4-Amino-1-hydroxy-2-oxo-1,8-naphthyridine-containing compounds having high potency against raltegravir-resistant integrase mutants of HIV-1

Article abstract of DOI:10.1021/jm5001908

There are currently three HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) approved by the FDA for the treatment of AIDS. However, the emergence of drug-resistant mutants emphasizes the need to develop additional agents that have improved efficacies against the existent resistant mutants. As reported herein, we modified our recently disclosed 1-hydroxy-2-oxo-1,2-dihydro-1,8- naphthyridine-3-carboxamides IN inhibitors to develop compounds that have improved efficacies against recombinant IN in biochemical assays. These new compounds show single-digit nanomolar antiviral potencies against HIV vectors that carry wild-type (WT) IN in a single round replication assay and have improved potency against vectors harboring the major forms of drug resistant IN mutants. These compounds also have low toxicity for cultured cells, which in several cases, results in selectivity indices (CC₅₀/EC₅₀) of greater than 10000. The compounds have the potential, with additional structural modifications, to yield clinical agents that are effective against the known strains of resistant viruses.

Full text of DOI:10.1021/jm5001908

Article
pubs.acs.org/jmc
4‑Amino-1-hydroxy-2-oxo-1,8-naphthyridine-Containing
Compounds Having High Potency against Raltegravir-Resistant
Integrase Mutants of HIV‑1
Xue Zhi Zhao,† Steven J. Smith,‡ Mathieu Metifiot,§,∥ Christophe Marchand,§ Paul L. Boyer,‡
́
Yves Pommier,§ Stephen H. Hughes,‡ and Terrence R. Burke, Jr.*,†
‡
†Chemical Biology Laboratory, and HIV Drug Resistance Program, Center for Cancer Research, NCI at Frederick, National
Institutes of Health, Building 376, Boyles Street, P.O. Box B, Frederick, Maryland 21702, United States
§Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
ABSTRACT: There are currently three HIV-1 integrase (IN) strand transfer inhibitors (INSTIs)
approved by the FDA for the treatment of AIDS. However, the emergence of drug-resistant mutants
emphasizes the need to develop additional agents that have improved efficacies against the existent
resistant mutants. As reported herein, we modified our recently disclosed 1-hydroxy-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamides IN inhibitors to develop compounds that have improved
efficacies against recombinant IN in biochemical assays. These new compounds show single-digit
nanomolar antiviral potencies against HIV vectors that carry wild-type (WT) IN in a single round
replication assay and have improved potency against vectors harboring the major forms of drug
resistant IN mutants. These compounds also have low toxicity for cultured cells, which in several cases, results in selectivity
indices (CC50/EC50) of greater than 10000. The compounds have the potential, with additional structural modifications, to yield
clinical agents that are effective against the known strains of resistant viruses.
INTRODUCTION
■
Acquired immunodeficiency syndrome (AIDS) is an infectious
disease caused by the human immunodeficiency virus (HIV).
Reverse transcriptase (RT), protease (PR), and integrase (IN)
are the three viral enzymes that are required for viral replication
and all three have been targeted by anti-AIDS therapeutics. IN
catalyzes the insertion of viral DNA into the host genome in
two sequential steps, termed “3′-processing” (3′-P) and “strand
transfer” (ST). The 3′-P reaction cleaves two nucleotides from
the 3′ end of the viral DNA, exposing a deoxycytosine residue
that is used in a nucleophilic attack on the host DNA in the ST
reaction. Both of these reactions involve two Mg2+ ions held in
place by three acidic residues Asp64, Asp116, and Glu152 that
collectively constitute the “DDE” motif.1 IN inhibitors are the
most recently developed class of anti-AIDS drugs. Merck’s
raltegravir (RAL, 1, Figure 1) (October 2007)2 and Gilead’s
elvitegravir (EVG) (August 2012)3 were the first two IN
Figure 1. Structures of HIV-1 integrase inhibitors described in the text.
Mg2+-chelating heteroatoms are shown in red, with the 4-position of
the 1-hydroxy-1,8-naphthyridin-2(1H)-one ring being indicated in
blue.
inhibitors to be approved by the FDA. The approved IN
inhibitors selectively block the ST step, and members of this
class of drugs are called “IN strand transfer inhibitors”
(INSTIs) because of their ability to preferentially block the
enzyme’s ST reaction relative to the 3′-P reaction.4 All of the
known INSTIs share important structural features, which
include a coplanar arrangement of three heteroatoms that
chelate the two catalytic Mg2+ ions, and a halobenzyl ring that
binds to the penultimate base (a deoxycytidine) adjacent to the
deoxyadenosine that lies at the 3′ end of the viral DNA after the
3′-P reaction. Binding of INSTIs blocks the ST reaction by
displacing the viral 3′-terminal deoxyadenosine from the
catalytic Mg2+ ions. Treatment with 1 and EVG selects for
resistant forms of HIV, and there is considerable cross-
resistance to these two drugs. GlaxoSmithKline’s dolutegravir
(DTG, 2, Figure 1)5,6 is a recently FDA-approved second-
Received: February 4, 2014
© XXXX American Chemical Society
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generation INSTI (August 2013), which shows improved
efficacies against RAL and EVG-resistant strains of HIV.7,8
However, 2 also selects for resistant strains of HIV.7 This
emphasizes the need to develop agents that can overcome
resistant strains of IN, including the emerging strains resistant
to 2. We recently reported that 1-hydroxy-1,8-naphthyridin-2H-
one-3-carboxamides, which include both 4-unsubstituted and 4-
hydroxyl-containing analogues (3 and 4, respectively, Figure 1),
potently inhibit wild-type (WT) IN in biochemical assays and
show good antiviral efficacies in single-round infection assays of
HIV-1 infectivity (Figure 1).9 Importantly, members of this
series retain good antiviral potency against a set of mutants
resistant to 1 in these latter assays. Here we describe structural
variation at the 4-position of compound 3, which yielded agents
of type 5 (Figure 1) that enhance their efficacy against
additional mutant forms of IN that are resistant to 1.
achieved via the heteroatom triad formed by the N-hydroxyl
group, the 2-oxo group, and the 8-naphthyridine nitrogen.
However, an important component of 3 and 4, not found in 6,
is a halobenzyl group, which is known to be important for
binding to IN by interacting with the penultimate deoxy-
cytosine in the 3′-end of enzyme-bound viral DNA.1 In the case
of 3 and 4, this binding function is served by a 2′,4′-
difluorobenzyl carboxamide group, which is appended at the 3-
position of the bicyclic nucleus. Previous work has shown that
the nature and pattern of halogen phenyl substitution can
significantly affect the potency of INSTIs.17 In developing 3
and 4, we found that a 2′,4′-difluorobenzyl moiety, which is
present in 2, was superior to the other halobenzyl rings we
tested.9 An important feature of 3 and 4 is that the carbonyl
oxygen of the 2′,4′-difluorobenzyl amide group may not be an
obligatory component of the metal-chelating triad. As a
consequence, there may be greater flexibility in this region of
the molecule than is found with inhibitors, such as 1, where the
halobenzyl amide carbonyl participates in Mg2+ chelation. This
flexibility is reminiscent of what is seen with 2, where the
flexibility of the haloamide component is thought to contribute
to the ability of 2 to maintain efficacy against certain forms of
IN that are resistant to 1.18−20
In our current work, we further modified the 1-hydroxy-1,8-
naphthyridin-2H-one nucleus by incorporating new function-
alities at the 4-position. In undertaking these efforts, we noted
that for RNase H inhibitors such as 6, an extended aryl
functionality increased their inhibitory potency.14 Therefore, we
began by preparing inhibitor 5a (Figure 2). In contrast to 6,
where the aryl functionality is attached through a methylene
unit, for reasons of synthetic simplicity, we employed a 4-amine
group in 5a. Subsequently, we prepared a series of analogues
(5a−5v) using an iterative process of design, synthesis,
biological evaluation, and redesign.
RESULTS AND DISCUSSION
■
Inhibitor Design. IN is a member of the polynucleotidyl
transferase class of enzymes that share similar catalytic
mechanisms.10 There is also a large body of data that describes
the known INSTIs, their efficacy against WT and drug-resistant
forms of HIV, and their interactions with prototype foamy virus
(PFV) IN. Accordingly, the structures of previously described
inhibitors can be used to aid the design of new anti-IN
compounds. This is exemplified by the development of 1 by
Merck, which can be traced to dihydroxypyrimidine carbox-
amide inhibitors of the hepatitis C virus NS5b RNA polymerase
(RNAP).10,11 Inhibitors of HIV-1 ribonuclease H (RNase H)
have been reported, which, like INSTIs, inhibit their target
enzyme by chelating two Mg2+ ions in the enzyme active site. In
RNase H, the Mg2+ ions are held in place by a “DEDD motif”
(D443, E478, D498, and D549).12−14 Compounds that inhibit
both IN and RNase H have been reported.15,16 By analogy to
the RNAP example, the known RNase H inhibitors might
provide insights that can be used to design improved INSTI
inhibitors. Accordingly, the design of our bicyclic 1-hydroxy-
1,8-naphthyridin-2H-one INSTIs (3 and 4)9 was guided by the
report that compounds such as the biaryl-containing 6 (Figure
2), which have submicromolar inhibitory potency against
Synthesis. Amidation of methyl ester 7 (obtained in three
steps from commercially available methyl 2-fluoronicoti-
nate)9,21 using 2,4-difluorobenzylamine gave the known
amide 8 in 70% yield (Scheme 1).9,21 Subsequent reaction
with toluenesulfonyl chloride produced the tosylated analogue
9 (93% yield), which was treated with a variety of amines to
provide 10a−10v (Scheme 1). A subset of these amines (10a−
10u) was converted to final products (5a−5u) by hydro-
genolysis of the N-benzyloxy group (H2/10% Pd·C). In the
case of final product 5p, acetylation of intermediate 10o to
yield 10p was done prior to debenzylation. For final product
5v, treatment of intermediate 10v with TFA yielded the free
amine 11 prior to debenzylation (Scheme 1).
Biological Evaluation. Compounds were evaluated in
biochemical assays using radiolabeled oligonucleotides to
measure their inhibitory potential in the 3′-P and ST
reactions.9,22 The initial series of compounds was designed to
examine the role of aromatic functionality at the 4-position.
The IN ST inhibitory potency of 5a (IC50 = 0.34 0.08 μM,
Table 1) was approximately 10-fold better than the value
previously reported for 6, which contains a similar 1,4-phenyl
group.14 The conformationally constrained biphenyl amine
analogue 5b was slightly less potent than 5a. In contrast,
introducing a 4′-nitrile or a 4′-amino group onto 5a (giving 5c
and 5d, respectively) slightly increased the potency of the
compound in the ST reaction relative to 5a. Importantly,
shortening the 4-substituent by removal of one phenyl ring
gave an approximate 19-fold enhancement in potency relative
to 5a (5e, ST IC50 = 0.018 0.006 μM, Table 1).
Figure 2. HIV-1 ribonuclease H (RNase H) inhibitors 6 and integrase
inhibitor 5a with the core metal-chelating 1-hydroxy-1,8-naphthyridin-
2(1H)-one system shown in red and the key 4-amino group indicated
in blue.
RNase H (IC50 = 0.64 μM), also have low micromolar
inhibitory potency against HIV-1 IN (ST IC50 = 2.4 μM).14 In
the case of 6, the reported EC50 value (half-maximal
concentration providing protection against viral-induced cell
death) in a HIV-1 HXB2 single-cycle viral replication assay in
HeLa P4-2 cells was 34 μM.14
Metal chelation by the 1-hydroxy-1,8-naphthyridin-2H-one
nucleus, which is common to 3, 4, and 6, can theoretically be
B
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Scheme 1. Synthesis of 4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides 5a−
a
5v
a
Reagents and conditions: (i) 2,4-diFBnNH2; (ii) TsCl, TEA, MeCN; (iii) R1R2NH or R1R2NH-HCl, DIEA, DMF; (iv) Ac2O, TEA, DCM; (v)
TFA, DCM; (vi) H2, 10% Pd·C, MeOH.
showed nanomolar ST inhibitory potencies against WT
enzyme, with 5d and 5e being significantly more potent
(EC50 = 6.3 2.4 and 14 1.9 nM, respectively) than other
members of the series (EC50 values >100 nM). Because all
compounds showed no cytotoxicity up to 250 μM), selectivity
indices (SI = CC50/EC50) were from at least 500 to greater than
approximately 40000 (Table 2). Of particular note, while
compound 5e was only slightly less potent against the WT
vector than what has been reported for 1 (EC50 = 4 2 nM), it
was significantly less susceptible to loss of efficacy against the
mutants: Y143R (2-fold loss versus a reported 54-fold loss for
1), N155H (8-fold loss versus a reported 39-fold loss for 1),
and G140S/Q148H (32-fold versus a reported 425-fold loss for
1).26 The large loss of potency incurred by 1 against the Y143R
mutant derives from a loss of π−π stacking of the inhibitor with
the Y143 phenyl ring. The ability of compounds in the current
series to retain good efficacy against the Y143R mutant
indicates that they do not have a similar interaction as 1 with
the aryl ring of Y143.
Table 1. Inhibitory Potencies of Carboxamides 5a−5e
a
Obtained Using an in Vitro IN Assay
We prepared an additional series of analogues (5f−5v) in
which several alkylamines were introduced at the 4-position
(Table 3). In biochemical assays in vitro, most of these
analogues exhibited low nanomolar inhibitory potencies in the
ST reaction. However, compounds 5i and 5k, which contained
cycloheptyl and n-butylphenyl substituents, respectively, had
ST IC50 values of 0.46 0.18 and 0.28 0.11 μM, respectively,
which were markedly elevated relative to other members of the
series. A third member of the series, having an (S)-ethyl N-
prolinate group, was also significantly less potent [(S)-5u, ST
IC50 = 0.31 0.04 μM] (Table 3).
a
Assays were performed using a gel-based protocol with Mg2+ cofactor
Antiviral potencies were determined for 5f−5v in cells
infected with viral vectors harboring WT and mutant forms of
IN (Table 4). Most compounds of the series displayed EC50
values in the low nanomolar range against the WT vector, with
a majority of the compounds showing single-digit nanomolar
potencies. These compounds also showed low cytotoxicity,
which resulted in good SI values, with several compounds
showing SI > 10000. Noteworthy exceptions were compounds
5g, 5h, and (S)-5u, which not only had significantly reduced
as describe in ref 22.
Antiviral potencies were evaluated in a cell-based assay using
lentiviral vectors carrying WT IN as well as mutant forms of IN
that are resistant to 1, Y143R, N155H, and the double mutant,
G140S/Q148H.23−25 In these assays, amine 5a was approx-
imately two orders-of-magnitude more potent against the WT
enzyme (EC50 = 372 63 nM, Table 2) than what has been
reported for 6 (32 μM).14 All members of the series (5a−5e)
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Table 2. Antiviral Potencies of Carboxamides 5a−5e in Cells Infected with HIV-1 Constructs Containing WT or Mutant IN
c
EC50 (FC, IN mutants )
a
b
d
compd
CC50 (μM)
EC50 (nM, WT)
Y143R
N155H
G140S/Q148H
SI
e
e
5a
5b
5c
5d
5e
>250
>250
>250
>250
>250
372 63
171 57
123 21
6.3 2.4
14 1.9
1×
3×
3×
16×
2×
N/A
N/A
>672
>1462
e
e
N/A
N/A
e
e
N/A
N/A
>2033
e
67×
8×
N/A
>39683
>17857
32×
a
b
Cytotoxic concentration resulting in 50% reduction in the level of ATP in human osteosarcoma (HOS) cells. Values obtained from cells infected
c
with lentiviral vector harboring WT IN. Cells were infected with viral constructs carrying IN mutations and indicated values correspond to the fold-
change (FC) in EC50 relative to WT. Selectivity index calculated as the ratio of CC50 to EC50. Not available.
d
e
lower). In some cases, the in vitro IC50 values for 5g and 5h
Table 3. Inhibitory Potencies of Carboxamides 5f−5v
a
were better than compounds such as 5i and 5k (460 and 280
nM, respectively, Table 3), which paradoxically exhibited good
EC50 values against the WT vector (12 3 and 50 13 nM,
respectively, Table 4). These data could indicate that being able
to form an intramolecular hydrogen bond between the 4-amino
group and the 3-carboxamide carbonyl oxygen has a more
important role for the activity of the compounds in an antiviral
assay done in cultured cells than in the biochemical assay done
in vitro.
The main objective of the current study was to derive
minimally cytotoxic inhibitors having good antiviral potency
against cells infected with WT virus, which also retained their
efficacy against viruses harboring mutant forms of IN that are
resistant to 1. As shown in Table 4, relative to their potencies
against WT, almost all members of the current series
maintained complete or nearly complete efficacy against virus
having the Y143R mutant. In addition, most members of the
series showed good retention of efficacy against virus having the
N155H and G140S/Q148H mutants (with a few exceptions,
10-fold or less loss of potency) (Table 4). Members of the
series also commonly exhibited high SI values, in the range of
four orders-of-magnitude.
On the basis of these data, we examined selected members of
the series (5o−5q and 5v) against a more extensive panel of
INSTI-resistant mutants that included R263 K and G118R
mutants, which have recently been identified through in vitro
selection studies with second-generation INSTIs.5 For
reference, we also included 1 and 2 as well as the parent 1-
hydroxy-1,8-naphthyridin-2H-ones (3 and 4), which formed
the starting points for the current series. Although 1 is potent
against viral vectors that carry WT IN (EC50 = 4 2 nM), it
shows extensive loss of antiviral efficacy against the mutants,
Obtained Using an in Vitro IN Assay
IC50 (μM)
compd
R1
R2
3′-processing
strand transfer
5f
−CH3
−CH3
−morpholino
−cycloheptyl
−CH2CH2Ph
−CH2(CH2)3Ph
−CH2(CH2)3CH3
−CH(CH3)2
−CH2CH2NH2
−CH2CH2OH
−CH2CH2OAc
−CH2CO2CH3
−H
−CH3
3.7 0.4
0.027 0.004
0.087 0.012
0.079 0.013
0.46 0.18
5g
21
2
5h
5i
77 12
13 1.1
−H
−H
−H
−H
−H
−H
−H
−H
−H
−H
5j
8.0 1.5
12 2.0
0.050 0.012
0.28 0.11
5k
5l
8.2 1.6
1.8 0.2
4.5 0.2
0.55 0.07
5.3 0.5
0.71 0.10
7.4 0.8
0.024 0.009
0.016 0.004
0.039 0.006
0.010 0.009
0.027 0.006
0.021 0.011
0.017 0.011
5m
5n
5o
5p
5q
(S)-5r
−NHCH(CH3)
CO2CH3
(R)-5r
(S)-5s
(R)-5s
(S)-5t
(R)-5t
−NHCH(CH3)
−H
−H
−H
−H
−H
5.8 0.6
16.7 1.4
13.5 1.0
5.8 0.5
4.4 0.5
0.027 0.005
0.010 0.002
0.0082 0.0015
0.0084 0.0032
0.013 0.04
CO2CH3
−NHCH(Ph)
CO2CH3
−NHCH(Ph)
CO2CH3
−NHCH(CH2OH)
CO2CH3
−NHCH(CH2OH)
CO2CH3
Y143R (EC50 = 162 16 nM; 41-fold loss), N155H (EC50
=
(S)-5u
−Pro-OEt
−H
86
6
0.31 0.04
154 33 nM; 38-fold loss), and G140S/Q148H (EC50 = 1900
300 nM; 475-fold loss). Compound 1 is more tolerant of the
G118R mutant (EC50 = 36 5 nM; 9-fold loss) and even less
affected by the R263 K mutant (EC50 = 9 4 nM; 2-fold loss)
(Table 5). In contrast, the recently FDA-approved second-
generation inhibitor 2, while showing similar potency to 1
5v
−H
2.5 0.3
0.019 0.002
a
Assays were performed using a gel-based protocol with Mg2+ cofactor
as describe in refs 9 and 22.
antiviral potencies (EC50 values of 268 8, 1200 260, and
590 72 nM, respectively) but also showed greater cytotoxicity
against WT vector (EC50 = 1.6
0.9 nM), exhibits a
significantly smaller loss of potency against vectors having the
mutants Y143R (EC50 = 4.3 1.2 nM; 3-fold loss), N155H
(EC50 = 3.6 1.3 nM; 2-fold loss), and G140S/Q148H (EC50
= 5.8 0.5 nM; 4-fold loss) (Table 5). However, as expected, 2
shows some loss of potency against virus having the R263 K
(CC50 values of 13
1.8, 8.4
3.2, and 18
7 μM,
respectively) (Table 4). These latter compounds are the only
members of the series having tertiary amines at the 4-position.
As such, these analogues would not be able to form internal
hydrogen bonds between their 4-amino groups and the 3-
carboxamide carbonyl oxygen. In spite of their poor antiviral
efficacies, the in vitro ST IC50 values for 5g and 5h (87 and 79
nM, respectively, Table 3) were only modestly elevated relative
to most other members of the series (typically 30 nM or
(EC50 = 11 3 nM; 7-fold loss) and G118R (EC50 = 13
5
nM; 8-fold loss) mutants.
Among the current series, the antiviral potencies of 5o−5q
are approximately equivalent to 1 against WT enzyme, with 5v
D
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Table 4. Antiviral Potencies of Carboxamides 5f−5v in Cells Infected with HIV-1 Constructs Containing WT or Mutant IN
c
EC50 (nM, IN mutants )
a
b
d
compd
CC50 (μM)
EC50 (nM, WT)
3.1 0.6
Y143R
N155H
G140S/Q148H
SI
5f
>250
13 1.8
8.4 3.2
68 8.7
>250
6.1 2.5 (2×)
273 52 (1×)
1800 870 (1.5×)
6.6 1.7 (0.55×)
18 2.6 (1×)
40 15 (0.80×)
8.8 2.7 (1×)
7.4 0.5 (1×)
57 13 (2×)
18 5.2 (6×)
2300 700 (9×)
3730 920 (3×)
14 4 (1×)
87 11 (28×)
6800 400 (25×)
6920 2000 (6×)
35 12 (3×)
>80645
49
5g
268
8
5h
1200 260
7
5i
12
14
3
8
5667
5j
79 5.2 (6×)
116 22 (2×)
12 4.5 (2×)
44 6.7 (6×)
119 3.4 (9×)
243 39 (5×)
71 0.14 (10×)
154 16 (21×)
>17857
2040
5k
102
8
50 13
7.4 1.4
7.2 3.0
35 11
5l
94 24
>250
12703
>34722
277
5m
e
e
5n
9.6 3.7
N/A
N/A
5o
24
3
5.2 0.6
4.5 1.5
3.8 1.2
4.2 1.6
11.3 4.7
4.6 1.8 (0.88×)
4.8 2.9 (1×)
4.6 2.2 (1×)
4.8 1.4 (1×)
8.1 1.7 (0.7×)
8.5 1.9 (0.9×)
8.2 1.8 (1 × )
11 4 (1 × )
25 4 (5×)
3.1 0.3 (0.69×)
19 7 (5×)
43 15 (8×)
35 14 (8×)
36 16 (9×)
141 20 (33×)
89 26 (8×)
4615
5p
>250
>55556
>65789
>59 524
>22 124
>27 778
>33 784
>25 773
>17 606
31
5q
>250
>250
>250
>250
>250
>250
>250
(S)-5r
(R)-5r
(S)-5s
(R)-5s
(S)-5t
(R)-5t
(S)-5u
5v
15.3 3.3 (4×)
27 9.9 (2×)
17.6 6.4 (2×)
22 4 (3 × )
29 8 (3 × )
71 5 (5 × )
9
2.4
7.4 3.3
9.7
55.6 6.2 (6×)
48 12 (6 × )
122 33 (13 × )
284 120 (20 × )
3
14.2 3.7
590 72
1.1 0.7
10.2 1.4 (0.7 × )
1660 920 (3 × )
2.5 0.6 (2 × )
e
e
18
7
N/A
N/A
>250
5.3 2.3 (5 × )
35 9 (32 × )
>227 273
a
b
Cytotoxic concentration resulting in 50% reduction in the level of ATP in human osteosarcoma (HOS) cells. Values obtained from cells infected
c
with lentiviral vector carrying WT IN. Cells were infected with viral constructs carrying IN mutations and indicated values correspond to the fold-
change (FC) in EC50 relative to WT. Selectivity index (SI) calculated as the ratio of CC50 to EC50. Not available.
d
e
Table 5. Antiviral Potencies of 5o−5q and 5v Compared with 1, 2, and Previously Reported Carboxamides 3 and 49 in Cells
Infected with HIV-1 Constructs Containing WT or Mutant IN
b
EC50 [nM/(FC), IN mutants ]
a
compd
EC50 (nM, WT)
Y143R
N155H
R263 K
G118R
G140S/Q148H
1
4
2
162 16 (41×)
4.3 1.2 (3×)
4.9 0.8 (1×)
11 2 (2×)
4.6 1.8 (0.88×)
4.8 2.9 (1×)
4.6 2.2 (1×)
2.5 0.6 (2×)
154 33 (38×)
3.6 1.3 (2×)
134 23 (26×)
31 8 (5×)
25 4 (5×)
3.1 0.3 (0.69×)
19 7 (5×)
9
4 (2×)
36 5 (9×)
13 5 (8×)
N/A
1900 300 (475×)
5.8 0.5 (4×)
438 121 (86×)
308 125 (50×)
43 15 (8×)
35 14 (8×)
36 16 (9×)
35 9 (32×)
2
1.6 0.9
5.1 1.9
6.2 2.9
5.2 0.6
4.5 1.5
3.8 1.2
1.1 0.66
11 3 (7×)
N/A
c
c
3
4
36 8 (6×)
26 (5×)
16 5 (4×)
26 8 (7×)
6.4 2.3 (6×)
107 8 (17×)
27 1 (5×)
44 11 (10×)
41 18 (11×)
16 5 (15×)
5o
5p
5q
5v
5.3 2.3 (5×)
a
b
Values obtained from cells infected with lentiviral vector harboring WT IN. Cells were infected with viral constructs carrying IN mutations and
c
indicated values correspond to the fold-change (FC) in EC50 relative to WT. Not available.
being slightly more potent (equivalent to 2) (Table 5).
Compounds 5p, 5q, and 5v also show fold-loss of potencies
that are approximately equivalent to 2 against the panel of
mutants, with the exception of 5v, which shows greater fold-loss
of potency against the mutant G140S/Q138H (32-fold).
However, because 5v has greater potency against the WT
virus than either 5p or 5q, the effective antiviral potencies
against virus having the G140S/Q138H mutant are approx-
imately equal for the three compounds (EC50 = 35 nM). A
summary analysis of Table 5 shows that 5p exhibits a profile
against the panel of mutants that is similar to 2. Because of its
slightly better potency against WT IN, compound 5v exhibits
the best overall performance among the new inhibitors, with
both absolute potencies and fold-loss of potencies against the
various mutant vectors that are similar to 2. The sole exception
is the G140S/Q148H mutant, where 5v suffers an approximate
7-fold loss of potency relative to 2. Importantly, 5v shows from
5- to 10-fold enhanced performance across the entire panel
relative to the starting compound 4. The standard assays are
performed in the presence of 5% fetal bovine serum (FBS). To
examine the potential effects of serum protein binding on their
antiviral potencies, compounds 5p, 5q, and 5v were evaluated
against the WT vector in the presence of increasing amounts
FBS (5%, 10%, and 15%). These experiments showed that the
antiviral potencies of the compounds were essentially
unchanged relative to values shown in Tables 4 and 5, which
were obtained in the presence of 5% fetal bovine serum protein
(data not shown).
The title compounds were originally designed to chelate the
two Mg2+ ions at the IN active site. However, HIV-1 reverse
transcriptase (RT) also contains two active sites, the polymer-
ase active site and the RNase H active site, each of which has
two bound Mg2+ ions. To examine whether representative
compounds of the series could also bind Mg2+ ions at one or
both of the active sites in RT, we tested 5p, 5q, and 5v, to see
whether they inhibited either the RNase H or the polymerase
activity of RT. Nevirapine, a well-known nonnucleoside reverse
transcriptase inhibitor (NNRTI), was included as a positive
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Figure 3. Polymerase and RNase H inhibition assays (A): The effects of 5p, 5q, and 5v on the DNA-dependent DNA polymerase activity of RT are
shown. Reactions were performed with WT RT in the presence of various concentrations of compounds (0, 0.02, 0.1, 0.5, 1, or 10 μM). Samples
were precipitated with EtOH and then fractionated on a 15% polyacrylamide sequencing gel. After electrophoresis, the gel was exposed to X-ray film.
RNase H inhibition assay (B,C,D): The effects of 5p, 5q, and 5v on the RNase H activity of RT are shown. The reactions were incubated for the
amount of time indicated (1, 5, 10, and 15 min) in the presence of the indicated concentration of the individual compounds (0, 0.1, 0.5, 1, and 10
μM). The size of intact RNA (full length) is 60 NT, as shown in the “No-RT” lane. The RNA fragments derived from the −17 and −8 families of
cleavages are shown.
control in the polymerase inhibition assays. The polymerase
inhibition assays show that all three compounds are able to
inhibit the DNA-dependent DNA polymerase activity of HIV-1
RT (Figure 3A). However, the data also show that the
compounds differ in their potency. All of the compounds are
less potent than nevirapine in the polymerase assay, Compound
5v the most potent, followed by 5p, then 5q. It is not clear at
this point whether the compounds are binding the Mg2+ ions at
the polymerase active site or binding within the NNRTI-
binding pocket near the polymerase active site, similar to
nevirapine. It is also possible that they may be binding at some
other site in HIV-1 RT.
The compounds were also tested for their ability to inhibit
the RNase H activity of RT. When RT binds an RNA−DNA
template/primer (T/P), the 3′ end of the DNA primer is
preferentially located at the polymerase active site; the RNase
H active site contacts the RNA template approximately 17 to
18 nucleotides (NT) from the polymerase active site.27 The
initial RNase H cleavage occurs approximately 17 NT from the
polymerase active site. These cleavages have been designated as
the −17 family of cleavages. The RT then alters its interactions
with the T/P, so that RNase H can make additional secondary
cleavages approximately 8 NT from the 3′ end of the primer
(−8 cleavages). The full length RNA is 60 NT in length. We
found that 5p, 5q, and 5v varied in their abilities to inhibit
RNase H activity (Figure 3B−D). However, the ranking of the
potencies of the compounds in the RNase H assay is different
from the polymerase assay (Figure 3A), which suggests that the
compounds are interacting with the RT in different places in
the two assays. The data show that 5q is the most potent
inhibitor of RNase H (it was the least potent compound in the
polymerase assay). Compound 5q did not completely block
RNase H activity, even at the highest concentration; a large
amount of full length RNA was present in the reaction that
contained 5q at the 10 μM concentration. It is also apparent
that more of the product of the −17 cleavages remained after
10 and 15 min as compared to the “no compound” controls.
This results from partial blocking the −8 cleavages. Similar
F
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Electrospray ionization−mass spectra (ESI-MS) were acquired with an
Agilent LC/MSD system equipped with a multimode ion source.
Purities of samples subjected to biological testing were assessed using
this system and shown to be ≥95%. High-resolution mass spectra
(HRMS) were acquired by LC/MS-ESI using an LTQ-Orbitrap-XL at
30K resolution.
General Procedure A. Preparation of 1-(Benzyloxy)-N-(2,4-
difluorobenzyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxa-
mide (10a−10v). A solution of p-methylbenzenesulfonate (9) (1.0
mmol), N-ethyl-N-isopropylpropan-2-amine (10 mmol) and amines
R1R2NH or R1R2NH·HCl (5.0 mmol) in DMF (2.0 mL) was heated
to 50 °C (1 h). The crude mixture was purified by CombiFlash silica
gel chromatography (hexanes and ethyl acetate) to provide amides
(10a−10v).
results were obtained with 5v, while 5p had very little effect on
RNase H activity. At the highest concentration, there were
subtle effects on the −8 cleavages, but the compound is
obviously less potent, in the RNase H assay, than the other two.
It is quite difficult to use the results of the in vitro assays to
estimate IC50 values. In the polymerase assay, it is clear that all
of the compounds are less potent than nevirapine. We did not
have a potent RNase H inhibitor that would allow us to make a
similar comparison in the RNase H inhibitor assay. Although it
is clear that the current compounds are primarily IN inhibitors,
because these compounds can inhibit both the RNase H and
polymerase activity of HIV-1 RT, they could serve as the
starting point for the synthesis of additional compounds that
would be specifically designed to inhibit these alternate targets.
In that regard, because there are no potent RNase H inhibitors,
and because many of the known RNase H inhibitors are
relatively toxic, using these compounds as leads to develop new
RNase H inhibitors is a potentially attractive option.
General Procedure B. Preparation of N-(2,4-Difluorobenzyl)-1-
hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides
(5a−5v). Amides (10a−10u or 11; 0.2 mmol) were dissolved in
MeOH (15 mL) and EtOAc (5 mL) and the solution degassed and
stirred at room temperature under H2 over Pd·C (10%, 0.2 mmol) (1
h). The mixture was filtered and the filtrate was concentrated, and the
resulting residue was purified by HPLC to provide amides (5a−5v).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-hydroxy-2-oxo-1,2-dihy-
dro-1,8-naphthyridine-3-carboxamide (8). A solution of methyl 1-
(benzyloxy)-4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carbox-
ylate (7)9 (1.0 g, 3.17 mmol) and (2,4-difluorophenyl)methanamine
(2.0 mL, 15.86 mmol) in DMF (3.0 mL) was subjected to microwave
irradiation with stirring (140 °C; 2 h). The resulting product mixture
was purified by CombiFlash silica gel chromatography to provide 8 as
CONCLUSION
■
Our current study examines the effects of introducing an amine
functionality at the 4-position of our previously reported 1-
hydroxy-1,8-naphthyridin-2H-ones (3 and 4).9 The focus of the
work was to enhance antiviral potency, with particular emphasis
on retaining efficacy against viruses harboring mutant forms of
IN that have been shown to be resistant to the first-generation
INSTI, 1. For reference we employed 2, which is a recently
FDA-approved second-generation INSTI with improved
performance against the known resistant mutants. Most
members of the series of new inhibitors (5) show single-digit
nanomolar antiviral potency against WT enzyme, with SI values
greater than 10000. As a whole, the family of new inhibitors
exhibited a smaller fold-change than 1 in antiviral assays that
employed IN mutants Y143R, N155H, and the double mutant
G140S/Q148H. Among the new inhibitors, compound 5p
showed a profile against the panel of mutants that was
comparable to 2, with 5v exhibiting the best overall absolute
performance among the new inhibitors, approximately 5- to 10-
fold enhancement relative to the starting compound 4.
Although 2 is more effective than first-generation INSTIs in
its ability to retain efficacy against resistant forms of IN, it has
been shown that 2 can select for resistant forms of the enzyme.
Therefore, there is a continuing need for the development of
new agents as potential alternatives to the currently approved
panel of three FDA-approved INSTIs. The structural class of
agents presented herein may represent an attractive platform
for developing such next-generation INSTIs.
1
a white solid (964 mg, 70% yield). H NMR (400 MHz, CDCl3) δ
10.35 (t, J = 5.5 Hz, 1H), 8.80 (dd, J = 4.7, 1.8 Hz, 1H), 8.49 (dd, J =
7.9, 1.8 Hz, 1H), 7.67 (dd, J = 7.6, 1.8 Hz, 2H), 7.42−7.36 (m, 4H),
7.31 (dd, J = 7.9, 4.7 Hz, 1H), 6.89−6.82 (m, 2H), 5.28 (s, 2H), 4.66
(d, J = 6.0 Hz, 2H). ESI-MS m/z: 438.1 (MH+).
1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-1,2-dihy-
dro-1,8-naphthyridin-4-yl 4-Methylbenzenesulfonate (9). To a
suspension of 8 (967 mg, 2.21 mmol) in CH3CN (10 mL) and
CH2Cl2 (2.0 mL) were added N-ethyl-N-isobutyl-2-methylpropan-1-
amine (2.81 mL, 13.27 mmol) and 4-methylbenzene-1-sulfonyl
chloride (1.27 g, 6.63 mmol), and the mixture was stirred at room
temperature (overnight). The resulting mixture was purified by
CombiFlash silica gel chromatography using solid loading to provide 9
1
as a colorless oil (1.21 g, 93% yield). H NMR (400 MHz, CDCl3) δ
8.76 (dd, J = 4.7, 1.7 Hz, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.23 (dd, J =
8.1, 1.7 Hz, 1H), 7.90−7.88 (m, 2H), 7.66−7.64 (m, 2H), 7.47−7.45
(m, 1H), 7.40−7.35 (m, 5H), 7.9−7.26 (m, 1H), 6.86−6.78 (m, 2H),
5.30 (s, 2H), 4.50 (d, J = 5.9 Hz, 2H). ESI-MS m/z: 592.1 (MH+).
4-([1,1′-Biphenyl]-4-ylamino)-1-(benzyloxy)-N-(2,4-difluoroben-
zyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (10a).
Treatment of 9 with [1,1′-biphenyl]-4-amine as outlined in general
1
procedure A, provided 10a as a colorless oil in 97% yield. H NMR
(400 MHz, CDCl3) δ 13.23 (s, 1H), 10.86 (t, J = 5.5 Hz, 1H), 8.62
(dd, J = 4.5, 1.5 Hz, 1H), 7.91 (dd, J = 8.2, 1.5 Hz, 1H), 7.71−7.69 (m,
2H), 7.58−7.34 (m, 7H), 7.28−7.23 (m, 5H), 7.09 (d, J = 8.5 Hz,
2H), 6.88 (dd, J = 8.3, 4.5 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 5.32 (s,
2H), 4.65 (d, J = 5.8 Hz, 2H). ESI-MS m/z: 589.2 (MH+).
EXPERIMENTAL SECTION
■
4-((9H-Fluoren-2-yl)amino)-1-(benzyloxy)-N-(2,4-difluorobenzyl)-
2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (10b). Treat-
ment of 9 with 9H-fluoren-2-amine as outlined in general procedure A,
1
General Synthetic. H and 13C NMR data were obtained on a
Varian 400 MHz spectrometer or a Varian 500 MHz spectrometer and
are reported in ppm relative to TMS and referenced to the solvent in
which the spectra were collected. Solvent was removed by rotary
evaporation under reduced pressure, and anhydrous solvents were
obtained commercially and used without further drying. Purification by
silica gel chromatography was performed using CombiFlash Rf 200i
with EtOAc−hexanes solvent systems. Preparative high pressure liquid
chromatography (HPLC) was conducted using a Waters Prep LC4000
system having photodiode array detection and Phenomenex C18
columns (catalogue no. 00G-4436-P0-AX, 250 mm × 21.2 mm 10
μm particle size, 110 Å pore) at a flow rate of 10 mL/min. Binary
solvent systems consisting of A = 0.1% aqueous TFA and B = 0.1%
TFA in acetonitrile were employed with gradients as indicated.
Products were obtained as amorphous solids following lyophilization.
1
provided 10b as a colorless oil in 99% yield. H NMR (400 MHz,
CDCl3) δ 13.31 (s, 1H), 10.87 (t, J = 5.7 Hz, 1H), 8.59 (ddd, J = 4.6,
1.7, 0.7 Hz, 1H), 7.85 (ddd, J = 8.3, 1.6, 0.7 Hz, 1H), 7.76−7.15 (m,
8H), 7.05 (dd, J = 8.1, 2.0 Hz, 1H), 6.88−6.84 (m, 3H), 6.83−6.78
(m, 2H), 6.72−6.66 (m, 2H), 5.33 (s, 2H), 4.65 (d, J = 5.8 Hz, 2H),
3.84 (s, 2H). ESI-MS m/z: 601.2 (MH+).
1-(Benzyloxy)-4-((4′-cyano-[1,1′-biphenyl]-4-yl)amino)-N-(2,4-di-
fluorobenzyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(10c). Treatment of 9 with 4′-amino-[1,1′-biphenyl]-4-carbonitrile as
outlined in general procedure A provided 10c as a yellow solid in 98%
1
yield. H NMR (400 MHz, CDCl3) δ 13.21 (s, 1H), 10.84 (t, J = 5.7
Hz, 1H), 8.66 (dd, J = 4.5, 1.6 Hz, 1H), 7.91 (dd, J = 8.2, 1.7 Hz, 1H),
7.74−7.60 (m, 4H), 7.55−7.53 (m, 2H), 7.44−7.38 (m, 4H), 7.13 (d, J
G
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= 8.5 Hz, 2H), 6.94 (dd, J = 8.3, 4.6 Hz, 1H), 6.88−6.80 (m, 2H),
6.78−6.75 (m, 2H), 5.33 (s, 2H), 4.65 (d, J = 5.8 Hz, 2H). ESI-MS m/
z: 614.2 (MH+).
1H), 8.68−8.66 (m, 1H), 8.31 (dd, J = 8.2, 1.5 Hz, 1H), 7.68 (dd, J =
7.5, 1.5 Hz, 2H), 7.39−7.33 (m, 3H), 7.26 (dd, J = 10.9, 3.9 Hz, 3H),
7.18−7.12 (m, 4H), 6.83−6.78 (m, 2H), 5.24 (s, 2H), 4.62 (d, J = 5.7
Hz, 2H), 3.68 (q, J = 6.3 Hz, 2H), 2.65 (t, J = 7.0 Hz, 2H), 1.81 (qd, J
= 6.9, 3.5 Hz, 4H). ESI-MS m/z: 569.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-2-oxo-4-(pentylamino)-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (10l). Treatment of 9
with pentan-1-amine as outlined in general procedure A, provided 10l
as a colorless oil in 95% yield. 1H NMR (400 MHz, CDCl3) δ 11.99 (t,
J = 4.6 Hz, 1H), 10.79 (t, J = 5.7 Hz, 1H), 8.68 (dd, J = 4.5, 1.2 Hz,
1H), 8.39 (dd, J = 8.2, 1.4 Hz, 1H), 7.68 (dd, J = 7.5, 1.5 Hz, 2H),
7.42−7.35 (m, 4H), 7.17 (dd, J = 8.2, 4.6 Hz, 1H), 6.86−6.78 (m,
2H), 5.25 (s, 2H), 4.63 (d, J = 5.7 Hz, 2H), 3.70 (td, J = 7.0, 5.1 Hz,
2H), 1.83−1.75 (m, 2H), 1.53−1.26 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H).
ESI-MS m/z: 507.2 (MH+).
4-((4′-Amino-[1,1′-biphenyl]-4-yl)amino)-1-(benzyloxy)-N-(2,4-di-
fluorobenzyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(10d). Treatment of 9 with benzidine as outlined in general procedure
1
A, provided 10d as a colorless oil in 97% yield. H NMR (400 MHz,
CDCl3) δ 13.20 (s, 1H), 10.83 (t, J = 5.8 Hz, 1H), 8.66−8.50 (m, 1H),
7.88 (dd, J = 8.4, 1.5 Hz, 1H), 7.69−7.67 (m, 2H), 7.44 (d, J = 8.4 Hz,
2H), 7.39−7.34 (m, 3H), 7.32−7.287 (m, 4H), 7.02 (d, J = 8.6 Hz,
2H), 6.86−6.80 (m, 2H), 6.72−6.67 (m, 2H), 5.29 (s, 2H), 4.62 (d, J
= 5.8 Hz, 2H). ESI-MS m/z: 604.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-2-oxo-4-(phenylamino)-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (10e). Treatment of 9
with aniline as outlined in general procedure A, provided 10e as a
1
yellow solid in 65% yield. H NMR (400 MHz, CDCl3) δ 13.19 (s,
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-(isopropylamino)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (10m). Treatment of
9 with propan-2-amine as outlined in general procedure A, provided
1H), 10.85 (t, J = 5.7 Hz, 1H), 8.61 (dd, J = 4.5, 1.7 Hz, 1H), 7.81 (dd,
J = 8.3, 1.6 Hz, 1H), 7.69 (dd, J = 7.8, 1.6 Hz, 2H), 7.43−7.36 (m,
4H), 7.31 (dd, J = 10.7, 4.9 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H), 7.06−
7.041 (m, 2H), 6.87−6.79 (m, 3H), 5.32 (s, 2H), 4.64 (d, J = 5.8 Hz,
2H). ESI-MS m/z: 513.1 (MH+).
1
10m as a colorless oil in 73% yield. H NMR (400 MHz, CDCl3) δ
11.66 (d, J = 8.9 Hz, 1H), 10.78 (t, J = 5.6 Hz, 1H), 8.71−8.67 (m,
1H), 8.28 (dd, J = 8.2, 1.6 Hz, 1H), 7.69−7.67 (m, 2H), 7.40−7.34
(m, 4H), 7.19−7.16 (m, 1H), 6.86−6.78 (m, 2H), 5.26 (s, 2H), 4.63
(d, J = 5.7 Hz, 2H), 4.24−4.17 (m, 1H), 1.40 (d, J = 6.3 Hz, 6H). ESI-
MS m/z: 479.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-(methylamino)-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (10f). Treatment of 9
with methanamine (2.0 M in THF) as outlined in general procedure
1
A, provided 10f as a white solid in 72% yield. H NMR (400 MHz,
tert-Butyl (2-((1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)ethyl)carbamate
(10n). Treatment of 9 with tert-butyl (2-aminoethyl)carbamate as
outlined in general procedure A, provided 10n as a white solid in 37%
CDCl3) δ 12.09 (d, J = 4.9 Hz, 1H), 10.83 (t, J = 5.6 Hz, 1H), 8.70
(dd, J = 4.6, 1.5 Hz, 1H), 8.47 (dd, J = 8.2, 1.6 Hz, 1H), 7.69 (dd, J =
7.7, 1.6 Hz, 2H), 7.41−7.35 (m, 4H), 7.20−7.17 (m, 1H), 6.86−6.78
(m, 2H), 5.26 (s, 2H), 4.61 (d, J = 5.8 Hz, 2H), 3.41 (d, J = 5.6 Hz,
3H). ESI-MS m/z: 451.1 (MH+).
1
yield. H NMR (400 MHz, CDCl3) δ 11.74 (s, 1H), 10.75 (t, J = 5.5
Hz, 1H), 8.69−8.56 (m, 1H), 8.34 (d, J = 7.9 Hz, 1H), 7.64 (dd, J =
7.4, 1.6 Hz, 2H), 7.36−7.31 (m, 4H), 7.17−7.13 (m, 1H), 6.82−6.74
(m, 2H), 5.20 (s, 2H), 5.09 (d, J = 18.3 Hz, 1H), 4.57 (d, J = 5.7 Hz,
2H), 3.77 (dd, J = 10.8, 5.4 Hz, 2H), 3.39 (dd, J = 11.4, 5.5 Hz, 2H),
1.36 (s, 9H). ESI-MS m/z: 580.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-(dimethylamino)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (10g). Treatment of
9 with dimethylamine hydrochloride as outlined in general procedure
1
A, provided 10g as a white solid in 91% yield. H NMR (400 MHz,
CDCl3) δ 8.61 (dd, J = 4.6, 1.7 Hz, 1H), 8.20 (dd, J = 8.1, 1.7 Hz, 1H),
8.05 (t, J = 5.9 Hz, 1H), 7.67 (dd, J = 7.5, 1.8 Hz, 2H), 7.55 (dd, J =
15.1, 8.5 Hz, 1H), 7.41−7.36 (m, 3H), 7.19 (dd, J = 8.1, 4.6 Hz, 1H),
6.88−6.78 (m, 2H), 5.22 (s, 2H), 4.66 (d, J = 6.0 Hz, 2H), 2.95 (s,
6H). ESI-MS m/z: 465.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-morpholino-2-oxo-1,2-di-
hydro-1,8-naphthyridine-3-carboxamide (10h). Treatment of 9 with
morpholine as outlined in general procedure A, provided 10h as a
colorless oil in 99% yield. 1H NMR (400 MHz, CDCl3) δ 8.48 (dd, J =
4.6, 1.6 Hz, 1H), 8.07−8.03 (m, 2H), 7.68−7.60 (m, 3H), 7.39 (dd, J
= 4.2, 2.2 Hz, 3H), 7.13 (dd, J = 8.0, 4.6 Hz, 1H), 6.89−6.80 (m, 2H),
5.08 (s, 2H), 4.64 (d, J = 5.9 Hz, 2H), 3.61−3.59 (m, 4H), 3.05−3.03
(m, 4H). ESI-MS m/z: 507.1 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-((2-hydroxyethyl)amino)-
2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (10o). Treat-
ment of 9 with 2-aminoethanol as outlined in general procedure A,
1
provided 10o as a white solid in 71% yield. H NMR (400 MHz,
CDCl3) δ 11.74 (t, J = 4.8 Hz, 1H), 10.80 (t, J = 5.7 Hz, 1H), 8.69−
8.68 (m, 1H), 8.41 (dd, J = 8.2, 1.0 Hz, 1H), 7.68 (dd, J = 7.3, 1.5 Hz,
2H), 7.41−7.35 (m, 4H), 7.19−7.15 (m, 1H), 6.86−6.78 (m, 2H),
5.24 (s, 2H), 4.61 (d, J = 5.7 Hz, 2H), 3.90−3.80 (m, 2H), 3.82 (dd, J
= 10.0, 5.1 Hz, 2H), 2.88 (bs, 1H). ESI-MS m/z: 481.1 (MH+).
2-((1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-1,2-
dihydro-1,8-naphthyridin-4-yl)amino)ethyl Acetate (10p). To a
solution of 10o (46 mg, 0.10 mmol) in CH2CL2 (3 mL) was added
triethylamine (32 μL, 0.23 mmol) and acetic anhydride (11 μL, 0.12
mmol) and the solution was stirred at room temperature (1.5 h).
Purification by CombiFlash silica gel chromatography provided 10p as
1-(Benzyloxy)-4-(cycloheptylamino)-N-(2,4-difluorobenzyl)-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (10i). Treatment
of 9 with cycloheptanamine as outlined in general procedure A,
1
a white solid (26 mg, 52% yield). H NMR (400 MHz, CDCl3) δ
1
11.93 (t, J = 5.2 Hz, 1H), 10.70 (t, J = 5.7 Hz, 1H), 8.67 (dd, J = 4.6,
1.6 Hz, 1H), 8.29 (dd, J = 8.2, 1.5 Hz, 1H), 7.64 (dt, J = 4.1, 2.2 Hz,
2H), 7.37−7.29 (m, 4H), 7.16 (dd, J = 8.2, 4.5 Hz, 1H), 6.83−6.75
(m, 2H), 5.22 (s, 2H), 4.58 (d, J = 5.8 Hz, 2H), 4.29 (t, J = 5.6 Hz,
2H), 3.89 (q, J = 5.5 Hz, 2H), 2.05 (s, 3H). ESI-MS m/z: 523.2
(MH+).
provided 10i as a colorless oil in 96% yield. H NMR (400 MHz,
CDCl3) δ 11.77 (d, J = 9.1 Hz, 1H), 10.77 (t, J = 5.7 Hz, 1H), 8.69
(dd, J = 4.6, 1.6 Hz, 1H), 8.24 (dd, J = 8.2, 1.5 Hz, 1H), 7.68 (dd, J =
7.8, 1.6 Hz, 2H), 7.41−7.34 (m, 4H), 7.19 (dd, J = 8.2, 4.5 Hz, 1H),
6.86−6.78 (m, 2H), 5.26 (s, 2H), 4.63 (d, J = 5.7 Hz, 2H), 4.01 (dq, J
= 13.2, 4.4 Hz, 1H), 2.13−2.05 (m, 2H), 1.82−1.74 (m, 4H), 1.62−
1.60 (m, 4H), 1.52−1.42 (m, 2H). ESI-MS m/z: 533.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-2-oxo-4-(phenethylamino)-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (10j). Treatment of 9
with 2-phenylethanamine as outlined in general procedure A, provided
Methyl (1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-
1,2-dihydro-1,8-naphthyridin-4-yl)glycinate (10q). Treatment of 9
with methyl 2-aminoacetate hydrochloride as outlined in general
1
procedure A, provided 10q as a white solid in 95% yield. H NMR
1
(400 MHz, CDCl3) δ 12.27 (t, J = 5.5 Hz, 1H), 10.70 (t, J = 5.6 Hz,
1H), 8.71 (dd, J = 4.6, 1.6 Hz, 1H), 8.22 (dd, J = 8.2, 1.6 Hz, 1H), 7.67
(dd, J = 7.8, 1.7 Hz, 2H), 7.44−7.35 (m, 4H), 7.20 (dd, J = 8.2, 4.6 Hz,
1H), 6.87−6.78 (m, 2H), 5.26 (s, 2H), 4.66 (d, J = 5.7 Hz, 2H), 4.44
(d, J = 5.6 Hz, 2H), 3.81 (s, 3H). ESI-MS m/z: 509.1 (MH+).
Methyl (1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-
1,2-dihydro-1,8-naphthyridin-4-yl)-L-alaninate [(S)-10r]. Treatment
of 9 with methyl (S)-2-aminopropanoate hydrochloride as outlined in
general procedure A, provided (S)-10r as a white solid in 93% yield.
1H NMR (400 MHz, CDCl3) δ 11.85 (d, J = 8.8 Hz, 1H), 10.67 (t, J =
10j as a colorless oil in 78% yield. H NMR (400 MHz, CDCl3) δ
12.06 (t, J = 4.7 Hz, 1H), 10.77 (t, J = 5.7 Hz, 1H), 8.67−8.66 (m,
1H), 8.30 (dd, J = 8.2, 1.6 Hz, 1H), 7.69−7.66 (m, 2H), 7.38−7.36
(m, 4H), 7.29−7.21 (m, 6H), 7.12 (dd, J = 8.2, 4.5 Hz, 1H), 6.88−
6.79 (m, 2H), 5.24 (s, 2H), 4.63 (d, J = 5.8 Hz, 2H), 3.96−3.91 (m,
2H), 3.08−3.05 (m, 2H). ESI-MS m/z: 541.2 (MH+).
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-2-oxo-4-((4-phenylbutyl)-
amino)-1,2-dihydro-1,8-naphthyridine-3-carboxamide (10k). Treat-
ment of 9 with 4-phenylbutan-1-amine as outlined in general
1
procedure A, provided 10k as a colorless oil in 93% yield. H NMR
(400 MHz, CDCl3) δ 11.99 (t, J = 4.6 Hz, 1H), 10.79 (t, J = 5.7 Hz,
5.6 Hz, 1H), 8.67−8.66 (m, 1H), 8.15 (dd, J = 8.2, 1.5 Hz, 1H), 7.63
H
dx.doi.org/10.1021/jm5001908 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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(dd, J = 7.4, 1.6 Hz, 2H), 7.37−7.31 (m, 4H), 7.17−7.14 (m, 1H),
6.83−6.75 (m, 2H), 5.23 (s, 2H), 4.64−4.58 (m, 1H), 4.61 (d, J = 6.3
Hz, 2H), 3.70 (s, 3H), 1.61 (d, J = 6.9 Hz, 3H). ESI-MS m/z: 523.2
(MH+).
Methyl (1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-
1,2-dihydro-1,8-naphthyridin-4-yl)-D-alaninate [(R)-10r]. Treatment
of 9 with methyl (R)-2-zminopropanoate hydrochloride as outlined in
general procedure A, provided (R)-10r as a white solid in 95% yield.
1H NMR (400 MHz, cdcl3) δ 11.85 (d, J = 8.8 Hz, 1H), 10.67 (t, J =
5.6 Hz, 1H), 8.67−8.66 (m, 1H), 8.15 (dd, J = 8.2, 1.5 Hz, 1H), 7.63
(dd, J = 7.4, 1.6 Hz, 2H), 7.37−7.317 (m, 4H), 7.17−7.14 (m, 1H),
6.83−6.75 (m, 2H), 5.23 (s, 2H), 4.64−4.58 (m, 1H), 4.61 (d, J = 6.3
Hz, 3H), 3.70 (s, 3H), 1.61 (d, J = 6.9 Hz, 3H). ESI-MS m/z: 523.2
(MH+).
Methyl (S)-2-((1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-
2-oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-2-phenylacetate
[(S)-10s]. Treatment of 9 with methyl (S)-2-amino-2-phenylacetate
hydrochloride as outlined in general procedure A, provided (S)-10s as
a white solid in 91% yield. 1H NMR (400 MHz, CDCl3) δ 12.76 (d, J
= 8.3 Hz, 1H), 10.65 (t, J = 5.7 Hz, 1H), 8.61 (dd, J = 4.6, 1.6 Hz,
1H), 8.01 (dd, J = 8.2, 1.6 Hz, 1H), 7.62 (dd, J = 7.6, 1.8 Hz, 2H), 7.51
(dd, J = 7.9, 1.2 Hz, 2H), 7.39−7.31 (m, 7H), 7.01 (dd, J = 8.2, 4.6 Hz,
1H), 6.83−6.75 (m, 2H), 5.53 (d, J = 8.3 Hz, 1H), 5.20 (s, 2H), 4.67
(qd, J = 15.3, 5.8 Hz, 2H), 3.69 (s, 3H). ESI-MS m/z: 585.2 (MH+).
Methyl (R)-2-((1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-
2-oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-2-phenylacetate
[(R)-10s]. Treatment of 9 with methyl (R)-2-amino-2-phenylacetate
hydrochloride as outlined in general procedure A, provided (R)-10s as
a white solid in 91% yield. 1H NMR (400 MHz, CDCl3) δ 12.76 (d, J
= 8.3 Hz, 1H), 10.64 (t, J = 5.6 Hz, 1H), 8.62−8.60 (m, 1H), 8.02−
7.96 (m, 2H), 7.61 (dd, J = 7.3, 1.8 Hz, 2H), 7.50 (d, J = 7.4 Hz, 2H),
7.43−7.24 (m, 7H), 7.01 (dd, J = 8.2, 4.5 Hz, 1H), 6.83−6.75 (m,
2H), 5.53 (d, J = 8.3 Hz, 1H), 5.20 (s, 2H), 4.66 (qd, J = 15.3, 5.7 Hz,
2H), 3.68 (s, 3H), 3.65 (d, J = 0.8 Hz, 4H). ESI-MS m/z: 585.2
(MH+).
5.7 Hz, 1H), 8.65 (dd, J = 4.6, 1.5 Hz, 1H), 8.28 (dd, J = 8.2, 1.5 Hz,
1H), 7.70−7.68 (m, 2H), 7.40−7.33 (m, 4H), 7.26−7.24 (m, 1H),
7.08 (dd, J = 8.2, 4.6 Hz, 1H), 6.84−6.76 (m, 2H), 6.47−6.45 (m,
2H), 5.26 (s, 2H), 4.73 (d, J = 6.1 Hz, 2H), 4.59 (d, J = 5.7 Hz, 2H),
3.79 (s, 3H), 3.76 (s, 3H). ESI-MS m/z: 587.2 (MH+), 609.2 (MNa+).
4-Amino-1-(benzyloxy)-N-(2,4-difluorobenzyl)-2-oxo-1,2-dihy-
dro-1,8-naphthyridine-3-carboxamide (11). To a solution of 10v (93
mg, 0.16 mmol) in CH2Cl2 (2.0 mL) was added TFA (1.0 mL), and
the mixture was stirred at room temperature (5 min) then
concentrated by rotary evaporation under reduced pressure.
Purification of the resulting residue by CombiFlash silica gel
chromatography provided 11 as a white solid (69 mg, 100% yield).
1H NMR (400 MHz, CDCl3) δ 10.62 (t, J = 5.7 Hz, 1H), 8.75 (dd, J =
4.6, 1.6 Hz, 1H), 8.04 (dd, J = 8.1, 1.6 Hz, 1H), 7.67 (dd, J = 7.7, 1.7
Hz, 2H), 7.38−7.35 (m, 4H), 7.24 (dd, J = 8.1, 4.6 Hz, 1H), 6.87−
6.78 (m, 2H), 5.26 (s, 2H), 4.63 (d, J = 5.8 Hz, 2H). ESI-MS: m/z
437.1 (M+H+).
4-([1,1′-Biphenyl]-4-ylamino)-N-(2,4-difluorobenzyl)-1-hydroxy-
2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (5a). Treat-
ment of 10a as described under general procedure B and purification
by preparative HPLC (linear gradient of 50% B to 90% B over 30 min;
retention time = 22.8 min) provided 5a as a white powder in 70%
1
yield. H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H), 10.60 (t, J =
5.8 Hz, 1H), 8.61 (dd, J = 4.5, 1.7 Hz, 1H), 7.86 (dd, J = 8.2, 1.7 Hz,
1H), 7.61−7.55 (m, 4H), 7.44−7.36 (m, 3H), 7.27 (t, J = 7.3 Hz, 1H),
7.20−7.15 (m, 1H), 7.13 (d, J = 8.5 Hz, 2H), 7.07 (dd, J = 8.2, 4.6 Hz,
1H), 7.02−6.97 (m, 1H), 4.46 (d, J = 5.7 Hz, 2H). ESI-MS m/z: 499.1
(MH+). HRMS calcd C28H21F2N4O3 [MH+], 499.1576; found,
499.1583.
4-((9H-Fluoren-2-yl)amino)-N-(2,4-difluorobenzyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (5b). Treatment
of 10b as described under general procedure B and purification by
preparative HPLC (linear gradient of 60% B to 80% B over 30 min;
retention time = 21.3 min) provided 5b as a white powder in 65%
1
yield. H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 10.68 (t, J =
Methyl (S)-2-((1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-
2-oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-3-hydroxypropa-
noate [(S)-10t]. Treatment of 9 with methyl (S)-2-amino-3-
hydroxypropanoate hydrochloride as outlined in general procedure
5.7 Hz, 1H), 8.59 (dd, J = 4.5, 1.6 Hz, 1H), 7.82 (dd, J = 8.2, 1.5 Hz,
1H), 7.79 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 7.5
Hz, 1H), 7.41 (dd, J = 15.3, 8.7 Hz, 1H), 7.31−7.29 (m, 2H), 7.24−
7.20 (m, 1H), 7.20−7.17 (m, 1H), 7.07 (dd, J = 8.1, 1.8 Hz, 1H),
7.02−6.97 (m, 2H), 4.47 (d, J = 5.8 Hz, 2H), 3.79 (s, 2H). ESI-MS m/
z: 511.1 (MH+). HRMS calcd C29H21F2N4O3 [MH+], 511.1576;
found, 511.1582.
1
A, provided (S)-10ts as a white solid in 83% yield. H NMR (400
MHz, CDCl3) δ 11.21 (d, J = 9.7 Hz, 1H), 10.66 (t, J = 5.6 Hz, 1H),
8.67 (dd, J = 4.5, 1.6 Hz, 1H), 8.30 (dd, J = 8.2, 1.5 Hz, 1H), 7.63 (dd,
J = 7.6, 1.8 Hz, 2H), 7.36−7.31 (m, 4H), 7.17 (dd, J = 8.2, 4.6 Hz,
1H), 6.83−6.75 (m, 2H), 5.22 (s, 2H), 4.60−4.57 (m, 3H), 3.99 (t, J =
4.8 Hz, 2H), 3.75 (s, 3H). ESI-MS m/z: 539.2 (MH+).
4-((4′-Cyano-[1,1′-biphenyl]-4-yl)amino)-N-(2,4-difluorobenzyl)-
1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(5c). Treatment of 10c as described under general procedure B and
purification by preparative HPLC (linear gradient of 30% B to 80% B
over 30 min; retention time = 22.3 min) provided 5c as a white
Methyl (R)-2-((1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-
2-oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-3-hydroxypropa-
noate [(R)-10t]. Treatment of 9 with methyl (R)-2-amino-3-
hydroxypropanoate hydrochloride as outlined in general procedure
1
powder in 39% yield. H NMR (400 MHz, DMSO-d6) δ 12.13 (s,
1H), 10.53 (t, J = 6.2 Hz, 1H), 8.64 (d, J = 4.4 Hz, 1H), 7.88 (d, J =
8.2 Hz, 1H), 7.83 (bs, 4H), 7.67 (d, J = 8.4 Hz, 2H), 7.43 (dd, J = 15.6,
8.3 Hz, 1H), 7.21−7.16 (m, 3H), 7.11 (dd, J = 9.0, 3.8 Hz, 1H), 7.00
(t, J = 8.9 Hz, 1H), 4.46 (d, J = 6.1 Hz, 2H). ESI-MS m/z: 524.1
(MH+). HRMS calcd C29H20F2N5O3 [MH+], 524.1529; found,
524.1521.
1
A, provided (R)-10t as a white solid in 97% yield. H NMR (400
MHz, CDCl3) δ 11.19 (d, J = 9.4 Hz, 1H), 10.67 (s, 1H), 8.68 (d, J =
4.2 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 6.0 Hz, 2H), 7.35−
7.32 (m, 4H), 7.17 (dd, J = 8.1, 4.5 Hz, 1H), 6.82−6.76 (m, 2H), 5.22
(s, 2H), 4.60−4.58 (m, 3H), 3.99 (t, J = 5.4 Hz, 2H), 3.75 (s, 3H).
ESI-MS m/z: 539.2 (MH+).
4-((4′-Amino-[1,1′-biphenyl]-4-yl)amino)-N-(2,4-difluorobenzyl)-
1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(5d). Treatment of 10d as described under general procedure B and
purification by preparative HPLC (linear gradient of 30% B to 70% B
over 30 min; retention time = 15.6 min) provided 5d as a white
Ethyl (1-(Benzyloxy)-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-
1,2-dihydro-1,8-naphthyridin-4-yl)-L-prolinate (10u). Treatment of
9 with ethyl (S)-pyrrolidine-2-carboxylate hydrochloride as outlined in
1
general procedure A, provided 10u as a white solid in 73% yield. H
1
NMR (400 MHz, CDCl3) δ 8.64 (dd, J = 4.6, 1.5 Hz, 1H), 8.34 (dd, J
= 8.0, 1.6 Hz, 1H), 8.16 (t, J = 5.8 Hz, 1H), 7.66 (dd, J = 7.5, 1.6 Hz,
2H), 7.49 (dd, J = 15.0, 8.5 Hz, 1H), 7.37−7.32 (m, 3H), 7.23−7.19
(m, 1H), 6.86−6.75 (m, 2H), 5.24 (q, J = 8.7 Hz, 2H), 4.62 (d, J = 3.4
Hz, 2H), 4.37 (dd, J = 8.5, 3.8 Hz, 1H), 4.01−3.94 (m, 2H), 3.76−
3.73 (m, 1H), 3.50−3.45 (m, 1H), 2.40−2.34 (m, 1H), 2.02−1.98 (m,
3H), 1.07 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 563.2 (MH+).
powder in 51% yield. H NMR (400 MHz, DMSO-d6) δ 12.42 (s,
1H), 10.89 (bs, 1H), 10.67 (t, J = 5.8 Hz, 1H), 8.62 (dd, J = 4.5, 1.6
Hz, 1H), 7.85 (dd, J = 8.2, 1.6 Hz, 1H), 7.47 (t, J = 7.9 Hz, 2H), 7.43−
7.40 (m, 3H), 7.23−7.18 (m, 1H), 7.09 (d, J = 8.6 Hz, 2H), 7.02−7.00
(m, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.48 (d, J = 5.9 Hz, 2H). ESI-MS
m/z: 514.1 (MH+). HRMS calcd C28H22F2N5O3 [MH+], 514.1685;
found, 514.1690.
1-(Benzyloxy)-N-(2,4-difluorobenzyl)-4-((2,4-dimethoxybenzyl)-
amino)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (10v).
Treatment of 9 with (2,4-dimethoxyphenyl)methanamine as outlined
in general procedure A, provided 10u as a colorless oil in 89% yield.
1H NMR (400 MHz, CDCl3) δ 12.00 (t, J = 6.0 Hz, 1H), 10.76 (t, J =
N-(2,4-Difluorobenzyl)-1-hydroxy-2-oxo-4-(phenylamino)-1,2-di-
hydro-1,8-naphthyridine-3-carboxamide (5e). Treatment of 10e as
described under general procedure B and purification by preparative
HPLC (linear gradient of 40% B to 90% B over 30 min; retention time
1
= 20.2 min) provided 5e as a white powder in 86% yield. H NMR
I
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Journal of Medicinal Chemistry
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(400 MHz, DMSO-d6) δ 12.39 (s, 1H), 10.86 (s, 1H), 10.65 (t, J = 5.7
Hz, 1H), 8.61 (dd, J = 4.5, 1.6 Hz, 1H), 7.75 (dd, J = 8.2, 1.7 Hz, 1H),
7.42 (dd, J = 15.4, 8.7 Hz, 1H), 7.27 (t, J = 7.8 Hz, 2H), 7.22−7.17 (m,
1H), 7.13−7.00 (m, 5H), 4.47 (d, J = 5.8 Hz, 2H). ESI-MS m/z: 423.1
(MH+). HRMS calcd C22H17F2N4O3 [MH+], 423.1263; found,
423.1269.
3H), 7.07 (d, J = 7.0 Hz, 1H), 7.01−6.97 (m, 1H), 4.46 (d, J = 5.7 Hz,
2H), 3.61 (bs, 2H), 2.54 (t, J = 6.9 Hz, 2H), 1.61−1.60 (m, 4H). ESI-
MS m/z: 479.2 (MH+). HRMS calcd C26H25F2N4O3 [MH+],
479.1889; found, 479.1894.
N-(2,4-Difluorobenzyl)-1-hydroxy-2-oxo-4-(pentylamino)-1,2-di-
hydro-1,8-naphthyridine-3-carboxamide (5l). Treatment of 10l as
described under general procedure B and purification by preparative
HPLC (linear gradient of 40% B to 80% B over 30 min; retention time
= 23.7 min) provided 5l as a yellow solid in 72% yield. 1H NMR (400
MHz, DMSO-d6) δ 10.95 (bs, 1H), 10.47 (t, J = 5.7 Hz, 1H), 8.63 (dd,
J = 4.5, 1.5 Hz, 1H), 8.54 (dd, J = 8.3, 1.5 Hz, 1H), 7.46−7.40 (m,
1H), 7.24 (dd, J = 8.2, 4.6 Hz, 1H), 7.19 (ddd, J = 10.5, 9.4, 2.6 Hz,
1H), 7.04−6.99 (m, 1H), 4.46 (d, J = 5.8 Hz, 2H), 3.60−3.57 (m,
2H), 1.57 (dd, J = 14.0, 7.0 Hz, 2H), 1.29−1.21 (m, 4H), 0.81 (t, J =
7.1 Hz, 3H). ESI-MS m/z: 417.1 (MH+). HRMS calcd C17H13F2N2O4
[MH+], 417.1733; found, 417.1734.
N-(2,4-Difluorobenzyl)-1-hydroxy-4-(isopropylamino)-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (5m). Treatment of 10m
as described under general procedure B and purification by preparative
HPLC (linear gradient of 40% B to 80% B over 30 min; retention time
= 17.0 min) provided 5m as a white solid in 80% yield. 1H NMR (400
MHz, DMSO-d6) δ 10.47 (bs, 1H), 10.42 (t, J = 5.9 Hz, 1H), 8.64 (dd,
J = 4.5, 1.6 Hz, 1H), 8.41 (dd, J = 8.2, 1.5 Hz, 1H), 7.44 (dd, J = 15.4,
8.6 Hz, 1H), 7.27 (dd, J = 8.2, 4.6 Hz, 1H), 7.22−7.17 (m, 1H), 7.02
(td, J = 8.6, 2.6 Hz, 1H), 4.46 (d, J = 5.8 Hz, 2H), 4.17−4.09 (m, 1H),
1.20 (s, 3H), 1.18 (s, 3H). ESI-MS m/z: 389.1 (MH+). HRMS calcd
C19H19F2N4O3 [MH+], 389.1420; found, 389.1422.
4-((2-Aminoethyl)amino)-N-(2,4-difluorobenzyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (5n). Treatment
of 10n as described under general procedure B and removal of tert-
butyl protection [TFA−CH2Cl2 (1:1, 2 mL), room temperature]
followed by purification using preparative HPLC (linear gradient of
0% B to 40% B over 30 min; retention time = 25.9 min) provided 5n
as a white solid in 61% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.65
(s, 1H), 9.88 (t, J = 5.9 Hz, 1H), 9.09 (s, 1H), 8.69 (dd, J = 4.5, 1.3
Hz, 1H), 8.48 (dd, J = 8.1, 1.4 Hz, 1H), 7.83 (bs, 2H), 7.65 (dd, J =
15.6, 8.8 Hz, 1H), 7.34 (dd, J = 8.1, 4.6 Hz, 1H), 7.27−7.21 (m, 1H),
7.08 (dd, J = 9.9, 7.3 Hz, 1H), 4.50 (d, J = 5.5 Hz, 2H), 3.68 (q, J = 5.9
Hz, 2H), 3.10 (bs, 2H). ESI-MS m/z: 390.1 (MH+). HRMS calcd
C18H18F2N5O3 [MH+], 390.1372; found, 390.1363.
N-(2,4-Difluorobenzyl)-1-hydroxy-4-((2-hydroxyethyl)amino)-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (5o). Treatment
of 10o as described under general procedure B and purification by
preparative HPLC (linear gradient of 20% B to 50% B over 30 min;
retention time = 21.6 min) provided 5o as a yellow solid in 70% yield.
1H NMR (400 MHz, DMSO-d6) δ 11.13 (bs, 1H), 10.49 (t, J = 5.5
Hz, 1H), 8.62 (dd, J = 4.5, 1.5 Hz, 1H), 8.55 (dd, J = 8.2, 1.6 Hz, 1H),
7.42 (dd, J = 15.3, 8.6 Hz, 1H), 7.23 (dd, J = 8.2, 4.5 Hz, 1H), 7.22−
7.16 (m, 1H), 7.02 (dd, J = 9.3, 7.7 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H),
3.68−3.65 (m, 2H), 3.56 (t, J = 5.2 Hz, 2H). ESI-MS m/z: 391.1
(MH+). HRMS calcd C18H17F2N4O4 [MH+], 391.1212; found,
391.1215.
2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-oxo-1,2-dihy-
dro-1,8-naphthyridin-4-yl)amino)ethyl Acetate (5p). Treatment of
10p as described under general procedure B and purification by
preparative HPLC (linear gradient of 30% B to 50% B over 30 min;
retention time = 23.6 min) provided 5p as a white powder in 79%
yield. 1H NMR (400 MHz, DMSO-d6) δ 10.55 (bs, 1H), 10.34 (t, J =
5.5 Hz, 1H), 8.69 (d, J = 4.5 Hz, 1H), 8.55 (d, J = 8.2 Hz, 1H), 7.52
(dd, J = 15.4, 8.6 Hz, 1H), 7.32 (dd, J = 8.1, 4.3 Hz, 1H), 7.27−7.22
(m, 1H), 7.07 (t, J = 7.4 Hz, 1H), 4.51 (d, J = 5.7 Hz, 2H), 4.19 (t, J =
5.1 Hz, 2H), 3.84 (d, J = 4.9 Hz, 2H), 2.00 (s, 3H). ESI-MS m/z:
433.1 (MH+). HRMS calcd C20H19F2N4O5 [MH+], 433.1318; found,
433.1308.
N-(2,4-Difluorobenzyl)-1-hydroxy-4-(methylamino)-2-oxo-1,2-di-
hydro-1,8-naphthyridine-3-carboxamide (5f). Treatment of 10f as
described under general procedure B and purification by preparative
HPLC (linear gradient of 20% B to 50% B over 30 min; retention time
1
= 26.4 min) provided 5f as a white powder in 63% yield. H NMR
(400 MHz, DMSO-d6) δ 10.19 (bs, 1H), 10.08 (bs, 1H), 8.59 (d, J =
3.8 Hz, 1H), 8.55 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 15.4, 8.5 Hz, 1H),
7.22 (dd, J = 8.0, 4.5 Hz, 1H), 7.18−7.13 (m, 1H), 7.00 (dd, J = 9.7,
7.6 Hz, 1H), 4.42 (d, J = 5.6 Hz, 2H), 3.12 (s, 3H). ESI-MS m/z:
361.1 (MH+). HRMS calcd C17H15F2N4O3 [MH+], 361.1107; found,
361.1105.
N-(2,4-Difluorobenzyl)-4-(dimethylamino)-1-hydroxy-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (5g). Treatment of 10g
as described under general procedure B and purification by preparative
HPLC (linear gradient of 20% B to 45% B over 30 min; retention time
= 21.6 min) provided 5g as a yellow solid in 89% yield. 1H NMR (400
MHz, DMSO-d6) δ 8.79 (t, J = 5.9 Hz, 1H), 8.57 (dd, J = 4.5, 1.5 Hz,
1H), 8.19 (dd, J = 8.0, 1.6 Hz, 1H), 7.70 (dd, J = 15.5, 8.7 Hz, 1H),
7.25 (dd, J = 8.0, 4.6 Hz, 1H), 7.18−7.13 (m, 1H), 7.02 (td, J = 8.5,
2.6 Hz, 1H), 4.38 (d, J = 5.8 Hz, 2H), 2.76 (s, 6H). ESI-MS m/z:
375.1 (MH+). HRMS calcd C18H17F2N4O3 [MH+], 375.1263; found,
375.1260.
N-(2,4-Difluorobenzyl)-1-hydroxy-4-morpholino-2-oxo-1,2-dihy-
dro-1,8-naphthyridine-3-carboxamide (5h). Treatment of 10h as
described under general procedure B and purification by preparative
HPLC (linear gradient of 20% B to 45% B over 30 min; retention time
= 20.5 min) provided 5h as a yellow solid in 82% yield. 1H NMR (400
MHz, DMSO-d6) δ 8.91 (t, J = 5.8 Hz, 1H), 8.62−8.61 (m, 1H), 8.23
(dd, J = 8.0, 1.6 Hz, 1H), 7.70 (dd, J = 15.4, 8.7 Hz, 1H), 7.30 (dd, J =
8.0, 4.6 Hz, 1H), 7.21−7.16 (m, 1H), 7.05 (td, J = 8.5, 2.2 Hz, 1H),
4.41 (d, J = 5.8 Hz, 2H), 3.67−3.65 (m, 4H), 2.98−2.96 (m, 4H). ESI-
MS m/z: 417.1 (MH+). HRMS calcd C20H19F2N4O4 [MH+],
417.1369; found, 417.1364.
4-(Cycloheptylamino)-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (5i). Treatment of
10i as described under general procedure B and purification by
preparative HPLC (linear gradient of 40% B to 80% B over 30 min;
retention time = 25.8 min) provided 5i as a yellow solid in 87% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.68 (bs, 1H), 10.45 (t, J = 5.7
Hz, 1H), 8.62 (dd, J = 4.5, 1.5 Hz, 1H), 8.35 (dd, J = 8.3, 1.5 Hz, 1H),
7.45−7.39 (m, 1H), 7.26 (dd, J = 8.2, 4.6 Hz, 1H), 7.17 (ddd, J = 10.5,
9.4, 2.6 Hz, 1H), 7.03−6.98 (m, 1H), 4.44 (d, J = 5.7 Hz, 2H), 4.02
(bs, 1H), 1.93−1.88 (m, 2H), 1.58−1.50 (m, 4H), 1.47−1.45 (m, 4H),
1.31−1.38 (s, 2H). ESI-MS m/z: 443.1 (MH+). HRMS calcd
C23H25F2N4O3 [MH+], 443.1889; found, 443.1897.
N-(2,4-Difluorobenzyl)-1-hydroxy-2-oxo-4-(phenethylamino)-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (5j). Treatment of 10j as
described under general procedure B and purification by preparative
HPLC (linear gradient of 40% B to 80% B over 30 min; retention time
= 22.0 min) provided 5j as a yellow solid in 93% yield. 1H NMR (400
MHz, DMSO-d6) δ 10.80 (bs, 1H), 10.36 (t, J = 5.8 Hz, 1H), 8.59 (dd,
J = 4.5, 1.5 Hz, 1H), 8.48 (dd, J = 8.3, 1.6 Hz, 1H), 7.42 (dd, J = 15.3,
8.6 Hz, 1H), 7.22−7.18 (m, 6H), 7.18−7.14 (m, 1H), 7.03−6.98 (m,
1H), 4.43 (d, J = 5.8 Hz, 2H), 3.82−3.78 (m, 2H), 2.87 (t, J = 7.3 Hz,
2H). ESI-MS m/z: 451.1 (MH+). HRMS calcd C24H21F2N4O3 [MH+],
451.1576; found, 451.1584.
N-(2,4-Difluorobenzyl)-1-hydroxy-2-oxo-4-((4-phenylbutyl)-
amino)-1,2-dihydro-1,8-naphthyridine-3-carboxamide (5k). Treat-
ment of 10k as described under general procedure B and purification
by preparative HPLC (linear gradient of 40% B to 80% B over 30 min;
retention time = 26.7 min) provided 5k as a yellow solid in 78% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.96 (bs, 1H), 10.48 (t, J = 5.5
Methyl 2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-oxo-
1,2-dihydro-1,8-naphthyridin-4-yl)amino)acetate (5q). Treatment
of 10q as described under general procedure B and purification by
preparative HPLC (linear gradient of 20% B to 50% B over 30 min;
retention time = 28.4 min) provided 5q as a white solid in 44% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.43 (brs, 1H), 10.09 (t, J = 5.7
Hz, 1H), 8.62 (dd, J = 4.6, 1.5 Hz, 1H), 8.51 (dd, J = 8.3, 1.6 Hz, 1H),
7.42 (dd, J = 15.4, 8.7 Hz, 1H), 7.25−7.16 (m, 4H), 7.12−7.07 (m,
J
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Hz, 1H), 8.62 (dd, J = 4.5, 1.3 Hz, 1H), 8.43 (dd, J = 8.2, 1.4 Hz, 1H),
7.50 (dd, J = 15.5, 8.7 Hz, 1H), 7.25 (dd, J = 8.2, 4.6 Hz, 1H), 7.20−
7.12 (m, 1H), 7.00 (td, J = 8.2, 2.4 Hz, 1H), 4.42 (s, 2H), 4.41 (s, 2H),
3.60 (s, 3H). ESI-MS m/z: 419.1 (MH+). HRMS calcd C19H17F2N4O5
[MH+], 419.1162; found, 419.1163.
Methyl (S)-2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)propanoate [(S)-5r].
Treatment of (S)-10r as described under general procedure B and
purification by preparative HPLC (linear gradient of 30% B to 50% B
over 30 min; retention time = 26.4 min) provided (S)-5r as a white
solid in 40% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.18 (t, J = 5.8
Hz, 1H), 10.02 (d, J = 8.4 Hz, 1H), 8.70 (dd, J = 4.5, 0.9 Hz, 1H), 8.46
(d, J = 8.2 Hz, 1H), 7.57 (dd, J = 15.4, 8.5 Hz, 1H), 7.34−7.31 (m,
1H), 7.23 (dd, J = 13.8, 6.1 Hz, 1H), 7.07 (td, J = 8.6, 2.7 Hz, 1H),
4.78−4.71 (m, 1H), 4.49 (qd, J = 15.5, 6.0 Hz, 2H), 3.61 (d, J = 0.5
Hz, 3H), 1.47 (d, J = 6.9 Hz, 3H). ESI-MS m/z: 433.1 (MH+). HRMS
calcd C20H19F2N4O5 [MH+], 433.1318; found, 433.1305.
Methyl (R)-2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)propanoate [(R)-5r].
Treatment of (R)-10r as described under general procedure B and
purification by preparative HPLC (linear gradient of 30% B to 50% B
over 30 min; retention time = 26.4 min) provided (R)-5r as a white
solid in 40% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.18 (t, J = 5.8
Hz, 1H), 10.02 (d, J = 9.6 Hz, 1H), 8.70 (dd, J = 4.6, 1.5 Hz, 1H), 8.46
(dd, J = 8.2, 1.5 Hz, 1H), 7.57 (dd, J = 15.5, 8.8 Hz, 1H), 7.33 (dd, J =
8.2, 4.6 Hz, 1H), 7.24 (ddd, J = 10.6, 9.4, 2.6 Hz, 1H), 7.10−7.05 (m,
1H), 4.74 (dq, J = 13.9, 6.9 Hz, 1H), 4.49 (qd, J = 15.2, 5.7 Hz, 2H),
3.61 (s, 3H), 1.47 (d, J = 6.9 Hz, 3H). ESI-MS m/z: 433.1 (MH+).
HRMS calcd C20H19F2N4O5 [MH+], 433.1318; found, 433.1303.
Methyl (S)-2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-2-phenylacetate
[(S)-5s]. Treatment of (S)-10s as described under general procedure B
and purification by preparative HPLC (linear gradient of 40% B to
70% B over 30 min; retention time = 22.6 min) provided (S)-5r as a
white solid in 51% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.72 (d, J
= 8.0 Hz, 1H), 10.57 (t, J = 5.9 Hz, 1H), 8.67 (dd, J = 4.5, 1.2 Hz,
1H), 8.46 (dd, J = 8.2, 1.4 Hz, 1H), 7.49 (dd, J = 15.4, 8.6 Hz, 1H),
7.37 (d, J = 4.2 Hz, 3H), 7.35−7.31 (m, 1H), 7.29−7.24 (m, 2H), 7.09
(dd, J = 9.8, 7.3 Hz, 1H), 6.01 (d, J = 8.1 Hz, 1H), 4.55 (d, J = 5.7 Hz,
2H), 3.61 (s, 3H). ESI-MS m/z: 495.1 (MH+). HRMS calcd
C25H21F2N4O5 [MH+], 495.1475; found, 495.1457.
Methyl (R)-2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-2-phenylacetate
[(R)-5s]. Treatment of (R)-10s as described under general procedure B
and purification by preparative HPLC (linear gradient of 40% B to
60% B over 30 min; retention time = 26.3 min) provided (R)-5s as a
white solid in 31% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.72 (d, J
= 8.3 Hz, 1H), 10.57 (t, J = 5.4 Hz, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.46
(d, J = 8.2 Hz, 1H), 7.49 (dd, J = 15.2, 8.6 Hz, 1H), 7.37−7.36 (m,
2H), 7.33 (dd, J = 8.1, 4.1 Hz, 1H), 7.29−7.24 (m, 2H), 7.09 (dd, J =
9.8, 7.1 Hz, 1H), 6.01 (d, J = 8.1 Hz, 1H), 4.55 (d, J = 5.6 Hz, 2H),
3.61 (s, 3H). ESI-MS m/z: 495.1 (MH+). HRMS calcd C25H21F2N4O5
[MH+], 495.1475; found, 495.1460.
(R)-5t as a white solid in 42% yield. 1H NMR (400 MHz, DMSO-d6)
δ 10.61 (d, J = 8.7 Hz, 1H), 10.37 (t, J = 5.6 Hz, 1H), 8.70 (dd, J = 4.5,
1.4 Hz, 1H), 8.38 (dd, J = 8.2, 1.4 Hz, 1H), 7.52 (dd, J = 15.6, 8.7 Hz,
1H), 7.32 (dd, J = 8.2, 4.6 Hz, 1H), 7.25 (ddd, J = 11.2, 10.6, 6.4 Hz,
1H), 7.07 (dd, J = 9.7, 7.3 Hz, 1H), 4.75 (dt, J = 8.8, 4.3 Hz, 1H), 4.51
(qd, J = 15.4, 5.8 Hz, 2H), 3.81 (qd, J = 11.0, 4.4 Hz, 2H), 3.63 (s,
3H). ESI-MS m/z: 449.1 (MH+). HRMS calcd C20H19F2N4O6 [MH+],
449.1267; found, 449.1255.
Ethyl (S)-1-(3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-oxo-
1,2-dihydro-1,8-naphthyridin-4-yl)pyrrolidine-2-carboxylate [(S)-
5u]. Treatment of (S)-10u as described under general procedure B
and purification by preparative HPLC (linear gradient of 40% B to
70% B over 30 min; retention time = 12.5 min) provided (S)-5u as a
yellow solid in 48% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.91 (t, J
= 5.9 Hz, 1H), 8.65 (dd, J = 4.6, 1.5 Hz, 1H), 8.39 (dd, J = 8.0, 1.6 Hz,
1H), 7.73 (dd, J = 15.4, 8.7 Hz, 1H), 7.34 (dd, J = 8.0, 4.6 Hz, 1H),
7.26−7.20 (m, 1H), 7.09 (td, J = 8.6, 2.4 Hz, 1H), 4.51 (dd, J = 15.1,
6.3 Hz, 1H), 4.38−4.30 (m, 2H), 3.95 (q, J = 7.1 Hz, 2H), 3.64 (dd, J
= 15.2, 6.4 Hz, 1H), 3.18 (dd, J = 12.8, 8.1 Hz, 1H), 2.11−1.93 (m,
1H), 1.92−1.87 (m, 2H), 1.80−1.76 (m, 1H), 1.03 (q, J = 7.0 Hz,
3H). ESI-MS m/z: 473.2 (MH+). HRMS calcd C23H23F2N4O5 [MH+],
473.1631; found, 473.1618.
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (5v). Treatment of 10v as
described under general procedure B and purification by preparative
HPLC (linear gradient of 30% B to 50% B over 30 min; retention time
= 19.5 min) provided 5v as a white solid in 47% yield. 1H NMR (400
MHz, DMSO-d6) δ 10.59 (t, J = 5.8 Hz, 1H), 8.66 (dd, J = 4.5, 1.4 Hz,
1H), 8.61 (dd, J = 8.1, 1.6 Hz, 1H), 7.36 (d, J = 6.6 Hz, 1H), 7.30 (dd,
J = 8.1, 4.6 Hz, 1H), 7.18 (d, J = 9.2 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H),
4.46 (d, J = 5.8 Hz, 2H). ESI-MS m/z: 347.1 (MH+). HRMS calcd
C16H13F2N4O3 [MH+], 347.0950; found, 347.0953.
Integrase Biochemical Assays. Determination of IN 3′- P and
ST inhibitory values using an in vitro assay IN reactions were carried
out using [γ-32P]-labeled DNA as previously described.9,22
Cellular Cytotoxicity Assays. Cytotoxicity was measured using
the human osteosarcoma cell line, HOS (Dr. Richard Schwartz,
Michigan State University, East Lansing, MI), by monitoring ATP
levels using a luciferase reporter assay as previously reported.9
Single-Round HIV-1 Infectivity Assays. As previously de-
scribed,9 the human embryonic kidney cell line 293T was transfected
with the pNLNgoMIVR−ΔLUC vector, which was made from
pNLNgoMIVR−ΔEnv.HSA by removing the HSA reporter gene and
replacing it with a luciferase reporter gene between the NotI and XhoI
restriction sites.28 VSV-g-pseudotyped HIV was produced transfecting
293T cells as described previously.29 On the day prior to transfection,
293T cells were plated on 100 mm diameter dishes at a density of 1.5
× 106 cells per plate. 293T cells were transfected with 16 μg of
pNLNgoMIVR−ΔLUC and 4 μg of pHCMV-g (obtained from Dr.
Jane Burns, University of California, San Diego) using the calcium
phosphate method. At approximately 6 h after the calcium phosphate
precipitate was added, the 293T cells were washed twice with
phosphate-buffered saline (PBS) and incubated with fresh media (48
h). The virus-containing supernatants were then harvested, clarified by
low-speed centrifugation, filtered, and diluted for preparation in
infection assays. On the day prior to the screen, HOS cells were
seeded in a 96-well luminescence cell culture plate at a density of 4000
cells in 100 μL per well. On the day of the screen, cells were treated
with the compounds from a concentration range of 10 μM to 0.0005
μM using 11 serial dilutions and then incubated at 37 °C (3 h). After
this incubation, 100 μL of virus stock diluted to achieve a maximum
luciferase signal between 0.2 and 1.5 RLUs was added to each well and
the plates were incubated at 37 °C (48 h). Infectivity was measured by
using the Steady-lite plus luminescence reporter gene assay system
(PerkinElmer, Waltham, MA). Luciferase activity was measured by
adding 100 μL of Steady-lite plus buffer (PerkinElmer) to the cells,
incubating at room temperature (20 min), and measuring
luminescence using a microplate reader. Activity was normalized to
infectivity in the absence of target compounds. KaleidaGraph (Synergy
Methyl (S)-2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-3-hydroxypropa-
noate [(S)-5t]. Treatment of (S)-10t as described under general
procedure B and purification by preparative HPLC (linear gradient of
30% B to 50% B over 30 min; retention time = 15.9 min) provided
(S)-5t as a white solid in 44% yield. 1H NMR (400 MHz, DMSO-d6) δ
10.72 (bs, 1H), 10.62 (bs, 1H), 10.37 (t, J = 5.9 Hz, 1H), 8.70 (dd, J =
4.5, 1.5 Hz, 1H), 8.38 (dd, J = 8.2, 1.5 Hz, 1H), 7.52 (dd, J = 15.4, 8.7
Hz, 1H), 7.31 (dd, J = 8.2, 4.6 Hz, 1H), 7.25 (ddd, J = 10.5, 9.4, 2.6
Hz, 1H), 7.10−7.06 (m, 1H), 4.77−4.72 (m, 1H), 4.51 (qd, J = 15.3,
5.6 Hz, 2H), 3.81 (qd, J = 11.1, 4.3 Hz, 2H), 3.63 (s, 3H). ESI-MS m/
z: 449.1 (MH+). HRMS calcd C20H19F2N4O6 [MH+], 449.1267;
found, 449.1256.
Methyl (R)-2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)-3-hydroxypropa-
noate [(R)-5t]. Treatment of (R)-10t as described under general
procedure B and purification by preparative HPLC (linear gradient of
25% B to 50% B over 30 min; retention time = 20.5 min) provided
K
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Software, Reading, PA) was used to perform regression analysis on the
data. EC50 values were determined from the fit model.
were fractionated on a 15% polyacrylamide sequencing gel. Products
were visualized by exposure to X-ray film.
Vector Constructs. pNLNgoMIVR−ΔEnv.LUC has been de-
scribed previously.28 The IN coding region was removed from
pNLNgoMIVR−ΔEnv.LUC (between KpnI and SalI sites) and
inserted between the KpnI and SalI sites of pBluescript II KS+.
Using this construct as the wild-type template, the following IN-
resistant mutants were prepared via the QuikChange II XL
(Stratagene, La Jolla, CA) site-directed mutagenesis protocol:
G118R, Y143R, Q148H, Q148 K, N155H, G140S + Q148H,
G140A + Q148 K, and E138 K + Q148 K. The following sense
with cognate antisense (not shown) oligonucleotides (Integrated DNA
Technologies, Coralville, IA) were used in the mutagenesis: G118R,
5′-GTACATACAGACAATCGCAGCAATTTCACCAGTAC-3′;
E138 K, 5′-GGCGGGGATCAAGCAGAAATTTGGCATTCCCTA-
3′; G140A, 5′-GGGGATCAAGCAGGAATTTGCCATTCCCTA-
CAATC-3′; G140S, 5′-GGGGATCAAGCAGGAATTTAG-
CATTCCCTACAATC-3′; Y143R, 5′-GCAGGAATTTGG-
CATTCCCCGCAATCCCCAAAGTCAAGGA-3′; Q148H, 5′-
CATTCCCTACAATCCCCAAAGTCATGGAGTAATAGAATCTA-
3′; Q148 K, 5′-CATTCCCTACAATCCCCAAAGTAAAGGAGTAA-
TAGAATCTATGAA-3′; N155H, 5′-CCAAAGTCAAGGAGTAATA-
GAATCTATGCATAAAGAATTAAAGAAAATTATAGGACA-3′.
The double mutation G140S + Q148H was constructed by using the
previously generated Q148H mutant and the appropriate oligonucleo-
tide for the second mutation, G140S. The double mutation G140A +
Q148 K was made by using the Q148 K mutant and the appropriate
oligonucleotide for the second mutation, G140A. The double
mutation E138 K + Q148 K was made by using the Q148 K mutant
and the appropriate oligonucleotide for the second mutation, E138 K.
The DNA sequence of each construct was verified independently by
DNA sequence determination. The mutant IN coding sequences from
pBluescript II KS+ were then subcloned into pNLNgo-
MIVR−ΔEnv.LUC (between the KpnI and SalI sites) to produce the
full-length mutant HIV-1 IN constructs. These DNA sequences were
checked independently by DNA sequence determination.
Reverse Transcriptase (RT) Assays. The assay used to determine
whether the compounds inhibit the of polymerase activity of RT has
been previously described.30 Briefly, the −47 sequencing primer (5′-
CGCCAGGGTTTTCCCAGTCACGAC-3′; New England Biolabs)
was 5′ end-labeled with [γ-32P] ATP and T4 polynucleotide kinase.
After purification, the labeled primer was annealed to single-stranded
M13mp18 DNA (1 μg of DNA for each sample to be assayed) by
heating and slow cooling. For each sample, 0.1 μg of wild-type RT
(approximately 17 nM final) added to the labeled primer template
(approximately 9.0 nM) in 25 mM Tris−HCl (pH 8.0), 75 mM KCl,
10 mM MgCl2, 100 μg of BSA per mL, and 10 mM CHAPS. The
reaction mixture was supplemented with 0.5 μM (each) of dATP,
dCTP, dGTP, and TTP. The compounds were added to give final
concentrations of 0, 0.02, 0.1, 0.5, 1, or 10 μM. The reactions were
allowed to proceed at 37 °C for 60 min and were stopped by the
addition of EDTA. The samples were precipitated by the addition of
two volumes of EtOH, fractionated by electrophoresis on a 6.0%
polyacrylamide gel, and the gel was autoradiographed. Reactions were
carried out in duplicate.
AUTHOR INFORMATION
Corresponding Author
*Phone: 301-846-5906. Fax: 301-846-6033. E-mail: burkete@
helix.nih.gov.
■
Present Address
∥Laboratoire MFP, CNRSUMR 5234, Universite
́
de
Bordeaux, 146 rue Leo Saignat, Bat 3A 3eme etage, 33076
́
̀
́
Bordeaux Cedex, France.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the Intramural Research
Program of the NIH, Center for Cancer Research, National
Institutes of Health, NCI at Frederick, and the Joint Science
and Technology Office of the Department of Defense and
funds from the Intramural AIDS Targeted Antiviral Program.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S.
Government.
ABBREVIATIONS USED
■
HIV-1, human immunodeficiency virus type 1; AIDS, acquired
immune deficiency syndrome; FDA, Food and Drug Admin-
istration; IN, integrase; RNase H, ribonuclease H; RT, reverse
transcriptase; NNRTI, nonnucleoside reverse transcriptase
inhibitor; NT, nucleotides; RAL, raltegravir; EVG, elvitegravir;
DTG, dolutegravir; 3′-P, 3′-processing; ST, strand transfer;
INSTIs, integrase strand transfer inhibitors; DNA, deoxyribo-
nucleic acid; IC50, half-maximum inhibitory concentration;
EC50, half maximal effective concentration; WT, wild-type;
DMF, dimethylformamide; HPLC, high-pressure liquid chro-
matography; HRMS, high-resolution mass spectrometry
REFERENCES
■
(1) Cherepanov, P.; Maertens, G. N.; Hare, S. Structural insights into
the retroviral DNA integration apparatus. Curr. Opin. Struct. Biol. 2011,
21, 249−256.
(2) Croxtall, J. D.; Scott, L. J. Raltegravir: in treatment-naive patients
with HIV-1 infection. Drugs 2010, 70, 631−642.
(3) Wills, T.; Vega, V. Elvitegravir: a once-daily inhibitor of HIV-1
integrase. Expert Opin. Invest. Drugs 2012, 21, 395−401.
(4) Marchand, C.; Maddali, K.; Metifiot, M.; Pommier, Y. HIV-1 IN
inhibitors: 2010 update and perspectives. Curr. Top. Med. Chem. 2009,
9, 1016−1037.
(5) Mesplede, T.; Quashie, P. K.; Wainberg, M. A. Resistance to HIV
integrase inhibitors. Curr. Opin. HIV AIDS 2012, 7, 401−408.
(6) Geretti, A. M.; Armenia, D.; Ceccherini-Silberstein, F. Emerging
patterns and implications of HIV-1 integrase inhibitor resistance. Curr.
Opin. Infect. Dis. 2012, 25, 677−686.
(7) Katlama, C.; Murphy, R. Dolutegravir for the treatment of HIV.
Expert Opin. Invest. Drugs 2012, 21, 523−530.
(8) Ballantyne, A. D.; Perry, C. M. Dolutegravir: first global approval.
Drugs 2013, 73, 1627−1637.
RNaseH Assays. This procedure has been previously described.30
Briefly, RNA oligonucleotide (5′-GGGGCCACUUUUUAAAA-
GAAAAGGGGGGACUGGAAGGGCUAAUUCACUCAC-3′) was
obtained from Dharmacon Research, Inc. The RNA oligonucleotide
was 5′-end labeled and was then annealed to a DNA oligonucleotide
(5′-GAGTGAATTAGCCCTTCCAGTCCC-3′) by heating and slow
cooling. A 0.2 μM concentration of the RNA/DNA hybrid was
suspended in 25 mM Tris (pH 8.0), 50 mM NaCl, 5.0 mM MgCl2,
100 μg of bovine serum albumin/mL, 10 mM CHAPS, and 1 U of
Superasin (Ambion). The compounds were added to the reactions to
give the following final concentrations (0, 0.1, 0.5, 1, and 10 μM). The
reaction volume was 12 μL. The reactions were initiated by the
addition of 50.0 ng of RT and were incubated at 37 °C. Aliquots were
removed at the indicated time points, and the reactions halted by
addition of 2× gel loading buffer (Ambion). The reaction products
(9) Zhao, X. Z.; Smith, S. J.; Metifiot, M.; Marchand, C.; Pommier,
Y.; Hughes, S. H.; Burke, T. R., Jr. 4-Amino-1-hydroxy-2-oxo-1,8-
naphthyridine-containing compounds having high antiviral potency
against cells harboring raltegravir-resistant integrase mutants. J. Med.
Chem. 2014, 57, 1573−1582.
L
dx.doi.org/10.1021/jm5001908 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(10) Summa, V.; Petrocchi, A.; Matassa, V. G.; Taliani, M.; Laufer,
R.; De Francesco, R.; Altamura, S.; Pace, P. HCV NS5b RNA-
dependent RNA polymerase inhibitors: From alpha, gamma-
diketoacids to 4,5-dihydroxypyrimidine- or 3-methyl-5- hydroxypyr-
imidinonecarboxylic acids. Design and synthesis. J. Med. Chem. 2004,
47, 5336−5339.
(11) Summa, V.; Petrocchi, A.; Matassa, V. G.; Gardelli, C.; Muraglia,
E.; Rowley, M.; Paz, O. G.; Laufer, R.; Monteagudo, E.; Pace, P. 4,5-
Dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidi-
none carboxamides are potent, selective HIV integrase inhibitors with
good pharmacokinetic rofiles in preclinical species. J. Med. Chem. 2006,
49, 6646−6649.
(12) Williams, P. D.; Venkatraman, S.; Langford, H. M.; Kim, B.;
Booth, T. M.; Grobler, J. A.; Staas, D.; Ruzek, R. D.; Embrey, M. W.;
Wiscount, C. M.; Lyle, T. A. Preparation of 1-hydroxynaphthyridin-
2(1H)-one derivatives as anti-HIV agents. (Merck & Co., Inc.)
WO2008010964A1, 2008.
(13) Su, H.-P.; Yan, Y.; Prasad, G. S.; Smith, R. F.; Daniels, C. L.;
Abeywickrema, P. D.; Reid, J. C.; Loughran, H. M.; Kornienko, M.;
Sharma, S.; Grobler, J. A.; Xu, B.; Sardana, V.; Allison, T. J.; Williams,
P. D.; Darke, P. L.; Hazuda, D. J.; Munshi, S. Structural basis for the
inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase
H active site-directed inhibitors. J. Virol. 2010, 84, 7625−7633.
(14) Williams, P. D.; Staas, D. D.; Venkatraman, S.; Loughran, H. M.;
Ruzek, R. D.; Booth, T. M.; Lyle, T. A.; Wai, J. S.; Vacca, J. P.;
Feuston, B. P.; Ecto, L. T.; Flynn, J. A.; Di, S. D. J.; Hazuda, D. J.;
Bahnck, C. M.; Himmelberger, A. L.; Dornadula, G.; Hrin, R. C.;
Stillmock, K. A.; Witmer, M. V.; Miller, M. D.; Grobler, J. A. Potent
and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-
1,8-naphthyridin-2(1H)-one scaffold. Bioorg. Med. Chem. Lett. 2010,
20, 6754−6757.
(15) Costi, R.; Metifiot, M.; Esposito, F.; Cuzzucoli Crucitti, G.;
Pescatori, L.; Messore, A.; Scipione, L.; Tortorella, S.; Zinzula, L.;
Marchand, C.; Pommier, Y.; Tramontano, E.; Di Santo, R. 6-(1-
Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of
recombinant HIV-1 integrase and ribonuclease H, synthesized by a
parallel synthesis approach. J. Med. Chem. 2013, 56, 8588−8598.
(16) Esposito, F.; Tramontano, E. Past and future. Current drugs
targeting HIV-1 integrase and reverse transcriptase-associated
ribonuclease H activity: single and dual active site inhibitors. Antivir.
Chem. Chemother. 2013, 23, 129−144.
Montgomery, D.; Molla, A.; Kempf, D. J.; Kohlbrenner, W. Inhibitors
of HCV NS5B polymerase: synthesis and structure−activity relation-
ships of N-1-heteroalkyl-4-hydroxyquinolon-3-yl-benzothiadiazines.
Bioor. Med. Chem. Lett. 2005, 15, 1577−1582.
(22) Zhao, X. Z.; Semenova, E. A.; Vu, B. C.; Maddali, K.; Marchand,
C.; Hughes, S. H.; Pommier, Y.; Burke, T. R., Jr. 2,3-Dihydro-6,7-
dihydroxy-1H-isoindol-1-one-based HIV-1 integrase inhibitors. J. Med.
Chem. 2008, 51, 251−259.
(23) Malet, I.; Delelis, O.; Valantin, M.-A.; Montes, B.; Soulie, C.;
Wirden, M.; Tchertanov, L.; Peytavin, G.; Reynes, J.; Mouscadet, J.-F.;
Katlama, C.; Calvez, V.; Marcelin, A.-G. Mutations associated with
failure of raltegravir treatment affect integrase sensitivity to the
inhibitor in vitro. Antimicrob. Agents Chemother. 2008, 52, 1351−1358.
(24) Delelis, O.; Thierry, S.; Subra, F.; Simon, F.; Malet, I.; Alloui, C.;
Sayon, S.; Calvez, V.; Deprez, E.; Marcelin, A.-G.; Tchertanov, L.;
Mouscadet, J.-F. Impact of Y143 HIV-1 integrase mutations on
resistance to raltegravir in vitro and in vivo. Antimicrob. Agents
Chemother. 2009, 54, 491−501.
(25) Metifiot, M.; Maddali, K.; Naumova, A.; Zhang, X.; Marchand,
C.; Pommier, Y. Biochemical and pharmacological analyses of HIV-1
integrase flexible loop mutants resistant to raltegravir. Biochemistry
2010, 49, 3715−3722.
(26) Metifiot, M.; Maddali, K.; Johnson, B. C.; Hare, S.; Smith, S. J.;
Zhao, X. Z.; Marchand, C.; Burke, T. R., Jr.; Hughes, S. H.;
Cherepanov, P.; Pommier, Y. Activities, crystal structures and
molecular dynamics of dihydro-1H-isoindole derivatives, inhibitors of
HIV-1 integrase. ACS Chem. Biol. 2013, 8, 209−217.
(27) Jacobo-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A. D.; Lu, X.;
Tantillo, C.; Williams, R. L.; Kamer, G.; Ferris, A. L.; Clark, P. Crystal
structure of human immunodeficiency virus type 1 reverse tran-
scriptase complexed with double-stranded DNA at 3.0 A resolution
shows bent DNA. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 6320−6324.
(28) Zhao, X. Z.; Maddali, K.; Metifiot, M.; Smith, S. J.; Vu, B. C.;
Marchand, C.; Hughes, S. H.; Pommier, Y.; Burke, T. R., Jr. Bicyclic
hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhib-
itors. Chem. Biol. Drug Des. 2012, 79, 157−165.
(29) Brachmann, A.; Koenig, J.; Julius, C.; Feldbruegge, M. A reverse
genetic approach for generating gene replacement mutants in Ustilago
maydis. Mol. Genet. Genomics 2004, 272, 216−226.
(30) Ivetac, A.; Swift, S. E.; Boyer, P. L.; Diaz, A.; Naughton, J.;
Young, J. A. T.; Hughes, S. H.; McCammon, J. A. Discovery of novel
inhibitors of HIV-1 reverse transcriptase through virtual screening of
experimental and theoretical ensembles. Chem. Biol. Drug Des. 2014,
83, 521−531.
(17) Zhao, X. Z.; Maddali, K.; Vu, B. C.; Marchand, C.; Hughes
Stephen, H.; Pommier, Y.; Burke Terrence, R., Jr. Examination of
halogen substituent effects on HIV-1 integrase inhibitors derived from
2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-ones and 4,5-dihydroxy-1H-
isoindole-1,3(2H)-diones. Bioorg. Med. Chem. Lett. 2009, 19, 2714−
2717.
(18) Kawasuji, T.; Johns, B. A.; Yoshida, H.; Taishi, T.; Taoda, Y.;
Murai, H.; Kiyama, R.; Fuji, M.; Yoshinaga, T.; Seki, T.; Kobayashi,
M.; Sato, A.; Fujiwara, T. Carbamoyl pyridone HIV-1 integrase
inhibitors. 1. Molecular design and establishment of an advanced two-
metal binding pharmacophore. J. Med. Chem. 2012, 55, 8735−8744.
(19) Kawasuji, T.; Johns, B. A.; Yoshida, H.; Weatherhead, J. G.;
Akiyama, T.; Taishi, T.; Taoda, Y.; Mikamiyama-Iwata, M.; Murai, H.;
Kiyama, R.; Fuji, M.; Tanimoto, N.; Yoshinaga, T.; Seki, T.; Kobayashi,
M.; Sato, A.; Garvey, E. P.; Fujiwara, T. Carbamoyl pyridone HIV-1
integrase inhibitors. 2. Bi- and tricyclic derivatives result in superior
antiviral and pharmacokinetic profiles. J. Med. Chem. 2013, 56, 1124−
1135.
(20) Johns, B. A.; Kawasuji, T.; Weatherhead, J. G.; Taishi, T.;
Temelkoff, D. P.; Yoshida, H.; Akiyama, T.; Taoda, Y.; Murai, H.;
Kiyama, R.; Fuji, M.; Tanimoto, N.; Jeffrey, J.; Foster, S. A.; Yoshinaga,
T.; Seki, T.; Kobayashi, M.; Sato, A.; Johnson, M. N.; Garvey, E. P.;
Fujiwara, T. Carbamoyl pyridone HIV-1 integrase inhibitors 3. A
diastereomeric approach to chiral nonracemic tricyclic ring systems
and the discovery of dolutegravir (S/GSK1349572) and (S/
GSK1265744). J. Med. Chem. 2013, 56, 5901−5916.
(21) Pratt, J. K.; Donner, P.; McDaniel, K. F.; Maring, C. J.; Kati, W.
M.; Mo, H.; Middleton, T.; Liu, Y.; Ng, T.; Xie, Q.; Zhang, R.;
M
dx.doi.org/10.1021/jm5001908 | J. Med. Chem. XXXX, XXX, XXX−XXX