Tryptophan Hydroxylase 1 (Tph-1)-Targeted Bone Anabolic Agents for

Article pubs.acs.org/jmc

Tryptophan Hydroxylase 1 (Tph-1)-Targeted Bone Anabolic Agents for Osteoporosis Hai-Jian Fu,† Yu-Ren Zhou,† Bei-Hua Bao,† Meng-Xuan Jia,† Yang Zhao,† Lei Zhang,† Jian-Xin Li,*,† Hai-Lang He,‡ and Xian-Mei Zhou*,‡ †

State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing 210093, P. R. China ‡ Department of Respiratory Medicine, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, 155 Hanzhong Road, Nanjing 210029, P. R. China S Supporting Information *

ABSTRACT: Tryptophan hydroxylase 1 (Tph-1), the principal enzyme for peripheral serotonin biosynthesis, provides a novel target to design anabolic agents for osteoporosis. Here, we present a design, synthesis of a novel series of ursolic acid derivatives under the guidance of docking technique, and bioevaluation of the derivatives using RBL2H3 cells and ovariectomized (OVX) rats. Of the compounds, 9a showed a potent inhibitory activity on serotonin biosynthesis. Further investigations revealed that 9a, as an efficient Tph-1 binder identified by SPR (estimated KD: 6.82 μM), suppressed the protein and mRNA expressions of Tph-1 and lowered serotonin contents in serum and gut without influence on brain serotonin. Moreover, oral administration of 9a elevated serum level of N-terminal propeptide of procollagen type 1 (P1NP), a bone formation marker, and improved bone microarchitecture without estrogenic side effects in ovariectomized rats. Collectively, 9a may serve as a new candidate for bone anabolic drug discovery.

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INTRODUCTION Osteoporosis is a metabolic bone disease characterized by low bone mass and structural deterioration of bone tissue associated with bone fragility and an increased vulnerability to low-trauma fractures. It is a major public health problem worldwide, especially among postmenopausal women, due to a dramatic reduction of estrogen level.1 Osteoporosis represents a considerable medical and socioeconomic burden for modern societies. In the United States, 10 million individuals over the age of 50 years, 8 million women and 2 million men, are estimated to already have the disease, and these numbers are expected to increase to about 14 million by 2020.2 For the year 2010, approximately 2.5 million new fractures due to osteoporosis occurred in Europe’s five largest countries (France, Germany, Italy, Spain, and United Kingdom) and Sweden alone, and direct cost of these fractures was estimated to be nearly €31 billion.3 In China, there are about 84 million osteoporosis patients currently.4 Bone remodeling is regulated by the balanced activities of bone-resorbing osteoclasts and bone-forming osteoblasts to maintain normal physiological structure and mineral content. © 2014 American Chemical Society

An imbalance that bone resorption exceeds bone formation in the bone remodeling process results in osteoporosis.4−6 Inhibition of osteoclast functions and enhancement of osteoblast activities are key therapeutical tools for osteoporosis. Osteoporosis drugs available on the current market are mainly divided into two categories: mostly antiresorptive agents, such as bisphosphonates,7 calcitonin, estrogen, or selective estrogen receptor modulators (SERMs).8 However, the antiresorptive drugs have limitations, in particular the fact that they also suppress new bone formation due to the coupling of osteoblast and osteoclast activities and consequently lead to a low bone turnover state.9 Anabolic agents act by stimulating formation of new bone, while the only approved drug on the current market is injectable forms of parathyroid hormone (rhPTH).10 Indeed, treatment with rhPTH is also limited to 24 months in the United States and 18 months in Europe due to the risk of increased prevalence of osteosarcoma.11 Although antiresorptive Received: February 12, 2014 Published: May 20, 2014 4692

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Fortunately, we discovered that UA displayed quite lower binding energy (ΔE, −10.22 kcal/mol) and inhibition constant value (Ki, 32.12 nM). To further improve the binding affinity, various UA derivatives were virtually designed, and their energy minimized three-dimensional conformers were docked into the crystal structure of the enzymatic domains of Tph-1 with its cofactor HBI. It is reported that water molecules in ligand-binding sites play a critical role in mediating the interactions between Tph-1 and its ligands and offer useful information on the process of pharmacophore construction,35,36 so all the water molecules in the crystal structure were remanined. The active site of Tph-1 is formed by a large binding pocket divided into two long channels: one is occupied by its cofactor HBI with the iron ion approximately situated at the intersection of the two channels, and the other is occupied by the amino acid substrate, where the amino acid moieties of inhibitors are bound (Supporting Information Figure 1a).36 As the ring A of UA occupies the binding pocket above HBI at the entrance to the catalytic site (Supporting Information Figure 1b), introduction of heterocyclic moieties to the ring A would affect its activity significantly. Besides, the O and N atoms of heterocyclic system may also form hydrogen bonds with residues of Tph-1 to gain additional potency. Therefore, we first introduced the heterocyclic moieties to the ring A at C-2 and C-3 positions. Next, we turned our attention to the 28-position of UA, pointing toward the pocket occupied by the amino acid substrate. This region is frequently used to gain affinity. As a general trend, introducing suitable substituents bearing hydrogen bond acceptors is important for binding to the pocket. Thus, the amino acids and other heterocyclic moieties were introduced to the 28-position of UA, which gave rise to series of UA derivatives. The typical docking results are shown in Table 1. The docking data suggested that introducing a heterocyclic ring (9) resulted in a lower binding energy (ΔE, −11.26 kcal/mol) and inhibition constant (Ki, 5.57 nM) compared with UA, as designed. Very interestingly, when conjugating amino acids and heterocyclic ring at C-28 position (−COOH) of UA, the binding energy decreased to −14.33 kcal/mol with the inhibition constant (Ki) 31.36 pM (9a). The above information suggested that the heterocyclic rings and amino acid moieties might be critical for a better binding affinity with Tph-1. Encouraged by the data, UA derivatives were synthesized to validate docking results and develop the lead molecules that can improve the potency against Tph-1. Chemistry. Guided by the docking results, the series of derivatives with heterocyclic rings fused at C-2 and C-3 positions and amino acids or heterocyclic segments at C-28 position were synthesized to seek reasonable Tph-1 inhibitors with the better bioactivity. The synthetic routes are outlined in Schemes 1−3. For the oxidation of UA (1) to prepare 3-keto UA (2), although Jones reagent has been shown to be very effective in some oxidation reactions with good yield, it causes serious environmental pollution.37 Thus, reagent H2O2/Na2WO4 with the phosphate buffer solution was used,38 and 2 was obtained in an excellent yield (86%) and good selectivity without much environmental pollution.39 Acetylation of the hydroxyl group of 1 with acetic anhydride in pyridine afforded 5.40 The indole derivatives 6 and 7 were synthesized by Fischer indolization of intermediate 2 with the phenylhydrazine hydrochloride in acetic acid. However, when treating 2 with 1,2-diamines and sulfur in

agents have been the backbone of osteoporosis management, as osteoporosis generally occurs in aging subjects bone remodeling or regeneration can be slower, development of anabolic agents for improving bone formation and bone repair has considerable social and economic impact. Serotonin (5-hydroxytryptamine, 5-HT), one of the most ancient neurotransmitter molecules, plays a key role in almost all living organisms, modulating central and peripheral functions through action on neurons and other cell types.12 Tryptophan hydroxylase (Tph) is the principal enzyme in the biosynthesis of serotonin (converts L-tryptophan into 5-hydroxy-tryptophan, the precursor to serotonin) and a primary rate-limiting factor in serotonergic functioning.13 In 2003, Walther reported the Tph exists two vertebrate isoforms: Tph-1 and Tph-2.14,15 Tph-1, a major enzyme related to serotonine biosynthesis in the periphery, is primarily expressed in the pinealocytes of the pineal gland and non-neuronal tissues such as enterochromaffin (EC) cells of the gut. Tph-2 is exclusively expressed in neuronal cells including dorsal raphe and myenteric plexus cells.16,17 Recent studies revealed that peripheral serotonin biosynthesized in EC cells of the gut (gutderived serotonin, GDS) is a powerful inhibitor of osteoblast proliferation.18−21 GDS enters the circulation but does not cross the blood−brain barrier.18,19 Further evidence demonstrated that a small molecule LP533401, a Tph-1 inhibitor, successfully increased bone mass in ovariectomized mice.21−24 Therefore, suppression of GDS biosynthesis via Tph-1 inhibitor provides a novel approach to design anabolic agents for osteoporosis treatment. As part of our ongoing effort in natural products based antiosteoporosis drug discovery, we tried to explore Tph-1targeted bone anabolic agents. First, a virtual screening of the potential Tph-1 inhibitor from natural products was conducted using the Tph-1 enzymatic domain with its cofactor 7,8-dihydro-L-biopterin (HBI) as a reference.21 Fortunately, ursolic acid (UA) showed quite good data with free binding energy (−10.22 kcal/mol) and inhibition constant (Ki, 32.12 nM). It was also reported that UA stimulated osteoblast differentiation in vitro.25 These results inspired us to explore the anabolic agents for osteoporosis treatment based on UA. UA, a natural product, is widely distributed throughout the plant kingdom26 and possesses various pharmacological properties such as anti-inflammatory,27 antioxidative,28 antitumor,29 antimicrobial, antiulcer, and antiwrinkle activities.30 A variety of structure modifications of UA have also been conducted.31−33 In this work, we have designed and synthesized three series of UA derivatives and evaluated their inhibitory activity on serotonin biosynthesis using RBL2H3 cells. Among the derivatives, 9a, a new compound, was further assayed for its inhibitory activities on protein and mRNA expressions of Tph-1 and Tph-2, and the binding affinity between 9a and Tph-1 was also analyzed with surface plasmon resonance (SPR) technique. Furthermore, the inhibitory activity on serotonin levels of brain, serum, and gut and the protective effect on bone loss of 9a were evaluated in an ovariectomized (OVX) rat model.

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RESULTS AND DISCUSSION Docking of Tph-1 and Ligands. Docking study is widely applied in the structure-based drug design and provides a practical strategy to identify new leads.34 Because the crystal structure of Tph-1 has been identified, to quickly elucidate the molecules that have binding affinity with Tph-1, we started a docking study with a number of natural products. 4693

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refluxing morpholine,41 the desired quinoxaline derivatives 8 and 9 were obtained with quite low yields (42%). Therefore, another synthetic protocol was tried. First, treatment of ketone 2 with excess of t-BuOK in t-BuOH and THF at room temperature under air condition overnight gave diketone 3.42 It is noteworthy that when the temperature increased to 40 °C, this reaction was completed within 40 min without any side products. Then, 3 was reacted with corresponding 1,2-diamines in refluxing ethanol and gave the target compounds 8 and 9 in excellent yields. The synthesis of pyrazole, isoxazole, and pyrimidine derivatives were performed as depicted in Scheme 2. Compound 4 was obtained by Claisen condensation of 2 with ethyl formate without protection of carboxyl group (C-28) under atmospheric condition in high yield.43 Treatment of 4 with hydrazine dihydrochloride, phenyl-hydrazine hydrochloride, 4-fluorophenylhydrazine hydrochloride, and hydroxylamine hydrochloride in EtOH under reflux for 2 h afforded pyrazole and isoxazole derivatives 15−18, respectively. Compound 19 was obtained by cleaving the isoxazole ring of 18. To obtain the intermediate 20, a number of experimental conditions were tried; unexpectedly, no product was provided. However, when anhydrous toluene equipped with a Stark water trap was used, the reaction went smoothly and effectively. Condensation of 20 with formamidine acetate in the presence of sodium methylate afforded 21, and demethylating with LiI in refluxing anhydrous DMF gave the desired pyrimidine derivative 22.44 With above UA analogues in hand, amino acid and heterocyclic moieties were further introduced into C-28 position as designed (Scheme 3). At first, the standard coupling reagent (HOBT/EDCI) was tested; unfortunately, the yields were quite low, most probably due to the steric hindrance at C-28. Then, C-28 chlorides of UA analogues were prepared by oxalyl chloride in anhydrous CH2Cl2, and then treatment with corresponding amino acid esters and heterocyclic moieties in the presence of triethylamine afforded corresponding amides 2a−c, 5a−c, 6a−c, 7a−c, 8a−c, 9a−c, 10−14, 15a−c, 16a−c, 17a−c, 18a−c, 19a−c, and 22a−c, respectively.

Table 1. Docking Results of Crystal Structure of Human Tph-1 to Typical UA Derivatives

Ki was obtained by the following equation: Ki = exp((ΔG × 1000)/ (Rcal × TK)), where ΔG is docking energy, Rcal is 1.98719, and TK is 298.15. bData is cited from ref 21. a

Scheme 1. Synthesis of Indole and Quinoxaline Derivativesa

Reagents and coditions: (a) acetic anhydride, acetic acid, pyridine, reflux, 5 h (78%); (b) NaH2PO4/Na2HPO4, Na2WO4, H2O2, DMA, 90 °C, 2 h (86%); (c) t-BuOK, t-BuOH, air, THF, 40 °C, 1 h (89%); (d) 4-chlorophenylhydrazine hydrochloride or 4-bromophenylhydrazine hydrochloride, acetic acid, reflux, 5 h (61−62%); (e) 4,5-dichlorobenzene-1,2-diamine or benzene-1,2-diamine, EtOH, reflux, 2 h (79−84%).

a

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Scheme 2. Synthesis of Pyrazole, Isoxazole, and Pyrimidine Derivativesa

a

Reagents and conditions: (f) CH3ONa, ethyl formate, benzene, rt, 12 h (80%); (g) hydrazine dihydrochloride, phenylhydrazine hydrochloride or 4-fluorophenylhydrazine hydrochloride, EtOH, reflux, 2 h (77−87%); (h) DMFDMA, toluene, reflux, 8 h (70%); (i) formamidine acetate, EtOH, reflux, 12 h (56%); (j) LiI, anhydrous DMF, reflux, 48 h (45%); (k) hydroxylamine hydrochloride, EtOH, reflux, 2 h (89%); (l) (i) Et2O, CH3ONa, rt, 12 h (89%); (ii) DDQ, EtOH, reflux, 5 h (88%).

method with fluorescence detector was established to analyze serotonin content of the cells. To examine whether the effects of the compounds upon serotonin biosynthesis were from their activity or cytotoxicity, a single dose cytotoxicity study of the UA derivatives on RBL2H3 cells by standard MTT assay (Table 2) was also conducted. As can be seen in Table 2, at 10 μM concentration, most of the UA derivatives lowered the cell serotonin level and showed an inhibitory effect on serotonin biosynthesis. Together with the cytotoxicity data, a structure−activity relationship (SAR) for the tested derivatives could be summarized. Acetylation or heterocyclization of UA without inserting amino acids at C-28 carboxyl (5−9, 15−19, and 22) did not show significant effect on serotonin biosynthesis compared with UA. Oxidation or acetylation of C-3 hydroxyl in addition to modification of C-28 carboxyl group with amino acids had a significant impact on the inhibitory activity (2a−c and 5a−b), while introducing phenylalanine moiety to 5 lowered the activity (5c). Indolization of ketone 2 with substitution of the C-28 carboxyl functionality by amino acids resulted in a reduction of the activity (6a−c and 7a−c). In most cases, the quinoxaline and pyrimidine six-membered heterocyclic ring derivatives that introduced amino acids at C-28 carboxyl displayed no effects on inhibitory activity (8a−c, 9b−c, 10, 22a−c). Interestingly, conjugates of glycine ethyl ester and quinoxaline derivatives could provide effective improvement of the activity without significant cytotoxicity (9a). However, the quinoxaline derivatives introduced with piperidine moieties displayed an increase activity but with higher cytotoxicity (11−14), which revealed that their effects blocking serotonin biosynthesis activity might be from their cytotoxicity. Pyrazole-extended derivatives gained more potent inhibitory activity by insertion of amino acids at C-28 carboxyl (15a−c), but this trend was

Scheme 3. Synthesis of Amino Acids and Heterocyclic Moieties Derivativesa

a

Reagents and conditions: (m) (i) oxalyl chloride, CH2Cl2, rt, 24 h; (ii) corresponding amines, Et3N, rt, CH2Cl2, 24 h (65−88%).

Inhibitory Activity on Tph-1-Mediated Serotonin Biosynthesis and Cytotoxicity. Because Tph-1 is the principal enzyme in the serotonin biosynthesis, serotonin level is an indicator of Tph-1 activity. Therefore, the inhibitory activity on serotonin biosynthesis of the synthesized compounds was initially evaluated using RBL2H3 cells, a Tph-1expressing serotonin-producing mast cell line.17,45,46 A HPLC 4695

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Table 2. Inhibitory Effects on Serotonin Biosythesis and Cytotoxicity of the Synthesized Compounds on RBL2H3 Cells compd Con. 1 2a 2b 2c 5 5a 5b 5c 6 6a 6b 6c 7 7a 7b 7c 8 8a 8b 8c 9 9a 9b 9c 10 11 IC50 9a

inhibitiona (%) 0.0 6.8 50.7 61.4 53.6 1.0 43.8 44.3 8.7 1.5 8.5 5.4 70.9 7.9 8.2 9.2 9.0 22.3 22.9 13.4 10.3 18.4 87.0 6.9 6.8 15.6 58.7

± 0.3 ± 1.4 ± 4.9**c ± 0.7**c ± 4.6**c ± 6.2 ± 0.7**c ± 0.1**c ± 0.4 ± 0.7 ± 0.5 ± 0.3 ± 0.3**c ± 9.1 ± 0.2 ± 0.2 ± 0.1 ± 2.6**c ± 0.3**c ± 0.1*b ± 0.1*b ± 6.5**c ± 2.3**c ± 0.2 ± 0.1 ± 1.2*b ± 2.6**c

cytotoxicity 0.0 10.0 1.7 2.6 1.1 4.1 1.6 6.6 12.4 2.9 14.7 15.0 1.6 3.7 1.7 3.0 16.0 2.2 2.0 0.7 7.7 3.5 1.0 0.5 12.4 6.2 51.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

compd

1.2 1.0*b 1.0 0.7 0.1 2.8 0.3 0.2 1.5*b 1.4 2.1*b 0.7*b 0.4 2.0 0.2 1.7 0.8*b 2.8 0.8 1.1 1.0 1.0 0.5 1.4 2.9*b 0.9 3.3**c

6.22

inhibitiona (%)

12 13 14 15 15a 15b 15c 16 16a 16b 16c 17 17a 17b 17c 18 18a 18b 18c 19 19a 19b 19c 22 22a 22b 22c

35.9 62.1 53.8 6.2 38.9 34.4 38.3 4.3 2.9 1.0 19.9 7.3 57.3 33.7 40.4 1.0 17.5 17.6 7.8 11.7 61.9 92.4 1.4 5.9 6.9 3.1 6.8

LP533401

0.4d

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8**c 0.1**c 0.7**c 2.0 1.3**c 1.1**c 0.2**c 1.7 1.3 0.1 0.3**c 2.0 2.0**c 1.6**c 0.8**c 0.1 0.1**c 0.4**c 0.1 0.9*b 0.3**c 0.2**c 1.0 0.2 0.3 0.2 0.4

cytotoxicity 59.5 52.1 31.3 0.1 0.4 0.9 1.9 2.4 1.2 1.8 0.9 1.5 15.8 1.0 2.0 2.1 11.1 17.7 0.8 4.3 50.1 58.1 5.4 4.3 1.9 10.3 2.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.7**c 1.9**c 4.4**c 1.3 1.5 1.3 1.5 2.0 0.5 2.0 1.3 1.1 1.7*b 0.8 0.3 1.3 0.8*b 2.6**c 3.6 1.1 1.6**c 1.2**c 2.0 1.1 3.0 1.4*b 0.4

Inhibition (%) = 100% − peak areas of the tested compound against the areas of the corresponding control group (%). All values are expressed as means ± S.D., n = 3. b*p < 0.05. c**p < 0.01 vs control. dData is cited from ref 25.

a

not observed in other similar five-membered heterocyclic ringfused derivatives (16a−c, 18a−c). Surprisingly, the inhibitory activity was improved in case of cyclization of ketone 4 with 4-fluorophenylhydrazine and introducing amino acids (17a−c) compared with 16a−c, which may be due to the fluorine moieties. Cleaving the isoxazole ring of 18a−b did elevate the inhibitory level on serotonin (19a−b), but they also showed higher cytotoxicity, similar to compounds 11−14. Above in vitro data clearly demonstrated that compound 9a exhibited the potent inhibitory activity without cytotoxicity. Further detailed test on its half inhibitory concentration (IC50) was performed (Supporting Information Figure 2a−b). As shown in Table 1, the IC50 of 9a was 6.22 μM, which was less potent than LP533401 (0.4 μM).23 Inhibitions on Protein and mRNA Expressions of Tph-1 and Tph-2. Tph-1 is abundantly expressed by RBL2H3 cells, and the conversion of L-tryptophan to 5-hydroxytryptophan catalyzed by Tph-1 is the rate-limiting step in the biosynthesis of serotonin. To understand whether 9a has the direct inhibitory effect on Tph-1, the expressions of protein and mRNA of Tph-1 were assayed. First, 9a at concentrations of 2, 5, and 10 μM was exposed to RBL2H3 cells, and the Tph-1 protein levels in the cells were measured with enzyme-linked immunosorbent assay (ELISA). As shown in Figure 1a, 9a at all concentrations significantly suppressed Tph-1 protein expression with a dosedependent manner. Next, we investigated whether 9a regulates Tph-1 mRNA expression with a quantitative real-time PCR (qRT-PCR) analysis.

As can be seen in Figure 1b, 9a at 10 μM significantly downregulated Tph-1 mRNA expression in the RBL2H3 cells in a time dependent manner. The above results revealed that 9a inhibited serotonin biosynthesis via suppressions of Tph-1 protein and mRNA expressions. To further elucidate whether 9a selectively inhibits Tph-1, we first tested whether compound 9a could inhibit serotonin biosynthesis in PC12 cell line that expresses Tph-2 endogenously.47 The results revealed that 9a inhibited serotonin synthesis catalyzed by Tph-2 in PC12 cells with an IC50 value of 45.51 μM (Supporting Information Figure 2c) without cytotoxicity (Supporting Information Figure 2d). Then, the effects of 9a on protein and mRNA expressions of Tph-2 were investigated. As shown in Figure 1c, 9a at 10 μM significantly lowered the proten expression of Tph-2. Although 9a displayed some suppression on the mRNA level of Tph-2, no statistical difference was observed. The data clearly indicated that 9a showed an approximately 7-fold more selective effect over Tph-1 compared to Tph-2 in cellular assays. Surface Plasmon Resonance Study. Surface plasmon resonance (SPR) detection, as a powerful technique, provides a well-established tool for studying the affinity and kinetics of biomolecular−protein interactions in real time.48 SPR biosensors can also exhibit some information-rich data on molecular recognition without labeling. Having identified 9a as a small molecule that significantly inhibited the Tph-1 protein and mRNA expressions, we next conducted an SPR analysis to verify the affinity of 9a toward Tph-1 and obtained a detailed 4696

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Figure 1. Inhibitory effects of 9a on the protein and mRNA expressions of Tph-1 and Tph-2. The protein levels of Tph-1 in RBL2H3 cells (a) and Tph-2 in PC12 cells (c) were measured with enzyme-linked immunosorbent assay (ELISA, Cusabio). The mRNA expressions of Tph-1 (b) and Tph-2 (d) were analyzed with qRT-PCR, and the cells were treated with 9a at 10 μM, or 0.1% DMSO only (Con.) for 24 and 48 h. The data were expressed as a fold change. Each value was expressed as means ± SD, n = 3. * p < 0.05, ** p < 0.01 vs Controls (Con.).

view of its interaction with the protein.49 Tph-1 was immobilized on Biacore sensor chip CM5 by standard amine coupling reaction, and the data were analyzed by BIAevaluation program.50 The results revealed that 9a efficiently interacted with the immobilized Tph-1 and demonstrated a concentration dependent response (Figure 2) and a clearly discernible

Tph-1.51 This observation supported that 9a might be an ideal inhibitor of Tph-1, which would contribute to the inhibition of serotonin biosynthesis. Docking Studies. To get a better comprehension of the detailed information on 9a and Tph-1 interactions at the atomic and energetic levels, 9a was docked into the enzymatic domains of Tph-1 with its cofactor HBI. As described in Figure 3a, 9a extends deeply into the binding site of Tph-1 and is quite close to the catalytic site in the hinge region formed by the residues (THR368, THR367, PHE318, GLU340, SER337, SER336, THR265, and HIS272).35 The same as LP533401, the amino acid moiety of 9a extends deeply into the binding pocket of Tph-1 catalytic domain, which is essential for mediating ligand−protein interactions (Figure 3a). However, the quinoxaline ring of 9a comes into close contact with the residue TYR235 near HBI of the Tph-1 catalytic domain and further makes strong hydrophobic contact with TYR235. Unlike 9a, the biphenyl ring of LP533401 is directed toward the solvent region, which indicates that biphenyl ring may be less important for achieving the affinity of ligand binding. As described in Figure 3b, the O atom of the glycine ethyl ester fragment of 9a could form a hydrogen bond with the N atom of THR367 with the bond distance of 2.7 Å. In addition, the O atom of the carboxyl group of 9a could generate two hydrogen bonds with two water molecules with the bond distance of 2.9 Å (undepicted in Figure 3). As known to all, the N···HO bond is much stronger than O−H···O,52,53 which indicates that the glycine ethyl ester fragment might play a crucial role in improving its potency against Tph-1. On the basis of the above docking results, we can speculate that these hydrogen bonds may be helpful to stabilize the interactions between 9a and Tph-1, and it might be the reason that 9a has

Figure 2. SPR analysis of the binding of compound 9a to Tph-1. Sensorgrams were obtained by injecting five different concentrations in running buffer: 50.00, 12.50, 6.25, 3.12, and 1.56 μM at the flow rate of 30 μL/min. The concentration of Tph-1 was approximately 5.00 μM. RU: resonance units.

exponential curve during both the association and dissociation phases (Supporting Information Figure 3). The sensorgram obtained for the 9a interacting with Tph-1 was fitted to a single-site biomolecular interaction model (A + B = AB) with rapid association rates and rapid dissociation rates. The binding affinity was represented to be a KD (6.82 μM), most probably due to the capability of specifically recognizing the immobilized 4697

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Figure 3. Binding model of 9a into the X-ray structure of Tph-1. (a) Binding mode of compound 9a (in cyan) and LP533401 (in pink) into the crystal structure of Tph-1 with its cofactor HBI (in magenta). (b) Binding of 9a to the active site of Tph-1. The amino acid interacting with 9a is shown in red. Only amino acids located within 4 Å of the bound ligand are displayed and labeled, and H-bonds are displayed as dashed black lines.

Figure 4. Inhibitory effects of compound 9a on serotonin biosynthesis in vivo. Serum (a) and brain (b) serotonin levels of sham-operated and ovariectomized (OVX) rats orally treated with vehicle or the indicated dose (0, 1, 10, and 20 mg/kg·d) of 9a for 30 days after ovariectomy, n = 8. (c) Serotonin levels in the gut. A separate animal study was conducted. The sham-operated and ovariectomized (OVX) rats were orally treated with vehicle or 9a at doses of 10 and 20 mg/kg per day for 7 days after ovariectomy, n = 5. All values are expressed as means ± SD, # p < 0.05 vs Sham. * p < 0.05; ** p < 0.01 vs OVX.

lower estimated free energy for binding (ΔE = −14.33 kcal mol−1) in this model. Effects on Serotonin Levels in Serum, Brain and Gut in Vivo. Circulating serotonin plays an important role in bone physiology and investigation of its effects on bone formation becomes even more important for tackling from the medical perspective.21 Increasing reports showed that inhibiting the biosynthesis of gut serotonin could prevent and cure ovariectomized (OVX) induced osteoporosis via an anabolic mode of action in mice and rats.21,54 Therefore, we used the OVX rat model to test whether compound 9a inhibits the biosynthesis of GDS in vivo. OVX rats were orally administrated with 9a at

doses of 1, 10, and 20 mg/kg per day, and the dosing period was 30 days after surgery. At the end of the experiment, the serotonin concentrations in serum were analyzed following the reported method.55 As shown in Figure 4a, OVX operation markedly increased the serum serotonin level, which was in agreement with other reported results.56 As expected, administration of 9a at all doses of 1, 10, or 20 mg/kg per day for 30 days resulted in a significantly decrease of serum serotonin concentration in a dose-dependent manner compared with the control group. Recent genetic studies have demonstrated that many medical disorders are associated with serotonin dysregulation,57 4698

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Figure 5. Preventive effect of 9a on bone loss in OVX rats. (a) Histomorphometric analysis of L2−L4 vertebra were performed on eXplore Locus SP scanner at 45 μm resolution and microcomputed tomography analysis of the spine were determined using eXplore Micro-View, version 2.0 (GE Healthcare). BMD = bone mineral density; BV/TV = bone volume over total volume; Tb.N = trabecular number; Tb.Th = trabecular thickness. Tb.Sp = trabecular space. All values are expressed as means ± SD, n = 8. # p < 0.05 vs Sham. * p < 0.05; ** p < 0.01 vs OVX.

especially, brain serotonin modulates very important central functions and exerts opposite influences on bone remodeling that decreases bone resorption.58 To understand the influences of 9a on the brain serotonin, the serotonin levels in brain homogenate (Figure 4b) were measured. The results demonstrated that 9a did not cause marked changes of serotonin levels in brain at all doses. As previously mentioned, peripheral serotonin is mainly synthesized by EC cells of the gut, therefore, the inhibitory activity of 9a on the serotonin level in the gut was examined. In a separate animal study, OVX rats were orally administrated with 9a at doses of 10 and 20 mg/kg once per day for 7 days, and the small intestines were collected. As shown in Figure 4c, serotonin levels in the small intestine (jejunum and ileum) were significantly lower in the 9a-treated groups, averaging 34 and 36% reductions at 10 and 20 mg/kg per day groups, respectively. As described previously, 9a also inhibited Tph-2 activity, and it is possible that the lack of a brain effect was related to failure to cross the blood−brain barrier. Therefore, the concentrations

of 9a in brain tissue of OVX rats treated with 9a for 7 days at the dose of 10 and 20 mg/kg per day were analyzed with LC-MS/MS method. As expected, 9a was not detected in brain tissue at both doses (limit of detection is 12.4 pg, LTQ Orbitrap XL), suggesting 9a did not cross the blood−brain barrier. Above results clearly indicated that although serotonin concentrations in serum and gut were decreased, brain serotonin content remained unaffected in 9a-treated OVX rats. In other words, 9a selectively inhibited serotonin biosynthesis in the gastrointestinal tract. Prevention of OVX-Induced Bone Loss. Although osteoporosis affects both older men and women, postmenopausal women have been the primary focus of osteoporosis.59 The OVX rat model is most commonly used in research on postmenopausal osteoporosis, in which ovariectomy results in an excess of bone resorption over bone formation initially and causes bone loss.60 As trabecular bone is one of the most severely affected sites in the OVX condition, the preventive effect of 9a on the deterioration of trabecular microarchitecture 4699

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Figure 6. Effects of 9a on serum bone turnover markers P1NP (a) and CTX-1 (b). P1NP and CTX-1 were measured with ELISA kits. All values are expressed as means ± SD, n = 8. # p < 0.05 vs Sham. * p < 0.05; ** p < 0.01 vs OVX.

histological observations using the light microscope (Supporting Information Figure 6a). The uteri of sham rats exhibited a smooth endometrial luminal epithelium (LE) and regular endometrial glands (GE). As expected, the uteri of OVX rats with 30-day administration of 9a at all doses exhibited no changes in the endometrial LE with undeveloped GE compared with those of untreated OVX rats (Supporting Information Figure 6b). Besides, morphological and pathological changes were not observed in the liver tissues (Supporting Information Figure 6c). Our current results demonstrated that inhibition of gut serotonin level correlated with the protection of bone loss. It is noteworthy that there has been at present some debate over the action of serotonin in bone. Contradictory to Yadav’s reports,21,22 Cai et al. did not find the correlation between change of serotonin content and bone mass in their mice models.66 The most likely reason is that they used different mice models, and furthermore serotonin possesses quite complex physiological actions in bone.67,68

as a result of OVX was evaluated. After 30 days treatment of OVX rats with 9a at doses of 1, 10, and 20 mg/kg per day, histomorphometric analyses of lumbar vertebrae were performed with microcomputed tomography (μCT)61,62 (Figure 5). As compared with the sham group, the trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and BMD were significantly reduced, and the trabecular separation (Tb.Sp) was significantly increased in OVX group. These data revealed that OVX operation significantly deteriorated bone microarchitecture (Figure 5a). PTH, the only clinically available bone anabolic drug for the treatment of osteoporosis was used as a positive control in the present study, and injection of PTH at dose of 20 μg/kg per day clearly improved the bone quality related parameters. Although oral treatment of 9a at 1 mg/kg per day only showed some improvement of bone parameters without statistically difference, 9a at dosages of 10 and 20 mg/kg markedly increased the bone quality related parameters including BMD (+22.6%) and BV/TV (+28.0%) compared with OVX group (Figure 5b−c). Ovariectomy is typically associated with reduction in trabecular separation and numbers,63 and the data also suggests that compound 9a at doses of 10 and 20 mg/kg per day exhibited increases in Tb.N (+43.3%), Tb.Th (+24.0%) and a decrease in Tb.SP (−24.9%), respectively (Figure 5d−f). Effects on Bone Turnover Markers. Serum N-terminal propeptide of procollagen type 1 (P1NP), as a typical bone formation marker, reflects the conversion of procollagen type 1 to type 1 collagen during bone formation.64 Thus, we further evaluated the serum P1NP levels. OVX operation increased the P1NP level, indicating OVX induced high turnover type osteoporosis (Figure 6a). As expected, PTH, as an anabolic drug, significantly increased the serum P1NP value. Excitingly, the same as PTH, 9a also markedly elevated the P1NP levels of OVX rats in a dose-dependent manner. As serum carboxy terminal telopeptide of collagen type I (CTX-1) level is an indicator of osteoclast numbers and bone resorption,65 we also analyzed the serum CTX-1 levels. The data demonstrated that OVX operation significantly increased the CTX-1 level. Some decrease were observed in 9a treated groups compared with OVX one while without statistically significant differences (Figure 6b). Above results clearly demonstrated that 9a rescued, through a bone anabolic fashion, OVX-induced bone loss in rats. Overall, our in vivo findings demonstrate that 9a inhibited GDS biosynthesis, increased the bone formation marker P1NP level, and prevent the OVX-induced bone loss. The uterus from treated and control animals were sectioned and stained for

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CONCLUSIONS

As Tph-1 is a primary enzyme on the serotonin biosynthesis, inhibition of Tph-1 leads to a decrease in circulating serotonin and an increase in bone formation, which provided a rational basis for the discovery of bone anabolic agents. Given our interest in the development of Tph-1 inhibitors as potential therapeutic strategy for osteoporosis, we explored the molecules with Tph-1 inhibitory activity. In this study, we synthesized a novel series of ursolic acid derivatives guided by the docking-based virtual screening and evaluated their activity on inhibition of serotonin biosynthesis through suppressing Tph-1 activity. Among the synthesized compounds, 9a with a lower binding energy and inhibition constant in the docking process displayed the potent inhibitory activity on serotonin biosynthesis via suppression of Tph-1 protein and mRNA expressions using RBL2H3 cells while without any cytotoxicity. Although 9a also inhibited Tph-2, but its potency was weaker than that of Tph-1. The in vitro activity was also confirmed with OVX rat model, in which the gut and circulating serotonin levels were significantly reduced compared with the control group. Very importantly, 9a exhibited no effect on the brain serotonin that exerts opposite influences on bone formation. The interaction between Tph-1 and 9a was analyzed with SPR and detailed docking study. Critically, 9a markedly improved bone trabecular microarchitecture and elevated the serum P1NP level, a bone formation marker, without hormone like side effects in vivo. Overall, our results established that 9a can 4700

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overnight and then the solvent was removed. The mixture was poured into 1 N HCl (200 mL) and extracted with EtOAc (200 mL × 3). The organic layer was dried with anhydrous Na2SO4 and concentrated to give the crude white solid. Then the residue was purified by a column chromatography (PE/EA, 5/1, v/v) to give 4 (3.85 g, 80%) as a white solid; mp 195−197 °C. 1H NMR (300 MHz, CDCl3) δ: 8.5 (s, 1H), 5.28 (s, 1H), 2.20 (d, J = 11.0 Hz, 1H), 2.04−1.76 (m, 5H), 1.65−1.52 (m, 6H), 1.48−1.42 (m, 6H), 1.26 (s, 6H), 1.11−1.06 (m, 6H), 1.03 (s, 3H), 1.01 (s, 3H), 0.95 (s, J = 5.3 Hz, 3H), 0.86 (d, J = 7.3 Hz, 3H), 0.66 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 217.5, 177.3, 172.1, 138.3, 136.1, 129.3, 128.3, 126.9, 126.1, 55.1, 53.5, 52.0, 47.7, 47.3, 46.7, 42.3, 39.3, 38.1, 37.1, 36.5, 34.0, 32.2, 30.7, 29.6, 27.7, 26.5, 24.7, 23.3, 21.3, 21.1, 19.4, 17.0, 16.4, 15.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C31H46NaO4, 505.3288; found, 505.3264. Synthesis of Compound 5. To a solution of 1 (456.44 mg, 1.00 mmol) in pyridine (40 mL) was added acetic anhydride (816.69 mg, 8.00 mmol) and DMAP (22.17 mg) at 115 °C for 4 h. Then the mixture was poured into 1 N NaHCO3 (200 mL) and extracted with EtOAc (200 mL × 3). The organic layer was dried with anhydrous Na2SO4 and concentrated to give the crude white solid. Then the residue was purified by a column chromatography (PE/EA, 6/1, v/v) to give 5 (392.55 mg, 78%) as a white solid; mp 190−192 °C. 1 H NMR (300 MHz, CDCl3) δ: 5.23 (s, 1H), 4.55−4.45 (m, 1H), 2.18 (d, J = 11.2 Hz, 1H), 2.05 (s, 3H), 1.95−1.82 (m, 3H), 1.73−1.58 (m, 6H), 1.57−1.41 (m, 6H), 1.38−1.26 (m, 4H), 1.07 (s, 3H), 0.96 (s, 3H), 0.95 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 5.5 Hz, 3H), 0.85 (s, 6H), 0.76 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 184.1, 171.0, 137.9, 125.7, 80.9, 55.2, 52.4, 47.9, 47.4, 41.8, 39.4, 39.0, 38.8, 37.7, 36.9, 36.7, 32.8, 30.6, 28.0, 24.0, 23.6, 23.2, 21.2, 18.1, 17.1, 17.0, 16.7, 15.5. HR-MS (m/z) [M + H]+ (ESI+) calcd for C32H51O4, 499.3782; found, 499.3788. Synthesis of Compounds 6 and 7. To a solution of 2 (454.23 mg, 1.00 mmol) in acetic acid (30 mL) was added 4-chlorophenylhydrazine hydrochloride (214.41 mg, 1.20 mmol) at 118 °C for 4 h. After cooling to room temperature, the solvents were removed and the residue was purified by a column chromatography (PE/EA, 4/1, v/v) to give 6 (341.99 mg, 61%) as a white solid; mp 212−214 °C. 1H NMR (300 MHz, CDCl3) δ: 7.42 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 4.5 Hz, 2H), 7.30 (s, 1H), 5.31 (s, 1H), 2.63 (d, J = 15.0 Hz, 1H), 2.06−1.84 (m, 2H), 2.06−1.84 (m, 4H), 1.75−1.67 (m, 3H), 1.54−1.36 (m, 6H), 1.25 (s, 6H), 1.10 (s, 3H), 1.04 (s, 3H), 0.94 (d, J = 5.4 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H), 0.82 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 183.6, 146.4, 140.6, 138.4, 137.8, 134.9, 130.3, 128.6, 125.7, 114.4, 54.4, 52.6, 48.0, 46.2, 42.0, 39.3, 39.0, 38.7, 37.9, 37.0, 36.6, 34.5, 32.3, 31.8, 30.6, 29.6, 29.3, 29.2, 27.9, 24.0, 23.3, 22.6, 22.3, 21.1, 19.0, 16.9, 16.8, 15.3, 14.0. HR-MS (m/z) [M + H]+ (ESI+) calcd for C36H49ClNO2, 562.3446; found, 562.3422. Compound 7 was synthesized in the same procedure as that of 6 (yield 62%); white solid; mp 198−200 °C. 1H NMR (300 MHz, CDCl3) δ: 7.75 (s, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.05 (dd, J = 8.5, 1.8 Hz, 1H), 5.36 (s, 1H), 2.74 (d, J = 15.0 Hz, 1H), 2.29−1.98 (m, 6H), 1.83−1.68 (m, 4H), 1.62−1.33 (m, 8H), 1.27 (s, 3H), 1.14 (s, 3H), 1.13 (s, 3H), 0.96 (d, J = 7.1 Hz, 3H), 0.87 (s, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.87 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 184.2, 142.4, 137.7, 134.4, 129.4, 126.0, 124.6, 121.0, 117.6, 111.2, 106.9, 53.1, 52.6, 48.1, 46.2, 42.1, 39.6, 39.1, 38.8, 38.0, 36.8, 36.7, 34.0, 32.4, 30.9, 30.6, 28.0, 24.1, 23.5, 23.3, 23.2, 21.15, 19.2, 17.0, 15.7. HR-MS (m/z) [M + H]+ (ESI+) calcd for C36H49BrNO2, 606.2941; found, 606.2969. Synthesis of Compounds 8 and 9. To a solution of 3 (468.22 mg, 1.00 mmol) in EtOH (40 mL) was added 4,5-dichlorobenzene1,2-diamine (211.07 mg, 1.20 mmol) at 85 °C for 5 h. After cooling to air temperature, the solvents were removed and the residue was purified by a column chromatography (PE/EA, 6/1, v/v) to give 8 (479.83 mg, 79%) as a white solid; mp 225−227 °C. 1H NMR (300 MHz, CDCl3) δ: 8.13 (s, 1H), 8.07 (s, 1H), 5.34 (s, 1H), 3.24 (d, J = 16.7 Hz, 1H), 2.65 (d, J = 10.2 Hz, 1H), 2.25 (d, J = 11.1 Hz, 1H), 2.15−1.91 (m, 5H), 1.81−1.64 (m, 6H), 1.60−1.44 (m, 5H), 1.39 (s, 3H), 1.35 (s, 3H), 1.14 (s, 3H), 0.96 (d, J = 6.0 Hz, 3H), 0.91 (s, 3H), 0.89 (d, J = 3.0 Hz, 3H), 0.88 (s, 3H).

rescue OVX induced bone loss in rats through a bone anabolic mechanism. We believe that this compound may serve as an excellent lead to discover potent bone anabolic agents, and more studies on lead optimization and druggability evaluation are ongoing.

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EXPERIMENTAL SECTION

Chemistry. All reagents were commercially available and used without further purification unless otherwise stated. All reactions were monitored by thin-layer chromatography (TLC) and column chromatography was carried on silica gel (Qingdao Haiyang Chemical Co., Ltd., 200−300 mesh). Melting points were determined using the X-4 melting point apparatus (Bei Jing Taike Co., Ltd.) and are uncorrected. 1H (300 MHz) NMR and 13C (75 MHz) NMR spectra were recorded on a Bruker DPX 300 MHz spectrometer at room temperature using CDCl3 as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts are given in δ values (ppm), and coupling constants (J) are given in Hz. Peak multiplicity abbreviations were indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad signal). HRMS were obtained on an Agilent 6540Q-TOF LC/MS spectrometer equipped with electrospray ionization (ESI) probe operating in positive or negative ion mode. The purity of all synthesized compounds was determined by HPLC method. HPLC analysis was carried out on a reversed-phase Thermo 10 mm × 4.6 mm, 5 μm C18 column maintained at ambient temperature with a flow rate of 0.4 mL/min and 10 μL samples were injected in all experiments. The retention time was expressed in min at the UV detection of 200, 202, and 254 nm. The mobile phase was composed of H2O/MeOH (1:9, v/v). According to HPLC analysis, the purity of all compounds is >95% (Supporting Information Table 1). Synthesis of Compound 2. To a solution of 1 (4.56 g, 0.01 mol) in DMA (100 mL) was added the H2O2 solution of NaH2PO4 (1.19 g, 0.01 mol), Na2HPO4 (1.42 g, 0.01 mol), and Na2WO4 (0.33 g, 0.01 mol) at 90 °C for 1 h. After cooling, the mixture was poured into water and extracted with EtOAc (200 mL × 3). The organic layer was dried with anhydrous Na2SO4 and concentrated to give the crude white solid. Then the residue was purified by a column chromatography (PE/EA, 6/1, v/v) to give 2 (3.91 g, 86%) as a white solid; mp 197−199 °C. 1H NMR (300 MHz, CDCl3) δ: 5.26 (t, J = 3.5 Hz, 1H), 2.61−2.47 (m, 1H), 2.43−2.31 (m, 1H), 2.22−2.20 (m, 1H), 2.05−1.82 (m, 5H), 1.69−1.67 (m, 4H), 1.57−1.38 (m, 6H), 1.38−1.27 (m, 5H), 1.25 (s, 3H), 1.08 (s, 3H), 1.05 (s, 3H), 1.02 (s, 3H), 0.94 (d, J = 5.6 Hz, 3H), 0.86 (d, J = 6.4 Hz, 3H), 0.82 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ: 217.66, 183.64, 137.98, 125.34, 55.15, 52.50, 47.89, 47.29, 46.66, 41.97, 39.05, 36.59, 34.05, 32.35, 31.82, 30.50, 29.59, 29.25, 27.89, 26.45, 23.95, 23.37, 22.58, 21.33, 21.05, 19.45, 16.89, 15.11, 14.01. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C30H46NaO3, 477.3339; found, 477.3353. Synthesis of Compound 3. To a solution of 2 (4.54 g, 10.00 mmol) in t-BuOH (240 mL) and THF (30 mL) was added t-BuOK (2.50 g, 22.28 mmol) at 50 °C under air for 1 h, then the solvents were removed and the residue was acidified with 1 N HCl. The mixture was extracted with EtOA, and the organic layer was washed with saturated brine, dried over Na2SO4, and concentrated to give the crude white solid. Then the residue was purified by a column chromatography (PE/EA, 5/1, v/v) to give 3 (4.04 g, 89%) as a white solid; mp 188−190 °C. 1H NMR (300 MHz, CDCl3) δ: 6.30 (s, 1H) 5.31 (s, 1H), 2.20 (d, J = 11.1 Hz, 1H), 1.96−1.88 (m, 2H), 1.71−1.65 (m, 3H), 1.64−1.60 (m, 2H), 1.55−1.45 (m, 4H), 1.37−1.32 (m, 2H), 1.09 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.92 (d, J = 5.8 Hz, 3H), 0.86 (d, J = 7.2 Hz, 3H), 0.82 (s, 3H), 0.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 196.7, 179.4, 158.1, 130.9, 130.5, 122.1, 52.8, 46.9, 46.4, 44.9, 42.5, 42.0, 41.9, 40.1, 38.0, 31.1, 30.6, 30.3, 29.6, 27.9, 25.5, 22.6, 21.4, 20.5, 19.0, 18.9, 18.3, 17.7, 16.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C30H44NaO4, 491.3132; found, 491.3122. Synthesis of Compound 4. To a solution of 2 (4.54 g, 10.00 mmol) and sodium methoxide (5.45 g, 0.10 mol) in benzene (120 mL) at 0 °C, the mixture was stirred at room temperature 4701

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Journal of Medicinal Chemistry

Article

C NMR (75 MHz, CDCl3) δ: 183.6, 162.5, 153.4, 140.9, 139.5, 138.0, 133.2, 133.0, 129.5, 128.7, 125.6, 53.3, 52.6, 49.4, 48.0, 45.4, 42.2, 40.5, 39.4, 39.1, 38.8, 36.8, 36.7, 32.2, 30.6, 28.0, 25.3, 24.1, 23.5, 23.3, 21.1, 20.2, 17.0, 16.8, 15.8. HR-MS (m/z) [M + H]+ (ESI+) calcd for C36H47Cl2N2O2, 609.3009; found, 609.3021. Compound 9 was synthesized in the same procedure as that of 8 (yield 84%); white solid; mp 213-215 °C. 1H NMR (300 MHz, CDCl3) δ: 8.06−7.91 (m, 2H), 7.72−7.59 (m, 2H), 5.35 (s, 1H), 3.28 (d, J = 16.6 Hz, 1H), 2.68 (d, J = 16.7 Hz, 1H), 2.26 (d, J = 11.2 Hz, 1H), 2.16−1.99 (m, 3H), 1.83−1.65 (m, 5H), 1.50−1.48 (m, 4H), 1.42 (s, 3H), 1.38 (s, 3H), 1.15 (s, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.94 (s, 3H), 0.91 (d, J = 7.5 Hz, 3H), 0.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 183.1, 161.4, 151.5, 142.3, 138.0, 129.3, 128.8, 127.3, 125.6, 52.4, 52.7, 48.9, 48.0, 45.4, 42.2, 40.4, 39.4, 39.1, 38.8, 36.8, 32.3, 30.6, 29.7, 28.0, 25.3, 24.1, 23.5, 23.3, 21.1, 20.2, 17.0, 16.8, 15.8. HR-MS (m/z) [M + H]+ (ESI+) calcd for C36H49N2O2, 541.3789; found, 541.3778. Synthesis of Compounds 15−18. To a solution of 4 (482.66 mg, 1.00 mmol) in EtOH (40 mL) was added hydrazine chloride (125.97 mg, 1.20 mmol) at 85 °C for 3 h. After cooling to air temperature, the solvents were removed and the residue was purified by a column chromatography (PE/EA, 6/1, v/v) to give 15 (365.90 mg, 77%) as a white solid; mp 237−239 °C. 1H NMR (300 MHz, CDCl3) δ: 9.13 (s, 1H), 7.19 (s, 1H), 5.31 (s, 1H), 2.59 (d, J = 14.7 Hz, 1H), 2.25 (d, J = 10.7 Hz, 1H), 2.10−1.97 (m, 4H), 1.76−1.66 (m, 4H), 1.56−1.44 (m, 3H), 1.29−1.27 (m, 3H), 1.26 (s, 3H), 1.16 (s, 3H), 1.11 (s, 3H), 0.95 (d, J = 5.9 Hz, 3H), 0.89 (d, J = 4.7 Hz, 3H), 0.84 (s, 3H), 0.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 182.9, 150.5, 138.1, 131.4, 125.6, 113.1, 53.3, 52.9, 48.0, 46.0, 42.1, 39.6, 39.1, 38.8, 38.3, 36.8, 36.4, 33.4, 32.4, 32.1, 31.2, 30.2, 29.7, 28.1, 24.1, 23.5, 21.2, 17.0, 15.3, 14.1. HR-MS (m/z) [M + H]+ (ESI+) calcd for C31H47N2O2, 479.3632; found, 479.3639. Compound 16−18 were synthesized in the same procedure as that of 15. Compound 16. Yield 87%; white solid; mp 208−210 °C. 1H NMR (300 MHz, CDCl3) δ: 7.44 (d, J = 5.4 Hz, 3H), 7.40−7.34 (m, 2H), 7.34 (s, 1H), 5.31 (s, 1H), 2.63 (d, J = 14.9 Hz, 1H), 2.25−2.09 (m, 2H), 2.07−1.83 (m, 5H), 1.73−1.64 (m, 4H), 1.53−1.44 (m, 3H), 1.43−1.32 (m, 4H), 1.25 (s, 3H), 1.10 (s, 3H), 1.03 (s, 3H), 0.96 (s, 3H), 0.95 (d, J = 7.5 Hz, 3H), 0.88 (d, J = 6.0 Hz, 3H), 0.82 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ: 183.6, 146.1, 142.2, 138.1, 137.7, 129.0, 128.9, 128.4, 125.8, 114.0, 54.5, 52.6, 48.0, 46.2, 42.0, 39.4, 39.0, 38.7, 37.9, 37.1, 36.6, 34.6, 32.4, 30.6, 29.6, 29.3, 27.9, 24.0, 23.3, 22.2, 21.1, 19.1, 16.9, 16.8, 15.3. HR-MS (m/z) [M + H]+ (ESI+) calcd for C37H51N2O2, 555.3945; found, 555.3924. Compound 17. Yield 87%; white solid; mp 229−241 °C. 1H NMR (300 MHz, CDCl3) δ: 7.40−7.31 (m, 3H), 7.16−7.09 (m, 2H), 5.32 (s, 1H), 2.64 (d, J = 15.0 Hz, 1H), 2.26−2.14 (m, 2H), 2.09−1.87 (m, 6H), 1.74−1.65 (m, 4H), 1.49 (s, 3H), 1.43−1.33 (m, 4H), 1.29−1.23 (m, 3H), 1.10 (s, 3H), 1.04 (s, 3H), 0.96 (s, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (s, 3H), 0.88 (d, J = 6.4 Hz, 3H), 0.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 183.6, 164.3, 161.0, 146.6, 138.1, 137.8, 130.9, 130.8, 125.7, 115.6, 115.3, 114.4, 54.5, 52.6, 48.0, 46.3, 42.1, 39.4, 39.1, 38.8, 38.0, 37.0, 36.7, 34.6, 32.4, 30.6, 29.3, 28.0, 24.0, 23.4, 23.3, 22.3, 21.1, 19.1, 17.0, 16.8, 15.3. HR-MS (m/z) [M + H]+ (ESI+) calcd for C37H50FN2O2, 573.3851; found, 573.3840. Compound 18. Yield 89%; white solid; mp 193−195 °C. 1H NMR (300 MHz, CDCl3) δ: 7.98 (s, 1H), 5.30 (s, 1H), 2.45 (d, J = 15.2 Hz, 1H), 2.22 (d, J = 11.4 Hz, 1H), 2.09−1.96 (m, 4H), 1.75−1.64 (m, 4H), 1.58−1.47 (m, 3H), 1.45−1.39 (m, 2H), 1.30 (s, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 0.95 (d, J = 6.1 Hz, 3H), 0.90 (s, 3H), 0.89 (d, J = 6.3 Hz, 3H), 0.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 184.3, 172.9, 150.0, 137.9, 125.4, 108.7, 53.4, 52.5, 47.9, 46.0, 42.0, 39.4, 39.0, 38.7, 38.5, 36.6, 35.5, 34.6, 32.1, 30.5, 28.7, 27.9, 26.8, 23.9, 23.4, 23.2, 21.2, 21.1, 18.7, 16.9, 16.8, 15.3. HR-MS (m/z) [M + H]+ (ESI+) calcd for C31H46NO3, 480.3472; found, 480.3469. Synthesis of Compound 19. To a solution of 18 (479.55 mg, 1.0 mmol) in MeOH (40 mL) was added NaOH (1.61 g, 0.04 mol) at 75 °C overnight. After cooling to air temperature, the solvents were removed and then the residue was acidified with 1 N HCl and

extracted with AcOEt (100 mL × 3). The organic layer was dried with anhydrous Na2SO4 and concentrated to give a white solid which was treated with DDQ (272.41 mg, 1.20 mmol) in EtOH at 78 °C for 5 h. After cooling, the mixture was concentrated and the residue was purified by a column chromatography (PE/EA, 6/1, v/v) to give 19 (419.96 mg, 88%) as a white solid; mp 210−212 °C. 1H NMR (300 MHz, CDCl3) δ: 7.76 (s, 1H), 5.32 (s, 1H), 2.24 (d, J = 11.5 Hz, 1H), 2.15−1.98 (m, 4H), 1.92−1.78 (m, 2H), 1.76−1.63 (m, 3H), 1.63−1.48 (m, 5H), 1.28−1.24 (m, 3H), 1.22 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 1.11 (s, 3H), 0.95 (d, J = 5.9 Hz, 3H), 0.88 (d, J = 6.0 Hz, 3H), 0.87 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 198.0, 183.2, 169.9, 138.8, 124.2, 114.8, 113.8, 52.6, 52.5, 47.9, 44.8, 42.3, 40.9, 40.4, 38.8, 38.7, 36.4, 32.3, 30.4, 29.6, 27.7, 23.8, 23.4, 23.0, 21.5, 21.0, 18.6, 18.0, 17.5, 16.9. HR-MS (m/z) [M + H]+ (ESI+) calcd for C31H44NO3, 478.3316; found, 478.3332. Synthesis of Compound 22. To a solution of 2 (4.55 g, 10.0 mmol) in toluene (50 mL) was added DMFDMA (7.0 mL, 0.05 mol) at 110 °C for 12 h. After cooling to air temperature, the solvents were removed to give crude product 20, which was used without further purification. To a solution of 20 in EtOH (50 mL) was added formamidine acetate (1.24 g, 0.05 mol) and sodium methoxide (540 mg, 10.0 mmol) at 78 °C for 8 h. After cooling to air temperature, the mixture was concentrated to give crude product 21, which was treated with lithium iodide (6.65 g, 50.0 mmol) in anhydrous DMF under reflux for 48 h. Then the solvents were removed and the residue was purified by a column chromatography (PE/EA, 2/1, v/v) to give 22 (1.03 g, 21%) as a white solid; mp 197−199 °C. 1 H NMR (300 MHz, CDCl3) δ: 9.01 (s, 1H), 8.31 (s, 1H), 5.33 (s, 1H), 2.72 (d, J = 15.8 Hz, 1H), 2.34−2.25 (m, 2H), 2.09−1.98 (m, 4H), 1.70−1.58 (m, 6H), 1.44−1.33 (m, 5H), 1.31 (s, 3H), 1.27 (s, 3H), 1.25 (s, 3H), 1.12 (s, 3H), 0.96−0.93 (m, 4H), 0.88 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 4.6 Hz, 3H), 0.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.0, 157.6, 138.3, 138.0, 125.6, 125.1, 112.6, 53.0, 52.9, 51.4, 48.1, 45.2, 44.6, 42.1, 39.2, 39.0, 38.8, 36.5, 35.2, 32.2, 30.6, 29.3, 27.9, 24.2, 23.4, 23.3, 22.3, 21.1, 20.2, 16.9, 16.7, 15.4, 15.2. HR-MS (m/z) [M + H]+ (ESI+) calcd for C32H47N2O2, 491.3632; found, 491.3629. General Procedure for the Synthesis of Compounds 2a−c, 5a−c, 6a−c, 7a−c, 8a−c, 9a−c, 10−14, 15a−c, 16a−c, 17a−c, 18a−c, 19a−c, and 22−c. To a solution of 2 (113.6 mg, 0.25 mmol) in DCM (25 mL) was added oxalyl chloride (314.83 mg, 2.50 mmol) at 0 °C. The reaction mixture was stirred overnight at room temperature. After concentrating in vacuo, to the mixture was added corresponding amino acid ester hydrochloride or heterocyclic moieties (41.87 mg, 0.30 mmol) and Et3N (101.19 mg, 1.00 mmol) and stirred for 5 h at room temperature. Then the mixture was concentrated and the residue was purified by a column chromatography (PE/EA, 5/1, v/v) to give the targeted compounds. Compound 2a. Yield 77%; white solid; mp 187−189 °C. 1H NMR (300 MHz, CDCl3) δ: 6.53 (t, J = 4.0 Hz, 1H), 5.43 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 4.07 (dd, J = 18.7, 5.2 Hz, 1H), 3.84 (dd, J = 18.7, 3.5 Hz, 1H), 2.60−2.33 (m, 2H), 2.06−1.95 (m, 4H), 1.91−1.57 (m, 6H), 1.49−1.38 (m, 6H), 1.29 (t, J = 7.2 Hz, 3H), 1.31−1.26 (m, 3H), 1.25 (s, 3H), 1.09 (d, J = 7.5 Hz, 3H), 1.03 (s, 3H), 0.95 (s, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.76 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 217.4, 177.9, 170.0, 139.0, 125.9, 61.3, 55.0, 53.6, 47.60, 47.2, 46.7, 42.3, 41.6, 39.9, 39.0, 38.9, 36.7, 36.4, 36.1, 34.0, 32.1, 30.7, 29.6, 27.7, 26.4, 24.7, 23.3, 21.2, 19.4, 17.1, 16.3, 15.1, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C34H53NNaO4, 562.3867; found, 562.3874. Compound 2b. Yield 70%; white solid; mp 205−207 °C. 1H NMR (300 MHz, CDCl3) δ: 6.34 (d, J = 6.8 Hz, 1H), 5.40 (t, J = 3.4 Hz, 1H), 4.69−4.44 (m, 1H), 3.71 (s, 3H), 2.61−2.47 (m, 1H), 2.44−2.30 (m, 1H), 2.06−1.92 (m, 4H), 1.66−1.77 (m, 2H), 1.65−1.56 (s, 6H), 1.52−1.42 (m, 5H), 1.37−1.24 (m, 6H), 1.10 (s, 3H), 1.09 (s, 3H), 1.04 (s, 6H), 0.96 (s, 3H), 0.95 (s, 3H), 0.94 (d, J = 5.6 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.75 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 217.5, 177.3, 173.5, 138.3, 126.1, 55.1, 53.7, 51.9, 50.6, 47.7, 47.2, 46.7, 42.2, 39.0, 38.9, 37.3, 36.5, 34.0, 32.3, 30.7, 29.5, 27.7, 26.5, 24.7, 23.2, 22.4, 21.9, 21.2, 20.9, 20.6, 19.4, 17.0, 16.5, 15.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C37H59NNaO4, 604.4336; found, 604.4350.

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dx.doi.org/10.1021/jm5002293 | J. Med. Chem. 2014, 57, 4692−4709

Journal of Medicinal Chemistry

Article

Compound 2c. Yield 66%; white solid; mp 212−214 °C. 1H NMR (300 MHz, CDCl3) δ: 7.29 (s, 1H), 7.11 (d, J = 6.5 Hz, 2H), 6.38 (d, J = 5.8 Hz, 1H), 5.28 (t, J = 3.8 Hz 1H), δ 4.80−4.67 (m, 1H), 3.69 (s, 3H), 3.14 (dd, J = 13.6, 6.2 Hz, 1H), 3.05 (dd, J = 13.7, 5.8 Hz, 1H), 2.61−2.44 (m, 1H), 2.44−2.28 (m, 1H), 2.04−1.77 (m, 6H), 1.65− 1.52 (m, 6H), 1.50−1.39 (m, 6H), 1.28−1.22 (m, 6H), 1.08 (d, J = 5.5 Hz, 3H), 1.07 (s, 3H), 1.03 (s, 3H), 1.01 (s, 3H), 0.95 (s, 3H), 0.85 (d, J = 6.4 Hz, 3H), 0.66 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 217.6, 177.4, 172.2, 138.4, 136.2, 129.3, 128.4, 127.0, 126.2, 55.2, 53.6, 52.1, 47.8, 47.4, 46.7, 42.3, 39.3, 38.2, 37.2, 36.6 34.1, 32.3, 30.8, 29.7, 27.8, 26.6, 24.8, 23.4, 21.4, 21.1, 19.5, 17.1, 16.4, 15.2. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C40H57NNaO4, 638.4180; found, 638.4148. Compound 5a. Yield 68%; white solid; mp 251−253 °C. 1H NMR (300 MHz, CDCl3) δ: 6.52 (t, J = 4.2 Hz, 1H), 5.37 (s, 1H), 4.55 (t, J = 7.2 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 4.03 (dd, J = 18.7, 5.2 Hz, 1H), 3.79 (dd, J = 18.7, 3.5 Hz, 1H), 2.01 (s, 3H), 1.99−1.88 (m, 4H), 1.61−1.54 (m, 4H), 1.53−1.45 (m, 4H), 1.28−1.21 (m, 3H), 1.25 (t, J = 7.2 Hz, 3H), 1.06 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.6 Hz, 2H), 0.82 (s, 3H), 0.68 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.0, 170.8, 170.0, 139.0, 126.1, 80.7, 61.2, 55.1, 53.5, 47.5, 47.3, 42.2, 41.6, 39.5, 39.4, 38.9, 38.2, 37.5, 36.8, 36.7, 32.5, 30.7, 27.9, 29.7, 24.7, 23.4, 23.3, 23.2, 21.14, 21.09, 18.0, 17.1, 16.6, 16.3, 15.4, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C36H57NNaO5, 606.4129; found, 606.4147. Compound 5b. Yield 66%; white solid; mp 224−226 °C. 1H NMR (300 MHz, CDCl3) δ: 6.35 (d, J = 7.0 Hz, 1H), 5.36 (s, 1H), 4.58− 4.45 (m, 2H), 3.69 (s, 3H), 2.04 (s, 3H), 1.98−1.91 (m, 3H), 1.73− 1.58 (m, 7H), 1.58−1.47 (m, 6H), 1.08 (s, 3H), 0.95 (s, 3H), 0.93 (s, 3H), 0.92 (d, J = 5.5 Hz, 3H), 0.91 (s, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.68 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.3, 173.4, 170.8, 138.3, 126.2, 80.7, 55.1, 53.6, 51.9, 50.6, 47.6, 47.4, 42.2, 39.53, 39.46, 38.9, 38.2, 37.5, 37.3, 36.7, 32.7, 30.7, 27.9, 27.7, 24.7, 24.6, 23.4, 23.2, 22.6, 22.3, 21.2, 21.1, 18.0, 17.1, 16.58, 16.53, 15.4. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C39H63NNaO5, 648.4598; found, 648.4609. Compound 5c. Yield 76%.; white solid; mp 191−193 °C. 1H NMR (300 MHz, CDCl3) δ: 7.28 (d, J = 6.2 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.13−7.03 (m, 2H), 6.39 (d, J = 6.2 Hz, 1H), 5.24 (s, 1H), 4.71 (q, J = 6.0 Hz, 1H), 4.53−4.41 (m, 1H), 3.66 (s, 3H), 3.08 (qd, J = 13.7, 5.9 Hz, 2H), 2.03 (s, 3H), 1.88−1.76 (m, 4H), 1.65−1.57 (m, 4H), 1.52−1.46 (m, 3H), 1.42 (s, 3H), 1.04 (s, 3H), 0.96−0.92 (m, 4H), 0.89 (s, 3H), 0.85 (s, 3H), 0.84 (d, J = 4.4 Hz, 1H), 0.59 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ: 177.4, 172.1, 170.9, 138.2, 136.2, 129.3, 128.3, 126.9, 126.3, 80.7, 55.2, 53.5, 52.0, 47.7, 47.4, 42.2, 39.6, 39.5, 39.0, 38.2, 38.1, 37.6, 37.2, 36.7, 32.6, 30.8, 29.6, 28.0, 27.7, 26.8, 24.7, 23.5, 23.2, 21.2, 21.1, 18.0, 17.1, 16.6, 16.3, 15.4. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C42H61NNaO5, 682.4442; found, 682.4458. Compound 6a. Yield 72%; white solid; mp 208−210 °C. 1H NMR (300 MHz, CDCl3) δ: 7.87 (s, 1H), 7.39 (d, J = 1.9 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 8.5, 2.0 Hz, 1H), 6.64−6.55 (m, 1H), 5.55 (s, 1H), 4.24 (q, J = 7.1 Hz, 2H), 4.12 (dd, J = 18.7, 5.3 Hz, 1H), 3.87 (dd, J = 18.7, 3.6 Hz, 1H), 2.76 (d, J = 15.0 Hz, 1H), 2.28−2.14 (m, 3H), 2.09−1.97 (m, 2H), 1.84−1.69 (m, 3H), 1.64−1.47 (m, 6H), 1.33 (t, J = 7.8 Hz, 3H), 1.33−1.29 (m, 3H), 1.28 (d, J = 6.1 Hz, 3H), 1.23 (s, 3H), 1.18 (s, 3H), 0.99 (s, 3H), 0.96 (d, J = 5.6 Hz, 3H), 0.95 (s, 3H), 0.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.2, 170.1, 142.5, 139.0, 134.5, 126.5, 124.6, 121.0, 117.3, 111.3, 106.8, 61.5, 53.9, 53.0, 47.8, 46.3, 42.5, 41.8, 39.8, 39.6, 39.0, 38.0, 37.0, 36.9, 34.1, 32.2, 30.9, 29.7, 27.9, 24.9, 23.5, 23.3, 21.2, 19.2, 17.2, 16.3, 15.7, 14.1. HRMS (m/z) [M + Na]+ (ESI+) calcd for C40H55ClN2NaO3, 669.3793; found, 669.3817. Compound 6b. Yield 67%; white solid; mp 251−253 °C. 1H NMR (300 MHz, CDCl3) δ: 7.86 (s, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.05 (dd, J = 8.5, 1.8 Hz, 1H), 6.42 (d, J = 7.0 Hz, 1H), 5.50 (t, J = 3.5 Hz 1H), 4.66−4.52 (m, 1H), 3.70 (s, 3H), 2.75 (d, J = 15.0 Hz, 1H), 2.24−2.15 (m, 2H), 2.06−1.97 (m, 2H), 1.85− 1.72 (m, 4H), 1.64−1.56 (m, 6H), 1.52−1.37 (m, 5H), 1.30 (s, 3H), 1.21 (s, 3H), 1.15 (s, 3H), 0.98 (s, 3H), 0.96 (d, J = 7.8 Hz, 6H),

0.95 (s, 3H), 0.94 (d, J = 4.5 Hz, 3H), 0.93 (s, 3H), 0.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.5, 173.5, 142.5, 138.3, 134.5, 129.4, 126.6, 124.5, 121.0, 117.5, 111.2, 106.8, 54.0, 53.1, 52.1, 50.8, 47.9, 46.3, 42.5, 42.4, 39.8, 39.7, 39.1, 38.0, 37.5, 36.9, 34.1, 32.4, 30.9, 27.9, 24.8, 23.4, 23.2, 22.7, 22.5, 21.2, 19.2, 17.2, 16.6, 15.7. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C43H61ClN2NaO3, 711.4263; found, 711.4282. Compound 6c. Yield 57%; white solid; mp 216−218 °C. 1H NMR (300 MHz, CDCl3) δ: 7.97 (s, 1H), 7.36 (d, J = 1.9 Hz, 1H), 7.29 (d, J = 7.2 Hz, 2H), 7.19 (d, J = 8.5 Hz, 1H), 7.12 (dd, J = 7.6, 1.6 Hz, 2H), 7.05 (dd, J = 8.5, 2.0 Hz, 1H), 6.47 (d, J = 6.3 Hz, 1H), 5.37 (s, 1H), 4.82−4.71 (m, 1H), 3.67 (s, 3H), 3.16 (dd, J = 13.7, 6.1 Hz, 1H), 3.07 (dd, J = 13.7, 5.7 Hz, 1H), 2.72 (d, J = 15.0 Hz, 1H), 2.22−1.98 (m, 4H), 1.92−1.69 (m, 5H), 1.52−1.38 (m, 6H), 1.28 (d, J = 5.5 Hz, 3H), 1.26 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 0.91 (s, 3H), 0.90 (d, J = 6.5 Hz, 3H), 0.72 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.5, 172.0, 142.5, 138.1, 136.1, 134.4, 129.3, 128.4, 126.9, 126.6, 124.4, 120.8, 117.4, 111.2, 106.6, 53.7, 53.5, 53.0, 52.1, 47.8, 46.2, 42.4, 39.7, 39.6, 39.0, 38.2, 37.8, 37.2, 36.8, 34.0, 32.2, 31.8, 30.8, 29.6, 27.8, 24.8, 23.3, 23.2, 21.1, 19.1, 17.1, 16.3, 15.6. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C46H59ClN2NaO3, 745.4106; found, 745.4137. Compound 7a. Yield 64%; white solid; mp 202−204 °C. 1H NMR (300 MHz, CDCl3) δ: 7.85 (s, 1H), 7.53 (s, 1H), 7.17 (d, J = 2.7 Hz, 2H), 6.58 (s, 1H), 5.53 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 4.10 (dd, J = 18.6, 5.3 Hz, 1H), 3.85 (dd, J = 18.8, 3.3 Hz, 1H), 2.74 (d, J = 15.0 Hz, 1H), 2.24−2.11 (m, 3H), 2.07−1.99 (m, 2H), 1.93−1.72 (m, 4H), 1.64−1.55 (m, 4H), 1.44−1.37 (m, 3H), 1.32−1.28 (m, 5H), 1.26 (s, 6H), 1.21 (t, J = 7.0 Hz, 3H), 1.16 (s, 3H), 0.98−0.94 (m, 6H), 0.94 (d, J = 5.4 Hz, 3H), 0.93 (s, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.2, 170.1, 142.3, 139.0, 134.7, 130.0, 126.5, 123.6, 120.6, 112.1, 111.7, 106.7, 61.5, 53.9, 53.0, 47.8, 46.2, 42.5, 41.7, 39.8, 39.6, 39.0, 37.9, 37.0, 36.8, 34.0, 32.2, 30.9, 29.7, 29.3, 27.9, 24.9, 23.5, 23.3, 22.7, 21.2, 19.2, 17.2, 16.3, 15.7, 14.1. HRMS (m/z) [M + Na]+ (ESI+) calcd for C40H55BrN2NaO3, 713.3288; found, 713.3307. Compound 7b. Yield 68%; white solid; mp 178−180 °C. 1H NMR (300 MHz, CDCl3) δ: 7.88 (s, 1H), 7.53 (s, 1H), 7.17 (s, 2H), 6.42 (d, J = 7.1 Hz, 1H), 5.50 (s, 1H), 4.63−4.55 (m, 1H), 3.70 (s, 3H), 2.75 (d, J = 15.0 Hz, 1H), 2.22−2.00 (m, 5H), 1.83−1.72 (m, 3H), 1.66− 1.59 (m, 7H), 1.51−1.40 (m, 5H), 1.30 (s, 3H), 1.26 (s, 6H), 1.21 (s, 3H), 1.14 (s, 3H), 0.98−0.93 (m, 3H), 0.96 (d, J = 8.0 Hz, 3H), 0.94 (d, J = 5.1 Hz, 3H), 0.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.5, 173.5, 142.3, 138.2, 134.7, 130.0, 126.6, 123.5, 120.6, 112.1, 111.7, 106.7, 54.0, 53.1, 52.1, 50.8, 47.9, 46.2, 42.5, 42.3, 39.8, 39.7, 39.1, 37.9, 37.5, 36.9, 34.0, 32.4, 31.9, 30.9, 29.4, 27.8, 24.8, 23.4, 23.2, 22.6, 22.4, 21.1, 19.2, 17.2, 16.6, 15.7, 14.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C43H61BrN2NaO3, 755.3758; found, 755.3775. Compound 7c. Yield 74%; white solid; mp 194−196 °C. 1H NMR (300 MHz, CDCl3) δ: 7.83 (s, 1H), 7.52 (s, 1H), 7.29 (d, J = 7.3 Hz, 2H), 7.16 (s, 1H), 7.12 (d, J = 7.8 Hz, 2H), 6.45 (d, J = 6.4 Hz, 1H), 5.37 (s, 1H), 4.80−4.69 (m, 1H), 3.67 (s, 3H), 3.15 (dd, J = 13.6, 6.1 Hz, 1H), 3.06 (dd, J = 13.7, 5.7 Hz, 1H), 2.72 (d, J = 15.0 Hz, 1H), 2.21−2.03 (m, 3H), 1.91−1.68 (m, 5H), 1.29−1.26 (m, 9H), 1.28 (d, J = 5.8 Hz, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 0.96 (s, 3H), 0.90−0.88 (m, 3H), 0.89 (d, J = 6.2 Hz, 3H), 0.86 (s, 3H), 0.71 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.5, 172.1, 142.3, 138.2, 136.2, 134.7, 130.0, 129.4, 128.4, 127.0, 126.6, 123.6, 120.6, 112.1, 111.7, 53.9, 53.5, 53.0, 52.1, 47.9, 46.2, 42.4, 39.8, 39.6, 39.1, 38.2, 37.9, 37.3, 36.8, 34.0, 32.3, 31.9, 30.9, 29.7, 29.5, 27.8, 24.8, 23.4, 23.2, 22.7, 21.2, 19.2, 17.1, 16.4, 15.7, 14.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C46H59BrN2NaO3, 789.3601; found, 789.3605. Compound 8a. Yield 68%; white solid; mp 215−217 °C. 1H NMR (300 MHz, CDCl3) δ: 8.15 (s, 1H), 8.08 (s, 1H), 6.53 (t, J = 4.4 Hz, 1H), 5.50 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.08 (dd, J = 18.7, 5.2 Hz, 1H), 3.85 (dd, J = 18.7, 3.6 Hz, 1H), 3.25 (d, J = 16.7 Hz, 1H), 2.66 (d, J = 16.8 Hz, 1H), 2.21−2.10 (m, 2H), 2.07−1.99 (m, 2H), 1.90− 1.78 (m, 3H), 1.73−1.63 (m, 4H), 1.59−1.51 (m, 4H), 1.41 (s, 3H), 1.40 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.28 (d, J = 7.2 Hz, 3H), 1.17 (s, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.90 (s, 3H), 0.82 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.9, 170.0, 162.3, 153.3, 140.7, 139.5, 139.0, 4703

dx.doi.org/10.1021/jm5002293 | J. Med. Chem. 2014, 57, 4692−4709

Journal of Medicinal Chemistry

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Compound 10. Yield 65%; white solid; mp 232−234 °C. 1H NMR (300 MHz, CDCl3) δ: 7.99 (d, J = 3.3 Hz, 2H), 7.76−7.62 (m, 2H), 7.07 (d, J = 5.2 Hz, 1H), 5.60 (s, 1H), 4.72−4.58 (m, 1H), 3.97 (d, J = 17.4 Hz, 1H), 2.71−2.50 (m, 3H), 2.42−2.29 (m, 2H), 2.11 (s, 3H), 2.03−1.79 (m, 5H), 1.62−1.46 (m, 6H), 1.40 (s, 3H), 1.37 (s, 3H), 1.20 (s, 3H), 1.04 (d, J = 5.7 Hz, 3H), 1.03 (d, J = 5.7 Hz, 3H), 0.83 (s, 6H). 13C NMR (75 MHz, CDCl3) δ: 178.3, 173.4, 162.0, 152.2, 142.4, 138.8, 138.3, 129.6, 129.0, 127.2, 125.9, 54.1, 53.4, 51.7, 48.0, 47.8, 45.4, 42.5, 40.4, 39.8, 39.6, 39.3, 37.6, 36.5, 32.4, 32.2, 30.8, 30.1, 29.7, 27.9, 25.2, 24.6, 23.5, 23.4, 21.3, 20.2, 17.6, 15.9, 15.5. HR-MS (m/z) [M + H]+ (ESI+) calcd for C41H58N3O3S, 672.4193; found, 672.4173. Compound 11. Yield 59%; white solid; mp 241−243 °C. 1H NMR (300 MHz, CDCl3) δ: 7.98 (dd, J = 6.6, 3.5 Hz, 2H), 7.65 (dd, J = 6.3, 3.4 Hz, 2H), 7.30 (s, 1H), 7.29 (s, 2H), 5.71 (d, J = 7.5 Hz, 1H), 5.35 (s, 1H), 3.84−3.68 (m, 1H), 3.56−3.41 (m, 2H), 3.26 (d, J = 16.4 Hz, 1H), 2.87−2.61 (m, 3H), 2.18−2.06 (m, 4H), 1.93−1.71 (m, 7H), 1.62−1.55 (m, 2H), 1.44 (s, 3H), 1.43 (s, 3H), 1.17 (s, 3H), 0.97 (s, 3H), 0.93 (s, 3H), 0.92 (d, J = 3.9 Hz, 1H), 0.91 (d, J = 2.9 Hz, 1H), 0.90 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.1, 161.0, 151.9, 142.1, 140.8, 139.6, 129.2, 128.8, 128.5, 128.2, 128.1, 127.0, 125.4, 63.1, 54.3, 53.5, 52.3, 52.1, 49.7, 47.9, 46.2, 45.4, 42.9, 40.3, 39.9, 39.5, 389.1, 37.6, 36.8, 32.3, 32.2, 31.8, 30.9, 29.7, 27.9, 25.3, 24.7, 23.5, 23.1, 21.2, 20.3, 17.3, 17.2, 15.9. HR-MS (m/z) [M + H]+ (ESI+) calcd for C48H65N4O, 713.5153; found, 713.5177. Compound 12. Yield 68%; white solid; mp 244−246 °C. 1H NMR (300 MHz, CDCl3) δ: 7.99 (dd, J = 6.3, 3.4 Hz, 2H), 7.66 (dd, J = 6.3, 3.4 Hz, 2H), 7.29 (s, 1H), 7.28 (s, 2H), 5.85 (s, 1H), 5.40 (s, 1H), 3.48 (s, 2H), 3.38−3.21 (m, 2H), 3.14−3.05 (m, 1H), 2.93−2.81 (m, 2H), 2.70 (d, J = 16.5 Hz, 1H), 2.30−2.06 (m, 3H), 1.98−1.79 (m, 6H), 1.74−1.54 (m, 6H), 1.44 (s, 6H), 1.27−1.23 (m, 3H), 1.18 (s, 3H), 0.95 (d, J = 5.9 Hz, 3H), 0.93 (s, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.88 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.9, 161.0, 151.8, 142.2, 140.8, 140.2, 129.3, 128.8, 128.6, 128.2, 128.1, 127.1, 125.3, 63.3, 54.2, 53.6, 53.5, 49.6, 47.8, 45.4, 42.8, 40.3, 39.9, 39.4, 39.1, 37.2, 37.1, 36.8, 36.0, 33.5, 32.3, 32.0, 30.9, 29.7, 27.8, 25.3, 24.9, 23.5, 23.2, 21.2, 20.3, 17.3, 16.7, 15.9. HR-MS (m/z) [M + H]+ (ESI+) calcd for C50H69N4O, 741.5466; found, 741.5490. Compound 13. Yield 66%; white solid; mp 226−228 °C. 1H NMR (300 MHz, CDCl3) δ: 8.04−7.91 (m, 2H), 7.65 (dd, J = 6.3, 3.5 Hz, 2H), 6.59 (s, 1H), 5.41 (s, 1H), 3.41−3.17 (m, 3H), 2.69 (d, J = 16.4 Hz, 1H), 2.46−2.29 (m, 6H), 2.21−2.10 (m, 2H), 1.96−1.74 (m, 5H), 1.66−1.55 (m, 6H), 1.44 (s, 3H), 1.43 (s, 3H), 1.17 (s, 3H), 0.95 (d, J = 5.9 Hz, 3H), 0.93 (d, J = 5.4 Hz, 3H), 0.92 (s, 3H), 0.89 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ: 177.7, 160.9, 151.8, 142.1, 140.7, 139.3, 128.7, 128.4, 128.0, 125.4, 57.0, 54.2, 54.0, 53.5, 49.5, 47.7, 45.3, 42.6, 40.3, 39.8, 39.4, 39.0, 37.2, 36.8, 35.8, 32.2, 32.0, 30.9, 29.6, 27.8, 25.9, 25.3, 24.7, 24.2, 23.4, 23.0, 21.1, 20.3, 17.2, 16.6, 15.7. HR-MS (m/z) [M + H]+ (ESI+) calcd for C43H63N4O, 651.4996; found, 651.5020. Compound 14. Yield 61%; white solid; mp 245−247 °C. 1H NMR (300 MHz, CDCl3) δ: 8.10−7.86 (m, 2H), 7.65 (dd, J = 6.3, 3.5 Hz, 2H), 6.39 (s, 1H), 5.38 (s, 1H), 3.75−3.71 (m, 3H), 3.54−3.36 (m, 1H), 3.25 (d, J = 16.4 Hz, 1H), 3.16−2.96 (m, 1H), 2.69 (d, J = 16.4 Hz, 1H), 2.47−2.36 (m, 5H), 2.18−1.96 (m, 4H), 1.87−1.64 (m, 7H), 1.44 (s, 3H), 1.43 (s, 3H), 1.17 (s, 3H), 0.95 (d, J = 5.5 Hz, 3H), 0.93 (s, 3H), 0.92 (d, J = 6.3 Hz, 3H), 0.88 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.7, 160.9, 151.8, 142.1, 140.8, 139.7, 128.7, 128.5, 128.0, 125.2, 66.8, 57.4, 53.9, 53.7, 53.4, 49.5, 47.6, 45.3, 42.6, 40.3, 39.7, 39.4, 39.0, 38.5, 37.4, 36.7, 32.2, 30.8, 27.7, 25.5, 25.3, 24.7, 23.4, 23.3, 21.1, 20.2, 17.2, 16.7, 15.7. HR-MS (m/z) [M + H]+ (ESI+) calcd for C43H63N4O2, 667.4946; found, 667.4964. Compound 15a. Yield 78%; white solid; mp 195−197 °C. 1H NMR (300 MHz, CDCl3) δ: 7.24 (s, 1H), 6.60 (t, J = 4.2 Hz, 1H), 5.46 (s, 1H), 4.20 (q, J = 7.1 Hz, 2H), 4.07 (dd, J = 18.2, 6.1 Hz, 1H), 3.83 (dd, J = 18.6, 3.4 Hz, 1H), 2.58 (d, J = 14.8 Hz, 1H), 2.09−1.98 (m, 6H), 1.77−1.65 (m, 3H), 1.57−1.44 (m, 5H), 1.32−1.28 (m, 5H), 1.27−1.19 (m, 6H), 1.30 (s, 3H), 1.26 (t, J = 7.3 Hz, 3H), 1.25 (d, J = 7.5 Hz, 3H), 1.11 (s, 3H), 0.94 (s, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.85 (s, 3H), 0.76 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.1, 170.2,

133.0, 132.8, 129.3, 128.7, 125.9, 61.3, 53.7, 53.2, 49.4, 47.7, 45.3, 42.4, 41.6, 40.4, 39.7, 39.3, 38.9, 36.7, 32.0, 30.7, 29.6, 29.2, 27.7, 25.2, 24.7, 23.3, 21.1, 20.1, 17.1, 16.1, 15.7, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C40H53Cl2N3NaO3, 716.3356; found, 716.3365. Compound 8b. Yield 76%; white solid; mp 209−211 °C. 1H NMR (300 MHz, CDCl3) δ: 8.13 (s, 1H), 8.07 (s, 1H), 6.35 (d, J = 7.1 Hz, 1H), 5.47 (s, 1H), 4.64−4.50 (m, 1H), 3.69 (s, 3H), 3.24 (d, J = 16.7 Hz, 1H), 2.65 (d, J = 16.7 Hz, 1H), 2.18−2.07 (m, 2H), 2.06−1.98 (m, 2H), 1.86−1.70 (m, 4H), 1.64−1.53 (m, 6H), 1.40 (s, 3H), 1.39 (s, 3H), 1.25 (s, 3H), 1.16 (s, 3H), 0.95 (d, J = 5.9 Hz, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.91 (d, J = 7.2 Hz, 3H), 0.80 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 173.5, 162.4, 153.4, 140.8, 139.6, 138.4, 133.1, 132.9, 129.4, 128.8, 126.2, 53.9, 53.3, 52.1, 50.7, 49.5, 47.9, 45.4, 42.6, 42.3, 40.5, 39.7, 39.5, 39.0, 37.4, 36.7, 32.2, 30.9, 29.6, 27.8, 25.3, 24.8, 24.7, 23.4, 23.2, 22.7, 22.4, 21.1, 20.2, 17.2, 16.5, 15.8. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C43H59Cl2N3NaO3, 758.3826; found, 758.3834. Compound 8c. Yield 71%; white solid; mp 250−252 °C. 1H NMR (300 MHz, CDCl3) δ: 8.14 (s, 1H), 8.08 (s, 1H), 7.29 (s, 2H), 7.14− 7.06 (m, 2H), 6.39 (d, J = 6.3 Hz, 1H), 5.34 (s, 1H), 4.81−4.70 (m, 1H), 3.66 (s, 3H), 3.14 (dd, J = 13.7, 6.1 Hz, 1H), 3.05 (dd, J = 13.7, 5.8 Hz, 1H), 2.14−1.81 (m, 4H), 1.76−1.66 (m, 5H), 1.58−1.47 (m, 4H), 1.40 (s, 3H), 1.39 (s, 3H), 1.25 (s, 3H), 1.13 (s, 3H), 0.97−0.94 (m, 3H), 0.91−0.86 (m, 3H), 0.88 (d, J = 5.7 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H), 0.72 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.9, 170.0, 162.3, 153.3, 140.7, 139.4, 139.0, 133.0, 132.8, 129.3, 128.7, 125.9, 61.3, 53.7, 53.2, 49.4, 47.7, 45.3, 42.4, 41.6, 40.4, 39.7, 39.3, 38.9, 36.9, 36.6, 32.1, 31.9, 30.7, 29.6, 29.2, 27.7, 25.2, 24.7, 23.3, 23.2, 21.1, 20.1, 17.1, 16.1, 15.7, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C46H57Cl2N3NaO3, 792.3669; found, 792.3688. Compound 9a. Yield 77%; white solid; mp 230−232 °C. 1H NMR (300 MHz, CDCl3) δ: 7.98 (dd, J = 6.5, 3.4 Hz, 2H), 7.65 (dd, J = 6.3, 3.4 Hz, 2H), 6.55 (s, 1H), 5.50 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.09 (dd, J = 18.6, 5.2 Hz, 1H), 3.84 (dd, J = 18.7, 3.5 Hz, 1H), 3.26 (d, J = 16.4 Hz, 1H), 2.69 (d, J = 16.4 Hz, 1H), 2.23−1.98 (m, 4H), 1.86− 1.64 (m, 5H), 1.60−1.55 (m, 2H), 1.44 (s, 3H), 1.43 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.26 (d, J = 7.3 Hz, 3H), 1.18 (s, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.92 (s, 3H), 0.82 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.0, 170.1, 161.0, 151.9, 142.1, 140.8, 139.1, 128.8, 128.5, 128.0, 126.2, 61.4, 53.8, 53.5, 49.6, 47.8, 45.4, 42.5, 41.7, 40.3, 39.8, 39.5, 39.0, 37.0, 36.8, 32.3, 32.1, 30.8, 29.7, 27.8, 25.3, 24.9, 23.4, 21.3, 20.2, 17.3, 16.2, 15.8, 14.1. HR-MS (m/z) [M + H]+ (ESI+) calcd for C40H56N3O3, 626.4316; found, 626.4337. Compound 9b. Yield 74%; white solid; mp 238−240 °C. 1H NMR (300 MHz, CDCl3) δ: 7.98 (dd, J = 6.6, 3.5 Hz, 2H), 7.72−7.56 (m, 2H), 6.37 (d, J = 7.1 Hz, 1H), 5.47 (s, 1H), 4.66−4.46 (m, 1H), 3.69 (s, 3H), 3.27 (d, J = 16.4 Hz, 1H), 2.69 (d, J = 16.4 Hz, 1H), 2.19− 2.14 (m, 1H), 2.05−1.96 (m, 2H), 1.86−1.69 (m, 5H), 1.64−1.53 (m, 6H), 1.44 (s, 3H), 1.43 (s, 3H), 1.25 (s, 3H), 1.17 (s, 3H), 0.96 (d, J = 5.9 Hz, 3H), 0.94 (s, 3H), 0.93 (d, J = 6.3 Hz, 3H), 0.92 (s, 3H), 0.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 173.5, 161.0, 151.9, 142.1, 140.8, 138.4, 128.8, 128.6, 128.0, 126.3, 53.9, 53.5, 52.1, 50.8, 49.6, 47.9, 45.5, 42.6, 42.3, 40.3, 39.8, 39.5, 39.0, 37.5, 36.8, 32.3, 30.9, 29.7, 27.8, 25.3, 24.8, 23.4, 23.3, 22.7, 22.4, 21.1, 20.3, 17.2, 16.5, 15.8. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C43H61N3NaO3, 690.4605; found, 690.4614. Compound 9c. Yield 63%; white solid; mp 233−235 °C. 1H NMR (300 MHz, CDCl3) δ: 7.98 (dd, J = 6.3, 3.4 Hz, 2H), 7.65 (dd, J = 6.3, 3.4 Hz, 2H), 7.30 (d, J = 6.5 Hz, 1H), 7.11 (d, J = 7.1 Hz, 2H), 6.41 (d, J = 6.3 Hz, 1H), 5.35 (s, 1H), 4.82−4.67 (m, 1H), 3.67 (s, 3H), 3.24 (d, J = 16.4 Hz, 1H), 3.15 (dd, J = 13.8, 6.0 Hz, 1H), 3.05 (dd, J = 13.7, 5.8 Hz, 1H), 2.66 (d, J = 16.6 Hz, 1H), 2.14−1.94 (m, 3H), 1.92−1.66 (m, 6H), 1.58−1.44 (m, 4H), 1.43 (s, 3H), 1.42 (s, 3H), 1.25 (s, 2H), 1.14 (s, 3H), 0.96 (s, 3H), 0.89 (s, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.72 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 172.1, 161.0, 151.9, 142.1, 140.8, 138.3, 136.2, 129.3, 128.8, 128.5, 128.4, 128.0, 127.0, 126.3, 53.7, 53.5, 52.1, 49.6, 47.8, 45.4, 42.5, 40.3, 39.7, 39.5, 39.1, 38.2, 37.2, 36.7, 32.2, 30.8, 29.7, 27.8, 25.3, 24.8, 23.3, 21.2, 20.2, 17.1, 16.3, 15.7. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C46H59N3NaO3, 724.4449; found, 724.4464. 4704

dx.doi.org/10.1021/jm5002293 | J. Med. Chem. 2014, 57, 4692−4709

Journal of Medicinal Chemistry

Article

2.63 (d, J = 14.9 Hz, 1H), 2.18−1.97 (m, 5H), 1.78−1.64 (m, 3H), 1.56−1.43 (m, 5H), 1.28 (t, J = 7.1 Hz, 3H), 1.32−1.24 (m, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.97 (d, J = 6.1 Hz, 3H), 0.92 (s, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.77 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.0, 170.1, 164.1, 160.8, 146.2, 138.9, 138.3, 130.8, 130.7, 126.3, 115.4, 115.2, 114.1, 61.4, 54.4, 53.8, 47.8, 46.2, 42.4, 41.6, 39.7, 39.4, 38.9, 37.9, 37.1, 36.9, 34.6, 32.1, 30.8, 29.6, 29.3, 27.8, 24.8, 23.3, 23.1, 22.3, 21.1, 19.0, 17.1, 16.1, 15.3, 14.0. HR-MS (m/z) [M + H]+ (ESI+) calcd for C41H57FN3O3, 658.4378; found, 658.4390. Compound 17b. Yield 74%; white solid; mp 192−194 °C. 1H NMR (300 MHz, CDCl3) δ: 7.38−7.31 (m, 3H), 7.14−7.07 (m, 2H), 6.37 (d, J = 7.1 Hz, 1H), 5.45 (s, 1H), 4.60−4.53 (m, 1H), 3.70 (s, 3H), 2.63 (d, J = 14.8 Hz, 1H), 2.16−1.98 (m, 5H), 1.85−1.64 (m, 5H), 1.60−1.45 (m, 7H), 1.30−1.21 (m, 5H), 1.11 (s, 3H), 1.03 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.93 (d, J = 4.7 Hz, 3H), 0.92 (s, 6H), 0.91 (d, J = 6.4 Hz, 3H), 0.75 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.3, 173.5, 164.1, 160.8, 146.2, 138.4, 138.2, 130.9, 130.7, 126.4, 115.4, 115.1, 114.2, 54.5, 53.8, 52.0, 50.7, 47.8, 46.3, 42.4, 42.2, 39.7, 39.5, 38.9, 37.9, 37.4, 37.1, 34.5, 32.4, 30.8, 29.6, 29.3, 27.8, 24.7, 23.3, 23.1, 22.6, 22.4, 22.3, 21.1, 19.0, 17.1, 16.5, 15.3. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C44H62FN3NaO3, 722.4667; found, 722.4675. Compound 17c. Yield 71%; white solid; mp 220−222 °C. 1H NMR (300 MHz, CDCl3) δ: 7.34 (s, 3H), 7.29 (s, 1H), 7.11 (d, J = 7.1 Hz, 3H), 6.41 (d, J = 5.9 Hz, 1H), 5.34 (s, 1H), 4.83−4.66 (m, 1H), 3.68 (s, 3H), 3.14 (dd, J = 13.7, 6.1 Hz, 1H), 3.05 (dd, J = 13.5, 5.6 Hz, 1H), 2.62 (d, J = 14.3 Hz, 1H), 2.10−1.93 (m, 3H), 1.87−1.60 (m, 6H), 1.51−1.43 (m, 3H), 1.28−1.22 (m, 3H), 1.09 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.89 (d, J = 4.6 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 172.0, 164.2, 160.9, 146.3, 138.3, 138.1, 136.1, 130.9, 130.8, 129.3, 128.3, 126.9, 126.4, 115.5, 115.2, 114.2, 54.5, 53.8, 53.4, 52.0, 47.8, 46.2, 42.3, 39.7, 39.4, 39.0, 38.1, 37.9, 37.2, 37.1, 34.5, 32.2, 30.8, 29.6, 29.3, 27.7, 24.7, 23.2, 23.1, 22.3, 21.0, 19.0, 17.0, 16.2, 15.3. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C47H60FN3NaO3, 756.4511; found, 756.4522. Compound 18a. Yield 85%; white solid; mp 187−189 °C. 1H NMR (300 MHz, CDCl3) δ: 7.97 (s, 1H), 6.52 (s, 1H), 5.46 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.06 (dd, J = 18.7, 5.1 Hz, 1H), 3.84 (dd, J = 18.7, 3.5 Hz, 1H), 2.44 (d, J = 15.1 Hz, 1H), 2.11−1.95 (m, 5H), 1.89−1.69 (m, 4H), 1.57−1.40 (m, 8H), 1.32−1.23 (m, 3H), 1.29 (d, J = 6.7 Hz, 3H), 1.28 (t, J = 7.4 Hz, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 0.95 (s, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.88 (s, 3H), 0.77 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.8, 172.8, 170.0, 150.0, 138.9, 125.8, 108.6, 61.3, 53.6, 53.3, 47.6, 45.9, 42.3, 41.6, 39.6, 39.4, 38.9, 38.4, 36.9, 35.5, 34.6, 31.9, 30.7, 29.5, 28.7, 27.7, 26.8, 24.7, 23.3, 23.1, 22.5, 21.2, 21.1, 18.7, 17.0, 16.1, 15.3, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C35H52N2NaO4, 587.3819; found, 587.3835. Compound 18b. Yield 82%; white solid; mp 231−233 °C. 1H NMR (300 MHz, CDCl3) δ: 7.98 (s, 1H), 6.34 (d, J = 7.1 Hz, 1H), 5.43 (s, 1H), 4.62−4.52 (m, 1H), 3.70 (s, 3H), 2.45 (d, J = 15.1 Hz, 1H), 2.10−1.92 (m, 5H), 1.85−1.69 (m, 4H), 1.62−1.47 (m, 7H), 1.42 (s, 3H), 1.30 (s, 3H), 1.20 (s, 3H), 1.11 (s, 3H), 0.95 (d, J = 6.2 Hz, 3H), 0.94 (s, 3H), 0.91 (d, J = 5.9 Hz, 3H), 0.89 (s, 3H), 0.75 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.3, 173.5, 172.9, 150.0, 138.3, 126.0, 108.7, 53.7, 53.4, 52.0, 50.6, 47.7, 46.0, 42.4, 42.2, 39.6, 39.5, 38.9, 38.5, 37.3, 35.5, 34.6, 32.1, 30.8, 28.7, 27.8, 26.8, 24.7, 24.6, 23.3, 23.1, 22.6, 22.3, 21.2, 21.0, 18.7, 17.0, 16.4, 15.4. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C38H58N2NaO4, 629.4289; found, 629.4298. Compound 18c. Yield 86%; white solid; mp 221−223 °C. 1H NMR (300 MHz, CDCl3) δ: 7.97 (s, 1H), 7.29 (s, 1H), 7.13−7.06 (m, 2H), 6.39 (d, J = 6.3 Hz, 1H), 5.31 (s, 1H), 4.78−4.69 (m, 1H), 3.67 (s, 3H), 3.14 (dd, J = 13.7, 6.0 Hz, 1H), 3.04 (dd, J = 13.7, 5.8 Hz, 1H), 2.42 (d, J = 15.1 Hz, 1H), 2.05−1.77 (m, 6H), 1.71−1.63 (m, 2H), 1.57−1.39 (m, 6H), 1.29 (s, 3H), 1.20 (s, 3H), 1.08 (s, 3H), 0.95 (d, J = 5.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.85 (s, 3H), 0.66 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ: 177.2, 172.8, 172.0, 150.0, 138.2, 136.1, 129.2, 128.3, 126.9, 126.0, 108.7, 53.5, 53.4, 53.3, 52.0, 47.7, 45.9, 42.2, 39.6, 39.5, 38.9, 38.4, 38.0, 37.1, 35.5, 34.6, 32.0, 30.8, 29.6, 28.7, 27.7, 24.7, 23.1, 21.3, 21.1, 18.6, 17.0, 16.2, 15.3. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C41H56N2NaO4, 663.4132; found, 663.4146.

149.3, 138.8, 133.9, 126.3, 112.2, 61.4, 53.7, 53.2, 47.5, 46.0, 42.4, 41.6, 39.7, 39.5, 38.9, 38.2, 36.9, 36.4, 33.2, 32.0, 31.2, 30.8, 29.6, 27.8, 24.8, 23.3, 23.1, 21.1, 19.0, 17.1, 16.1, 15.2, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C35H53N3NaO3, 586.3979; found, 586.3993. Compound 15b. Yield 54%; white solid; mp 206−208 °C. 1H NMR (300 MHz, CDCl3) δ: 7.24 (s, 1H), 6.38 (d, J = 7.1 Hz, 1H), 5.44 (s, 1H), 4.61−4.54 (m, 1H), 3.70 (s, 3H), 2.60 (d, J = 14.8 Hz, 1H), 2.09−1.98 (m, 4H), 1.63−1.41 (m, 9H), 1.30 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 0.96 (s, 3H), 0.93 (d, J = 6.2 Hz, 3H), 0.92 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.87 (s, 3H), 0.76 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 173.5, 149.2, 138.1, 133.6, 126.4, 112.0, 53.7, 53.2, 52.6, 52.0, 51.7, 50.6, 47.7, 45.9, 43.9, 42.3, 42.1, 39.6, 39.5, 38.9, 38.2, 33.2, 31.1, 29.5, 24.6, 23.8, 22.9, 22.6, 22.3, 22.2, 21.7, 21.0, 17.0, 16.4, 15.2. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C38H59N3NaO3, 628.4449; found, 628.4466. Compound 15c. Yield 52%; white solid; mp 217−219 °C. 1H NMR (300 MHz, CDCl3) δ: 7.23 (s, 2H), 7.10 (d, J = 6.1 Hz, 3H), 6.46 (d, J = 5.1 Hz, 1H), 5.31 (s, 1H), 4.80−4.67 (m, 1H), 3.66 (s, 3H), 3.14 (dd, J = 13.2, 5.5 Hz, 1H), 3.05 (dd, J = 13.5, 5.6 Hz, 1H), 2.57 (d, J = 14.8 Hz, 1H), 2.06−1.94 (m, 5H), 1.70−1.41 (m, 9H), 1.30 (s, 3H), 1.26 (d, J = 6.1 Hz, 3H), 1.20 (s, 3H), 1.08 (s, 3H), 0.94 (s, 3H), 0.86 (d, J = 6.7 Hz, 3H), 0.88−0.81 (m, 3H), 0.67 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 172.1, 149.2, 138.0, 136.1, 134.1, 129.3, 128.3, 126.9, 126.4, 112.2, 60.3, 53.6, 53.5, 53.2, 52.0, 47.7, 46.0, 42.3, 39.5, 38.2, 37.1, 36.4, 33.2, 32.2, 31.1, 29.6, 27.7, 24.7, 23.8, 23.1, 21.1, 19.0, 17.0, 16.2, 15.2, 14.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C41H57N3NaO3, 662.4292; found, 662.4308. Compound 16a. Yield 67%; white solid; mp 246−248 °C. 1H NMR (300 MHz, CDCl3) δ: 7.42 (d, J = 3.7 Hz, 3H), 7.37 (d, J = 4.6 Hz, 2H), 7.33 (s, 1H), 6.56 (s, 1H), 5.49 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.07 (dd, J = 18.8, 5.0 Hz, 1H), 3.83 (dd, J = 18.7, 3.2 Hz, 1H), 2.63 (d, J = 14.8 Hz, 1H), 2.18−1.99 (m, 5H), 1.89−1.65 (m, 4H), 1.51−1.43 (m, 5H), 1.32−1.22 (m, 3H), 1.28 (t, J = 7.1 Hz, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.94 (d, J = 5.3 Hz, 3H), 0.92 (s, 3H), 0.91 (d, J = 6.8 Hz, 1H), 0.77 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 178.0, 170.1, 146.0, 142.3, 138.9, 138.1, 129.0, 128.8, 128.4, 126.3, 113.9, 61.3, 54.5, 53.8, 47.7, 46.2, 42.4, 41.6, 39.7, 39.4, 38.9, 37.9, 37.1, 36.9, 34.6, 32.1, 30.8, 29.3, 27.8, 24.8, 23.3, 23.1, 22.2, 21.1, 19.1, 17.1, 16.1, 15.3, 14.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C41H57N3NaO3, 662.4292; found, 662.4284. Compound 16b. Yield 66%; white solid; mp 224−226 °C. 1H NMR (300 MHz, CDCl3) δ: 7.44−7.40 (m, 3H), 7.39−7.34 (m, 2H), 7.33 (s, 1H), 6.37 (d, J = 7.1 Hz, 1H), 5.46 (s, 1H), 4.60−4.53 (m, 1H), 3.70 (s, 3H), 2.64 (d, J = 14.9 Hz, 1H), 2.17−1.97 (m, 5H), 1.78−1.55 (m, 7H), 1.54−1.44 (m, 5H), 1.41−1.23 (m, 5H), 1.11 (s, 3H), 1.04 (s, 3H), 1.00 (s, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.93 (s, 6H), 0.92 (s, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.75 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 173.5, 146.0, 142.3, 138.1, 129.0, 128.8, 128.4, 126.5, 114.0, 54.5, 53.9, 52.0, 50.7, 47.8, 46.3, 42.4, 42.3, 39.7, 39.5, 39.0, 37.9, 37.4, 37.2, 34.6, 32.4, 30.8, 29.3, 27.8, 24.8, 23.3, 23.1, 22.6, 22.4, 22.2, 21.1, 19.1, 17.1, 16.5, 15.3. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C44H63N3NaO3, 704.4762; found, 704.4736. Compound 16c. Yield 71%; white solid; mp 250−252 °C. 1H NMR (300 MHz, CDCl3) δ: 7.46−7.40 (m, 3H), 7.38 (d, J = 5.5 Hz, 2H), 7.33 (s, 1H), 7.29 (s, 1H), 7.10 (d, J = 6.2 Hz, 2H), 6.43 (d, J = 6.3 Hz, 1H), 5.34 (s, 1H), 4.83−4.68 (m, 1H), 3.68 (s, 3H), 3.14 (dd, J = 13.6, 6.0 Hz, 1H), 3.05 (dd, J = 13.7, 5.7 Hz, 1H), 2.62 (d, J = 14.9 Hz, 1H), 2.17−1.96 (m, 4H), 1.91−1.79 (m, 2H), 1.77−1.60 (m, 3H), 1.52−1.44 (m, 4H), 1.31−1.20 (m, 6H), 1.08 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.4, 172.0, 146.1, 142.2, 138.1, 138.0, 136.1, 129.3,129.0, 128.9, 128.4, 128.3, 126.9, 126.5, 114.0, 54.5, 53.7, 53.4, 52.1, 47.8, 46.2, 42.3, 39.7, 39.4, 39.0, 38.1, 37.9, 37.2, 37.1, 34.6, 32.3, 30.8, 29.6, 29.3, 27.7, 24.7, 23.2, 22.2, 21.1, 19.0, 17.0, 16.2, 15.3. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C47H61N3NaO3, 738.4605; found, 738.4648. Compound 17a. Yield 80%; white solid; mp 207−209 °C. 1H NMR (300 MHz, CDCl3) δ: 7.39−7.30 (m, 3H), 7.16−7.05 (m, 2H), 6.55 (t, J = 4.1 Hz, 1H), 5.49 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.07 (dd, J = 18.7, 5.2 Hz, 1H), 3.84 (dd, J = 18.7, 3.5 Hz, 1H), 4705

dx.doi.org/10.1021/jm5002293 | J. Med. Chem. 2014, 57, 4692−4709

Journal of Medicinal Chemistry

Article

δ: 177.3, 172.5, 172.1, 157.3, 156.7, 138.4, 136.1, 129.3, 128.4, 127.3, 126.9, 126.0, 53.5, 52.8, 52.0, 47.7, 45.6, 42.3, 39.5, 39.1, 39.0, 38.1, 37.6, 35.7, 32.0, 30.9, 30.8, 29.6, 29.4, 27.6, 24.7, 23.6, 23.2, 22.6, 21.0, 19.8, 17.0, 16.3, 15.1, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C42H57N3NaO3, 674.4292; found, 674.4312. Binding Mode Evaluation and Binding Energy Calculation. The crystal structure of human Tph-1 was obtained from RCSB Protein Data Bank (PDB code: 1mlw). Autodock 4.2 and PyRx 0.5 programs were employed to the virtual screening, and the docked models were analyzed using PyMOL. We used Perl scripts to identify the polar and apolar interactions of the amino acid residue side chains with a distance of 4.0 Å between atoms. Serotonin Biological Assays in Vitro. RBL2H3 cells (Tph-1expressing cells, purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science) were seeded at 5 × 104 cells per well using 24-well plates and cultured for 4 h in α-MEM media supplemented with 10% fetal bovine serum (FBS). Thereafter, the cells were treated with the each tested compounds at the final concentration of 10 μM or vehicle. Forty-eight h later, the media was removed then to each well was added 100 μL of RIPA lysis buffer and 1.9 mL of mobile phase that is composed of 0.1 mM NaH2PO4/ acetonitrile (98:2, v/v). After centrifugation at 1600g for 15 min, the supernatant was subjected to HPLC (Shimadzu Prominence LC-20A) equipped with a fluorometric detector, the excitation and emission wavelengths were set at 290 and 330 nm, respectively. HPLC analysis was carried out on a reversed-phase Waters 10 mm × 4.6 mm, 5 μm C18 column maintained at ambient temperature with a flow rate of 0.4 mL/min, and 5 μL of samples were injected in all experiments. Furthermore, data analysis was performed using the ratio of the integrated peak areas of the test compounds treated groups to the areas of the corresponding control group. PC12 (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science) cells were cultured in D-MEM media supplemented with 10% fetal bovine serum (FBS), using the same assay as described above. It was further tested at six different concentrations (50.0, 40.0, 30.0, 20.0, 10.0, and 5.0 μM) in duplicate to generate inhibition curves. IC50 values were determined by nonlinear regression analysis using program Prism software (GraphPad Software Inc., San Diego, CA). Cytotoxicity Assay of UA Derivatives on RBL2H3 and PC12 Cells. The single-dose cytotoxicity study of the UA derivatives upon RBL2H3 and PC12 cells was measured by standard MTT assay. The cells with media containing 0.1% DMSO were used as control. Then 20 μL of MTT (5 mg/mL) reagent was added 4 h before the end of culture. Then 90 μL of lysis buffer (10% SDS, 50% DMF, pH 7.2) was added to each well for 6 h, and the absorbance value at 570 nm was collected by microplate reader. The percentage of cell death was determined using the following formula: cytotoxicity (%) = (1 − [compounds (OD570) − background (OD570)]/[control (OD570) − background (OD570)]) × 100. Tph-1 and Tph-2 Levels. For measurements of Tph-1 and Tph-2 productions, RBL2H3 cells (5 × 104 cells per well) or PC12 cells (5 × 104 cells per well) were seeded using 24-well plates and cultured for 4 h in α-MEM or D-MEM media containing 10% fetal bovine serum (FBS), respectively. Compound diluents or vehicle were added into the well at the concentrations of 0, 2.0, 5.0, and 10.0 μM. At the end of incubation, the supernatant of cell lysate was collected and assayed immediately, with the microplate reader measuring absorbance at 450 nm using corresponding ELISA kits. In all of the cell cultures, 9a was dissolved in DMSO followed by dilution with appropriate medium to desired concentrations, and DMSO final concentration was 0.1%. DMSO at 0.1% was added to normal control groups and showed no effects on cells. All the cultures were kept in a CO2 incubator under moist condition of 5% CO2 in air at 37 °C. Quantitative Real-Time Polymerase Chain Reaction (qRTPCR). Total RNA was extracted using TRI-zol reagent from RBL-2H3 or PC12 cells treated with vehicle or indicated concentration of compound 9a. The mRNA expressions of Tph-1 and Tph-2 were quantified on Mx 3005P (Stratagene) using SYBR green dye and normalized with

Compound 19a. Yield 78%; white solid; mp 211−213 °C. 1H NMR (300 MHz, CDCl3) δ: 7.75 (s, 1H), 6.45 (t, J = 4.3 Hz, 1H), 5.46 (t, J = 3.3 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.03 (dd, J = 18.7, 5.0 Hz, 1H), 3.87 (dd, J = 18.7, 3.8 Hz, 1H), 2.20−1.97 (m, 4H), 1.89−1.70 (m, 4H), 1.43−1.38 (m, 3H), 1.30−1.24 (m, 3H), 1.28 (t, J = 7.2 Hz, 3H), 1.26 (d, J = 6.3 Hz, 3H), 1.20 (s, 3H), 1.19 (s, 3H), 1.12 (s, 3H), 1.11 (s, 3H), 0.97−0.94 (m, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 198.0, 177.6, 169.8, 139.7, 124.6, 114.8, 113.8, 61.4, 53.6, 52.4, 47.6, 44.7, 42.6, 41.6, 41.0, 40.4, 40.3, 39.5, 38.8, 36.9, 32.1, 31.8, 30.7, 29.6, 29.2, 27.7, 27.6, 24.6, 23.2, 22.6, 21.5, 21.0, 18.6, 17.9, 17.1, 17.0, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C35H50N2NaO4, 585.3663; found, 585.3665. Compound 19b. Yield 73%; white solid; mp 209−211 °C. 1H NMR (300 MHz, CDCl3) δ: 7.75 (s, 1H), 6.27 (d, J = 7.2 Hz, 1H), 5.44 (s, 1H), 4.61−4.51 (m, 1H), 3.69 (s, 3H), 2.16−2.01 (m, 3H), 1.88−1.64 (m, 5H), 1.64−1.46 (m, 9H), 1.20 (s, 3H), 1.19 (s, 3H), 1.12 (s, 3H), 1.10 (s, 3H), 0.94 (d, J = 6.3 Hz, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.79 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 198.0, 177.1, 173.6, 169.9, 139.1, 124.7, 114.8, 113.8, 53.7, 52.5, 52.0, 50.6, 47.8, 44.8, 42.7, 42.1, 41.0, 40.5, 40.4, 39.5, 38.9, 37.3, 32.3, 30.7, 27.7, 27.6, 24.8, 24.5, 23.1, 22.6, 22.3, 21.5, 21.0, 18.6, 17.9, 17.3,17.1. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C38H56N2NaO4, 627.4132; found, 627.4143. Compound 19c. Yield 69%; white solid; mp 227−229 °C. 1H NMR (300 MHz, CDCl3) δ: 7.72 (s, 1H), 7.30 (d, J = 6.2 Hz, 1H), 7.10 (d, J = 6.4 Hz, 2H), 6.30 (d, J = 6.4 Hz, 1H), 5.30 (s, 1H), 4.79−4.66 (m, 1H), 3.68 (s, 3H), 3.14 (dd, J = 13.7, 5.9 Hz, 1H), 3.03 (dd, J = 13.6, 6.0 Hz, 1H), 2.14−1.86 (m, 4H), 1.83−1.75 (m, 2H), 1.57−1.43 (m, 6H), 1.41−1.29 (m, 3H), 1.18 (d, J = 5.5 Hz, 3H), 1.17 (s, 3H), 1.12 (s, 3H), 1.07 (s, 3H), 0.94 (s, 3H), 0.85 (d, J = 6.3 Hz, 3H), 0.69 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 198.0, 177.1, 172.1, 169.9, 139.0, 136.1, 129.2, 128.4, 127.0, 124.7, 114.8, 113.8, 53.5, 53.3, 52.4, 52.1, 47.7, 44.7, 42.6, 41.0, 40.4, 40.3, 39.4, 38.9, 38.0, 37.1, 32.2, 30.7, 27.7, 27.5, 24.5, 23.1, 23.0, 21.5, 21.0, 18.6, 17.9, 17.1, 17.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C41H54N2NaO4, 661.3976; found, 661.3983. Compound 22a. Yield 59%; white solid; mp 205−207 °C. 1H NMR (300 MHz, CDCl3) δ: 9.01 (s, 1H), 8.29 (s, 1H), 6.53 (s, 1H), 5.48 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 4.06 (dd, J = 18.0, 4.4 Hz, 1H), 3.85 (dd, J = 18.7, 1.6 Hz, 1H), 2.71 (d, J = 15.7 Hz, 1H), 2.35 (d, J = 15.7 Hz, 1H), 2.15−2.02 (m, 3H), 1.78−1.66 (m, 3H), 1.47−1.38 (m, 4H), 1.32−1.28 (m, 3H), 1.27−1.24 (m, 3H), 1.26 (t, J = 7.4 Hz, 3H), 1.25 (d, J = 4.9 Hz, 3H), 1.24 (s, 3H), 1.14 (s, 3H), 0.96 (s, 3H), 0.90 (d, J = 6.7 Hz, 3H), 0.84 (s, 3H), 0.80 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 177.9, 172.4, 170.1 157.3, 156.7, 139.1, 127.3, 125.9, 61.4, 53.8, 52.8, 47.7, 45.6, 42.5, 42.2, 41.6, 39.7, 39.4, 39.1, 38.9, 36.9, 35.8, 32.0, 31.8, 29.6, 29.2, 27.7, 24.8, 23.6, 22.6, 21.1, 19.8, 17.1, 16.2, 15.1, 14.0. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C36H53N3NaO3, 598.3979; found, 598.3994. Compound 22b. Yield 59%; white solid; mp 205−207 °C. 1H NMR (300 MHz, CDCl3) δ: 9.02 (s, 1H), 8.31 (s, 1H), 6.35 (d, J = 7.2 Hz, 1H), 5.46 (t, J = 3.4 Hz, 1H), 4.61−4.55 (m, 1H), 3.70 (s, 3H), 2.73 (d, J = 15.8 Hz, 1H), 2.36 (d, J = 15.8 Hz, 1H), 2.14−2.02 (m, 4H), 1.85−1.69 (m, 4H), 1.66−1.55 (m, 5H), 1.55−1.44 (m, 5H), 1.32 (s, 3H), 1.28 (s, 3H), 1.14 (s, 3H), 0.96 (d, J = 5.2 Hz, 3H), 0.95 (s, 3H), 0.93 (s, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.86 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 177.4, 173.6, 156.7, 138.5, 126.1, 53.9, 53.0, 52.1, 50.8, 47.9, 45.7, 42.6, 42.3, 39.8, 39.5, 39.3, 39.0, 37.5, 35.9, 32.3, 31.1, 30.9, 27.8, 24.9, 24.8, 23.7, 23.5, 23.2, 22.7, 22.4, 21.2, 20.0, 17.2, 16.6, 15.2. HR-MS (m/z) [M + Na]+ (ESI+) calcd for C39H59N3NaO3, 640.4449; found, 640.4465. Compound 22c. Yield 53%; white solid; mp 219−221 °C. 1H NMR (300 MHz, CDCl3) δ: 9.02 (s, 1H), 8.30 (s, 1H), 7.29 (s, 1H), 7.11 (d, J = 6.2 Hz, 2H), 6.39 (d, J = 6.2 Hz, 1H), 5.33 (s, 1H), 4.80−4.69 (m, 1H), 3.67 (s, 3H), 3.14 (dd, J = 13.6, 6.1 Hz, 1H), 3.05 (dd, J = 13.6, 5.6 Hz, 1H), 2.70 (d, J = 15.9 Hz, 1H), 2.33 (d, J = 15.8 Hz, 1H), 2.06−2.01 (m, 2H), 1.94−1.78 (m, 2H), 1.74−1.60 (m, 5H), 1.44− 1.36 (m, 4H), 1.31 (s, 3H), 1.27−1.25 (m, 5H), 1.26 (d, J = 5.5 Hz, 3H), 1.10 (s, 3H), 0.95 (s, 3H), 0.90−0.84 (m, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.82 (s, 3H), 0.70 (s, 3H). 13C NMR (75 MHz, CDCl3) 4706

dx.doi.org/10.1021/jm5002293 | J. Med. Chem. 2014, 57, 4692−4709

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Article

β-actin. Primer sequences of Tph-1 were as follows: forward primer 5′-CCTCAGAGGAGACGGTTCAGAAA-3′ and reverse primer 5′-TCTCAGCT GCCCATCTTGCT-3′. Tph-2 primers were 5′-TAAATACTGGGCCA GGAGAGG-3′ and 5′-AGTGTCTTTGCCGCTTCTCTT-3′. Binding Affinity of Compound 9a with Tph-1. All experiments were carried out on a Biacore T200 instrument (GE Healthcare, Uppsala, Sweden) at 15 °C. Tph-1 was covalently immobilized on parallel channels of a CM5 sensor chip according to the manufacturer′s protocol. The sensor chip surface was activated by a 5 min injection of 0.4 M N-ethyl-N′-(3-(dimethylamino)propyl) carbodiimide and 0.1 M N-hydroxysuccinimide. The compound 9a was serially (2-fold) diluted in the running buffer AMF (containing 10 mM Hepes (pH 7.4), 3 mM EDTA, 150 mM NaCl, 0.005% Surfactant P20, 10 μM GDP, 4 mM MgCl2, 1 mM dithiothreitol, 10 mM NaF, 10 mM MgCl2) supplemented with 0.1% DMSO. The solution of 9a was injected for 40 s at the flow rate of 5.0 μL/min, and the dissociation was followed for 90 s at 15 °C. Furthermore, buffer blanks were used as the positive control and double referencing. All data were corrected for nonspecific binding and Global fitting using the BIA-evaluation program. The experiments were performed more than three times. In Vivo Experiments and Bone Microcomputed Tomography Analysis. The 12-week-old female rats (220 ± 20 g each) were obtained from the Jiangning Animal Farm (Nanjing, China). Animals were housed in a climate-controlled room, 12 h light/dark photoperiod. All the animals had free access to food and water. To adapt to the new environment, the rats were held for 1 week before experiments. The rats were randomly divided into six groups (n = 8) as follows: a sham-operation + vehicle (5% ethanol and 95% distilled water, Sham group), OVX + vehicle (OVX group), OVX + orally administrated 9a at doses of 1.0 (OVX + 1 group), 10.0 (OVX + 10 group), and 20.0 (OVX + 20 group) (mg/kg per day), respectively, and OVX + PTH with intraperitoneal injection of PTH at 20 μg/kg per day (PTH group). Rats were anaesthetized with pentobital sodium (20 g/L in saline solution 2.5 mL/kg body weight) intraperitoneally, and bilateral ovariectomies (OVX operation) were conducted on the OVX, 9a, and PTH groups. A sham operation, during which the ovaries were touched with forceps, was performed on the sham group. Three days after surgery, rats were treated with vehicle, compound 9a, or PTH once daily for 30 days. At the end of the experiment, whole blood samples were collected and centrifuged at 3000 rpm for 10 min and the serum was kept at −20 °C until analyses. Then, the rats were killed by CO2 asphyxiation, and brain, lumbar vertebrae, livers, and uteri were collected for further measurements. Analysis of Serotonin Levels in Serum, Brain, and Gut. To 100 μL of serum was added 100 μL off 5% HClO4 and centrifuged at 1600g for 15 min. The supernatant was diluted with mobile phase composed of 0.1 mM NaH2PO4/acetonitrile (98:2, v/v) and subjected to HPLC system equipped with a fluorometric detector. The brain tissue (about 2.0 g) was weighed, and 1 mL of 5% HClO4 and 9 mL of mobile phase composed of 0.1 mM NaH2PO4/acetonitrile (97:3, v/v) were added. After being centrifuged at 1600g for 10 min, the supernatant was subjected to HPLC system. HPLC analysis was carried out on a reversed-phase Waters 10 mm × 4.6 mm, 5 μm C18 column maintained at ambient temperature with a flow rate of 0.4 mL/min, and 5 μL samples were injected in all experiments. The excitation and emission wavelengths set at 290 and 330 nm, respectively. The serotonin contents in serum and brain were analyzed following the reported method.59 The 12-week-old female rats (220 ± 20 g each) were used for gut serotonin content analysis. The rats were randomly divided into four groups (n = 5) as follows: a sham-operation + vehicle (5% ethanol and 95% distilled water, Sham group), OVX + vehicle (OVX group), OVX + orally administrated 9a at doses of 10.0 (OVX + 10 group), and 20.0 (OVX + 20 group) (mg/kg)/day, respectively. Three days after surgery, rats were treated with vehicle and compound 9a once daily for 7 days. At the end of the experiment, small intestines were collected kept at −20 °C until analysis.23 The intestine tissue (about 3.18 g) was weighed and 1.0 mL of 5% HClO4 and 9 mL of mobile phase composed of 0.1 mM NaH2PO4/acetonitrile (97:3, v/v) were

added. After being centrifuged at 1600g for 10 min, the supernatant was subjected to HPLC system. Analysis of Compound 9a Content in Brain. The brain homogenate (about 2.0 g) was weighed, and 2.0 mL of 5% HClO4 and 2.0 mL of ethyl acetate were added. After being centrifuged at 1600g for 10 min, the organic layer was isolated and dried by a Termovap sample concentrator. The obtained residue was dissolved in 10.0 mL of methanol filtered with membrane (0.22 μm). The filtered samples were directly subjected to LC−MS measurement (LTQ Orbitrap XL, Thermo Scientific). μCT Analysis of Bone. Rat lumbar vertebrae (L2−L4) were collected for the measurement of bone microarchitectural parameters. μCT experiments were performed on the eXplore Locus SP scanner (GE HealthCare), and bone histomorphometric analysis was carried out on eXplore Micro-View, version 2.0, according to standard criteria. P1NP and CTX-1 Analysis. The circulating levels of P1NP or CTX-1 were assayed by ELISA (Cusabio) kitss following the manufacturer’s instructions. Data were analyzed by one-way ANOVA followed by Dunnett’s test or Student’s t-test when appropriate. Probability values of 0.05 or less were considered to be statistically significant. All statistic analyses were performed with SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The animal study was approved by the Jiangsu Animal Care and Use Committee, and all of the protocols complied with the national and institutional rules regarding animal experiments.

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ASSOCIATED CONTENT

S Supporting Information *

Analytical HPLC data on all test compounds, chemical and biological experimental details, and 1H and 13C NMR spectra of new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*For J.-X. L.: phone, +86-25-83686419; E-mail, [email protected]. *For X.-M. Z.: phone, +86-25-86617141; E-mail, [email protected]. 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 This work was supported by National Natural Science Foundation of China (21272114, 91313303), and the National Natural Science Fund for Creative Research Groups (21121091).

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ABBREVIATIONS USED UA, ursolic acid; BMD, bone mineral density; SERMs, selective estrogen receptor modulators; rhPTH, parathyroid hormone; EC, enterochromaffin cells; 5-HT, 5-hydroxytryptamine; Tph1, tryptophan hydroxylase 1; GDS, gut-derived serotonin; HBI, 7,8-dihydro-L-biopterin; SPR, surface plasmon resonance; OVX, ovariectomized; μCT, microcomputed tomography; BV, bone volume; TV, total volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; P1NP, N-terminal propeptide of procollagen type 1; CTX-1, carboxy terminal telopeptide of collagen type I; LE, luminal epithelium; GE, endometrial glands 4707

dx.doi.org/10.1021/jm5002293 | J. Med. Chem. 2014, 57, 4692−4709

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