Design and synthesis of photoactivatable aryl diketo acid-containing HIV-1 integrase inhibitors as potential affinity probes

Article abstract of DOI:10.1016/j.bmcl.2003.12.064

Aryl diketo acids (ADKs) represent an important new class of HIV-1 integrase (IN) inhibitors. In order to facilitate examination of the structural basis underlying IN·ADK interaction, biphenyl ketone and phenyl azide photophores were incorporated into ADK structures. Of particular note is the novel dual utilization of azide and phenyketone moieties for both enzyme recognition and for crosslinking. The resulting analogues maintained low micromolar inhibitory potency against IN in recombinant in vitro assays. These potential HIV-1 integrase photoaffinity labels may provide useful tools for studying enzyme interactions of the ADK inhibitor class.

Full text of DOI:10.1016/j.bmcl.2003.12.064

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1205–1207
Design and synthesis of photoactivatable aryl diketo
acid-containing HIV-1 integrase inhibitors as potential
affinity probes
Xuechun Zhang,a Christophe Marchand,b Yves Pommierb and Terrence R. Burke, Jr.a,*
aLaboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, PO Box B, Bldg.376 Boyles St,.
Frederick, MD 21702-1201, USA
bLaboratory of Molecular, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg.37,
Rm.5C68, Bethesda, MD 20892, USA
Received 15 October 2003; revised 5 December 2003; accepted 12 December 2003
Abstract—Aryl diketo acids (ADKs) represent an important new class of HIV-1integrase (IN) inhibitors. In order to facilitate
examination of the structural basis underlying INꢀ ADK interaction, biphenyl ketone and phenyl azide photophores were incorpo-
rated into ADK structures. Of particular note is the novel dual utilization of azide and phenyketone moieties for both enzyme
recognition and for crosslinking. The resulting analogues maintained low micromolar inhibitory potency against IN in recombinant
in vitro assays. These potential HIV-1integrase photoaffinity labels may provide useful tools for studying enzyme interactions of
the ADK inhibitor class.
Published by Elsevier Ltd.
1. Introduction
for similar derivation as photoaffinity labels. We have
previously reported biphenyl ketone-containing deriva-
tives of coumarin compounds (1a and b, Fig. 1) as HIV-
Along with reverse transcriptase (RT) and protease
(PR), integrase (IN) is an enzyme critically required for
replication of the human immunodeficiency virus type-1
(HIV-1). Although all three enzymes represent targets
for anti-AIDS therapeutic development,1 unlike the first
two enzymes for which drugs are currently marketed,
IN has yet to yield an FDA-approved inhibitor.
Recently a promising new class of aryl diketo (ADK)
inhibitors has emerged,2,3 however a lack of structural
information regarding binding of these inhibitors to IN
has frustrated the structure-based design of next-gen-
eration agents.4,5 Wide precedence exists for the appli-
cation of photoaffinity labeling to elucidate ligand
enzyme interactions.6 Among photophores, biphenyl
ketones and aryl azides have shown particular use as
photo-activatable handles in variety of biologically
relevant systems.7 The high level of interest in ADK
family inhibitors, coupled with the paucity of informa-
tion regarding the structural basis of their interaction
with IN, makes them particularly attractive candidates
8
1IN photoaffinity labels. ADK family IN inhibitors,
which are typified by compounds 2a and b, exhibit
high HIV-1binding affinity that is dependent on both
the nature and location of substituents on the ADK
phenyl ring. For the unsubstitued analogue 2a, inhibi-
tion of HIV-1IN catalyzed strand transfer (ST) 9 occurs
with an IC50 value of 25 mM.10 Introduction of sub-
stituents at the 3-position can enhance inhibitory
potency, as exemplified by 2b (IC50=<0.10 mM)11 and
3 (IC50=0.35 mM).10 Accordingly, the phenyl 3-position
was chosen as the site to incorporate biphenyl ketone
and azide photo-activatable functionality. This resulted
* Corresponding author. Tel.: +1-301-846-5906; fax: +1-301-846-
6033; e-mail: tburke@helix.nih.gov
Figure 1. Structures of HIV-1IN inhibitors and photoaffinity labels.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2003.12.064

1206
X.Zhang et al./ Bioorg.Med.Chem.Lett.14 (2004) 1205–1207
Scheme 1. Reagents and conditions: (i) N-methoxy-N-methylacetamide, n-BuLi, THF, ꢂ78ꢁ C (75% yield); (ii) diethyl oxalate, NaH, toluene, 60ꢁ C
(quantitative); (iii) 1N NaOH, dioxane:H 2O, rt (53% yield).
in selection of diketo acids 4–6 as target compounds
(Fig. 1). Of note, compounds 4 and 6 both utilize phenyl
rings of the parent DKA itself as integral components
of their respective biphenyl ketone and aryl azide pho-
tophores, while compound 5 appends biphenyl ketone
functionality as a separate entity onto the parent DKA
structure. This latter substitution may be viewed as
replacing the benzylic phenyl group of ADK inhibitor 3
with the photophore.
biphenyl ketone moieties onto 9 and 12 was achieved
either by elaborating the phenyl ADK phenyl ring as
one half of the biphenyl ketone (Scheme 1), or by
appending a preformed diphenyl ketone via the ether
linkage using ((4-bromomethyl)phenyl) phenyl ketone
(11, Scheme 2). Synthesis of 3-(azidophenyl) methyl
ketone (15) was accomplished from the corresponding
3-aminophenyl methyl ketone precursor (14, Scheme 3).
3. Biological evaluation
2. Synthesis
Integrase functions in a two step manner: (1) Initial
removal of a dinucleotide unit from the 30-ends of the
viral DNA (termed ‘30-processing’ or 30-P) and (2) Inte-
gration of the 30-processed strands into the host DNA
(termed ‘strand transfer’ or ST). Using viral long term-
inal repeat oligonucleotide sequences as substrate, an in
Preparation of final ADKs 4–6,12 relied on condensa-
tion of appropriate phenyl methyl ketones 9, 12 and 15
with diethyl oxalate to yield ethyl esters 10, 13 and 16,
respectively (Schemes 1–3). Base-catalyzed hydrolysis
provided the title free acids 3–5. Incorporation of
13
vitro recombinant HIV-1integrase assay was used to
evaluate the ability of final products 4–6 to inhibit HIV-
1integrase. The assay provides IC 50 values against both
30-P and ST processes. This capability is important since
as a class, ADK family inhibitors are distinguished by
their selective inhibition of ST versus 30-P catalysis.10,14
Results indicate that all three compounds inhibit ST
processes with IC50 values in the low micromolar range.
The more potent biphenyl ketone-based inhibitor 4,
exhibits approximately 10-fold higher affinity than its
counterpart 5, while aryl azide-containing 6 has an IC50
value between these two. Consistent with other mem-
bers of the AKD family, selective inhibition against ST
is observed relative to 30-P (Table 1).
Scheme 2. Reagents and conditions: (i) K2CO3, 1-(3-hydroxyphenyl)-
ethane-1-one, DMF, 70 ꢁC (56% yield); (ii) diethyl oxalate, NaH,
toluene, 60 ꢁC (quantitative); (iii) 1N NaOH, dioxane:H 2O, rt (66%
yield).
4. Conclusions
Reported herein is the design and synthesis of novel
ADK family HIV-1integrase inhibitors that contain
functionality intended to allow photo-activated cross
coupling. Of particular note is the novel dual utilization
of azide and phenyketone moieties for both enzyme
recognition and for crosslinking. These agents may
potentially prove useful as photoaffinity labels in studies
designed to gain greater understanding of the manner in
which ADK family inhibitors interact with HIV-1IN.
Such information may aid the structure-based design of
next generation agents.
Scheme 3. Reagents and conditions: (i) (a) NaNO2, HCl, H2O, 0 ꢁC;
(b) NaN3, 0 ꢁC (83% yield); (ii) diethyl oxalate, NaH, toluene, 60 ꢁC
(quantitative); (iii) 1N NaOH, dioxane:H 2O, rt (70% yield).
Table 1. Inhibition of HIV-1integrase as measured in an in vitro
assay
IC50 (mM)
Compd
30-P
ST
Acknowledgements
4
5
6
>100
>100
>100
2.7, 2.1
19.0, 29.0
5.3, 5.2
Appreciation is expressed to Dr. James Kelley of the
LMC for mass spectral analysis.

X.Zhang et al./ Bioorg.Med.Chem.Lett.14 (2004) 1205–1207
1207
References and notes
Y.; Nicklaus, M.; Pommier, Y.; Burke, T. R., Jr J.Med.
Chem. 2003, 45, 3184.
1. Pommier, Y.; Marchand, C.; Neamati, N. Antiviral Res.
2000, 47, 139.
2. Goldgur, Y.; Craigie, R.; Cohen, G. H.; Fujiwara, T.;
Yoshinaga, T.; Fujishita, T.; Sugimoto, H.; Endo, T.;
Murai, H.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 13040.
3. Espeseth, A. S.; Felock, P.; Wolfe, A.; Witmer, M.;
Grobler, J.; Anthony, N.; Egbertson, M.; Melamed, J. Y.;
Young, S.; Hamill, T.; Cole, J. L.; Hazuda, D. J. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 11244.
4. Neamati, N.; Marchand, C.; Winslow, H. E.; Pommier,
Y. Antiretroviral Ther. 2001, 87.
5. Young, S. D. Curr.Opin.Drug Disc.Dev. 2001, 4, 402.
6. Jacoby, W. B.; Wilchek, M. Affinity Labeling in Meth.
Enzymol.; Colowick, S. P., Kaplan, N. O., Eds.; Academic
Press, Inc.: New York, NY, 1977; Vol. 46.
7. Dorman, G. Photoaffinity Labeling in Biological Signal
Transduction in Bioorganic Chemistry of Signal Trans-
duction; Waldmann, H., Ed.; Springer-Verlag: Berlin,
2001, Vol. 211, pp 169–229.
11. Wai, J. S.; Egbertson, M. S.; Payne, L. S.; Fisher, T. E.;
Embrey, M. W.; Tran, L. O.; Melamed, J. Y.; Langford,
H. M.; Guare, J. P., Jr.; Zhuang, L.; Grey, V. E.; Vacc,
J. P.; Holloway, M. K.; Naylor-Olsen, A. M.; Hazuda,
D. J. F. P. J.; Wolfe, A. L.; Stillmock, K. A.; Schleif,
W. A.; Gabryelski, L. J.; Young, S. D. J.Med.Chem.
2000, 43, 4923.
12. Analytical data for final products: 2-hydroxy-4-oxo-4-(3-
(phenylcarbonyl)phenyl)but-2-enoic acid (4) (obtained as a
white solid following HPLC purification) mp 93ꢁC (dec),
1H NMR (DMSO-d6) d 8.35 (1H, d, J=8.5 Hz), 8.29 (1H,
s), 8.01(1H, dd, J=6 Hz, 1.5 Hz), 7.78–7.57 (6H, m), 7.08
(1H, s), FAB-MS (ꢂVE) m/z: 295 (MꢂH); 2-hydroxy-4-
oxo-4-(3-((4-(phenylcarbonyl)phenyl)methoxy)phenyl)but-
2-enoic acid (5) mp 107–109ꢁC, 1H NMR (DMSO-d6) d
7.74–7.68 (4H, m), 7.65–7.59 (5H, m), 7.53–7.50 (2H, t,
J=8 Hz), 7.46 (1H, t, J=8 Hz), 7.32 (1H, d, J=8 Hz),
7.05 (1H, s), 5.30 (2H, s), FAB-MS (ꢂVE) m/z: 401(MꢂH);
4-(3-azidophenyl)-2-hydroxy-4-oxobut-2-enoic acid (6) mp
118–120 ꢁC, 1H NMR (DMSO-d6) d 7.87 (1H, d, J=8
Hz), 7.68 (1H, t, J=2 Hz), 7.59 (1H, t, J=7.8 Hz), 7.43
(1H, m), 7.09 (1H, s). FAB-MS (ꢂVE) m/z: 232 (MꢂH).
13. Marchand, C.; Neamati, N.; Pommier, Y. Methods Enzy-
mol. 2001, 340, 624.
8. Zhao, H.; Neamati, N.; Pommier, Y.; Burke, T. R., Jr.
Heterocycles 1997, 45, 2277.
9. 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.
10. Pais, G. C. G.; Zhang, X.; Marchand, C.; Neamati, N.;
Cowansage, K.; Svarovskaia, E. S.; Pathak, V. K.; Tang,
14. Marchand, C.; Zhang, X.; Pais, G. C. G.; Cowasange, K.;
Neamati, N.; Burke, T. R., Jr.; Pommier, Y. J.Biol.
Chem. 2002, 277, 12596.