Development of second-generation small-molecule RhoA inhibitors with enhanced water solubility, tissue potency, and significant in vivo efficacy

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DOI: 10.1002/cmdc.201402386

Development of Second-Generation Small-Molecule RhoA

Inhibitors with Enhanced Water Solubility, Tissue Potency,

and Significant in vivo Efficacy

[

a]

[b]

[a]

[b]

[c]

Sheng Ma, Jing Deng, Baoli Li, Xiujiang Li, Zhaowei Yan, Jin Zhu, Gang Chen,

[c]

[b, d]

[a]

[b]

Zhong Wang,* Hualiang Jiang,

Liyan Miao,* and Jian Li*

RhoA, a member of the Rho GTPases, is involved in a variety of

cellular functions and could be a suitable therapeutic target

for the treatment of cardiovascular diseases. However, few

small-molecule RhoA inhibitors have been reported. Based on

our previously reported lead compounds, 32 new 2-substituted

quinoline (or quinoxaline) derivatives were synthesized and

tested in biological assays. Six compounds showed high RhoA

inhibitory activities, with IC₅₀values of 1.17–1.84 mm. Among

these, (E)-3-(3-(ethyl(quinolin-2-yl)amino)phenyl)acrylic acid

(26d) demonstrated noticeable vasorelaxation effects against

phenylephrine-induced contraction in thoracic aorta artery

rings, and compound 26b had good water solubility and

showed significant in vivo efficacy, which was similar to that of

5-(1,4-diazepane-1-sulfonyl)isoquinoline (fasudil) in a subarach-

noid hemorrhage–cardiovascular model. To the best of our

knowledge, compound 26b is the first example of a small-

molecule RhoA inhibitor with potent in vivo efficacy, which

could serve as a good lead for designing cardiovascular

agents.

(

26b) and (E)-3-(3-(butyl(quinolin-2-yl)amino)phenyl)acrylic acid

Introduction

RhoA, as one of the most well-characterized members of the

Rho GTPases, plays important roles in multiple cellular process-

es, including cytoskeletal rearrangement, gene expression, and

membrane trafficking, as well as cell adhesion, migration, dif-

The RhoA/ROCK pathway is the main regulator in the patho-

genesis of sustained smooth muscle cell contraction and vaso-

[4,5]

spasm.

Activation of RhoA/ROCK inhibits myosin light chain

phosphatase (MLCP), followed by direct phosphorylation of

[

1]

[5]

ferentiation, proliferation, and apoptosis. Like all members of

the GTPases, RhoA acts as a molecular switch, cycling between

the active GTP-bound form and the inactive GDP-bound

MLC and facilitation of contraction. Thus, inhibition of the

RhoA pathway has been considered an appealing target for

pharmacological treatment of numerous cardiovascular (CVS)

[

2]

[6,7]

form. Active GTP-bound RhoA interacts with downstream ef-

fector proteins, including the most well-characterized Rho-

disorders.

Moreover, RhoA has been reported as a potential

[8]

[9]

target for the treatment of cancer, nervous system diseases,

[3]

[10]

associated kinases (ROCK), to regular cellular functions.

and asthma.

Subarachnoid hemorrhage (SAH), or bleeding into the sub-

arachnoid space, is a devastating cerebrovascular disease that

threatens the health of people throughout the world. Approxi-

mately 85% cases of spontaneous SAH are due to the rupture

of an intracranial aneurysm, although it can also be caused by

+

[

a] S. Ma, Z. Yan, Prof. L. Miao

Department of Clinical Pharmacology Research Laboratory

The First Affiliated Hospital of Soochow University

88 Shi Zhi Street, Suzhou 215006 (China)

1

[11]

trauma. Despite recent improvements in knowledge about

E-mail: miaolysuzhou@163.com

SAH pathophysiology and angioplasty, the molecular mecha-

nism of SAH is still unclear. New evidence has revealed that

early brain injury (EBI), cortical spreading depolarization (CSD),

and impaired microcirculatory function could be important

+

[

b] Dr. J. Deng, B. Li, X. Li, Dr. J. Zhu, Prof. H. Jiang, Prof. J. Li

Shanghai Key Laboratory of New Drug Design, School of Pharmacy

East China University of Science and Technology

30 Mei Long Road, Shanghai 200237 (China)

1

E-mail: jianli@ecust.edu.cn

[12]

players in SAH-induced injury. However, cerebral vasospasm

[

c] G. Chen, Prof. Z. Wang

Department of Neurosurgery

The First Affiliated Hospital of Soochow University

88 Shi Zhi Street, Suzhou 215006 (China)

remains one of the primary poor outcomes after SAH.

As a ROCK inhibitor, fasudil (5-(1,4-diazepane-1-sulfonyl)iso-

quinoline) has been marketed in Japan for some time for the

1

[13]

E-mail: w13306208761@hotmail.com

treatment of cerebral vasospasms occurring after SAH. Fasu-

dil significantly decreased vasospasm not only via an inhibition

of smooth muscle contraction but also through up-regulation

of endothelial nitric oxide synthase (eNOS), which was proven

to be a safe and useful drug for patients with ruptured cere-

[

d] Prof. H. Jiang

Drug Discovery and Design Center

Shanghai Institute of Materia Medica, Chinese Academy of Sciences

55 Zu Chong Zhi Road, Shanghai 201203 (China)

5

+

[

] These authors contributed equally to this work.

[

14]

bral aneurysms.

In addition, some evidence suggests that

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201402386.

[15]

statins could be ROCK inhibitors and that some statins (e.g.,

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Figure 1. Discovery of first-generation small-molecule RhoA inhibitors.

Results and Discussion

pitavastatin and simvastatin) may decrease the risk of sympto-

matic vasospasm caused by SAH, especially in animal models,

but no robust effect on clinical outcomes has been demon-

Design of second-generation inhibitors

[

16]

strated. These evidence support the idea that RhoA/ROCK in-

hibitors could be potential therapeutics for cerebral vaso-

spasms after SAH. However, very limited efforts have been de-

Through structure-based virtual screening and chemical modi-

fication, compounds 3a,b and 4 were previously reported as

first-generation small-molecule RhoA inhibitors from our

group. These compounds were used as leads for designing

second-generation small-molecule RhoA inhibitors. In this

study, to further improve the inhibitory activity and physico-

chemical properties of the lead compounds, chemical modifi-

cations were performed, and two sets of analogues were pre-

pared. Firstly, with the help of molecular modeling in the previ-

ous study, we obtained two-dimensional (Figure 2A) and

[17–24]

voted toward the development of direct RhoA inhibitors.

Toward this end, we previously reported the first-generation

small-molecule RhoA inhibitors through structure-based virtual

screening in conjunction with chemical synthesis and bioas-

[

22]

says. Starting from lead compound 1 (Figure 1), it was found

that compound 2, with a simplified structure (one amino sub-

stituent) retained the RhoA inhibitory activity with an inhibito-

ry rate similar to that of lead

compound 1. After further struc-

tural modification, three com-

pounds (3a,b and 4) were found

to have high RhoA inhibitory ac-

tivities, with IC₅₀values of 1.24–

2.05 mm. Compounds 3a,b also

had noticeable vasorelaxation ef-

fects against phenylephrine (PE)-

induced contraction in thoracic

aorta artery rings and therefore

served as leads for the second-

generation small-molecule RhoA

inhibitors in this study.

[22]

Figure 2. Predicted binding pose of 3b in the GNP binding site of RhoA.

In total, 32 new compounds

were designed, synthesized, and

tested with biological assays. Six compounds (26b, 26d, 26i–j,

three-dimensional (Figure 2B) interaction schemes of docked

poses of 3b in the GNP binding site of RhoA. We found that

the aniline nitrogen atom was completely exposed to solvent.

Please refer to ref. [22] for details regarding molecular docking.

Thus, we designed and synthesized 22 analogues with various

substitutions at the aniline nitrogen atom (compounds 26a–r,

Table 1; compounds 27a–d, Table 2) to estimate if the substitu-

tion on the aniline nitrogen atom would affect physicochemi-

cal properties and inhibitory activity. Secondly, we replaced the

functional group acid on the side chain with the bioisosteres

and designed 10 analogues (28a–b, 29a–b, 30a–b, 31a–b,

and 32a–b; Table 3).

26l, and 26q) were found to have high RhoA inhibitory activi-

ties, and two compounds (26b and 26d) showed significant

inhibitory effects against PE-induced contraction in the thora-

cic aorta artery, approximately twofold more potent than that

of the first-generation small-molecule RhoA inhibitors discov-

ered previously. Among these, compound 26b showed good

water solubility and promising therapeutic effects, similar to

that of fasudil, in in vivo animal models.

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Table 1. Structure and RhoA inhibitory activity of compounds 3a, 3b,

and 26a–r.

Table 3. Structure and RhoA inhibitory activity of compounds 28a–b,

29a–b, 30a–b, 31a–b, and 32a–b.

2

[a]

Compd

X

R

Inhibition [%]

42

1

[a]

Compd

X

R

Inhibition [%]

3

2

a

b

N

H

78

90

52

86

63

92

48

73

79

70

CH

28a

CH

6a

6b

6c

6d

6e

6 f

6g

6h

Me

Et

nPr

nBu

iPr

CH

28b

N

57

71

2

cPr

cPent

cHex

2

9a

9b

CH

2

6i

CH

89

2

6j

CH

Bz

85

51

N

76

6k

2

6l

CH

N

(CH

2

)

2

3

2

OH

OTBDMS

90

77

59

66

65

81

58

6m

6n

6o

6p

6q

6r

30a

CH

N

71

67

77

65

[

b]

CH

Et

2

COOH

CONH

2

3

0b

N

CH

2

COOH

[a] RhoA inhibition was determined by cell-based assay at 2.5 mm; data

represent a single determination. [b] TBDMS=tert-butyldimethylsilyl.

31a

CH

N

3

1b

2a

Table 2. Structure and RhoA inhibitory activity of compounds 4 and

2

7a–d.

CH

N

55

49

1

[a]

Compd

X

R

Inhibition [%]

32b

4

N

H

Et

CH

Et

CH

76

62

69

73

62

27a

27b

27c

27d

CH

N

[

a] RhoA inhibition was determined at 2.5 mm; data represent a single

2

COOH

determination.

N

2

[

a] RhoA inhibition was determined at 2.5 mm; data represent a single

of the esters with LiOH provided carboxylic acid products.

Compounds 27a–d were synthesized using a similar process

as for compounds 26a–r (Scheme 2).

determination.

Synthesis

Compounds 28a–b were synthesized using the approach

outlined in Scheme 3. 3-Nitrobenzaldehyde 16 was treated

Scheme 1 depicts the synthetic route for the preparation of

compounds 26a–r. 2-Quinoxalinol 5a or 2-quinoxalinone 5b

were chlorinated with phenyl phosphonic dichloride (BPOD) to

yield the corresponding aryl chlorides, 6a or 6b. Esterification

with hydroxylamine hydrochloride using Pd(OAc) as a catalyst

2

in DMSO/H O to give amide 17. Stirring at reflux with N,N-di-

2

methylformamide dimethyl acetal (DMF-DMA) gave compound

18. Cyclization of compound 18 with hydrazine hydrate afford-

of 3-nitrocinnamic acid 7, followed by reduction with SnCl in

ed compound 19, followed by reduction with SnCl to give the

2

ethanol, gave aniline 9. Coupling of 6a–b and aniline 9 afford-

ed compounds 10a–b, which were substituted with different

haloalkanes, giving the esters of compounds 26a–r. Hydrolysis

desired aniline, 20. Compound 20 was then coupled with 6a–

b in DMF to give products 28a–b.

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Esterification of 6-amino-2-

naphthoic acid 21, followed by

coupling with 6a--b in DMF,

gave esters 23a–b, which were

hydrolyzed using LiOH at room

temperature to give carboxylic

acid products 29a–b (Scheme 4).

Treatment of 10a–b and 14a–

b

with hydroxylamine and

sodium methoxide in MeOH at

0

8C gave products 30a–b and

1a–b (Scheme 5). The coupling

3

reaction of 6a–b and 24 gave

compounds 25a–b, which were

converted into phosphate prod-

ucts 32a–b using diethyl chloro-

phosphate, triethylamine (TEA),

and

4-dimethylaminopyridine

(

DMAP) in THF at room tempera-

ture (Scheme 6).

Scheme 1. Reagents and conditions: a) BPOD, reflux, 12 h, 81–85%; b) H

2

SO

4

, EtOH, reflux, 12 h, 81%; c) SnCl

O, MeOH, 408C, 16 h,

2

,

In total, 32 compounds (26a–

r, 27a–d, 28a–b, 29a–b, 30a–b,

EtOH, reflux, 12 h, 98%; d) DMF, reflux, 12 h, 51–81%; e) NaH, DMF, RT, 12 h; f) LiOH·H

0–95% (over steps e and f).

2

1

31a–b, and 32a–b) were de-

signed and synthesized; their

chemical structures are shown in

Tables 1–3. These compounds

were synthesized through the

routes outlined in Schemes 1–6,

and the details for synthetic pro-

cedures and structural character-

izations are described in the Ex-

perimental Section. All com-

pounds were confirmed to be

ꢀ

95% pure (Supporting Infor-

mation Table 1S).

Water solubility

Scheme 2. Reagents and conditions: a) H

RT, 12 h; d) LiOH, MeOH, RT, 12 h, 4–71% (over steps c and d).

2

SO

4

, EtOH, reflux, 12 h, 85%; b) DMF, reflux, 12 h, 58–62%; c) NaH, DMF,

Attention was given to the low

water solubility of compound

3

a. The crystal packing caused

by the molecular planarity of the

quinoxaline and aniline in com-

pound 3a was deemed likely to

cause the low solubility of

ꢁ

1 [27]

4

3 mgmL .

Therefore, we

speculated that the solubility

would probably be improved by

introducing different substitu-

ents at the aniline nitrogen. The

hypothesis was supported by

melting point changes indicative

of solubility changes. Introduc-

ing an ethyl substituent at the

aniline nitrogen gave a dramatic

decrease in the melting point

from >3008C (compound 3b) to

Scheme 3. Reagents and conditions: a) NH

2

OH·HCl, Cs

2

CO

3

, Pd(OAc)

2 2

, DMSO, H O, reflux, 12 h, 57%; b) DMF-DMA,

reflux, 5 h, 79%; c) AcOH, N ·H O, reflux, 12 h, 36%; d) SnCl

2

H

4

2

, EtOH, reflux, 12 h, 96%; e) DMF, reflux, 12 h,

49–59%.

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Biological activities

Inhibition of RhoA

For the primary assay, the inhibi-

tory activities of synthetic com-

pounds against RhoA were eval-

uated by using the inhibitory

rate at a single concentration.

Using

small-molecule RhoA inhibitors

3a–b and 4) developed in our

three

first-generation

(

2 4 2

Scheme 4. Reagents and conditions: a) H SO , EtOH, reflux, 12 h, 88%; b) DMF, reflux, 12 h, 58–74%; c) LiOH·H O,

group as positive controls, the

percent inhibitions of com-

pounds 26a–r, 27a–d, 28a–b,

MeOH, RT, 12 h, 74–84%.

29a–b, 30a–b, 31a–b, and 32a–

b were measured at 2.5 mm. The

results are summarized in

Tables 1–3. Of the synthetic de-

rivatives tested, six compounds

(

26b, 26d, 26i–j, 26l, and 26q)

displayed high inhibitory activi-

ties against RhoA (expressed as

an inhibitory rate at 2.5 mm

ꢀ

80%) with half maximal inhibi-

tory concentration (IC ) values

50

2 2

Scheme 5. Reagents and conditions: a) MeONa, NH OH, MeOH, H O, 08C, 1 h, 34–98%.

of 1.17 to 1.84 mm (Table 4), cal-

culated by fitting the dose–

Table 4. Isometric contraction measurements and water solubility of se-

lected compounds.

[

a]

ꢁ1

Compd

IC50 [mm]

Solubility [mgmL ]

3

2

a

123.2ꢂ8.9

70.9ꢂ1.3

73.4ꢂ2.6

43

639

92

6b

6d

6i

6j

6l

[

b]

26

20

0

701

[c]

–

[c]

6q

20

3

Scheme 6. Reagents and conditions: a) DMF, reflux, 12 h, 58–64%; b) diethyl-

chlorophosphate, TEA, DMAP, THF, RT, 16 h, 33–78%.

[c]

fasudil

[

a] Data represent the meanꢂSD of three independent experiments.

b] Percent inhibition at 150 mm. [c] Not determined.

8

3–878C (compound 26b). The solubility increased almost 14-

ꢁ

1

ꢁ1

fold, from 43 mgmL for 3a to 639 mgmL for 26b, by substi-

tution at the aniline nitrogen (Table 4). The lower solubility of

compound 26d than that of 26b may be due to the more lip-

ophilic butyl group. In addition, introduction of the polar

groups to the molecules would lower their logP values, which

also could lead to improved solubility (compound 26i). Com-

pound 26i could also be a salt form for both the acidic group

response curves using

a logistic derivative equation in

Origin 8.0. To make a direct comparison, the methods used in

[22]

this study were the same as previously reported.

By analyzing the data in Tables 1–3 and Table 5, we found

that all six good candidate inhibitors were from Table 1, and

the inhibitory activities were comparable with those of the

first-generation small-molecule RhoA inhibitors. The inhibitory

activity of the most potent compound, 26d (IC =1.17ꢂ

(

acid) and basic groups (tertiary amine) in the structure, which

could explain its good water solubility.

5

0

.19 mm, Table 5), was slightly increased over that of lead com-

pound 3b (IC =1.24ꢂ0.08 mm, Table 5), demonstrating that

50

a substituent on the aniline nitrogen could be tolerated for

cellular potency.

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General observation in SAH-CVS model

Table 5. RhoA inhibitory activity of selected compounds.

[

a]

[b]

Compd

Inhibition [%]

IC50 [mm]

There were no significant differences in body weight and tem-

perature among the experimental groups (data not shown).

After induction of SAH, all animals stopped breathing for

3

4

2

a

b

78

90

76

86

92

89

85

90

81

1.51ꢂ0.13

1.24ꢂ0.08

2.05ꢂ0.07

1.66ꢂ0.28

1.17ꢂ0.19

1.26ꢂ0.13

1.84ꢂ0.24

1.32ꢂ0.17

1.81ꢂ0.19

~

30 s. Fifteen rats died after blood injection, and the mortality

6b

6d

6i

6j

6l

in SAH animals was 15/46 (33%).

Morphometric vasospasm

6q

Compound 26b, the most potent and soluble compound, was

selected to evaluate the relaxant effect on the cerebral vaso-

spasm rat SAH-CVS model. The measured luminal cross-sec-

tional perimeters of basilar arteries (BAs) in each group are

shown in Table 6. Microscopic observation of the BA vaso-

[

a] RhoA inhibition was determined at 2.5 mm; data represent a single

determination. [b] Data represent the meanꢂSD of three independent

experiments.

Effect against PE-induced contraction in thoracic aorta artery

rings

Table 6. Therapeutic effect of compound 26b in the cerebral vasospasm

rat SAH-CVS model.

To test the vasorelaxation effects of the inhibitors against PE-

induced contraction in thoracic aorta artery rings, lead com-

pound 3a and six other compounds displaying good inhibitory

activities against RhoA (26b, 26d, 26i–j, 26l, and 26q) were

evaluated. In all vessels submaximally contracted with 100 nm

PE, compounds 3a, 26b, and 26d (0–150 mm) strongly inhibit-

[a]

Group

Dose [mm]

N

IP [mm]

control

SAH

SAH+vehicle

fasudil

–

15.2

6

4

6

472.50ꢂ53.47

386.25ꢂ11.25

390.93ꢂ20.60

468.83ꢂ43.53

(

1 equiv)

ed tension development in

a dose-dependent fashion

2

(

2

(

2

(

6b

low dose)

6b

medium dose)

6b

high dose)

23.7

(1.56 equiv)

95.0

(6.25 equiv)

380

(25 equiv)

4

6

5

406.25ꢂ16.97

464.00ꢂ50.46

475.60ꢂ63.79

[

a] The inner perimeter (IP) of the basilar arteries was measured (see

Experimental Section for details); data represent the meanꢂSD of two

independent experiments.

spasm of control rats and rats subjected to SAH with different

treatments was shown in Figure 4. Moderate arterial narrowing

and reduction of the intima in the SAH group and vehicle

group were observed (Figure 4B,C). The inner perimeter of BAs

in the above two groups became smaller than in the control

group (386.25ꢂ11.25 mm and 390.93ꢂ20.60 mm vs. 472.50ꢂ

Figure 3. Effects of increasing concentrations of 3a, 26b, 26d, 26j, 26i, 26l,

6q, and DMSO on PE-induced submaximal contraction in isolated arterial

rings. Values are the means ꢂSD (n=3).

2

53.47 mm, p<0.05). Compared with SAH and vehicle groups,

the difference between the inner perimeter of the BAs in the

fasudil group (468.83ꢂ43.53 mm), the 26b medium dose

group (464.00ꢂ50.46 mm) and the 26b high dose group

(475.60ꢂ63.79 mm) was statistically significant (p<0.05), but

the slight increase observed in the 26b low dose group

(406.25ꢂ16.97 mm) was not statistically significant (p>0.05),

indicating that in certain concentration ranges, 26b showed

relaxant effects in a dose-dependent fashion. Furthermore,

even at a medium dose (6.25 equiv of fasudil), 26b showed

similar relaxant effects on cerebral vasospasm to fasudil (p>

0.05). To the best of our knowledge, compound 26b is the first

example of small-molecule RhoA inhibitor with significant in

vivo efficacy, which could serve as a good lead for designing

more potent therapeutics for vasospasms after SAH.

(

Figure 3), with maximal inhibitory rates of 71.0ꢂ7.1%, 85.4ꢂ

2

.4%, and 92.9ꢂ6.1%, respectively (150 mm, n=3, Figure 3),

while compounds 26i–j, 26l, and 26q showed weak vasorelax-

ation effects. The IC₅₀values of compounds 26b and 26d were

7

0.9ꢂ1.3 mm and 73.4ꢂ2.6 mm, respectively, which were ap-

proximately twofold more potent than that of the lead com-

pound 3a (IC =123.2ꢂ8.9 mm, Table 4). These encouraging

50

results may be the result of the better solubility of compounds

2

6b and 26d. The inconsistency between the water solubility

and vasorelaxation effects of compound 26i was complicated,

and needs to be further investigated (Table 4). It indicated that

chemical modification strategy focusing on water solubility in

this study was efficient and valuable for improving pharmaco-

logical potencies.

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provide an experimental basis

for the relationship between

RhoA protein and vasospasms

after SAH.

Experimental Section

Chemistry

General: Reagents (chemicals)

were purchased from Lancaster,

Alfa Aesar, J&K, Acros, and Shang-

hai Chemical Reagent Co. and

were used without further purifica-

tion. HSGF 254 (150–200 mm thick-

ness; Yantai Huiyou Co., China)

was used for analytical thin-layer

chromatography (TLC). Yields were

not optimized. Melting points

were measured in capillary tubes

on an SGW X-4 melting point ap-

paratus without correction. Nuclear

magnetic resonance (NMR) spec-

Figure 4. Microscopic observation of the BA vasospasm of control rats (A) and rats subjected to SAH with different

treatments. Moderate vasospasm could be detected in the SAH group (B) and SAH+ vehicle group (C), showing

luminal narrowing, increased wall thickness, and corrugation of the tunica intima. The inner perimeter of BAs was

significantly increased in the SAH+medium dose of 26b group (E), the SAH+high dose of 26b group (F) and

SAH+fasudil group (G), but slightly increased in the low dose of 26b group (D) (100ꢁ magnification).

Structure–activity relationships (SAR)

troscopy was performed on a Bruker AMX-400 NMR. Chemical

shifts were reported in parts per million (ppm, d) downfield from

tetramethylsilane. Proton coupling patterns were described as sin-

glet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Low-

and high-resolution mass spectra (LRMS and HRMS) were given

with electric and electrospray ionization (EI and ESI) produced by

a Finnigan MAT-95 and LCQ-DECA spectrometer.

SAR analysis of a set of 32 compounds provided important in-

sight into the essential structural requirements for effective

RhoA inhibition. An analysis of the data shown in Tables 1–3

revealed some preliminary SARs for compounds 26a–r, 27a–d,

28a–b, 29a–b, 30a–b, 31a–b, and 32a–b: 1) various substitu-

ents (including steric, electronic, and hydrophobic groups) on

the aniline nitrogen had a limited influence on the inhibitory

activity of compounds (Table 1, 3b vs. 26a–r); 2) substituents

on the aniline nitrogen could significantly improve physico-

chemical properties of compounds (e.g., solubility) by breaking

molecular planarity (Table 5, 3a vs. 26b); 3) the further explo-

ration of an amino substituent on the quinoxaline/quinoline

ring-like “acid bioisosteres” did not result in any improvements

2-Chloroquinoline (6a): Quinolin-2-ol (2.0 g, 0.014 mol) was added

to phenylphosphonic dichloride (20 mL). The mixture was stirred at

reflux for 12 h at 1108C under N . After the reaction was cooled to

2

room temperature, it was slowly poured into ice water with contin-

ued stirring. After the product was re-dissolved in solvent, it was

removed by filtration and washed with water to afford 6a (yield:

1

.911 g, 85%): H NMR (400 MHz, CDCl ): d=8.14 (d, J=8.6 Hz, 1H,

3

ArH), 8.08 (d, J=8.5 Hz, 1H, ArH), 7.83 (d, J=8.2 Hz, 1H, ArH),

7.80–7.73 (m, 1H, ArH), 7.58 (m, 1H, ArH), 7.41 ppm (d, J=8.6 Hz,

1H, ArH).

(

Table 3) and carboxylic acid was still the best amino substitu-

ent for good activity (Table 3, 3b vs. 28a or 30a or 32a); and

) between the quinoxaline (3a) and quinoline (3b) frame-

4

2

-Chloroquinoxaline (6b): Compound 6b was prepared from 2-

works, the quinoline ring was optimal for improving biological

quinoxalinone in the same manner as described for the prepara-

1

activities (26b vs. 26q).

tion of 6a (yield: 1.830 g, 81%): H NMR (400 MHz, CDCl ): d=8.54

3

(s, 1H, ArH), 7.92–7.81 (m, 1H, ArH), 7.80–7.72 (m, 1H, ArH), 7.63–

7

.50 ppm (m, 2H, ArH).

Conclusions

Ethyl 3-nitrocinnamate (8): 3-Nitrocinnamic acid (2.0 g, 0.01 mol)

was dissolved in 20 mL EtOH, and 1 mL concentrated sulfuric acid

was added slowly to the reaction. The mixture was stirred at reflux

We proceeded with research on RhoA inhibitors and discov-

ered second-generation small-molecule RhoA inhibitors using

chemical synthesis and bioassays. Based on the structures of

lead compounds 3a–b and 4, chemical modifications were

performed. In total, 32 new compounds were synthesized and

tested with biological assays. Finally, six compounds (26b,

for 12 h under N . After the reaction was cooled to room tempera-

2

^t^u^r^e^,ⁱ^t^w^a^s^c^oⁿ^c^eⁿ^t^r^a^t^e^dⁱⁿ^v^a^c^u^o^,^aⁿ^d^s^a^t^u^r^a^t^e^d^N^a^H^C^O3 was

added to make the reaction alkaline. The mixture was extracted

with EtOAc (20 mL), and the organic layer was dried over MgSO₄,

filtered, and concentrated in vacuo. Then, the residue was purified

26d, 26i–j, 26l, and 26q) were found to show high RhoA in-

by flash chromatography on silica gel to afford compound 8 (yield:

hibitory activities, and two compounds (26b and 26d) showed

significant inhibitory effects against PE-induced contraction in

thoracic aorta artery rings. Compound 26b showed promising

therapeutic effects which were similar to those of fasudil in an

SAH-CVS model. On the basis of in vitro and in vivo biological

results, compound 26b could serve as a good lead for design-

ing more potent cardiovascular drug. The results in this study

1

.84 g, 81%): H NMR (400 MHz, CDCl ): d=7.62 (d, J=15.8 Hz, 1H,

3

ArH), 7.20 (t, J=7.6 Hz, 1H, ArH), 6.98 (d, J=7.8 Hz, 1H, ArH), 6.88

(s, 1H, ArH), 6.76 (d, J=8.1 Hz, 1H, CH), 6.40 (d, J=16.2 Hz, 1H,

CH), 4.28 (q, J=7.2 Hz, 2H, CH ), 1.36 ppm (t, J=7.2 Hz, 3H, CH ).

2

3

(E)-Ethyl-3-(3-aminophenyl) acrylate (9): Ethyl 3-nitrocinnamate 8

(2.0 g, 0.009 mol) was added to EtOH (40 mL), followed by stan-

nous chloride (10.35 g, 0.054 mol). The mixture was stirred at reflux

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for 12 h. After the reaction was cooled to room temperature, it

7.56–7.53 (m, 2H, ArH), 7.39–7.36 (m, 1H, ArH), 7.28–7.25 (m, 1H,

ArH), 6.61 (d, J=16.0 Hz, 2H, CH), 4.14 (dd, J=14.1, 7.1 Hz, 2H,

CH ), 1.21 ppm (t, J=6.9 Hz, 3H, CH ); MS (ESI+): m/z=319.1 [M+

was concentrated in vacuo, and saturated NaHCO was added to

3

make the mixture alkaline, which was then extracted with EtOAc

2

+

3

+

(

20 mL). The organic layer was dried over MgSO , filtered, concen-

H] ; HRMS (ESI+): m/z [M+H] calcd for C H N O : 319.1447,

4

20 19

2

trated in vacuo, and purified by flash chromatography on silica gel

found: 319.1441.

1

to afford 9 (yield: 1.698 g, 98%): H NMR (400 MHz, CDCl ): d=7.62

3

(E)-3-(3-(Propyl(quinolin-2-yl)amino)phenyl)acrylic acid (26c):

(

7

(

d, J=16.0 Hz, 1H, ArH), 7.20 (t, J=7.8 Hz, 1H, ArH), 6.98 (d, J=

.6 Hz, 1H, ArH), 6.88 (s, 1H, ArH), 6.76 (d, J=7.9 Hz, 1H, CH), 6.40

d, J=16.0 Hz, 1H, CH), 4.28 (q, J=7.1 Hz, 2H, CH ), 3.76 (s, 2H,

Compound 26c was prepared from 10a and 1-iodopropane in the

same manner as described for the preparation of 26a (yield:

2

1

3

98 mg, 95% over two steps): mp: 73–768C; HPLC: 99%; H NMR

NH ), 1.36 ppm (t, J=7.1 Hz, 3H, CH ).

2

3

(

400 MHz, [D ]acetone): d=7.85 (d, J=9.1 Hz, 1H, ArH), 7.75–7.62

6

(

E)-Ethyl 3-(3-(quinolin-2-ylamino)phenyl)acrylate (10a): 2-Chlor-

(m, 5H, ArH), 7.56 (t, J=7.7 Hz, 2H, ArH), 7.41 (d, J=7.7 Hz, 1H,

ArH), 7.24 (t, J=7.0 Hz, 1H, ArH), 6.69 (d, J=9.1 Hz, 1H, CH), 6.61

oquinoline (6a, 200 mg, 0.001 mol) and (E)-ethyl-3-(3-aminophenyl)

acrylate (9, 230 mg, 0.001 mol) were dissolved in 2 mL DMF. The

(d, J=16.1 Hz, 1H, CH), 4.16–4.11 (m, 2H, CH ), 1.84–1.69 (m, 2H,

2

mixture was stirred at reflux for 12 h at 1108C under N . After the

reaction was cooled to room temperature, it was poured into

water and extracted with EtOAc (20 mL), and the combined organ-

CH ), 0.97 ppm (t, J=7.4 Hz, 3H, CH ); MS (ESIꢁ): m/z=331.2

2

3

ꢁ

[MꢁH] ; HRMS (ESIꢁ): m/z [MꢁH] calcd for C H N O : 331.1447,

21

19

2

found: 331.1452.

ic layers were dried over MgSO , filtered, concentrated in vacuo,

4

(

E)-3-(3-(Butyl(quinolin-2-yl)amino)phenyl)acrylic acid (26d):

and purified by flash chromatography on silica gel to afford 10a

1

Compound 26d was prepared from 10a and 1-iodobutane in the

same manner as described for the preparation of 26a (yield:

3

(

yield: 194 mg, 51%): H NMR (400 MHz, CDCl ): d=7.95 (d, J=

3

8

7

.9 Hz, 1H, ArH), 7.88 (s, 1H, ArH), 7.84 (d, J=8.4 Hz, 1H, ArH),

.76–7.60 (m, 4H, ArH), 7.35 (m, 2H, ArH), 7.24 (d, J=7.7 Hz, 1H,

1

90 mg, 90% over two steps): mp: 69–728C; HPLC: 98%; H NMR

(400 MHz, [D ]acetone): d=7.89 (d, J=8.7 Hz, 1H, ArH), 7.70 (dd,

6

ArH), 6.96 (d, J=8.9 Hz, 1H, CH), 6.48 (d, J=16.0 Hz, 1H, CH), 4.31

J=20.7, 10.4 Hz, 5H, ArH), 7.59 (t, J=7.7 Hz, 2H, ArH), 7.45 (d, J=

.7 Hz, 1H, ArH), 7.27 (t, J=7.3 Hz, 1H, ArH), 6.70 (d, J=9.2 Hz, 1H,

(

q, J=7.1 Hz, 2H, CH ), 1.38 ppm (t, J=7.1 Hz, 3H, CH ).

2 3

7

(E)-Ethyl 3-(3-(quinoxalin-2-ylamino)phenyl)acrylate (10b): Com-

CH), 6.63 (d, J=16.0 Hz, 1H, CH), 4.24 (s, 2H, CH ), 1.78–1.69 (m,

2

pound 10b was prepared from 2-chloroquinoxaline (6b) in the

2H, CH ), 1.51–1.38 (m, 2H, CH ), 0.95 ppm (t, J=7.4 Hz, 3H, CH );

2

3

ꢁ

same manner as described for the preparation of 10a (yield:

MS (ESIꢁ): m/z=345.2 [MꢁH] ; HRMS (ESIꢁ): m/z [MꢁH] calcd for

1

3

13 mg, 81%): H NMR (400 Hz, CDCl ): d=8.45 (s, 1H, ArH), 8.02

C H N O 345.1603, found: 345.1609.

3

22 21

2

(

7

s, 1H, ArH), 7.94 (d, J=8.0 Hz, 1H, ArH), 7.80–7.84 (m, 2H, ArH),

.71 (d, J=16.0 Hz, 1H, ArH), 7.65 (t, J=7.6 Hz, 1H, ArH), 7.49 (t,

J=8.0 Hz, 1H, ArH), 7.39 (t, J=8.0 Hz, 1H, ArH), 7.25 (d, J=7.6 Hz,

H, CH), 6.48 (d, J=16.0 Hz, 1H, CH), 4.30 (q, 2H, CH ), 1.36 ppm

(

E)-3-(3-(Isopropyl(quinolin-2-yl)amino)phenyl)acrylic acid (26e):

Compound 26e was prepared from 10a and 2-iodopropane in the

same manner as described for the preparation of 26a (yield:

1

2

1

59 mg, 37% over two steps): mp: 100–1038C; HPLC: 99%;

(

t, J=7.2 Hz, 3H, CH₃).

1

H NMR (400 MHz, [D ]DMSO): d=7.81 (t, J=8.2 Hz, 2H, ArH), 7.63

6

(

E)-3-(3-(Methyl(quinolin-2-yl)amino)phenyl)acrylic acid (26a):

(dd, J=20.5, 11.3 Hz, 5H, ArH), 7.57–7.51 (m, 1H, ArH), 7.28 (d, J=

7.7 Hz, 1H, ArH), 7.21 (t, J=7.6 Hz, 1H, ArH), 6.61 (d, J=15.9 Hz,

NaH was added portionwise to a solution of (E)-ethyl 3-(3-(quinoxa-

lin-2-ylamino)phenyl)acrylate (10a, 0.7 mmol) in DMF (4 mL). The

mixture was stirred for 15 min in an ice bath under N , then iodo-

methane (600 mL) was added dropwise, and stirring was continued

for 12 h at room temperature. After the reaction was completed,

the mixture was extracted with water (10 mL) and EtOAc (20 mL).

1H, CH), 6.20 (d, J=9.1 Hz, 1H, CH), 5.61–5.34 (m, 1H, CH),

+

2

1.15 ppm (d, J=6.8 Hz, 6H, CH ); MS (EI) m/z 332.2 (M ), 317.1

3

+

(100%); HRMS (EI) m/z [M] calcd for C H N O : 332.1525, found:

21

20

2

332.1525, 317.1294 (100%).

(E)-3-(3-((Cyclopropylmethyl)(quinolin-2-yl)amino)phenyl)acrylic

The organic layer was dried over MgSO and filtered. The filtrate

4

acid (26 f): Compound 26 f was prepared from 10a and (bromo-

methyl)cyclopropane in the same manner as described in the prep-

was concentrated in vacuo and then purified by flash column chro-

matography on silica gel to afford 11 a, which was used directly in

the next step without further purification.

aration of 26a (yield: 188 mg, 43% over two steps): mp: 96–998C;

1

HPLC: 96%; H NMR (400 MHz, [D ]DMSO): d=7.91 (d, J=8.1 Hz,

6

Compound 11 a (199 mg, 0.0006 mol) and lithium hydroxide

1H, ArH), 7.78–7.60 (m, 5H, ArH), 7.55 (dt, J=15.4, 7.5 Hz, 2H, ArH),

7.40 (d, J=7.7 Hz, 1H, ArH), 7.27 (d, J=6.5 Hz, 1H, ArH), 6.61 (t, J=

(

43 mg) were dissolved in MeOH (4 mL), which was stirred over-

night at 408C. After the reaction was completed, it was concentrat-

ed in vacuo, and aqueous HCl was added, which acidified the mix-

ture to pH 5.5. The precipitate was collected to afford 26a (yield:

12.0 Hz, 2H, CH), 3.98 (d, J=6.8 Hz, 2H, CH ), 1.27–1.15 (m, 1H,

2

CH), 0.40 (d, J=6.7 Hz, 2H, CH ), 0.17 ppm (q, J=5.1 Hz, 2H, CH );

2

ꢁ

MS (ESIꢁ): m/z=343.2 [MꢁH] ; HRMS (ESIꢁ): m/z [MꢁH] calcd for

C H N O : 343.1447, found: 343.1452.

22 19 2 2

6

0 mg, 14% over two steps): mp: 103–1068C; HPLC: 100%;

1

H NMR (400 MHz, [D ]acetone): d=7.94 (d, J=9.2 Hz, 1H, ArH),

6

(E)-3-(3-((Cyclopentylmethyl)(quinolin-2-yl)amino)phenyl)acrylic

7

.75 (dd, J=17.3, 8.0 Hz, 4H, ArH), 7.66 (d, J=7.6 Hz, 1H, ArH),

.60 (dd, J=16.5, 8.2 Hz, 2H, ArH), 7.48 (d, J=7.6 Hz, 1H, ArH),

.31 (t, J=7.4 Hz, 1H, ArH), 6.85 (d, J=9.3 Hz, 1H, CH), 6.64 (d, J=

acid (26g): Compound 26g was prepared from 10a and iodome-

thylcyclopentane in the same manner as described for the prepara-

7

1

tion of 26a (yield: 100 mg, 21% over two steps): mp: 102–1058C;

6.0 Hz, 1H, CH), 3.68 ppm (s, 3H, CH ); MS (ESI+): m/z=305.1

3

+

1

+

HPLC: 97%; H NMR (400 MHz, CD

3

OD): d=7.92 (d, J=9.3 Hz, 1H,

[M+H] ; HRMS (ESI+): m/z [M+H] calcd for C H N O :

19 17 2 2

ArH), 7.72 (dd, J=22.9, 7.1 Hz, 3H, ArH), 7.60 (dt, J=15.3, 8.1 Hz,

H, ArH), 7.42 (d, J=7.8 Hz, 1H, ArH), 7.31 (t, J=7.3 Hz, 1H, ArH),

3

05.1290, found: 305.1285.

4

(E)-3-(3-(Ethyl(quinolin-2-yl)amino)phenyl)acrylic acid (26b):

6.74 (d, J=9.3 Hz, 1H, CH), 6.55 (d, J=16.0 Hz, 1H, CH), 4.17 (d, J=

Compound 26b was prepared from 10a and iodoethane in the

7.6 Hz, 2H, CH ), 2.35 (dt, J=15.0, 7.6 Hz, 1H, CH), 1.74 (td, J=12.4,

2

same manner as described for the preparation of 26a (yield:

8.5 Hz, 4H, CH ), 1.64–1.50 (m, 2H, CH ), 0.91 ppm (d, J=9.8 Hz,

2

1

ꢁ

113 mg, 57% over two steps): mp: 83–878C; HPLC: 99%; H NMR

2H, CH ); MS (ESIꢁ): m/z=371.2 [MꢁH] ; HRMS (ESIꢁ): m/z

2

ꢁ

(

400 MHz, [D ]DMSO): d=7.91 (s, 1H, ArH), 7.75–7.60 (m, 5H, ArH),

[MꢁH] calcd for C H N O : 371.1760, found: 371.1765.

6

24 23

2

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+

(E)-3-(3-((Cyclohexylmethyl)(quinolin-2-yl)amino)phenyl)acrylic

H] ; HRMS (ESI+): m/z [M+H] calcd for C H N O : 335.1396,

20

19

2

3

acid (26h): Compound 26h was prepared from 10a and cyclohex-

found: 335.1390.

ylmethyl bromide in the same manner as described for the prepa-

(

E)-3-(3-((3-Hydroxypropyl)(quinolin-2-yl)amino)phenyl)acrylic

ration of 26a (yield: 111 mg, 23% over two steps): mp: 87–908C;

1

acid (26m): Compound 26m was prepared from 10a and 3-iodo-

propanol in the same manner as described for the preparation of

2

9

HPLC: 95%; H NMR (400 MHz, [D ]DMSO): d=7.87 (d, J=9.2 Hz,

6

1

7

1

2

H, ArH), 7.66 (d, J=7.6 Hz, 1H, ArH), 7.60 (d, J=8.2 Hz, 1H, ArH),

.50 (dt, J=15.2, 9.3 Hz, 4H, ArH), 7.31 (t, J=13.3 Hz, 2H, ArH),

.23 (t, J=6.9 Hz, 1H, ArH), 6.64 (d, J=9.1 Hz, 1H, CH), 6.52 (d, J=

6a (yield: 216 mg, 49% over two steps): mp: 166–1698C; HPLC:

1

7%; H NMR (400 MHz, CD OD): d=7.81 (d, J=9.1 Hz, 1H, ArH),

3

7

.63 (d, J=7.9 Hz, 1H, ArH), 7.59 (d, J=7.8 Hz, 2H, ArH), 7.54 (d,

5.9 Hz, 1H, CH), 4.02 (d, J=6.6 Hz, 2H, CH ), 1.67 (dd, J=49.2,

2

J=8.3 Hz, 3H, ArH), 7.48 (t, J=7.7 Hz, 1H, ArH), 7.33 (d, J=7.5 Hz,

1H, ArH), 7.23 (t, J=7.4 Hz, 1H, ArH), 6.63 (d, J=9.2 Hz, 1H, CH),

6.48 (d, J=16.0 Hz, 1H, CH), 4.25 (t, J=6.3 Hz, 2H, CH ), 3.63 (t, J=

5.9 Hz, 7H, CH , CH), 1.25 (d, J=10.2 Hz, 1H, CH ), 1.13 ppm (t,

2

ꢁ

J=9.4 Hz, 3H, CH ); MS (ESIꢁ): m/z=385.2 [MꢁH] ; HRMS (ESIꢁ):

2

ꢁ

2

m/z [MꢁH] calcd for C H N O : 385.1916, found: 385.1922.

25

2

5

.8 Hz, 2H, CH ), 1.83–1.75 ppm (m, 2H, CH ); MS (ESIꢁ): m/z=

2

ꢁ

(E)-3-(3-((2-(Piperidin-1-yl)ethyl)(quinolin-2-yl)amino)phenyl)-

347.1 [MꢁH] ; HRMS (ESIꢁ): m/z [MꢁH] calcd for C21

H N O :

19 2 3

acrylic acid (26i): Compound 26i was prepared from 10a and 2-

347.1396, found: 347.1401.

piperidinoethylchloride hydrochloride in the same manner as de-

(

E)-3-(3-((2-((tert-Butyldimethylsilyl)oxy)ethyl)(quinolin-2-yl)ami-

no)phenyl)acrylic acid (26n): Compound 26n was prepared from

0a and tert-butyl(2-iodoethoxy)dimethylsilane in the same

manner as described for the preparation of 26a (yield: 102 mg,

scribed for the preparation of 26a (yield: 239 mg, 63% over two

1

steps): mp: 82–868C; HPLC: 98%; H NMR (400 MHz, CD OD): d=

3

1

7

.83 (d, J=9.1 Hz, 1H, ArH), 7.68 (d, J=8.4 Hz, 1H, ArH), 7.61 (d,

J=7.7 Hz, 1H, ArH), 7.57–7.50 (m, 2H, ArH), 7.45 (dt, J=15.4,

.8 Hz, 2H, ArH), 7.36 (d, J=16.0 Hz, 1H, ArH), 7.30–7.21 (m, 2H,

ArH), 6.58 (d, J=9.1 Hz, 1H, CH), 6.44 (d, J=16.0 Hz, 1H, CH), 4.42

1

0% over two steps): mp: 124–1278C; HPLC: 99%; H NMR

7

(400 MHz, [D ]DMSO): d=7.92 (d, J=9.2 Hz, 1H, ArH), 7.73 (s, 1H,

6

ArH), 7.70 (d, J=7.4 Hz, 1H, ArH), 7.67–7.54 (m, 4H, ArH), 7.50 (t,

J=7.7 Hz, 1H, ArH), 7.42 (d, J=8.1 Hz, 1H, ArH), 7.30–7.23 (m, 1H,

ArH), 6.70 (d, J=9.1 Hz, 1H, CH), 6.57 (d, J=16.0 Hz, 1H, CH), 4.17

(

t, J=5.8 Hz, 2H, CH ), 3.35 (t, J=5.8 Hz, 2H, CH ), 1.83 (dd, J=

2 2

1

2.4, 7.1 Hz, 4H, CH ), 0.79 ppm (d, J=9.5 Hz, 6H, CH ); MS (ESIꢁ):

2

m/z=400.2 [MꢁH] ; HRMS (ESIꢁ): m/z _[_M_ꢁ_H_]ꢁ calcd for

ꢁ

(t, J=6.3 Hz, 2H, CH ), 3.93 (t, J=6.3 Hz, 2H, CH ), 0.82 (s, 9H, CH ),

2

3

C H N O : 400.2025, found: 400.2031.

2

5

26

3

2

+

0

.00 ppm (s, 6H, CH ); MS (ESI+): m/z=449.2 [M+H] ; HRMS

3

+

(ESI+): m/z [M+H] calcd for C H N O Si: 449.2260, found:

(E)-3-(3-(Benzyl(quinolin-2-yl)amino)phenyl)acrylic acid (26j):

26 33 2 3

4

49.2255.

Compound 26j was prepared from 10a and benzyl bromide in the

same manner as described for the preparation of 26a (yield:

(

E)-3-(3-((Carboxymethyl)(quinolin-2-yl)amino)phenyl)acrylic acid

26o): Compound 26o was prepared from 10a and methyl bro-

3

52 mg, 45% over two steps): mp: 98–1028C; HPLC: 100%;

1

H NMR (400 MHz, [D ]DMSO): d=7.93 (d, J=9.0 Hz, 1H, ArH),

6

moacetate in the same manner as described for the preparation of

6a (yield: 31 mg, 10% over two steps): mp: 184–1878C; HPLC:

7

1

6

.72–7.63 (m, 2H, ArH), 7.59 (d, J=8.6 Hz, 1H, ArH), 7.51 (dd, J=

5.8, 8.0 Hz, 3H, ArH), 7.39 (t, J=7.9 Hz, 1H, ArH), 7.31 (t, J=

.8 Hz, 3H, ArH), 7.27–7.19 (m, 3H, ArH), 7.14 (t, J=7.4 Hz, 1H,

2

1

00%; H NMR (400 MHz, [D ]acetone): d=7.99 (d, J=9.1 Hz, 1H,

6

ArH), 7.85 (s, 1H, ArH), 7.82–7.75 (m, 2H, ArH), 7.73–7.66 (m, 2H,

ArH), 7.65–7.54 (m, 3H, ArH), 7.32 (t, J=7.4 Hz, 1H, ArH), 6.87 (d,

J=9.1 Hz, 1H, CH), 6.61 (d, J=16.0 Hz, 1H, CH), 4.92 ppm (s, 2H,

CH ); MS (ESI+): m/z=349.1 [M+H] ; HRMS (ESI+): m/z [M+H]

calcd for C H N O : 349.1188, found: 349.1183.

ArH), 6.77 (d, J=9.1 Hz, 1H, CH ), 6.48 (d, J=16.0 Hz, 1H, CH ),

2

+

5

(

.39 ppm (s, 2H, CH ); MS (EI) m/z 380.2 (M ), 380.2(100%); HRMS

2

EI) m/z [M] calcd for C H N O : 380.1525, found: 380.1525,

25 20 2 2

+

2

3

80.1524 (100%).

20

17

2

4

(

E)-3-(3-((Pyridin-2-ylmethyl)(quinolin-2-yl)amino)phenyl)acrylic

(

E)-3-(3-((2-Amino-2-oxoethyl)(quinolin-2-yl)amino)phenyl)acrylic

acid (26k): Compound 26k was prepared from 10a and 2-(bromo-

methyl)pyridine hydrochloride in the same manner as described in

the preparation of 26a (yield: 184 mg, 38% over two steps): mp:

1

acid (26p): Compound 26p was prepared from 10a and 2-iodo-

acetamide in the same manner as described for the preparation of

2

1

6a (yield: 136 mg, 26% over two steps): mp: 189–1938C; HPLC:

1

16–1198C; HPLC: 96%; H NMR (400 MHz, [D ]DMSO): d=8.52 (d,

6

00%; H NMR (400 MHz, [D ]DMSO): d=7.95 (d, J=9.0 Hz, 1H,

6

J=4.4 Hz, 1H, ArH), 8.01 (d, J=9.2 Hz, 1H, ArH), 7.84 (s, 1H, ArH),

ArH), 7.76 (s, 1H, ArH), 7.72 (d, J=7.8 Hz, 1H, ArH), 7.60 (dd, J=

7

.75 (dd, J=11.1, 8.0 Hz, 2H, ArH), 7.64–7.52 (m, 6H, ArH), 7.49 (d,

J=6.3 Hz, 2H, ArH), 7.28 (d, J=6.3 Hz, 1H, ArH), 6.88 (d, J=9.1 Hz,

H, CH), 6.56 (d, J=16.0 Hz, 1H, CH), 5.49 ppm (s, 2H, CH ); MS

1

3

9

5.2, 7.4 Hz, 3H, ArH), 7.53 (t, J=6.9 Hz, 1H, ArH), 7.52–7.44 (m,

H, ArH), 7.27 (t, J=7.3 Hz, 1H, ArH), 7.07 (s, 1H, ArH), 6.77 (d, J=

1

2

.2 Hz, 1H, CH), 6.54 (d, J=16.0 Hz, 1H, CH), 4.63 ppm (s, 2H, CH );

ꢁ

2

(

ESIꢁ): m/z=380.1 [MꢁH] ; HRMS (ESIꢁ): m/z [MꢁH] calcd for

+

MS (ESI+): m/z=348.1 [M+H] ; HRMS (ESI+): m/z [M+H] calcd

for C H N O : 348.1348, found: 348.1343.

C H N O : 380.1399, found: 380.1405.

2

4

18

3

2

20

18

3

(

E)-3-(3-((2-Hydroxyethyl)(quinolin-2-yl)amino)phenyl)acrylic acid

26l): Tetrabutylammonium fluoride (1 mL) was added to a solution

(

E)-3-(3-(Ethyl(quinoxalin-2-yl)amino)phenyl)acrylic acid (26q):

Compound 26q was prepared from 10b and methyl trifluoro-

methane sulfonate in the same manner as described for the prepa-

ration of 26a (yield: 140 mg, 59% over two steps): mp: 157–

1

ArH), 7.82 (d, J=8.1 Hz, 2H, ArH), 7.70 (t, J=7.5 Hz, 2H, ArH), 7.67–

7

1

J=7.0 Hz, 3H, CH ); MS (ESI+): m/z=320.1 [M+H] ; HRMS (ESI+):

m/z [M+H] calcd for C H N O : 320.1399, found: 320.1394.

of 26n (193 mg, 0.4 mmol) in dry THF (4 mL). The resulting reac-

tion mixture was stirred at reflux overnight at room temperature.

The solvent was removed under reduced pressure, then the resi-

due was purified by flash chromatography on silica gel to afford

2

1

608C; HPLC: 100%; H NMR (400 MHz, [D ]DMSO): d=8.15 (s, 1H,

6

1

6l (yield: 69 mg, 48%): mp: 195–1988C; HPLC: 100%; H NMR

.54 (m, 3H, ArH), 7.46 (d, J=8.2 Hz, 2H, ArH, CH), 6.64 (d, J=

(

400 MHz, [D ]DMSO): d=7.91 (d, J=9.1 Hz, 1H, ArH), 7.74 (s, 1H,

6

6.0 Hz, 1H, CH), 4.10 (dd, J=14.2, 7.1 Hz, 2H, CH ), 1.22 ppm (t,

2

ArH), 7.70 (d, J=7.9 Hz, 1H, ArH), 7.61 (dd, J=13.0, 7.9 Hz, 3H,

ArH), 7.56 (d, J=7.0 Hz, 1H, ArH), 7.51 (t, J=7.7 Hz, 1H, ArH), 7.42

+

3

+

19

18

3

2

(

9

d, J=8.1 Hz, 1H, ArH), 7.26 (t, J=7.2 Hz, 1H, ArH), 6.67 (d, J=

.1 Hz, 1H, CH), 6.57 (d, J=16.0 Hz, 1H, CH), 4.13 (t, J=6.2 Hz, 2H,

CH ), 3.71 ppm (t, J=6.2 Hz, 2H, CH ); MS (ESI+): m/z=335.1 [M+

(E)-3-(3-((Carboxymethyl)(quinoxalin-2-yl)amino)phenyl)acrylic

acid (26r): Compound 26r was prepared from 10b and methyl

2

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bromoacetate in the same manner as described for the preparation

7.0 Hz, 1H, ArH), 7.74–7.67 (m, 1H, ArH), 7.67–7.56 (m, 1H, ArH),

7.50–7.38 (m, 2H, ArH), 7.26 (dd, J=17.2, 9.5 Hz, 3H, ArH), 4.06 (q,

J=7.0 Hz, 2H, CH ), 2.88 (t, J=7.5 Hz, 2H, CH ), 2.58 (t, J=7.6 Hz,

of 26a (yield: 90 mg, 27% over two steps): mp: 200–2038C; HPLC:

1

7

00%; H NMR (400 MHz, [D ]acetone): d=8.37 (s, 1H, ArH), 7.95 (s,

6

2

H, ArH), 7.87 (d, J=8.2 Hz, 1H, ArH), 7.75 (t, J=10.9 Hz, 3H, ArH),

.66 (dd, J=14.1, 7.0 Hz, 3H, ArH), 7.49 (t, J=7.5 Hz, 1H, CH), 6.64

2H, CH ), 1.20 ppm (t, J=7.0 Hz, 3H, CH ); MS (ESI+): m/z=322.2

2

+

3

+

[M+H] ; HRMS (ESI+): m/z [M+H] calcd for C H N O :

1

9

20

3

2

(

3

d, J=16.1 Hz, 1H, CH), 4.86 ppm (s, 2H, CH ); MS (ESI+): m/z=

72.1 [M+Na] ; HRMS (ESI+): m/z [M+Na] calcd for C H N O :

19 15 3 4

72.0960, found 372.0955.

322.1556, found: 322.1550.

2

+

3

-(3-((Carboxymethyl)(quinoxalin-2-yl)amino)phenyl)propanoic

acid (27d): Compound 27d was prepared from 14b and methyl

Ethyl 3-(3-aminophenyl)propanoate (13): Compound 13 was pre-

bromoacetate in the same manner as described for the preparation

pared from 3-(3-aminophenyl)propionic acid in the same manner

as described for the preparation of 8 (yield: 1.501 g, 85%): H NMR

of 26a (yield: 28 mg, 9% over two steps): mp: 204–2078C; HPLC:

1

100%; H NMR (400 MHz, [D ]acetone): d=8.29 (s, 1H, ArH), 7.86

6

(

400 MHz, CDCl ): d=7.18–7.04 (m, 1H, ArH), 6.65 (d, J=7.7 Hz,

H, ArH), 6.59 (d, J=6.3 Hz, 2H, ArH), 4.15 (q, J=7.1 Hz, 2H, CH₂),

(d, J=8.1 Hz, 1H, ArH), 7.71 (d, J=8.3 Hz, 1H, ArH), 7.65 (t, J=

7.6 Hz, 1H, ArH), 7.53–7.48 (m, 2H, ArH), 7.48–7.45 (m, 1H, ArH),

7.42 (d, J=7.8 Hz, 1H, ArH), 7.35 (d, J=7.4 Hz, 1H, ArH), 4.79 (s,

2H, CH ), 3.01 (t, J=7.6 Hz, 2H, CH ), 2.71 ppm (t, J=7.6 Hz, 2H,

3

1

3

1

.52 (s, 2H, NH ), 2.92–2.84 (m, 2H, CH ), 2.70–2.52 (m, 2H, CH ),

2

.26 ppm (dd, J=9.6, 4.7 Hz, 3H, CH₃).

2

+

CH ); MS (ESI+): m/z=374.1 [M+Na] ; HRMS (ESI+): m/z [M+

2

Ethyl 3-(3-(quinolin-2-ylamino)phenyl)propanoate (14a): Com-

pound 14a was prepared from 13 in the same manner as de-

scribed for the preparation of 10a (yield: 294 mg, 58%): H NMR

+

Na] calcd for C H N O : 374.1117, found: 374.1111.

19

17

3

4

1

3-Nitrobenzamide (17): 3-Nitrobenzaldehyde (16, 0.007 mol), hy-

droxylamine hydrochloride (56 mg, 0.008 mol), and cesium carbon-

ate (259 mg, 0.008 mol) were added to thionyl chloride (1.5 mL)

and water (0.5 mL). The mixture was stirred for 7 h at 1008C, and

then palladium diacetate (8 mg) was added to the reaction. The re-

action was stirred for another 12 h. After the reaction was cooled

to room temperature, it was poured into water and extracted with

EtOAc (20 mL). The combined organic layers were dried over

MgSO₄and filtered. The filtrate was concentrated in vacuo and

(

400 MHz, CDCl ): d=7.92 (d, J=8.9 Hz, 1H, ArH), 7.78 (d, J=

3

8

4

7

.4 Hz, 1H, ArH), 7.64 (d, J=8.0 Hz, 1H, ArH), 7.59 (dd, J=11.2,

.1 Hz, 1H, ArH), 7.44 (d, J=8.0 Hz, 1H, ArH), 7.40 (s, 1H, ArH),

.33–7.27 (m, 2H, ArH), 6.98 (d, J=8.9 Hz, 1H, ArH), 6.93 (d, J=

.5 Hz, 1H, ArH), 4.14 (q, J=7.1 Hz, 2H, CH ), 2.97 (t, J=7.8 Hz, 2H,

2

CH ), 2.66 (t, J=7.8 Hz, 2H, CH ), 1.24 ppm (t, J=7.1 Hz, 3H, CH ).

2

3

Ethyl 3-(3-(quinoxalin-2-ylamino)phenyl)propanoate(14b): Com-

pound 14b was prepared from 13 in the same manner as de-

scribed for the preparation of 10a (yield: 313 mg, 62%): H NMR

(

0

then purified by flash chromatography on silica gel to afford 17

1

(

yield: 63 mg, 57%): H NMR (400 MHz, CD OD): d=8.78–8.72 (m,

3

400 MHz, [D ]acetone): d=8.41 (s, 1H, ArH), 7.91 (dd, J=8.2,

6

.9 Hz, 1H, ArH), 7.83 (s, 1H, ArH), 7.79 (dd, J=8.2, 0.7 Hz, 1H,

1

H, ArH), 8.41 (m, J=8.2, 2.2, 1.0 Hz, 1H, ArH), 8.30–8.25 (m, 1H,

ArH), 7.74 ppm (t, J=8.0 Hz, 1H, ArH).

ArH), 7.66 (dd, J=8.0, 1.6 Hz, 1H, ArH), 7.64–7.55 (m, 2H, ArH),

7

.47–7.38 (m, 1H, ArH), 7.26 (t, J=8.0 Hz, 1H, ArH), 6.92 (d, J=

(E)-N-((Dimethylamino)methylene)-3-nitrobenzamide (18): 3-Ni-

trobenzamide (17, 0.002 mol) was dissolved in N,N-dimethylforma-

mide dimethyl acetal (DMF-DMA, 2 mL), and the mixtures was

stirred at reflux for 5 h at 1058C. After the reaction was cooled to

room temperature, it was concentrated in vacuo and washed with

.6 Hz, 1H, ArH), 4.19–4.12 (m, 2H, CH ), 2.97 (t, J=7.7 Hz, 2H,

2

CH ), 2.68 (t, J=7.7 Hz, 2H,CH ), 1.27–1.21 ppm (m, 3H, CH ).

2

3

-(3-(Ethyl(quinolin-2-yl)amino)phenyl)propanoic acid (27a).

Compound 27a was prepared from 14a and iodoethane in the

same manner as described for the preparation of 26a (yield:

1

pentane to afford 18 (yield: 391 mg, 79%): H NMR (400 MHz,

CDCl ): d=9.17–9.09 (m, 1H, ArH), 8.72 (s, 1H, CH), 8.61 (d, J=

3

1

2

04 mg, 49% over two steps): HPLC: 99%; H NMR (400 MHz,

7

.7 Hz, 1H, ArH), 8.37 (m, 1H, ArH), 7.63 (t, J=7.9 Hz, 1H, ArH),

CD OD): d=7.80 (d, J=9.2 Hz, 1H, ArH), 7.73 (d, J=8.5 Hz, 1H,

ArH), 7.63 (d, J=8.0 Hz, 1H, ArH), 7.60–7.52 (m, 1H, ArH), 7.43 (t,

J=7.7 Hz, 1H, ArH), 7.29–7.19 (m, 3H, ArH), 7.15 (d, J=6.8 Hz, 1H,

3

.32 (s, 3H, CH ), 3.28 ppm (s, 3H, CH ).

3

3-(3-Nitrophenyl)-4H-1,2,4-triazole

(19):

Compound

18

ArH), 6.62 (d, J=9.2 Hz, 1H, ArH), 4.16 (q, J=7.0 Hz, 2H, CH ), 2.98

(0.0014 mol) was added to acetic acid glacial (4 mL), followed by

the addition hydrazine hydrate (150 mg). The mixture was stirred

2

(

7

t, J=7.5 Hz, 2H, CH ), 2.64 (t, J=7.5 Hz, 2H, CH ), 1.28 ppm (t, J=

2 2

+

.1 Hz, 3H, CH ); MS (ESI+): m/z=321.2 [M+H] ; HRMS (ESI+):

for 12 h at 908C under N . After the reaction was completed, it was

3

2

+

m/z [M+H] calcd for C H N O : 321.1603, found: 321.1598.

cooled to room temperature, concentrated in vacuo, and filtered.

The filter cake was dissolved in EtOAc and washed with saturated

20

21

2

3

-(3-((Carboxymethyl)(quinolin-2-yl)amino)phenyl)propanoic

NaHCO (2ꢁ10 mL) to afford 19 as a white crystalline solid (yield:

3

acid (27b): Compound 27b was prepared from 14a and methyl

bromoacetate in the same manner as described for the preparation

1

9

1 mg, 36%): H NMR (400 MHz, CDCl ): d=9.02 (s, 1H, ArH), 8.49

3

(d, J=7.8 Hz, 1H, ArH), 8.40 (s, 1H, ArH), 8.31 (d, J=8.0 Hz, 1H,

of 26a (yield: 12 mg, 4% over two steps): mp: 139–1428C; HPLC:

ArH), 7.68 ppm (t, J=8.0 Hz, 1H, ArH).

1

00%; H NMR (400 MHz, [D ]acetone): d=7.93 (d, J=9.1 Hz, 1H,

6

ArH), 7.78–7.65 (m, 2H, ArH), 7.59 (t, J=7.6 Hz, 1H, ArH), 7.49–7.39

m, 2H, ArH), 7.34 (d, J=7.5 Hz, 1H, ArH), 7.29 (t, J=7.3 Hz, 2H,

ArH), 6.81 (d, J=9.1 Hz, 1H, ArH), 4.80 (s, 2H, CH ), 2.99 (t, J=

3-(4H-1,2,4-Triazol-3-yl)aniline (20): Compound 20 was prepared

(

from 19 in the same manner as described for the preparation of 9

1

(yield: 310 mg, 96%): H NMR (400 MHz, CDCl ): d=9.34 (s, 1H,

2

3

7

3

.6 Hz, 2H, CH ), 2.69 ppm (t, J=7.6 Hz, 2H, CH ); MS (ESI+): m/z=

ArH), 8.63 (d, J=7.7 Hz, 1H, ArH), 8.51 (s, 1H, ArH), 8.47 (d, J=

8.0 Hz, 1H, ArH), 7.71 ppm (t, J=8.1 Hz, 1H, ArH).

2

+

2

+

51.1 [M+H] ; HRMS (ESI+): m/z [M+H] calcd for C H N O :

20

19

2

4

51.1345, found: 351.1339.

N-(3-(4H-1,2,4-Triazol-3-yl)phenyl)quinolin-2-amine (28a): Com-

pound 28a was prepared from 20 in the same manner as de-

3

-(3-(Ethyl(quinoxalin-2-yl)amino)phenyl)propanoic acid (27c):

Compound 27c was prepared from 14b and iodoethane in the

same manner as described in the preparation of 26a (yield:

scribed for the preparation of 10a (yield: 185 mg, 59%): mp: 186–

1

1888C; HPLC: 98%; H NMR (400 MHz, [D ]acetone): d=8.76 (s, 1H,

6

1

42 mg, 71% over two steps): mp: 155–1578C; HPLC: 100%;

ArH), 8.38 (s, 1H, ArH), 8.29 (d, J=8.0 Hz, 1H, ArH), 8.07 (d, J=

8.9 Hz, 1H, ArH), 7.83 (d, J=8.3 Hz, 1H, ArH), 7.76 (t, J=8.0 Hz, 2H,

1

H NMR (400 MHz, [D ]DMSO): d=8.07 (s, 1H, ArH), 7.81 (d, J=

6

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ArH), 7.64–7.59 (m, 1H, ArH), 7.46 (t, J=7.8 Hz, 1H, ArH), 7.38–7.28

(E)-N-Hydroxy-3-(3-(quinolin-2-ylamino)phenyl)acrylamide (30a):

(

m, 1H, ArH), 7.20–7.06 ppm (m, 1H, ArH); MS (ESI+): m/z=288.1

Sodium methoxide (100 mg sodium, 8 mL MeOH) and hydroxyl-

+

[M+H] ; HRMS (ESI+): m/z [M+H] calcd for C H N : 288.1249,

amine solution (50% in H O, 2 mL) was added to a solution of 10a

17

14

5

2

found: 288.1244.

(0.63 mmol) in MeOH (5 mL). The resulting reaction mixture was

stirred at 08C. The solvent was removed under reduced pressure

and was then poured into water and acidified to pH 5.5 by 1 n HCl

to dissolve the solid, which was filtered, washed with water, and

N-(3-(4H-1,2,4-Triazol-3-yl)phenyl)quinoxalin-2-amine

Compound 28b was prepared from 20 in the same manner as de-

scribed for the preparation of 10a (yield: 88 mg, 49%): mp: 261–

(28b):

air-dried to afford 30a (yield: 107 mg, 56%): mp: 173–1778C;

1

2

648C; HPLC: 98%; H NMR (400 MHz, [D ]acetone): d=9.28 (s, 1H,

1

6

HPLC: 98%; H NMR (400 MHz, [D ]acetone): d=8.82 (s, 1H, NH),

6

ArH), 8.76 (s, 1H, ArH), 8.61 (s, 1H, ArH), 8.28 (d, J=7.9 Hz, 1H,

ArH), 7.91 (d, J=8.2 Hz, 1H, ArH), 7.83 (d, J=8.2 Hz, 2H, ArH),

8

.56 (s, 1H, NH), 8.07 (d, J=8.9 Hz, 1H, ArH), 7.95 (d, J=7.7 Hz, 1H,

ArH), 7.84 (d, J=8.3 Hz, 1H, ArH), 7.75 (d, J=7.9 Hz, 1H, ArH), 7.63

dd, J=15.9, 8.1 Hz, 2H, ArH), 7.35 (dt, J=17.5, 7.4 Hz, 3H, ArH),

.22 (d, J=7.5 Hz, 1H, ArH), 7.10 (d, J=8.9 Hz, 1H, CH), 6.65 ppm

7

.72–7.66 (m, 1H, ArH), 7.54–7.47 ppm (m, 2H, ArH); MS (ESI+):

(

7

+

m/z=289.1 [M+H] ; HRMS (ESI+): m/z [M+H] calcd for

C H N : 289.1202, found: 289.1196.

+

1

6

13

6

(d, J=15.6 Hz, 1H, CH); MS (ESI+): m/z=306.1 [M+H] ; HRMS

+

(ESI+): m/z [M+H] calcd for C H N O : 306.1243, found:

18 16 3 2

Ethyl 6-amino-2-naphthoate (22): Compound 22 was prepared

3

06.1237.

from 6-amino-2-naphthoic acid in the same manner as described

1

for the preparation of 8 (yield: 1.042 g, 88%): H NMR (400 MHz,

(E)-N-Hydroxy-3-(3-(quinoxalin-2-ylamino)phenyl)acrylamide

(30b): Compound 30b was prepared from 10b in the same

CDCl ): d=8.45 (s, 1H, ArH), 7.95 (dd, J=8.6, 1.7 Hz, 1H, ArH),

3

7

2

.78–7.74 (m, 1H, ArH), 7.59 (d, J=8.7 Hz, 1H, ArH), 7.03–6.93 (m,

H, ArH), 4.41 (q, J=7.1 Hz, 2H, CH ), 4.03 (s, 2H, NH ), 1.43 ppm

manner as described for the preparation of 30a (yield: 65 mg,

1

2

34%): mp: 228–2328C; HPLC: 95%; H NMR (400 MHz, [D ]DMSO):

6

(

t, J=7.1 Hz, 3H, CH₃).

d=10.94 (s, 1H, OH), 10.11 (s, 1H, NH), 9.11 (s, 1H, NH), 8.60 (s, 1H,

ArH), 8.44 (s, 1H, ArH), 7.88 (t, J=6.7 Hz, 3H, ArH), 7.69 (t, J=

Ethyl 6-(quinolin-2-ylamino)-2-naphthoate (23a): Compound 23a

was prepared from 22 and 6a in the same manner as described in

the preparation of 10a (yield: 155 mg, 74%): H NMR (400 MHz,

7

1

.7 Hz, 1H, ArH), 7.50 (dd, J=12.0, 3.7 Hz, 2H, ArH), 7.43 (t, J=

.9 Hz, 1H, ArH), 7.23 (d, J=7.8 Hz, 1H, CH), 6.52 ppm (d, J=

1

+

5.6 Hz, 1H, CH); MS (ESI+): m/z=329.1 [M+Na] ; HRMS (ESI+):

CDCl ): d=8.55 (s, 1H, ArH), 8.38 (d, J=1.9 Hz, 1H, ArH), 8.05 (dd,

+

3

m/z [M+Na] calcd for C H N O : 329.1014, found: 329.1009.

17

14

4

2

J=8.6, 1.6 Hz, 1H, ArH), 8.02–7.98 (m, 1H, ArH), 7.91 (t, J=8.7 Hz,

N-Hydroxy-3-(3-(quinolin-2-ylamino)phenyl)propanamide (31a):

Compound 31a was prepared from 14a in the same manner as

2

7

1

H, ArH), 7.82 (d, J=8.6 Hz, 1H, ArH), 7.69 (d, J=8.0 Hz, 1H, ArH),

.68–7.58 (m, 2H, ArH), 7.39–7.34 (m, 1H, ArH), 7.07 (d, J=8.9 Hz,

described for the preparation of 30a (yield: 172 mg, 90%): mp:

H, ArH), 4.44 (q, J=7.1 Hz, 2H, CH ), 1.45 ppm (t, J=7.1 Hz, 3H,

2

1

8

5–898C; HPLC: 99%; H NMR (400 MHz, [D ]DMSO): d=10.44 (s,

CH₃).

6

1

H, OH), 9.38 (s, 1H, NH), 8.73 (s, 1H, NH), 8.05 (d, J=8.9 Hz, 1H,

Ethyl 6-(quinoxalin-2-ylamino)-2-naphthoate (23b): Compound

ArH), 7.89 (d, J=8.0 Hz, 1H, ArH), 7.80–7.67 (m, 3H, ArH), 7.58 (t,

J=7.6 Hz, 1H, ArH), 7.29 (t, J=7.4 Hz, 1H, ArH), 7.24 (t, J=7.8 Hz,

1H, ArH), 7.06 (d, J=8.9 Hz, 1H, ArH), 6.81 (d, J=7.4 Hz, 1H, ArH),

2.83 (t, J=7.8 Hz, 2H, CH ), 2.31 ppm (t, J=7.8 Hz, 2H, CH ); MS

2

3b was prepared from 22 and 6b in the same manner as de-

1

scribed in the preparation of 10b (yield: 120 mg, 58%): H NMR

400 MHz, CDCl ): d=8.61 (s, 1H, ArH), 8.56 (d, J=8.8 Hz, 2H, ArH),

(

3

2

+

8

7

.09 (dd, J=8.5, 1.6 Hz, 1H, ArH), 7.98 (dd, J=8.5, 3.6 Hz, 2H, ArH),

.92 (d, J=8.3 Hz, 1H, ArH), 7.88 (d, J=8.7 Hz, 1H, ArH), 7.75–7.64

(ESI+): m/z=308.1 [M+H] ; HRMS (ESI+): m/z [M+H] calcd for

C H N O : 308.1399, found: 308.1394.

18

3

2

(

1

m, 2H, ArH), 7.59–7.51 (m, 1H, ArH), 4.45 (q, J=7.1 Hz, 2H, CH₂),

.46 ppm (t, J=7.1 Hz, 3H, CH₃).

N-Hydroxy-3-(3-(quinoxalin-2-ylamino)phenyl)propanamide

31b): Compound 31b was prepared from 14b in the same

manner as described for the preparation of 30a (yield: 188 mg,

(

6

-(Quinolin-2-ylamino)-2-naphthoic acid (29a): LiOH·H O (30 mg)

2

1

9

8%): mp: 213–2178C; HPLC: 99%; H NMR (400 MHz, [D ]DMSO):

was added to a solution of 23a (0.4 mmol) in MeOH (5 mL). The re-

sulting reaction mixture was stirred at reflux for 12 h at 408C. Sol-

vent was removed under reduced pressure and was acidified to

pH 5.5 by 1 n HCl to dissolve the solid, which was filtered, washed

with water, and air-dried to afford 29a (yield: 106 mg, 84%): mp:

3

1

6

d=10.43 (s, 1H, OH), 9.91 (s, 1H, NH), 8.74 (s, 1H, NH), 8.56 (s, 1H,

ArH), 7.88 (dd, J=16.7, 8.0 Hz, 2H, ArH), 7.76 (d, J=7.0 Hz, 2H,

ArH), 7.66 (t, J=7.6 Hz, 1H, ArH), 7.48 (t, J=7.5 Hz, 1H, ArH), 7.29

(t, J=7.8 Hz, 1H, ArH), 6.89 (d, J=7.2 Hz, 1H, ArH), 2.85 (t, J=

1

7

3

.8 Hz, 2H, CH ), 2.35–2.29 ppm (m, 2H, CH ); MS (ESI+): m/z=

2 2

15–3188C; HPLC: 97%; H NMR (400 MHz, [D ]DMSO): d=9.90 (s,

6

+

31.1 [M+Na] ; HRMS (ESI+): m/z [M+Na] calcd for C H N O :

H, NH), 9.09 (s, 1H, ArH), 8.52 (s, 1H, ArH), 8.17 (d, J=8.8 Hz, 1H,

17 16

4

2

31.1171, found: 331.1165.

ArH), 8.07 (d, J=8.9 Hz, 1H, ArH), 7.95 (s, 2H, ArH), 7.88 (t, J=

.0 Hz, 2H, ArH), 7.81 (d, J=8.1 Hz, 1H, ArH), 7.67 (t, J=7.7 Hz, 1H,

ArH), 7.38 (t, J=7.4 Hz, 1H, ArH), 7.20 ppm (d, J=8.9 Hz, 1H, ArH);

9

(3-(Quinolin-2-ylamino)phenyl)methanol (25a): Compound 25a

was prepared from 3-amino-benzenemethanol and 6a in the same

+

MS (ESI+): m/z=315.1 [M+H] ; HRMS (ESI+): m/z [M+H] calcd

for C H N O : 315.1134, found: 315.1128.

manner as described for the preparation of 10a (yield: 646 mg,

2

0

15

2

1

6

4%): H NMR (400 MHz, [D ]acetone): d=8.60 (s, 1H, NH), 8.01 (d,

6

J=8.9 Hz, 2H, ArH), 7.97 (s, 1H, ArH), 7.76 (d, J=8.3 Hz, 1H, ArH),

.71 (d, J=7.8 Hz, 1H, ArH), 7.59 (dd, J=11.2, 4.1 Hz, 1H, ArH), 7.29

t, J=7.8 Hz, 2H, ArH), 7.07 (d, J=8.9 Hz, 1H, ArH), 6.99 (d, J=

7.5 Hz, 1H, ArH), 4.66 ppm (s, 2H, CH ).

6

2

-(Quinoxalin-2-ylamino)-2-naphthoic acid (29b): Compound

9b was prepared from 23b in the same manner as described for

7

(

the preparation of 29a (yield: 81 mg, 74%): mp: 331–3348C;

HPLC: 100%; H NMR (400 MHz, [D ]DMSO): d=10.46 (s, 1H, ArH),

1

2

6

9

8

7

.10 (s, 1H, ArH), 8.75 (s, 1H, ArH), 8.60 (s, 1H, ArH), 8.17 (d, J=

(

2

3-(Quinoxalin-2-ylamino)phenyl)methanol (25b): Compound

.9 Hz, 1H, ArH), 8.09–7.90 (m, 4H, ArH), 7.84–7.77 (m, 1H, ArH),

5b was prepared from 24 and 6b in the same manner as de-

+

.61 ppm (t, J=7.6 Hz, 1H, ArH); MS (ESI+): m/z=316.1 [M+H] ;

1

scribed in the preparation of 10b (yield: 444 mg, 58%): H NMR

(400 MHz, [D ]acetone): d=8.66 (s, 1H, NH), 8.07 (d, J=8.6 Hz, 1H,

ArH), 7.99 (s, 1H, ArH), 7.78 (d, J=8.5 Hz, 1H, ArH), 7.73 (d, J=

+

HRMS (ESI+): m/z [M+H] calcd for C H N O : 316.1086, found:

19

14

3

2

6

3

16.1086.

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7

1

.7 Hz, 1H, ArH), 7.61 (dd, J=11.4, 4.3 Hz, 1H, ArH), 7.32 (t, J=

.6 Hz, 2H, ArH), 7.10 (d, J=8.8 Hz, 1H, ArH), 7.03 (d, J=7.6 Hz,

H, ArH), 4.68 ppm (s, 2H, CH₂).

acid (LPA) and dimethyl sulfoxide (DMSO), were purchased from

Sigma. (R)-(ꢁ)-phenylephrine hydrochloride (PE) was purchased

from Wako Chemicals. The RhoA G-LISA activation assay biochem

kit was purchased from Cytoskeleton.

Diethyl 3-(quinolin-2-ylamino)benzyl phosphate (32a): Diethyl

chlorophosphate (232 mL) was added dropwise to a solution of

2

minopyridine (20 mg) in dry THF (4 mL) at room temperature

under N . The mixture was stirred for 16 h at room temperature.

After the reaction was completed, it was poured into saturated

KHSO solution, which was extracted with EtOAc (20 mL). The or-

ganic layer was washed twice with saturated. NaHCO and twice

with saturated NaCl, dried over MgSO , and filtered. The filtrate

Animals: The animal use and care protocols, including all operation

procedures, were approved by the Animal Care and Use Commit-

tee of Soochow University and conformed to the Guide for the

Care and Use of Laboratory Animals by the National Institute of

Health, China. Male Sprague–Dawley rats weighing 300–350 g

were purchased from the Animal Center of the Chinese Academy

of Sciences (Shanghai, China). They were acclimated in a humidified

room and maintained on a standard pellet diet at the Animal

Center of Soochow University for at least 7 days. The temperature

in both the feeding room and the operation room was maintained

at 25ꢂ18C.

5a (209 mg, 0.8 mmol), triethylamine (332 mL), and 4-dimethyla-

2

4

3

4

was concentrated in vacuo, then purified by flash chromatography

on silica gel to afford 32a (yield: 240 mg, 78%): HPLC: 97%;

1

H NMR (400 MHz, [D ]acetone): d=8.31 (d, J=9.0 Hz, 1H, ArH),

6

7

.89 (d, J=8.0 Hz, 2H, ArH), 7.86–7.74 (m, 2H, ArH), 7.70 (s, 1H,

RhoA activation assay: RhoA activity was measured by a RhoA acti-

ArH), 7.48 (dd, J=17.0, 8.3 Hz, 2H, ArH), 7.30 (d, J=9.0 Hz, 2H,

ArH), 5.12 (d, J=8.2 Hz, 2H, CH ), 4.15–4.05 (m, 4H, CH ), 1.28 ppm

[22]

vation assay as described previously.

Briefly, when HBVSMCs

2

+

reached 70% confluence, the cells were starved overnight in

serum-free DMEM (0% FBS). Then, 1.25 mL of chemical compound

or DMSO (final concentration of compound: 2.5 mm) was added to

the cells. After an additional 1 h of incubation at 378C, cells were

(

t, J=6.8 Hz, 6H, CH ); MS (ESI+): m/z=387.1 [M+H] ; HRMS

3

+

(

ESI+): m/z [M+H] calcd for C H N O P: 387.1474, found:

20

24

2

4

3

87.1468.

ꢁ

1

Diethyl 3-(quinoxalin-2-ylamino)benzyl phosphate (32b): Com-

pound 32b was prepared from 25b in the same manner as de-

scribed for the preparation of 32a (yield: 205 mg, 33%): HPLC:

treated with or without LPA (5 mgmL ) for 3 min. Following treat-

ment, the cells were washed in ice-cold PBS and lysed. Thereafter,

RhoA activity was measured from the cell lysates using the absorb-

ance-based RhoA G-LISA activation assay biochem kit (Cytoskele-

ton) according to the manufacturer’s protocol. Active RhoA was

detected using indirect immune detection, followed by a colorimet-

ric reaction, measured by absorbance at 490 nm. The inhibitory

rate (IR) was calculated using Equation (1), in which L represents

DMSO+LPA, C represents compound+LPA, and D is DMSO.

1

9

1

8

8%; H NMR (400 MHz, [D ]acetone): d=9.28 (s, 1H, NH), 8.56 (s,

H, ArH), 8.20 (s, 1H, ArH), 8.07 (d, J=8.1 Hz, 1H, ArH), 7.88 (d, J=

.2 Hz, 1H, ArH), 7.83 (d, J=8.3 Hz, 1H, ArH), 7.67 (t, J=7.6 Hz, 1H,

6

ArH), 7.49 (t, J=7.5 Hz, 1H, ArH), 7.40 (t, J=7.8 Hz, 1H, ArH), 7.12

d, J=7.5 Hz, 1H, ArH), 5.11 (d, J=8.1 Hz, 2H, CH ), 4.11 (q, J=

(

2

7

3

.3 Hz, 4H, CH ), 1.29 ppm (t, J=7.0 Hz, 6H, CH ); MS (ESI+): m/z=

88.1 [M+H] ; HRMS (ESI+): m/z [M+H] calcd for C H N O P:

88.1426, found: 388.1421.

2

3

+

19

23

3

4

OD

ꢁOD

L

C

IR ¼

ð1Þ

OD ꢁOD

L

D

Water solubility

Isometric contraction measurement: Isometric contraction was mea-

sured in rings from the thoracic aortic rings as previously de-

For each suspension, 2 mg of compound was weighed, followed

by the addition of 1.5 mL ultra-pure water, then the mixture was

sealed and shaken for 24 h. The suspensions were centrifuged at

[25]

scribed. Briefly, isolated aortic rings, equilibrated with modified

K-H and 95% O , 5% CO at 378C, were initially placed under the

2

optimal resting tension of 1.5 g for 60 min until a stable rest ten-

sion was achieved. The pharmacological effects of compounds

were studied on rings submaximally contracted with 0.1 mm PE.

Once a steady contraction was observed, one aorta ring was treat-

ed with compound (100 mm, dissolved in 100% DMSO) added in

1

2000 rpm (Eppendorf Centrifuge 5430) for 10 min to separate

solid materials from the solution. The supernatant was not re-

tained. After filtration through a filter membrane (0.45 mm), the

concentration of the filtrate was determined by HPLC analysis.

An Agilent 1260 liquid chromatography system (Agilent Technolo-

gies, Germany) with a UV detector monitored at 280 nm was used.

^C^o^m^p^o^uⁿ^d^s^w^e^r^e^s^e^p^a^r^a^t^e^d^b^y^u^sⁱⁿ^g^a^r^e^v^e^r^s^e^d^-^p^h^a^s^e^C¹8 analyti-

cal column (Zorbax Eclipse Plus, 4.6ꢁ150 mm, 5 mm particle size).

Mobile phases A and B consist of water with 0.1% TFA and aceto-

nitrile, respectively. An optimized gradient elution was used for

HPLC separation: 0–3 min, 30% B; 3–13 min, 10% B; 13–15 min,

a

cumulative manner (final concentration of 30 mm–150 mm),

whereas another ring of each vessel received only the same

volume of solvent (DMSO, 3 mL–15 mL) and was used as a paired

time control. The concentration of compound was increased once

the maximal relaxant effect of the preceding concentration was re-

corded. Isometric tension was recorded by an analogue-to-digital

system (ALCB10 MPA2000, Shanghai Alcott Biotech, Shanghai,

China) connected to a desktop computer.

ꢁ

1

3

0% B. The flow rate used was 1.0 mLmin . The column tempera-

ture was kept at 258C. Samples were injected (injection volume:

0 mL) on to the column.

Subarachnoid hemorrhage (SAH) model: SAH was induced by the

1

double-hemorrhage pre-chiasmatic injection model in rats as previ-

[26]

ously described. Briefly, after the rats were anesthetized with 4%

ꢁ

1

chloral hydrate (400 mgkg body weight), they were positioned

prone in a stereotactic frame. After careful disinfection, a midline

scalp incision was made, and a 1 mm hole was drilled 7.5 mm an-

terior to the bregma in the midline, at angle of 308 caudally. Non-

heparinized, freshly autologous blood (0.2 mL) from the femoral

artery was then injected aseptically into the pre-chiasmatic cistern

through this burr hole over a period of 30 s. Immediately after the

injection of blood, the hole was sealed with wax to prevent leak-

Biology

General: All solvents and reagents of reagent grade or ultra-pure

quality were purchased commercially and used without further pu-

rification. Dulbecco’s modified Eagle’s medium (DMEM) and fetal

bovine serum (FBS) were purchased from Invitrogen. Human brain

vascular smooth muscle cells (HBVSMCs) were purchased from Sci-

enCell. Phenylephrine (PE), as well as oleoyl-l-a-lysophosphatidic

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age. The animals were tilted at a 308 for 30 min with their heads

down, in a prone position, to permit pooling of blood around the

BA. Afterward, the rats were returned to their cages, the room tem-

perature was kept at 25ꢂ18C, and 20 mL of 0.9% NaCl was inject-

ed subcutaneously to prevent dehydration. A second blood injec-

tion was administered 48 h after the first injection by the same

procedure.

Keywords: cardiovascular agents

subarachnoid hemorrhage · water solubility · vasorelaxation

·

RhoA inhibitors

·

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8

), SAH+fasudil group (n=8), SAH+low dose of 26b group (n=

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5 mm (medium dose), and 380 mm (high dose), respectively. The

first SAH induction was defined as day 1. A volume of 5 mL of the

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622.

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The authors gratefully acknowledge financial support from the

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[

21222211, 21372001, 91313303, and 81202394), the Ministry of

Education of China’s Program for New Century Excellent Talents

in University (grant no. NCET-12-0853), the Fundamental Re-

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Received: September 8, 2014

410 , 463–469.

Published online on && &&, 0000

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Whoa RhoA! Second-generation small-

molecule RhoA inhibitors were obtained

by structural modifications to the ani-

line nitrogen and acid side chain of lead

compounds. Among 32 compounds

synthesized and tested in biological

assays, one compound was identified as

the most potent and water soluble,

showing good in vitro and in vivo

efficacy.

S. Ma, J. Deng, B. Li, X. Li, Z. Yan, J. Zhu,

G. Chen, Z. Wang,* H. Jiang, L. Miao,*

J. Li*

&& – &&

Development of Second-Generation

Small-Molecule RhoA Inhibitors with

Enhanced Water Solubility, Tissue

Potency, and Significant in vivo

Efficacy

ꢀ

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 0000, 00, 1 – 15

&15

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Article Doi

Development of second-generation small-molecule RhoA inhibitors with enhanced water solubility, tissue potency, and significant in vivo efficacy

DOI: 10.1002/cmdc.201402386

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Article abstract of DOI:10.1002/cmdc.201402386

Full text of DOI:10.1002/cmdc.201402386

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