1 H), 3.95 (s, 3 H); MS (EI, Pos) m/z 320, 318 (M)+, 289, 287, 225, 223, 69 (base peak). Methyl 4-Isobutyl-3-methoxybenzoate (21h). The same procedure as 17 was performed using 20h (3.0 g, 9.6 mmol), isobutylmagnesium bromide (2 mol/L in Et2O, 5.7 mL, 11.5 mmol), Fe(acac)3 (169 mg, 0.48 mmol), dry THF (50 mL), and dry NMP (5 mL) to yield 21h (1.104 g, 52% yield) as a pale yellow oil. TLC Rf = 0.42 (hexane/EtOAc, 8/1); 1H NMR (300 MHz, CDCl3) δ 7.56 (dd, J = 7.5, 1.5 Hz, 1 H), 7.48 (d, J = 1.5 Hz, 1 H), 7.12 (d, J = 7.5 Hz, 1 H), 3.90 (s, 3 H), 3.86 (s, 3 H), 2.52 (d, J = 7.0 Hz, 2 H), 1.84−2.00 (m, 1 H), 0.89 (d, J = 7.0 Hz, 6 H); MS (FAB, Pos, matrix = glycerin + mNBA) m/z 223 (M + H)+. Methyl 2-Chloro-4-isobutylbenzoate (21i). The same procedure as 17 was performed using 20i (637 mg, 2.0 mmol), isobutylmagnesium bromide (2 mol/L in Et2O, 1.2 mL, 2.4 mmol), Fe(acac)3 (35 mg, 0.10 mmol), dry THF (12 mL), and dry NMP (1.2 mL) to yield 21i (322 mg, 71% yield) as a colorless oil. TLC Rf = 0.48 (hexane/EtOAc, 10/1); 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J = 8.0 Hz, 1 H), 7.24 (d, J = 1.5 Hz, 1 H), 7.08 (dd, J = 8.0, 1.5 Hz, 1 H),

3.92 (s, 3 H), 2.48 (d, J = 7.5 Hz, 2 H), 1.81−1.96 (m, 1 H), 0.91 (d, J = 6.5 Hz, 6 H); MS (EI, Pos) m/z 228, 226 (M)+, 186, 184 (base peak). Methyl 4-Chloro-2-methylbenzoate (23j). To MeOH (6 mL) was added SOCl2 (0.64 mL, 8.8 mmol) at 0 °C. After stirring for 10 min at 0 °C, 4-chloro-2-methylbenzoic acid 22 (1.0 g, 5.9 mmol) and MeOH (4 mL) were added to the reaction mixture at 0 °C. The reaction mixture was warmed to ambient temperature, and MeOH (10 mL) was added to the mixture. The resulting solution was allowed to stir at ambient temperature for 17.5 h. A solution of MeOH (4 mL) and SOCl2 (0.64 mL, 8.8 mmol) was added to the reaction mixture at 0 °C. The resulting solution was allowed to stir at ambient temperature for 2 days. After checking consumption of the starting material, the reaction mixture was concentrated under reduced pressure. To the residue were added ice and saturated NaHCO3-aq. The mixture was extracted with EtOAc twice and combined organic layer was washed with water and brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 23j (1.20 g, quantitative yield) as a colorless oil. TLC Rf = 0.56 (hexane/EtOAc, 5/ 1); 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 8.0 Hz, 1 H), 7.18− 7.25 (m, 2 H), 3.88 (s, 3 H), 2.58 (s, 3 H); MS (EI, Pos) m/z 184 (M)+, 153, 84 (base peak). Methyl 4-Isobutyl-2-methylbenzoate (24j). The same procedure as 17 was performed using methyl 23j (490 mg, 2.7 mmol), isobutylmagnesium bromide (2 mol/L in Et2O, 1.6 mL, 3.2 mmol), Fe(acac)3 (47 mg, 0.13 mmol), dry THF (15 mL), and dry NMP (1.5 mL) to yield crude 24j (590 mg, ∼50% of starting material was remained) as a pale yellow oil, which was used for the next reaction without purification. Ethyl 2-Fluoro-4-isobutylbenzoate (24k). The same procedure as 17 was performed using ethyl 4-chloro-2-fluorobenzoate 23k (1.0 g, 5.0 mmol), isobutylmagnesium bromide (2 mol/L in Et2O, 3.0 mL, 6.0 mmol), Fe(acac)3 (87 mg, 0.25 mmol), dry THF (30 mL), and dry NMP (3 mL) to yield 24k (1.37 g, quantitative yield) as a yellow oil. TLC Rf = 0.56 (hexane/EtOAc, 5/1); 1H NMR (300 MHz, CDCl3) δ 7.83 (t, J = 8.0 Hz, 1 H), 6.97 (dd, J = 8.0, 1.5 Hz, 1 H), 6.91 (dd, J = 12.0, 1.5 Hz, 1 H), 4.38 (q, J = 7.0 Hz, 2 H), 2.50 (d, J = 7.0 Hz, 2 H), 1.85−1.94 (m, 1 H), 1.39 (t, J = 7.0 Hz, 3 H), 0.91 (d, J = 7.0 Hz, 6 H); MS (EI, Pos) m/z 224 (M)+, 179, 154 (base peak). 4-Ethyl-2-hydroxybenzaldehyde (27m). To a stirred solution of 3-ethylphenol 26m (3.0 g, 24.6 mmol) in DMF (20 mL) were added MOMCl (2.05 mL, 27.0 mmol) and K2CO3 (6.80 g, 49.0 mmol) at ambient temperature. After stirring for 19 h at ambient temperature, MOMCl (0.50 mL, 6.6 mmol) and DMF (20 mL) were added to the reaction mixture, which was heated at 50 °C and stirred for 9.5 h. The reaction mixture was poured into cold water, which was extracted with (EtOAc/hexane, 3/1) twice. The combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield 1-ethyl-3-(methoxymethoxy)benzene (1.50 g, 45% yield) as a pale yellow oil. TLC Rf = 0.42 (hexane/EtOAc, 10/ 1); 1H NMR (300 MHz, CDCl3) δ 7.21−7.16 (m, 1 H), 6.87−6.82 (m, 3 H), 5.16 (s, 2 H), 3.48 (s, 3 H), 2.62 (q, J = 7.5 Hz, 2 H), 1.23 (t, J = 7.5 Hz, 3 H). The same procedure as 18 was performed using 1-ethyl-3(methoxymethoxy)benzene (800 mg, 5.3 mmol), t-BuLi (1.6 mol/L in n-pentane, 4.0 mL, 6.4 mmol), n-hexane (15 mL), and DMF (0.62 mL, 8.0 mmol) to yield 27m (490 mg, 52% yield) as a yellow oil. TLC Rf = 0.28 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 10.44 (s, 1 H), 7.77 (d, J = 8.0 Hz, 1 H), 7.03 (d, J = 1.0 Hz, 1 H), 6.93 (dd, J = 8.0, 1.0 Hz, 1 H), 5.30 (s, 2 H), 3.53 (s, 3 H), 2.68 (q, J = 7.5 Hz, 2 H), 1.26 (t, J = 7.5 Hz, 3 H). 2-(Methoxymethoxy)-4-propylbenzoic Acid (27n). To a solution of 3-n-propylphenol 26n (10.0 g, 73.0 mmol) in dry THF (150 mL) were added i-PrNEt2 (38.4 mL, 220 mmol) and MOMCl (13.9 mL, 184 mmol) at 0 °C. After the reaction mixture was stirred for 15 h at 100 °C, ice/sat. NH4Cl-aq was added to the reaction mixture. The mixture was extracted with MTBE twice. And combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 19525



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To a stirred solution of crude methyl 2-chloro-6-propylnicotinate (2.14 g, 10.0 mmol) in dry MeOH (5 mL) was added a solution of NaOMe in MeOH (28% w/w, 5.8 g, 30.0 mmol) at ambient temperature. After the reaction mixture was stirred for 18 h at ambient temperature and for 6 h at 50 °C, the reaction mixture was poured into NH4Cl-aq. The mixture was extracted with MTBE. The organic layer was washed with brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield methyl 2-methoxy-6propylnicotinate 34q (267 mg, 13% yield in 2 steps) as a yellow oil. TLC Rf = 0.86 (hexane/EtOAc, 1/1). Methyl 4-Methoxy-6-propylnicotinate (34r). The same procedure as 17 was performed using 33r (2.40 g, 11.6 mmol), npropylmagnesium bromide (1.0 mol/L in THF, 23.0 mL, 23.0 mmol), Fe(acac)3 (43 mg, 0.12 mmol), THF (10 mL), and NMP (2 mL) to yield crude methyl 4-chloro-6-propylnicotinate (1.62 g, 65% yield) as a yellow oil. TLC Rf = 0.58 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 8.95 (s, 1 H), 7.25 (s, 1 H), 3.95 (s, 3 H), 2.71−2.85 (m, 2 H), 1.67−1.86 (m, 2 H), 0.97 (t, J = 7.3 Hz, 3 H); MS (FAB, Pos) m/ z 216, 214 (M + H)+. The same procedure as 34q was performed using methyl 4-chloro6-propylnicotinate (50 mg, 0.23 mmol), NaOMe in MeOH (28% w/ w, 89 mg, 0.46 mmol), and dry MeOH (0.5 mL) to yield methyl 4methoxy-6-propylnicotinate 34r (15 mg, 31% yield) as a colorless oil. TLC Rf = 0.45 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 8.84 (s, 1 H), 6.73 (s, 1 H), 3.95 (s, 3 H), 3.90 (s, 3 H), 2.72−2.82 (m, 2 H), 1.68−1.88 (m, 2 H), 0.98 (t, J = 7.4 Hz, 3 H); MS (EI) m/z 209 (M)+, 208, 194, 181 (base peak). 1-(Chloromethyl)-3-methoxy-2-methylbenzene (36x). The same procedure as 9f was performed using 3-methoxy-2-methylbenzoic acid 35x (5.0 g, 30.0 mmol), a solution of BH3·THF complex (1.2 mol/L, 40.0 mL, 48 mmol), and dry THF (50 mL) to yield (3methoxy-2-methylphenyl)methanol (4.6 g, quantitative yield) as a pale yellow powder. TLC Rf = 0.29 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.18 (t, J = 7.5 Hz, 1 H), 6.99 (d, J = 7.5 Hz, 1 H), 6.83 (d, J = 7.5 Hz, 1 H), 4.70 (d, J = 5.0 Hz, 2 H), 3.84 (s, 3 H), 2.23 (s, 3 H), 1.49 (t, J = 5.0 Hz, 1 H). To a stirred solution of (3-methoxy-2-methylphenyl)methanol (1.0 g, 6.7 mmol) in CCl4 (6.4 mL, 67 mmol) was added PPh3 (2.6 g, 10 mmol) at ambient temperature. After the reaction mixture was stirred for 1 h at 70 °C and 14 h at 55 °C, precipitates were removed by filtration. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield 36x (1.1 g, 98% yield) as a pale yellow oil. TLC Rf = 0.63 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.15 (t, J = 8.0 Hz, 1 H), 6.94 (d, J = 8.0 Hz, 1 H), 6.84 (d, J = 8.0 Hz, 1 H), 4.61 (s, 2 H), 3.83 (s, 3 H), 2.28 (s, 3 H). 4-(Chloromethyl)-2-methoxy-1-methylbenzene (36y). The same procedure as 9h was performed using methyl 3-methoxy-4methylbenzoate 35y (5.0 g, 27.8 mmol), LiAlH4 (1.27 g, 33.3 mmol), and dry THF (110 mL) to yield (3-methoxy-4-methylphenyl)methanol (4.1 g, 98% yield) as a pale yellow oil. TLC Rf = 0.19 (hexane/EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.11 (d, J = 7.5 Hz, 1 H), 6.87 (s, 1 H), 6.84 (d, J = 7.5 Hz, 1 H), 4.66 (s, 2 H), 3.85 (s, 3 H), 2.21 (s, 3 H). The same procedure as 36x was performed using (3-methoxy-4methylphenyl)methanol (3.50 g, 23.0 mmol), CCl4 (22.0 mL, 230 mmol), and PPh3 (7.84 g, 30.0 mmol) to yield 36y (3.30 g, 84% yield) as a colorless oil. TLC Rf = 0.68 (hexane/EtOAc, 5/1); 1H NMR (300 MHz, CDCl3) δ 7.09 (d, J = 7.0 Hz, 1 H), 6.87−6.84 (m, 2 H), 4.57 (s, 2 H), 3.84 (s, 3 H), 2.21 (s, 3 H). 4-(3-Methoxy-2-methylphenyl)butanoic Acid (37x). To 36x (1.1 g, 6.5 mmol) were added ethyl acrylate (1.06 mL, 10.0 mmol), nBu3N (3.1 mL, 13 mmol), and Pd(OAc)2 (44 mg, 0.20 mmol) at ambient temperature. After stirring for 14 h at 95 °C, cold water, 1 mol/L HCl-aq, and EtOAc were consecutively added to the reaction mixture. The insoluble was removed through Celite, and the filtrate was extracted with EtOAc twice. The combined organic layer was washed with 1 mol/L HCl-aq, water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure. The residue was

(methoxymethoxy)-3-propylbenzene (14.3 g, quantitative yield) as a brown oil. It was used for the next reaction without purification. TLC Rf = 0.64 (hexane/EtOAc, 10/1); 1H NMR (300 MHz, CDCl3) δ 7.19 (dd, J = 8.5, 7.5 Hz, 1 H), 6.81−6.89 (m, 3 H), 5.17 (s, 2 H), 3.48 (s, 3 H), 2.56 (t, J = 7.5 Hz, 2 H), 1.57−1.70 (m, 2 H), 0.94 (t, J = 7.5 Hz, 3 H); MS (EI, Pos) m/z 180 (M)+ (base peak), 150. The same procedure as 18 was performed using 1-(methoxymethoxy)-3-propylbenzene (2.0 g (content 1.85 g, 10.3 mmol)), t-BuLi (1.56 mol/L in n-pentane, 7.2 mL, 11.3 mmol), dry n-hexane (30 mL), and CO2 gas instead of DMF to yield 27n (1.86 g, 81% yield) as a pale yellow powder. TLC Rf = 0.40 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 1 H), 7.07 (d, J = 1.5 Hz, 1 H), 7.00 (dd, J = 8.0, 1.5 Hz, 1 H), 5.42 (s, 2 H), 3.57 (s, 3 H), 2.63 (t, J = 7.5 Hz, 2 H), 1.59−1.74 (m, 2 H), 0.96 (t, J = 7.5 Hz, 3 H); MS (EI, Pos) m/z 224 (M)+ (base peak), 180, 162 (base peak), 134. Methyl 4-Butyl-2-methoxybenzoate (29). The same procedure as 16 was performed using methyl 4-hydroxy-2-methoxybenzoate 28 (6.0 g, 32.9 mmol), Tf2O (6.65 mL, 39.5 mmol), pyridine (3.73 mL, 46.1 mmol), CH2Cl2 (80 mL), and THF (10 mL) to yield methyl 2methoxy-4-{[(trifluoromethyl)sulfonyl]oxy}benzoate (8.55 g, 83% yield) as a pale yellow oil. TLC Rf = 0.59 (hexane/EtOAc, 2/1); 1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 8.5 Hz, 1 H), 6.91 (dd, J = 8.5, 2.5 Hz, 1 H), 6.87 (d, J = 2.5 Hz, 1 H), 3.93 (s, 3 H), 3.91 (s, 3 H). The same procedure as 17 was performed using methyl 2-methoxy4-{[(trifluoromethyl)sulfonyl]oxy}benzoate (300 mg, 0.95 mmol), nbutylmagnesium bromide (0.84 mol/L in THF, 1.35 mL, 1.1 mmol), Fe(acac)3 (34 mg, 0.095 mmol), THF (3 mL), and NMP (0.3 mL) to yield 29 (56 mg, 26% yield) as a colorless oil. TLC Rf = 0.41 (hexane/ EtOAc, 6/1); 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 8.0 Hz, 1 H), 6.80−6.77 (m, 2 H), 3.90 (s, 3 H), 3.87 (s, 3 H), 2.63 (t, J = 8.0 Hz, 2 H), 1.65−1.55 (m, 2 H), 1.42−1.31 (m, 2 H), 0.93 (t, J = 7.0 Hz, 3 H). Methyl 4-(2-Hydroxy-2-methylpropyl)-2-methoxybenzoate (31). To a stirred solution of i-Pr2NH (10.0 mL, 76.0 mmol) in dry THF (90 mL) was added n-BuLi (1.6 mol/L in n-hexane, 47.5 mL, 76 mmol) at −35 °C. After the reaction mixture was stirred for 30 min at −35 °C, the reaction mixture was cooled to −78 °C and DMPU (27 mL) and then a solution of methyl 2-methoxy-4-methylbenzoate 30 (9.12 g, 50.7 mmol) in THF (9 mL) were added to the mixture. After the reaction mixture was stirred for 2 h at −78 °C, acetone (5.6 mL, 76 mmol) was added to the mixture. After the reaction mixture was stirred for 1.5 h at 0 °C, the reaction mixture was poured into NH4Claq. The mixture was extracted with EtOAc twice. The combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel to yield crude 31 (2.5 g, 21% yield) as a colorless oil. TLC Rf = 0.26 (hexane/EtOAc, 1/1). Methyl 2,6-Dichloronicotinate (33q). To a stirred solution of 2,6-dichloronicotinic acid 32q (8.70 g, 45.3 mmol) in MeOH (50 mL) and PhMe (30 mL) was added H2SO4 (0.5 mL) at ambient temperature. After the reaction mixture was stirred for 18 h at 110 °C, the reaction mixture was concentrated under reduced pressure, the residue was triturated in water, and precipitate was collected. Resulting powder was washed with hot hexane to yield 33q (7.30 g, 78% yield) as a pale yellow solid. TLC Rf = 0.68 (hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) δ 8.16 (d, J = 8.2 Hz, 1 H), 7.36 (d, J = 8.2 Hz, 1 H), 3.96 (s, 3 H); MS (EI, Pos) m/z 207, 205 (M)+, 174 (base peak). Methyl 4,6-Dichloronicotinate (33r). The same procedure as 33q was performed using 4,6-dichloronicotinic acid 32r (4.36 g, 22.7 mmol), MeOH (30 mL), PhMe (20 mL), and a few drops of H2SO4 to yield 33r (2.40 g, 51% yield) as a yellow powder. TLC Rf = 0.70 (hexane/EtOAc, 3/1). Methyl 2-Methoxy-6-propylnicotinate (34q). The same procedure as 17 was performed using 33q (5.00 g, 24.3 mmol), npropylmagnesium bromide (1.0 mol/L in THF, 29.0 mL, 29.0 mmol), Fe(acac)3 (850 mg, 2.4 mmol), THF (40 mL), and NMP (10 mL) to yield crude methyl 2-chloro-6-propylnicotinate (4.38 g, 85% yield) as a pale yellow oil. TLC Rf = 0.50 (hexane/EtOAc, 3/1); MS (FAB, Pos, glycerin + m-NBA) m/z 216, 214 (M + H)+. 9526



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purified by chromatography on silica gel to yield crude mixture of ethyl (3E)-4-(3-methoxy-2-methylphenyl)but-3-enoate and ethyl (2E)-4-(3methoxy-2-methylphenyl)but-2-enoate (922 mg, crude 60% yield) as a yellow oil. TLC Rf = 0.30 (hexane/EtOAc, 10/1). To a suspension of Pd−C (wet, 10% w/w, 200 mg) in MeOH (3 mL) was added a mixture of ethyl (3E)-4-(3-methoxy-2methylphenyl)but-3-enoate and ethyl (2E)-4-(3-methoxy-2methylphenyl)but-2-enoate (922 mg, crude 3.9 mmol) at ambient temperature. After stirring for 3 h at ambient temperature under H2 atmosphere, the insoluble was removed through Celite and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel to yield mixture of ethyl 4-(3-methoxy-2methylphenyl)butanoate and methyl 4-(3-methoxy-2-methylphenyl)butanoate (455 mg, crude 49% yield) as a colorless oil. TLC Rf = 0.30 (hexane/EtOAc, 10/1). To a solution of mixture of ethyl 4-(3-methoxy-2-methylphenyl)butanoate and methyl 4-(3-methoxy-2-methylphenyl)butanoate (455 mg, ∼1.9 mmol) in MeOH (5 mL) was added 2 mol/L NaOH-aq (5.0 mL, 10.0 mmol) at ambient temperature. After stirring for 12 h at ambient temperature, THF (5 mL) was added to the reaction mixture to make it homogeneous. After stirring for 4 h at ambient temperature, cold 2 mol/L HCl-aq (6.0 mL, 12.0 mmol) was added to the reaction mixture. Organic solvents were removed under reduced pressure, and the mixture was extracted with EtOAc twice. The combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 37x (395 mg, 30% yield in 3 steps) as a white powder. TLC Rf = 0.15 (hexane/EtOAc, 3/ 1); 1H NMR (300 MHz, CDCl3) δ 7.09 (t, J = 8.0 Hz, 1 H), 6.76 (d, J = 8.0 Hz, 1 H), 6.73 (d, J = 8.0 Hz, 1 H), 3.82 (s, 3 H), 2.70−2.65 (m, 2 H), 2.44−2.39 (m, 2 H), 2.17 (s, 3 H), 1.96−1.86 (m, 2 H). 4-(3-Methoxy-4-methylphenyl)butanoic Acid (37y). The same procedure as 37x was performed three times using 36y (1.1 g, 6.5 mmol), ethyl acrylate (1.06 mL, 10.0 mmol), n-Bu3N (3.1 mL, 13 mmol), and Pd(OAc)2 (88 mg, 0.40 mmol) under microwave irradiation (50 W, 70 °C, 2 min, 70 W, 100 °C, 10 min, and 100 W, 130 °C, 20 min) to yield a mixture of ethyl (3E)-4-(3-methoxy-4methylphenyl)but-3-enoate and ethyl (2E)-4-(3-methoxy-4methylphenyl)but-2-enoate (3.10 g, 68% yield) as a yellow oil. TLC Rf = 0.41 (hexane/EtOAc, 5/1). The same procedure as 37x was performed using a mixture of ethyl (3E)-4-(3-methoxy-4-methylphenyl)but-3-enoate and ethyl (2E)-4-(3methoxy-4-methylphenyl)but-2-enoate (3.10 g, 13.2 mmol), Pd−C (wet, 10% w/w, 600 mg), and EtOH (33 mL) to yield ethyl 4-(3methoxy-4-methylphenyl)butanoate (3.17 g, quantitative yield) as a pale yellow oil. TLC Rf = 0.46 (hexane/EtOAc, 5/1). The same procedure as 37x was performed using ethyl 4-(3methoxy-4-methylphenyl)butanoate (3.1 g, 13.2 mmol), 2 mol/L NaOH-aq (20 mL, 40 mmol), EtOH (15 mL), and THF (15 mL) to yield 37y (2.79 g, quantitative yield) as a white powder. TLC Rf = 0.28 (hexane/EtOAc, 2/1); 1H NMR (300 MHz, CDCl3) δ 7.03 (d, J = 7.0 Hz, 1 H), 6.68−6.63 (m, 2 H), 3.81 (s, 3 H), 2.64 (t, J = 7.5 Hz, 2 H), 2.38 (t, J = 7.5 Hz, 2 H), 2.18 (s, 3 H), 2.01−1.92 (m, 2 H). 6-Methoxy-5-methyl-3,4-dihydro-1(2H)-naphthalenone (38x). To a suspension of 37x (395 mg, 1.9 mmol), a few drops of dry DMF in dry CH2Cl2 (5 mL) were added (COCl)2 (0.25 mL, 2.8 mmol) at 0 °C. After stirring for 20 min at ambient temperature, the reaction mixture was concentrated under reduced pressure and dry PhMe (10 mL) was added to the residue. To the resulting solution was added SnCl4 (0.27 mL, 2.3 mmol) at 0 °C. After stirring for 20 min at ambient temperature, cold water was added to the reaction mixture at 0 °C. The mixture was extracted with EtOAc twice. The combined organic layer was washed with water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure to yield 38x (382 mg, quantitative yield) as a pale yellow powder. TLC Rf = 0.41 (hexane/ EtOAc, 3/1); 1H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 8.5 Hz, 1 H), 6.83 (d, J = 8.5 Hz, 1 H), 3.89 (s, 3 H), 2.87 (t, J = 6.0 Hz, 2 H), 2.59 (dd, J = 7.0, 6.0 Hz, 2 H), 2.07−2.18 (m, 5 H); MS (EI, Pos) m/z 190 (M)+ (base peak), 162. 6-Methoxy-7-methyl-3,4-dihydro-1(2H)-naphthalenone (38y). The same procedure as 38x was performed using 37y (2.79 g

(content 2.76 g, 13.2 mmol)), (COCl)2 (0.25 mL, 2.8 mmol), dry DMF (0.020 uL), dry CH2Cl2 (30 mL) in acid chloride formation reaction and SnCl4 (1.9 mL, 16 mmol) and dry PhMe (10 mL) in Friedel−Crafts type cyclization reaction to yield 38y (1.70 g, 67% yield) as a pale yellow powder. TLC Rf = 0.33 (hexane/EtOAc, 3/1); 1 H NMR (300 MHz, CDCl3) δ 7.83 (s, 1 H), 6.61 (s, 1 H), 3.88 (s, 3 H), 2.92 (t, J = 6.0 Hz, 2 H), 2.56−2.63 (m, 2 H), 2.20 (s, 3 H), 2.06− 2.17 (m, 2 H); MS (EI, Pos) m/z 190 (M)+, 162 (base peak). 1,5-Dimethyl-7,8-dihydronaphthalen-2-yl Methyl Ether (39x). To a solution of 38x (360 mg, 1.9 mmol) in dry THF (6 mL) was added a solution of MeMgCl (3.0 M in THF, 1.3 mL, 3.8 mmol) at 0 °C. After the reaction mixture was stirred for 1.5 h at 0 °C, ice/sat. NH4Cl-aq was added to the reaction mixture. To the mixture was added a solution of HCl-aq (1 mol/L) to pH 1. After the reaction mixture was stirred for 100 min at ambient temperature, the reaction mixture was extracted with EtOAc twice. The combined organic extract was washed with HCl-aq (1 mol/L), water, brine and dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified by chromatography on silica gel with (hexane/EtOAc) to yield 39x (306 mg, 86% yield) as a white powder. TLC Rf = 0.71 (hexane/EtOAc, 10/1); 1H NMR (300 MHz, CDCl3) δ 7.09 (d, J = 8.5 Hz, 1 H), 6.70 (d, J = 8.5 Hz, 1 H), 5.75−5.71 (m, 1 H), 3.83 (s, 3 H), 2.75−2.70 (m, 2 H), 2.27−2.19 (m, 2 H), 2.17 (s, 3 H), 2.04−2.02 (m, 3 H). 3,5-Dimethyl-7,8-dihydronaphthalen-2-yl Methyl Ether (39y). The same procedure as 39x was performed using 38y (1.49 g, 7.8 mmol), a solution of MeMgCl (3.0 M in THF, 6 mL, 18 mmol), and dry THF (18 mL) to yield 39y (1.42 g, 96% yield) as a pale yellow oil. TLC Rf = 0.42 (hexane/EtOAc, 20/1); 1H NMR (300 MHz, CDCl3) δ 7.01 (s, 1 H), 6.64 (s, 1 H), 5.72−5.68 (m, 1 H), 3.83 (s, 3 H), 2.75−2.70 (m, 2 H), 2.26−2.20 (m, 5 H), 2.03−2.01 (m, 3 H). Biological Assays. Ca2+ Influx Assay. Human S1P1, S1P2, or S1P3 gene overexpressing CHO-K1 cells were cultured in Ham’s F12 medium (manufactured by GibcoBRL) containing 10% FBS (fetal bovine serum), penicillin/streptomycin, and blasticidin (5 μg/mL). The cultured cells were incubated in a 5 μM Fura2-AM solution (Ham’s F12 medium containing 10% of FBS, 20 mM HEPES buffer (pH 7.4), and 2.5 mM probenecid) at 37 °C for 60 min. After washing once with Hanks solution containing 20 mM HEPES buffer (pH 7.4) and 2.5 mM probenecid, the plate was soaked in the same solution. Then, the plate was set on a fluorescent drug screening system (FDSS 6000; Hamamatsu Photonics K. K.), and the intracellular calcium ion concentration was measured without stimulation for 30 s. A test compound (final concentration of 0.1 nM to 30 μM, dimethylsulfoxide (DMSO) solution) was added, and S1P (final concentration of 100 nM) was added 5 min thereafter. Then, the increase in the intracellular calcium ion concentration was measured before and after the addition of S1P at intervals of 3 s (excitation wavelengths of 340 and 380 nm, fluorescence wavelength of 500 nm). The potency of the compound against each S1P receptor was determined by using the peak value due to S1P stimulation in a well containing DMSO as a substitute for the evaluated compound as a control value (A), comparing the value before the addition of the evaluated compound with the increased value (B) in the fluorescent ratio after the addition, and calculating the increase ratio (%) in the intracellular calcium ion [Ca 2+ ] i concentration as increase ratio (%) = (B/A) × 100. Increase ratios of the compound at individual concentrations were determined, and the EC50 value was calculated. cAMP Accumulation Assay. Cells expressing S1P1 or S1P5 receptors were incubated with the compounds in the presence of 50 μmol/L forskolin and 1 mmol/L IBMX for 30 min at 37 °C. After lysing the cells, accumulation of cAMP was measured by ELISA (cAMP-Screen 96-well 200 assay kit, Applied Biosystems). The EC50 values of compounds evaluated for the inhibitory effects on cAMP accumulation were determined using Prism version 4.02 (GraphPad Software) based on the results from three independent experiments. Peripheral Lymphocyte Lowering (PLL) Test in Mice. Test compounds were orally administered to male BALB/c mice (Charles River Laboratories, Japan, Inc., 6-week-old at the time of use). At 4 or 24 h after the administration, the blood was collected from the aorta 9527



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abdominalis under ether anesthesia. The peripheral blood lymphocyte count was measured with an automated hematology analyzer (SF3000, manufactured by Sysmex Corporation). Evaluation was made by setting the average lymphocyte count in a vehicle-administered group (vehicle group) as 100% and calculating the percentage of vehicle from the average lymphocyte count of each test compound-administered group. On the basis of the test compound doses and percentages of vehicle with the doses, the dose of the compound required for lowering the lymphocyte count to 50% was calculated as ED50. Animal studies were conducted in compliance with the “Guidelines for Animal Studies”, established by Research Headquarters, Ono Pharmaceutical Co., Ltd. CIA Model in Rats. Arthritis was induced by immunizing with bovine type II collagen emulsified in incomplete Freund’s adjuvant on days 0 and 7 to female Lewis rats. Arthritis was not induced in normal control group. Arthritis efficacy was evaluated by using arthritis score characterized by edema and/or erythema in the paws. Compound 13n (0.03 or 0.1 mg/kg) or 0.5% MC was administered orally once daily from day 0 to day 27. Arthritis scores were expressed as the mean value plus standard error for the normal control (n = 5), control (n = 10), 13n 0.03 mg/kg (n = 9), 13n 0.1 mg/kg (n = 9). The steel test was used to compare the scores between the control and 13n groups, with a p-value of less than 5%.

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rat sphingosine-1-phosphate; RRMS, relapsing−remitting multiple sclerosis; S1P, sphingosine-1-phosphate; TM, transmembrane domain; TMAD, N,N,N′,N′-tetramethylazodicarboxamide; T1/2, half-life

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(1) (a) Cuvillier, O.; Pirianov, G.; Kleuser, B.; Vanek, P. G.; Coso, O. A.; Gutkind, S.; Spiegel, S. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 1996, 381, 800−803. (b) Lee, M.-J.; Thangada, S.; Claffey, K. P.; Ancellin, N.; Liu, C. H.; Kluk, M.; Volpi, M.; Sha’afi, R. I.; Hla, T. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 1999, 99, 301−312. (c) Marsolais, D.; Rosen, H. Chemical modulators of sphingosine-1phosphate receptors as barrier oriented therapeutic molecules. Nat. Rev. Drug Discovery 2009, 8, 297−307. (d) Rosen, H.; GonzalezCabrera, P. J.; Sanna, M. G.; Brown, S. Sphingosine 1-phosphate receptor signaling. Annu. Rev. Biochem. 2009, 78, 743−768. (e) Rivera, R.; Chun, J. Biological effects of lysophospholipids. Rev. Physiol. Biochem. Pharmacol. 2006, 160, 25−46. (f) Mutoh, T.; Rivera, R.; Chun, J. Insights into the pharmacological relevance of lysophospholipid receptors. Br. J. Pharmacol. 2012, 165, 829−844. (g) Kihara, Y.; Maceyka, M.; Spiegel, S.; Chun, J. Lysophospholipid receptor nomenclature review: IUPHAR Review 8. Br. J. Pharmacol. 2014, 171, 3575−3594. (h) Pyne, N. J.; Pyne, S. Sphingosine 1-phosphate and cancer. Nat. Rev. Cancer 2010, 10, 489−503. (2) (a) Fujita, T.; Hirose, R.; Yoneta, M.; Sasaki, S.; Inoue, K.; Kiuchi, M.; Hirase, S.; Chiba, K.; Sakamoto, H.; Arita, M. Potent immunosuppressants, 2-alkyl-2-aminopropane-1,3-diols. J. Med. Chem. 1996, 39, 4451−4459. (b) Quesniaux, V.; Fullard, L.; Arendse, H.; Davison, G.; Markgraaff, N.; Auer, R.; Ehrhart, F.; Kraus, G.; Schuurman, H.-J. A novel immunosuppressant, FTY720, induces peripheral lymphodepletion of both T- and B cells and immunosuppression in baboons. Transplant Immunol. 1999, 7, 149−157. (c) Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K. R. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. 2002, 277, 21453−21457. (d) Mullershausen, F.; Zecri, F.; Cetin, C.; Billich, A.; Guerini, D.; Seuwen, K. Persistent signaling induced by FTY720phosphate is mediated by internalized S1P1 receptors. Nat. Chem. Biol. 2009, 5, 428−434. (e) Brinkmann, V.; Billich, A.; Baumruker, T.; Heining, P.; Schmouder, R.; Francis, G.; Aradhye, S.; Burtin, P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discovery 2010, 9, 883−897. (f) Graul, A. I.; Cruces, E. The year’s new drugs & biologics, 2010. Drugs Today 2011, 47, 27−51. (g) Rio, J.; Comabella, M.; Montalban, X. Multiple sclerosis: current treatment algorithms. Curr. Opin. Neurol. 2011, 24, 230−237. (3) Matloubian, M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.; Xu, Y.; Brinkmann, V.; Allende, M. L.; Proia, R. L.; Cyster, J. G. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004, 427, 355−360. (4) (a) Budde, K.; Schmouder, R. L.; Brunkhorst, R.; Nashan, B.; Lücker, P. W.; Mayer, T.; Choudhury, S.; Skerjanec, A.; Kraus, G.; Neumayer, H. H. First human trial of FTY720, a novel immunomodulator, in stable renal transplant patients. J. Am. Soc. Nephrol. 2002, 13, 1073−1083. (b) Tedesco-Silva, H.; Mourad, G.; Kahan, B. D.; Boira, J. G.; Weimar, W.; Mulgaonkar, S.; Nashan, B.; Madsen, S.; Charpentier, B.; Pellet, P.; Vanrenterghem, Y. FTY720, a novel immunomodulator: efficacy and safety results from the first phase 2A study in de novo renal transplantation. Transplantation 2005, 79, 1553−1560. (c) Scott, L. J. Fingolimod: a review of its use in the management of relapsing-remitting multiple sclerosis. CNS Drugs 2011, 25, 673−698. (5) Sanna, M. G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.; Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H. Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00785. Preparation of [3H]-13n for in vitro DMPK studies, pharmacokinetic studies in rats and monkeys, in vitro DMPK studies, and effects of 13n on heart rate and lymphocyte counts in unrestrained conscious monkeys (PDF) Molecular formula strings for 6, 7, and 13a−v and some data (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+81) 75 961 1151. Fax: (+81) 75 962 9314. ORCID

Haruto Kurata: 0000-0002-9009-3428 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Masashi Minami, Akira Akimoto, Tomoyuki Shono, Masaki Tsujimura, Naoya Matsumura, Natsuko Tokuda, and Kazutoyo Sato for helpful discussions. We also thank Renee Mosi, Ph.D., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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ABBREVIATIONS USED BA, bioavailability; CLtot, total clearance; Fe(acac)3, iron(III) acetylacetonate; hMS, human microsome; hPPB, human plasma protein binding; hS1P, human sphingosine-1-phosphate; NaHMDS, sodium hexamethyldisilazane; Pd−C, palladium on carbon; PLL, peripheral lymphocyte lowering; rS1P, 9528



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Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine

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