Efficient synthesis and utilization of phenyl-substituted heteroaromatic carboxylic acids as aryl diketo acid isosteres in the design of novel HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.047

Three new types of aryl diketo acid (ADK) isosteres were designed by conversion of the biologically labile 1,3-diketo unit into heteroaromatic motif such as isoxazole, isothiazole, or 1H-pyrazole to improve the physicochemical property of ADK-based HIV-1 integrase (IN) inhibitors. The synthesis of the heteroaromatic carboxylic acids was established by employing phenyl β-diketoester or benzaldehyde as the starting material and 1,3-dipolar cycloaddition as the key reaction. Of the compounds tested, the 3-benzyloxyphenyl-substituted isoxazole carboxylic acid displayed the best IN inhibitory and antiviral activities, with N-hydroxylamidation enhancing the in vitro and in vivo potency. These findings are important for further optimization of ADK-based IN inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.07.047

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4521–4524
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis and utilization of phenyl-substituted heteroaromatic
carboxylic acids as aryl diketo acid isosteres in the design of novel HIV-1
integrase inhibitors
Li-Fan Zeng a, Hu-Shan Zhang a, Yun-Hua Wang b, Tino Sanchez c, Yong-Tang Zheng b, Nouri Neamati c,
Ya-Qiu Long a,
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China
b Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, China
c Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new types of aryl diketo acid (ADK) isosteres were designed by conversion of the biologically labile
1,3-diketo unit into heteroaromatic motif such as isoxazole, isothiazole, or 1H-pyrazole to improve the
physicochemical property of ADK-based HIV-1 integrase (IN) inhibitors. The synthesis of the heteroaro-
matic carboxylic acids was established by employing phenyl b-diketoester or benzaldehyde as the start-
ing material and 1,3-dipolar cycloaddition as the key reaction. Of the compounds tested, the 3-
benzyloxyphenyl-substituted isoxazole carboxylic acid displayed the best IN inhibitory and antiviral
activities, with N-hydroxylamidation enhancing the in vitro and in vivo potency. These findings are
important for further optimization of ADK-based IN inhibitors.
Received 10 June 2008
Revised 9 July 2008
Accepted 11 July 2008
Available online 15 July 2008
Keywords:
HIV-1 integrase inhibitor
Bioisostere
Heteroaromatic carboxylic acid
Isoxazole
Ó 2008 Elsevier Ltd. All rights reserved.
Isothiazole
1H-Pyrazole
Bioavailability
Antiviral effect
The Pol gene of retrovirus HIV-1 encodes three essential en-
zymes—protease (PR), reverse transcriptase (RT), and integrase
(IN). IN mediates the integration of proviral DNA into the cell gen-
ome, which is essential for retroviral replication and chronic infec-
tion, and homologous enzymes are lacking in the human host.1
Thus IN has become an attractive target for anti-HIV therapeutics.
The function of IN is achieved in a stepwise fashion by endonucleo-
lytic processing of proviral DNA (termed 30-processing or 30-P)
within a cytoplasmic preintegration complex, followed by translo-
cation of the complex into the nuclear compartment where inte-
gration of 30-processed proviral DNA into host DNA occurs in a
reaction referred to as strand transfer.
Among an impressive number of synthetic IN inhibitors re-
ported, the aryl diketo acids (ADK) are the most promising and ad-
vanced class which selectively inhibit the strand transfer reaction
in extracellular assays and exhibit good antiviral effects against
HIV-infected cells.2–4 Because diketo acid is biologically labile, a
variety of bioisosteres were developed such as 8-hydroxy-1,6-
naphthyridine, 4-oxo-1,4-dihydroquinoline-3-carboxylic acid, and
6-oxo-1,6-dihydro pyrimidin-5-olate whose derivatives were sub-
sequently developed into clinical candidates (Fig. 1). The applica-
tion of the ADK isosteres facilitated the FDA-approval of the first
IN inhibitor, MK-0518 (Raltegravir), into a successful anti-HIV
drug.5
The emergence of viral strains resistant to clinically studied IN
inhibitors and the dynamic nature of the HIV-1 genome demand
a continued effort toward the discovery of novel inhibitors to keep
a therapeutic advantage over the virus. A major focus of our labo-
ratories is to design a new class of ADK bioisosteres by conversion
of the biologically labile diketo moiety into metabolically stable
heteroaromatic group. In this study, we designed and synthesized
three types of phenyl-substituted heteroaromatic carboxylic acids
(Fig. 2). The phenyl substitution and the relative orientation of the
phenyl and the carboxylic acid functionality were examined with
respect to the effect on the IN inhibition and HIV replication.
The phenyl-substituted isoxazole- or 1H-pyrazole-3-carboxylic
acids were conveniently synthesized by coupling the aryl diketo
* Corresponding author. Tel./fax: +86 21 50806876.
E-mail address: yqlong@mail.shcnc.ac.cn (Y.-Q. Long).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.047

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L.-F. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4521–4524
Figure 1. The structures of ADK-derived IN inhibitors clinically studied. The dashed circle indicated the moiety of diketo acid isostere.
Figure 2. The design of phenyl-substituted heteroaromatic carboxylic acids as aryl diketo acid isosteres.
ester with hydroxylamine or hydrazine under different conditions.
As depicted in Scheme 1, the variously substituted acetophenones
were oxalylated by dimethyl oxalate in the presence of sodium
tert-butoxide, affording b-diketoester 6a,b in high yields.6 Treat-
ment of the aryl b-diketoester with hydroxylammonium chloride
in refluxing methanol yielded isoxazole ester 7a,b,7 which was
hydrolyzed to give 5-phenylisoxazole-3-carboxylic acid 1a,b. Fur-
ther treatment of isoxazole ester 7 with hydroxylammonium chlo-
ride and diluted solution of NaOH generated 5-phenyl-N-
hydroxyisoxazole-3-carboxamide 8. Toward another direction,
the aryl diketo ester 6 was readily converted into pyrazole ester
by coupling with hydrazine in the presence of acetic acid,8 which
was then hydrolyzed into 5-phenyl-1H-pyrazole-3-carboxylic
acids 3a,b.
Contrary to 5-phenyl-isoxazole-3-carboxylic acid, the synthesis
of 3-phenyl-isoxazole-5-carboxylic acid adopted a different strat-
egy by employing a 1,3-dipolar cycloaddition of benzonitrile N-
oxide with methyl propiolate as the key step. As shown in Scheme
2, starting from the substituted benzaldehyde, the treatment with
hydroxylammonium chloride and sodium acetate afforded benzal-
Scheme 1. General synthetic route toward 5-phenyl-isoxazole-3-carboxylic acid and 5-phenyl-1H-pyrazole-3-carboxylic acid. Reagents and conditions: (a) t-BuONa,
(CH3OOC)2, THF, 0 °C–rt; (b) NH2OHꢀHCl, CH3OH, reflux, 3 h; (c) 1 N NaOH, THF, 60 °C, 1 h; (d) 12.5 equiv, 1 N NaOH, 10 equiv, NH2OHꢀHCl, CH3OH, rt, 2 h; (e) hydrazine, acetic
acid, reflux, 1 h; (f) hydroxylammonium chloride, 2 N NaOH, methanol, 80 °C, 1 h.

L.-F. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4521–4524
4523
Scheme 2. Synthetic approach to 3-phenyl-isoxazole-5-carboxylic acid. Reagents and condition: (a) NH2OHꢀHCl, CH3COONa, methanol/H2O, reflux, 8 h; (b) NCS, cat. pyridine,
THF, 60 °C, 30 min; (c) TEA, rt, 10 min; (d) methyl propiolate, rt; (e) 2 N NaOH, methanol, 80 °C, 1 h.
dehyde oxime 10. The oxime was chlorinated with NCS (N-chloro-
succinimide) and a catalytic amount of pyridine in THF at 60 °C un-
der nitrogen atmosphere. The reaction mixture was treated with
Et3N in THF over 10 min followed by addition of methyl propiolate,
during which the benzonitrile N-oxide 12 was generated in situ.
The 1,3-dipolar cycloaddition of benzonitrile N-oxide with methyl
propiolate yielded the isoxazole ester 13,9 which was hydrolyzed
to produce 3-phenylisoxazole-5-carboxylic acid 2.
Similarly, the synthesis of isothiazole-5/4-carboxylic acid in-
volved a 1,3-dipolar cycloaddition reaction of nitrile sulfide gener-
ated by thermal decarboxylation of the corresponding 1,3,4-
oxathiazole-2-one, as shown in Scheme 3. The substituted phen-
yloxathiazolone 15 was readily prepared by heating substituted
benzamide 14 with chlorocarbonylsulfenyl chloride in refluxing
dioxane for 4.5 h. And the benzamide 14 was directly converted
from the substituted benzaldehyde by one-pot tandem reaction,10
whereby the benzaldehyde reacted with iodine in 25% ammonia
water at room temperature followed by addition of urea hydrogen
peroxide and K2CO3 to produce the corresponding amide. The cou-
pling of nitrile sulfide with methyl propiolate afforded almost
equal ratio isomers 16 and 17, which were distinguished by the
chemical shift of the hydrogen on the isothiazole ring.11 The iso-
thiazole-5/4-carboxylic acid esters were hydrolyzed to the corre-
sponding acids 4 and 5, respectively.
the electron-donating group was preferred over the electron-with-
drawing group for the phenyl-substituted heteroaromatic carbox-
ylic acids as IN inhibitors. In accord, the 5-(3-
(benzyloxy)phenyl)isoxazole-3-carboxylic acid (compound 1a)
exhibited an IC50 value of 68
while 5-(4-nitrophenyl)isoxazole-3-carboxylic acid was 1.4-fold
less potent (compound 1b, IC50 = 97 M).
lM against strand transfer reaction,
l
On the other hand, the relative location of the phenyl ring and
the carboxylic acid functionality on the heteroaromatic ring was
important for the potency. The 5-phenylisoxazole-3-carboxylic
acid (compounds 1a,b) was a superior scaffold to the 3-phenylisox-
azole-5-carboxylic acid (compounds 2a,b) for IN inhibition, thus
the 5-(3-(benzyloxy)phenyl)isoxazole-3-carboxylic acid (1a,
IC50 = 68
lM) was more potent than the 3-(3-(benzyl-
oxy)phenyl)isoxazole-5-carboxylic acid (2a, IC50 = 81
l
M) in inhib-
iting the strand transfer reaction. Further modification on 5-
phenylisoxazole-3-carboxylic acid by carboxyl amidation with
hydroxylamine resulted in a 2.5-fold enhancement on the strand
transfer inhibitory potency (8a, IC50 = 27
compound 1a.
lM) relative to the parent
The secondary active structure in this series was 5-phenyl-1H-
pyrazole-3-carboxylic acid, of which the 3-benzyloxyphenyl-
substituted analog displayed moderate activity against the strand
transfer reaction (3a, IC50 = 100 lM). However, the introduction
HIV-1 integrase inhibitory activity of phenyl-substituted heteroar-
omatic carboxylic acids. We examined the inhibitory activity of the
new class of ADK isosteres on IN catalytic function. As shown in
Table 1, no inhibition against 30-processing reaction was observed
of sulfur atom in the heteroaromatic ring caused a substantial loss
of the IN inhibitory activity. The resulting isothiazole-5/4-carbox-
ylic acids were almost inactive against IN regardless of the substi-
tution and spatial arrangement of the phenyl ring.
for the three types of ADK isosteres at the concentration of 100
l
M,
Antiviral potency against HIV-1IIIB in C8166 cell cultures of the phe-
nyl-substituted heteroaromatic carboxylic acids. Although the IN
inhibitory activity was not as potent as we expected, these ADK
isosteres exert potent to moderate inhibition on the cytopathic ef-
fect of HIV-1 in infected C8166 cells, which indicated that the iso-
stere surrogate did possess better bioavailability. Furthermore, the
which is consistent with the general character of aryl diketo acid
functioning as selective strand transfer inhibitors. Of these hetero-
aromatic carboxylic acids, the phenyl-substituted isoxazole-car-
boxylic acids exhibited best potency against the strand transfer
reaction. With respect to the substitution effect on the phenyl ring,
Scheme 3. The synthetic route to 3-phenylisothiazole-5/4-carboxylic acid. Reagents and conditions: (a) 1.1 equiv I2, 25% ammonia solution, rt, 1 h; then urea hydrogen
peroxide, K2CO3, THF, rt, 2–4 h; (b) chlorocarbonylsulfenyl chloride, dioxane, reflux, 4.5 h; (c) methyl propiolate, xylene, reflux, 26 h; (d) THF, 1 N NaOH, rt, 2 h.

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L.-F. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4521–4524
Table 1
The IN inhibitory activity and antiviral effect of the substituted phenyl heteroaromatic acids and derivatives
a
Compound
R
X
Y
R0
Inhibition of HIV-1 integrase (IC50
)
Anti-HIV-1 activityb
TIe
c
d
30-Processing (
lM)
Strand transfer (
lM)
EC50
(l
M)
CC50 (lM)
1a
1b
8a
2a
3a
3b
9b
4a
5a
3-BnO
4-NO2
3-BnO
3-BnO
3-BnO
4-NO2
4-NO2
3-BnO
3-BnO
O
O
O
N
N
N
N
N
N
N
N
N
O
N
N
N
S
3-COOH
3-COOH
3-CONHOH
5-COOH
3-COOH
3-COOH
3-COOMe
5-COOH
4-COOH
>100
>100
>100
>100
>100
>100
>100
>100
>100
68
97
27
91
206
>854
136
282
640
>857
>809
246
2.27
>4.3
2.17
39.25
2.31
>238
>3.2
4.76
>11
199
62.8
7.2
277
3.6
253
51.7
58
81
100
>100
>100
>100
>100
S
>642
a
HIV-1 integrase inhibitory activity was measured according to the procedure described in Ref. 12.
Anti-HIV-1 data represent the mean values of two separate experiments.
b
c
Effective concentration required to protect C8166 cells against the cytopathogenicity of HIV-1 by 50%.13
Cytostatic concentration required to reduce C8166 cell proliferation by 50% tested by MTT method.13
Therapeutic index (TI) is a ratio of the CC50 value/EC50 value.
d
e
three types of novel ADK isosteres displayed weak cytotoxicity
(CC50 fell within 200–800 M), resulting in high therapeutic index.
Acknowledgments
l
For the 5-phenylisoxazole-3-carboxylic acid series, the antiviral
effect was correlated with the IN inhibitory activity. The best IN
inhibitor, that is, 5-(3-(benzyloxy)phenyl)-N-hydroxyisoxazole-3-
National Natural Science Foundation of China (30672528), Sci-
ence and Technology Commission of Shanghai Municipality
(07QH14018), Key Scientific and Technological Projects of China
(2004BA719A14) and Yunnan province (2004NG12) are greatly
appreciated for the financial supports.
carboxamide (8a) exhibited an EC50 value of 62.8 lM. However,
the 5-phenyl-1H-pyrazole-3-carboxylic acid and 3-phenylisoxaz-
ole-5-carboxylic acid displayed potent antiviral activity though
they behaved as weak IN inhibitors. The best anti-HIV-potency
was exhibited by 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylic acid
(3b) and 3-(3-(benzyloxy)phenyl)isoxazole-5-carboxylic acid (2a)
References and notes
1. Asante-Appiah, E.; Skalka, A. M. Antivir. Res. 1997, 36, 139.
2. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev. Drug Discov. 2005, 4, 236.
3. Pais, G. C. G.; Burke, T. R. Drugs Future 2002, 27, 1101.
with an EC50 value of 3.6 and 7.2 lM, respectively. More impor-
tantly, the two active compounds possessed significantly low cyto-
toxicities with a TI value of >238 and 39, respectively. Even the
isothiazole-5/4-carboxylic acid class which was inactive against
IN exerted moderate activity to protect C8166 cells from HIV-1
4. Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock, K.; Grobler, J. A.;
Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau, C.; Miller, M. D. Science 2000, 287,
646.
5. Evering, T. H.; Markowitz, M. Expert Opin. Investig. Drugs 2008, 17, 413.
6. (a) Jiang, X.-H.; Song, L.-D.; Long, Y.-Q. J. Org. Chem. 2003, 68, 7555; (b) Jiang, X.-
H.; Long, Y.-Q. Chin. J. Chem. 2004, 22, 978.
7. Pei, Y.; Wichham, B. O. S. Tetrahedron Lett. 1993, 34, 7509.
8. Van Herk, T.; Brussee, J.; van den Nieuwendijk, A. M. C. H.; van der Klein, P. A.
M.; Ijzerman, A. P.; Stannek, C.; Burmeister, A.; Lorenzen, A. J. Med. Chem. 2003,
46, 3945.
9. Cha, M. Y.; Choi, B. C.; Kang, K. H.; Pae, A. N.; Choi, K. I.; Cho, Y. S.; Koh, H. Y.;
Lee, H.-Y.; Jungc, D.; Kong, J. Y. Bioorg. Med. Chem. Lett. 2002, 12, 1327.
10. Shie, J.-J.; Fang, J.-M. J. Org. Chem. 2003, 68, 1158.
infection (4a, EC50 = 51.7 lM; 5a, EC50 = 58 lM) with low cytotox-
icity. On the one hand, the good antiviral potency of the heteroar-
omatic carboxylic acid series might be attributable to the improved
bioavailability of the bioisosteres as we anticipated; on the other
hand, the inconsistency between IN inhibitory activity and the
antiviral effect might involve multiple targeting in the HIV-1 life
cycle. Thus, further investigation on the mechanism of 5-phenyl-
1H-pyrazole-3-carboxylic acid and 3-phenylisoxazole-5-carboxylic
acid as anti-HIV agents is under way.
In conclusion, we designed and synthesized three types of ADK
isosteres by conversion of the 1,3-diketo unit into isoxazole, 1H-
pyrazole, and isothiazole moieties. The resulting 5-(3-(benzyl-
oxy)phenyl)-N-hydroxyisoxazole-3-carboxamide (8a) displayed
an IC50 value of 27 lM against IN, whereas the best antiviral effect
was exhibited by 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylic acid
(3b) and 3-(3-(benzyloxy)phenyl)isoxazole-5-carboxylic acid (2a)
with an EC50 value of 3.6 and 7.2 lM, respectively. The phenyl-
substituted heteroaromatic carboxylic acids afforded advanta-
geous features of improved antiviral potency and decreased cyto-
toxicity with high therapeutic index, providing promising new
approach and scaffold to develop potent anti-HIV agents.
11. Brownsort, P. A.; Paton, R. M. J. Chem. Soc., Perkin Trans. I 1987, 2339.
12. Integrase Assays procedure: To determine the extent of 30-processing and strand
transfer, wild-type IN was preincubated at a final concentration of 200 nM
with the inhibitor in reaction buffer (50 mM NaCl, 1 mM Hepes pH 7.5, 50
EDTA, 50 dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/ml
bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethylsulfoxide, and
25 mM MOPS pH 7.2) at 30 °C for 30 min. An aliquot (5 l) was
lM
lM
l
electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris–borate,
pH 8.3, 2 mM EDTA, 20% acrylamide, 8 M urea). Gels were dried, exposed in a
PhosphorImager cassette, and analyzed using a Typhoon 8610 Variable Mode
Imager (Amersham Biosciences) and quantitated using ImageQuant 5.2.
Percent inhibition (% I) was calculated using the following equation:
%
I = 100 ꢁ [1 ꢂ (D ꢂ C)/(N ꢂ C)], where C, N, and D are the fractions of 21-mer
substrate converted to 19-mer (30-processing product) or strand transfer
products for DNA alone, DNA plus IN, and IN plus drug, respectively. The IC50
values were determined by plotting the logarithm of drug concentration versus
percent inhibition to obtain concentration that produced 50% inhibition.
13. Wang, J. H.; Tam, S. C.; Huang, H.; Ouyang, D. Y.; Wang, Y. Y.; Zheng, Y. T.
Biochem. Biophys. Res. Commun. 2004, 317, 965.