Novel HIV-1 integrase inhibitors derived from quinolone antibiotics

Article abstract of DOI:10.1021/jm0600139

The viral enzyme integrase is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents a remaining target for antiretroviral drugs. Here, we describe the modification of a quinolone antibiotic to produce the novel integr

Full text of DOI:10.1021/jm0600139

1506
J. Med. Chem. 2006, 49, 1506-1508
Novel HIV-1 Integrase Inhibitors Derived from
Quinolone Antibiotics
Motohide Sato,† Takahisa Motomura,† Hisateru Aramaki,†
Takashi Matsuda,† Masaki Yamashita,† Yoshiharu Ito,†
Hiroshi Kawakami,† Yuji Matsuzaki,† Wataru Watanabe,†
Kazunobu Yamataka,† Satoru Ikeda,† Eiichi Kodama,‡
Masao Matsuoka,‡ and Hisashi Shinkai*,†
Central Pharmaceutical Research Institute, JT Inc.,
1-1 Murasaki-cho, Takatsuki, Osaka, 569-1125, Japan, and Institute
for Virus Research, Kyoto UniVersity, 53 Shogoin Kawaramachi,
Sakyo-ku, Kyoto, 606-8507, Japan
ReceiVed January 6, 2006
Abstract: The viral enzyme integrase is essential for the replication
of human immunodeficiency virus type 1 (HIV-1) and represents a
remaining target for antiretroviral drugs. Here, we describe the
modification of a quinolone antibiotic to produce the novel integrase
inhibitor JTK-303 (GS 9137) that blocks strand transfer by the viral
enzyme. It shares the core structure of quinolone antibiotics, exhibits
an IC50 of 7.2 nM in the strand transfer assay, and shows an EC50 of
0.9 nM in an acute HIV-1 infection assay.
Human immunodeficiency virus type 1 (HIV-1) integrase,
along with HIV-1 reverse transcriptase and HIV-1 protease, is
an essential enzyme for retroviral replication and represents an
important target for interrupting the viral replication cycle.1
HIV-1 integrase first catalyzes removal of the terminal dinucle-
otide from each 3′-end of viral DNA (3′-processing) and
subsequently mediates joining of the 3′-end of the viral DNA
to host DNA (strand transfer).2 Reverse transcriptase inhibitors
and protease inhibitors have already made significant advances
in antiretroviral therapy but cannot achieve complete suppression
and risk producing resistant HIV-1.3,4 On the other hand, despite
numerous attempts to develop integrase inhibitors, only the
diketo acid class of compounds is at an advanced stage of
development and no integrase inhibitors have yet been approved
for therapeutic use.1,5-7 Here, we report that the core structure
of quinolone antibiotics can be used as an alternative to the
diketo acid class of HIV-1 integrase inhibitors and how this
finding led to a novel quinolone integrase inhibitor, JTK-303
(GS 9137).
Figure 1. Structures of the diketo acid family and its new bioisoster.
All bioisosters of the diketo acid motif have the three
functional groups that mimic a ketone, enolizable ketone, and
carboxyl oxygen and can have a coplanar conformation (Figure
1). Therefore, we designed the structure of 4-quinolone-3-
glyoxylic acid 6 as a new scaffold that maintained the copla-
narity of diketo acid functional groups (Figure 1). Interestingly,
not the 4-quinolone-3-glyoxlic acid 6 but its precursor 4-qui-
nolone-3-carboxylic acid 7 showed integrase inhibitory activity.
The 4-quinolone-3-carboxylic acid 7 only had two functional
groups, a â-ketone and a carboxylic acid, which were coplanar.
This result showed that the coplanar monoketo acid motif in
4-quinolone-3-carboxylic acid 7 could be an alternative to the
diketo acid motif and provided novel insight into the structural
requirements and the binding mode of this type of inhibitor.
Quinolone 7 had an IC50 of 1.6 µM in the strand transfer assay,
and structural modification of 7 led to a far more potent integrase
inhibitor 12 with stronger antiviral activity (Table 1). Introduc-
tion of 2-fluoro and 3-chloro substituents into the distal benzene
ring of 7 (8) led to a significant improvement of its inhibition
of strand transfer (IC50 ) 44 nM) and to the appearance of
antiviral activity (EC50 ) 0.81 µM). Compound 9, bearing a
hydroxyethyl group at the 1-position of the quinolone ring, was
1.8-fold more potent at inhibiting strand transfer (IC50 ) 24
nM) and displayed about 11-fold stronger antiviral activity (EC50
) 76 nM) than 8. Introduction of a methoxy group at the
7-position of the quinolone ring of 9 (10) led to a significant
improvement of its inhibition of strand transfer (IC50 ) 9.1 nM)
and of antiviral activity (EC50 ) 17.1 nM). Compound 11,
bearing an isopropyl group at the 1S-position of the hydroxyethyl
moiety, was about 3-fold more potent at inhibiting strand transfer
(IC50 ) 8.2 nM) and about 10-fold stronger at inhibiting HIV
The diketo acid moiety (γ-ketone, enolizable R-ketone, and
carboxylic acid) was believed to be essential for the inhibitory
activity of this series of integrase inhibitors,8 and the structures
of diketotriazole 2,6 diketotetrazole 3,9 diketopyridine 4,10 and
7,11
7-carbonyl-8-hydroxy-(1,6)-naphthyridine 5
were reported
to be bioisosters of the diketo acid pharmacophore (Figure 1).
The carboxylic acid could be replaced with not only acidic
bioisosters, such as tetrazole and triazole, but also by a basic
heterocycle bearing a lone pair donor atom, such as a pyridine
ring. It has been reported that the heteroaromatic nitrogen in
the pyridine ring mimics the corresponding carboxyl oxygen
in the diketo acid as a Lewis base equivalent.11 The enolizable
ketone at the R-position of diketo acids can be replaced with a
phenolic hydroxyl group, indicating that the R-enol form of each
diketo acid is its biologically active coplanar conformation.11
* To whom correspondence should be addressed. Phone: +81 72 681
9700. Fax: +81 72 681 9725. E-mail: hisashi.shinkai@ims.jti.co.jp.
† JT Inc.
‡ Kyoto University.
10.1021/jm0600139 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/15/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1507
Scheme 1a
Table 1. Summary of the Structural Optimization Process for
Quinolone Integrase Inhibitorse
a The strand transfer assay was performed according to the method of
Hazuda14 with some modifications.15 b Antiviral activity was measured by
the acute HIV-1 infection assay16 with some modifications.17 c Prepared
according to the reported method.18 d Available from Wako Pure Chemical
e Data are given as the mean ( SD (n ) 3).
a Reagents and conditions: (a) 1-iodo-4-nitrobenzene, PdCl2(Ph3P)2, THF,
reflux; (b) Zn, AcOH; (c) diethyl ethoxymethylenemalonate, toluene, reflux;
(d) Ph2O, 250 °C; (e) NaOH, EtOH/H2O, reflux; (f) TBSOCH2CH2Br,
K2CO3, DMF, 80 °C; (g) TBAF, THF; (h) NIS, H2SO4; (i) SOCl2, DMF,
toluene, reflux; (j) ethyl 3-(dimethylamino)acrylate, THF, 50 °C; (k) (S)-
valinol, THF; (l) K2CO3, DMF, 70 °C; (m) TBSCl, imidazole, DMF; (n)
13, Pd(dba)2, trifurylphosphine, THF, reflux; (o) NaOH, i-PrOH/H2O, reflux;
(p) NaOMe, MeOH, reflux.
replication (EC50 ) 7.5 nM) than 9, although introduction of
an isopropyl group at the 1R-position of the hydroxyethyl moiety
could not enhance inhibitory acitivity. Introduction of both a
methoxy group at the 7-position of the quinolone ring and an
isopropyl group at the 1S-position of the hydroxyethyl moiety
of 9 (12) led to a synergistic improvement of antiviral acitivity
(EC50 ) 0.9 nM), but there was no additive or synergistic
improvement in the inhibition of HIV-1 integrase (IC50 ) 7.2
nM). This may be due to the condition of the strand transfer
assay using 5 nM of target DNA that influences potencies of
inhibitors.
Preparation of the quinolone analogues (7-12) is shown in
Scheme 1. Palladium-catalyzed coupling of 3-chloro-2-fluo-
robenzylzinc bromide 13, which was derived from the corre-
sponding benzylbromide, with 1-iodo-4-nitrobenzene or 1-iodo-
2-methoxy-4-nitrobenzene (Negishi coupling) and subsequent
reduction of the nitro group gave the aniline 15 or 16.
Condensation of 15, 16, or commercially available 14 with
diethyl ethoxymethylenemalonate and subsequent thermal cy-
clization of the aminoacrylate products in diphenyl ether led to
the quinolone esters 17, 18 and 19.19 Hydrolysis of 17 and 18
gave 7 and 8, respectively. N-Alkylation of 18 or 19 with the
tert-butyldimethylsilyl (TBS) ether of 2-hydroxyethylbromide
and subsequent hydrolysis of the ethyl ester and TBS ether
resulted in 9 or 10. After 5-iodonation of 2-fluorobenzoic acids
20 and 21, the acid chlorides of 20 and 21 were coupled with
ethyl 3-(dimethylamino)acrylate to produce the acrylates 22 and
23, respectively. Substitution with (S)-valinol and subsequent
cyclization with potassium carbonate and protection of the
alcohol with TBS ether gave the quinolones 24 and 25,
respectively.20 Negishi coupling of 24 and 25 with 13 led to
the quinolone esters 26 and 27, respectively. Hydrolysis of 26
gave 11. Hydrolysis of 27 and subsequent methoxylation with
sodium methoxide produced 12.
In summary, modification of quinolone antibiotics, which did
not show HIV-1 integrase inhibitory activity (Table 1), led to
discovery of the coplanar monoketo acid motif in their scaffold,
4-quinolone-3-carboxylic acid, as an alternative to the diketo
acid motif. These novel quinolone integrase inhibitors were
structurally optimized in the highly potent 12, which had little
antibacterial activity although it still retained the core structure
of quinolone antibiotics. Compound 12 was much more potent
at inhibiting integrase-catalyzed strand transfer processes than
3′-processing reactions, as previously reported for compounds
of the diketo acid class.12,13 This indicates that it probably
inhibits HIV-1 integrase via a mechanism similar to that of
diketo acids, although there is no direct evidence (such as
cocrystal data) that the coplanar monoketo acid motif shows
the same mode of binding to the enzyme as the diketo acid

1508 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Letters
(13) Sechi, M.; Derudas, M.; Dallocchio, R.; Dessi, A.; Bacchi, A.; Sannia,
L.; Carta, F.; Palomba, M.; Ragab, O.; Chan, C.; Shoemaker, R.;
Sei, S.; Dayam, R.; Neamati, N. Design and synthesis of novel indole
â-diketo acid derivatives as HIV-integrase inhibitors, J. Med. Chem.
2004, 47, 5298-5310.
(14) Hazuda, D. J.; Hastings, J. C.; Wolfe, A. L.; Emini, E. A. A novel
assay for the DNA strand-transfer reaction of HIV-1 integrase.
Nucleic Acids Res. 1994, 22, 1121-1122.
motif. Clinical studies of the novel quinolone integrase inhibitor
12 (GS 9137) are currently being conducted by Gilead Sciences.
Acknowledgment. We thank H. Isoshima and K. Kondo
for sample preparation; S. Kato for comments on the manuscript;
and S. Ishiguro, J. Haruta, and M. Kano for support.
Supporting Information Available: Analytical data for 5 and
7-12. This material is available free of charge via the Internet at
http://pubs.acs.org.
(15) Donor DNA (which was processed at the 3′ end of the strand and
biotinylated at the 5′ end) was immobilized on streptavidin-coated
microtiter plates. Recombinant integrase (300 nM) was assembled
on the immobilized donor DNA (0.5 pmol per well) in 100 µL of
reaction buffer (30 mM 3-(N-morpholino)propanesulfonic acid
(MOPS), 5 mM MgCl2, 3 mM dithiothreitol (DTT), 0.1 mg/mL
bovine serum albumin (BSA), 5% glycerol, 10% DMSO, 0.01%
Tween-20) by incubation for 60 min at 37 °C. Then excess enzyme
was removed and a test compound was added. The strand transfer
reaction was initiated by addition of target DNA (5 nM), which was
labeled at the 3′ end with digoxigenin. After incubation at 37 °C for
10 min, the plates were washed with phosphate-buffered saline (PBS)
containing 0.1% Tween-20. The digoxigenin-labeled products were
detected using anti-digoxigenin-peroxidase (POD) Fab fragments
(Roche Diagnostics) and a POD substrate, tetramethylbenzidine
(TMB). Then 100 µL of anti-digoxigenin-POD Fab fragment solution
was added to each well, and the plates were incubated at 37 °C for
60 min. After the mixture was washed with PBS containing 0.1%
Tween-20, 100 µL of the POD substrate (TMB) was added to each
well, and the plates were incubated at room temperature. The
colorimetric reaction was stopped by addition of 100 µL of 0.5 M
H2SO4, and the absorbance was measured at 450 nm by a microplate
reader (SPECTRA max 340, Molecular Devices).
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JM0600139