Synthesis and in vitro anti-hepatitis B virus activities of some ethyl 6-bromo-5-hydroxy-1H-indole-3-carboxylates

DOI: 10.1016/j.bmc.2005.08.041

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Bioorganic and Medicinal Chemistry p. 911 - 917 (2006)

Update date:2022-09-26

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Authors:

Chai, Huifang

Zhao, Yanfang

Zhao, Chunshen

Gong, Ping

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Article abstract of DOI:10.1016/j.bmc.2005.08.041

A series of ethyl 6-bromo-5-hydroxy-1H-indole-3-carboxylates, 8a-11v, were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities in 2.2.15 cells. The selective indexes of inhibition on replication of HBV DNA of compounds 11s (>8.7) a

Full text of DOI:10.1016/j.bmc.2005.08.041

Bioorganic & Medicinal Chemistry 14 (2006) 911–917

Synthesis and in vitro anti-hepatitis B virus activities of some

ethyl 6-bromo-5-hydroxy-1H-indole-3-carboxylates

Huifang Chai, Yanfang Zhao, Chunshen Zhao and Ping Gong^*

School of Pharmceutical Engineering, Shenyang Pharmceutical University, Shenyang 110016, PR China

Received 18 July 2005; revised 17 August 2005; accepted 18 August 2005

Available online 23 September 2005

Abstract—A series of ethyl 6-bromo-5-hydroxy-1H-indole-3-carboxylates, 8a–11v, were synthesized and evaluated for their anti-

hepatitis B virus (HBV) activities in 2.2.15 cells. The selective indexes of inhibition on replication of HBV DNA of compounds

11s (>8.7) and 11t (10.8), which were introduced halogen on the phenyl ring at position 2, were greater than those of the other

evaluated compounds including lamivudine (7.0). Compounds 9e, 9h, 9l, and 11v exhibited signiﬁcant anti-HBV activities, and

the IC₅₀values on replication of HBV DNA of these compounds were 3.6, 6.37, 5.2, and 5.4 lg/ml, respectively, which were far

more potent than the positive control lamivudine 228 lg/ml.

1. Introduction

Hepatitis B virus (HBV)-infected hepatitis is one of the

common most infectious diseases in the world. More

than 400 million people worldwide are chronically

infected by the hepatitis B virus.¹HBV infection causes

liver diseases such as cirrhosis and maybe eventually

hepatocellular carcinoma. Current clinical therapies for

HBV infections with interferon-a, lamivudine, and riba-

virin have limited eﬃcacy in a signiﬁcant proportion of

patients and often result in severe side eﬀects.^2–4Thus, it

is urgently needed to develop more eﬀective and reliable

therapeutics for the treatment of HBV infection.

Figure 1. Structure of arbidol.

In this paper, we report the design and synthesis of

several new ethyl 6-bromo-5-hydroxy-1H-indole-3-car-

boxylate derivatives, which were mainly modiﬁed at

position 1, 4 on indole cycle and diﬀerent substitutions

on the phenyl ring on position 2, and we evaluate their

in vitro anti-HBV activities.

Ethyl

6-bromo-5-hydroxy-1H-indole-3-carboxylates

displayed a variety of biological eﬀects, such as antiviral

eﬀects, immunostimulative, and interferon induced

activity.^5,6Their representive agent is arbidol (Fig. 1),

which was launched in Russia for the prophylaxis and

treatment of acute respiratory viral infections, acting

as an inhibitor of virus entry and membrane fusion.^7,8

The biological properties of ethyl 6-bromo-5-hydroxy-

1H-indole-3-carboxylates raised our interest to focus

on their derivatives as potential anti-HBV agents.

2. Results and discussion

2.1. Synthetic approach

The title compounds, ethyl 6-bromo-5-hydroxy-1H-in-

dole-3-carboxylate derivatives, were obtained as de-

scribed in Scheme 1. The structures of compounds

8a–11v are listed in Table 1.

The key intermediate 1-alkyl-5-hydroxy-2-methyl-1H-in-

dole-3-carboxylate 3 was synthesized from commercially

available 1 and an appropriate alkyl substituted amine,⁹

such as methylamine and cyclopropylamine, followed by

Keywords: Anti-hepatitis B virus activity; Ethyl 6-bromo-5-hydroxy-

1H-indole-3-carboxylates; Synthesis.

Corresponding author. Tel./fax: +86 24 2398642; e-mail:

gongpinggp@126.com

doi:10.1016/j.bmc.2005.08.041

912

H. Chai et al. / Bioorg. Med. Chem. 14 (2006) 911–917

Scheme 1. Synthesis of target compounds. Reagents and conditions: (a) R₁NH_2,rt; (b) 1,4-benzoquinone/ClCH₂CH₂Cl, reﬂux; (c) CH₃COCl/

pyridine/CH₃COCH₃, 40–45 °C; (d) Br₂/CCl₄, reﬂux; (e) R₂-substituted thiophenol/KOH/CH₃OH, rt; (f) 37% HCHO/dimethyl amine/CH₃COOH,

60 °C-reﬂux; (g) appropriate amines/CH₃CH₂OH, 78 °C-reﬂux; (h) hydrogen peroxide/acetic acid, 0 °C; (i) 37% HCHO/appropriate amines/

CH₃COOH, 60 °C-reﬂux; (j) hydrogen peroxide/acetic acid, 0 °C.

Table 1. Structures of compounds 8a–11v

Compound

Methyl

m-Methoxy

2-Pyridinyl

Hydrogen

p-Fluoro

2-Methylimidazolyl

2-Aminoethanethio

Imidazolyl

Sulfur

Cyclopropyl

Methyl

Sulﬁnyl

Imidazolyl

3⁰,4⁰-Diﬂuoro

p-Nitryl

Methyl

Hydrogen

3⁰,4⁰-Diﬂuoro

m-Methoxy

Hydrogen

p-Fluoro

2-Methylimidazolyl

Guanidinyl

Methyl

Cyclopropyl

Methyl

Guanidinyl

2-Aminoethanethio

Cyclopropyl

Methyl

p-Fluoro

2-Pyridinyl

m-Methoxy

Hydrogen

p-Fluoro

Cyclopropyl

Methyl

Triazolyl

4⁰,5⁰-Dicyanoimidazolyl

Dimethylamino

Morpholino

11p

11q

11r

11s

11t

11u

11v

Methyl

Cyclopropyl

Methyl

2-Pyridinyl

p-Fluoro

m-Chloro

p-Fluoro

3⁰,4⁰-Diﬂuoro

Morpholino

Pyrrolidinyl

4-Methyl piperazinyl

Methyl

Cyclopropyl

Nentizescu condensation of 2 and 1,4-benzoquinone.¹⁰

To protect the hydroxy group on position 5, acetyl chlo-

ride was added into the solution of 3 and pyridine, and

gave 4 in a yield of 85%. By the two steps of bromination

with bromine and reaction with electron-withdrawing

group or electron-donating group substituted thiophe-

nol, 4 was transformed into 6.

on potential anti-HBV activities. So we introduced het-

eroaromatic amines or other bases, for example, imidaz-

ole, methyl- or cyano-substituted imidazole, triazole,

2-aminosulfanyl, and so on, at position 4. But the

above-mentioned Mannich bases could not be prepared

directly by the standard Mannich reaction of 6 with

those amines. We obtained 7 ﬁrst by the Mannich reac-

tion of 6 with dimethylamine, according to the proce-

dure of Grinev and Pershin.¹¹Then the mixture of 7

and appropriate base in anhydrous ethanol was reﬂuxed

It was of interest to examine whether the diversity of the

Mannich basic group on position 4 could have an eﬀect

H. Chai et al. / Bioorg. Med. Chem. 14 (2006) 911–917

913

for 4–6 h, ultimately resulting in the formation of com-

pounds 8a–b. To relieve the cellular toxicity and increase

the antiviral activity, the sulﬁdes on some analogues of 8

were further oxidized into sulﬁnyl by hydrogen peroxide

in glacial acetic acid and gave compounds 9c–o.

57.6 lg/ml) were less eﬀective than 9e, 9h, 9l, and

11v, their potencies were still 8 or 4 times of the con-

trol. SIs of 11t and 11s (>8.7, 10.8) were the highest

in all tested compounds. All of these compounds pos-

sessed ﬂuoro or chloro on the phenyl ring at position

2. By contrast, compounds 8a, 9i, and 9n only dis-

played suppressant properties on the production of

HBsAg and HBeAg. However, 9c, 9j, and 11p were

ineﬀective in HBV-infected 2.2.15 cells. These results

suggested that the compounds substituted on the phen-

yl ring of position 2 possessed more anti-HBV potency

than those non-substituted compounds. Particularly,

the electron-withdrawing groups such as halogen were

more in favor of enhancing activities than the elec-

tron-donating groups, except for nitro (9t).

Various Mannich bases were introduced at position 4

directly by the Mannich reaction of 6 with secondary

amines such as dimethylamine, morpholine, N-methyl-

piperazine and pyrrolidine, and gave 10.¹¹The oxidation

of 10 was undertaken to yield target compounds 11p–v

ultimately.

2.2. Biological activity test

All target compounds 8a–11v were evaluated for their

cytotoxicities and anti-HBV activities, namely the ability

to inhibit the replication of HBV DNA and the produc-

tion of HBsAg and HBeAg in HBV-infected 2.2.15 cells.

The results are summarized in Table 2.

The introduction of sulﬁnyl, instead of sulﬁde, did re-

duce the cytotoxicities and kept up or increased the

anti-HBV activities in addition (compounds 9m vs 8b;

9i vs 8a).

As shown in Table 2, half of the evaluated compounds

exhibited inhibitory eﬀects on HBV and were superior

to lamivudine.

Diﬀerent Mannich basic functionalities introduced at

position 4 seemed to have few inﬂuence on antiviral

activities. But 11u (4-pyrrolidinomethyl derivative) and

9o (4⁰,5⁰-dicyanoimidazolylmethyl derivative) did not

have inhibitory eﬀects at the relevant maximum intoxic

concentration.

Of these compounds, 9e showed the most potent in vi-

tro anti-HBV activity (IC₅₀: 3.6 lg/ml). Its analogues

9h, 9l, and 11v also exhibited signiﬁcant eﬃcacy on

HBV, their IC₅₀on the replication of HBV DNA being

6.37, 5.2, and 5.4 lg/ml, respectively, which were far

more potent than the positive control lamivudine

228 lg/ml. The selective indexes of 9e, 9h, and 11v

(5.8, 6.28, and 6.35) were comparable to that of lami-

vudine (7.0). Although 11t and 11s (IC₅₀: 26.64 lg/ml,

Compared with the 1-methyl compound (9k, IC₅₀:

29.30 lg/ml), cyclopropyl at position 1 (9l, IC₅₀:

5.2 lg/ml) could increase the antiviral eﬀects, whereas

the cytotoxicity was enhanced correspondingly. TC₅₀

values of 9k and 9l were 125 and 18.52 lg/ml,

respectively.

Table 2. Anti-HBV activities of compounds 8a–11v

Compound

TC₅₀(lg/ml)^a

HbsAg

IC₅₀(lg/ml)^b

HBeAg

IC₅₀(lg/ml)^b

DNA replication

SI^c

IC₅₀(lg/ml)^b

SI^c

6.35

8.0

2.66

—

2.39

—

3.29

—

4.86

—

192.45

55.56

21.11

40.0

—

11.4

3.6

—

4.87

5.8

—

99.21

40.0

8.0

49.06

—

2.02

—

22.49

6.37

—

4.4

6.28

2.54

—

3.15

—

111.11

125

—

62.16

—

2.01

—

29.30

5.2

26.84

—

4.27

18.52

125

3.63

4.66

—

111.1

28.74

96.23

346.68

>500

288.68

346.68

34.35

1600

61.48

—

1.81

—

33.77

—

11p

11q

—

19.80

—

11r

11s

61.3

—

5.6

—

57.6

26.64

—

>8.7

10.8

—

11t

11u

—

11v

Lamivudine

—

5.4

6.35

7.0

228

^aTC₅₀is 50% cytotoxic concentration in 2.2.15 cells.

^bIC₅₀is 50% inhibitory concentration.

^cSelective index (SI: TC₅₀/IC₅₀).

914

H. Chai et al. / Bioorg. Med. Chem. 14 (2006) 911–917

3. Conclusion

4.1.2.1. Ethyl 6-bromo-4-[(2-methyl-1-imidazolyl)-

methyl]-5-hydroxy-1-methyl-2-[(3-methoxy-phenyl)sulf-

anylmethyl-1H-indole-3-carboxylate (8a). To a solution

of 2-methyl-1-imidazole (0.03 mol) in anhydrous

ethanol (50 ml) was added ethyl 6-bromo-4-[(dimethyla-

mino)methyl]-5-hydroxy-1-methyl-2-[(3-methoxyphenyl)sulf-

anylmethyl]-1H-indole-3-carboxylate (0.01 mol). The

reaction mixture was further stirred with reﬂuxing until

a mass of solid appeared in the solution. The precipitate

was ﬁltered, washed with acetone (15 ml), and dried in

vacuo. Then a white powder 8a (4.9 g) was ultimately

obtained. mp: 177–179 °C; ¹H NMR (DMSO-d₆): d

1.30 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 2.35 (s, 3H, 2-

CH₃of imidazolyl), 3.65 (s, 3H, –OCH₃), 3.72 (s, 3H,

–NCH₃), 4.16 (q, 2H, J = 7.2Hz, –OCH₂CH₃), 4.67 (s,

2H, –SCH₂–), 4.81 (s, 2H, –CH₂N), 6.33–6.57 (m,

2H, 4,5-2H of imidazolyl), 6.92–7.56 (m, 4H, –PhH),

7.75 (s, 1H, –UH), 9.85 (br s, 1H, –OH); MS: m/z

544.0, 546.0 [MH⁺].

In summary, we synthesized a series of new ethyl 6-bro-

mo-5-hydroxy-1H-indole-3-carboxylate derivatives and

examined their anti-HBV activities and cytotoxicities.

Most of them exhibited better bioactivities against

HBV in 2.2.15 cells. According to the above results,

the following conclusions can be made:

1. Substitutions of the phenyl ring at position 2 can

improve anti-HBV activities. Particularly the

electron-withdrawing groups such as ﬂuoro and

chloro contributed more anti-HBV eﬀects than the

electron-donating methoxy group.

2. The oxidation of sulﬁde into sulﬁnyl reduced the cel-

lular toxicities and kept up or increased the anti-HBV

activities.

3. Derivatives having diﬀerent Mannich basic function-

alities at the 4 position did not show signiﬁcant

distinctness of anti-HBV eﬀects, though the elec-

tron-withdrawing group on the basic group could

decrease it.

4. The steric changes of the alkyl group (C 6 3) at posi-

tion 1 have a slight inﬂuence on the antiviral potency

of compounds.

4.1.2.2. Ethyl 6-bromo-4-(2-aminoethylsulfanylmeth-

yl)-5-hydroxy-1-methyl-2-[(2-pyridinyl)sulfanylmethyl]-

1H-indole-3-carboxylate (8b). mp: 183–185 °C; ¹H NMR

(DMSO-d₆): d 1.30 (t, 3H, J = 6.9 Hz, –OCH₂CH₃),

2.94–3.10 (m, 4H, –SCH₂CH₂–), 3.77 (s, 3H, –NCH₃),

4.27 (q, 2H, J = 6.9 Hz, –OCH₂CH₃), 4.45 (s, 2H,

–SCH₂–), 4.91 (d, 2H, J = 3 Hz, –CH₂N), 7.14–7.69 (s,

4H, –pyridinyl H), 7.74 (s, 1H, –UH), 7.96 (br s, 2H,

–NH₂), 8.71 (br s, 1H, –OH); MS: m/z 510.0, 512.0 [MH⁺].

Moreover, being the ﬁrst report on ethyl 6-bromo-5-hy-

droxy-1H-indole-3-carboxylates serving as anti-HBV

agents, these results propose a lead compound in the re-

search and development of anti-HBV and similar virus-

infected hepatitis.

4.1.3. General procedure for the synthesis of compounds

9c–11v. Appropriate analogues of 8 or 10 (0.01 mol)

were ﬁrst dissolved in glacial acetic acid and water

(25 ml, 10:1), and hydrogen peroxide (0.011 mol) was

successively dropped into the solution at 0 °C. After stir-

ring at 0 °C for 6–8 h, the solvent was evaporated in vac-

uo, and 50 ml of water was added in one portion. The

resultant mixture was adjusted to pH 12 with sodium

hydroxide and extracted with methylene chloride. The

organic phase was dried over sodium sulfate and evapo-

rated in vacuo to yield a yellow oil in most circumstanc-

es. Then, 50 ml of ether was poured into the yellow oil.

The solution was cooled at 0 °C and the precipitate was

ﬁltered and dried to give 9c–11v.

4. Experimental

4.1. Chemistry

All melting points were obtained with a Veego melting

point apparatus and are uncorrected. Proton (¹H) nucle-

ar magnetic resonance spectroscopy was performed

using Bruker ARX-300, 300 MHz spectrometers with

Me₄Si as an internal standard. Mass spectra (MS) were

taken in ESI mode on Agilent 1100 LC–MS.

4.1.1. Ethyl 5-acetoxy-1-alkyl-2-methy-1H-lindole-3-car-

boxylate (4). To 500 ml of acetone were added

0.35 mol of 3 and 0.7 mol pyridine, and then 0.7 mol

of acetyl chloride was added dropwise with good

stirring under 40–45 °C. The resulting solution was

stirred at this temperature for 4–5 h and then cooled.

The solution was poured into 500 ml water, and the

precipitate was collected by ﬁltration and washed with

water.

4.1.3.1. Ethyl 6-bromo-1-cyclopropyl-5-hydroxy-4-(1-

imidazolylmethyl)-2-(phenylsulﬁnylmethyl)-1H-indole-3-

carboxylate (9c). Ethyl 6-bromo-1-cyclopropyl-5-hy-

droxy-4-(1-imidazolyl methyl)-2-(phenylsulfanylmeth-

yl)-1H-indole-3-carboxylate (0.01 mol) was dissolved in

glacial acetic acid and water (25 ml, 10:1), and then

hydrogen peroxide (0.011 mol) was dropped into the

solution at 0 °C. After stirring at 0 °C for 6–8 h, the sol-

vent was evaporated in vacuo, and 50 ml of water was

added in one portion. The solution was adjusted to

pH 12 with sodium hydroxide and extracted with meth-

ylene chloride. The organic phase was dried over sodium

sulfate and evaporated in vacuo to yield a yellow oil.

Then 50 ml of ether was poured into the yellow oil.

The solution was cooled at 0 °C and the precipitate

was ﬁltered and dried to give white powder 9c (5.3 g).

mp: 197–199 °C; ¹H NMR (CDCl₃): d 0.80 (m, 2H,

–CH₂–N–CH(CH₂)₂), 1.25 (m, 1H, –CH₂–N–CH(CH₂)₂),

4.1.2. General procedure for the synthesis of compounds

8a–8b. To a solution of imidazole or 2-methyl-1-imidaz-

ole or aminoethanethiol (0.03 mol) in anhydrous etha-

nol (50 ml) was added 7 (0.01 mol) at 78 °C with

constant stirring. The solid was dissolved about 1 h lat-

er. The reaction mixture was further stirred with reﬂux-

ing until a mass of solid appeared in the solution. The

precipitate was ﬁltered and dried to give the desired

compounds 8a–8b.

H. Chai et al. / Bioorg. Med. Chem. 14 (2006) 911–917

915

1.35 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 1.62 (m, 2H, –CH₂–

N–CH(CH₂)₂), 4.24 (q, 2H, J = 7.2 Hz, –OCH₂CH₃),

5.09 (s, 2H, –CH₂N), 5.38 (s, 2H, –sulﬁnyl-CH₂–),

7.18–7.23 (t, 2H, 4,5-2H of imidazolyl), 7.29–7.43 (m,

5H, –PhH), 7.56 (s, 1H, 2-H of imidazolyl), 7.66 (s, 1H,

–UH), 10.20 (br s, 1H, –OH); MS: m/z 542.0, 544.0

[MH⁺].

4.1.3.7. Ethyl 6-bromo-5-hydroxy-4-[(2-methyl-1-imid-

azolyl)methyl]-1-methyl-2-[(3-methoxyphenyl)sulﬁnylm-

ethyl]-1H-indole-3-carboxylate (9i). mp: 179–181 °C; H

NMR (DMSO-d₆):

1.17 (t, 3H, J = 7.2 Hz,

–OCH₂CH₃), 2.34 (s, 3H, 2-CH₃of imidazolyl), 3.53 (s,

3H, –OCH₃), 3.67 (s, 3H, –NCH₃), 4.07 (q, 2H,

J = 7.2 Hz, –OCH₂CH₃), 4.83 (s, 2H, –CH₂N), 5.56 (s,

2H, –sulﬁnyl-CH₂–), 6.31–6.54 (m, 2H, 4,5-2H of imidaz-

olyl), 6.92–7.76 (m, 4H, –PhH), 7.88 (s, 1H, –UH); MS:

m/z 560.1, 562.1 [MH⁺].

4.1.3.2. Ethyl 6-bromo-1-cyclopropyl-5-hydroxy-4-(1-

imidazolylmethyl)-2-[(4- ﬂuorophenyl)sulﬁnylmethyl]-1H-

indole-3-carboxylate (9d). mp: 200–202 °C; ¹H NMR

(CDCl₃): d 0.90–1.00 (m, 2H, –CH₂–N–CH(CH₂)₂),

1.20 (m, 2H, –CH₂–N–CH(CH₂)₂), 1.36 (t, 3H,

J = 7.2 Hz, –OCH₂CH₃), 2.47 (m, 1H, –CH₂–N–

CH(CH₂)₂), 4.32 (q, 2H, J = 7.2 Hz, –OCH₂CH₃),

4.74 (s, 2H, –CH₂N), 5.91 (s, 2H, –sulﬁnyl-CH₂–),

6.95–7.15 (m, 4H, –PhH), 7.38–7.55 (m, 3H, 3H of

imidazolyl), 7.71 (s, 1H, –UH); MS: m/z 560.0, 562.0

[MH⁺].

4.1.3.8. Ethyl 6-bromo-1-cyclopropyl-4-(guanidinylm-

ethyl)-5-hydroxy-2-(phenylsulﬁnylmethyl)-1H-indole-3-

carboxylate (9j). mp: 192–194 °C; ¹H NMR (DMSO-d₆):

d 0.98 (m, 2H, –CH₂–N–CH(CH₂)₂), 1.13 (m, 2H, –CH₂–

N–CH(CH₂)₂), 1.23 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 2.92

(m, 1H, –CH₂–N–CH(CH₂)₂), 4.19 (q, 2H, J = 7.2 Hz,

–OCH₂CH₃), 4.51 (s, 2H, –sulﬁnyl-CH₂–), 5.38 (s, 2H,

–CH₂N), 6.60 (2H, –NH₂), 6.94 (1H, –C@NH),7.19

(m, 5H, –PhH), 7.54 (s, 1H, –UH); MS: m/z 533.0, 535.1

[MH⁺].

4.1.3.3. Ethyl 6-bromo-1-cyclopropyl-2-[(3,4-diﬂuor-

ophenyl)sulﬁnylmethyl]-5-hydroxy-4-(1-imidazolylmethyl)-

1H-indole-3-carboxylate (9e). mp: 193–195 °C; ¹H

NMR (DMSO-d₆): d 0.95–1.04 (m, 2H, –CH₂–N–

CH(CH₂)₂), 1.21 (m, 2H, –CH₂–N–CH(CH₂)₂), 1.35

(t, 3H, J = 7.2 Hz, –OCH₂CH₃), 2.77 (m, 1H, –CH₂–

N–CH(CH₂)₂), 4.15 (q, 2H, J = 7.2 Hz, –OCH₂CH₃),

4.83 (s, 2H, –CH₂N), 5.71 (s, 2H, –sulﬁnyl-CH₂–),

6.67–7.39 (m, 3H, –PhH), 7.54–7.69 (m, 3H, 3H of

imidazolyl), 7.72 (s, 1H, –UH); MS: m/z 578.1, 580.1

[MH⁺].

4.1.3.9. Ethyl 6-bromo-4-(guanidinomethyl)-5-hy-

droxy-1-methyl-2-[(4-ﬂuorophenyl)sulﬁnylmethyl]-1H-in-

dole-3-carboxylate (9k). mp: 188–190 °C; ¹H NMR

(DMSO-d₆): d 1.36 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

3.62 (s, 3H, –NCH₃), 4.28 (q, 2H, J = 7.2 Hz,

–OCH₂CH₃), 4.65 (s, 2H, –CH₂N), 4.78 (s, 2H, –sulﬁ-

nyl-CH₂–), 6.68 (2H, –NH₂), 6.92 (1H, –C@NH), 7.00–

7.44 (m, 4H, –PhH), 7.47 (s, 1H, –UH); MS: m/z 525.0,

527.0 [MH⁺].

4.1.3.4. Ethyl 6-bromo-5-hydroxy-4-(1-imidazolylmeth-

4.1.3.10. Ethyl 6-bromo-1-cyclopropyl-4-(guanidinom-

ethyl)-5-hydroxy-2-[(4-ﬂuorophenyl)sulﬁnylmethyl]-1H-

indole-3-carboxylate (9l). mp: 194–196 °C; ¹H NMR

yl)-1-methyl-2-[(4-cyanophenyl)

sulﬁnylmethyl]-1H-

indole-3-carboxylate (9f). mp: 189–191 °C; ¹H NMR

(DMSO-d₆): d 1.17 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

3.68 (s, 3H, –NCH₃), 4.07 (q, 2H, J = 7.2 Hz,

–OCH₂CH₃), 4.95 (s, 2H, –CH₂N), 5.68 (s, 2H, –sulﬁ-

nyl-CH₂–), 6.72–7.36 (m, 4H, –PhH), 7.69–7.72 (m, 3H,

3H of imidazolyl), 7.84 (s, 1H, –UH); MS: m/z 561.0,

563.0 [MH⁺].

(CDCl₃):

0.90 (m, 2H, –N–CH(CH₂)₂), 1.24

(m, 2H, –N–CH(CH₂)₂), 1.32 (t, 3H, J = 7.2 Hz,

–OCH₂CH₃), 2.69 (m, 1H, –N–CH(CH₂)₂), 4.23 (q,

2H, J = 7.2 Hz, –OCH₂CH₃), 4.75 (s, 2H, –CH₂N),

4.85 (s, 2H, –sulﬁnyl-CH₂–), 6.80 (2H, –NH₂), 7.32

(1H, –C@NH), 7.39–7.60 (m, 4H, –PhH), 7.83 (s,

1H, –UH), 9.28 (br s, 1H, –OH); MS: m/z 551.0,

553.0 [MH⁺].

4.1.3.5. Ethyl 6-bromo-5-hydroxy-4-[(2-methyl-1-imid-

azolyl)methyl]-1-methyl-2-(phenyl-sulﬁnylmethyl)-1H-

indole-3-carboxylate (9g). mp: 186–188 °C; ¹H NMR

(DMSO-d₆): d 1.39 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

2.33 (s, 3H, 2-CH₃of imidazolyl), 3.54 (s, 3H,

–NCH₃), 4.06 (q, 2H, J = 7.2 Hz, –OCH₂CH₃), 4.80

(s, 2H, –CH₂N), 5.54 (s, 2H, –sulﬁnyl-CH₂–),

6.29–6.54 (m, 2H, 4,5-2H of imidazolyl), 7.44–7.64

(m, 5H, –PhH), 7.87 (s, 1H, –UH); MS: m/z 530.1,

532.0 [MH⁺].

4.1.3.11. Ethyl 6-bromo-4-[(2-aminoethyl)sulfanylm-

ethyl]-5-hydroxy-1-methyl-2-[(2-pyridinyl)sulﬁnylmethyl]-

1H-indole-3-carboxylate (9m). mp: 190–192 °C; ¹H NMR

(DMSO-d₆): d 1.32 (t, 3H, J = 6.9 Hz, –OCH₂CH₃),

2.97–3.16 (m, 4H, –SCH₂CH₂–), 3.74 (s, 3H, –NCH₃),

4.24 (q, 2H, J = 6.9 Hz, –OCH₂CH₃), 4.48 (d, 2H,

J = 3 Hz, –CH₂N), 5.11 (s, 2H, –sulﬁnyl-CH₂–), 7.18–

7.64 (s, 4H, –pyridinyl H), 7.79 (s, 1H, –UH), 7.99 (br s,

2H, –NH₂), 8.91 (br s, 1H, –OH); MS: m/z 526.1, 528.1

[MH⁺].

4.1.3.6. Ethyl 6-bromo-5-hydroxy-2-[(3,4-diﬂuorophe-

nyl)sulﬁnylmethyl]-4-[(2-methyl-1- imidazolyl)methyl]-1-

methyl-1H-indole-3-carboxylate (9h). mp: 182–184 °C;

¹H NMR (DMSO-d₆): d 1.07 (t, 3H, J = 7.2 Hz,

–OCH₂CH₃), 2.31 (s, 3H, 2-CH₃of imidazolyl), 3.69

(s, 3H, –NCH₃), 3.98 (q, 2H, J = 7.2 Hz, –OCH₂CH₃),

4.89 (s, 2H, –CH₂N), 5.48 (s, 2H, –sulﬁnyl-CH₂–),

6.24–6.52 (m, 2H, 4,5-2H of imidazolyl), 7.26–7.61 (m,

3H, –PhH), 7.90 (s, 1H, –UH); MS: m/z 566.0, 568.0

[MH⁺].

4.1.3.12. Ethyl 6-bromo-1-cyclopropyl-5-hydroxy-4-(1-

triazolylmethyl)-2-[(3-methoxyphenyl)sulﬁnylmethyl]-1H-

indole-3-carboxylate (9n). mp: 201–203 °C; ¹H NMR

(CDCl₃): d 0.89 (m, 2H, –N–CH(CH₂)₂), 1.16 (m, 2H,

–N–CH(CH₂)₂), 1.47 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

2.26 (m, 1H, –N–CH(CH₂)₂), 3.50 (s, 3H, –OCH₃),

4.40 (q, 2H, J = 7.2 Hz, –OCH₂CH₃), 4.90 (s, 2H,

–CH₂N), 6.10 (s, 2H, –sulﬁnyl-CH₂–), 6.84 (2H, 2H

916

H. Chai et al. / Bioorg. Med. Chem. 14 (2006) 911–917

of triazolyl), 6.99–7.96 (m, 4H, –PhH), 8.62 (s, 1H,

–UH), 9.60 (br s, 1H, –OH); MS: m/z 573.0, 575.0 [MH⁺].

4.1.3.19. Ethyl 6-bromo-2-[(4-ﬂuorophenyl)sulﬁnylm-

ethyl]-5-hydroxy-1-methyl -4-(pyrrolidinomethyl)-1H-in-

dole-3-carboxylate (11u). mp: 196–198 °C; ¹H NMR

(CDCl₃): d 1.42 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

1.90 (s, 4H, 4H of pyrrolidinyl), 2.75 (s, 4H, 4H of

pyrrolidinyl),3.49 (s, 3H, –NCH₃), 4.28 (q, 2H,

J = 7.2 Hz, –OCH₂CH₃), 4.31 (d, 2H, –NCH₂–),

4.60 (s, 2H, –sulﬁnyl-CH₂–), 7.13–7.49 (m, 4H,

–PhH), 7.51 (s, 1H, –UH); MS: m/z 536.9, 538.9

[MH⁺].

4.1.3.13. Ethyl 6-bromo-4-(4,5-dicyano-1-imidazolylm-

ethyl)-5-hydroxy-1-methyl-2-[(3-methoxyphenyl)sul-

ﬁnylmethyl]-1H-indole-3-carboxylate (9o). mp: 198–200 °C;

¹H NMR (DMSO-d₆): d 1.19 (t, 3H, J = 7.2 Hz,

–OCH₂CH₃), 3.52 (s, 3H, –OCH₃), 3.64 (s, 3H, –NCH₃),

4.09 (q, 2H, J = 7.2 Hz, –OCH₂CH₃), 4.84 (s, 2H, –

CH₂N), 5.87 (s, 2H, –sulﬁnyl-CH₂–), 6.94 (m, 1H, 1H

of 4,5-dicyano-1-imidazolyl), 7.00–7.90 (m, 4H, –PhH),

7.97 (s, 1H, –UH), 9.34 (br s, 1H, –OH); MS: m/z 596.0,

598.0 [MH⁺].

4.1.3.20. Ethyl 6-bromo-1-cyclopropyl-2-[(3,4-diﬂuor-

ophenyl)sulﬁnylmethyl]-5-hydroxy-4-(4-methylpiperazi-

nomethyl)-1H-indole-3-carboxylate (11v). mp: 191–

4.1.3.14. Ethyl 6-bromo-4-(dimethylaminomethyl)-5-

hydroxy-1-methyl-2-(phenylsulﬁnylmethyl)-1H-indole-3-

carboxylate (11p). mp: 178–180 °C; ¹H NMR (CDCl₃): d

1.49 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 2.88 (m, 6H,

–N(CH₃)₂), 3.34 (s, 3H, –NCH₃), 4.41 (q, 2H,

J = 7.2 Hz, –OCH₂CH₃), 4.82 (s, 2H, –CH₂N), 5.33

(s, 2H, –sulﬁnyl-CH₂–), 7.44–7.54 (m, 5H, –PhH), 7.56

(s, 1H, –UH), 10.00 (br s, 1H, –OH); MS: m/z 493.0,

495.0 [MH⁺].

193 °C; H NMR (CDCl₃): d 0.93–1.07 (m, 2H, –CH₂–

N–CH(CH₂)₂), 1.22 (m, 2H, –CH₂–N–CH(CH₂)₂),

1.32 (s, 4H, 4H of piperazinyl), 1.42 (t, 3H, J = 7.2 Hz,

–OCH₂CH₃), 2.32 (s, 4H, 4H of piperazinyl), 2.83

(m, 1H, –CH₂–N–CH(CH₂)₂), 4.19 (s, 3H, 4-CH₃of

piperazinyl), 4.32 (q, 2H, J = 7.2 Hz, –OCH₂CH₃),

4.68 (s, 2H, –CH₂N), 4.80 (s, 2H, –sulﬁnyl-CH₂–),

6.99–7.28 (m, 3H, –PhH), 7.71 (s, 1H, –UH); MS: m/z

610.0, 612.0 [MH⁺].

4.1.3.15. Ethyl 6-bromo-4-(dimethylaminomethyl)-2-

[(4-ﬂuorophenyl)sulﬁnylmethyl]-5-hydroxy-1-methyl-1H-

indole-3-carboxylate (11q). mp: 181–183 °C; H NMR

4.2. Biological assay

4.2.1. In vitro anti-HBV assays. The antiviral activities

of compounds 8a–11v against HBV in 2.2.15 cells

were evaluated by methods reported elsewhere. The

in vitro anti-HBV activities included the ability to

inhibit the production of HBsAg and HBeAg and

the replication of HBV DNA in HBV-infected 2.2.15

cells. For the antiviral analyses, conﬂuent cultures of

2.2.15 cells were maintained on 96-well ﬂat-bottomed

tissue culture plates in RPMI 1640 medium with 2%

fetal bovine serum.^12,13Cultures were treated with

eight consecutive daily doses of the test compounds

and lamivudine (purchased by Glaxo & Welcome

Co.). The cell control was set up. Medium was chan-

ged daily with fresh test compounds and positive con-

trol. HBV nucleic acid and protein levels were

measured eight days after the ﬁrst treatment. Extracel-

lular HBV surface (HBsAg) and e (HBeAg) antigen

levels produced from 2.2.15 cells were evaluated by

semiquantitative enzyme immunoassay (EIA) methods

using commercial kits (HBsAg, Abbott Laboratories;

HBeAg, Diasorin, Inc.) as previously described.¹⁴

Intracellular HBV DNA levels were measured by

quantitative Southern blot hybridization.¹³The IC₅₀

and selected index of the evaluated compounds and

lamivudine were calculated, respectively.

(CDCl₃): d 1.43 (t, 3H, J = 7.2 Hz, –OCH₂CH₃), 2.40

(m, 6H, –N(CH₃)₂), 3.50 (s, 3H, –NCH₃), 4.22 (q, 2H,

J = 7.2 Hz, –OCH₂CH₃), 4.35 (s, 2H, –CH₂N), 4.60

(s, 2H, –sulﬁnyl-CH₂–), 7.13–7.50 (m, 4H, –PhH), 7.51

(s, 1H, –UH); MS: m/z 511.0, 513.0 [MH⁺].

4.1.3.16. Ethyl 6-bromo-4-(dimethylaminomethyl)-5-

hydroxy-1-cyclopropyl-2-[(2-pyridinyl)- sulﬁnylmethyl]-

1H-indole-3-carboxylate (11r). mp: 176–178 °C; ¹H

NMR (DMSO-d₆): d 0.91 (m, 2H, –N–CH(CH₂)₂),

1.18 (m, 2H, –N–CH(CH₂)₂), 1.42 (t, 3H, J = 7.2 Hz,

–OCH₂CH₃), 2.29 (m, 1H, –N–CH(CH₂)₂), 2.38 (m,

6H, –N(CH₃)₂), 3.87 (q, 2H, J = 7.2 Hz, –OCH₂CH₃),

4.62 (s, 2H, –CH₂N), 5.54 (s, 2H, –sulﬁnyl-CH₂–),

7.15–7.60 (m, 4H, –pyridinyl H), 7.78 (s, 1H, –UH),

8.85 (br s, 1H, –OH); MS: m/z 520.1, 522.1 [MH⁺].

4.1.3.17. Ethyl 6-bromo-2-[(4-ﬂuorophenyl)sulﬁnylm-

ethyl]-5-hydroxy-4- (morphorinomethyl) -1-methyl-1H-in-

dole-3-carboxylate (11s). mp: 195–197 °C; ¹H NMR

(DMSO-d₆): d 1.31 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

2.44 (s, 4H, 4H of morpholino), 3.57 (s, 3H, –NCH₃),

3.62 (s, 4H, 4H of morpholino), 4.07 (q, 2H,

J = 7.2 Hz, –OCH₂CH₃), 4.17 (d, 2H, –NCH₂–), 4.78

(s, 2H, –sulﬁnyl-CH₂–), 7.36–7.74 (m, 4H, –PhH), 7.81

(s, 1H, –UH); MS: m/z 552.9, 554.9 [MH⁺].

4.2.2. Cytotoxicity assay. Cytotoxicity induced by the

test compounds in cultures of 2.2.15 cells was also

determined. Brieﬂy, 2.2.15 cells were grown to conﬂu-

ence in 96-well ﬂat-bottomed tissue culture plates and

treated with test compound (in 0.2 mL culture medi-

um/well) as described above. Untreated control cul-

tures were maintained on each 96-well plate.

Toxicity was determined by measuring neutral red

dye uptake, as determined from the absorbance at

510 nm relative to untreated cells, at 24 h following

day 9 of treatment.

4.1.3.18. Ethyl 6-bromo-2-[(3-chlorophenyl)sulﬁnylm-

ethyl]-5-hydroxy-4-(morphorinomethyl)-1-methyl-1H-in-

dole-3-carboxylate (11t). mp: 188–190 °C; ¹H NMR

(DMSO-d₆): d 1.30 (t, 3H, J = 7.2 Hz, –OCH₂CH₃),

2.49 (s, 4H, 4H of morpholino), 3.56 (s, 3H, –NCH₃),

3.60 (s, 4H, 4H of morpholino), 4.08 (q, 2H,

J = 7.2 Hz, –OCH₂CH₃), 4.16 (d, 2H, –NCH₂–), 4.81

(s, 2H, –sulﬁnyl-CH₂–), 7.38–7.65 (m, 4H, –PhH), 7.74

(s, 1H, –UH); MS: m/z 568.9, 570.9 [MH⁺].

H. Chai et al. / Bioorg. Med. Chem. 14 (2006) 911–917

917

5. Triﬁmov, F.A.; Tsyshkova, N.G.; Bogdanova, N.S. et al.

US 5198552, 1993-4-30, A61K31/405.

Acknowledgment

6. Leneva, I. A.; Fadeeva, N. I.; Fedykina, I. T. Abst 187, 7th

international conference on Antiviral research, 1994.

7. Drugs of the future, 1992, 17, 1079–1081.

8. Glushkov, R. G.; Guskova, T. A., et al. Vestn. Ross.

Akad. Med. Nauk. 1993, 3, 36–40.

9. Clickman, S. A.; Cope, A. C. J. Am. Chem. Soc. 1946, 67,

1017.

10. Monti, S. A. J. Org. Chem. 1966, 31, 2669.

11. Grinev, A. N.; Pershin, G. N. WO 9008135, 1990.

12. Sells, M. A.; Zelent, A. Z.; Shvartsman, M.; Acs, G.

J. Virol. 1988, 62, 2836–2844.

This work was supported by a grant from the National

Natural Science Foundation of China (20372047).

References and notes

1. Ching, L. L.; Vlad, R.; Man-Fung, Y.; Thierry, P. Lancet

2003, 362, 2089–2094.

2. De clercq, E. J. Clin. Virol. 2004, 30, 115–133.

3. Ganem, D.; Prince, A. M. N. Engl. J. Med. 2004, 350,

1118–1129.

13. Korba, B. E.; Gerin, J. L. Antivir. Res. 1992, 19, 55–70.

14. Korba, B. E.; Gerin, J. L. Antivir. Res. 1995, 28,

225–242.

4. Humphries, J. C.; Dixon, J. S. Intervirology 2003, 46, 413–

420.

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