Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00596

Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.

Full text of DOI:10.1021/acs.jmedchem.7b00596

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Article
Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors
Xue Zhi Zhao, Steven J Smith, Daniel P Maskell, Mathieu Métifiot, Valerie E Pye, Katherine Fesen,
Christophe Marchand, Yves Pommier, Peter Cherepanov, Stephen H Hughes, and Terrence R Burke
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00596 • Publication Date (Web): 24 Jul 2017
Downloaded from http://pubs.acs.org on July 24, 2017
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Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors
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Xue Zhi Zhao, Steven J. Smith, Daniel P. Maskell, Mathieu Métifiot, Valerie E. Pye,
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Katherine Fesen, Christophe Marchand, Yves Pommier, Peter Cherepanov, Stephen H.
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1,
Hughes and Terrence R. Burke, Jr. *
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Chemical Biology Laboratory and HIV Dynamics and Replication Program, Center for
Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD
21702 and 3Developmental Therapeutics Branch and Laboratory of Molecular
Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of
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Health, Bethesda, MD 20892 and Chromatin Structure and Mobile DNA, The Francis Crick
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Institute, London, NW1 1AT, UK and Imperial College London, St-Mary’s Campus, Norfolk
Place, London, W2 1PG, UK.
___________________________________________
*
Corresponding Author: Tel: 301-846-5906, Fax: 301-846-6033
E-mail: burkete@helix.nih.gov
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ABSTRACT
Integrase mutations can reduce effectiveness of the first-generation FDA-approved
integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The
second-generation agent, dolutegravir (DTG) has enjoyed considerable clinical success;
however, resistance-causing mutations that diminish the efficacy of DTG have appeared.
Our current findings support and extend the substrate envelope concept that broadly
effective INSTIs can be designed by filling the envelope defined by the DNA substrates.
Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides
as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly
effective INSTIs can be developed using this scaffold. Herein, we report an extended
investigation of 6-substituents as well the first examples of 7-substituted analogs of this
scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents
at the 6-position that are highly effective, with the best compound (6p) retaining better
efficacy against a broad panel of known INSTI resistant mutants than any analogs we have
previously described.
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INTRODUCTION
HIV-1 integrase (IN) plays a key role in the viral life cycle, inserting the double-stranded
DNA that is generated by reverse transcription of the viral RNA genome into the genome of
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the host cell. Integration is essential for viral replication, and for this reason, IN is a
therapeutic target for the treatment of HIV infections. To date, three HIV IN antagonists
have been approved for clinical use: raltegravir (RAL, 1), elvitegravir (EVG, 2) and
dolutegravir (DTG, 3) (Figure 1).2-4 These drugs belong to a class of compounds called
integrase strand transfer inhibitors (INSTIs) because they inhibit DNA strand transfer (ST),
the second step of integration catalyzed by IN, rather than the first step, 3’-processing
reaction (3’-P).5-8 Development of drug resistance mutations is a common problem in
antiviral therapy and not surprisingly, mutations affecting the susceptibility of the virus to
RAL and EVG have rapidly emerged.9-11 However, the second-generation inhibitor, DTG
retains potency against some but not all RAL/EVG resistant HIV variants.12-16 Therefore, the
development of new small molecules that have minimal toxicity and improved efficacy
against the existing resistant mutants remains an important research objective.17
Retroviral integration is mediated by IN multimers that are assembled on the viral
DNA ends forming a stable synaptic complex, also referred to as the intasome. 18-21 The
INSTIs only bind to the active site of IN when the processed viral DNA ends are
appropriately bound to the intasome.8, 22 The way in which INSTIs bind to the intasome was
elucidated by solving crystal structures of the orthologous retroviral IN from the prototype
foamy virus (PFV).19, 23, 24 The INSTIs are “interfacial” inhibitors; they bind to the active site
of IN and interact with the bound viral DNA following the 3’-processing step.8, 19, 25
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Essential structural features that contribute to the binding of INSTIs include an array of
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three heteroatoms (highlighted in red, Figure 1) that chelate the two catalytic Mg ions in
the IN active site and a halobenzyl side chain (halophenyl portion highlighted in blue,
Figure 1) that stacks with the penultimate nucleotide (a deoxycytidine) at the 3’ end of the
viral DNA.8, 19 We have recently shown that the 1-hydroxy-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide motif (4) can serve a useful platform for developing HIV-1
IN inhibitors that retain high efficacy against the RAL/EVG-resistant mutants.26, 27 We
initially examined the properties of a series of analogs related to structure 4 by varying the
substituents at the 4-position. Our objective was to identify compounds that retain efficacy
against the mutations Y143R, N155H and Q148H/G140S, which have been associated with
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clinical resistance to RAL and some of these mutations also play a role in the development
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of resistance against DTG. This approach yielded compounds including 4a–d, which are
approximately equivalent to RAL in their potency against recombinant wild-type (WT)
HIV-1 IN in biochemical assays. However, the small molecules also showed improved
antiviral efficacies against the Y143R and N155H mutants in cell-based assays.26, 27
Although antiviral efficacies against the Q148H/G140S double mutant were also improved
relative to RAL, the new compounds were inferior to DTG, prompting us to continue our
developmental efforts.
Structural studies using the PFV intasome have revealed that the tricyclic system of
DTG is sufficiently extended to make contacts with G187 in the β4–α2 loop of PFV IN (G118
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in IN). It has been argued that the interactions with this region may contribute to the
improved properties of DTG and other second-generation INSTIs.4, 23, 29, 30 Therefore, we
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considered that adding functionality to either the 6- or 7-positions of 4 could interact with
the same region of the catalytic site (highlighted in green and cyan, respectively in the
structures of DTG and 4, Figure 1). In a preliminary work, we modified the 6-position of 4
and showed that adding linear side chains bearing terminal hydroxyl groups can improve
antiviral efficacies against the Q148H/G140S double mutant to levels approaching that of
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DTG. Furthermore, depending on the 6-substituent, compounds could retain essentially
all of their antiviral potency against a more extensive panel of HIV-1 based vectors that
carry the major DTG-resistant IN mutants, including the G118R, T66I, E92Q, R263K, H51Y
single mutants and the H51Y/R263K double mutant.17, 32-34 These data have two important
implications: First, 6-substituents can have an important role in maintaining antiviral
efficacy against resistant mutant forms of IN. Second, compounds that are broadly effective
against mutant forms of IN bind in ways that involve substrate mimicry and when bound,
fit within the "substrate envelope".31, 35 However, the data were confined to a small number
of 6-modificaitons that had limited chemical diversity. Given the potential promise of 6-
substituents, we felt that it was important to more thoroughly examine this position and
accordingly, we describe here an extensive set of 6-modifications (designated as the 6
series analogs, Figure 1). We were also interested in examining substituents at the 7-
position of 4, since these could potentially afford an alternate way to engage G118 in the
β4–α2 loop of IN. We present the first examination of compounds that have modifications
at the 7-position (designated as the 5 series analogs). To provide a more complete SAR
study, we have also introduced various functionalities at the 4-position in combination
with various 6- and 7-substitutents. These efforts have resulted in one of the most potent
and effective compounds that we have developed to date. This work sheds additional light
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on enzyme-inhibitor interactions that can contribute to retention of efficacy against strains
of virus that contain mutant forms of IN resistant to the approved INSTIs.
RESULTS AND DISCUSSION
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Chemistry. To prepare our 7-substituted analogs 5a–i, we employed 2,6-dichloronicotinic
acid (7) as starting material. The 6-chloro group of 7 was converted into the final 7-
substituent in two ways. In one route, the 6-chloro moiety was displaced with methoxyl
t
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(
KO Bu in MeOH) and this was followed by esterification of the 3-carboxy group to yield 8
(Scheme 1). In the second route, the 3-carboxyl group of 7 was first converted directly to
the 3-methyl ester. In both cases, the 2-chloro group was then displaced with BnONH to
2
give the corresponding 2-benzyloxyamine-containing derivatives (10a and 9, respectively).
The 6-chloro-substituents of the second series 9 were transformed into the corresponding
6-piperidinyl and 6-morpholino derivatives (10b and 10c, respectively) by heating with
DMF solutions of piperidine or morpholine. Using a procedure similar to those we
previously reported (Scheme 1),26, 27, 31 intermediates 10a–c were transformed to the 4-
hydroxy (5a–c), 4-amino (5d–f) and 4-[2-(methyl glycinate)] (5g–i) final products.
(Insert Scheme 1)
We transformed the 6-bromo analogs (17a–c, Scheme 2) into a variety of 6-
substituted INSTIs by coupling alkynes and alkenes using Sonogashira and Heck coupling
chemistries.31 In addition to the 6-substituted products with a 4-hydroxyl (6a), we
prepared analogs having a free amine at the 4-position (6b–p) as well as analogs with 4-[2-
(methyl glycinate)]amine (6q) and 4-(2-hydroxyethyl)amine (6r–t) (Scheme 2).
(Insert Scheme 2)
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Evaluation of New Derivatives Using In Vitro IN Catalytic Assays and Single-Round
Virus Replication Assays. We designed compounds 5a–i to examine the effects of
substituents at the 4-position (hydroxyl, amine, or methyl glycinate) in combination with
modifications of the 7-position (methoxyl, piperidinyl or morpholino). We evaluated the
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compounds by in vitro IN activity assays that utilize P-radiolabeled oligonucleotides
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(
Table 1). We also tested the potency of the compounds in single-round replication assays
that employ HIV-1 vectors carrying WT IN. We evaluated the antiviral potencies of a subset
of the compounds against HIV-1 vectors carrying the canonical Y143R, N155H and
Q148H/G140S mutations that are associated with high-level HIV-1 resistance to RAL and
virological failure in patients (Table 2).17, 32-34, 38, 39 As we recently reported, compounds
that lack substituents at the 6- and 7-positions and harboring a hydroxyl group at the 4-
position (5'a) showed lower potencies in the ST assay and poorer antiviral potencies than
the corresponding 4-amino-containing compound (5’d) (Tables 1 and 2).26, 27 In our
current work, we found that a compound with a 4-[2-(methyl glycinate)] modification of
the 4-amino group (5’g) retained its enzymatic potency in these assays relative to 5’d.
Bulky substituents at the 7-position (5b, 5c, 5e, 5f, 5h and 5i, Table 1) diminished potency
by three-orders-of-magnitude relative to the corresponding unsubsituted analogs (5’a, 5’d
and 5’g, respectively). Although a 7-methoxyl group was better tolerated (compare with 5a,
5d and 5g), in all cases there was a reduction in the antiviral potency (Table 2).
(
Insert Table 1)
Insert Table 2)
(
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We recently described a series of structurally related 4-amino-containing INSTIs,
which possess 6-substituents consisting of primary hydroxyl or sulfone groups tethered by
31
alkyl chains of various lengths. The chemical diversity of these substituents was limited.
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To extend the diversity of the modifications at the 6-position, our current study examines
analogs containing tethered acetoxy (6b), benzyloxy (6c), cyclohexyl (6d), phenyl (6e and
6
f), dimethylamino (6g), carboxyl (6i), methyl ester (6p) and carboxamido having a variety
of alkylamide groups (6j – 6o) groups (Tables 1and 2). Several of the resulting compounds
retained low-nanomolar IC50 values in ST reactions. N,N-Dimethylpropylamine (6g, ST IC50
=
14 nM), propanoic acid (6i) propanoic amides (6j – o) and propanoic acid methyl ester
(
6p), as well as the reverse ester 6b displayed IC50 values of 10 nM or less in the ST assay.
Tethering a phenyl ring with an ethylene chain maintained good inhibitory potency (6f, ST
IC50 = 14 nM). However, appending phenyl or cyclohexyl groups by increasing spacer
lengths reduced potency by as much as three orders-of magnitude: three methylenes
(cyclohexyl-containing 6d, ST IC50 = 3.3 ꢀM), four methylenes (phenyl-containing 6e, ST
IC50 = 2.0 ꢀM) or longer (6c, ST IC50 = 3.3 ꢀM) (Table 1). This result contrasts with our
previous finding that appending a phenyl ring using a three-unit chain that contains a
sulfone moiety can result in retention of high potency. In the latter case, a co-crystal
structure with the PFV intasome revealed that the sulfone group causes the phenyl ring to
adopt an unusual π–π stacking orientation, in which it is folded up under the naphthyridine
ring system and fill the catalytic space more completely.31
Most 6-substituted analogs in our current study showed low nanomolar potencies
against the WT enzyme in the in vitro ST assay and against a WT HIV-1 vector in the single-
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round infectivity assay (Tables 1 and 2, respectively). However, for the 6-tethered 2-
hydroxyethylamides (6n and 6o), in vitro ST inhibitory potencies and antiviral potencies
against a vector that replicates using WT IN differed considerably (IC50 < 10 nM as
compared to EC50 ≥ 200 nM). Additionally, while analogs 6c, 6d and 6e showed micromolar
ST-inhibitory potencies in the in vitro assay, they were significantly more potent in the
antiviral assay. The reasons for these discrepancies are not clear. Among the remaining
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compounds, 6p, with a 6-(CH
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CO
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CH substituent, exhibited the best antiviral profile
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against the panel of resistant mutants (Table 2). This derivative displayed a modest
improvement in antiviral potency against a vector carrying a WT IN (EC50 = 0.67 nM) as
compared to 6’b, which had been the most promising compound in work we recently
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) OH group and a primary amine at the 4-
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position. Adding 4-[2-(methyl glycinate)] or 2-hydroxyethyl substituents to the 4-amino
group of 6’b did not improve the inhibitory profiles (compounds 6q and 6r, respectively),
nor were the antiviral potencies improved by the addition of a 2-hydroxyethyl substituent
to the 4-amino group of 6p to yield 6t (Tables 2). Most 6-subsituted analogs were not
cytotoxic within the range tested (Table 2). Exceptions were found with 6c, 6d and 6e,
which also showed anomalous discrepancies between their in vitro and antiviral potencies
and with 6f and 6g. The reasons for the greater cytotoxicities of these analogs are not clear.
An important component of our current study is to test the effects of substituents at
the 7-position. Given proximity to the metal-chelating 8-aryl nitrogen, we were uncertain of
what the effects such modifications would have. Bulky groups at the 7-position (5b, 5c, 5e,
5f, 5h and 5i) resulted in from two- to three-orders of magnitude loss of inhibitory potency
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against the WT enzyme in vitro. These latter data were consistent with the bulky
substituents causing a disruption of essential interactions with the catalytic metal ions. Of
greater interest was our finding that 7-OCH substituents were well tolerated against WT
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enzyme in in vitro assays (see 5a, 5d and 5g, Table 1).
Potencies of the New Compounds Against an Extended Set of Drug Resistant IN
Mutants. We selected a subset of the analogs (6b, 6p, 6r, 6s and 6t) for additional
evaluation in single-round viral replication assays using HIV-1 vectors harboring single
G118R, T66I, E92Q, R263K, H51Y, or the double H51Y/R263K substitutions associated
with DTG resistance (Table 3).17, 32-34, 38, 39 For comparison, we also included 6’b, which was
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one of the most broadly effective of our previously reported inhibitors. The new
compounds showed good antiviral potencies against the entire panel of mutants. Although
several compounds appear to be promising, the most potent analog (by a small margin)
was the 6-methyl 3-propanoate-containing 6p, which showed a slightly better inhibitory
profile against the first panel of mutants (Table 2), while also retaining greater inhibitory
potency than DTG against the second, more extended panel of mutants (Table 4).
(
Insert Table 3)
Insert Table 4)
(
Crystal Structures of the PFV Intasome in Complex with 5’g, 5g and 6p. To understand
how some of the current analogs interact with the active site of IN, we soaked PFV
intasome crystals in the presence of 5'g, 5g or 6p and refined the resulting structures
(Table 5). We recently described a crystal structure of 5’d bound to the active site of PFV
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intasome. The inhibitor 5’g bound to PFV IN differs from the previously reported 5’d
structure only in having a N-(methyl 2-glycinate)amine moiety at its 4-position, and,
predictably, the two compounds bind to the active site in a very similar fashion.
Superposition with the DTG-bound structure confirmed that 8-naphthyridine nitrogen and
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1
-N-hydroxyl of 5’g take the place of the 6-oxo and 7-hydroxyl heteroatoms of DTG. The
naphthyridine 2-oxo carbonyl of 5’g corresponds to the ring 8-oxo carbonyl of DTG (Figure
2
3
2A). However, the 4-N-(methyl 2-glycinate)amine group of 5’g extends into the region
which is occupied by the first base of the scissile dinucleotide in viral DNA prior to 3’-P
(designated A18 in the crystal structure, Figure 2A), which may be related to the ability of
8
the compound to inhibit the 3’-P reaction. Therefore, an extended form of substrate
mimicry may arise from the modification at the 4-position of 5’g, which was not seen in the
3
1
co-crystal structure of 5’d. The idea that inhibitors that remain within the "substrate
envelope" are particularly effective against resistant forms of the target enzyme was
proposed by Schiffer and colleagues and is based on the idea that to be able to support viral
replication, any mutant form of a viral enzyme must be capable of binding its normal
3
5
substrate(s). Thus, if the bound inhibitor remains within an envelope defined by the
substrate(s), the inhibitor is expected to retain some efficacy against mutant forms of the
4
0
enzyme. Even though the original hypothesis was based on work with protease inhibitors,
it seems likely that this principle can be applied broadly to anti-viral compounds that bind
at the active sites of essential viral enzymes. The 4-N-(methyl 2-glycinate) amine group of
5
’g is situated near (within 5 Å) to the side chains of Y212 and P214 (residues
corresponding to Y143 and P145 in HIV-1 IN) and to the adenosine base of the 3' terminal
viral DNA residue (A17), which adopts two alternative conformations in the crystal
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structure (Figure 2B). The interactions with the side chains are much less extensive in the
cases of DTG (3) and 5’d. Extension into this region of the catalytic site is also seen with
several highly potent INSTIs having diverse structures (see Figure S1 in Supporting
Information).
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(Insert Figure 2)
Prior structural studies of the PFV intasome showed that the tricyclic system of DTG
makes contacts with G187 in the β4–α2 loop of PFV IN (G118 in IN) and it has been argued
that the interactions with this region may contribute to the improved properties of DTG
and other second-generation INSTIs. 4, 23, 29, 30 As has already been discussed, we thought
that it might be possible to make useful contacts with the same region of the catalytic site
by adding functionality to the 6- and 7-positions of 4. To this end, we introduced
substituents at the 6- and 7-positions of 4 that could potentially recapitulate some of the
desirable features of DTG (highlighted in green and cyan, respectively in the structures of
DTG and 4, Figure 1). We obtained the crystal structure of 6p bound to the PFV intasome,
because it represents the most potent 6-substituted analog in our current study. The
structure revealed that the 6-methyl 3-propanoate side chain of the compound is situated
within the region defined by an envelope defined by both the pre-3'-P viral DNA and the
host target DNA substrate (Figure 3). This is consistent with our recent report that 6-
substituents of the compounds exhibiting the best antiviral profiles against the panel of
resistant mutants bind within an envelope defined as the confluence of viral DNA
3
1
substrates. The structure also shows that the methyl carboxylate group of 6p makes van
der Waals interactions with the β4–α2 loop of IN, becoming sandwiched between main
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chain atoms of Q186 and G187 (residues corresponding to HIV-1 N117 and G118,
respectively) and the side chain of Y212 (Figure 4).
(
Insert Figure 3)
Insert Figure 4)
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(
To understand the effects of introducing a 7-OCH
structure of 5g bound to the PFV intasome and overlaid the corresponding structure of
DTG bound to the PFV intasome (Figure 4). We observed that the 7-OCH group of 5g is
approximately 3.6 Å from the G187 residue in the β4–α2 loop, which compares favorably
3
substituent, we solved the
3
with DTG and 6p (approximately 3.9 Å and 3.7 Å, respectively, Figure 4A). The 7-OCH
3
group of 5g did not appear to significantly alter the position of the metal chelating
heteroatoms of 5g relative to 6p. However, it is not immediately obvious why 5g is more
susceptible to the N155H mutation compared to 6p or to the more closely related 5'g. Since
the N155 residue of HIV-1 IN corresponds to N224 in PFV IN, the X-ray crystal structure
with the mutation N224H in PFV IN did show that the side chain of His224 interacts with a
phosphoryl oxygen atom in viral DNA and that INSTI binding is accompanied by a loss of
24
this contact. The associated energetic penalty may explain the loss potency of some
compounds against N155H HIV-1 IN. In the co-crystal structure of PFV intasome with 5g,
the location of this phosphoryl oxygen is nearly identical to that seen for 6p, 5'g and DTG
(Figure 4C). The abilities of some INSTIs to re-adjust in the active site of IN may help
explain variations among in their susceptibilities of the different INSTIs to the N155H
mutation.23, 24, 41
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CONCLUSIONS
Earlier, we reported that the 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-
carboxamide scaffold represents a potentially attractive platform for the development of
INSTIs, that retain good antiviral efficacy against a panel of viral vectors carrying resistant
mutant forms of IN.26, 27 More recently, we began an exploration of the effects of modifying
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the 6-position of our earlier compounds. We found that 6-substituents may play an
important role in enhancing the maintenance of antiviral efficacy against drug-resistant
mutant forms of IN. Furthermore, the most broadly effective compounds are substrate
mimics that fit within the "substrate envelope." Our original purpose in synthesizing 6-
substituents was to create additional interactions within areas of the catalytic region
accessed by the third ring of DTG. However, the range of functionality reported in the prior
paper was quite limited, consisting of primary hydroxyl groups tethered by linear
polymethylene chains of varying lengths, and a single tethered phenyl sulfone. We now
report a much more extensive exploration of functionalities at the 6-position, as well as the
first examination of derivatives with modifications at the 7-position. We also tested
whether the compounds could be improved by introduced by varying functionalities at the
4-position.
We observed that -OCH groups are somewhat better tolerated than bulky
3
subsituents at the 7-position when examined in vitro against WT enzyme. However,
compounds with 7-OMe groups showed significantly reduced antiviral potency when
tested against viral vectors that carry the N155H mutation. We found that a variety of
substituents can be added at the 6-position, which enhance the antiviral potencies of the
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compounds against the major RAL, EVG and DTG-resistant IN mutants. Many of these
compounds are not cytotoxic at the tested concentrations, and the best analog, 6p, is the
most effective in terms of its ability to inhibit the entire spectrum of IN mutants of any of
the compounds we have described. The crystal structure of the PFV intasome with 6p
bound supports the idea that interactions with the β4–α2 loop are important and that
compounds that bind within the envelope defined by both the viral and host DNA
substrates helps the compounds retain broad efficacy. We found that the 4-amine
substituent in 5'g accesses a region of the active site of IN that would normally be occupied
by the base of the T+2 adenosine of the viral DNA prior to the 3’-P cleavage, providing
further support that binding within an envelope defined by the DNA substrates helps the
compounds retain broad efficacy. Thus, our results will inform the design and development
of next-generation INSTIs with broader efficacy against the known drug resistant mutants.
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EXPERIMENTAL SECTION
1
13
General Procedures. Proton ( H) and carbon ( C) NMR spectra were recorded 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 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 (Cat. No. 00G-4436-P0-AX, 250 mm × 21.2 mm 10 µm particle size, 110 Å pore) at
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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. Electrospray ionization-mass
spectrometric (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 spectrometric (HRMS)
were acquired by LC/MS-ESI using LTQ-Orbitrap-XL at 30K resolution.
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General Procedure I for the Synthesis of Carboxamides (5a−i and 6a−t). Benzyl
protected compounds (13, 15, 16 and 19) (0.1 mmol) were suspended in methanol (10 mL)
and ethyl acetate (3 mL). Equivalent of Pd/C (10%) was added. The mixture was stirred at
room temperature under hydrogen. When the starting material was disappeared (TLC), the
crude mixture was filtered and washed by methanol. The filtrate was concentrated and
purified by HPLC to provide final carboxamides (5a−i and 6a−t).
N-(2,4-Difluorobenzyl)-1,4-dihydroxy-7-methoxy-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide (5a). Treatment of 13a as outlined in general procedure I,
and purification by preparative HPLC (with a linear gradient of 30% B to 90% B over 30
1
min; retention time = 25.5 min) provided 5a as a white fluffy solid (22% yield). H NMR
6
(400 MHz, DMSO-d ) δ 11.00 (s, 1H), 10.42 (d, J = 5.8 Hz, 1H), 8.29 (dd, J = 8.7, 0.9 Hz, 1H),
7.48 (dd, J = 15.3, 8.6 Hz, 1H), 7.30 – 7.25 (m, 1H), 7.10 (dd, J = 9.8, 7.3 Hz, 1H), 6.83 – 6.81
(m, 1H), 4.62 (d, J = 6.0 Hz, 2H), 4.02 (s, 3H). ESI-MS m/z: 378.1 (MH+).
N-(2,4-Difluorobenzyl)-1,4-dihydroxy-2-oxo-7-(piperidin-1-yl)-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (5b). Treatment of 13b as outlined in general
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procedure I, and purification by preparative HPLC (with a linear gradient of 50% B to 90%
B over 30 min; retention time = 24.0 min) provided 5b as a white fluffy solid (13% yield).
1H NMR (400 MHz, DMSO-d
6
) δ 10.52 (bs, 1H), 10.36 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 9.1 Hz,
10
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H), 7.41 (dd, J = 15.3, 8.7 Hz, 1H), 7.25 – 7.19 (m, 1H), 7.04 (dd, J = 9.3, 6.7 Hz, 1H), 6.82 (d,
J = 9.2 Hz, 1H), 4.54 (d, J = 5.8 Hz, 2H), 3.74 (s, 4H), 1.61 (bs, 2H), 1.52 (bs, 4H). ESI-MS m/z:
31.2 (MH+).
4
N-(2,4-Difluorobenzyl)-1,4-dihydroxy-7-morpholino-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide (5c). Treatment of 13c as outlined in general procedure I,
and purification by preparative HPLC (with a linear gradient of 30% B to 90% B over 30
1
min; retention time = 24.3 min) provided 5c as a white fluffy solid (30% yield). H NMR
(400 MHz, DMSO-d ) δ 10.60 (s, 1H), 10.36 (t, J = 6.0 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.41
6
(dd, J = 15.3, 8.6 Hz, 1H), 7.25 – 7.19 (m, 1H), 7.06 – 7.02 (m, 1H), 6.83 (d, J = 9.1 Hz, 1H),
.54 (d, J = 6.0 Hz, 2H), 3.73 (s, 4H), 3.65 (t, J = 4.5 Hz, 4H). ESI-MS m/z: 433.1 (MH+).
4
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-7-methoxy-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide (5d). Treatment of 16a as outlined in general procedure I,
and purification by preparative HPLC (with a linear gradient of 30% B to 75% B over 30
1
min; retention time = 22.4 min) provided 5d as a white fluffy solid (37% yield). H NMR
(400 MHz, DMSO-d
6
) δ 10.60 (t, J = 5.6 Hz, 1H), 10.51 (bs, 1H), 8.48 (d, J = 8.8 Hz, 1H), 7.38
(dd, J = 15.4, 8.7 Hz, 1H), 7.22 – 7.17 (m, 1H), 7.02 (td, J = 8.5, 2.5 Hz, 1H), 6.72 (d, J = 8.8 Hz,
H), 4.46 (d, J = 5.7 Hz, 2H), 3.94 (s, 3H). ESI-MS m/z: 377.1 (MH+).
1
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-7-(piperidin-1-yl)-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (5e). Treatment of 16b as outlined in
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general procedure I, and purification by preparative HPLC (with a linear gradient of 50% B
to 70% B over 30 min; retention time = 20.6 min) provided 5e as a white fluffy solid (49%
1
yield). H NMR (400 MHz, DMSO-d
6
) δ 10.63 (t, J = 5.8 Hz, 2H), 8.19 (d, J = 9.2 Hz, 1H), 7.36
0
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(dd, J = 15.4, 8.7 Hz, 1H), 7.21 – 7.16 (m, 1H), 7.02 (t, J = 9.0 Hz, 1H), 6.75 (d, J = 9.2 Hz, 1H),
.44 (d, J = 5.7 Hz, 2H), 3.69 (bs, 4H), 1.59 (bs, 2H), 1.50 (bs, 4H). ESI-MS m/z: 430.2 (MH+).
4
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-7-morpholino-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (5f). Treatment of 16c as outlined in general
procedure I, and purification by preparative HPLC (with a linear gradient of 30% B to 65%
1
B over 30 min; retention time = 24.0 min) provided 5f as a white fluffy solid (28% yield). H
NMR (400 MHz, DMSO-d ) δ 10.62 (t, J = 5.7 Hz, 1H), 10.27 (bs, 1H), 10.19 (bs, 1H), 8.26 (d,
6
J = 9.1 Hz, 1H), 7.36 (dd, J = 15.4, 8.7 Hz, 1H), 7.21 – 7.16 (m, 1H), 7.02 (td, J = 8.2, 2.0 Hz,
1H), 6.76 (d, J = 9.1 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 3.66 (d, J = 3.3 Hz, 8H). ESI-MS m/z:
4
32.1 (MH+).
Methyl 2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-7-methoxy-2-oxo-
,2-dihydro-1,8-naphthyridin-4-yl)amino)acetate (5g). Treatment of 15a as outlined in
1
general procedure I, and purification by preparative HPLC (with a linear gradient of 30% B
to 80% B over 30 min; retention time = 23.7 min) provided 5g as a white fluffy solid (20%
1
yield). H NMR (400 MHz, DMSO-d
6
) δ 11.17 (bs, 1H), 10.61 (bs, 1H), 10.45 (t, J = 5.7 Hz,
1H), 8.31 (d, J = 9.0 Hz, 1H), 7.45 (dd, J = 15.3, 8.7 Hz, 1H), 7.22 – 7.17 (m, 1H), 7.02 (td, J =
.5, 1.6 Hz, 1H), 6.65 (d, J = 8.9 Hz, 1H), 4.48 (d, J = 5.4 Hz, 2H), 4.45 (d, J = 5.9 Hz, 2H), 3.94
8
(
s, 3H), 3.63 (s, 3H). ESI-MS m/z: 449.1 (MH+).
Methyl 2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-2-oxo-7-(piperidin-
1-yl)-1,2-dihydro-1,8-naphthyridin-4-yl)amino)acetate (5h). Treatment of 15b as
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outlined in general procedure I, and purification by preparative HPLC (with a linear
gradient of 50% B to 70% B over 30 min; retention time = 22.9 min) provided 5h as a white
1
fluffy solid (29% yield). H NMR (400 MHz, DMSO-d
6
) δ 11.45 (bs, 1H), 10.70 (t, J = 5.7 Hz,
10
11
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1
H), 7.98 (d, J = 9.4 Hz, 1H), 7.39 (dd, J = 15.3, 8.7 Hz, 1H), 7.22 – 7.16 (m, 1H), 7.02 (td, J =
.6, 2.1 Hz, 1H), 6.65 (d, J = 9.4 Hz, 1H), 4.45 (bs, 2H), 4.44 (s, 2H), 3.70 (bs, 4H), 3.64 (s, 3H),
.60 (bs, 3H), 1.51 (bs, 3H). ESI-MS m/z: 502.2 (MH+).
Methyl 2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-7-morpholino-2-
oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)acetate (5i). Treatment of 15c as
outlined in general procedure I, and purification by preparative HPLC (with a linear
gradient of 30% B to 70% B over 30 min; retention time = 26.0 min) provided 5i as a white
1
fluffy solid (18% yield). H NMR (400 MHz, DMSO-d
6
) δ 11.43 (bs, 1H), 10.66 (t, J = 5.8 Hz,
1H), 10.31 (bs, 1H), 8.06 (d, J = 9.3 Hz, 1H), 7.40 (dd, J = 15.4, 8.7 Hz, 1H), 7.21 – 7.16 (m,
1
H), 7.02 (dd, J = 9.8, 7.4 Hz, 1H), 6.66 (d, J = 9.3 Hz, 1H), 4.47 (d, J = 5.4 Hz, 2H), 4.45 (d, J =
.7 Hz, 2H), 3.66 (d, J = 5.2 Hz, 8H), 3.63 (s, 3H). ESI-MS m/z: 504.2 (MH+).
5
N-(2,4-Difluorobenzyl)-1,4-dihydroxy-6-(5-hydroxypentyl)-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (6a). Treatment of 1-(benzyloxy)-6-bromo-
N-(2,4-difluorobenzyl)-4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
1
7a31 with commercially available pent-4-yn-1-ol as outlined in general procedures J and I
and purification by preparative HPLC (linear gradient of 30% B to 70% B over 30 min;
1
retention time = 23.8 min) provided 6a as a white fluffy solid (46% yield, two steps). H
NMR (500 MHz, DMSO-d
6
) δ 10.96 (bs, 1H), 10.52 (t, J = 6.1 Hz, 1H), 8.70 (d, J = 2.0 Hz, 1H),
8.26 (d, J = 2.0 Hz, 1H), 7.48 (dd, J = 15.3, 8.6 Hz, 1H), 7.30 – 7.25 (m, 1H), 7.09 (td, J = 8.5,
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2
2
2
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5
5
5
6
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.5 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 3.37 (t, J = 6.5 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 1.65 –
+
1.59 (m, 2H), 1.46 – 1.42 (m, 2H), 1.35 – 1.28 (m, 2H). ESI-MS m/z: 434.1 (MH ). HRMS
+
calcd C21
H
22
F
2
N
O
3 5
[MH ], 434.1522; found, 434.1507.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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8
9
0
1
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3
4
5
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7
8
9
0
3-(5-Amino-6-((2,4-difluorobenzyl)carbamoyl)-8-hydroxy-7-oxo-7,8-dihydro-
1,8-naphthyridin-3-yl)propyl Acetate (6b). Treatment of 19a as outlined in general
procedure I and purification by preparative HPLC (linear gradient of 30% B to 70% B over
1
30 min; retention time = 20.6 min) provided 6b as a white fluffy solid (71% yield). H NMR
(500 MHz, DMSO-d
6
) δ 10.69 (t, J = 5.7 Hz, 1H), 8.60 (s, 1H), 8.57 (s, 1H), 7.42 (dd, J = 15.6,
7.5 Hz, 1H), 7.24 (t, J = 9.9 Hz, 1H), 7.07 (t, J = 8.5 Hz, 1H), 4.52 (d, J = 5.5 Hz, 2H), 4.05 (t, J =
6.3 Hz, 2H), 2.75 (t, J = 7.9 Hz, 2H), 2.00 (s, 3H), 1.97 (d, J = 7.7 Hz, 2H). ESI-MS m/z: 447.1
+
+
(MH ). HRMS calcd C21
H
21
F
2
N
4
O [MH ], 447.1475; found, 447.1477.
5
6-(5-Amino-6-((2,4-difluorobenzyl)carbamoyl)-8-hydroxy-7-oxo-7,8-dihydro-
1
,8-naphthyridin-3-yl)hexyl Benzoate (6c). Protection of 1-(benzyloxy)-N-(2,4-
difluorobenzyl)-4-((2,4-dimethoxybenzyl)amino)-6-(6-hydroxyhex-1-yn-1-yl)-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamide31 (50 mg, 0.073 mmol) using benzoic
anhydride (25 mg, 0.11 mmol) in pyridine (0.5 mL) (room temperature, 2 h) provided 6-(8-
(benzyloxy)-6-((2,4-difluorobenzyl)carbamoyl)-5-((2,4-dimethoxybenzyl)amino)-7-oxo-
7,8-dihydro-1,8-naphthyridin-3-yl)hex-5-yn-1-yl benzoate as a white solid (56 mg, 96%
1
yield). [ H NMR (500 MHz, CDCl
3
) δ 11.99 (t, J = 5.8 Hz, 1H), 10.69 (t, J = 5.8 Hz, 1H), 8.67 (d,
J = 1.4 Hz, 1H), 8.34 (d, J = 1.7 Hz, 1H), 8.07 – 8.05 (m, 2H), 7.39 – 7.68 (m, 2H), 7.57 (t, J =
.4 Hz, 1H), 7.45 (t, J = 7.8 Hz, 2H), 7.40 – 7.36 (m, 4H), 7.23 (d, J = 8.9 Hz, 1H), 6.86 – 6.79
m, 2H), 6.48 – 6.46 (m, 2H), 5.26 (s, 2H), 4.75 (d, J = 5.9 Hz, 2H), 4.60 (d, J = 5.6 Hz, 2H),
7
(
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.41 (t, J = 6.4 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 2.54 (t, J = 7.0 Hz, 2H), 1.99 – 1.93 (m, 2H),
+
1.84 – 1.78 (m, 2H). ESI-MS m/z: 787.3 (MH ).] After treatment as outlined in general
procedure H and I and purification by preparative HPLC (linear gradient of 55% B to 75%
10
11
12
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15
16
17
18
19
20
21
22
23
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45
46
47
48
49
50
51
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53
54
55
56
57
58
59
60
B over 30 min; retention time = 25.4 min), 6c was provided as a white fluffy solid (45%
1
yield, two steps). H NMR (500 MHz, DMSO-d
6
) δ 10.69 (t, J = 5.7 Hz, 1H), 8.58 (d, J = 1.9 Hz,
1H), 8.54 (s, 1H), 7.94 (dd, J = 8.4, 1.3 Hz, 2H), 7.64 (dt, J = 8.7, 1.3 Hz, 1H), 7.53 – 7.50 (m,
2H), 7.42 (dd, J = 15.3, 8.5 Hz, 1H), 7.26 – 7.21 (m, 1H), 7.06 (dd, J = 9.8, 7.3 Hz, 1H), 4.51 (d,
J = 5.6 Hz, 2H), 4.26 (t, J = 6.5 Hz, 2H), 2.70 – 2.67 (m, 2H), 1.73 – 1.66 (m, 4H), 1.45 – 1.42
+
O
[MH+],
(
m, 2H), 1.39-1.38 (m, 2H). ESI-MS m/z: 551.2 (MH ). HRMS calcd C29
29
H F
2
N
4
5
551.2089; found, 551.2089.
4-Amino-6-(3-cyclohexylpropyl)-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (6d). Treatment of 19b as outlined in
general procedure I and purification by preparative HPLC (linear gradient of 50% B to 80%
B over 30 min; retention time = 27.5 min) provided 6d as a white fluffy solid (40% yield).
1H NMR (500 MHz, DMSO-d
15.5, 8.6 Hz, 1H), 7.20 – 7.15 (m, 1H), 7.00 (t, J = 8.5 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 2.59
(t, J = 7.7 Hz, 2H), 1.63 – 1.52 (m, 7H), 1.16 – 1.00 (m, 6H), 0.82 – 0.75 (m, 2H). ESI-MS m/z:
6
) δ 10.63 (t, J = 5.8 Hz, 1H), 8.51 (s, 1H), 8.47 (s, 1H), 7.36 (dd, J
=
+
+
4
71.2 (MH ). HRMS calcd C25
H
29
F
2
N
4
O [MH ], 471.2202; found, 471.2204.
3
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-6-(4-phenylbutyl)-1,2-
dihydro-1,8-naphthyridine-3-carboxamide (6e). Treatment of 19c as outlined in
general procedure I and purification by preparative HPLC (linear gradient of 40% B to 80%
B over 30 min; retention time = 26.0 min) provided 6e as a white fluffy solid (27% yield).
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4
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5
5
5
5
5
5
5
6
1H NMR (500 MHz, DMSO-d
) δ 10.69 (t, J = 5.8 Hz, 1H), 8.58 (s, 1H), 8.54 (s, 1H), 7.42 (dd, J
6
= 15.5, 8.5 Hz, 1H), 7.26 (t, J = 7.6 Hz, 2H), 7.24-7.21 (m, 1H), 7.19-7.14 (m, 3H), 7.07 (t, J =
8.5 Hz, 1H), 4.51 (d, J = 5.7 Hz, 2H), 2.72 (t, J = 7.3 Hz, 2H), 2.62 (t, J = 7.5 Hz, 2H), 1.69 (dd, J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
=
14.8, 7.1 Hz, 2H), 1.65 – 1.53 (m, 2H). ESI-MS m/z: 479.2 (MH ). HRMS calcd C26
H
25
F
2
N
O
4 3
+
[
MH ], 479.1889; found, 479.1880.
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-2-oxo-6-phenethyl-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (6f). Treatment of 19d as outlined in general
procedure I and purification by preparative HPLC (linear gradient of 40% B to 80% B over
1
30 min; retention time = 22.5 min) provided 6f as a white fluffy solid (65% yield). H NMR
(500 MHz, DMSO-d ) δ 10.69 (bs, 1H), 10.51 (bs, 1H), 8.61 (s, 1H), 8.53 (s, 1H), 7.45-7.42
6
(m, 1H), 7.35-7.32 (m, 1H), 7.27 (d, J = 7.0 Hz, 1H), 7.23 (d, J = 6.7 Hz, 2H), 7.20-7.17 (m, 1H),
+
7.08-7.05 (m, 1H), 4.52 (d, J = 5.5 Hz, 2H), 2.99 (bs, 4H). ESI-MS m/z: 451.1 (MH ). HRMS
+
calcd C24
H
21
F
2
N
O
4 3
[MH ], 451.1576; found, 451.1576.
4-Amino-N-(2,4-difluorobenzyl)-6-(3-(dimethylamino)propyl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6g). Treatment of 19e as outlined
in general procedure I and purification by preparative HPLC (linear gradient of 30% B to
55% B over 30 min; retention time = 17.2 min) provided 6g as a white fluffy solid (52%
1
yield). H NMR (500 MHz, DMSO-d
6
) δ 10.68 (t, J = 5.8 Hz, 1H), 10.57 (s, 1H), 8.62 (s, 1H),
8.57 (s, 1H), 7.42 (dd, J = 15.5, 8.5 Hz, 1H), 7.27 – 7.22 (m, 1H), 7.07 (dd, J = 9.8, 7.3 Hz, 1H),
4.52 (d, J = 5.8 Hz, 2H), 3.06 (d, J = 7.7 Hz, 2H), 2.78 (s, 6H), 2.74 (t, J = 7.5 Hz, 2H), 2.01 (dt, J
+
+
=
15.7, 7.8 Hz, 2H). ESI-MS m/z: 432.2 (MH ). HRMS calcd C21
H
24
F
2
N
5
O [MH ], 432.1842;
3
found, 432.1824.
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-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-(2-hydroxyethoxy)propyl)-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6h). Treatment of 19f as outlined
in general procedure I and purification by preparative HPLC (linear gradient of 30% B to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
0% B over 30 min; retention time = 20.5 min) provided 6h as a white fluffy solid (86%
1
yield). H NMR (500 MHz, DMSO-d
6
) δ 10.69 (t, J = 5.8 Hz, 1H), 8.59 (d, J = 1.9 Hz, 1H), 8.56
(d, J = 1.9 Hz, 1H), 7.52 – 7.35 (m, 1H), 7.24 (ddd, J = 10.5, 9.4, 2.6 Hz, 1H), 7.07 (ddd, J =
10.4, 8.1, 2.2 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H), 3.51 (t, J = 5.3 Hz, 2H), 3.41 (dt, J = 7.5, 5.7 Hz,
+
4H), 2.76 – 2.73 (m, 2H), 1.92 – 1.86 (m, 2H). ESI-MS m/z: 449.1 (MH ). HRMS calcd
+
C
21
H
23
F
2
N
O
4 5
[MH ], 449.1631; found, 449.1631.
3-(5-Amino-6-((2,4-difluorobenzyl)carbamoyl)-8-hydroxy-7-oxo-7,8-dihydro-
,8-naphthyridin-3-yl)propanoic Acid (6i). Treatment of 19g with aqueous NaOH and
1
then as outlined in general procedure I and purification by preparative HPLC (with a linear
gradient of 30% B to 50% B over 30 min; retention time = 19.5 min) provided 6i as a white
1
fluffy solid (80% yield). H NMR (400 MHz, DMSO-d
6
) δ 10.63 (t, J = 5.4 Hz, 1H), 8.68 – 8.66
(m, 1H), 8.63 – 8.60 (m, 1H), 8.57 (d, J = 1.9 Hz, 1H), 8.53 (d, J = 1.9 Hz, 1H), 7.40 – 7.34 (m,
1H), 7.21 – 7.16 (m, 1H), 7.04 – 6.99 (m, 1H), 4.47 (d, J = 5.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H),
+
O
[MH+],
2.62 (t, J = 7.6 Hz, 2H). ESI-MS m/z: 419.1 (MH ). HRMS calcd C19
H
17 2
F N
4
5
419.1162; found, 419.1147.
4-Amino-N-(2,4-difluorobenzyl)-6-(3-(dimethylamino)-3-oxopropyl)-1-
hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6j) and (E)-4-Amino-
N-(2,4-difluorobenzyl)-6-(3-(dimethylamino)-3-oxoprop-1-en-1-yl)-1-hydroxy-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6k). Treatment of 18e as outlined
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2
2
2
2
2
2
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3
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3
3
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3
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4
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5
5
5
5
5
5
5
5
5
6
in general procedure I and H and purification by preparative HPLC (with a linear gradient
of 30% B to 45% B over 30 min) provided 6j and 6k as white fluffy solids. For 6j: retention
1
time = 20.7 min. H NMR (400 MHz, DMSO-d
6
) δ 10.62 (t, J = 5.8 Hz, 1H), 10.46 (brs, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
1
2
.56 (d, J = 1.8 Hz, 1H), 8.50 (d, J = 1.8 Hz, 1H), 7.36 (dd, J = 15.4, 8.6 Hz, 1H), 7.20 – 7.14 (m,
H), 7.00 (td, J = 8.6, 1.9 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 2.90 (s, 3H), 2.84 (t, J = 7.4 Hz, 2H),
+
.74 (s, 3H), 2.65 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 446.2 (MH ). HRMS calcd C21
H
22
F
2
N
O
5 4
+
1
[MH ], 446.1634; found, 446.1617. For 6k: retention time = 24.1 min. H NMR (400 MHz,
DMSO-d ) δ 10.63 (s, 1H), 10.53 (t, J = 5.8 Hz, 1H), 8.94 (s, 2H), 7.49 (d, J = 15.4 Hz, 1H),
.37 (dd, J = 12.2, 5.3 Hz, 1H), 7.31 (d, J = 15.5 Hz, 1H), 7.21 – 7.15 (m, 1H), 7.01 (td, J = 8.9,
6
7
2
.3 Hz, 1H), 4.46 (d, J = 5.7 Hz, 2H), 3.14 (s, 3H), 2.89 (s, 3H). ESI-MS m/z: 444.1 (MH+).
+
HRMS calcd C21
H
20
F
2
N
5
O [MH ), 444.1478; found, 444.1475.
4
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-(isopropylamino)-3-
oxopropyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6l) and (E)-4-
Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-(isopropylamino)-3-oxoprop-1-en-1-
yl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6m). Treatment of 18f as
outlined in general procedure I and H and purification by preparative HPLC (with a linear
gradient of 30% B to 45% B over 30 min) provided 6l and 6m as white fluffy solids. For 6l:
1
retention time = 23.0 min. H NMR (400 MHz, DMSO-d
6
) δ 10.62 (t, J = 5.7 Hz, 1H), 10.46
(brs, 1H), 8.50 (d, J = 1.8 Hz, 1H), 8.47 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.36 (dd, J = 15.4, 8.7
Hz, 1H), 7.20-7.14 (m, 1H), 7.02-6.98 (m, 1H), 4.45 (d, J = 5.7 Hz, 2H), 3.76-3.68 (m, 1H),
2.84 (t, J = 7.5 Hz, 2H), 2.36 (t, J = 7.6 Hz, 2H), 0.90 (d, J = 6.6 Hz, 6H). ESI-MS m/z: 460.2
+
+
(
MH ). HRMS calcd C22
H
24
F
2
N
O
5 4
[MH ], 460.1791; found, 460.1772. For 6m: retention
1
time = 27.9 min. H NMR (400 MHz, DMSO-d
6
) δ 10.63 (s, 1H), 10.53 (t, J = 5.8 Hz, 1H), 8.84
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(
s, 1H), 8.82 (s, 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 15.8 Hz, 1H), 7.37-7.33 (m, 1H),
7.20-7.15 (m, 1H), 7.00 (td, J = 8.6, 2.3 Hz, 1H), 6.66 (d, J = 15.9 Hz, 1H), 4.46 (d, J = 5.7 Hz,
+
2
H), 3.91 (td, J = 13.3, 6.6 Hz, 1H), 1.05 (d, J = 6.6 Hz, 6H). ESI-MS m/z: 458.2 (MH ). HRMS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
calcd C22
H
22
F
2
N
O
5 4
[MH ], 458.1634; found, 458.1628.
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-((2-hydroxyethyl)amino)-3-
oxopropyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6n) and (E)-4-
Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-((2-hydroxyethyl)amino)-3-oxoprop-
1
-en-1-yl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6o). Treatment of
8g as outlined in general procedure I and H and purification by preparative HPLC (with a
1
linear gradient of 20% B to 50% B over 30 min) provided 6n and 6o as white fluffy solids.
1
For 6n: retention time = 22.1 min). H NMR (400 MHz, DMSO-d
6
) δ 8.56 (d, J = 1.9 Hz, 1H),
8.49 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 15.4, 8.6 Hz, 1H), 7.25-7.19 (m, 1H), 7.06 (dd, J = 9.4, 7.7
Hz, 1H), 4.50 (s, 2H), 3.32 (t, J = 6.1 Hz, 2H), 3.08 (t, J = 6.1 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H),
+
+
2
.48 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 462.1 (MH ), 484.1 (MNa ). HRMS calcd C21
H
22
F
2
N
O
5 5
+
1
[MH ], 462.1584; found, 462.1562. For 6o: retention time = 23.9 min. H NMR (400 MHz,
DMSO-d ) δ 10.63 (s, 1H), 10.53 (t, J = 5.8 Hz, 1H), 8.85 (s, 1H), 8.83 (s, 1H), 8.09 (t, J = 5.8
Hz, 1H), 7.43 (d, J = 15.8 Hz, 1H), 7.36 (dd, J = 15.4, 8.6 Hz, 1H), 7.20-7.15 (m, 1H), 7.03-6.98
6
(
m, 1H), 6.73 (d, J = 15.9 Hz, 1H), 4.65 (brs, 1H), 4.46 (d, J = 5.6 Hz, 2H), 3.40 (brs, 2H), 3.20
+
+
(
dd, J = 11.9, 6.0 Hz, 2H). ESI-MS m/z: 460.1 (MH ), 482.1 (MNa ). HRMS calcd C21
H
20
F
2
N
O
5 5
+
[
MH ], 460.1427; found, 460.1421.
Methyl 3-(5-Amino-6-((2,4-difluorobenzyl)carbamoyl)-8-hydroxy-7-oxo-7,8-
dihydro-1,8-naphthyridin-3-yl)propanoate (6p). Treatment of 19g as outlined in
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
general procedure I and purification by preparative HPLC (linear gradient of 30 % B to 65%
B over 30 min; retention time = 23.4 min) provided 6p as a white fluffy solid (72% yield).
1H NMR (500 MHz, DMSO-d
6
) δ 10.68 (t, J = 5.8 Hz, 1H), 8.62 (d, J = 1.4 Hz, 1H), 8.58 (s, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
2
.42 (dd, J = 15.3, 8.6 Hz, 1H), 7.26 – 7.22 (m, 1H), 7.07 (t, J = 8.5 Hz, 1H), 4.51 (d, J = 5.8 Hz,
H), 3.58 (s, 3H), 2.95 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 7.6 Hz, 2H). ESI-MS m/z: 433.1 (MH+).
+
HRMS calcd C20
H
19
F
2
N
4
O [MH ], 433.1318; found, 433.1311.
5
Methyl 2-((3-((2,4-Difluorobenzyl)carbamoyl)-1-hydroxy-6-(5-
hydroxypentyl)-2-oxo-1,2-dihydro-1,8-naphthyridin-4-yl)amino)acetate (6q).
Treatment of 18i as outlined in general procedure I and purification by preparative HPLC
(linear gradient of 30% B to 60% B over 30 min; retention time = 20.5 min) provided 6q as
1
a white fluffy solid (82% yield). H NMR (500 MHz, DMSO-d
6
) δ 10.22 (bs, 1H), 10.05 (bs,
1H), 8.48 (s, 1H), 8.22 (s, 1H), 7.52 (dd, J = 15.6, 8.6 Hz, 1H), 7.24 – 7.11 (m, 1H), 7.00 (t, J =
8.5 Hz, 1H), 4.41 (t, J = 5.5 Hz, 4H), 3.59 (s, 3H), 3.32 (t, J = 6.5 Hz, 4H), 2.62 (t, J = 7.6 Hz,
2
H), 1.61 – 1.47 (m, 2H), 1.45 – 1.33 (m, 2H), 1.32 – 1.18 (m, 2H). ESI-MS m/z: 505.2 (MH+).
+
HRMS calcd C24
H
27
F
2
N
O
4 6
[MH ], 505.1893; found, 505.1889.
N-(2,4-Difluorobenzyl)-1-hydroxy-4-((2-hydroxyethyl)amino)-6-(5-
hydroxypentyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (6r). Treatment
of 18j as outlined in general procedure I and purification by preparative HPLC (linear
gradient of 25% B to 40% B over 30 min; retention time = 24.5 min) provided 6r as a white
1
fluffy solid (22% yield). H NMR (500 MHz, DMSO-d
6
) δ 10.98 (s, 1H), 10.52 (s, 1H), 8.54 (s,
1H), 8.37 (s, 1H), 7.50 – 7.47 (m, 1H), 7.24 (t, J = 10.0 Hz, 1H), 7.07 (t, J = 8.4 Hz, 1H), 4.50 (d,
J = 5.6 Hz, 2H), 3.70 (bs, 2H), 3.61 (bs, 2H), 3.38 (t, J = 6.5 Hz, 2H), 2.69 (t, J = 7.4 Hz, 2H),
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.63 – 1.60 (m, 2H), 1.46 – 1.44 (m, 2H), 1.33- 1.24 (m, 2H). ESI-MS m/z: 477.2 (MH+).
+
HRMS calcd C23
H
26
F
2
N
O
4 5
[MH ], 477.1944; found, 477.1944.
5
-(6-((2,4-Difluorobenzyl)carbamoyl)-8-hydroxy-5-((2-hydroxyethyl)amino)-
-oxo-7,8-dihydro-1,8-naphthyridin-3-yl)pentyl Acetate (6s). The mixture of 18j (74
mg, 0.13 mmol), triethylamine (109 µL, 0.79 mmol) and acetic anhydride (37 µl, 0.40 mmol)
in CH Cl (3 mL) was stirred at room temperature (3 h). The crude mixture was purified by
silica gel chromatography to provide 5-(8-(benzyloxy)-6-((2,4-difluorobenzyl)carbamoyl)-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
2
2
5-((2-hydroxyethyl)amino)-7-oxo-7,8-dihydro-1,8-naphthyridin-3-yl)pent-4-yn-1-yl
1
acetate as a colorless oil (31 mg, 39% yield). [ H NMR (500 MHz, CDCl
3
) δ 12.00 (brs, 1H),
10.69 (t, J = 5.6 Hz, 1H), 8.31 (s, 1H), 7.67 (d, J = 6.9 Hz, 2H), 7.41 – 7.36 (m, 4H), 6.86 – 6.80
(m, 2H), 5.24 (s, 2H), 4.62 (d, J = 5.6 Hz, 2H), 4.35 (t, J = 5.4 Hz, 2H), 3.94 (dd, J = 10.6, 5.3 Hz,
2H), 3.83 (t, J = 5.9 Hz, 2H), 2.60 (t, J = 7.0 Hz, 2H), 2.11 (s, 3H), 1.90 (p, J = 6.5 Hz, 2H). ESI-
+
MS m/z: 605.2 (MH ).] This compound was treated as outlined in general procedure I and
purifide by preparative HPLC (linear gradient of 30% B to 55% B over 30 min; retention
1
time = 24.1 min) to provide 6s as a white fluffy solid (12% yield, two steps from 18j). H
NMR (500 MHz, DMSO-d
6
) δ 10.56 (brs, 1H), 10.29 (brs, 1H), 8.53 (s, 1H), 8.30 (s, 1H), 7.53
– 7.48 (m, 1H), 7.22 (t, J = 9.9 Hz, 1H), 7.07 – 7.03 (m, 1H), 4.48 (d, J = 5.8 Hz, 2H), 4.17 –
4.15 (m, 2H), 3.81 (d, J = 4.5 Hz, 2H), 3.38 – 3.36 (m, 2H), 2.68 (t, J = 7.4 Hz, 2H), 1.97 (s, 3H),
1.64 – 1.57 (m, 2H), 1.47 – 1.41 (m, 2H), 1.33 – 1.29 (m, 2H). ESI-MS m/z: 519.2 (MH+),
41.1 (MNa+).
5
Methyl 3-(6-((2,4-Difluorobenzyl)carbamoyl)-8-hydroxy-5-((2-
hydroxyethyl)amino)-7-oxo-7,8-dihydro-1,8-naphthyridin-3-yl)propanoate (6t).
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Treatment of 18k as outlined in general procedure I and purification by preparative HPLC
(linear gradient of 30% B to 50% B over 30 min; retention time = 21.0 min) provided 6t as
1
a white fluffy solid (20% yield). H NMR (500 MHz, DMSO-d
6
) δ 11.09 (bs, 1H), 10.54 (t, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.7 Hz, 1H), 8.57 (d, J = 1.8 Hz, 1H), 8.43 (d, J = 1.7 Hz, 1H), 7.46 (dd, J = 15.4, 8.6 Hz, 1H),
.26 – 7.21 (m, 1H), 7.09 – 7.05 (m, 1H), 4.50 (d, J = 5.7 Hz, 2H), 3.71 (s, 2H), 3.61 (t, J = 5.1
7
Hz, 2H), 3.58 (s, 3H), 2.96 (t, J = 7.4 Hz, 2H), 2.73 (t, J = 7.4 Hz, 2H). ESI-MS m/z: 477.1
+
+
(MH ). HRMS calcd C23
H
26
F
2
N
4
O [MH ], 477.1580; found, 477.1581.
5
Methyl 2-Chloro-6-methoxynicotinate (8). Commercial available 2,6-
Dichloronicotinic acid 7 (3.1 g, 16 mmol) was added to the mixture of potassium t-butoxide
(5.7 g, 48 mmol) in methanol (75 mL). The reaction mixture was stirred (65 °C, 24 h). The
reaction mixture was concentrated and acidified using concentrated aqueous HCl. The
crude mixture was filtered. The formed solid was collected to provide 2-chloro-6-
36
1
methoxynicotinic acid as a white solid (2.8 g, 94% yield). [ H NMR (400 MHz, DMSO-d ) δ
6
1
3
1
3.33 (bs, 1H), 8.19 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 3.92 (s, 3H). C NMR (101
MHz, DMSO-d ) δ 165.52, 164.54, 147.69, 143.80, 120.02, 109.82, 54.86. ESI-MS m/z: 188.0
6
+
(
MH ).] 2-Chloro-6-methoxynicotinic acid (2.8 g, 15 mmol) was suspended in thionyl
chloride (20 mL). The suspension was stirred and refluxed (3 h). The reaction mixture was
concentrated. The residue was mixed with methanol (20 mL). The mixture was refluxed (3
h) and concentrated. The crude residue was purified by silica gel chromatography to
1
provide 8 as a colorless oil (2.5 g, 81% yield). H NMR (400 MHz, CDCl
3
) δ 7.89 (d, J = 8.5 Hz,
1
3
1
H), 6.48 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H), 3.70 (s, 3H). C NMR (101 MHz, CDCl
3
) δ 164.56,
164.25, 148.96, 142.60, 118.11, 108.96, 54.30, 52.09.
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General Procedure A for the Synthesis of Methyl 2-((Benzyloxy)amino)-
nicotinates (9 and 10a). 2-Chloronicotinates (7 and 8) (12 mmol) were mixed with O-
benzylhydroxylamine (48 mmol) and DIEA (36 mmol). The reaction mixture was stirred
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
110 °C, 18 h). The crude mixture was purified by silica gel chromatography to provide
methyl 2-((benzyloxy)amino)-nicotinates (9 and 10a).
Methyl 2-((Benzyloxy)amino)-6-chloronicotinate (9). Treatment of methyl 2,6-
dichloronicotinate, [which was prepared from commercially available 2,6-dichloronicotinic
acid 7 in 91% yield based on known method.42 1H NMR (400 MHz, CDCl
) δ 8.10 (d, J = 8.1
) δ 163.95, 152.88,
49.74, 142.46, 125.12, 122.79, 52.94.], as outlined in general procedure A provided 9 as a
3
1
3
Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H). C NMR (101 MHz, CDCl
3
1
1
colorless oil (55% yield). H NMR (400 MHz, CDCl
3
) δ 10.08 (s, 1H), 8.07 – 7.97 (m, 1H),
1
3
7.52 – 7.46 (m, 2H), 7.41 – 7.25 (m, 3H), 6.77 – 6.68 (m, 1H), 5.03 (s, 2H), 3.80 (s, 3H). C
NMR (101 MHz, CDCl
3
) δ 166.13, 159.29, 155.57, 142.02, 135.92, 129.17(2C), 128.45(3C),
1
14.10, 104.88, 78.29, 52.26. ESI-MS m/z: 293.1 (MH+).
Methyl 2-((Benzyloxy)amino)-6-methoxynicotinate (10a). Treatment of 8 as
1
outlined in general procedure A provided 10a as a colorless oil (51% yield). H NMR (400
MHz, CDCl
.16 (d, J = 8.6 Hz, 1H), 5.09 (s, 2H), 3.99 (s, 3H), 3.78 (s, 3H). C NMR (101 MHz, CDCl
166.82, 166.70, 160.47, 142.09, 136.36, 128.81 (2C), 128.42 (2C), 128.26, 101.26, 98.90,
8.39, 53.74, 51.67.
General Procedure B for the Synthesis of 6-Substituted Methyl 2-
(Benzyloxy)amino)-nicotinates (10b and 10c). Methyl 2-((benzyloxy)amino)-6-
chloronicotinate (9) (1 mmol) was dissolved in DMF (1 mL). Piperidine or morphiline (4
3
) δ 10.22 (s, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.47 – 7.45 (m, 2H), 7.38 – 7.29 (m, 3H),
1
3
6
3
) δ
7
(
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