A series of 5-(5,6)-dihydrouracil substituted 8-hydroxy-[1,6]naphthyridine- 7-carboxylic acid 4-fluorobenzylamide inhibitors of HIV-1 integrase and viral replication in cells

Article abstract of DOI:10.1016/j.bmcl.2005.06.105

Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibit

Full text of DOI:10.1016/j.bmcl.2005.06.105

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4550–4554
A series of 5-(5,6)-dihydrouracil substituted
8-hydroxy-[1,6]naphthyridine-7-carboxylic acid
4-fluorobenzylamide inhibitors of HIV-1 integrase
and viral replication in cells
Mark W. Embrey,a,* John S. Wai,a Timothy W. Funk,a Carl F. Homnick,a
Debbie S. Perlow,a Steven D. Young,a Joseph P. Vacca,a Daria J. Hazuda,b
Peter J. Felock,b Kara A. Stillmock,b Marc V. Witmer,b Gregory Moyer,c
William A. Schleif,c Lori J. Gabryelski,c Lixia Jin,d I-Wu Chen,d
Joan D. Ellis,d Bradley K. Wong,d Jiunn H. Lin,d Yvonne M. Leonard,a
Nancy N. Tsoue and Linghang Zhuanga
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Drug Metabolism and Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
eDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 29 April 2005; revised 30 June 2005; accepted 30 June 2005
Available online 15 August 2005
Abstract—Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-
carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in
inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent
antiviral agent than 1, with a CIC95 of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats
and dogs.
Ó 2005 Elsevier Ltd. All rights reserved.
The etiological agent of acquired immune-deficiency syn-
drome (AIDS) is the human immunodeficiency virus type
1 (HIV-1). One of the possible targets for chemotherapeu-
tic intervention is a virally encoded enzyme, HIV inte-
grase, which catalyzes the insertion of double-stranded
viral DNA into the host cellÕs DNA. Integration is a
three-step process consisting of the assembly of the viral
DNA onto the integrase enzyme, endonucleolytic pro-
cessing of the viral DNA, and strand transfer of the pro-
cessed viral DNA into the host cellÕs DNA.1
diketoacid pharmacophore in our early integrase inhib-
itors.2,3 Further research led to the discovery of the
N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-car-
boxamide 1 as a potent structural backbone for inte-
grase inhibition (Fig. 1). Compound 1 inhibits the
strand transfer process of HIV-1 integration with an
IC50 of 33.4 nM.3,4 It inhibits the replication of HIV-1
in cell culture with a CIC95 of 1250 nM (assay run with
10% fetal bovine serum (FBS)).5 In the presence of 50%
normal human serum (NHS) a 4-fold drop in potency
(CIC95 = 5000 nM) is observed due to the binding of
drug to serum protein (99.2%).6
Recently our laboratory reported the discovery of 8-hy-
droxy-[1,6]naphthyridine as a viable replacement to the
We attempted to incorporate polar heterocycles into
compound 1 for improvement in both intrinsic potency
and physical properties. The bromonaphthyridine 2 was
readily accessible in quantities of several grams (Scheme
1).7 Reacting 2 with methyl acrylate, under FuÕs Heck
Keywords: HIV-1 integrase inhibitors; Naphthyridine.
*
Corresponding author. Tel.: +1 215 652 0881; fax: +1 215 652
3971; e-mail: mark_embrey@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.105

M. W. Embrey et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4550–4554
4551
Figure 1. Evolution of 1.
Scheme 1. Reagents and conditions: (a) Pd[P(t-Bu)3]2, Cy2NMe, P(t-Bu)3, Pd2(dba)3, methyl acrylate, 1,4-dioxane, sealed tube, 120 °C; (b) R,R0-urea
(50–260 eq), melt 165 °C.
reaction conditions,8 provided the a,b-unsaturated
methyl ester 3. The latter served as the common interme-
diate toward various heterocycles.
shifts in the presence of human serum, but the (+) enan-
tiomer displays slightly better activity against HIV-1
replication in cells versus the (ꢀ) enantiomer (CIC95 of
93.8 nM vs. 125 nM) (Table 1).
For example, melting 3 with urea at 165 °C in a sealed
tube provided dihydrouracil 4.9 Compound 4 inhibits
the strand transfer reaction of HIV-1 integrase with an
IC50 of 10 nM and inhibits the replication of virus in cell
culture with a CIC95 of 55 nM (Table 1). In the presence
of 50% NHS, no drop in potency is observed, due to the
lower protein binding nature of 4 (92.1%). Compound 4
is not cytotoxic at concentrations up to 10 lM.10 Reso-
lution of the enantiomers by chiral HPLC separation
provided the (+) and (ꢀ) enantiomers, 8 and 9, respec-
tively. Both enantiomers exhibit similar potency in the
in vitro assay. In the cell-based assay, neither compound
Treating 3 with methylurea and subsequent separation
of the reaction products by preparative HPLC provided
the regio-isomers 5 and 6 (as determined by NOE exper-
iments) in poor yield. Although potency is similar in the
in vitro assay, in the cellular anti-viral assay, 5 is four-
fold more potent than 6 with a CIC95 of 15.6 nM in
the presence of 50% NHS (Table 1).
Similarly, treating 3 with N,N0-dimethyl urea provided
7. This compound maintains inhibitory activity against
HIV-1 integrase strand transfer with an IC50 of

4552
M. W. Embrey et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4550–4554
Table 1. Inhibition of HIV-1 integrase catalytic activities and HIV-1 replication in cells by a series of 5-(dihydrouracil or uracil) substituted
8-hydroxy-[1,6]naphthyridine-7-carboxylic acid 4-fluorobenzylamides
Compound
R
R0 Optical purity Inhibition of strand
transfer IC50 (nM)a,b (with 10% FBS)c
Antiviral activity CIC95(nM) Antiviral activity CIC95 % Binding to human
(nM) (with 50% NHS)c plasma proteind
1
—
—
—
32.8 7.8 (n = 6)
1250 (n = 3)
5000 (n = 2)
99.2
Dihydrouracil
4
H
H
Racemic
10(n = 8)
53.7 25.1 (n = 8)
15.6 (n = 1)
31.3 (n = 2)
55.7 22.6 (n = 8)
15.6 (n = 1)
62.5 (n = 2)
92.1
5
H
Me
Me Racemic
Racemic
12.5 4.3 (n = 4)
12.8 3.0 (n = 4)
8.7 1.9(n = 6)
11 1.7 (n = 4)
13.3 3.7 (n = 4)
11.3 1.9 (n = 3)
35 5(n = 2)
6
H
7
Me Me Racemic
18.6 6.1 (n = 16)
93.8 31.3 (n = 2)
125 (n = 2)
60.7 7.4 (n = 17)
93.8 31.3 (n = 2)
125 (n = 2)
94.3
8
H
H
H
H
(+)97.2%ee
(ꢀ)92.8%ee
9
10
11
Me Me (+) 100%ee
Me Me (ꢀ)93.1%ee
41.7 14.7 (n = 3)
20.1 7.1 (n = 7)
166.7 58.9 (n = 3)
40.2 14.1 (n = 7)
96.0
95.0
Uracil
14
Me Me
27.5 13.0 (n = 4)
250 (n = 1)
1000 (n = 1)
97.8
a See Refs. 3,4.
b The lower limit of activity of this assay is ꢁ10 nM.
c See Ref. 5.
d See Ref. 6.
8.7 nM (Table 1). Compound 7 has a CIC95 of 18.6 nM
in 10% FBS, which increases to 60.7 nM in the presence
of 50% NHS, due to its higher protein binding of 94.3%.
Chiral HPLC separation of the racemic 7 provided the
(+) and (ꢀ) enantiomers, 10 and 11 respectively. The
(ꢀ) enantiomer 11 displays better cellular activity
(CIC95 of 20.1 nM with 10% FBS and 40.2 nM with
50% NHS) as compared to the (+) enantiomer 10
(CIC95 of 41.7 nM with 10% FBS and 166.7 nM with
50% NHS) even though 11 is less active in the enzyme
assay (IC50 = 35 nM vs. IC50 = 11.3 nM for 10) (Table
1). When 11 is dosed to rats as the sodium salt, it dis-
plays good pharmacokinetics with a clearance of
9.76 mL/min/kg, a Cmax of 4.81 lM, and an absolute
oral bioavailability of 28.0%. Similarly when dosed to
dogs, 11 displays good pharmacokinetics with a clear-
ance of 3.47 mL/min/kg, a Cmax of 1.58 lM, and a
bioavailability of 41.5%.11
Figure 2. X-ray crystal structure of 7.
would be expected to orient perpendicular to the plane
of the naphthyridine ring and as such, the uracil ring
would not be pointed toward the 4-flourobenzyl amide.
Attempts to oxidize the dihydrouracil to the uracil were
unsuccessful. The compound 14 was synthesized by cou-
pling the iodide 12 with the dimethyl uracil zinc iodide
13 using the method of Knochel (Scheme 2).13
Additional features of 7 were revealed by X-ray crystal-
lographic structure determination (Fig. 2).12 The naph-
thyridine ring is positioned in
a
pseudo-axial
orientation relative to the dihydrouracil ring. The naph-
thyridine and the exocyclic amide are in an extended co-
planar conformation. The dihydrouracil ring is almost
planar, orientates perpendicular to the naphthyridine
ring, and points toward the 4-fluorobenzyl amide.
Compared to the saturated analogs, the unsaturated
uracil analog 14 is less active in the enzyme assay
(IC50 = 27.5 nM). This decrease in activity is also more
significantly reflected in the cell-based assay with a
The unsaturated N,N0-dimethyluracil analog 14 was also
synthesized. In this case, the plane of the uracil ring

M. W. Embrey et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4550–4554
4553
Scheme 2. Reagents and conditions: (a) Pd2(dba)3, (2-furyl)3P, DMA, 80 °C.
Jolly, S. M.; Wai, J. S.; Zhuang, L.; Fisher, T. E.; Embrey,
M. W.; Guare, J. P., Jr.; Egbertson, M. S.; Vacca, J. P.;
Huff, J. R.; Felock, P. J.; Witmer, M. V.; Stillmock, K. A.;
Danovich, R.; Grobler, J.; Miller, M. D.; Espeseth, A. S.;
Jin, L.; Chen, I.-W.; Lin, J.; Kassahun, K.; Ellis, J. D.;
Wong, B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.;
Cortese, R.; Emini, E.; Summa, V.; Holloway, W. A.;
Young, S. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
11233.
CIC95 of 250 nM (with 10% FBS) and a CIC95 of
1000 nM with 50% NHS (PB = 97.8%) (Table 1).
This observation suggests that sp3 hybridized linkages
off of the 5-position of naphthyridine-based inhibitors
will provide more potent analogs against viral replica-
tion in cells. Further research was done in this area by
our group, which will be reported at a later date.
4. Hazuda, D. J.; Felock, P.; Hastings, J. C.; Pramanik, B.;
Wolfe, A. J. Virol. 1997, 71, 7005.
In summary, a potent series of dihydrouracil heterocy-
cles were synthesized that displays nanomolar inhibition
of HIV-1 replication in a cell-based assay with little shift
in efficacy in the presence of human serum. This repre-
sents a 100-fold improvement in potency over the
unsubstituted analog 1. In addition, the (ꢀ) enantiomer
of the dimethylated dihydrouracil 11 displays good
pharmacokinetic properties when dosed in rats and in
dogs.
5. Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.;
McDaniel, S. L.; Darke, P. L.; Zugay, J.; Quintero, J. C.;
Blahy, O. M.; Roth, E.; Sardana, V. V.; Schlabac, A. J.;
Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M.
K.; Lin, J.; Chen, I.-W.; Vastag, K.; Ostovic, D.; Ander-
son, P. S.; Emini, E. E.; Huff, J. R. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 4096.
6. Analytical HPLC/UV detection based assay that measures
the ability of a compound to bind with human plasma
(primarily Albumin) in pH 7.4 buffer at RT.
7. Anthony, N. J.; Gomez, R. P.; Young, S. D.; Egbertson,
M.; Wai, J. S.; Zhuang, L.; Embrey, M.; Tran, L.;
Melamed, J. Y.; Langford, H. M.; Guare, J. P.; Fisher, T.
E.; Jolly, S. M.; Kuo, M. S.; Perlow, D. S.; Bennett, J. J.;
Funk, T. W. Preparation of (poly)azanaphthalenyl carb-
oxamides as HIV integrase inhibitors. PCT Int. Appl.
(2002), 434 pp. WO 0230930 A2 20020418.
Acknowledgments
The authors are grateful to Melissa S. Egbertson and
H. Marie Langford for helpful discussions and their
colleagues in the analytical chemistry group for protein
binding data.
8. Littke, A. F.; Fu, G. C. J. Am. Chem. Soc. 2001, 123, 6989,
To an oven-dried sealed tube under nitrogen, was added 2
(1.51 g, 4.01 mmol) and 1,4-dioxane (7 mL), and the
mixture was bubbled with nitrogen for 10 min. Methyl
acrylate (3.5 mL, 38.9 mmol), Cy2NMe (1.08 mL,
5.04 mmol), P(t-Bu)3 (1.35 mL of a 50 mg/mL soln in
1,4-dioxane, 0.33 mmol), Pd2(dba)3 (116 mg, 0.13 mmol),
and Pd[P(t-Bu)3]2 (60 mg, 0.12 mmol) were added. The
reaction was sealed and heated at 120 °C overnight. The
reaction was diluted with ethyl acetate, filtered through
celite, washed with water three times, dried over Na2SO4,
and concentrated. Recrystallizing in ethanol afforded the
References and notes
1. (a) Chiu, T. K.; Davies, D. R. Curr. Top. Med. Chem.
2004, 4, 965; (b) Anthony, N. J. Curr. Top. Med. Chem.
2004, 4, 979; (c) Pommier, Y.; Johnson, A. A.; Marchard,
C. Nat. Rev. Drug Disc. 2005, 4, 236.
2. 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.; 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. J. Med. Chem. 2000,
43, 4923.
3. (a) Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. E.;
Egbertson, M. S.; Payne, L. S.; Guare, J. P., Jr.; Vacca, J.
P.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock,
K. A.; Witmer, M. V.; Moyer, G.; Schleif, W. A.;
Gabryelski, L. J.; Leonard, Y. M.; Lynch, J. J., Jr.;
Michelson, S. R.; Young, S. D. J. Med. Chem. 2003, 46,
453; (b) Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.;
1
ester 3 in 55% yield. H NMR (400 MHz, CDCl3) d 13.58
(s, 1H), 9.21 (dd, J = 4.2, 1.3 Hz, 1H), 8.63 (d, J = 8.6 Hz,
1H), 8.42 (br t, J = 5.1, 1H), 8.31 (d, J = 15.4 Hz, 1H),
7.71 (dd, J = 8.6, 4.2 Hz, 1H), 7.40 (dd, J = 8.3, 5.4 Hz,
2H), 7.08 (t, J = 8.5 Hz, 2H), 7.01 (d, J = 15.4 Hz, 1H),
4.72 (d, J = 6.4 Hz, 2H), 3.87 (s, 3H).
9. Lee, C. K.; Shim, J. Y. Bull. Korean Chem. Soc. 1991, 12,
343, To stirred, molten urea (50 g) heated in an oil bath at
165 °C under an atmosphere of nitrogen, 3 (1.20 g,
3.15 mmol) was added. The resultant mixture was stirred
at the same temperature for 2.5 h. The product mixture

4554
M. W. Embrey et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4550–4554
was dissolved in water and the solution was acidified to
13. Stevenson, T. M.; Prasad, A. S. B.; Citineni, J. R.; Knochel,
P. Tetrahedron Lett. 1996, 37, 8375, To a mixture of tri-(2-
furyl)phosphine (33 mg, 0.14 mmol), Pd(dba)3 (41 mg,
0.07 mmol), and N-(4-fluorobenzyl)-8-hydroxy-5-iodo-
1,6-naphthyridine-7-carb-oxamide 12 (300 mg, 0.71 mmol)
in anhydrous N,N-dimethylacetamide (1 mL) under nitro-
gen, a solution of 1,3-dimethyluracil-6-zinc iodide 13
[prepared by treatment of zinc powder (0.44 g, 6.76 mmol)
in anhydrous N,N-dimethylacetamide (2 mL) successively
with 1,2-dibromoethane (23 lL, 0.27 mmol), trimethylsilyl
chloride (25 lL, 0.20 mmol), and 6-iodo-1,3-dimethylura-
cil14 (0.60 g, 2.25 mmol) at 80 °C for 15 min] was added. The
resultant mixture was heated at 80 °C for 1 h, and treated
with an aqueous solution of Na2EDTA and diluted with
chloroform. The precipitated solid was filtered, dissolved in
DMSO, and subjected to HPLC purification on C-18
stationary phase eluting with water/acetonitrile/TFA
mobile phase. Lyophilization of appropriate fractions
provided 14 in 60% yield. 1H NMR (400 MHz, DMSO-
d6) d 13.97 (br s, 1H), 9.82 (t, J = 6.2 Hz, 1H), 9.22 (br d,
J = 4.1 Hz, 1H), 8.60 (br d, J = 8.3 Hz, 1H), 7.85 (dd,
J = 8.3, 4.1 Hz, 1H), 7.43 (br t, J = 7.7 Hz, 2H), 7.17 (br t,
J = 8.8 Hz, 2H), 5.91 (s, 1H), 4.58 (dd, J = 14.7, 6.2 Hz,
1H), 4.49 (dd, J = 14.7, 6.2 Hz, 1H), 3.29 (s, 3 H), 3.03
(s, 3H).
pH 5–6. The resultant precipitate was filtered, dissolved in
DMSO, and subjected to HPLC purification on C-18
stationary phase eluted with water/acetonitrile/TFA
mobile phase. Lyophilization of appropriate fractions
1
provided compound 4 in 44% yield. H NMR (400 MHz,
ꢁ5:1 CDCl3-DMSO-d6) d 13.35 (s, 1H), 9.19 (br d, 1H),
8.68–8.59 (m, 2H), 8.14 (br s, 1H), 7.71 (dd, J = 8.6,
4.2 Hz, 1H), 7.42 (dd, J = 8.4, 5.5 Hz, 2H), 7.06 (t,
J = 8.6 Hz, 2H), 5.36 (br d, 1H), 4.65 (br s, 2H), 3.03 (br
s, 2H), 2.59 (br s, 1H)
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.
11. Compound was dosed intravenously in dimethyl sulfoxide
(DMSO) at a concentration of 2 mg/kg in rats and at
1 mg/kg in dogs. Compound was dosed orally in aqueous
solution (with 0.5% methylcellulose) at a concentration of
10 mg/kg in rats and at 1 mg/kg in dogs.
12. Crystallographic data (excluding structure factors) for 7
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number CCDC
264140. Copies of the data can be obtained free of charge,
on application to CCDC, 12 Union Rd, Cambridge CB2
1EZ, UK [fax: +44(00)1223-336033 or e-mail: deposit@
ccdc.cam.ac.uk].
14. Saito, I.; Ikehira, H.; Matsuura, T. J. Org. Chem. 1986, 51,
5148.