A potent and orally active HIV-1 integrase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2006.11.080

A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.

Full text of DOI:10.1016/j.bmcl.2006.11.080

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1392–1398
A potent and orally active HIV-1 integrase inhibitor
Melissa S. Egbertson,a,* H. Marie Moritz,a Jeffrey Y. Melamed,a Wei Han,a
Debra S. Perlow,a Michelle S. Kuo,a Mark Embrey,a Joseph P. Vacca,a
Matthew M. Zrada,a Amanda R. Cortes,a Audrey Wallace,a Yvonne Leonard,a
Daria J. Hazuda,b Michael D. Miller,b Peter J. Felock,b Kara A. Stillmock,b
Marc V. Witmer,b William Schleif,c Lori J. Gabryelski,c Gregory Moyer,c
Joan D. Ellis,e Lixia Jin,e Wei Xu,d Matthew P. Braun,e Kellem Kassahun,e
Nancy N. Tsouf and Steven D. Younga
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 Viral Vaccine Research, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Pharmaceutical Research, Merck Research Laboratories, West Point, PA 19486, USA
eDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
fDepartment of Medicinal Chemistry, Rahway, NJ 07065, USA
Received 22 September 2006; revised 20 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good
pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Replication of the human immunodeficiency virus
(HIV) is dependent upon the enzyme HIV Integrase
(IN), one of three essential enzymes encoded in the viral
genome. HIV IN catalyzes the insertion of the viral gen-
ome into the host genome. HIV IN subunits assemble
onto double stranded viral DNA and process the 30 ends
to prepare the strand for integration. The pre-integra-
tion complex then enters the nucleus where it catalyzes
the DNA insertion (strand transfer) step. Inhibitors of
viral IN strand transfer inhibit HIV-1 viral replication.1
HIV IN thus presents an attractive chemotherapeutic
target for the treatment of HIV infection and AIDS.
cess catalyzed by HIV-1 IN (IC50 = 10 nM, Table 1)
and has shown excellent antiviral activity in cell culture
in the absence of human serum proteins (IC95 = 15 nM,
n = 237).2 Addition of 50% normal human serum
(NHS) to the cell culture results in a 7-fold loss of efficacy
(IC95 = 102 nM, n = 237), presumably as a result of the
compound’s high affinity for serum protein (99% protein
bound).3 Compounds with greater potency against HIV-1
in the presence of 50% NHS were sought with the goal of
improving efficacy and of lowering dose.
In our effort to decrease the affinity of 1 for serum pro-
tein, we were aware of work by Whitesides and co-work-
ers, who have shown that self-assembling monolayers
containing alkylated amide side chains demonstrate re-
duced binding to proteins like fibrinogen and lysozyme
compared to monolayers containing alkyl side chains.7
We explored substitution of the benzyl moiety of com-
pound 1 with alkylated amides to lower protein binding.
The 2-position of the benzyl phenyl was targeted after
substitution at the 3- and 4-positions with polar groups
resulted in losses of potency relative to 1 (R.P. Gomez,
N.J. Anthony, S.D. Young, unpublished data). To allow
Compound 1, L-870810, an 8-hydroxy-1,6-naphthyri-
dine, is the first HIV IN inhibitor to significantly de-
crease viral load and increase CD4 cell count in
treatment naive or experienced patients infected with
HIV. It is a potent inhibitor of the strand transfer pro-
Keywords: HIV; AIDS; Integrase; Strand transfer; 1,6-Naphthyridine.
*
Corresponding author. Tel.: +1 215 652 5318; fax: +1 215 652
3971; e-mail: melissa_egbertson@merck.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.080

M. S. Egbertson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1392–1398
Table 1. Efficacy and physical properties of compound 1
1393
The 2-position acetamide analogs 5a and 5b retain
intrinsic potency relative to the unsubstituted benzyl
parent 7 (Table 2). For these compounds a reduction
in protein binding and logP relative to parent 7 is also
observed. For example 5b, these changes are accompa-
nied by improved antiviral activity with a smaller shift
(4-fold vs 8-fold) observed between the low (FBS) and
high (NHS) protein versions of the cell assay.
Inhibition of
Antiviral
% Protein LogPd equiv
strand transfer activity in bindingc
sol pH 7.4e
(mg/mL)
a
IC50 (nM)
Attachment of a polar group directly to the 2-position of
the benzyl ring was explored next. We decided to prepare
analogs containing the 4-fluoro group, which is important
for potency in HIV IN inhibitors.10 The first polar
groups to be explored were sulfonamide and sulfone.
Amide bond formation with the amines 2-methane-
sulfonamide-4-fluoro benzylamine and 2-mono- and
di-methylaminosulfonyl-4-fluorobenzylamine11 using the
conditions described for 5a and 5b gave compounds 8a,b
and 9 (Table 3).
cell culture
b
IC95 (nM)
FBS NHS
10
15
102
99
2.1
0.00021
a The inhibition of strand transfer assay was performed with recom-
binant HIV-IN (0.1 lM) preassembled on immobilized oligonucleo-
tides using 0.5 nM DNA.2b Values are means of at least two
experiments (for compound 1, n = 5), lower limit of accuracy ꢀ5 nM,
standard deviation is ꢀ2-fold.
b Antiviral activity was assessed by measuring the decrease in HIV-1
p24 core antigen in MT-4 human T-lymphoid cells/HIV-1IIIb cul-
tured in the presence of increasing concentrations of inhibitor.
Antiviral activity in cell culture (IC95) is the drug concentration
which inhibits 95% viral growth relative to control in cell culture.
Low protein conditions (FBS): run with 10% fetal bovine serum, high
protein conditions (NHS): run with 50% normal human serum. For
all compounds n = 2 or greater unless otherwise noted. For com-
pound 1 n = 237. Lower limit ꢀ7 nM. Standard deviation ꢀ2-fold.4
The results for the 2-sulfonamide and sulfone analogs
are summarized in Table 3. The sulfonamides 8a and
8b were less active than 1 in strand transfer and showed
only modest decreases in protein binding (0–3%). These
small changes did not greatly affect the compound activ-
ity in cell culture in the presence of NHS relative to 1.
The methyl sulfone compound 9 was equipotent to 1
in strand transfer and showed an encouraging 2-fold
improvement in cell culture in the presence of NHS
(antiviral IC95 49 vs 102 nM for 1). The protein binding
was decreased approximately 2% relative to 1. All three
compounds showed decreases in logP; the decrease was
greatest for 9 (1.6 vs 2.1 for 1).
c
% Protein binding ultrafiltration method, see Ref. 3.
d LogP measurement, see Ref. 5.
e
Equilibrium solubility assay, see Ref. 6.
maximum flexibility in the nature of the final amide, our
synthesis attempted to incorporate the functional group
as an ester-substituted benzylamine that, after coupling
with the naphthyridine core, could be transformed into
a variety of amides 5 (Scheme 1). Pre-activation of the
acid 38,9 with EDT/HOBt followed by treatment with
the benzylamine 2 and base gave intermediate 4 in good
yield and minimized loss of the benzylamine to the side
product 6. Compounds 5a and 5b were prepared in
ꢀ30% yield in three steps.
Incorporation of a 2-amide group required another syn-
thetic approach, as we anticipated that 4-fluoro-2-car-
boxy benzylamine ester 10 would undergo facile
intramolecular cyclization to the 5-membered lactam
11 under the conditions of the EDT-mediated peptide
coupling (Scheme 2).
We therefore settled on masking the benzylamine, incor-
porating the desired amide at the 2-position, and then
unveiling the benzylamine to do the coupling. Two
routes were worked out in this fashion. In the first route,
the benzylic nitrogen was introduced as bis-tert-butoxy-
carbonyl amine, then palladium-catalyzed carbonylative
amide formation was used to give amide 15 as a mixture
of mono- and bis-BOC products (Scheme 3). The course
of the carbonylation reaction was sensitive to the
amount of methylamine present, with greater than six-
teen equivalents being necessary to inhibit the formation
of the oxalate product 16. Deprotection of the amine
with HCl gave the stable hydrochloride salt 17a, which
can be stored as a solid at room temperature for months
without significant degradation. The overall yield of 17a
after four steps was 40%.
O S
N
O
O S
O
N
N
N
H
N
HO
N
+
NH3
Oxalate-
N
O
OH
O
O
OH
O
3
O
O
4
a
2
b,c
O S
O
N
N
H
N
NH
N
R= H, Me
O
R
O
OH
O
N
5a R = Me
5b R = H
6
The second route was developed to avoid complications
associated with scaling up the palladium reaction. Bro-
mination of the commercially available ester 1812 to give
19 was followed by displacement of the bromine with
bis-tert-butoxycarbonyl amine to give 20 (Scheme 4).
Scheme 1. Preparation of analogs in Table 2. Reagents and conditions:
(a) EDT, HOBt, DMF, followed by 3 and NMM, rt (52%); (b) 1 N aq.
NaOH, (8 equiv) /THF/MeOH, 1:1:1 vol, 50 ꢁC (71%); (c) RNHMe,
BOP reagent, TEA, DMF, rt (80%).

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M. S. Egbertson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1392–1398
Table 2. SAR of analogs including amide side chainsa,b,c,d
Compound
R
Inhibition of strand
a
transfer IC50 (nM)
Antiviral activity in cell
b
culture IC95 (nM)
PBd (%)
LogP c
10% FBS
50% NHS
7
10
125
1000
99
1.9
5a
5b
14
7
167
109
1000e
94
92
1.0
1.0
438
a The inhibition of strand transfer assay was performed with recombinant HIV-IN (0.1 lM) preassembled on immobilized oligonucleotides using
0.5 nM DNA.2b Values are means of at least two experiments (for compound 1, n = 5), lower limit of accuracy ꢀ5 nM, standard deviation is ꢀ2-
fold.
b Antiviral activity was assessed by measuring the decrease in HIV-1 p24 core antigen in MT-4 human T-lymphoid cells/HIV-1IIIb cultured in the
presence of increasing concentrations of inhibitor. Antiviral activity in cell culture (IC95) is the drug concentration which inhibits 95% viral growth
relative to control in cell culture. Low protein conditions (FBS): run with 10% fetal bovine serum, high protein conditions (NHS): run with 50%
normal human serum. For all compounds n = 2 or greater unless otherwise noted. For compound 1 n = 237. Lower limit ꢀ7 nM. Standard
deviation ꢀ2-fold.4
c % Protein binding ultrafiltration method, see Ref. 3.
d LogP measurement, see Ref. 5.
e n = 1.
The amide of interest could then be readily formed by
heating the ester with the required amine in methanol
under pressure. Deprotection with HCl gas gave the sta-
ble amine hydrochloride salts 17a or 17b in good yield
(overall yield 56% for four steps).
culture in the presence of NHS is therefore improved
6-fold relative to 1 to give an IC95 of 16 nM.
An analysis of the amide’s and sultam’s effects on the
potency and physical properties of the naphthyridine
template is shown in Table 4.14 The 2-methylamide
has a minimal impact on intrinsic potency. The strand
transfer potency of the unsubstituted naphthyridine 22
is similar to that of the 2-methylamido-substituted ana-
log 23 and, likewise, unsubstituted 1 has the same poten-
cy as its 2-methylamido-substituted analog 21b. The
2-methylamido group lowers the logP of 22 by 1 log
unit and of 1 by 0.5 log units. The unsubstituted naph-
thyridine 22 is highly protein bound (99%). Addition to
22 of either the polar 2-methylamido group to give 23 or
the sultam to give 1 results in a very small or undetect-
able change in the protein binding (0–2%). However,
combination of both groups in compound 21b has a
synergistic effect and leads to a ꢀ7% decrease in protein
binding relative to 22. This particular combination of
protein binding and logP provides improved cell culture
activity in this series.
The final compounds were prepared by pre-activating
acid 3 with EDT/HOAT, then coupling with the amine
hydrochlorides 17a and 17b, and base. The major prod-
ucts were the desired naphthyridine amides 21a and 21b,
(Scheme 5) and the formation of 11 was minimized
(yield 65%).
The results for the 2-amide analogs are shown in Table 3.
Even though 21a is less potent than 1 in the strand transfer
assay (Table 3) and less potent in cell culture in FBS, the
lower protein binding (90% vs 99% for 1) contributes to
improved antiviral properties in the presence of NHS
(63 vs 102 nM for 1).
The mono-methylamide 21b retains intrinsic activity
against IN (strand transfer IC50 = 10 nM) and shows a
decrease in protein binding (92% vs 99% for compound
1). Antiviral activity in cell culture under low protein
conditions improves 2-fold relative to 1 (IC95 = 7 vs
14 nM), and only a 2-fold loss of activity is observed
in the presence of 50% NHS. The antiviral activity in cell
The lower protein binding of 21b was confirmed with a
more accurate ultracentrifugation protocol using 14C
radiolabeled inhibitor. Under these conditions a 10-fold
increase in free fraction is observed between radiolabeled

M. S. Egbertson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1392–1398
Table 3. SAR of analogs including polar side chainsa,b,c,d
1395
Compound
R
H
Inhibition of strand
a
transfer IC50 (nM)
Antiviral activity in cell
cultureb
PBc (%)
LogPd
10% FBS
50% NHS
1
10
30
15
16
102
94
99
97
2.1
1.9
8a
8b
9
45
10
<8e
12
125
49
99
98
90
1.9
1.6
1.0
21a
110
23
63
21b
10
7
16
92
1.6
a The inhibition of strand transfer assay was performed with recombinant HIV-IN (0.1 lM) preassembled on immobilized oligonucleotides using
0.5 nM DNA.2b Values are means of at least two experiments (for compound 1, n = 5), lower limit of accuracy ꢀ5 nM, standard deviation is ꢀ2-
fold.
b Antiviral activity was assessed by measuring the decrease in HIV-1 p24 core antigen in MT-4 human T-lymphoid cells/HIV-1IIIb cultured in the
presence of increasing concentrations of inhibitor. Antiviral activity in cell culture (IC95) is the drug concentration which inhibits 95% viral growth
relative to control in cell culture. Low protein conditions (FBS): run with 10% fetal bovine serum, high protein conditions (NHS): run with 50%
normal human serum. For all compounds n = 2 or greater unless otherwise noted. For compound 1 n = 237. Lower limit ꢀ7 nM. Standard
deviation ꢀ2-fold.4
c % Protein binding ultrafiltration method, see Ref. 3.
d LogP measurement, see Ref. 5.
e n = 1.
Scheme 2. Free base cyclization to lactam.
21b and 1 (15% vs 1.5%).13,15 The solubility of 21b at pH
7.4 is 2.3 lg/mL compared to 0.21 lg/mL for 1.6
The pharmacokinetic profiles of 21b are comparable to
those of 1 in rat, dog, and rhesus (Table 5).16 Figure 1
shows the pharmacokinetic profile of 21b sodium salt
Scheme 3. Synthesis of compound 17a. Reagents and conditions: (a)
NBS, CCl4 benzoyl peroxide (71%); (b) NaH, HN(BOC)2, DMF 50 ꢁC
in rats after oral dosing at 10 mg/kg in 1% aqueous
methylcellulose. At 24 h, the concentration of drug
remaining (78 25 nM) is still ꢀ5-fold greater than
the IC95 for antiviral activity in cell culture (16 nM).
(88%); (c) Pd(OAc)2, dppf, DIEA, DMF, CO(g) 75 psi, NH2Me > 16
equiv 70 ꢁC (ꢀ65% mono plus bis-BOC products); (d) HCl (g) EtOAc,
ꢁ78 to 0 ꢁC (quant.).
tial mutant viruses. Figure 2 shows the comparative
resistance profile of compounds 1 and 21b against virus
containing site-directed mutations. These mutations
A desirable feature of a HIV IN drug candidate is the
ability to display good antiviral activity against poten-

1396
M. S. Egbertson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1392–1398
Table 5. Compounds 1 and 21b pharmacokinetic profilea
Pharmacokinetics, Na+ saltsa
F (%)b T1/2 iv (h)c
Species
Cld
(mL/min/kg)
1
21b
1
21b
1
21b
Rat
Dog
41
24
51
45
65
23
1.2
2.9
0.7
1.2
1.8
1.4
2.8
2.0
6.6
8.6
2.8
Rhesus
18.3
Scheme 4. Alternative synthesis of compounds 17. Reagents and
conditions: (a) NBS, CCl4, benzoyl peroxide (71%); (b) NaH,
HN(BOC)2, DMF, 0 ꢁC (88%); (c) MeNH2, MeOH sealed tube,
70 ꢁC, (95%); (d) HCl (g) EtOAc, ꢁ78 to 0 ꢁC (95%).
a For dose and methods see Ref. 16.
b %F, percent bioavailability after oral administration.
c T1/2
half-life of concentration in hours after intravenous (iv)
,
administration.
d Cl, clearance in mL/min/kg after iv administration.
were observed to arise from serial passage in cell culture
under exposure to diketoacid IN inhibitors and 1.2b
Compound 1 is somewhat susceptible to virus with
mutations in the active-site residues F121 and N155
(4- and 12-fold loss in efficacy observed relative to wild
type). Compound 21b retains excellent activity against
all these mutations, with only 2- to 4-fold shifts in the
IC50 observed against the mutants compared to wild
type virus.
The combination of favorable properties found in 21b
prompted us to learn more about its structure. Figure 3
shows the X-ray structure of 21b.17 The amide bond
Scheme 5. Preparation of compounds 21. Reagents and conditions: (a)
1.3 equiv EDC, 1.3 equiv HOAT; (b) 1.1 equiv amine, 1.1 equiv
Hunig’s base (65%).
Table 4. Group effects on antiviral potency and protein binding are additivea,b,c,d
Compound
R1
R2
Inhibition strand
a
transfer IC50 (nM)
Antiviral activity in cell
b
culture (NHS) IC95 (nM)
Protein bindingc
Log P d
22
23
H
CONHMe
H
H
33
51
5000
1042
99
98
2.4
1.4
1
H
10
10
102
16
99
92
2.1
1.6
21b
CONHMe
a The inhibition of strand transfer assay was performed with recombinant HIV-IN (0.1 lM) preassembled on immobilized oligonucleotides using
0.5 nM DNA.2b Values are means of at least two experiments (for compound 1, n = 5), lower limit of accuracy ꢀ5 nM, standard deviation is ꢀ2-fold.
b Antiviral activity was assessed by measuring the decrease in HIV-1 p24 core antigen in MT-4 human T-lymphoid cells/HIV-1IIIb cultured in the
presence of increasing concentrations of inhibitor. Antiviral activity in cell culture (IC95) is the drug concentration which inhibits 95% viral growth
relative to control in cell culture. Low protein conditions (FBS): run with 10% fetal bovine serum, high protein conditions (NHS): run with 50%
normal human serum. For all compounds n = 2 or greater unless otherwise noted. For compound 1 n = 237. Lower limit ꢀ7 nM. Standard
deviation ꢀ2-fold.4
c
% Protein binding ultrafiltration method, see Ref. 3.
d
LogP measurement, see Ref. 5.

M. S. Egbertson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1392–1398
1397
Rat PK 21b
group with a torsion angle of 49ꢁ. The sultam nitrogen
is sp3 hybridized, and the sultam ring is perpendicular
to the plane of the naphthyridine.
100
10
experiment 1
experiment 2
experiment 3
Average
In summary, optimization of 1 has led to 21b
(L-900564), an inhibitor of HIV IN, that has excellent cell
potency in the presence of NHS (IC95 NHS = 16 nM,
n = 15), excellent activity against mutants raised to
HIV IN inhibitors, and a very good pharmacokinetic
profile. Compound 21b was selected for further pre-clin-
ical evaluation.
1
0.1
0.01
Acknowledgments
0
5
10
15
20
25
30
Time (Hours)
The authors thank Peter Maligres and Michael Palucki
of Rahway Process and John Wai of West Point Medic-
inal Chemistry for helpful discussions, Carl Homnick
and Graham Smith for analytical support, Charles W.
Ross and Steve Pitzenberger for mass spec and NMR
support, and Jim Guare, Randy Newton, Joe Paw-
luczyk, and Rick Woodward of the West Point Medici-
nal Chemistry Synthetic Services Team for timely
supplies of intermediates.
Figure 1. Pharmacokinetic profile for 21b sodium salt dosed orally in
rats (10 mg/kg, 1% aq methylcellulose suspension).16
12
T661*
10
8
V151I"
F121Y"
6
T125K"
4
T66I/M154I*!
T66I/S153Y^!
N155S^*!
References and notes
2
1. For a recent review on the structure and function of HIV-1
IN, see: (a) Chiu, T. K.; Davies, D. R. Curr. Top. Med.
Chem. 2004, 4, 965; (b) Pommier, Y.; Johnson, A. A.;
Marchand, C. Nat. Rev. Drug Disc. 2005, 4, 236; For a
recent review on HIV-1 IN inhibitors, see: (c) Anthony, N.
J. Curr. Top. Med. Chem. 2004, 4, 979.
0
1
21b
Figure 2. Comparative resistance profile of 1 and 21b with HIV-1
containing site-directed IN mutations. Numbers denote shift in IC50
relative to wild type HIV-1 as derived from single cycle infectivity
assays. ^Viruses selected by serial passage in the presence of diketo-
acids. ‘‘Viruses selected by serial passage in cell culture in the presence
of 1. *Viruses which exhibit >50% reduction in specific infectivity
(impaired replication capacity).2b
2. (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.;
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, M. K.;
Young, S. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
11233; (c) Little, S.J.; Drusano, G.; Schooley, R.; Haas,
D.W.; Kumar, P.; Hammer, S.; McMahon, D.; Squires,
K.; Asfour, R.; Richman, D.; Chen, J.; Saah, A.; Leavitt,
R.; Hazuda, D.; Nguyen, B.-Y. for Protocol 004 Study
Team, 12th Conference on Retroviruses and Opportunis-
tic Infections, Feb. 22–25, 2005, Boston, MA, Abstract
161.
3. Human plasma binding. Compound is dissolved in pH 7.4
buffer (NaH2PO4/Na2HPO4) to give a 5 · 10ꢁ5 M solution
and filtered through a 0.45-mm filter. 4.5 mL is diluted
with 0.5 mL, pH 7.4 buffer to give a control solution.
4.5 mL is diluted with 0.5 mL human plasma (final plasma
conc. = 5.8 · 10ꢁ5 M) to give the test solution. The control
and test solutions are pipetted into Centrifreeꢂ ultrafil-
tration devices and centrifuged. The control and test
Figure 3. ORTEP representation of 21b. Non-hydrogen atoms are
represented by ellipsoids corresponding to 30% probability. Hydrogen
atoms have been drawn at an arbitrary size.
linking the benzyl group to the naphthyridine is approx-
imately coplanar with the naphthyridine and aligned to
hydrogen bond with the phenolic oxygen. The monom-
ethyl amide is twisted out of the plane of the benzyl
ultrafiltrates are assayed by HPLC compared to
a
5 · 10ꢁ5 M reference solution of compound dissolved in

1398
M. S. Egbertson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1392–1398
MeOH. KD = [Cf (HPd ꢁ Ci + Cf)]/(Ci ꢁ Cf) (calcd). %
Felock, P. J.; Wolfe, A. L.; Stillmock, K. A.; Schleif, W.
A.; Gabryelski, L. J.; Young, S. D. J. Med. Chem. 2000,
43, 4923.
Binding = 100 [(Ci ꢁ Cf)/Ci] (obsd). % Binding = 100
*
*
[HP/(HP + KD)] (calcd) where Ci = initial compound
concentration (from control); Cf = final compound concen-
tration (from plasma average); HP = assumed initial human
plasma concentration (5.8 · 10ꢁ4 M); HPd = assumed
diluted human plasma concentration (5.8 · 10ꢁ5 M); KD,
dissociation constant.
11. Perlow, D. S.; Kuo, M. S.; Moritz, H. M.; Wai, J. S.;
Egbertson, M. S. Syn. Comm. 2007, 37, in press.
12. Maybridge Chemicals.
13. Protein binding determination with radiolabeled material.
An ultracentrifugation method was used to determine the
unbound fraction of radiolabeled compound in human
plasma. Plasma (pH adjusted to 7.4) was treated with
compound to yield final concentrations of 1, 5, and 20 lM.
Following incubation at 37 ꢁC for 30 min, aliquots of
plasma were transferred immediately to 1.5 mL polyallo-
mer centrifuge tubes. The tubes were centrifuged at 37 ꢁC
4. 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.
5. UV detection method determining the relative concentra-
tion of a methanol solution of a compound partitioned
between an octanol and pH 7.4 (KH2PO4/NaOH) water
layer. HPLC methods are based on the maximum absor-
bance of each compound. LogP = log (Octanol HPLC
area)(Octanol dilution)/(Buffer HPLC area)(buffer
dilution).
6. Solubility determination protocol. A suspension of crystal-
line drug in 10 mM, pH 7.4, sodium phosphate buffer was
allowed to equilibrate at room temperature for 5 days.
Aliquots of the suspension were centrifuged at 15,000 rpm
for 15 min. The solution drug concentration determined
by HPCL at day 2 and 5 is taken as the solubility value.
7. Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R.
E.; Yan, L.; Whitesides, G. M. J. Am. Chem. Soc. 2000,
122, 8303.
8. 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 IN inhibitors. PCT Int. Appl. (2002),
434 pp. WO 0230930 A2 20020418.
and 180,000g overnight (ꢀ18 h) using
a high-speed
centrifuge (Beckman OptimaTM, Model TLX). A six- or
eight-place, fixed-angle rotor (TLA-100.3 or TLA-100.4)
was used. Aliquots (0.2 mL each) of the supernatant were
removed sequentially. Radioactivity in supernatants and
original plasma samples was determined by liquid scintil-
lation spectrometry with quench correction by external
standardization. The percent unbound fraction (fu) was
calculated as follows: fu (%) = dpm in protein-free section/
dpm in original plasma · 100%.
14. Compounds 22 and 23 were prepared in a manner similar
to that described for 21b. See Ref. 9 for experimental
details.
15. Compounds 1 and 21b containing 14C at the 8-carboxy
carbon were prepared by methods outlined in reference 8,
using methyl N-[(4-methylphenyl)sulfonyl]glycinate labeled
with 14C at glycine carbonyl carbon to give 14C labeled 3,
which was coupled as described above to give the final
compounds.
16. The sodium salts were ground in a mortar and pestle, and
dosed orally as an aqueous solution/suspension in 1% aq
methylcellulose in rats (10 mg/kg). In dogs and rhesus
monkeys 0.5% aq methylcellulose suspensions were dosed
at 1 mg/kg. Compound was dosed intravenously dissolved
in dimethylsulfoxide (DMSO) at concentrations of 2 mg/
kg in rats and 1 mg/kg in dogs and rhesus.
17. Crystallographic data (excluding structure factors) for 21b
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number
619397. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road,
Cambridge CB@ 1EZ, UK [Fax: +44 1223 336033 or
email: depost@ccdc.cam.ac.uk].
9. Egbertson, M.; Langford, H. M.; Melamed, J. Y.; Wai, J.
S.; Han, W.; Perlow, D. S.; Zhuang, L.; Embrey, M.;
Young, S. D. N-(substituted benzyl)-8-hydroxy-1,6-naph-
thyridine-7-carboxamides useful as HIV IN inhibitors
US20050176955 A1.
10. 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.;