Development of the next generation of HIV-1 integrase inhibitors: Pyrazolone as a novel inhibitor scaffold

Article abstract of DOI:10.1016/j.bmcl.2010.08.057

HIV-1 integrase (IN), one of the essential enzymes in HIV infection, has been validated as a target for HIV treatment. While more than 20 drugs have been approved by the FDA to treat HIV/AIDS, only one drug, Raltegravir (1), was approved as an IN inhibitor. The rapid mutation of the virus, which leads to multidrug resistant HIV strains, presents an urgent need to find potent compounds that can serve as second-generation IN inhibitors. The pyrazolone scaffold, predicted by a computational modeling study using GS-9137(2) as a pharmacophoric model, has shown to inhibit the IN catalytic activities in low micromolar range. We have synthesized various analogs based on the pyrazolone scaffold and performed SAR studies. This paper will showcase the up-to-date result of this scaffold as a promising HIV-1 IN inhibitor.

Full text of DOI:10.1016/j.bmcl.2010.08.057

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6854–6857
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of the next generation of HIV-1 integrase inhibitors:
Pyrazolone as a novel inhibitor scaffold
Victor Hadi a, Yung-Hyo Koh a, Tino Wilson Sanchez b, Danielle Barrios b, Nouri Neamati b,
,
⇑
Kyung Woon Jung a,
⇑
a Loker Hydrocarbon Research Institute, Department of Chemistry, University of Southern California, Los Angeles, CA 90089, United States
b Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
HIV-1 integrase (IN), one of the essential enzymes in HIV infection, has been validated as a target for HIV
treatment. While more than 20 drugs have been approved by the FDA to treat HIV/AIDS, only one drug,
Raltegravir (1), was approved as an IN inhibitor. The rapid mutation of the virus, which leads to multidrug
resistant HIV strains, presents an urgent need to find potent compounds that can serve as second-gener-
ation IN inhibitors. The pyrazolone scaffold, predicted by a computational modeling study using GS-
9137(2) as a pharmacophoric model, has shown to inhibit the IN catalytic activities in low micromolar
range. We have synthesized various analogs based on the pyrazolone scaffold and performed SAR studies.
This paper will showcase the up-to-date result of this scaffold as a promising HIV-1 IN inhibitor.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 19 July 2010
Accepted 11 August 2010
Available online 17 August 2010
Keywords:
HIV
Integrase inhibition
Pyrazolone scaffold
Acquired immunodeficiency syndrome (AIDS) is a debilitating
disease affecting more than 39 million people worldwide. Since the
disease was first detected in 1981, more than 25 million people have
died of AIDS.1 Consequently, the World Health Organization recog-
nizes HIV as the fourth largest cause of death globally.1 As the most
recent advancement toward eradication of this pandemic, a highly
successful combination therapy of three HIV drugs known as HAART
(highly active antiretroviral therapy) is considered as the best treat-
ment against AIDS so far. However, increasing drug resistance due to
rapid mutation of this retrovirus has prompted the faster develop-
ment of many reserved drugs against resistant viral strains.
HIV-1 integrase (IN) has been validated to be a crucial target for
shutting down the HIV reproductive cycle. This viral enzyme
catalyzes the insertion of proviral DNA into the host genome in
two-step processes: (1) 30-processing step that removes two nucle-
otides from the 30-hydroxyl end of its viral DNA and (2) strand
transfer step, which joins 30-end of the viral DNA onto the host
DNA by nucleophilic addition. Additionally, the presences of diva-
lent metals such as Mg2+ or Mn2+ are required for the catalysis of
these two steps.2
rently in late-stage clinical trials.4 GS-9137 showed highly potent
antiretroviral activity in both treatment-naive and experienced
patients in the Phase II clinical trials. Like other IN inhibitors, resis-
tant mutants against this drug have been identified in the catalytic
core domain of the IN.5
Although the recent success in the discovery and the clinical
development of IN inhibitors as novel antiretroviral agents provided
us a new choice in the treatment of HIV/AIDS, multidrug resistant
HIV-1 strains have emerged as a result of the dynamic nature of
the HIV genome coupled with the requirement of a sustained anti-
retroviral treatment regimen in chronic HIV-1 patients. Once the first
generation of IN inhibitors becomes commonly used in the clinic, the
emergence of resistant HIV-1 virus strains containing mutations in
the IN is inevitable. Therefore, alternative second generation agents
with improved resistance profiles are urgently needed.
It has been our goal to design and discover novel and structur-
ally diverse second-generation IN inhibitors that show favorable
inhibition profiles against known resistant mutant strains. We
quickly became interested in the work of Dayam et al.6 who
Two compounds demonstrate effective inhibition of IN (Fig. 1).
Presently the only FDA approved drug is Raltegravir (MK-0518, 1),
which selectively inhibits the strand transfer step and has shown
an excellent efficacy in HIV-1 patients.3 Another IN inhibitor,
GS-9137 (Elvitegravir, 2), a quinolone 3-carboxylic acid, is cur-
⇑
Corresponding authors. Tel.: +1 323 442 2341; fax: +1 323 442 1390 (N.N.);
Tel.: +1 213 740 8768; fax: +1 213 821 4096 (K.W.J.).
Figure 1. Examples of clinically used HIV-1 integrase inhibitors.
E-mail addresses: neamati@usc.edu (N. Neamati), kwjung@usc.edu (K.W. Jung).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.057

V. Hadi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6854–6857
6855
Table 1
reported a series of potential lead compounds for IN inhibitor by
HIV-1 integrase inhibitory activity of compound 4 analogs
using structure-based pharmacophore model derived from quino-
lone 3-carboxylic acids (such as in GS-9137). Among the com-
pounds, the pyrazolone 3 is the most potent in inhibiting IN and
its IC50 is in the low micromolar range.
Compds R1
30-Processing
IC50 M)
Strand transfer
(l
IC50
(lM)
The preliminary study of this pyrazolone scaffold prompted us
to develop a more diversified library that includes phenyl-substi-
tuted pyrazolone compounds. This pyrazolone can be modified
on three possible sites (R1, R2, and R3) as shown in Figure 2. The
R1 modification involves the benzylidene ring and its benzyloxy
substituent of compound 3, which is believed to bind to the hydro-
phobic region in the active site of IN. The R2 modification will
change the functional group in the proximity of the pyrazolone’s
carbonyl group, which is crucial for metal chelation inside the en-
zyme active site. Lastly, R3 modification could tune the electronic
factors on the pyrazolone core ring.
For modification on the benzylidene ring of the pyrazolone, the
following 4a–4g analogs were synthesized as depicted in Scheme
1. This modification will determine the factors needed for increas-
ing binding affinity to the metal ion inside the IN active site. They
are as follows: the position of the methoxy group, the position of
the benzyloxy group, and the linker space joining two aromatic
rings. Starting with 3-bromoaniline (7), the corresponding hydra-
zine was synthesized quantitatively via a diazotiation reaction fol-
lowed by tin (II) chloride reduction. The resulting hydrazine was
reacted with trifluoroacetoacetate in boiling ethanol to yield the
pyrazolone 8, which was then reacted with various aromatic alde-
hydes (R1CHO)7a in boiling water to yield the desired analogs bear-
ing different benzylidene tethers.
4a
89 10
>100
34
2
4b
4c
4d
4e
>100
61
50
83
7
8
5
15
30
40
3
7
9
4f
>100
22
11
3
1
4g
21
1
These compounds were subjected to in vitro testing, and the re-
sults (Table 1) showed that compound 4g was the most potent
compound in this R1 modified library with an IC50 of 11
for inhibition on strand transfer activity.
1 lM
Since the second type of alteration is crucial for the metal che-
lation ability of pyrazolone compounds, the choice of starting
hydrazines should be important. Cyclization of various hydrazines
9a–9o with ethyl trifluoroacetoacetate in boiling ethanol yielded
the corresponding pyrazolone intermediates 10a–10o, which were
then treated with substituted benzaldehyde 11 to generate the R2
modified pyrazolone analogs (Scheme 2). Unfortunately, several
attempts to make pyrazolones with an ortho-halophenyl R2 substi-
tuent (Cl and Br) were not successful due to decomposition of the
intermediates when refluxing in boiling water. Also, the syntheses
of pyrazolones with heterocyclic R2 groups (such as pyridine,
pyrimidine, and tetrazole) were not as straightforward as those
of their phenyl-substituted pyrazolone counterparts. The last con-
densation step to synthesize these heterocyclic pyrazolones failed
to generate the desired products even after prolonged reaction
time.
The in vitro results of R2 modified pyrazolones are summarized
in Table 2. The presence of larger substituents (–Ph and –I) was
more favored than the smaller substituents (–F and –CF3). Meta
Figure 2. Three possible sites of modification in the pyrazolone scaffold.
Scheme 1. Synthesis of R1 modified pyrazolone compounds. Reagents and condi-
tions: i) NaNO2, HCl, 0 °C, then SnCl2.2H2O; ii) Ethyl trifluoroacetoacetate, EtOH,
reflux; iii) R1CHO, H2O, reflux.
Scheme 2. Synthesis of R2 modified pyrazolone compounds. Reagents and condi-
tions: i) ethyl trifluroacetoacetate, EtOH, reflux; ii) 3-(4-fluorobenzyloxy)-4-
methoxybenzaldehyde (11), H2O, reflux.

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V. Hadi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6854–6857
Table 2
HIV-1 integrase inhibitory activity of compound 5 analogs
Compds
R2
30-Processing
IC50 M)
Strand transfer
IC50 M)
(
l
(l
5a
5b
5c
5d
5e
5f
5g
5h (=4e)
5i
5j
5k
5l
Phenyl
m-Tolyl
>100
>100
90 13
>100
52 13
>100
88 24
>100
>100
m-Trifluoromethylphenyl
m-Phenylphenyl
m-Nitrophenyl
m-Fluorophenyl
m-Chlorophenyl
m-Bromophenyl
m-Iodophenyl
3,5-Dichlorophenyl
p-Bromophenyl
p-Iodophenyl
24
16
19
4
5
2
60 0.1
Scheme 3. Synthesis of R3 modified pyrazolone compounds. Reagents and condi-
tions: i) (for 12a-12c), methyl acetoacetate, methyl propionacetate, or methyl
trifluoroacetoacetate in boiling EtOH; (for 12d), methyl cyanoacetoacetate in EtOH,
then trifluoroacetic anhydride; (for 12e), dimethylacetone-1,3-dicarboxylate in
AcOH, 100 °C; (for 12g), diethyl acetylenedicarboxylate, K2CO3 in EtOH; ii) 3-(4-
fluorobenzyloxy)-4-methoxybenzaldehyde (11), H2O, reflux; (for 4f and 4h) LiOH,
MeOH/THF/H2O.
30
40
35
12
25
69
6
9
3
1
6
9
83
5
>100
>100
82
1
>100
>100
>100
>100
5m
5n
5o
p-Carboxylphenyl
p-Sulfonyamidophenyl
Benzothiazolyl
41 12
>100
27
6
a better inhibitory activity (ST IC50 of 11 lM). Puzzled with this
result, we elongated the tether of the second benzyl group as in
compound 14b. Finally, switching the position of the fluorobenzyl
group provided compound 14d which has the lowest ST IC50 so far
substitution was slightly more preferred to the para substitution.
The best compound in this group was 5j, which has IC50 value of
(3 0.4 lM).
Utilizing Autodock Vina8 to validate this experimental finding
(Table 4), we began checking the lowest energy conformations of
compounds 14a–14d. Compound 14d was the only compound
with the anticipated conformation, where the carbonyl in the pyr-
azolone core was aligned closely to metal cofactor, so it would dis-
rupt the IN activity toward cleaving DNA strand and/or transferring
its viral DNA by sequestering the active site of IN (Fig. 3a). On the
other hand, the rest of optimized compounds (14a–14c) interacted
with metal ion through their carboxylate moieties. We postulated
that the absence of the methyl on the benzylidene ring gave rise to
extra stabilization possibly through hydrogen bonding donor inter-
action with His67, while the presence of the carboxylate functional
group also serves as a hydrogen bond acceptor from Asn155 as
shown in Figure 3b. The nitro substituent gave an extra stabiliza-
tion through hydrogen bond acceptor with Gln148.
12
1
l
M for the strand transfer reaction.
The role of the last modification is to investigate the effect of the
5-substituent of the pyrazolone ring on IN inhibition. Table 3 lists
many analogs bearing different R3 substituents on the ring. We
predicted that variation of the electronic nature of the pyrazolone
through electron donating (NHC(@O)CF3), electron withdrawing
(CF3, CO2H, CO2Et), or weakly interacting substituents (Me and
Et) may assist in determination of the electronic factor necessary
for binding toward the metal inside the IN active site. In general,
synthesis of this analog was accomplished by cyclization of 12 with
a variety of ethyl acetate derivatives to generate intermediates 13,
followed by sequential condensation to form pyrazolones 6. The
compounds 6a–6c which contain different aliphatic carbon R3
groups (e.g., Me, Et, and CF3) were synthesized from the reaction
of m-bromophenyl hydrazine with methyl acetoacetate, methyl
propionoacetate, and methyl trifluoroaceto-acetate, respectively,
followed by subsequent condensation with 11. For the synthesis
of 6d, 12 was condensed with ethyl cyanoacetate7b, acylated with
trifluoroacetic anhydride to generate 13d, and then condensed fur-
ther with 11 to give 6d. The similar two-step protocol was carried
out for the synthesis of 6e–6h utilizing but-2-ynedioic acid diethyl
ester and dimethylacetonedicarboxylate7c as depicted in Scheme 3.
The presence of electron donating or electron neutral group did
not increase the inhibitory activity against the IN. However, the
presence of electron withdrawing group such as CF3 and CO2H in-
creased the inhibitory activity. Compound 6h exhibited the most
In conclusion, we have exploited SAR studies of pyrazolone
scaffold for IN which was discovered through pharmacophore
models. The compound 14d exhibited single-digit micromolar
activity against IN strand transfer process. This work is expected
to provide helpful information for the discovery of novel IN
inhibitor.
Table 4
HIV-1 integrase inhibitory activity of second round optimized compounds
Compds
R1
30-
Processing transfer
Strand
potent analog with ST IC50 of 19
3 lM.
To conclude the optimization study, we combined the best fea-
tures obtained from the prior optimization studies. The combina-
tion of these features for each modification effort yielded a
second prototype compound 14a, which after bioassay did not give
IC50
(lM) IC50 (lM)
14a
14b
15
11
Table 3
60 14
14
10
3
4
HIV-1 integrase inhibitory activity of compound 6 analogs
Compds
R3
30-Processing
IC50 M)
Strand transfer
IC50 M)
(
l
(l
6a
6b
6c (=5h)
6d
6e
6f
6g
6h
Methyl
Ethyl
>100
>100
>100
>100
14c
14d
95
21
7
8
1
Trifluoromethyl
Trifluoroacetamide
Methyl homoacetate
Homoacetate
Ethyl carboxylate
Carboxylate
83
5
40
9
>100
>100
>100
>100
>100
100
57
0.4
88 11
19
55
5
3

V. Hadi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6854–6857
6857
Figure 3. (A) Predicted binding conformations of compound 14d inside the HIV-1 integrase active site (modified PDB1QS4). The lowest energy conformation is represented as
a solid stick representation, while the rests are the other possible conformation. The docking analyses were performed around the green sphere which represents the metal
active site. (B) Predicted protein–ligand interaction between compound 14d and HIV-integrase. Three residues (H67, N155, and Q148) show plausible interactions with this
docked compound.
Grinsztein, B.; Nguyen, B. Y.; Katlama, C.; Getell, J. M.; Lazzatin, A.; Vittecoq, D.;
Gonzalez, C. J.; Chen, J.; Harvey, C. M.; Isaacs, R. D. Lancet 2007, 369, 1261.
Acknowledgment
4. (a) Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.; Yamashita, M.; Ito, Y.;
The authors thank General Clinical Research Center Grant
(M01-RR000043) and the National Institute of Health (S10
RR025432) for generous financial support.
Kawakami, H.; Matsuzaki, Y.; Watanabe, W.; Yamataka, K.; Ikeda, S.; Kodama, E.;
Matsuoka, M.; Shinkai, H. J. Med. Chem. 2006, 49, 1506; (b) DeJesus, E.; Berger,
D.; Markowitz, M.; Cohen, C.; Kawkins, T.; Ruene, P.; Elion, R.; Farthing, C.;
Zhong, L.; Cheng, A. K.; McColl, D.; Kearney, B. P. J. Acquir. Immune Defic. Syndr.
2006, 43, 1; (c) Sato, M.; Kawakami, H.; Motomura, T.; Aramaki, H.; Matsuda, T.;
Yamashita, M.; Ito, Y.; Matsuzaki, Y.; Yamataka, K.; Ikeda, S.; Shinkai, H. J. Med.
Chem. 2009, 52, 4869.
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