Structure-activity relationship study of phenylpyrazole derivatives as a novel class of anti-HIV agents

Article abstract of DOI:10.1016/j.bmcl.2013.06.026

The structure-activity relationship of phenylpyrazole derivative 1 was investigated for the development of novel anti-HIV agents. Initial efforts revealed that the diazenyl group can be replaced by an aminomethylene group. In addition, we synthesized various derivatives by the reductive amination of benzaldehydes with 5-aminopyrazoles and carried out parallel structural optimization on the benzyl group and the pyrazole ring. This optimization led to a six-fold more potent derivative 32j than the lead compound 1, and this derivative has a 3′,4′-dichloro-(1,1′-biphenyl)-3-yl group.

Full text of DOI:10.1016/j.bmcl.2013.06.026

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4557–4561
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of phenylpyrazole derivatives
as a novel class of anti-HIV agents
Tsukasa Mizuhara a, Takayuki Kato b, Atsushi Hirai b, Hideki Kurihara b, Yasuhiro Shimada b,
Masahiko Taniguchi b, Hideki Maeta b, Hiroaki Togami c, Kazuya Shimura c, Masao Matsuoka c,
Shiho Okazaki a, Tomoki Takeuchi a, Hiroaki Ohno a, Shinya Oishi a, Nobutaka Fujii a,
⇑
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Pharmaceutical & Healthcare Research Laboratories, FUJIFILM Corporation, Kaisei, Kanagawa 258-8577, Japan
c Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationship of phenylpyrazole derivative 1 was investigated for the development
of novel anti-HIV agents. Initial efforts revealed that the diazenyl group can be replaced by an aminom-
ethylene group. In addition, we synthesized various derivatives by the reductive amination of benzalde-
hydes with 5-aminopyrazoles and carried out parallel structural optimization on the benzyl group and
the pyrazole ring. This optimization led to a six-fold more potent derivative 32j than the lead compound
1, and this derivative has a 30,40-dichloro-(1,10-biphenyl)-3-yl group.
Received 15 May 2013
Revised 5 June 2013
Accepted 11 June 2013
Available online 19 June 2013
Keywords:
Anti-HIV agent
Phenylpyrazole
Ó 2013 Elsevier Ltd. All rights reserved.
Human immunodeficiency virus (HIV) infection remains one of
the most serious threats to public health that causes acquired
immunodeficiency syndrome,1 in which progressive failure of the
immune system allows life-threatening opportunistic infections.
According to estimates in the UNAIDS Report 2012, ꢀ34 million
people worldwide are living with HIV and ꢀ2.5 million people
are newly infected with HIV.2 This global health threat has trig-
gered intensive drug discovery efforts for a number of anti-HIV
drugs, including nucleoside reverse transcriptase inhibitors (NNR-
TIs), non-nucleoside reverse transcriptase inhibitors (NNRTI), pro-
tease inhibitors (PIs), fusion inhibitors, integrase inhibitors and CC
chemokine receptor type 5 (CCR5) antagonists.3 Highly active anti-
retroviral therapy involving co-administration of NRTIs, NNRTIs
and PIs suppresses HIV replication to control disease progression
in HIV-infected patients.4 However, the emergence of drug-resis-
tant HIV variants5 and drug-related adverse effects such as dyslipi-
demia6 hinder treatment to control chronic HIV replication. To
overcome these problems, the development of novel effective
anti-HIV agents with fewer adverse effects are needed.
During the course of our research project for the development
of novel anti-HIV agents by random screening of an in-house small
molecule library,7 we identified a phenyldiazenylpyrazole com-
pound 1 that showed submicromolar anti-HIV activity (Fig. 1). To
the best of our knowledge, the biological activities of the phen-
yldiazenylpyrazole derivatives have not been documented. Herein,
we report the structure–activity relationship study from a lead
compound 1, including substitution of the diazo linkage, and the
modification of the right benzene and left N-phenylpyrazole parts.
The structure–activity relationship study began by improving
the stability of the diazo linkage between the amidobenzene and
phenylpyrazole groups, because diazo compounds can be exten-
sively metabolized by intra- and extra-cellular enzymatic compo-
nents of intestinal bacteria.8 To explore the biomimetic
equivalents of the diazo structure, we designed a styrene deriva-
tive 2 and an aminomethylene derivative 3 (Fig. 1).
Synthesis of compound 2 is outlined in Scheme 1. Pyrazole
derivative 5 was obtained from the condensation of (Z)-3-amino-
2-(2-chloroacetyl)but-2-enenitrile 4 and phenylhydrazine. Sepa-
rately, FeCl3-mediated reduction of the nitro group of compound
7 followed by acylation provided propionamidophenol 9, which
was subjected to formylation using hexamethylenetetramine to af-
ford aldehyde 10.9 Benzyl-protected aldehyde 11 was obtained by
alkylation of 10 with benzyl bromide. Reaction of aldehyde 11
using phosphonium salt 6 gave an olefin 12. Subsequent exposure
of compound 12 to TMSI provided the desired styrene derivative 2.
Abbreviations: HIV, human immunodeficiency virus; NNRTI, non-nucleoside
reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI,
protease inhibitor.
⇑
Corresponding author. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail address: nfujii@pharm.kyoto-u.ac.jp (N. Fujii).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.026

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T. Mizuhara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4557–4561
Table 1
Me
CN
H
N
Structure–activity relationship of the
linkage part
N
1
N
O
N
µ
M
EC50 = 0.32 0.040
N
OH
a
µ
M
CC 50 = 10
Compound
EC50 (lM)
Cl
1
2
3
0.32 0.04
0.44 0.12
0.36 0.11
Substitution with Potential Biomimetic
Equivalents of the Diazo Structure
a
EC50 values represent the concen-
tration of compound required to inhibit
the HIV infection by 50%. The data were
obtained from three independent
experiments.
Me
CN
Me
CN
H
N
H
N
N
N
O
O
N
N
N
OH
H
OH
Table 2
Structure–activity relationships of the benzylamine moiety
Cl
Cl
Me CN
2
3
R1
R2
N
Figure 1. Structures of lead compound 1 and the designed derivatives 2 and 3.
N
N
H
R3
Me
CN
CN
R
N
a
g
Compound
R1
R2
R3
EC50 (lM)
a
Me
N
Cl
NH2
O
3
NHCOEt
NHCOEt
NHCOEt
NHCOEt
NHCOEt
NHCOMe
NHCO-n-Pr
NHCO-n-pentyl
CONHEt
OH
H
Cl
Cl
Cl
H
Me
Cl
Cl
Cl
Cl
0.36 0.11
>10
>10
Me
CN
NHCOEt
R
24a
24b
24c
24d
24e
24f
24g
24h
4
N
OMe
OH
OH
OH
OH
OH
OH
5 (R = Cl)
N
b
>10
>10
6 (R = P+Ph3Cl-)
Cl
>1.0b
3.3 0.89
>10
R
12 (R = OBn)
2 (R = OH)
OH
h
Cl
NHCOEt
Cl
R
e
>1.0b
CHO
a
7 (R = NO2)
8 (R = NH2·HCl)
9 (R = NHCOEt)
EC50 values represent the concentration of the compound required to inhibit the
c
d
10 (R = OH)
11 (R = OBn)
HIV-1 infection by 50%. The data were obtained from three independent
f
experiments.
b
Cytotoxicity was observed at 10 lM.
Me
CN
Me
CN
NHCOEt
OH
Me
EtO
CN
CN
i
j
N
N
NH2
N
N
N
H
these derivatives 24a–h started with the synthesis of various benz-
aldehydes 20 (Scheme 2). Briefly, nitration of 3-chlorobenzoic acid
15 followed by subsequent functional group transformations
including Fe-mediated reduction, acylation, reduction of the car-
boxylic group and PCC oxidation gave aldehyde 20a. Formylation
of 7 using hexamethylenetetramine gave a phenol 20e, which
was subjected to subsequent O-methylation to give a methoxy-
benzaldehyde 20b. Nitration of 4-hydroxy-3-methyl-benzalde-
hyde 21 gave compound 20d. Condensation of a salicylic acid 22
with ethylamine followed by formylation furnished aldehyde
20h. Reactions of benzaldehydes 20a–e11 and 20h with 5-amino-
pyrazole 14 gave benzylamine derivatives 24a,h and 25b–e. FeCl3-
mediated reduction of benzylamines 25b–e followed by acylation
afforded derivatives 24b–g.
Cl
13
14
3
Scheme 1. Synthesis of compounds 2 and 3. Reagents and conditions: (a) Ph-
NHNH2, AcOH, MeOCH2CH2OH, 105 °C; (b) PPh3, toluene, 110 °C; (c) FeCl3,
NH2NH2ꢁH2O, activated carbon, i-PrOH, 80 °C, then 4 M-HCl/1,4-dioxane; (d)
EtCOCl, pyridine, THF, rt; (e) hexamethylenetetramine, AcOH, 130 °C; (f) BnBr,
K2CO3, CHCl3/MeOH, 60 °C; (g) n-BuLi, THF, 0 °C, then 11, THF, rt; (h) TMSI, MeCN,
80 °C; (i) Ph-NHNH2, EtOH, rt; (j) 10, PPTS, toluene, reflux, then LiBH4, THF, 0 °C to
rt.
Compound 3 was also synthesized by using benzaldehyde 10. The
treatment of phenylhydrazine with acrylonitrile 13 gave 5-amino-
pyrazole derivative 14. The reductive amination of aldehydes 10
with 14 provided the desired aminomethylene derivative 3.
With two derivatives having potentially equivalent linkers
available, we then began our investigation for the structure–activ-
ity relationships of lead compound 1 for anti-HIV activity (Table 1).
The bioevaluation of compounds 2 and 3 by the NCK assay10 re-
vealed that styrene derivative 2 and aminomethylene derivative
3 had comparable anti-HIV activity to the original compound 1
Removal of the 4-hydroxy or 3-chloro group on the benzene
ring resulted in a loss of anti-HIV activity (24a and 24c, EC50 >10 -
l
M) (Table 2). Substitution with either a 4-methoxy or 3-methyl
group also led to loss of anti-HIV activity (24b and 24d, EC50 >10 -
M), indicating that the 4-hydroxy group and the 3-chloro group
l
are essential functional groups for anti-HIV activity. In the study
of the N-acyl group at the 5-propionamido moiety, a significant de-
crease in anti-HIV activity was observed by substitution of the 5-
propionamido group with the shorter acetamido group (24e,
(EC50 = 0.44 and 0.36 lM, respectively). These results encouraged
us to extend the structural optimization from the potent aminom-
ethylene compound 3.
EC50 >1.0
and hexanamido groups (24g) (EC50 >10
transformation of the propionamido group with the ethylcarba-
moyl group did not improve anti-HIV activity (24h, EC50 >1.0 M).
Of note, compounds 24e and 24h showed cytotoxicity at 10 M.
l
M), and the longer butyramido (24f, EC50 = 3.30
lM)
First, to investigate the structural requirements of the right 3-
chloro-4-hydroxy-5-propionamidobenzylamine part in compound
3, a variety of derivatives 24a–h with modification on the benzene
ring were designed and synthesized (Table 2). The synthesis of
lM). The retroamide
l
l

T. Mizuhara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4557–4561
4559
Table 3
Cl
Cl
R
Cl
NHCOEt
Structure–activity relationship of the pyrazole moiety
a
d
R1
R2
NHCOEt
OH
CO2H
15
CO2H
R
N
16 (R = NO2)
17 (R = NH2)
18 (R = NHCOEt)
19 (R = CH2OH)
20a (R = CHO)
N
N
b
e
H
R3
c
Cl
OR
OH
OH
Compound
R1
R2
R3
EC50 (lM)
a
Cl
NO2
Me
Me
a
NO2
f
7
3
Me
H
Me
Me
Me
Et
CN
CN
H
CN
CN
CN
CN
CO2Me
Ph
Ph
Ph
Me
0.36 0.11
>1.0b
>10
29a
29b
29c
29d
29e
29f
29g
CHO
20e (R = H)
20b (R = Me)
CHO
21
CHO
20d
>10
g
c-hexyl
>1.0b
>10
Ph
Ph
Ph
OH
Ph
Me
>10
OH
Cl
Cl
CONHEt
f
>1.0b
R
a
EC50 values represent the concentration of the compound required to inhibit the
HIV-1 infection by 50%. The data were obtained from three independent
CHO
20h
22 (R = CO2H)
23 (R = CONHEt)
experiments.
h
b
Cytotoxicity was observed at 10 lM.
Me
CN
Me
CN
R1
R2
R1
R2
i
i or j
N
N
R1
N
R2
NH2
R1
N
R2
N
N
H
N
14
N
R1
R2
H
NHCOEt
OH
a
b
Cl
R3
N
N
N
EtO
CN
H
R3
R3
13 (R1 = Me, R2 = CN)
24a (R1 = NHCOEt; R2 = H)
24h (R1 = CONHEt; R2 = OH)
25b-e (R1 = NO2)
Cl
27a (R1 = H, R2 = CN)
k
l
28a-g (R3 = Ph, Me, c-hexyl)
29a-g
31a-q
26b-e (R1 = NH2·HCl)
24b-d (R1 = NHCOEt)
(R1 = Me, R2 = H)
30a-q (R3 = Ar)
27b
27e (R1 = Et, R2 = CN)
27f (R1 = Ph, R2 = CN)
Me
CN
Me
CN
(R1 = Me, R2 = CO2Me)
27g
NH2·HCl
OH
NHCO-R
m
N
N
Me
CN
Me
CN
N
N
N
N
OH
H
H
NHCOEt
OH
NHCOEt
OH
N
N
c
Cl
Cl
N
N
N
N
H
H
Cl
Cl
26e
24e (R = Me)
(R = n-Pr)
24f
Br
Ar
(R = n-pentyl)
24g
31c
32a-m
Scheme 2. Synthesis of compounds 24a–h with a variety of substituted benzyl-
amine moieties. Reagents and conditions: (a) H2SO4, HNO3, 0 °C to rt; (b) Fe, NH4Cl,
i-PrOH, H2O, 90 °C; (c) EtCOCl, Et3N, THF, 0 °C to rt; (d) EtOCOCl, Et3N, THF, ꢂ10 °C,
then NaBH4, MeOH, 0 °C to rt; (e) PCC, CH2Cl2, rt; (f) hexamethylenetetramine,
AcOH, 130 °C; (g) MeI, K2CO3, DMF, rt; (h) CBr4, Et3N, EtNH2, PPh3, CH2Cl2, rt; (i) 20,
PPTS, toluene, reflux, then LiBH4, THF, 0 °C to rt; (j) 20, TsOH, toluene, reflux, then
NaBH4, EtOH, 0 °C to rt; (k) FeCl3, NH2NH2ꢁH2O, activated carbon, i-PrOH, 80 °C, then
4 M-HCl/1,4-dioxane; (l) EtCO2H, EDC, HOBt, (i-Pr)2NEt, DMF, 45 °C; (m) RCO2H,
EDC, HOBt, (i-Pr)2NEt, DMF, 45 °C.
Scheme 3. Synthesis of compounds 29a–g, 31a–q, and 32a–m with a variety of N-
arylpyrazole and N-alkylpyrazole moieties. Reagents and conditions: (a) R3-NHNH2,
EtOH, rt; (b) 10, PPTS, toluene, reflux, then LiBH4, THF, 0 °C to rt; (c) Ar-B(OH)2 or
Ar-Bpin, Pd(dba)2, PCy3, K3PO4, 1,4-dioxane, H2O, 80 °C.
meta-position of the N-phenyl ring with methyl (31b), fluoro
(31d), or phenyl groups (31g) resulted in comparable or more po-
tent anti-HIV activity (EC50 = 0.37, 0.33 and 0.13 lM, respectively)
to that of compound 3, whereas a significant decrease in anti-HIV
activity was observed following the introduction of a methoxy
(31a), nitro (31f), or acetamido group (31h). Anti-HIV activity of
bromo (31c) or trifluoromethyl (31e) derivatives could not be
determined because of their significant cytotoxicity. The methyl
modification at the ortho- and para-positions (31i and 31j) afforded
We next examined substituent effects on the left pyrazole part
of compound 3 (Tables 3–5). A variety of 5-aminopyrazoles 28a–g
and 30a–q were prepared by reaction of 3-ethoxyacrylonitrile
derivatives (13 and 27) with aryl- or alkyl-hydrazines (Scheme 3).
Reductive amination of 28a–g and 30a–q with benzaldehyde 10
provided the series of pyrazole derivatives 29a–g and 31a–q. 1-
Biaryl-5-aminopyrazole derivatives 32a–m were synthesized using
a Suzuki–Miyaura cross-coupling reaction of compound 31c with
aryl boronic acid or the pinacol ester.
reduced anti-HIV activities (EC50 >1.0
3,5-dichlorophenyl group showed no antiviral activity (EC50 >1.0 -
M). In addition, replacement of the phenyl group with naphthyl
(31l and 31m), 3-pyridyl (31o), or quinolinyl group (31p and
31q) failed to improve anti-HIV activity (EC50 >1.0 M). 2-Pyridyl
lM). Compound 31k with a
l
l
Removal of the 3-methyl or 4-cyano group on the pyrazole 3 re-
derivative 31n showed unexpected levels of cytotoxicity.
duced anti-HIV activity (29a and 29b, EC50 >1.0 lM), as shown in
The optimization studies indicated that the introduction of a
biphenyl-3-yl group on the pyrazole moiety (31g) effectively im-
proved the anti-HIV activity. To develop more potent anti-HIV
agents, we extended the structure–activity relationship investiga-
tions to the modified aryl groups onto the N-phenylpyrazole moi-
Table 3. In addition, no potent derivatives were obtained by all of
our attempts including substitutions of the N-phenyl group with
alkyl groups (29c and 29d), substitutions of the 3-methyl group
with either an ethyl (29e) or phenyl group (29f), and substitution
of the 4-cyano group with a methoxycarbonyl group (29g).
We turned to examine the structure–activity relationship of the
N-aryl moiety on the pyrazole (Table 4). The modification at the
ety (Table 5). We designed and synthesized
a variety of
methoxyphenyl, tolyl or chlorophenyl derivatives (32a–i). Among

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T. Mizuhara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4557–4561
Table 4
Table 5
Structure–activity relationship of the N-arylpyrazole moiety
Structure–activity relationship of the N-biarylpyrazole moiety
Me
CN
NHCOEt
OH
N
N
N
Ar
H
R
Cl
a
Compound
Ar
EC50 (lM)
a
Compound
Ar
EC50 (lM)
R
32a
32b
32c
R = OMe
R = Me
R = Cl
>0.1c
>0.1c
3
31a
R = H
R = OMe
R = Me
R = Br
0.36 0.11
>10
0.25 0.13b
31b
31c
31d
31e
31f
0.37 0.11b
>0.1c
32d
32e
32f
R = OMe
R = Me
R = Cl
>0.1c
R
0.058 0.007c
R
R = F
0.33 0.12b
>0.1c
>0.1c
R = CF3
R = NO2
R = Ph
>1.0b
32g
32h
32i
R = OMe
R = Me
R = Cl
>0.1c
0.097 0.040c
31g
31h
0.13 0.03c
>10
>0.1c
R = NHCOCH3
R
Cl
32j
0.047 0.011c
31i
>1.0b
Cl
Me
32k
32l
>0.1c
>1.0b
Me
31j
>1.0b
>1.0b
N
31k
32m
>0.1c
Cl
Cl
N
a
EC50 values represent the concentration of the compound required to inhibit the
>1.0b
HIV-1 infection by 50%. The data were obtained from three independent
31l
experiments.
b
Cytotoxicity was observed at 10
Cytotoxicity was observed at 1.0
l
l
M.
M.
c
31m
31n
31o
31p
>1.0b
>0.1c
>10
In conclusion, we have designed and synthesized a series of
phenylpyrazole derivatives for the development of novel anti-HIV
agents. The structure–activity relationship study demonstrated
that the aminomethylene group can be used as an appropriate isos-
teric unit of the diazo linkage in the lead compound 1. The substit-
uents on the right phenol part and left pyrazole ring are essential
for the potent anti-HIV activity. The optimization study on N-phen-
ylpyrazoles led to the identification of a potent 30,40-dichloro-(1,10-
biphenyl)-3-yl derivative 32j12 which exhibited six-fold more po-
tent anti-HIV activity when compared with compound 1. Although
the target molecules remain unclear, further optimization of these
compounds to improve the potency and selectivity should provide
a therapeutic approach to HIV infection.
N
N
>1.0b
N
31q
>10
Acknowledgements
N
a
EC50 values represent the concentration of the compound required to inhibit the
This work was supported by Health and Labor Science Research
Grants (Research on HIV/AIDS, Japan); Grants-in-Aid for Scientific
Research and Platform for Drug Discovery, Informatics, and Struc-
tural Life Science from MEXT, Japan. We thank Ms. Kumiko Hiyama
and Dr. Kazunobu Takahashi for their technical supports. T.M. and
T.T. are grateful for JSPS Research Fellowships for Young Scientists
and Strategic Young Researcher Overseas Visits Program for Accel-
erating Brain Circulation.
HIV-1 infection by 50%. The data were obtained from three independent
experiments.
b
Cytotoxicity was observed at 10
Cytotoxicity was observed at 1.0
l
l
M.
M.
c
these, significantly improved anti-HIV activity was observed in 3-
tolyl (32e) and 4-tolyl (32h) derivatives (EC50 = 0.058 and
0.097
(32j) also exhibited more potent anti-HIV activity when compared
with compound 31g (EC50 = 0.047 M). In contrast, naphthyl (32k),
lM, respectively). Additionally, the 3,4-dichloro derivative
References and notes
l
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activity. Of note, three potent derivatives (32e, 32h and 32j) exhib-
ited moderate cytotoxicity at 1.0
lM; therefore, improvement in
the selectivity index is needed by further optimizations.

T. Mizuhara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4557–4561
4561
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12. Compound 32j: 1H NMR (400 MHz, CDCl3) d: 1.27–1.24 (m, 4H), 2.33 (s, 3H),
2.49–2.42 (m, 2H), 4.55 (d, J = 5.9 Hz, 2H), 4.64 (t, J = 5.9 Hz, 1H), 7.08 (d,
J = 2.0 Hz, 1H), 7.74–7.35 (m, 9H). HRMS (FAB): m/z calcd for C27H23Cl3N5O2
[M+H]+ 554.0917; found: 554.0916.
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