Design and synthesis of 8-hydroxy-[1,6]naphthyridines as novel inhibitors of HIV-1 integrase in vitro and in infected cells

Article abstract of DOI:10.1021/jm025553u

Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC₅₀ of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 μM. It does not exhibit cytotoxicity in cell culture

Full text of DOI:10.1021/jm025553u

J . Med. Chem. 2003, 46, 453-456
453
Letters
Design a n d Syn th esis of
8-Hyd r oxy-[1,6]Na p h th yr id in es a s Novel
In h ibitor s of HIV-1 In tegr a se in Vitr o
a n d in In fected Cells
Linghang Zhuang,*,† J ohn S. Wai,†
Mark W. Embrey,† Thorsten E. Fisher,†
Melissa S. Egbertson,† Linda S. Payne,†
J ames P. Guare, J r.,† J oseph P. Vacca,†
Daria J . Hazuda,§ Peter J . Felock,§ Abigail L. Wolfe,§
Kara A. Stillmock,§ Marc V. Witmer,§
Gregory Moyer,§ William A. Schleif,§
Lori J . Gabryelski,§ Yvonne M. Leonard,‡
J oseph J . Lynch, J r.,‡ Stuart R. Michelson,† and
Steven D. Young†
F igu r e 1. Structures of diketoacid 1 and catechol 2.
for the enzyme inhibitory activity of these inhibitors;4b
therefore, efforts were directed toward seeking viable
replacements for this critical pharmacophore. In this
paper, we describe the design and syntheses of a set of
analogues that led to the discovery of 8-hydroxy-[1,6]-
naphthyridine as a suitable replacement for the 1,3-
diketoacid motif.
Departments of Medicinal Chemistry, Antiviral Research,
and Pharmacology, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received J une 10, 2002
Abstr a ct: Naphthyridine 7 inhibits the strand transfer of the
integration process catalyzed by integrase with an IC50 of 10
nM and inhibits 95% of the spread of HIV-1 infection in cell
culture at 0.39 µM. It does not exhibit cytotoxicity in cell
culture at e12.5 µM and shows a good pharmacokinetic profile
when dosed orally to rats. The antiviral activity of 7 and its
effect on integration were confirmed using viruses with specific
integrase mutations.
Resu lts a n d Discu ssion . On the basis of the SAR
from the 1,3-diketoacid HIV integrase inhibitors,4a the
following assumptions for their biologically active con-
formation were made. The 1,3-diketoacid moiety eno-
lizes at the R-position, and the resultant conjugated Z-4-
oxo-2-hydroxy-2-butenoic acid side chain is coplanar
with the central benzene ring (Figure 1). It was hypoth-
esized that a similar arrangement of key hydroxyl
groups could be achieved by a 1,2-catechol and by
incorporating an oxygen bridge to maintain coplanarity
of the two phenyl rings (see 2, Figure 1).5 Compound 2
inhibits the strand transfer step of the HIV-1 integra-
tion process with an IC50 value of 0.6 µM (Table 1).6 This
result establishes that the R-enol and carboxylic acid
moiety of the diketoacid side chain can be replaced with
a 1,2-catechol and provides support for the proposed
active conformation of the 4-aryl-1,3-diketoacids.
In tr od u ction . Human immunodeficiency virus-type
1 (HIV-1) is the etiological agent of acquired immune
deficiency syndrome (AIDS). Current antiretroviral
therapies target two viral enzymes, HIV-1 reverse
transcriptase and HIV protease, to interrupt the viral
replicative cycle.1,2 The HIV-1 genome encodes a third
enzyme, HIV integrase, a target yet to be exploited for
antiviral therapy.3 Integrase catalyzes the integration
of double-stranded viral DNA into the host cell’s ge-
nomic DNA. Integration consists of three biochemical
steps: the assembly of integrase on viral DNA, endo-
nucleolytic cleavage of the first two nucleotides from
each 3′ terminal of the viral DNA, and strand transfer
of the recessed viral DNA to the host cell DNA. Recently,
diketoacids such as 1 (Figure 1)4 were reported by our
laboratory to be selective inhibitors of the strand
transfer reaction. Although these compounds effectively
prevent viral DNA integration and inhibit HIV-1 rep-
lication in cell culture,4a the presence of the biologically
labile 1,3-diketoacid moiety in the molecules is a concern
for development of these compounds as chemotherapeu-
tic agents. The 1,3-diketoacid functionality is essential
For further modification of 2, three factors were taken
into consideration (Figure 2). First, 1,2-catechols are
biologically labile,7 so it was necessary to replace one
of the hydroxyl groups in 2 with other suitable func-
tionality while maintaining activity against integrase.
On the basis of our earlier SAR,8a the 2-hydroxyl group
in 2 was replaced with a heteroaromatic nitrogen
(Figure 2). This aromatic nitrogen is a Lewis base
equivalent of the corresponding carboxylate oxygen in
diketoacid 1. Second, coplanar polyaromatic ring sys-
tems in general exhibit poor physical properties as
therapeutic agents. As such, the oxygen bridge in 2 was
removed. Third, to allow for adoption of coplanar
conformations between the central benzene ring and the
heteroarylpyridine, a heteroatom was incorporated at
an appropriate position of the heteroaryl ring. This
avoids the unfavorable conformational bias presented
in their carbon counterparts (see 5, Figure 2).5 These
* To whom correspondence should be addressed. Address: Depart-
ment of Medicinal Chemistry, WP14-3, Merck Research Laboratories,
P.O. Box 4, West Point, PA 19486. Phone: 215-652-3584. Fax: 215-
652-3971. E-mail: linghang_zhuang@merck.com.
† Department of Medicinal Chemistry.
§ Department of Antiviral Research.
‡ Department of Pharmacology.
10.1021/jm025553u CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/09/2003

454 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Letters
Ta ble 1. Inhibition of HIV-1 Integrase Catalytic Activities and
HIV-1 Replication in Cells by Compounds 1-7 (Bn ) Benzyl)
F igu r e 2. Structures of furanopyridine 3, naphthyridine 4,
and quinoline 5. A CH-CH interaction in 5 prevents the
central phenyl ring and the quinoline ring from a adopting
coplanar conformation.
to the central benzene ring led to quinoline 6 (Table 1),5
which exhibits an IC50 of 50 nM in the strand transfer
assay and is comparable to that of naphthyridine 4 (40
nM). These results are in accordance with the earlier
assumption that the ability of these inhibitors to adopt
a coplanar arrangement of the central phenyl ring (or
pyridine ring) and the naphthyridine ring (or quinoline
ring) is important for the inhibitory activity of these
inhibitors. It is also noteworthy to point out that
8-hydroxy-[1,6]naphthyridine 4 is significantly less cy-
totoxic than the corresponding 8-hydroxyquinolines 5
and 6 (12.50 µM vs 1.25 and 2.50 µM, respectively) in
the cell assay.9,10 This may be related to the observation
that unsubstituted 8-hydroxy-[1,6]naphthyridine exhib-
its significantly weaker affinity for metal ions versus
unsubstituted 8-hydroxyquinoline.11
a
Assays were performed with recombinant HIV-1 integrase (0.1
µM) preassembled on immobilized oligonucleotides. Inhibitors were
added after assembly and washings. For details, see ref 6. Cell
culture inhibitory concentrations (CIC95) are defined as those that
inhibited by g95% the spread of HIV-1 infection in susceptible
cell culture, using the HIV-1 IIIb variant and MT-4 T-lymphoid
cells. For details, see ref 9. c See ref 10.
b
considerations led to the design and synthesis of fura-
nopyridine 3 and [1,6]naphthyridine 4.5
Further elaboration of 4 led to naphthyridine 7,
bearing a sultam substitution at the 3 position of the
central phenyl ring. Naphthyridine 7 exhibits an IC50
of 10 nM in the strand transfer assay; it is essentially
as potent as diketoacid 1. In a single-cycle HIV-1
infectivity assay,4c 7 exhibits significant loss in potency
against HIV-1-containing integrase mutations previ-
ously shown to confer resistance to the 1,3-diketoacid
inhibitors (EC50 > 0.8 µM; T66I, S153Y) versus the wild-
type virus (EC50 ) 0.14 µM; NL4-3). This result dem-
Furanopyridine 3 (IC50 ) 1000 nM) is found to be 100-
fold less potent than the diketoacid 1 (IC50 ) 10 nM) in
the strand transfer assay (Table 1).6 On the other hand,
naphthyridine 4 (IC50 ) 40 nM) exhibits good potency
in the same assay and is only 4-fold less potent than 1
(Table 1). The corresponding quinoline 5 exhibits an IC50
of 370 nM in this assay and is thus 9-fold less potent
than naphthyridine 4. Shifting the aromatic nitrogen
Sch em e 1a
a
Reagents: (a) Br2, AlCl3 (cat.), 1,4-dioxane; (b) i-Pr2NEt, CH3CN, CBZCl, 76% for steps a and b; (c) Pd(II)(PPh3)2Cl2, Et3N, CO (250
psi), EtOH, 50%; (d) NaHMDS, THF; (e) 48% HBr, CH3CN, air oxidation, 42% for steps d and e; (f) SOCl2, toluene, 98%; (g) LiN3, DMSO;
(h) 5% Pt/C, H2, EtOH, 51% for steps g and h.

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 455
Sch em e 2a
duced with sodium borohydride to afford benzyl alcohol
16 in 77% yield. Alcohol 16 was converted to its
corresponding bromide, which was then reacted with
γ-sultam16 to give 17 in 74% yield. Compound 17 was
subjected to Heck reaction conditions with butyl vinyl
ether followed by acid hydrolysis to provide methyl
ketone 18 in 90% yield.17
In summary, starting with diketoacid 1, novel bioi-
sosteric diketoacid equivalents were designed and pre-
pared. This led to the discovery of 8-hydroxy-[1,6]-
naphthyridine as a suitable replacement for the 1,3-
diketoacid moiety in our earlier HIV-1 integrase
inhibitors. Among them, naphthyridine 7 exhibits excel-
lent potency and a good pharmacokinetic profile. Fur-
ther work on 8-hydroxy-[1,6]naphthyridine-derived HIV-1
integrase inhibitors is in progress.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for preparation of compounds 2-7. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
a
Reagents: (a) n-BuLi, PhCHO, Et2O, -78 °C; (b) Et3SiH,
(1) Recent reviews on HIV-1 reverse transcriptase inhibitors: (a)
Hogberg, M.; Morrison, I. HIV-1 non-nucleoside reverse tran-
scriptase inhibitors. Expert Opin. Ther. Pat. 2000, 10, 1189-
1199. (b) J onckheere, H.; Anne, J .; De Clercq, E. The HIV-1
reverse transcription (RT) process as target for RT inhibitors.
Med. Res. Rev. 2000, 20, 129-154.
BF3‚OEt2, CH2Cl2, 0 °C, 82% for steps a and b; (c) n-BuLi, DMF,
Et2O, -78 °C; (d) NaBH4, MeOH, 77% for steps c and d; (e) PPh3,
CBr4, CH2Cl2, 75%; (f) γ-sultam, K2CO3, CH3CN, reflux, 99%; (g)
butyl vinyl ether, Pd(II)(OAc)2, Tl(I)OAc, DPPP, Et3N, DMF, 100
°C, 1 N HCl, THF, 90%.
(2) Recent reviews on HIV-1 protease inhibitors: (a) Lebon, F.;
Ledecq, M. Approaches to the design of effective HIV-1 protease
inhibitors. Curr. Med. Chem. 2000, 7, 455-477. (b) Vacca, J . P.;
Condra, J . H. Clinically effective HIV-1 protease inhibitors. Drug
Discovery Today 1997, 2, 261-272.
(3) Recent reviews on biology of HIV-1 integrase: (a) Esposito, D.;
Craigie, R. HIV integrase structure and function. Adv. Virus Res.
1999, 52, 319-333. (b) Asante-Appiah, E.; Skalka, A. M. HIV-1
integrase: structural organization, conformational changes, and
catalysis. Adv. Virus Res. 1999, 52, 351-369. For a recent review
on HIV-1 integrase inhibitors, see the following. Young, S. D.
Inhibition of HIV-1 integrase by small molecules: the potential
for a new class of AIDS chemotherapeutics. Curr. Opin. Drug
Discovery Dev. 2001, 4, 402-410.
onstrates that the antiviral activity of compound 7 is
due to its effect on integrase and suggests that it inhibits
HIV-1 integrase in a mechanism similar to that of the
diketoacids reported earlier. Naphthyridine 7 exhibits
a CIC95 of 0.39 µM in the cell assay, 1-fold more potent
than 1. Furthermore, it does not show cytotoxicity at
concentrations up to 12.5 µM.10 When naphthyridine 7
is administered intravenously to rats at 2 mg/kg,12 it
exhibits a half-life (T1/2) of 9.7 h with a moderate
clearance rate (Clp) of 2.98 mL min-1 kg-1. At 10 mg/
kg dose given orally to rats,12 7 reaches the peak plasma
level (Cmax) of 1.17 µM at 60 min and maintains a
plasma concentration greater than 0.8 µM throughout
the first 6 h.
The synthesis of [1,6]naphthyridine 4 is shown in
Scheme 1. The known methyl ketone 84a was monobro-
minated to give bromoketone 9, which was treated
subsequently with amine 11 and benzyl chloroformate
(CBZCl) to afford 12 in 76% yield overall. Amine 11 was
prepared from alcohol 1013 via chlorination, azide
displacement, and platinum-catalyzed hydrogenation in
50% yield. Palladium-catalyzed carbonylation of 12
under 250 psi of carbon monoxide in ethanol provided
ester 13 (50%).14 Treatment of 13 with sodium bis-
(trimethylsilyl)amide provided the Dieckmann cycliza-
tion intermediate. Subsequent CBZ protecting group
removal with 48% hydrobromic acid and in situ air
oxidation furnished [1,6]naphthyridine 4 in 42% yield.
The naphthyridine 7 was prepared in a similar manner
starting from sultam-substituted methyl ketone 18.
Preparation of ketone 18 is shown in Scheme 2. Mono-
lithiation of 1,3,5-tribromobenzene 14 followed by treat-
ment with benzaldehyde provided the corresponding
benzylic alcohol. This alcohol was reduced with a
mixture of triethylsilane and boron trifluoride diethyl
etherate to give 15 in overall 82% yield.15 Dibromoben-
zene 15 was monolithiated and treated with N,N-
dimethylformamide. The resultant aldehyde was re-
(4) (a) 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., J r.; Zhuang, L.; Grey, V. E.; Vacca, J . P.; Holloway,
M. K.; Naylor-Olsen, A. M.; Hazuda, D. J .; Felock, P. J .; Wolfe,
A. L.; Stillmock, K. A.; Schleif, W. A.; Gabryelski, L. J .; Young,
S. D. 4-Aryl-2,4-dioxobutanoic acid inhibitors of HIV-1 integrase
and viral replication in cells. J . Med. Chem. 2000, 43, 4923-
4926. (b) Grobler, J . A.; Stillmock, K.; Hu, B.; Witmer, M.; Felock,
P.; Espeseth, A.; Wolfe, A.; Egbertson, M. S.; Bourgeois, M.;
Melamed, J . Y.; Wai, J . S.; Young, S. D.; Vacca, J . P.; Hazuda,
D. J . Diketo acid inhibitor mechanism and HIV-1 integrase:
Implications for metal binding in the active site of phospho-
transferase enzymes. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
6661-6666. (c) Hazuda, D. J .; Felock, P.; Witmar, M.; Wolfe,
A.; Stillmock, K.; Grobler, J . A.; Espeseth, A.; Gabryelski, L.;
Schleif, W.; Blau, C.; Miller, M. D. Inhibitors of strand transfer
that prevent integration and inhibit HIV-1 replication in cells.
Science 2000, 287, 646-650.
(5) Preparations of compounds 2-7 are in Supporting Information.
(6) Hazuda, D. J .; Felock, P.; Hastings, J . C.; Pramanik, B.; Wolfe,
A. Differential divalent cation requirements uncouple the as-
sembly and catalytic reactions of human immunodeficiency virus
type 1 integrase. J . Virol. 1997, 71, 7005-7011. The assay was
performed essentially as described with the following modifica-
tions introduced to increase sensitivity. Donor DNA biotinylated
on the 5′ end of the strand processed by integrase was im-
mobilized onto streptavidin-coated microtiter plates (Black
Reactibind, Pierce) using a 1.5-pmol well. Recombinant integrase
was assembled onto the immobilized donor oligonucleotide in
reaction buffer (20 mM Hepes, pH 7.6, 40 mM NaCl, 5 mM
â-mercaptoethanol, 50 µg/mL bovine serum albumin) containing
25 mM MnCl2. Excess enzyme was removed, and the complexes
were washed extensively prior to the addition of the target DNA
substrate. Target DNA oligonucleotides were labeled on each 3′
end with FITC. Strand transfer reactions were performed in 2.5
mM MgCl2 using 0.5 nM target substrate as indicated. After
strand transfer, the FITC-labeled products were detected using
an anti-FITC antibody conjugated with alkaline phosphatase
(Boehringer Mannheim) and a chemiluminescence substrate

456 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
(Sapphire II, Tropix). The assay was performed in final
Letters
a
by HIV-1 p24 core antigen ELISA. Control cultures in the
absence of inhibitor were fully infected at 4 days.
(10) Cytotoxicity is evaluated by visual inspection of the culture for
cytopathic effects distinguished as gross morphological changes,
growth pattern change, and metabolic change as indicated by
lack of change in the medium pH indicator. With inhibitor 7,
all cells remain viable after 4 days. During the 4-day incubation
period, MT-4 cells will typically double at least 4 times. No effect
on cell numbers was observed with 7 at up to 12.5 µM.
(11) Albert, A.; Hampton, A. Analogues of 8-hydroxyquinoline having
additional cyclic nitrogen atoms. Part II. Further preparations,
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(12) The compound was dosed to rats intravenously in DMSO at 1.6
mg/mL. The compound was dosed to rat orally in aqueous
solution with 1% of methylcellulose and 1% of DMSO at a 3.0
mg/mL.
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concentration of 10% DMSO. To specifically evaluate inhibition
of strand transfer, compounds were added after assembly, just
prior to the addition of the target DNA.
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4096-4100. 95% cell culture inhibitory concentrations (CIC95
)
are defined as those that inhibited by g95% the spread of HIV-1
infection in susceptible cell culture. MT-4 human T-lymphoid
cells were maintained in RPMI 1640 medium containing 10%
heat-inactivated fetal bovine serum. Cells were infected en
masse at low multiplicity (0.01) using HIV-1 strain IIIb and were
incubated for 24 h. At this time, cells were washed and
distributed into 96-well microtiter dishes. Serial 2-fold dilutions
of inhibitor were added to the wells, and the cultures were
maintained for three additional days. Virus spread was assessed
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