Tryptophan hydroxylase 1 (Tph-1)-targeted bone anabolic agents for osteoporosis

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 K_D: 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.

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,⁷calcitonin, estrogen, or selective estrogen

receptor modulators (SERMs).⁸However, 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.⁹Anabolic 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.¹¹Although antiresorptive

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.¹Osteoporosis 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 2020.²For 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.³In 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.

Received: February 12, 2014

Published: May 20, 2014

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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.

Fortunately, we discovered that UA displayed quite lower

binding energy (ΔE, −10.22 kcal/mol) and inhibition constant

value (K_i, 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,^35,36so 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).³⁶As the ring A of UA occupies the

binding pocket above HBI at the entrance to the catalytic site

(Supporting Information Figure 1b), introduction of hetero-

cyclic 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 hydro-

gen 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 (K_i, 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 (K_i)

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.

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.¹³In 2003,

Walther reported the Tph exists two vertebrate isoforms:

Tph-1 and Tph-2.^14,15Tph-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.^16,17Recent studies revealed that

peripheral serotonin biosynthesized in EC cells of the gut (gut-

derived serotonin, GDS) is a powerful inhibitor of osteoblast

proliferation.¹⁸⁻²¹GDS enters the circulation but does not cross

the blood−brain barrier.^18,19Further evidence demonstrated

that a small molecule LP533401, a Tph-1 inhibitor, successfully

increased bone mass in ovariectomized mice.²¹⁻²⁴Therefore,

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-1-

targeted bone anabolic agents. First, a virtual screening of the

potential Tph-1 inhibitor from natural products was con-

ducted using the Tph-1 enzymatic domain with its cofactor

7,8-dihydro-^L-biopterin (HBI) as a reference.²¹Fortunately,

ursolic acid (UA) showed quite good data with free binding

energy (−10.22 kcal/mol) and inhibition constant (K_i, 32.12 nM).

It was also reported that UA stimulated osteoblast diﬀerentiation

in vitro.²⁵These 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,²⁷antioxidative,²⁸antitumor,²⁹antimicrobial,

antiulcer, and antiwrinkle activities.³⁰A variety of structure

modiﬁcations of UA have also been conducted.³¹⁻³³In 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.

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.³⁷Thus, reagent H₂O₂/Na₂WO₄with

the phosphate buﬀer solution was used,³⁸and 2 was obtained in

an excellent yield (86%) and good selectivity without much environ-

mental pollution.³⁹Acetylation of the hydroxyl group of 1 with

acetic anhydride in pyridine aﬀorded 5.⁴⁰The indole derivatives

6 and 7 were synthesized by Fischer indolization of inter-

mediate 2 with the phenylhydrazine hydrochloride in acetic

acid. However, when treating 2 with 1,2-diamines and sulfur in

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.³⁴Because 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.

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reﬂuxing morpholine,⁴¹the 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.

Table 1. Docking Results of Crystal Structure of Human

Tph-1 to Typical UA Derivatives

The synthesis of pyrazole, isoxazole, and pyrimidine deriva-

tives 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 atmos-

pheric condition in high yield.⁴³Treatment of 4 with hydrazine

dihydrochloride, phenyl-hydrazine hydrochloride, 4-ﬂuorophenyl-

hydrazine 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 hetero-

cyclic 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.

a

K_iwas obtained by the following equation: K_i= exp((ΔG × 1000)/

(R_cal× TK)), where ΔG is docking energy, R_calis 1.98719, and TK is

298.15. Data is cited from ref 21.

b

a

Scheme 1. Synthesis of Indole and Quinoxaline Derivatives

a

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%).

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a

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

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.

Scheme 3. Synthesis of Amino Acids and Heterocyclic

Moieties Derivatives

a

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

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 com-

pounds was initially evaluated using RBL2H3 cells, a Tph-1-

expressing serotonin-producing mast cell line.^17,45,46A HPLC

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Table 2. Inhibitory Eﬀects on Serotonin Biosythesis and Cytotoxicity of the Synthesized Compounds on RBL2H3 Cells

a

compd

inhibition (%)

cytotoxicity

0.0 1.2

compd

12

inhibition (%)

cytotoxicity

c

Con.

1

0.0 0.3

6.8 1.4

35.9 0.8**

62.1 0.1**

53.8 0.7**

6.2 2.0

59.5 2.7**

52.1 1.9**

31.3 4.4**

0.1 1.3

b

c

10.0 1.0*

1.7 1.0

2.6 0.7

1.1 0.1

4.1 2.8

1.6 0.3

6.6 0.2

12.4 1.5*

2.9 1.4

14.7 2.1*

15.0 0.7*

1.6 0.4

3.7 2.0

1.7 0.2

3.0 1.7

16.0 0.8*

2.2 2.8

2.0 0.8

0.7 1.1

7.7 1.0

3.5 1.0

1.0 0.5

0.5 1.4

12.4 2.9*

6.2 0.9

13

c

2a

2b

2c

5

50.7 4.9**

61.4 0.7**

53.6 4.6**

1.0 6.2

14

c

15

c

15a

15b

15c

16

38.9 1.3**

34.4 1.1**

38.3 0.2**

4.3 1.7

2.9 1.3

1.0 0.1

0.4 1.5

c

0.9 1.3

c

5a

5b

5c

6

43.8 0.7**

44.3 0.1**

8.7 0.4

1.5 0.7

8.5 0.5

5.4 0.3

1.9 1.5

c

2.4 2.0

b

16a

16b

16c

17

1.2 0.5

1.8 2.0

b

c

6a

6b

6c

7

19.9 0.3**

7.3 2.0

0.9 1.3

1.5 1.1

c

b

70.9 0.3**

17a

17b

17c

18

57.3 2.0**

33.7 1.6**

40.4 0.8**

1.0 0.1

15.8 1.7*

c

7.9 9.1

8.2 0.2

9.2 0.2

9.0 0.1

1.0 0.8

2.0 0.3

2.1 1.3

c

7a

7b

7c

8

b

c

b

18a

18b

18c

19

17.5 0.1**

17.6 0.4**

7.8 0.1

11.1 0.8*

c

22.3 2.6**

22.9 0.3**

13.4 0.1*

10.3 0.1*

18.4 6.5**

87.0 2.3**

6.9 0.2

6.8 0.1

17.7 2.6**

0.8 3.6

c

8a

8b

8c

9

b

11.7 0.9*

61.9 0.3**

92.4 0.2**

1.4 1.0

5.9 0.2

6.9 0.3

3.1 0.2

6.8 0.4

4.3 1.1

b

c

19a

19b

19c

22

50.1 1.6**

58.1 1.2**

5.4 2.0

c

9a

9b

9c

10

11

IC₅₀

9a

4.3 1.1

b

22a

22b

22c

1.9 3.0

b

15.6 1.2*

58.7 2.6**

10.3 1.4*

c

51.1 3.3**

2.6 0.4

d

6.22

LP533401

0.4

a

Inhibition (%) = 100% − peak areas of the tested compound against the areas of the corresponding control group (%). All values are expressed as

b

c

d

means S.D., n = 3. *p < 0.05. **p < 0.01 vs control. Data is cited from ref 25.

not observed in other similar ﬁve-membered heterocyclic ring-

fused 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 IC₅₀of 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 dose-

dependent manner.

As can be seen in Figure 1b, 9a at 10 μM signiﬁcantly down-

regulated 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 endo-

genously.⁴⁷The results revealed that 9a inhibited serotonin

synthesis catalyzed by Tph-2 in PC12 cells with an IC₅₀value

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.⁴⁸SPR bio-

sensors can also exhibit some information-rich data on mole-

cular 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

Next, we investigated whether 9a regulates Tph-1 mRNA ex-

pression with a quantitative real-time PCR (qRT-PCR) analysis.

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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.⁴⁹Tph-1 was immobi-

lized on Biacore sensor chip CM5 by standard amine coupling

reaction, and the data were analyzed by BIAevaluation

program.⁵⁰The results revealed that 9a eﬃciently interacted

with the immobilized Tph-1 and demonstrated a concentra-

tion dependent response (Figure 2) and a clearly discernible

Tph-1.⁵¹This 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).³⁵The 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 quinoxa-

line 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,^52,53which

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 K_D(6.82 μM), most probably

due to the capability of speciﬁcally recognizing the immobilized

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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.

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.⁵⁵As shown in Figure 4a, OVX operation

markedly increased the serum serotonin level, which was in

agreement with other reported results.⁵⁶As expected, admini-

stration 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.

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.²¹Increasing reports showed that inhibiting the bio-

synthesis of gut serotonin could prevent and cure ovariectom-

ized (OVX) induced osteoporosis via an anabolic mode of

action in mice and rats.^21,54Therefore, 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

Recent genetic studies have demonstrated that many

medical disorders are associated with serotonin dysregulation,⁵⁷

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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.⁵⁸To understand the inﬂuences

of 9a on the brain serotonin, the serotonin levels in brain homo-

genate (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.

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 con-

centrations 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, postmeno-

pausal 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.⁶⁰As 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

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

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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.

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 statisti-

cally 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 reduc-

tion in trabecular separation and numbers,⁶³and 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.⁶⁴Thus, 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.

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.⁶⁶The

most likely reason is that they used diﬀerent mice models, and

furthermore serotonin possesses quite complex physiological

actions in bone.^67,68

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

As serum carboxy terminal telopeptide of collagen type I

(CTX-1) level is an indicator of osteoclast numbers and

bone resorption,⁶⁵we 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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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 Na₂SO₄and 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

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.

1

solid; mp 195−197 °C. H 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 Na₂SO₄and 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.

¹H 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.

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

1

uncorrected. H (300 MHz) NMR and ¹³C (75 MHz) NMR spectra

were recorded on a Bruker DPX 300 MHz spectrometer at room

temperature using CDCl₃as 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 abbrevia-

tions 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 H₂O₂solution 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 Na₂SO₄and 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

1

solid; mp 197−199 °C. H 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).

¹³C 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

1

solid; mp 188−190 °C. H NMR (300 MHz, CDCl₃) δ: 6.30 (s, 1H)

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-dichlorobenzene-

1,2-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).

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

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¹³C 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.

extracted with AcOEt (100 mL × 3). The organic layer was dried with

anhydrous Na₂SO₄and 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 tem-

perature, 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.

¹H 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 temp-

erature. 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 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).

¹³C 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 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 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.

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.

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

4702

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.

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. HR-

MS (m/z) [M + Na]⁺(ESI⁺) calcd for C₄₀H₅₅BrN₂NaO₃, 713.3288;

found, 713.3307.

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 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 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).

¹³C 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. HR-

MS (m/z) [M + Na]⁺(ESI⁺) calcd for C₄₀H₅₅ClN₂NaO₃, 669.3793;

found, 669.3817.

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,

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),

4703

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

Journal of Medicinal Chemistry

Article

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 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 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

C₄₆H₅₇C_l2N₃NaO₃, 792.3669; found, 792.3688.

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 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 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).

¹³C 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 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 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 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.

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,

4704

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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.

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 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 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 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 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 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 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).

¹³C 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.

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

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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.

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-1-

expressing cells, purchased from Shanghai Institutes for Biological

Sciences, Chinese Academy of Science) were seeded at 5 × 10⁴cells

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 con-

centration 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 con-

centrations (50.0, 40.0, 30.0, 20.0, 10.0, and 5.0 μM) in duplicate to

generate inhibition curves. IC₅₀values 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 × 10⁴cells per well) or PC12 cells

(5 × 10⁴cells 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 CO₂incubator under moist condition of 5% CO₂in air

at 37 °C.

Quantitative Real-Time Polymerase Chain Reaction (qRT-

PCR). Total RNA was extracted using TRI-zol reagent from RBL-2H3

or PC12 cells treated with vehicle or indicated concentration of com-

pound 9a. The mRNA expressions of Tph-1 and Tph-2 were quantiﬁed

on Mx 3005P (Stratagene) using SYBR green dye and normalized with

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

Journal of Medicinal Chemistry

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′-AGTGT-

CTTTGCCGCTTCTCTT-3′.

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% HClO₄and

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.

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) supple-

mented 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 Tomog-

raphy 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 CO₂asphyxiation, 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% HClO₄and 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% HClO₄and 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.²³The intestine tissue (about

3.18 g) was weighed and 1.0 mL of 5% HClO₄and 9 mL of mobile

phase composed of 0.1 mM NaH₂PO₄/acetonitrile (97:3, v/v) were

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.

AUTHOR INFORMATION

■

Corresponding Authors

*For J.-X. L.: phone, +86-25-83686419; E-mail, lijxnju@nju.edu.cn.

*For X.-M. Z.: phone, +86-25-86617141; E-mail,

xianmeizhou@aliyun.com.

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.

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).

ABBREVIATIONS USED

■

UA, ursolic acid; BMD, bone mineral density; SERMs, selective

estrogen receptor modulators; rhPTH, parathyroid hormone;

EC, enterochromaﬃn cells; 5-HT, 5-hydroxytryptamine; Tph-

1, 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

Journal of Medicinal Chemistry

Article

(22) Yadav, V. K.; Ryu, J.-H.; Suda, N.; Tanaka, K. F.; Gingrich, J. A.;

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Tryptophan hydroxylase 1 (Tph-1)-targeted bone anabolic agents for osteoporosis

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