4-Aryl-2,4-dioxobutanoic acid inhibitors of HIV-1 integrase and viral replication in cells [1]

Full text of DOI:10.1021/jm000176b

© Copyright 2000 by the American Chemical Society
Volume 43, Number 26
December 28, 2000
Communications to the Editor
istry and structure-activity relationships (SAR) of a
series of diketo acids derived from 1.
4-Ar yl-2,4-d ioxobu ta n oic Acid In h ibitor s
of HIV-1 In tegr a se a n d Vir a l Rep lica tion
in Cells
J ohn S. Wai,*,† Melissa S. Egbertson,†
Linda S. Payne,† Thorsten E. Fisher,†
Mark W. Embrey,† Lekhanh O. Tran,†
J effrey Y. Melamed,† H. Marie Langford,†
J ames P. Guare, J r.,† Linghang Zhuang,†
Vanessa E. Grey,† J oseph P. Vacca,†
M. Katharine Holloway,‡ Adel M. Naylor-Olsen,‡
Daria J . Hazuda,§ Peter J . Felock,§ Abigail L. Wolfe,§
Kara A. Stillmock,§ William A. Schleif,§
Lori J . Gabryelski,§ and Steven D. Young†
Resu lts a n d Discu ssion . Replacing the central
pyrrole ring of 1 with a series of aromatic systems
having various substitution patterns provided a quick
survey of the optimum relative orientation of the benzyl
and diketo acid side chains. This variable is a function
of the angle between the two lines extended from the
benzyl and diketo acid side chains into the aromatic
systems (Table 1). In the preliminary survey, the set of
compounds prepared did not have a fluoro substituent
on the distal benzene ring as in lead compound 1. The
intrinsic potency of these inhibitors increases as the
angle of bisection increases from 60° to 118° (Table 1,
compounds 2-5). Compound 5 has a 1,3-disubstituted
central benzene ring, and its activity appears to exceed
the sensitivity limits of the strand-transfer enzyme
assay at 0.1 µM.3 This increase in potency against HIV
integrase translates into improvement in inhibitory
activity against replication of HIV-1 in cell culture.4 No
cytotoxicity was observed with these inhibitors at
concentrations up to 50 µM as determined by cell
viability.5 As the angle of bisection increases further, a
gradual loss in both inhibitory activities against the
enzyme and HIV-1 replication in cell culture was
observed (Table 1, compounds 6-8).
Departments of Medicinal Chemistry, Molecular Systems,
and Antiviral Research, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received April 19, 2000
In tr od u ction . Human immunodeficiency virus type
1 (HIV-1) is the etiological agent of acquired immune-
deficiency syndrome (AIDS). The unique nature of the
replicative cycle of HIV-1 provides many potential
targets for chemotherapeutic intervention. One of these,
the viral integrase, catalyzes the insertion of the provi-
ral DNA into the genome of the host cell. Integration is
a multistep process which includes three different
biochemical processes: assembly of proviral DNA on
integrase, endonucleolytic processing of the proviral
DNA, and strand transfer of the proviral DNA to host
cell DNA.1 Recently, diketo acid derivative 1 was
reported to be a selective inhibitor of the strand-transfer
process. This compound effectively prevents proviral
DNA integration and inhibits HIV-1 replication in cell
culture.2 In this Communication, we describe the chem-
The significant difference in cell potency of 1 versus
the corresponding desfluoro analogue 3 (CIC95 9.6 µM
vs 42.0 µM, respectively) prompted us to substitute
different positions of the distal benzene ring of com-
pound 5. Table 2 summarizes the results. Introduction
of a chloro substituent at the 2′-position of the distal
* To whom correspondence should be addressed at: Department of
Medicinal Chemistry, WP14-3, Merck Research Laboratories, P.O. Box
4, West Point, PA 19486. Tel: 215-652-3020. Fax: 215-652-3971.
E-mail: john_wai@merck.com.
† Department of Medicinal Chemistry.
‡ Department of Molecular Systems.
§ Department of Antiviral Research.
10.1021/jm000176b CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/28/2000

4924 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Communications to the Editor
Ta ble 1. Inhibition of HIV-1 Integrase Catalytic Activities and
Ta ble 2. Inhibition of HIV-1 Integrase Catalytic Activities and
HIV-1 Replication in Cells by a Series of Diketo Acids
HIV-1 Replication in Cells by a Series of Diketo Acids
a
Each angle of bisection is an average of 10-26 determinations
based on X-ray coordinates of similarly substituted heterocyclic/
aromatic compounds. Assays were performed with recombinant
b
HIV-1 integrase (0.1 µM) preassembled on immobilized oligonucle-
otides. Inhibitors were added after assembly and washings. For
details see ref 3. c 95% Cell culture inhibitory concentrations
(CIC95) are defined as those which inhibited by 95% the spread of
HIV-1 infection in cell culture, using the HIV-1IIIb variant and
MT-4 T-lymphoid cells. For details see ref 4.
a
b
See ref 3. See ref 4.
Sch em e 1a
a
Reagents: (a) PhMgBr, THF; (b) Et3SiH, BF3‚Et2O, CH2C12; (c) n-BuLi, CH3CON(OCH3)CH3, THF; (d) (CO2CH3)2, NaOCH3, THF,
then NaOH, H2O; (e) 1,3-dibromobenzene, n-BuLi, THF; (f) Zn(CN)2, (Ph3P)4Pd, DMF; (g) EtOH or i-PrOH, KHMDS, THF; (h) CH3MgI,
benzene.

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4925
Ta ble 3. Inhibition of HIV-1 Integrase Catalytic Activities and
HIV-1 Replication in Cells by a Series of Diketo Acids
whether the improvement in antiviral activity is due
to increased intrinsic potency or to a change in physical
properties which improves cell penetration.
Scheme 1 depicts the chemistry employed in the
preparation of this series of 3-benzylphenyl diketo acids.
For the preparation of compounds 15-17, treatment of
the appropriately substituted 3-bromobenzaldehyde 20
with phenylmagnesium bromide, followed by exposure
of the resulting crude adduct to triethylsilane in the
presence of boron trifluoride etherate,6 provided the
corresponding 3-benzylphenyl bromide 21. Bromide 21
was then lithiated, and the resulting solution was
treated with N-methoxy-N-methylacetamide to provide
ketone 22.7 Treatment of 22 with dialkyl oxalate and
sodium alkoxide provided the intermediate ester ad-
ducts,8 which were hydrolyzed in situ to provide the
target diketo acids. For the preparation of compounds
5 and 9-14, the required bromide 21 was prepared by
treatment of benzaldehyde or a suitably substituted
halobenzaldehyde 23 with 3-bromo-l-lithiobenzene, fol-
lowed by exposure of the resulting crude adduct to
triethylsilane in the presence of boron trifluoride ether-
ate. Further elaboration as described above provided the
target compounds. For the synthesis of the 2-ethoxy and
2-isoproxy analogues 18 and 19, the required acetophe-
none was prepared in three steps (Scheme 1, steps f-h).
Treatment of 3-benzyl-6-fluoro-l-bromobenzene with
zinc cyanide in the presence of tetrakis(triphenylphos-
phine)palladium(0) provided nitrile 24.9 Compound 24
was exposed to a mixture of an alcohol and KHMDS10
and then to methylmagnesium iodide to provide the
appropriately substituted ketone 22.11
Con clu sion . In summary, modification of a screening
lead 1 provided a series of potent 3-benzylphenyl diketo
acid based HIV-1 integrase inhibitors. The most active
compounds from this series inhibit replication of HIV-1
in cell culture at CIC95 0.10-0.62 µM. This result
represents a 100-fold improvement in potency versus
the lead 1. Furthermore, compound 19 is only 2-fold less
potent than the protease inhibitor indinavir (CIC95 0.05
µM) in the same assay (Table 3). Cytotoxicity was not
observed in cell culture at concentrations up to 50 µM.
Further work on this approach to new antiviral agents
to treat HIV infection will be reported in due course.
a
b
See ref 3. See ref 4.
benzene ring leads to a slight improvement in inhibition
of HIV replication. Chloro substitution at the 3′-position
has no effect, while at the 4′-position it is not well-
tolerated (Table 2, compounds 5 vs 9-11). Similarly,
only a slight improvement in inhibition of HIV replica-
tion was observed with the introduction of a 2′- or 4′-
fluoro substituent, and a moderate loss in potency was
observed when it was introduced at the 3′-position
(Table 2, compounds 5 vs 12-14). In these compounds
(5, 9-14), the benzyl group and diketo acid side chains
are spread further apart than in pyrrole analogues (1,
3), and the benzyl group may have already extended
into the region responsible for the potency enhancement
observed with a fluorine substitution in the pyrrole
series.
Effect of substitution on the central benzene ring was
investigated. Introduction of a methoxy group at the 3-
or 4-position of the central benzene ring leads to a drop
in antiviral activity (Table 3, compounds 15 and 16).
However, introduction of a 2-methoxy group leads to a
significant improvement in potency against replication
of HIV-1 in cell culture (Table 3, compound 17, CIC95
0.62 µM, vs compound 5, CIC95 1.11 µM). Further
improvement is observed with ethoxy and isopropoxy
substitutions (Table 3, compounds 18 and 19). Since the
activity of inhibitors 5 and 17-19 exceeds the limit of
detection of the enzyme assay, it is difficult to ascertain
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and elemental analyses. This material is available free
of charge via the Internet at http://pubs.acs.org.
Refer en ces
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Integrase: Structural organization, conformational changes, and
catalysis. Adv. Virus Res. 1999, 52, 351-369. For a recent review
on HIV-1 integrase inhibitors, see: Pommier, Y.; Neamati, N.
Inhibitors of Human Immunodeficiency Virus Integrase. Adv.
Virus Res. 1999, 52, 427-458.
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Assembly and Catalytic Reactions of Human Immunodeficiency
Virus Type 1 Integrase. J . Virol. 1997, 71, 7005-7011. Assays

4926 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Communications to the Editor
were performed with recombinant HIV-1 integrase (0.1 µM)
preassembled on immobilized oligonucleotides. Inhibitors were
either added during assembly without washing or after assembly
and washings. Inhibition was determined in relation to the
integrase control reaction (without inhibitor) performed in
quadruplicate and averaged. All samples were background
subtracted. In each case, there was no difference between either
assay format as anticipated for strand-transfer inhibitors (see
ref 2a).
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were maintained for
3 additional days. Virus spread was
assessed by HIV-1 p24 core antigen ELISA. Control cultures in
the absence of inhibitor were fully infected at 4 days.
J M000176B