Repositioning HIV-1 integrase inhibitors for cancer therapeutics: 1,6-naphthyridine-7-carboxamide as a promising scaffold with drug-like properties

Article

pubs.acs.org/jmc

Repositioning HIV‑1 Integrase Inhibitors for Cancer Therapeutics:

1,6-Naphthyridine-7-carboxamide as a Promising Scaﬀold with

Drug-like Properties

Li-Fan Zeng,^†,∥Yong Wang,^†,∥Roza Kazemi,^‡Shili Xu,^‡Zhong-Liang Xu,^†Tino W. Sanchez,^‡

Liu-Meng Yang,^§Bikash Debnath,^‡Srinivas Odde,^‡Hua Xie,^†Yong-Tang Zheng,^§Jian Ding,^†

Nouri Neamati,*^,‡and Ya-Qiu Long*^,†

^†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China

^‡Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California,

1985 Zonal Avenue, Los Angeles, California 90089, United States

^§Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province,

Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

ABSTRACT: Among a large number of HIV-1 integrase (IN) inhibitors, the 8-hydroxy-[1,6]naphthyridines (i.e., L-870,810)

were one of the promising class of antiretroviral drugs developed by Merck Laboratories. In spite of its remarkable potency and

eﬃcacy, unfortunately upon completion of phase I clinical studies, development of L-870,810 was halted. Because of its desirable

pharmacological and pharmaceutical properties we were intrigued to design novel analogues of L-870,810 with goals to (1)

improve upon limitations of naphthyridine-7-carboxamides as antiviral agents and (2) to reposition their use as innovative

cytotoxic agents for cancer therapeutics. Herein, we report on the design and synthesis of a series of 1,6-naphthyridine-7-

carboxamides with various substitutions at the 5- and 8-positions. All the new 5-substituted-8-hydroxy-[1,6]naphthyridines were

potent IN inhibitors and the 5-substituted-8-amino-[1,6]naphthyridines were signiﬁcantly cytotoxic. Further optimization of the

5,8-disubstituted-[1,6]naphthyridines with structural variation on 7-carboxamide delivered novel compounds with signiﬁcant

cytotoxicity in a panel of cancer cell lines and eﬀective inhibition against select oncogenic kinases.

are often dropped from the radar screen. One such agent,

L-870,810, showed favorable pharmacological and pharmaceut-

ical properties but was proven not to be an ideal candidate for

antiviral drug development.^6,7Because of our previous success

in repositioning our earlier discovered hydrazide class of IN

inhibitors^8,9we hypothesized that 1,6-naphthyridine-7-carbox-

amides could serve as a promising new scaﬀold for further opti-

mization into cytotoxic compounds useful as anticancer thera-

peutics (Figure 1). In fact, it was previously reported that the

1,6-naphthyridines inhibit FGFR-1 and VEGFR-2 kinases when

substituted at positions 2 and 7 by amino and 3 by aryl groups,

suggesting that various substitutions on this active scaﬀold

could confer diverse biological activities.^10,11Indeed, L-870,

810 was used as an eﬀective prototype to develop a series of

INTRODUCTION

■

Over the past several years, the concept of drug repositioning

(aka drug repurposing, reproﬁling, and indication switch:

ﬁnding a new use for an existing drug) has gained considerable

attention. This concept has also been considered for target repo-

sitioning, and it is expected to produce many more surprising

new drugs for diﬀerent diseases.^1,2Drug repositioning is most

eﬀective when it is applied during all phases of clinical develop-

ment. Historically, this approach was accomplished through

serendipitous discoveries and unintended side eﬀects, however,

computational repositioning strategies are becoming increas-

ingly popular because the beneﬁt of such discoveries can be

enormous.³⁻⁵

Quite often, when a drug fails early phase clinical develop-

ment there is no further appetite to investigate potential oﬀ-

targets and reasons for failure. In cases when a backup com-

pound wins the competition for development, the earlier drugs

Received: May 21, 2012

Published: October 26, 2012

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Figure 1. Repurposing HIV-1 IN inhibitors 5,8-substitutions of 1,6-naphthyridine-7-carboxamide into novel cytotoxic agents useful for preclinical

development for cancer treatment.

structurally novel CGRP antagonists with good pharmacoki-

netic properties.¹²

followed by a Dieckmann cyclization in the presence of sodium

methoxide yielded the core structure 8-hydroxy-1,6-naphthyr-

idine 6. During the Mitsunobu reaction, the use of anhydrous

MgSO₄as an additive accelerated this reaction, probably due to

the contribution to the anhydrous reaction condition that was

beneﬁcial for the subsequent cyclization reaction in a one-

pot reaction. Further bromination at the 5-position and amida-

tion at the 7-position aﬀorded the key intermediate I-1, from

which 5-amino substituted- and 8-amino substituted-1,6-

naphthyridine-7-carboxamides were obtained via nucleophilic

aromatic substitution by various amines. Speciﬁcally, I-1 was

heated to 135 °C with hexane-1,6-diamine or 2-(1H-indol-3-yl)-

ethanamine in DMPU to aﬀord I-7 and I-8, respectively, and

the direct reﬂuxing of I-1 in diﬀerent amino alcohol provided

I-9−I-11. The 5-sulfonamide substituted analogues (I-4−I-6) were

synthesized by reﬂuxing I-1 in pyridine with diﬀerent sulfonamides

in the presence of cuprous oxide. In addition, structurally

diverse analogues with variation on 8-amino substituent (I-12−

I-20, II-1−II-15) were synthesized from the 8-mesylate or

tosylate intermediate (I-2 or I-3) by nucleophilic substitution

of various amines in reﬂuxing THF solution.

The 7-substituted analogues with 4-aminocyclohexylamino

group held constant at the 8-position (III-1−III-16) were syn-

thesized in a similar fashion by incorporating various amines

into the 7-carboxamide position [III-(1−16)-8] followed by instal-

lation of the trans-cyclohexane-1,4-diamino at the 8-position. The

7-carboxylate substituted analog (III-17) was synthesized anal-

ogously by direct amination at the 8-position of the intermediate

10. However, the 7,8-substituents interchanged analogue, III-18,

was synthesized in a diﬀerent reaction sequence, i.e., 8-nucleophilic

aromatic substitution occurred prior to the 7-amidation because the

cyclohexane-1,4-diamino group was suitable for introducing into

the 7-carboxamide position at the ﬁnal step of the synthesis.

New 1,6-Naphthyridine Derivatives are Potent IN

Inhibitors and Display Signiﬁcant Antiviral Activities.

We ﬁrst evaluated the IN inhibitory activity of these synthetic 5,8-

disubstituted-1,6-naphthyridine derivatives. Interestingly, the sub-

stitutions at the 5- and 8-positions were found to play diﬀer-

ent roles in the bioactivity of the 1,6-naphthyridine chemotype.

As shown in Table 1, the 5-substitution on the 8-hydroxy-1,6-

naphthyridine-7-carboxamide just tuned the IN strand transfer

activity (I-1−I-11), while the 8-substitution on the 1,6-naphthyr-

idine-7-carboxamide was critical for retaining the IN inhibitory activity

(compounds I-12 to I-20 vs I-1 to I-11). It is clear that the pre-

sence of the hydroxyl group at the 8-position is essential for strand

transfer activity, which is consistent with the original design of

8-hydroxy-1,6-naphthyridine-7-carboxamide scaﬀold as the bio-

isostere of the aryl diketoacid active structure.²⁸The conversion

of hydroxyl into methylsulfonate (I-2) or 4-methylbenzenesulfo-

nate (I-3) resulted in a remarkable decrease in the potency of

HIV integrase (IN) is an attractive target for the develop-

ment of anti-AIDS drugs because it is an essential enzyme for

the replication of the virus and has no cellular counterparts.¹³

IN catalyzes the integration of viral DNA into the host genome

in a two-step process, 3′-processing and strand transfer. Among

a large number of synthetic IN inhibitors,¹⁴aryl diketoacid and

its bioisosteres are the most developed class that selectively inhibit

the strand transfer step by metal chelation mechanism.¹⁵The

8-hydroxy-7-carboxy-1,6-naphthyridine serves as the bioisostere of

aryl diketoacid with better bioavailability, aﬀording potent in vivo

antiviral potency.⁷Previous 1,6-naphthyridine-based SAR studies

only focused on the substitutions at the 5- and 7-position with

respect to the IN inhibition.^6,7,16−28These later studies led to the

discovery of highly potent IN inhibitors; however, to our knowl-

edge none of these compounds were pursued as anticancer

agents. Other earlier reports included the use of similar 1,6-

naphthyridines as potent and selective inhibitors of human cyto-

megalovirus.²⁹⁻³¹The above-mentioned antitumor 1,6-naphthyr-

idines employed a diﬀerent substitution pattern with ureido at the

2-position, aryl at the 3-position, and amino at the 7-position.

Herein, we designed, synthesized, and provided biological

activities of a novel 1,6-naphthyridine-7-carboxamide-based

focused library with variation on the 5- and 8-substituents

(Figure 1). Our study shows that 1,6-naphthyridine scaﬀold is

especially suitable for optimization of compounds that not only

inhibit the catalytic activities of IN but also display signiﬁcant cyto-

toxicity. This latter property was further pursued to develop novel

agents useful for development as potential anticancer agents.

RESULTS AND DISCUSSION

■

Chemistry. An eﬃcient synthesis to build the 1,6-naphthyridine-

7-carboxyamide-based focused library with variation on 5- and 8-

substitution was established by modifying the reaction con-

ditions reported previously.³²As depicted in Scheme 1, starting

from quinolinic anhydride, treatment with reﬂuxing isopropanol

provided the mono acid 2, which was converted to the corre-

sponding acyl chloride 3 by reﬂuxing with thionyl chloride. An

optimization was made on this transformation due to the trouble-

some workup with a large amount of thionyl chloride and the

hydrolysis of the acyl chloride 3 under moisture produced by

the thionyl chloride. By employing dichloromethane as a sol-

vent and thionyl chloride (1.5 equiv) as a reagent, we performed

the transformation with ease and good yield. Consequently,

reﬂuxing the solution of acid 2 (1 equiv) and thionyl chloride

(1.5 equiv) in dichloromethane with a catalytic amount of

DMF smoothly aﬀorded the acyl chloride 3 that was reduced to

the alcohol 4 with sodium borohydride. Subsequent Mitsunobu

reaction with the phenylsulfonamide of glycine methyl ester

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a

Scheme 1. General Synthetic Route toward the 5,8-Substituted 1,6-Naphthyridine-7-carboxamides

a

Reagents and conditions: (a) i-PrOH, reﬂux, 16 h, 78%; (b) SOCl₂(1.5 equiv), CH₂Cl₂, cat. DMF, reﬂux, 3 h; (c) NaBH₄, THF, 0 °C, 40−50 min,

overall yield 73% for two steps; (d) TsNHCH₂COOCH₃, DEAD, PPh₃, anhydrous MgSO₄, THF, 0 °C, 2 h; (e) NaOMe, CH₃OH, 0 °C to rt, 3 h,

overall yield 65% for two steps; (f) NBS, CH₂Cl₂, rt, 1 h, 85%; (g) 4-ﬂuoro-benzylamine or other amines, toluene, reﬂux, 20 h, 65−98%, or 1N

LiOH, methanol, reﬂux, 5 h, then bis(trichloromethyl)carbonate, DIPEA, aniline, DMF, 0 °C, 2h (for the preparation of (III-15)-8); (h) TsCl or

MsCl, TEA, CH₂Cl₂, 5 h, 90%; (i) NHR₁R₂, THF, reﬂux, 8 h, 50−95%; (j) R′SO₂NRH, pyridine, cuprous oxide, reﬂux, 16 h, 35−65%; (k) alcohol

amine (neat), 130 °C, 9 h (for the preparation of I-9−I-11); RNH₂, DMPU, 135 °C, 48 h (for the preparation of I-7, I-8); (l) tert-butyl (1r,4r)-4-

aminocyclohexylcarbamate, Et₃N, THF, reﬂux, 8 h, 70%; then piperidine, 110 °C, 48 h, 89%; ﬁnally, TFA, DCM, 2h, rt, 95%; (m)1N LiOH, THF, rt,

12 h; then Boc-protected amine, EDCI, HOBT, DIPEA, DCM, 3 h; ﬁnally, TFA, DCM, 2 h.

3′-processing and strand transfer. Furthermore, the replacement of

the hydroxyl group with an amino substitution at the 8-position caused

a substantial loss of IN inhibitory activity (I-12−I-20) that might be

attributed to the loss of the chelation binding motif of the β-keto−

enol, an essential pharmacophore for strand transfer inhibition.³³

When the 8-hydroxy substituent was held constant, the amino

substitution at the 5-position of 8-hydroxy-1,6-naphthyridine-7-

carboxamide was generally beneﬁcial for antiviral activity

(Table 1, I-4−I-11), conferring submicromolar IC₅₀values in

inhibiting the IN strand transfer reaction. Furthermore, all these

5-amino-substituted 8-hydroxy-1,6-naphthyridine-7-carboxamides

displayed potent antiviral activity and a high therapeutic index.

It is worth noting that the structurally simple 2-hydroxy ethyl

amino turned out to be an optimal substituent for both

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the HIV-1 IN inhibitory activity of 1,6-naphthyridine shed new

light on the design and understanding of the 1,6-naphthyridine-

based IN inhibitors, which will be helpful for the evolution of

this scaﬀold into clinically useful antiviral drugs.

Table 1. Inhibition of HIV-1 Integrase Catalytic Activities

and Antiviral Activities of the Lead Compounds

Active Compounds Chelate Divalent Metals on the IN

Active Site. Several active and inactive compounds were docked

on the IN catalytic site using GOLD (Cambridge Crystallographic

Data Center, Cambridge, UK) to explore mechanisms behind

structure−activity relationships. Given the lack of a full length

crystal structure of HIV-1 IN, a homology model was built

based on the X-ray crystal structure of prototype foamy virus

(PFV) intasome (PDB: 3OYA),^34,35a retro-lentivirus belonging

to the same viral genus as HIV. Binding modes of Raltegravir,

I-1, I-4 (L-970,810), I-9, I-12, and I-14 are shown in Figure 2.

Like raltegravir, deprotonated hydroxyl groups of active com-

pounds, I-1, I-4, and I-9, formed strong metal chelating inter-

actions with two Mg²⁺ions along with Asp64, Asp116, and

Glu152. Phenyl groups of these compounds formed stack-

ing interactions with DC16 from DNA. Sulfonyl oxygen atoms

of I-4 forms H-bonds with Asn117. Thus, these compounds

potentially displace the reactive 3′OH of DNA from the active

site to cause deactivation of the intasome. Although I-12 docked

in a similar fashion as I-1, the N-methyl group of I-12 created

steric hindrance into the binding site to generate weaker inter-

actions with Mg²⁺ions. Another inactive compound, I-14, was

docked completely in the reverse position. Substitution with

benzyl on the NH group blocked chelating interaction with

Mg²⁺ions. These observations are further comﬁrmed by docking

scores. The GOLD score of these active compounds are around

100, while that of inactive compounds are less than 70.

Repositioning of Novel Derivatives for Anticancer

Properties. Because the 5,8-substitutions were shown to steer

the biological proﬁle of 1,6-naphthyridine-7-carboxamide scaﬀold,

we were interested to test the 5,8-substituted 1,6-naphthyridine-

7-carboxamides in a panel of cancer cell lines to better under-

stand their biological activities. Interestingly, some of the 8-

aliphatic diamino substituted analogues that were totally inactive

in our IN assay displayed distinct cytotoxicity (Table 2, I-18,

I-20). However, the 1,6-naphthyridine-7-carboxamides bearing

aryl or alkyl substituted monoamino groups at the 8-position

were not cytotoxic (i.e., I-13−I-17). On the other hand, the

5-sulfoxamide substituted 8-hydroxy-1,6-naphthyridines displayed

low micromolar cytotoxicity against the tested tumor cell lines

(i.e., I-4-6), whereas the 5-alkylamino substituted counterparts

were inactive in inhibiting the cell growth even at the concen-

tration of 20 μM (I-8-11). Because of these encouraging pre-

liminary observations, further SAR and structural optimization

were conducted around the 8-amino substituted-1,6-naphthyr-

idine-7-carboxamide scaﬀold to select compounds for repurpos-

ing in our cancer program.

Our initial cytotoxicity data indicated that the diamino struc-

ture was an important functionality at the 8-position of 1,6-

naphthyridine-7-carboxamide for maintaining antiproliferative

activity (exempliﬁed by I-18, I-20 vs I-12−I-17). Therefore,

during the lead optimization campaign, the open chain alkyl

diamino, the aryl diamino, and the aliphatic cyclic diamino groups

were examined as the 8-substituent in the second generation

of the cytotoxic 1,6-naphthyridine series (Figure 3). As shown in

Table 3, the aliphatic cyclic diamino structures generally exhibited

signiﬁcant antiproliferative activity against a panel of cancer cell

lines with the 4-aminopiperidine and trans-1,4-diaminocyclohex-

ane being of special interest (II-10, II-13). When the terminal

amino group was protected by an acyl group (II-3, II-5, II-9, II-11),

a

IC₅₀: the drug concentration that produced 50% inhibition of the

b

enzyme function. EC₅₀: eﬀective concentration required to protect

C8166 cells against the cytopathogenicity of HIV-1 by 50%. CC₅₀:

cytotoxic concentration required to reduce C8166 cell proliferation by

50% tested by MTT method. TI: therapeutic index is a ratio of the

c

d

e

CC₅₀value/EC₅₀value. ND: not determined.

3′-processing and the strand transfer inhibition comparable with

the sulfonamide substituent in L-870,810 (I-9−I-11 vs I-4).

Moreover, the chirality of the 2-methyl on the 1-hydroxypropan-

2-yl-amino substituent had a subtle eﬀect on the antiviral potency

with S-conﬁguration being favored (I-10 vs I-9, I-11). How-

ever, the sulfonamide-substituted analogues displayed an overall

superior antiviral eﬀect in terms of the EC₅₀value and therapeutic

index. Our investigation on the eﬀect of the 5,8-substitution on

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Figure 2. Binding modes of Raltegravir, I-1, I-4, I-9, I-12, and I-14 on the modeled IN active site based on the X-ray crystal structure of prototype

foamy virus (PFV) intasome (PDB: 3OYA).^34,35

the loss of the free N−H resulted in decreased potency. This

suggests that the amino substituent bearing a free terminal

amino group at the 8-position is important for the cytotoxicity

of the 1,6-naphthyridine scaﬀold. Setting trans-1,4-diamino-

cyclohexane as the privileged structure at the 8-position, the

optimal substituent at 5-position was simply explored. The re-

placement of the original bromo group by a hydrogen (II-1),

piperidine (II-14), or sultam (II-15) at the 5-position all led to

a loss of cytotoxicity to a similar extent, although the amino sub-

stituent at the 5-position was beneﬁcial for the IN inhibition.

Therefore, the (1r,4r)-4-aminocyclohexylamino and the bromo

groups were maintained at positions 8 and 5, respectively, for the

following SAR study of the third generation series.

As antiproliferative agents, four representative compounds I-

18, II-6, II-10, and II-13, were selected for colony formation

assay. Encouragingly, these cellular active 8-amino-1,6-naph-

thyridine-7-carboxamides signiﬁcantly inhibited the colony forma-

tion of highly invasive breast cancer cells MDA-MB-435 (Figure 4),

consistent with their antiproliferative activity. Especially, the most

potent compound II-13 signiﬁcantly blocked the colony forma-

tion of MDA-MB-435 cells in a dose-dependent manner, with a

submicromolar IC₅₀value.

Starting from the evolved compound II-13, further structural

optimization eﬀort was focused on the 7-carboxamide sub-

stitution of the 1,6-naphthyridine framework while the (1r,4r)-

4-aminocyclohexylamino moiety at the 8-position was held con-

stant. As shown in Table 4, the third generation of cytotoxic

8-((4-aminocyclohexyl)amino)-1,6-naphthyridine-7-carboxamide

series were synthesized and assessed with structural variation

on the 7-carboxamide portion. The original 4-ﬂuorobenzylamino

group was examined with various substitutions on the phenyl ring

or the methylene bridge (III-1−III-10). Furthermore, the eﬀect

of heteroaromatic or aliphatic cyclic amine or the methoxy replace-

ment at 7-carboxamide (III-11−III-13, III-16, III-17) on cyto-

toxicity was investigated. The chain length of the arylamino

moiety was investigated as well (i.e., III-14, III-15 vs II-13).

Interestingly, this scaﬀold well tolerated the structural variation

on the arylamino group at the 7-carboxamide position. Except

for the rigid (S)-1-(4-ﬂuorophenyl)ethanamino, the furan-2-

ylmethanamino, cyclohexanamino, and methoxy substitutions

(III-4, III-11, III-16, III-17) that were detrimental to the anti-

proliferative activity, all other structural displacement exerted

slight impact on the inhibitory potency against various tumor

cell lines. The electron-donating group and halogen were favored

substitutions on the benzyl ring. In addition, the interchange of

8-((4-aminocyclohexyl)amino and 7-N-(4-ﬂuorobenzyl) carbox-

amide (III-18 vs II-13) marginally decreased cytotoxicity. It is clear

that N-(halogen substituted benzyl)carboxamide at 7-position is an

important motif for cytotoxicity of 8-((4-aminocyclohexyl)amino-

1,6-naphthyridine chemotype.

Select 8-Amino-1,6-naphthyridine-7-carboxamides Arrest

Cells in G₀/G₁and Show Multiple Kinase Inhibitory Activity.

8-Amino-1,6-naphthyridine-7-carboxamide represents a new class

of antiproliferative agents and warrants further investigation.

Compounds II-6, II-10, and II-13 completely blocked colony

formation of highly aggressive MDA-MB-435 cancer cells at

10 μM (Figure 4A). The IC₅₀value range for II-13 in the same

cell line was between 0.1 and 1 μM (Figure 4B). I-18 blocked

cells in G₀/G₁as early as 12 h, and this block persisted till 48 h

with 61% of cells in G₀/G₁versus 36% in control cells (Figure 4C).

Similarly, II-13 arrested the cells in G₀/G₁(Figure 4D).

So far, starting from L-870810, we established a focused

library based on the 1,6-naphthyridine core structure with varia-

tions on 5,7,8-substitutions and discovered a new class of anti-

proliferative agents. Our initial target identiﬁcation eﬀort was

directed at the tyrosine kinases important for cancer progression.

Some representative 5,8-substituted-1,6-naphthyridine-7-car-

boxamides that were active in our cytotoxicity assays were

chosen to test their inhibitory activities against in-house tyro-

sine kinases including EGFR, erbB-2, c-Src, and KDR (Table 5).

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Table 2. Cytotoxicity of the First Generation 8-Substituted-

1,6-naphthyridine-7-carboxamide Derivatives

Table 3. Cytotoxicity of the Second Generation 8-Amino

Substituted-1,6-naphthyridine-7-carboxamide Derivatives

with Variation on 8-Amino Substitution

a

IC₅₀: Inhibitory concentrations at 50% in μM were measured by

MTT assay after 72 h continuous drug exposure. Standard deviations

are from three independent experiments run in triplicate. Entries with

two values are from two independent experiments run in triplicate.

a

IC₅₀: Inhibitory concentrations at 50% in μM were measured by

MTT assay after 72 h continuous drug exposure. Standard deviations

are from three independent experiments run in triplicates. ND: not

determined.

b

ND: not determined.

naphthyridine-7-carboxamide displayed eﬀective inhibition

against the EGFR, c-Src, and/or KDR kinases at the concen-

tration of 10 μM (III-2, III-8, III-9). Furthermore, the sub-

stitution at the 7-carboxamide was found to signiﬁcantly aﬀect

The 8-hydroxy-5-amino substituted analogues (I-4, I-7) were

inactive in the kinase assay, whereas some 8-((1,4)-trans-4-

aminocyclohexyl)amino)-5-bromo-N-(substituted-benzyl)-1,6-

Figure 3. Structural optimization of hit compounds for their cytotoxic property with focus on amino substituent at the position 8.

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Figure 4. 8-Amino derivatives of 1,6-naphthyridine-7-carboxamide decrease cell proliferation and arrest cells in G₀/G₁phase. Colony formation assay

was performed using highly invasive MDA-MB-435 cells treated continuously for 7 days with I-18, II-6, II-10, and II-13, at 10 μM (A) and at various

doses with II-13 (B). DNA content was analyzed in propidium iodide stained MDA-MB-435. Cells were treated with 2 × IC₅₀values of I-18 (C) and

II-13 (D) at indicated time. Dark-blue bars represent the G₀/G₁fraction, light-cyan bars represent cells in S phase, and brown bars correspond to

cells in the G₂/M phase of the cell cycle.

the kinase inhibitory activity although these 7-variously sub-

stituted analogues all displayed marked antiproliferative eﬀect

on the tumor cell lines tested. The phenethylamino or anilinyl

or 1,4-trans-cyclohexyldiamino substitution at the 7-carboxamide

portion resulted in no inhibition against these kinases (III-14, III-

15, III-18). Therefore, the 8-((1,4)-trans-4-aminocyclohexyl)-

amino)-5-bromo-N-(substituted-benzyl)-1,6-naphthyridine-7-

carboxamides are a new class of pan-tyrosine kinase inhibitors.

The chloro or alkoxy substituent at 4′- or 2′-position of the benzyl

group was beneﬁcial for the kinase inhibition, consistent with the

antiproliferative activity in the tumor cells.

On the basis of the promising kinase inhibitory activity results,

further kinase proﬁling assays were conducted against 31 oncology

kinases (Figure 5).³⁶Three compounds, III-2, III-8, and III-9,

which were active in the preliminary tyrosine kinase screening,

were selected for the kinase selectivity assay. Interestingly, the 2′-

chloro and 2′,4′-dichloro substituted N-benzyl-1,6-naphthyridine-

7-carboxamide derivatives (III-8, III-9) displayed signiﬁcant inhi-

bition against several oncogenic kinases such as IGF-1R, FGFR1,

Aurora-A, LCK, and Abl(T315I) at 10 μM, whereas the 4′-

methoxy substituted analogue (III-2) showed moderate inhibition

against KDR, Met, FGFR1, Tie2, and IGF-1R kinases, as shown in

Figure 5. Structural variation on the phenyl ring at 7-carboxamide

was responsible for kinase selectivity and inhibitory potency.

This suggests that the 8-((1,4)-trans-4-aminocyclohexyl)amino)-

1,6-naphthyridine-7-carboxamide is an attractive scaﬀold for further

structural optimization and development into targeted cancer

therapeutics.

CONCLUSIONS

■

Starting from a well-known 8-hydroxy-[1,6]naphthyridine scaﬀold,

we successfully identiﬁed novel IN inhibitors with signiﬁcant anti-

viral activities. Furthermore, by varying the substituent at the

8-position, we showed for the ﬁrst time that a subset of this class of

compounds is quite useful for repurposing as innovative cytotoxic

agents. The lead compounds disclosed in this study show sig-

niﬁcant cytotoxicity in a panel of cancer cell lines, arrest cells in

G₀/G₁, and inhibit select kinases. Several of these compounds are

interesting and warrant further preclinical development, and the

results presented in this study validate our previous studies in

repurposing select IN inhibitors for anticancer development. In-

depth mechanistic studies are currently in progress in our laboratory

and will be published in due course.

EXPERIMENTAL SECTION

■

Materials, Chemicals, and Enzymes. All compounds were

dissolved in DMSO, and the stock solutions were stored at −20 °C.

The γ-[³²P]ATP was purchased from either Amersham Biosciences or

ICN. The expression system for the wild-type IN was a generous gift

of Dr. Robert Craigie, Laboratory of Molecular Biology, NIDDK, NIH

(Bethesda, MD).

Preparation of Oligonucleotide Substrates. The oligonucleo-

tides 21top, 5′-GTGTGGAAAATCTCTAGCAGT-3′, and 21bot,

5′-ACTGCTAGAGATTTTCCACAC-3′, were purchased from Norris

Cancer Center Microsequencing Core Facility (University of Southern

California) and puriﬁed by UV shadowing on polyacrylamide gel. To

analyze the extent of 3′-processing and strand transfer using 5′-end-

labeled substrates, 21top was 5′-end-labeled using T₄polynucleotide

kinase (Epicenter, Madison, WI) and γ-[³²P]ATP (Amersham Biosciences

or ICN). The kinase was heat-inactivated, and 21bot was added in 1.5 M

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Table 4. Cytotoxicity of the Third Generation 8-Amino

Substituted-1,6-naphthyridine-7-carboxamide Derivatives

with Variation on the 7-Carboxamide Moiety

Table 5. Inhibition of EGFR, ErbB2, Src, and KDR Kinase

Activities by Selected 5,8-Substituted-1,6-naphthyridine-7-

carboxamide Derivatives

a

% kinase inhibition at 10 μM for each compound

compd

I-4

EGFR

ErbB2

Src

KDR

b

1

30

10

0

8

11

ND

1

13

23

15

17

18

7

ND

I-7

ND

10

0

II-1

II-4

II-6

0

II-7

7

8

II-10

II-12

II-13

III-1

III-2

III-3

III-6

III-8

III-9

III-14

III-15

III-18

Lapatinib

5

1

9

0

ND

7

29

13

ND

76

ND

70

90

ND

0

4

21

20

51

0

ND

2

0

ND

34

41

ND

22

59

68

0

13

0

5

3

c

96

92

c

PP2

94

c

SU11248

76

a

The inhibitory rate of tyrosine kinase in the presence of the ﬁxed

concentration of the drug was detected using enzyme-linked-immu-

b

nosorbent assay (ELISA) in the presence of 10 μM of ATP. ND: not

c

determined. The reference compound for the kinase assay: Lapatinib

for EGFR and ErbB-2, PP2 for Src, SU11248 for KDR, tested at 10 μM.

(USA Scientiﬁc, Ocala, FL) to separate annealed double-stranded

oligonucleotide from unincorporated material.

IN Assays. To determine the extent of 3′-processing and strand

transfer, wild-type IN was preincubated at a ﬁnal concentration of

200 nM with the inhibitor in reaction buﬀer (50 mM NaCl, 1 mM

HEPES, pH 7.5, 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol

(w/v), 7.5 mM MnCl₂, 0.1 mg/mL bovine serum albumin, 10 mM

2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 mM MOPS,

pH 7.2) at 30 °C for 30 min. Then, a 20 nM concentration of the

5′-end ³²P-labeled linear oligonucleotide substrate was added, and incu-

bation was continued for an additional 1 h. Reactions were quenched

by the addition of half the sample volume (8 μL) of loading dye (98%

deionized formamide, 10 mM EDTA, 0.025% xylene cyanol, and

0.025% bromophenol blue). An aliquot was electrophoresed on a

denaturing 20% polyacrylamide gel (0.09 M tris-borate, pH 8.3, 2 mM

EDTA, 20% acrylamide, and 8 M urea).

Gels were dried, exposed in a PhosphorImager cassette, and analyzed

using a Typhoon 8610 variable mode imager (Amersham Biosciences,

Piscataway, NJ) and quantitated using ImageQuant 5.2. The percent

inhibition (% I) was calculated using the following equation

%I = 100 × [1 − (D − C)/(N − C)]

(1)

where C, N, and D are the fractions of 21-mer substrate converted to

19-mer (3′-processing product) or strand transfer products for DNA

alone, DNA plus IN, and IN plus drug, respectively. The IC₅₀values

were determined by plotting the logarithm of drug concentration

versus percent inhibition to obtain the concentration that produced

50% inhibition.

a

IC₅₀: inhibitory concentrations at 50% in μM were measured by MTT

assay after 72 h continuous drug exposure. Standard deviations are from

three independent experiments run in triplicate. ND: not determined.

Antiviral Assay.³⁷C8166 cells were maintained in RPMI-1640

supplemented with 10% heat-inactivated fetal calf serum (Gibco). The

cells used in all experiments were in log-phase growth.

b

excess. The mixture was heated at 95 °C, allowed to cool slowly

to room temperature, and processed through a spin 25 minicolumn

Syncytium Reduction Assay. In the presence of 100 μL of various

concentrations of compounds, C8166 cells (4 × 10⁵mL⁻¹) were

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bromide (MTT) method. Brieﬂy, cells were seeded on a 96-well microplate

in the absence or presence of various concentrations of compounds in

triplicate and incubated at 37 °C in a humid atmosphere of 5% CO₂for

72 h. The supernatants were discarded, and MTT reagent (5 mg/mL in

PBS) was added to each well and then incubated for 4 h and 100 μL of

50% DMF-20% SDS was added. After the formazan was dissolved com-

pletely, the plates were read on a Bio-Tek EL x 800 ELISA reader at

595/630 nm. The cytotoxic concentration that caused the reduction of

viable cells by 50% (CC₅₀) was calculated from dose−response curve. TI

(therapeutic index) value is a ratio of the CC₅₀value/EC₅₀value.

Binding Mechanism Prediction. Given the lack of a full length

crystal structure of HIV-1 IN, a homology model was built based on

the X-ray crystal structure of prototype foamy virus (PFV) intasome

(PDB: 3OYA),^34,35a retro-lentivirus belonging to the same viral genus

as HIV. Select compounds were docked in the catalytic binding site of

the homology model of IN reported earlier³⁸using GOLD (genetic opti-

mization for ligand docking) software package, version 4.0 (Cambridge

Crystallographic Data Centre, Cambridge, U.K.). GOLD uses a genetic

algorithm (GA) to explore the conformational space of a compound

inside the binding site of a protein.^39,40The active site was deﬁned as

the collection of protein residues with a sphere of 15 Å radius. Before

docking diﬀerent possible stereoisomers, ionized forms and con-

formations of ligands were prepared by LigPrep (Schrodinger, LLC).⁴¹

Ionization option, Epik,⁴²was used to ionize ligands at pH 7.0 0.2.

Docking studies were performed using the standard default settings

with 100 GA runs on each molecule. For each of the 100 independent

GA runs, a maximum of 100000 operations were performed on a set of

ﬁve groups with a population of 100 individuals. With respect to ligand

ﬂexibility special care has been taken by including options such as

ﬂipping of ring corners, amides, pyramidal nitrogens, secondary and

tertiary amines, and rotation of carboxylate groups, as well as torsion

angle distribution and postprocess rotatable bonds as default. The

annealing parameters were used as default cutoﬀ values of 3.0 Å for

hydrogen bonds and 4.0 Å for van der Waals interactions. Hydro-

phobic ﬁtting points were calculated to facilitate the correct starting

orientation of the compound for docking by placing the hydrophobic

atoms appropriately in the corresponding areas of the active site.

When the top three solutions attained root-mean-square deviation

(rmsd) values within 1.5 Å, docking was terminated. GOLD-Score, a

scoring function of the software, is a dimensionless ﬁtness value that

takes into account the intra- and intermolecular hydrogen bond-

ing interaction energy, van der Waals energy, and ligand torsion energy.^39,40

Cell Culture. MDA-MB-435 and MCF7 breast cancer, LNCaP

prostate cancer, and HT29 colon cancer cell lines were obtained from

Dr. Alan Epstein (USC) or purchased from the American Type Cell

Culture (Manassas, VA). HCT116 p53^+/+and HCT116 p53^−/−colon

cancer cell lines were kindly provided by Dr. Bert Vogelstein (Johns

Hopkins University, Baltimore, MD). Cell lines were maintained in the

appropriate growth media (DMEM for MDA-MB-435, MCF7 and

LNCaP, RPMI for the HT29 and HCT116 cell lines) containing 10%

heat-inactivated fetal bovine serum and supplemented with 2 mM

L-gutamine at 37 °C in a humidiﬁed atmosphere of 5% CO₂. For sub-

culture and experiments, cells were washed with 1× PBS, detached

using 0.025% Trypsin-EDTA (Cellgro, Mediatech, Mannassas, VA),

collected in growth media, and centrifuged. All experiments were per-

formed in growth media using subconﬂuent cells in the exponential

growth phase. For use in tissue culture experiments, compounds were

prepared at 10 mM concentration in sterile dimethylsulfoxide (DMSO)

(EMD Chemicals, Gibbstown, NJ) and stored at −20 °C when not

in use.

Figure 5. Inhibition rate of compounds III-2, III-8, and III-9 against

31 oncogenic kinases at the drug concentration of 10 μM and at the

K_mvalue for ATP for each kinase, by using KinaseProﬁler techniques.

All data are expressed as mean values of two independent experiments.

Cytotoxicity Assay. Cytotoxicity was assessed by MTT assay as

previously described.⁴³Cells were seeded in 96-well tissue culture treated

dishes and allowed to adhere overnight. Cells were subsequently treated

with a continuous exposure to drugs for 72 h. An MTT solution was

added to each well to give a ﬁnal concentration of 0.3 mg/mL MTT.

Cells were incubated with MTT for 3−4 h at 37 °C. After removal of the

supernatant, DMSO was added and the absorbance was read at 570 nm.

All assays were done in triplicate. The IC₅₀was then determined for each

drug from a plot of log (drug concentration) versus percentage of cell kill.

infected with HIV-1_IIIBat a multiplicity of infection (MOI) of 0.06.

The ﬁnal volume per well was 200 μL. AZT was used as a positive

control. After 3 days of culture, the cytopathic eﬀect (CPE) was mea-

sured by counting the number of syncytia (multinucleated giant cell)

in each well under an inverted microscope. Percentage inhibition of

syncytial cell number in treated culture to that in infected control

culture and 50% eﬀective concentration (EC₅₀) was calculated.

Cytotoxicity Assay. : The cellular toxicity of compounds on C8166

cells was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium

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Colony Formation Assay. Colony formation assay was performed

as previously described to further assess drug toxicity.⁴⁴Brieﬂy, cells

were seeded in 6-well tissue culture dishes at a density of 500 cells per

well in growth media and allowed to adhere overnight. Cells were

subsequently treated with varying concentrations of compound for

24 h. Following treatment, monolayers were washed with 1× PBS and

incubated in growth media for a period of 7−10 days, allowing suﬃ-

cient time for colonies to form in control wells. To visualize the extent

of colony formation, cells were ﬁxed and stained in a 2% solution of

crystal violet containing 1% glutaraldehyde. Excess stain was removed

through multiple washes in distilled water and allowed to air-dry. Stained

plates were imaged using Quantity One software running on the VersaDoc

imaging platform (BioRAD).

Cell Cycle Analysis. Cells were seeded in 100 mm tissue culture

dishes at a density of 1 × 10⁶cells/plate in growth media and allowed

to adhere overnight. The following day cells were treated with 2 × IC₅₀

concentrations of tested compounds or DMSO alone as vehicle

control for 12−48 h. After treatment, cells were detached with trypsin,

and both media and cells were collected by centrifugation. Cells

were washed and resuspended in 1× PBS prior to ﬁxation in ethanol

overnight at −20 °C. Fixed cells were treated with 10 μg/mL RNase A

(Sigma-Aldrich, St. Louis, MO) and stained in a 50 μg/mL solution of

propidium iodide (Sigma-Aldrich, St. Louis, MO). DNA content was

determined by ﬂow cytometry using the BD LSR II (BD Biosciences,

San Jose, CA) equipped with a 488 nM Sapphire argon-ion laser and

PE emission detector.

Solvent system I: A, 0.05% TFA in water; B, methanol, gradient

30−90% of solvent B in A over 20 min at 2 mL/min;

Solvent system II: A, 0.05% TFA in water; C, 0.05% TFA in 90%

acetonitrile in water, gradient 30−90% C in A over 20 min at 2 mL/min.

Pyridine-2,3-dicarboxylic Acid 2-Isopropyl Ester (2). A

suspension of quinolinic anhydride (48.1 g, 0.32 mol) in 100 mL of

2-propanol was warmed to reﬂux for 16 h, cooled to room tempera-

ture, and concentrated in vacuo. The resulting yellow solid was recrys-

tallized from ethyl acetate to give the desired monoester 2 as a white

solid (52.7 g, 78.3%); mp 140−141 °C. ¹H NMR (CDCl₃, 300 MHz):

δ 8.87 (d, J = 4.2 Hz, 1H), 8.35 (d, J = 7.8 Hz, 1H), 7.55 (dd, J = 5.1,

8.1 Hz, 1 H), 5.36 (septet, J = 6.3 Hz, 1H), 1.41 (d, J = 6.3 Hz, 6H).

3-Hydroxymethyl-pyridine-2-carboxylic Acid Isopropyl

Ester (4). To a solution of 2 (5.0 g, 23.9 mmol) in 25 mL of DCM

was added thionyl chloride (2.5 mL, 35.85 mmol) and a catalytic

amount of DMF (0.5 mL, 0.53 mmol). The suspension mixture

was heated to reﬂux for 3 h and then concentrated in vacuum. The

remaining thionyl chloride was removed by dissolving the residue in

THF and then concentrating the resultant solution. This process was

repeated twice. The resulting chloride was dissolved in 20 mL of THF

and cooled to 0 °C. Sodium borohydride (1.2 g, 31.58 mmol) was

added, and the reaction mixture was stirred at 0 °C for 40−50 min.

The reaction solution was carefully poured into ice (foaming) and

extracted with CH₂C1₂. The organic phase was dried over Na₂SO₄and

concentrated in vacuum. Puriﬁcation by ﬂash chromatography gave

1

4 as a yellow oil. (3.4 g, overall yield 73% for two steps). H NMR

(CDC1₃, 300 MHz) δ 8.69 (dd, J = 1.5, 4.7 Hz, 1H), 7.88 (dd, J = 1.5,

7.7 Hz, 1H), 7.46 (dd, J = 4.7, 7.8 Hz, 1 H), 5.35 (septet, J = 6.4 Hz,

1H), 4.81 (m, 2 H), 1.45 (d, J = 6.3 Hz, 6H). EI-MS m/z: 195 (M)⁺.

Isopropyl 3-((N-(2-Methoxy-2-oxoethyl)-4-methylphenylsul-

fonamido)methyl)picolinate (5). Isopropyl 3-(hydroxymethyl)

pyridine-2-carboxylate (1.734 g, 8.89 mmol), methyl 2-(tosylamino)acetate

(2.163 g, 8.89 mmol), anhydrous MgSO₄(1.066 g, 8.89 mmol), and

triphenylphosphine (3.499 g, 13.338 mmol) were dissolved in dry THF

(100 mL) and cooled to 0 °C under nitrogen. DEAD (2.165 mL,

13.338 mmol) in 10 mL of dry THF was added dropwise. The ice bath

was removed, and the solution was stirred for 2 h at rt followed by

concentration in vacuo. The red−orange oil was directly used for the

next reaction without further puriﬁcation.

Tyrosine Kinase Assay. The activity of tyrosine kinases were

detected using enzyme-linked-immunosorbent assay (ELISA). Brieﬂy,

20 μg/mL Poly(Glu,Tyr)_4:1(Sigma, St. Louis, MO) was precoated in

96-well ELISA plates as substrate. Each well was treated with 50 μL of

10 μmol/L ATP solution, which was diluted in kinase reaction buﬀer

(50 mM HEPES pH 7.4, 20 mM MgCl₂, 0.1 mM MnCl₂, 0.2 mM

Na₃VO₄, 1 mM DTT). Then 1 μL of various concentrations of test

compounds or reference compound dissolved in DMSO were added

to each reaction well. Experiments at each concentration were per-

formed in duplicate. The reaction was initiated by adding tyrosine

kinase diluted in kinase reaction buﬀer. After incubation at 37 °C for

60 min, the wells were washed three times with phosphate buﬀered

saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 μL of anti-

phosphotyrosine (PY99) antibody (1:1000, Santa Cruz Biotechnology,

Santa Cruz, CA) diluted in T-PBS containing 5 mg/mL BSA was added,

and the plate was incubated at 37 °C for 30 min. After the plate was

washed three times, 100 μL of horseradish peroxidase-conjugated goat

antimouse IgG (1:2000, Calbiochem, SanDiego, CA) diluted in T-PBS

containing 5 mg/mL BSA was added and the plate was incubated

at 37 °C for 30 min. The plate was washed, and then 100 μL of cit-

rate buﬀer (0.1 M, pH5.5) containing 0.03% H₂O₂and 2 mg/mL

o-phenylenediamine was added and samples were incubated at room

temperature until color emerged. The reaction was terminated by

adding 50 μL of 2 M H₂SO₄, and the plate was read using a multiwell

spectrophotometer (VERSAmax, Molecular Devices, Sunnyvale, CA,

USA) at 492 nm. The inhibitory rate (%) was calculated with the

formula: [1 −(A₄₉₂treated/A₄₉₂control)] × 100%.

Methyl 8-Hydroxy-1,6-naphthyridine-7-carboxylate (6). To

the solution of the crude product 5 (8.89 mmol) in dry methanol

(50 mL) at 0 °C was added sodium methoxide (1.681 g, 31.123 mmol)

slowly. After addition, the reaction mixture was stirred at rt for 3 h. The

solvent was removed in vacuo to obtain red−orange oil, which was

partitioned between water (20 mL) and ethyl acetate (20 mL). The

organic layer was back-extracted with saturated sodium bicarbonate

solution. The pH of the aqueous layer was adjusted to 7, and then the

aqueous layer was extracted with methylene chloride. The organic

layer was dried over sodium sulfate. Puriﬁcation by ﬂash chromatog-

raphy aﬀorded 6 as an oﬀ-white solid. (1.19 g, 65% two steps); mp

1

179−180 °C. H NMR (CDC1₃, 300 MHz): δ 11.79 (s, 1H), 9.20

(s, 1H), 8.85 (s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 7.71 (dd, J = 4.1, 8.2 Hz,

1H), 4.12 (s, 3H).

Methyl 5-Bromo-8-hydroxy-1,6-naphthyridine-7-carboxy-

late (7). To a solution of 6 (34 mg, 0.167 mmol) in CH₂Cl₂(1 mL)

at room temperature was added NBS (30 mg, 0.167 mmol). The reac-

tion mixture was stirred for 1 h. The solids were collected by ﬁltra-

tion and dried in vacuo to aﬀord 7 (30 mg, yield 64%) as a white solid.

¹H NMR (DMSO-d₆, 300 MHz): δ 9.26 (dd, J = 1.5, 4.2 Hz, 1H), 8.59

(dd, J = 1.6, 8.4 Hz, 1H), 8.00 (dd, J = 4.2, 8.4 Hz, 1 H), 3.94 (s, 3 H).

5-Bromo-8-hydroxy-N-(4-ﬂuorobenzyl)-1,6-naphthyridine-

7-carboxamide (I-1). A slurry of 7 (25 mg, 0.0883 mmol) and

4-ﬂuorobenzylamine (12 mg, 0.0883 mmol) in toluene (2 mL) were

heated at reﬂux for 20 h. Upon cooling to rt, the resulting solids were

collected by ﬁltration and washed with methanol (0.5 mL) to aﬀord

I-1 as a white solid (yield 78%); mp 178−180 °C. ¹H NMR (300 MHz,

CDC1₃): δ 13.31 (s, 1H), 9.20 (dd, J = 1.4, 4.2 Hz, 1H), 8.54 (dd,

J = 1.4, 8.8 Hz, 1H), 8.17 (m, 1H), 7.74 (dd, J = 4.4, 8.6 Hz, 1H),

7.38 (dd, J = 5.4, 8.4 Hz, 2H), 7.07 (t, J = 8.4 Hz, 2H), 4.67 (d, J = 6.3

Hz, 2H). EI-MS m/z: 375 (M)⁺, 377 (M + 2)⁺. Anal. Calcd for

General Synthetic Methods. Unless otherwise stated, the ¹H

NMR spectra were recorded on a Varian 300 MHz or 400 MHz spec-

trometer and ¹³C NMR spectra on a Varian Mercury 100 MHz spec-

trometer. The data are reported in parts per million relative to TMS

and referenced to the solvent. Melting points (uncorrected) were

determined on a Buchi-510 capillary apparatus. The MS and HRMS

(ESI) spectra were obtained on an APEXIII 7.0 T FTMS mass spec-

trometer. Rotate power was detected by P-1030 (A012360639) auto-

matic polarimeter. The ﬂash column chromatography was performed

on silica gel H (10−40 μm). Anhydrous solvents were obtained by

standard procedures. The purity of all target compounds was ≥95%

determined by elemental analysis or analytical HPLC. Elemental ana-

lyses were obtained using Elementar Vario EL II elemental analyzer.

Analytical HPLC was performed on JASCO LC-1500 with Vydac

C18 reversed-phase column (10 mm × 250 mm) and UV detector at

254 nm, using two solvent systems as indicated below.

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C₁₆H₁₁BrFN₃O₂: C 51.08, H 2.95, N 11.17. Found: C 51.45, H 3.05,

N 11.03.

to 35:65 over 30 min, with 0.05% TFA, UV detector, 254 nm) gave I-7

as a red solid, yield 30%; mp 88−91 °C. ¹H NMR (300 MHz, CDC1₃)

δ 12.31 (s, 1H), 9.06 (d, J = 4.2 Hz, 1H), 8.27 (m, 1H), 8.25 (d,

J = 8.3 Hz, 1H), 8.15 (s, 1H), 7.51 (dd, J = 5.4, 8.6 Hz, 1H), 7.35

(m, 2H), 7.04 (t, J = 8.6 Hz, 2H), 4.65 (d, J = 6.2 Hz, 2H). EI-MS

m/z: 439 (M)⁺, ESI-MS m/z: 440 (M + 1)⁺. Analytical HPLC t_R=

5.650 min, 100% (solvent system I); t_R= 13.500 min, 98.36% (solvent

system II).

5-(2-(2H-Indol-3-yl)ethylamino)-N-(4-ﬂuorobenzyl)-8-hy-

droxy-1,6-naphthyridine-7-carboxamide (I-8). 2-(1H-Indol-3-yl)-

ethanamine (240 mg, 1.5 mmol) was added to a solution of I-1 (112.5 mg,

0.3 mmol) in DMPU (5 mL). After heating at 135 °C for 48 h, the solu-

tion was cooled to room temperature, diluted with dichloromethane,

and then washed by saturated aqueous NH₄Cl and brine. Usual work-

up followed by puriﬁcation by ﬂash chromatography on silica gel

(DCM:methanol = 100:1) aﬀorded I-8 as a red solid, yield 38%; mp

101−103 °C. ¹H NMR (CDCl₃, 300 MHz) δ 12.35 (s, 1H), 9.05 (dd,

1H, J = 1.5, 4.2 Hz), 8.64 (s, 1H), 8.25 (m, 1H), 7.96 (d, 1H, J =

8.4 Hz), 7.60 (d, 1H, J = 7.8 Hz), 7.45−7.30 (m, 4H), 7.17−6.98

(m, 5H), 5.25 (brs, 1H), 3.79 (m, 2H), 3.16 (t, 2H, J = 6.9 Hz). EI-MS

m/z: 455 (M)⁺. Anal. HPLC t_R= 19.692 min, 100% (solvent system I);

t_R= 13.667 min, 98.97% (solvent system II).

N-(4-Fluorobenzyl)-8-hydroxy-5-(2-hydroxyethylamino)-1,6-

naphthyridine-7-carboxamide (I-9). I-1 (37.5 mg, 0.13 mmol) was

added to 2-aminoethanol, then heated to 130 °C for 9 h, and the

solution was cooled to room temperature, diluted with dichloro-

methane, and then washed by saturated aqueous NH₄Cl and brine and

dried over Na₂SO₄, and then the solution was removed under vacuum.

The residue was puriﬁed by RP-HPLC (Vydac C18 column (10 mm ×

250 mm), R_t= 7.35 min, gradient (water:nitrile = 70:30 to 10:90 over

30 min, with 0.05% TFA, UV detector, 254 nm), giving I-9 as a yellow

solid, yield 51%; mp 121−124 °C. ¹H NMR (300 MHz, CD₃COCD₃)

δ 9.08 (m, 1H), 8.72 (m, 1H), 7.67(m, 1H), 7.45 (m, 2H), 7.09 (t, J =

8.1 Hz, 2H), 4.63 (s, 2H), 3.68 (m, 2H), 3.58 (m, 2H). EI-MS m/z:

356 (M)⁺. Anal. HPLC t_R= 14.483 min, 100% (solvent system I); t_R=

9.95 min, 97.43% (solvent system II).

Toluene-4-sulfonic Acid 5-Bromo-7-(4-ﬂuoro-benzylcarba-

moyl)-1,6-naphthyridin-8-yl Ester (I-3). p-Toluenesulfonyl chlo-

ride (268 mg,1.4 mmol) was added to a solution of I-1 (283 mg,

0.75 mmol) and triethylamine (161.92 mg, 1.6 mmol) in chloroform

(5 mL) over 10 min in 50 °C.The solution was stirred for 5 h and then

washed by saturated aqueous NH₄Cl and brine and dried over Na₂SO₄,

and the solution was removed under vacuum. Puriﬁcation by ﬂash chro-

matography (DCM:methanol = 40:1) to give I-3 as a white solid.

(360 mg, 91%); mp 152−154 °C. ¹H NMR (CDCl₃): δ 9.03 (dd, 1H,

J = 1.5, 4.2 Hz), 8.58 (dd, 1H, J = 1.5, 8.7 Hz), 7.96 (m, 1H), 7.94

(d, 2H, J = 8.1 Hz), 7.69 (dd, 1H, J = 4.2, 8.7 Hz), 7.36 (m, 2H), 7.32

(d, 2H, J = 8.1 Hz), 7.04 (t, 2H, J = 8.7 Hz), 4.60 (d, J = 6.6 Hz, 2H),

2.47 (s, 3H). EI-MS m/z: 530 (M + 1)⁺, 532 (M + 3)⁺. Anal. Calcd

for C₂₄H₂₀BrFN₄O: C 52.09, H 3.23, N 7.92. Found: C 51.94, H 3.17,

N 7.94.

7-(4-Fluorobenzylcarbamoyl)-5-bromo-1,6-naphthyridin-8-

yl Methanesulfonate (I-2). I-2 was prepared according to the same

procedure described for I-3. White solid, yield 83%; mp 150−152 °C.

¹H NMR (300 MHz, CDC1₃) δ 9.32 (d, J = 4.2 Hz, 1H), 8.62 (d, J =

8.7 Hz, 1H), 8.22 (m, 1H), 7.79 (dd, J = 4.2, 9.0 Hz, 1H), 7.38 (m,

2H), 7.05 (t, J = 8.4 Hz, 1H), 4.68 (d, J = 5.7 Hz, 2H), 3.70 (s, 1H).

EI-MS m/z: 454 (M + 1)⁺, 456 (M + 3)⁺. Anal. Calcd. for

C₁₇H₁₃BrFN₃O₄S·AcOEt: C 46.50, H 3.90, N 7.75. Found: C 47.44, H

3.75, N 8.10.

5-(1,1-Dioxido-1,2-thiazinan-2-yl)-N-(4-ﬂuorobenzyl)-8-hy-

droxy-1,6-naphthyridine-7-carboxamide (I-4). I-4 was prepared

according to the same procedure described in patent WO 2002030931

1

A2, giving a yellow solid, yield 65%. H NMR (300 MHz, CDC1₃) δ

13.34 (br, 1H), 9.19 (dd, J = 1.5, 4.2 Hz, 1H), 8.61 (d, J = 1.5, 8.7 Hz,

1H), 7.96 (m, 1H), 7.69 (dd, J = 4.8, 8.7 Hz, 1H), 7.37 (m, 2H), 7.07

(t, J = 8.6 Hz, 2H), 4.67 (m, 2H), 3.93 (m, 2H), 3.30 (t, J = 6.3 Hz,

2H), 2.45−2.36 (m, 2H), 2.18−2.15 (m, 1H), 1.85−1.82 (m, 1H).

EI-MS m/z: 430 (M)⁺. Anal. HPLC t_R= 17.04 min, 100% (solvent

system I); t_R= 12.35 min, 99.45% (solvent system II).

N-(4-Fluorobenzyl)-8-hydroxy-5-(N-(3-(methylsulfonamido)-

propyl)methylsulfonamido)-1,6-naphthyridine-7-carboxamid

(I-5). N-(3-Methanesulfonylamino-propyl)-methanesulfonamide (115 mg,

0.5 mmol) was added to a solution of I-1 (187.5 mg, 0.5 mmol) and

cuprous oxide (86 mg, 0.6 mmol) in pyridine (5 mL). After reﬂuxing

for 10 h, the solution was evaporated at vacuum, diluted with dichloro-

methane, and then ﬁltered to remove the solids. The ﬁltrate was stirred

with a slurry of EDTA (400 mg) in water (50 mL). After 16 h, the

dichloromethane extracts were dried over Na₂SO₄. After ﬁltering, the

solution was concentrated under vacuum. The residue was puriﬁed by

ﬂash chromatography on silica gel (DCM:methanol = 50:1) to give I-5

(S)-N-(4-Fluorobenzyl)-8-hydroxy-5-((1-hydroxypropan-2-

yl)amino)-1,6-naphthyridine-7-carboxamide (I-10). I-10 was

prepared according to the same procedure described for I-9, giving a

yellow solid, yield 41%; mp 117−120 °C. ¹H NMR (300 MHz,

CDCl₃) δ 9.23 (d, J = 7.5 Hz, 1H), 9.07 (d, J = 3.9 Hz, 1H), 8.13 (m,

1H), 7.73 (dd, J = 3.9, 7.5 Hz, 1H), 7.32 (m, 2H), 7.02 (t, J = 8.4 Hz,

2H), 4.64 (d, J = 5.7 Hz, 2H), 4.16 (m, 1H), 3.96 (d, J = 4.8 Hz, 2H),

1.39 (d, J = 6.0 Hz, 3H). EI-MS m/z: 370 (M)⁺. HPLC t_R= 15.317

min, 100% (solvent system I); t_R= 10.258 min, 95.61% (solvent

system II); [α]²_D²= +10.2° (c = 2.4200 g/100 mL, EtOH).

(R)-N-(4-Fluorobenzyl)-8-hydroxy-5-((1-hydroxypropan-2-yl)-

amino)-1,6-naphthyridine-7-carboxamide (I-11). I-11 was pre-

pared according to the same procedure described for I-9, giving a

yellow solid, yield 33%; mp 115−118 °C. ¹H NMR (300 MHz,

CDCl₃) δ 9.23 (d, J = 7.5 Hz, 1H), 9.07 (d, J = 3.9 Hz, 1H), 8.13 (m,

1H), 7.73 (dd, J = 3.9, 7.5 Hz,1H), 7.32 (m, 2H), 7.02 (t, J = 8.4 Hz,

2H), 4.64 (d, J = 5.7 Hz, 2H), 4.16 (m, 1H), 3.96 (d, J = 4.8 Hz, 2H),

1.39 (d, J = 6.0 Hz, 3H). EI-MS m/z: 370 (M)⁺. HPLC t_R= 15.808 min,

100% (solvent system I); t_R= 10.108 min, 98.94% (solvent system II);

[α]²_D²= −20.1° (c = 2.1050 g/100 mL, EtOH).

1

as a yellow solid, yield 54%; mp 205−208 °C. H NMR (300 MHz,

CD₃OD) δ 9.13 (d, J = 3.3 Hz, 1H), 8.80 (d, J = 7.8 Hz, 1H), 7.86

(dd, J = 3.6, 8.7 Hz, 1H), 7.44 (m, 2H), 7.07 (t, J = 9.0 Hz, 2H), 4.70

(s, 2H), 4.05 (m, 2H), 3.10 (m, 2H), 3.08 (s, 3H), 2.90 (s, 3H), 2.08−

1.94 (m, 2H). EI-MS m/z: 525 (M)⁺. Anal. HPLC t_R= 14.54 min,

100% (solvent system I); t_R= 10.84 min, 99.79% (solvent system II).

N-(4-Fluorobenzyl)-8-hydroxy-5-(N-(6-(methylsulfonamido)-

hexyl)methylsulfonamido)-1,6-naphthyridine-7-carboxamide

(I-6). I-6 was prepared according to the same procedure described

1

for I-5, giving a yellow solid, yield 35%; mp 178−182 °C. H NMR

5-Bromo-8-methylamino-N-(4-ﬂuorobenzyl)-1,6-naphthyri-

dine-7-carboxamide (I-12). Methylamine (5 mL) in alcohol was

added to a solution of I-3 (26 mg, 0.05 mmol) and triethylamine (15 mg,

0.15 mmol) in THF (5 mL). After reﬂuxing for 8 h, the solution

was evaporated at vacuum, and the residue was diluted with dichloro-

methane, then washed by saturated aqueous Na₂CO₃, saturated

aqueous NH₄Cl, and brine, respectively. Usual workup and puriﬁcation

by ﬂash chromatography on silica gel (petroleum:ethyl acetate = 8:1)

(300 MHz, CDC1₃) δ 13.51 (br, 1H), 9.20 (dd, J = 1.5, 4.2 Hz, 1H),

8.69 (d, J = 1.5, 8.7 Hz, 1H), 8.01 (t, J = 6.0 Hz, 1H), 7.73 (dd, J = 4.5,

8.4 Hz, 1H), 7.36 (m, 2H), 7.05 (t, J = 8.6 Hz, 2H), 4.69 (d, J =

6.0 Hz, 2H), 4.36 (m, 1H), 3.81 (t, J = 6.9 Hz, 2H), 3.10 (m, 2H),

3.01 (s, 3H), 2.91 (s, 3H), 1.5−1.2 (m, 8H). EI-MS m/z: 567 (M)⁺.

Anal. HPLC t_R= 16.117 min, 98.56% (solvent system I); t_R= 6.192 min,

96.20% (solvent system II).

N-(4-Fluorobenzyl)-5-((6-formamidohexyl)amino)-8-hy-

droxy-1,6-naphthyridine-7-carboxamide (I-7). Hexane-1,3-di-

amine (167 mg, 1.44 mmol) was added to a solution of I-1 (100 mg,

0.27 mmol) in DMPU (5 mL). After the solution was heated under

135 °C for 48 h, the solution was cooled to room temperature and the

usual workup was performed. Puriﬁcation by RP-HPLC (Vydac C18

column (10 mm × 250 mm), t_R= 19 min, gradient (water:nitrile = 70:30

1

gave I-12 as a yellow solid, yield 52%; mp: 136−140 °C. H NMR

(CDCl₃, 300 MHz): δ 8.92 (d, 1H, J = 3 Hz), 8.40 (d, 1H, J = 8.4 Hz),

8.28 (m,1H), 7.57 (dd, 1H, J = 4.5,8.4 Hz), 7.34 (m, 2H), 7.03 (t, 2H,

J = 8.5 Hz), 4.59 (d, 2H, J = 6.0 Hz), 3.61 (s, 3H). EI-MS m/z: 388

(M − 1)⁺, 390 (M + 1)⁺. Anal. HPLC t_R= 21.750 min, 100% (solvent

system I); t_R= 16.758 min, 99.57% (solvent system II).

9502

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Journal of Medicinal Chemistry

Article

5-Bromo-8-(2-hydroxy-ethylamino)-N-(4-ﬂuorobenzyl)-1,6-

naphthyridine-7-carboxamide (I-13). I-13 was prepared according

to the same procedure described for I-12, giving a yellow solid, yield

790 (M + 2)⁺. Anal. HPLC t_R= 25.642 min, 95.27% (solvent system

I); t_R= 5.933 min, 100% (solvent system II).

8-((2-Aminoethyl)amino)-5-bromo-N-(4-ﬂuorobenzyl)-1,6-

naphthyridine-7-carboxamide (I-20). I-20 was prepared according

to the same procedure described for I-18, giving a yellow solid, yield

1

54%; mp 103−105 °C. H NMR (CDCl₃, 300 MHz): δ 8.92 (d, 1H,

J = 2.4 Hz), 8.46 (d, 1H, J = 8.4 Hz), 8.34 (m, 1H), 7.62 (dd, 1H, J =

4.5, 8.4 Hz), 7.34 (m, 2H), 7.03 (t, 2H, J = 8.5 Hz), 4.60 (d, 2H, J =

6.3 Hz), 4.23 (t, 2H, J = 4.8 Hz), 3.94 (t, 2H, J = 4.8 Hz). EI-MS m/z:

418 (M)⁺, 420 (M + 2)⁺. Anal. HPLC t_R= 19.575 min, 100% (solvent

system I); t_R= 6.517 min, 100% (solvent system II).

1

50%; mp 82−84 °C. H NMR (CDCl₃, 300 MHz): δ 9.73 (m, 1H),

8.94 (d, 1H, J = 3 Hz), 8.40 (d, 1H, J = 8.4 Hz), 8.30 (m, 1H), 7.57

(dd, 1H, J = 4.5, 8.4 Hz), 7.33 (m, 2H), 7.02 (t, 2H, J = 8.4 Hz), 4.59

(d, 2H, J = 6.6 Hz), 4.19 (q, 2H, J = 6.3 Hz), 3.09 (t, 2H, J = 6.3 Hz).

EI-MS m/z: 417 (M)⁺, 419 (M + 2)⁺. Anal. HPLC t_R= 19.475 min,

100% (solvent system I); t_R= 11.792 min, 99.29% (solvent system II).

8-((3-Aminopropyl)amino)-N-(4-ﬂuorobenzyl)-1,6-naphthyr-

idine-7-carboxamide (II-1). II-1 was prepared according to the

same procedure described for I-18, giving a light-yellow solid, yield

73%; mp 62−64 °C. ¹H NMR (300 MHz, CDCl₃): δ 9.59 (d, 1H, J =

7.5 Hz), 8.91 (dd, 1H, J = 1.5, 3.9 Hz), 8.62 (t, 1H, J = 6.3 Hz), 8.19

(s, 1H), 8.08 (dd, 1H, J = 1.5, 8.4 Hz), 7.48 (dd, 1H, J = 4.2, 8.4 Hz),

7.34 (m, 2H), 7.02 (t, 2H, J = 8.7 Hz), 4.60 (d, 2H, J = 6.3 Hz), 4.23

(q, 2H, J = 6.6 Hz), 2.91 (t, 2H, J = 6.6 Hz), 2.00 (s, 2H). EI-MS m/z:

353 (M)⁺. Anal. HPLC t_R= 18.167 min, 100% (solvent system I); t_R=

12.742 min, 98.29% (solvent system II).

8-Benzylamino-5-bromo-N-(4-ﬂuorobenzyl)-1,6-naphthyri-

dine-7-carboxamide (I-14). I-14 was prepared according to the

same procedure described for I-12, giving a green−yellow solid, yield

1

57%; mp 142−144 °C. H NMR (CDCl₃, 300 MHz): δ 8.94 (d, 1H,

J = 4.8 Hz), 8.41 (d, 1H, J = 8.1 Hz), 8.31 (m, 1H),7.59 (dd, 1H, J =

4.5, 8.7 Hz), 7.40−7.19 (m, 7H), 7.04 (t, 2H, J = 8.7 Hz), 5.40 (s, 2H),

4.60 (d, 2H, J = 6.3 Hz). Anal. HPLC t_R= 23.867 min, 100% (solvent

system I); t_R= 19.550 min, 100% (solvent system II).

5-Bromo-8-((S)-1-phenyl-ethylamino)-N-(4-ﬂuorobenzyl)-

1,6-naphthyridine-7-carboxamide (I-15). I-15 was prepared

according to the same procedure described for I-12, giving a yellow

1

solid, yield 64%; mp 102−104 °C. H NMR (CDCl₃, 300 MHz):

δ 8.88 (d, 1H, J = 4.2 Hz), 8.33 (d, 1H, J = 8.4 Hz), 8.33 (m, 1H),7.51

(dd, 1H, J = 4.2, 8.4 Hz), 7.40−7.35 (m, 4H), 7.21 (t, 2H, J = 7.5 Hz),

7.12 (d, 1H, J = 7.2 Hz), 7.05 (t, 2H, J = 8.7 Hz), 6.45 (q, 1H, J =

6.9 Hz), 4.69−4.61 (m, 2H), 1.65 (d, 3H, J = 6.9 Hz). Anal. HPLC

t_R= 23.800 min, 100% (solvent system I); t_R= 18.608 min, 96.93%

(solvent system II).

5-Bromo-8-((R)-1-phenyl-ethylamino)-N-(4-ﬂuorobenzyl)-

1,6-naphthyridine-7-carboxamide (I-16). I-16 was prepared

according to the same procedure described for I-12, giving a green−

yellow solid, yield 55%; mp 107−108 °C. ¹H NMR (CDCl₃, 300 MHz):

δ 8.88 (d, 1H, J = 4.2 Hz), 8.33 (d, 1H, J = 8.4 Hz), 8.33 (m, 1H),7.51

(dd, 1H, J = 4.2, 8.4 Hz), 7.40−7.35 (m, 4H), 7.21 (t, 2H, J = 7.5 Hz),

7.12 (d, 1H, J = 7.2 Hz), 7.05 (t, 2H, J = 8.7 Hz), 6.45 (q, 1H, J =

6.9 Hz), 4.69−4.61 (m,2H), 1.65 (d, 3H, J = 6.9 Hz). EI-MS m/z: 478

(M)⁺, 480 (M + 2)⁺. Anal. HPLC t_R= 23.983 min, 96.18% (solvent

system I); t_R= 18.967 min, 100% (solvent system II).

5-Bromo-N-(4-ﬂuorobenzyl)-8-((4-ﬂuorobenzyl)amino)-1,6-

naphthyridine-7-carboxamide (I-17). I-17 was prepared according

to the same procedure described for I-12, giving a yellow solid, yield

68%; mp 136−138 °C. ¹H NMR (CDCl₃, 300 MHz): δ 8.93 (dd, 1H,

J = 1.5, 4.2 Hz), 8.42 (dd, 1H, J = 1.5, 8.4 Hz), 8.33 (m, 1H), 7.59 (dd,

1H, J = 4.2, 8.4 Hz), 7.38−7.32 (m, 4H), 7.04 (t, 2H, J = 8.4 Hz), 6.96

(t, 2H, J = 8.4 Hz), 5.35 (s, 2H), 4.60 (d, 2H, J = 6.6 Hz). EI-MS m/z:

482 (M)⁺, 484 (M + 2)⁺. Anal. HPLC t_R= 24.492 min, 100% (solvent

system I); t_R= 19.458 min, 100% (solvent system II).

Methyl 2-((5-Bromo-7-((4-ﬂuorobenzyl)carbamoyl)-1,6-

naphthyridin-8-yl)amino)acetate (II-2). II-2 was prepared accord-

ing to the same procedure described for I-18, giving a green−yellow

1

solid, yield 71%; mp 184−185 °C. H NMR (300 MHz, CDCl₃):

δ 10.17 (br, 1H), 8.86 (dd, 1H, J = 1.5, 4.2 Hz), 8.43 (dd, 1H, J = 1.5,

8.4 Hz), 8.34 (t, 1H, J = 6.0 Hz), 7.58 (dd, 1H, J = 4.2, 8.4 Hz), 7.36

(m, 2H), 7.04 (t, 2H, J = 8.7 Hz), 4.82 (s, 2H), 4.63 (d, 2H, J = 6.3

Hz), 3.73 (s, 3H). EI-MS m/z: 446 (M)⁺, 448 (M + 2)⁺. Anal. HPLC

t_R= 21.025 min, 100% (solvent system I); t_R= 16.158 min, 100%

(solvent system II).

5-Bromo-N-(4-ﬂuorobenzyl)-8-((3-(methylsulfonamido)-

propyl)amino)-1,6-naphthyridine-7-carboxamide (II-3). II-3 was

prepared according to the same procedure described for I-18, aﬀording

1

a yellow solid, yield 85%; mp 153−155 °C. H NMR (300 MHz,

CDCl₃): δ 9.21 (m, 1H), 9.11 (dd, 1H, J = 1.8, 4.2 Hz), 8.53 (dd, 1H,

J = 1.5, 8.4 Hz), 8.40 (t, 1H, J = 6.3 Hz), 7.68 (dd, 1H, J = 4.2,

8.4 Hz), 7.35 (m, 2H), 7.05 (t, 2H, J = 8.7 Hz), 6.61 (t, 1H, J =

6.0 Hz), 4.61 (d, 2H, J = 6.0 Hz), 4.14 (m, 2H), 3.42 (q, 2H, J =

6.0 Hz), 2.91 (s, 3H), 2.01 (m, 2H). EI-MS m/z: 509 (M)⁺, 511 (M + 2)⁺.

Anal. HPLC t_R= 20.833 min, 100% (solvent system I); t_R= 6.067 min,

99.46% (solvent system II).

5-Bromo-8-((3-(dimethylamino)propyl)amino)-N-(4-ﬂuoro-

benzyl)-1,6-naphthyridine-7-carboxamide (II-4). II-4 was pre-

pared according to the same procedure described for I-18, aﬀording a

1

light-yellow solid, yield 91%; mp 60−62 °C. H NMR (300 MHz,

8-(3-Amino-propylamino)-5-bromo-N-(4-ﬂuorobenzyl)-1,6-

naphthyridine-7-carboxamide (I-18). Propane-1,3-diamine (22 mg,

0.3 mmol) was added to a solution of I-3 (53 mg, 0.1 mmol) and

triethylamine (30 mg, 0.3 mmol) in THF (5 mL). After reﬂuxing for

8 h, the solution was evaporated at vacuum, and the residue was diluted

with dichloromethane, washed by saturated aqueous Na₂CO₃, satu-

rated aqueous NH₄Cl, and brine, and dried over Na₂SO₄. The ﬁltrate

was concentrated under vacuum. Puriﬁcation by ﬂash chromatography

on basic aluminum oxide (DCM:methanol = 50:1) aﬀorded I-18 as a

yellow solid, yield 58%; mp 78−80 °C. ¹H NMR (CDCl₃, 300 MHz):

δ 9.60 (t, 1H, J = 5.4 Hz), 8.97 (d, 1H, J = 3 Hz), 8.42 (d, 1H, J =

8.4 Hz), 8.32 (t, 1H, J = 6.0 Hz), 7.60 (dd, 1H, J = 4.5, 8.4 Hz), 7.36

(m, 2H), 7.04 (t, 2H, J = 8.7 Hz), 4.60 (d, 2H, J = 6.6 Hz), 4.19 (q,

2H, J = 6.3 Hz), 3.96 (t, 2H, J = 6.6 Hz), 1.93 (m, 2H). EI-MS m/z:

431(M)⁺, 433 (M + 2)⁺. Anal. HPLC t_R= 19.083 min, 100% (solvent

system I); t_R= 12.928 min, 100% (solvent system II).

CDCl₃): δ 9.74 (t, 1H, J = 6.6 Hz), 8.94 (dd, 1H, J = 1.8, 4.2 Hz), 8.42

(dd, 1H, J = 1.5, 8.4 Hz), 8.31 (t, 1H, J = 6.3 Hz), 7.68 (dd, 1H, J =

4.2, 8.4 Hz), 7.35 (m, 2H), 7.04 (t, 2H, J = 8.7 Hz), 4.61 (d, 2H, J =

6.0 Hz), 4.17 (q, 2H, J = 6.6 Hz), 2.54 (m, 2H), 1.96 (m, 2H). EI-MS

m/z: 459 (M)⁺, 461 (M + 2)⁺. Anal. HPLC t_R= 18.867 min, 100%

(solvent system I); t_R= 12.808 min, 100% (solvent system II).

8-((3-Benzamidopropyl)amino)-5-bromo-N-(4-ﬂuoroben-

zyl)-1,6-naphthyridine-7-carboxamide (II-5). II-5 was prepared

according to the same procedure described for I-18, giving a brown

1

solid, yield 57%; mp 144−146 °C. H NMR (300 MHz, CDCl₃):

δ 9.63 (br, 1H), 8.83 (dd, 1H, J = 1.8, 4.2 Hz), 8.43 (dd, 1H, J = 1.5,

8.4 Hz), 8.34 (t, 1H, J = 6.3 Hz), 7.75 (d, 2H, J = 6.9 Hz), 7.57 (dd,

1H, J = 4.2, 8.4 Hz), 7.50−7.32 (m, 5H), 7.04 (t, 2H, J = 8.7 Hz), 6.68

(br, 1H), 4.59 (d, 2H, J = 6.6 Hz), 4.26 (t, 2H, J = 6.6 Hz), 3.66 (q,

2H, J = 6.3 Hz), 2.08 (m, 2H). EI-MS m/z: 535 (M)⁺, 537 (M + 2)⁺.

Anal. HPLC t_R= 6.408 min, 100% (solvent system I); t_R= 6.208 min,

100% (solvent system II).

8,8′-(Propane-1,3-diylbis(azanediyl))bis(5-bromo-N-(4-ﬂuo-

robenzyl)-1,6-naphthyridine-7-carboxamide) (I-19). I-19 was

prepared according to the same procedure described for I-12, giv-

8-((6-Aminohexyl)amino)-5-bromo-N-(4-ﬂuorobenzyl)-1,6-

naphthyridine-7-carboxamide (II-6). II-6 was prepared according

to the same procedure described for I-18, giving a green−yellow solid,

1

ing a yellow solid, yield 75%; mp 169−171 °C. H NMR (CDCl₃,

1

300 MHz): δ 9.72 (2H, brs), 8.82 (dd, 2H, J = 1.8, 4.2 Hz), 8.35 (dd,

2H, J = 1.8, 8.4 Hz), 8.22 (m, 2H), 7.50 (dd, 2H, J = 4.2, 8.4 Hz),

7.35 (m, 4H), 7.03 (t, 4H, J = 8.5 Hz), 4.57 (d, 4H, J = 6.0 Hz), 4.35

(t, 4H, J = 6.6 Hz), 2.15 (t, 2H, J = 6.6 Hz). EI-MS m/z: 788 (M)⁺,

yield 48%; mp 108−112 °C. H NMR (300 MHz, CDCl₃): δ 9.65

(br, 1H), 8.91 (dd, 1H, J = 1.8, 4.2 Hz), 8.38 (dd, 1H, J = 1.5, 8.4 Hz),

8.30 (t, 1H, J = 6.3 Hz), 7.56 (dd, 1H, J = 4.2, 8.4 Hz), 7.35 (m, 2H), 7.03

(t, 2H, J = 8.7 Hz), 4.60 (d, 2H, J = 6.6 Hz), 4.26 (m, 2H), 2.75 (m, 2H),

9503

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Journal of Medicinal Chemistry

Article

1.8−1.15 (m, 8H). EI-MS m/z: 473 (M)⁺, 475 (M + 2)⁺. Anal. HPLC

t_R= 19.558 min, 100% (solvent system I); t_R= 13.508 min, 96.32%

(solvent system II).

4.60 (d, 2H, J = 6.0 Hz), 2.76 (m, 1H), 2.18 (m, 2H), 1.92 (m, 4H),

1.42 (m, 4H). EI-MS m/z: 471 (M)⁺, 473 (M + 2)⁺. HRMS calcd for

C₂₂H₂₃BrFN₅O (M)⁺: 471.1070. Found: 471.1063. Anal. HPLC t_R=

5.325 min, 93.63% (solvent system I); t_R= 12.692 min, 99.60% (sol-

vent system II).

8-(4-Aminophenylamino)-5-bromo-N-(4-ﬂuorobenzyl)-1,6-

naphthyridine-7-carboxamide (II-7). II-7 was prepared according

to the same procedure described for I-18, giving a carmine solid, yield

46%; mp 190−192 °C. ¹H NMR (300 MHz, CDCl₃): δ 10.64 (s, 1H),

8.73 (dd, 1H, J = 1.8, 4.2 Hz), 8.44 (dd, 1H, J = 1.5, 8.4 Hz), 8.38

(t, 1H, J = 6.3 Hz), 7.54 (dd, 1H, J = 4.2, 8.4 Hz), 7.36 (m, 2H), 7.03

(t, 2H, J = 8.7 Hz), 6.80 (d, 2H, J = 8.4 Hz), 6.56 (d, 2H, J = 8.4 Hz),

4.60 (d, 2H, J = 6.6 Hz). EI-MS m/z: 465 (M)⁺, 467 (M + 2)⁺. Anal.

HPLC t_R= 16.067 min, 100% (solvent system I); t_R= 10.667 min,

99.18% (solvent system II).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-N-(4-ﬂuorobenzyl)-5-

(piperidin-1-yl)-1,6-naphthyridine-7-carboxamide (II-14). Fol-

lowing the procedure as the preparation of compound I-18, the

reaction of tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate (65 mg,

0.3 mmol) with I-3 (53 mg, 0.1 mmol) and triethylamine (30 mg,

0.3 mmol) gave tert-butyl ((1r,4r)-4-((5-bromo-7-((4-ﬂuorobenzyl)-

carbamoyl)-1,6-naphthyridin-8-yl)amino)cyclohexyl)carbamate as a

1

yellow solid, yield 70%. H NMR (300 MHz, CDCl₃): δ 9.65 (d, 1H,

8-(3-Aminophenylamino)-5-bromo-N-(4-ﬂuorobenzyl)-1,6-

naphthyridine-7-carboxamide (II-8). II-8 was prepared according

to the same procedure described for I-18, giving a yellow solid, yield

44%; mp 167−169 °C. ¹H NMR (300 MHz, CDCl₃): δ 10.54 (s, 1H),

8.85 (dd, 1H, J = 1.8, 4.2 Hz), 8.47 (dd, 1H, J = 1.5, 8.4 Hz), 8.38

(t, 1H, J = 6.3 Hz), 7.59 (dd, 1H, J = 4.2, 8.4 Hz), 7.35 (m, 2H), 7.04

(t, 2H, J = 8.7 Hz), 6.95 (t, 1H, J = 8.0 Hz), 6.36 (d, 1H, J = 8.0 Hz),

6.28 (m, 2H), 4.61 (d, 2H, J = 6.3 Hz), 3.56 (br, 2H). EI-MS m/z: 465

(M)⁺, 467 (M + 2)⁺. Analytical HPLC t_R= 16.292 min, 100% (solvent

system I); t_R= 11.067 min, 100% (solvent system II).

J = 7.5 Hz), 8.93 (dd, 1H, J = 1.5, 3.9 Hz), 8.42 (dd, 1H, J = 1.5,

8.4 Hz), 8.30 (m, 1H), 7.59 (dd, 1H, J = 4.2, 8.4 Hz), 7.35 (m, 2H), 7.04

(t, 2H), 4.96 (m, 1H), 4.60 (d, 2H, J = 6.3 Hz), 4.38 (m, 1H), 3.48

(m, 1H), 2.20 (m, 2H), 2.06 (m, 2H), 1.45 (s, 9H), 1.32 (m, 4H).

EI-MS m/z: 571 (M)⁺, 573 (M + 2)⁺.

tert-Butyl ((1r,4r)-4-((5-bromo-7-((4-ﬂuorobenzyl)carbamoyl)-1,6-

naphthyridin-8-yl)amino)cyclohexyl)carbamate (29 mg, 0.05 mmol)

was added to a solution of piperidine (1 mL). After heating at 110 °C

for 48 h, the solution was cooled to room temperature, diluted with

dichloromethane, and then washed with saturated aqueous NH₄Cl and

brine. Usual workup followed by puriﬁcation by ﬂash chromatography

on silica gel (DCM:methanol = 100:1) aﬀorded tert-butyl ((1r,4r)-4-

((7-((4-ﬂuorobenzyl)carbamoyl)-5-(piperidin-1-yl)-1,6-naphthyridin-

8-yl)amino)cyclohexyl)carbamate as a yellow solid, yield 89%. ¹H

NMR (300 MHz, CDCl₃): δ 8.93 (d, 1H, J = 1.5 Hz), 8.53 (m, 1H),

8.36 (dd, 1H, J = 1.5, 8.4 Hz), 7.47 (dd, 1H, J = 4.2, 8.4 Hz), 7.34 (m,

2H), 7.03 (m, 2H), 4.74 (m, 1H), 4.62 (d, 2H, J = 6.0 Hz), 4.30 (m,

1H), 3.74 (m, 1H), 3.46 (m, 1H), 3.11 (m, 4H), 2.14 (m, 2H), 1.84

(m, 4H), 1.69 (m, 4H), 1.44 (s, 9H), 1.45−1.23 (m, 4H).

tert-Butyl 4-((5-Bromo-7-((4-ﬂuorobenzyl)carbamoyl)-1,6-

naphthyridin-8-yl)amino)piperidine-1-carboxylate (II-9). II-9

was prepared according to the same procedure described for I-18,

giving a yellow solid, yield 75%; mp 134−137 °C. ¹H NMR (300 MHz,

CDCl₃): δ 8.96 (dd, 1H, J = 1.5, 3.9 Hz), 8.44 (dd, 1H, J = 1.5, 8.4 Hz),

8.32 (t, 1H, J = 6.0 Hz), 7.61 (dd, 1H, J = 4.2, 8.4 Hz), 7.35 (m, 2H),

7.04 (t, 2H, J = 8.7 Hz), 5.18 (m, 1H), 4.60 (d, 2H, J = 6.0 Hz), 3.99

(m, 2H), 3.05 (m, 2H), 2.06 (m, 2H), 1.62 (m, 2H), 1.45 (s, 9H).

EI-MS m/z: 557 (M)⁺, 559 (M + 2)⁺. Anal. HPLC t_R= 23.967 min,

100% (solvent system I); t_R= 19.517 min, 100% (solvent system II).

5-Bromo-N-(4-ﬂuorobenzyl)-8-(piperidin-4-ylamino)-1,6-

naphthyridine-7-carboxamide (II-10). II-10 was prepared accord-

ing to the same procedure described for I-18, giving a green−yellow

The resultant tert-butyl ((1r,4r)-4-((7-((4-ﬂuorobenzyl)carbamoyl)-

5-(piperidin-1-yl)-1,6-naphthyridin-8-yl)amino)cyclohexyl)carbamate

was deprotected in 20% TFA−DCM solution to give the target

1

compound II-14 as a yellow solid, yield 95%. H NMR (300 MHz,

1

solid, yield 46%; mp >200 °C. H NMR (300 MHz, CDCl₃): δ 8.94

CDCl₃): δ 8.94 (d, 1H, J = 2.7 Hz), 8.70 (m, 1H), 8.54 (m, 1H), 8.35

(d, 1H, J = 9.0 Hz), 7.46 (dd, 1H, J = 3.9, 8.1 Hz), 7.34 (m, 2H), 7.03

(m, 2H), 4.75 (m, 1H), 4.62 (d, 2H, J = 6.3 Hz), 3.11 (m, 4H), 2.75

(m, 1H), 2.13 (m, 2H), 2.05 (m, 3H), 1.72 (m, 5H), 1.45−1.23 (m,

4H). EI-MS m/z: 476 (M)⁺. Anal. HPLC t_R= 13.843 min, 96.19%

(solvent system I); t_R= 6.577 min, 99.29% (solvent system II).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-(1,1-dioxido-1,2-

thiazinan-2-yl)-N-(4-ﬂuorobenzyl)-1,6-naphthyridine-7-car-

boxamide (II-15). Following the procedure as the preparation of

compound I-3, the treatment of I-4 (43 mg, 0.1 mmol) with methyl-

benzenesulfonyl chloride (75 mg, 0.4 mmol) and triethylamine (40 mg,

0.4 mmol) gave 5-(1,1-dioxido-1,2-thiazinan-2-yl)-7-((4-ﬂuorobenzyl)-

carbamoyl)-1,6-naphthyridin-8-yl 4-methylbenzenesulfonate as a white

(dd, 1H, J = 2.1, 4.2 Hz), 8.46 (dd, 1H, J = 1.8, 8.4 Hz), 8.32 (t, 1H,

J = 6.0 Hz), 7.63 (dd, 1H, J = 4.2, 8.4 Hz), 7.33 (m, 2H), 7.02 (t, 2H,

J = 8.7 Hz), 5.27 (m, 1H), 4.56 (d, 2H, J = 6.0 Hz), 3.39 (m, 2H), 2.31

(m, 2H), 1.84 (m, 2H). EI-MS m/z: 457 (M)⁺, 459 (M + 2)⁺. Anal.

HPLC t_R= 6.292 min, 100% (solvent system I); t_R= 12.658 min,

100% (solvent system II).

tert-Butyl 4-(5-Bromo-7-(4-ﬂuorobenzylcarbamoyl)-1,6-

naphthyridin-8-yl)piperazine-1-carboxylate (II-11). II-11 was

prepared according to the same procedure described for I-18, giving

1

a yellow solid, yield 64%; mp 118−122 °C. H NMR (300 MHz,

CDCl₃): δ 9.02 (dd, 1H, J = 1.5, 4.2 Hz), 8.47 (dd, 1H, J = 1.8, 8.4 Hz),

8.18 (t, 1H, J = 8.7 Hz), 7.59 (dd, 1H, J = 4.2, 8.4 Hz), 7.35 (m, 2H),

6.99 (t, 2H, J = 8.7 Hz), 4.61 (d, 2H, J = 6.3 Hz), 3.59 (s, 4H), 3.47

(s, 4H), 1.49 (s, 9H). EI-MS m/z: 543 (M)⁺, 545 (M + 2)⁺. Anal. HPLC

t_R= 20.483 min, 100% (solvent system I); t_R= 14.767 min, 99.35%

(solvent system II).

1

solid, yield 82%. H NMR (300 MHz, CDCl₃): δ 8.92 (d, 1H, J =

3.9 Hz), 8.60 (d, 1H, J = 8.4 Hz), 7.93 (d, 2H, J = 8.1 Hz), 7.59 (dd, 1H,

J = 4.2, 8.7 Hz), 7.49 (m, 1H), 7.36 (m, 4H), 7.05 (t, 2H, J = 8.7 Hz),

4.63 (d, 2H, J = 6.0 Hz), 4.03 (m, 2H), 3.31 (m, 2H), 2.47 (s, 3H), 2.36

(m, 2H), 1.66 (m, 2H). EI-MS m/z: 584 (M)⁺.

5-Bromo-N-(4-ﬂuorobenzyl)-8-(piperazin-1-yl)-1,6-naph-

thyridine-7-carboxamide (II-12). II-12 was prepared according to

the same procedure described for I-18, giving a brown solid, yield

38%; mp 191−195 °C. ¹H NMR (300 MHz, CDCl₃): δ 9.07 (dd, 1H,

J = 1.5, 4.2 Hz), 8.59 (t, 1H, J = 6.0 Hz), 8.53 (dd, 1H, J = 1.5,

8.4 Hz), 7.63 (dd, 1H, J = 4.2, 8.4 Hz), 7.39 (m, 2H), 7.04 (t, 2H, J =

8.7 Hz), 4.66 (d, 2H, J = 6.3 Hz), 3.59 (t, 4H, J = 4.5 Hz), 3.13 (t, 4H,

J = 4.5 Hz), 2.74 (br, 1H). EI-MS m/z: 443 (M)⁺, 445 (M + 2)⁺. Anal.

HPLC t_R= 19.808 min, 100% (solvent system I); t_R= 12.050 min,

96.48% (solvent system II).

II-15 was prepared in a similar fashion as described for I-18 from

the precursor above, giving a yellow solid, yield 54%; mp 173−176 °C.

¹H NMR (300 MHz, CDCl₃): δ 9.65 (d, 1H, J = 7.8 Hz), 8.92 (m,

1H), 8.48 (dd, 1H, J = 1.5, 8.7 Hz), 8.07 (m, 1H), 7.54 (dd, 1H, J =

4.2, 8.4 Hz), 7.34 (dd, 2H, J = 5.4, 8.4 Hz), 7.04 (t, 2H, J = 8.4 Hz),

5.00 (m, 1H), 4.56 (m, 2H), 3.92 (m, 1H), 3.74 (m, 1H), 3.24 (m,

2H), 2.75 (m, 1H), 2.40 (m, 1H), 2.20 (m, 4H), 1.90 (m, 2H), 1.65

(m, 2H), 1.43 (m, 4H). EI-MS m/z: 526 (M)⁺. Anal. HPLC t_R

=

17.875 min, 95.23% (solvent system I); t_R= 7.375 min, 100% (solvent

system II).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(4-ﬂuoro-

benzyl)-1,6-naphthyridine-7-carboxamide (II-13). II-13 was

prepared according to the same procedure described for I-18, giving

a light-yellow solid, yield 81%; mp 147−152 °C. ¹H NMR (300 MHz,

CDCl₃): δ 9.63 (d, 1H, J = 7.5 Hz), 8.94 (dd, 1H, J = 1.5, 3.9 Hz),

8.40 (dd, 1H, J = 1.5, 8.4 Hz), 8.33 (t, 1H, J = 6.0 Hz), 7.58 (dd, 1H,

J = 4.2, 8.4 Hz), 7.35 (m, 2H), 7.04 (t, 2H, J = 8.7 Hz), 4.97 (m, 1H),

N-Benzyl-5-bromo-8-hydroxy-1,6-naphthyridine-7-carboxa-

mide (III-1-8). III-1-8 was prepared in a similar fashion as described

for I-1, giving a white solid (yield: 72%). ¹H NMR (300 MHz, CDCl₃)

δ 9.19 (d, J = 4.2 Hz, 1H), 8.54 (d, J = 7.8 Hz, 1H), 8.21 (m, 1H), 7.74

(dd, J = 4.2, 7.8 Hz, 1H), 7.40−7.29 (m, 5H), 4.70 (d, J = 6.0 Hz, 2H).

9504

dx.doi.org/10.1021/jm300667v | J. Med. Chem. 2012, 55, 9492−9509

Journal of Medicinal Chemistry

Article

7-(Benzylcarbamoyl)-5-bromo-1,6-naphthyridin-8-yl 4-

Methylbenzenesulfonate (III-1-9). III-1-9 was prepared in a similar

fashion as described for I-3, giving a white solid (yield: 83%). ¹H NMR

(300 MHz, CDCl₃) δ 9.04 (dd, J = 1.5, 4.2 Hz, 1H), 8.56 (d, J = 1.8,

7.8 Hz, 1H), 7.93 (m, 3H), 7.68 (dd, J = 4.5, 8.7 Hz, 1H), 7.38−7.30

(m, 7H), 4.62 (d, J = 6.0 Hz, 2H), 2.46 (s, 3H).

(yield: 82%). ¹H NMR (300 MHz, CDCl₃) δ 13.30 (s, 1H), 9.17 (dd,

J = 1.5, 4.2 Hz, 1H), 8.53 (dd, J = 1.5, 8.4 Hz, 1H), 8.03 (d, J = 8.1 Hz,

1H), 7.71 (dd, J = 4.2, 8.4 Hz, 1H), 7.40 (m, 2H), 7.06 (d, J = 8.7 Hz,

2H), 5.30 (m, 1H), 1.67 (d, J = 6.9 Hz, 3H).

(S)-5-Bromo-7-((1-(4-ﬂuorophenyl)ethyl)carbamoyl)-1,6-

naphthyridin-8-yl 4-methylbenzenesulfonate (III-4-9). III-4-9

was prepared according to the same procedure described for I-3, giving

8-(((1r,4r)-4-Aminocyclohexyl)amino)-N-benzyl-5-bromo-

1,6-naphthyridine-7-carboxamide (III-1). III-1 was prepared in a

similar fashion as described for I-18, giving a yellow solid, yield 56%;

1

a white solid (yield: 79%). H NMR (300 MHz, CDCl₃) δ 9.01 (dd,

J = 1.2, 4.2 Hz, 1H), 8.56 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H),

7.84 (m, 1H), 7.67 (dd, J = 4.5, 8.7 Hz, 1H), 7.40 (m, 2H), 7.28 (d, J =

8.7 Hz, 2H), 5.26 (m, 1H), 2.45 (s, 3H), 1.60 (d, J = 6.9 Hz, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-((S)-1-(4-

ﬂuorophenyl)ethyl)-1,6-naphthyridine-7-carboxamide (III-4).

III-4 was prepared according to the same procedure described for

1

mp 123−126 °C. H NMR (300 MHz, CDCl₃): δ 9.62 (d, 1H, J =

7.5 Hz), 8.94 (dd, 1H, J = 1.5, 3.9 Hz), 8.41 (d, 1H, J = 8.4 Hz), 8.31

(m, 1H), 7.58 (dd, 1H, J = 4.2, 8.4 Hz), 7.37−7.27 (m, 5H), 4.97 (m,

1H), 4.63 (d, 2H, J = 6.0 Hz), 2.76 (m, 1H), 2.18 (m, 1H), 1.92 (m,

4H), 1.42 (m, 4H). EI-MS m/z: 453 (M)⁺, 455 (M + 2)⁺. Anal. HPLC

t_R= 5.8 min, 100% (solvent system I); t_R= 8.79 min, 95.72% (solvent

system II).

1

I-18, giving a yellow solid, yield 66%; mp 129−131 °C. H NMR

(300 MHz, CDCl₃): δ 9.57 (d, 1H, J = 7.5 Hz), 8.93 (dd, 1H, J = 1.5,

3.9 Hz), 8.41 (dd, 1H, J = 1.5, 8.4 Hz), 8.21 (m, 1H), 7.58 (dd, 1H, J =

4.2, 8.4 Hz), 7.37 (m, 2H), 7.01 (t, 2H, J = 8.7 Hz), 5.24 (m, 1H), 4.97

(m, 1H), 4.93 (m, 1H), 2.76 (m, 1H), 2.18 (m, 2H), 1.92 (m, 2H),

1.60 (d, 3H, J = 6.9 Hz), 1.42 (m, 4H). EI-MS m/z: 485 (M)⁺, 487

(M + 2)⁺. Anal. HPLC t_R= 14.31 min, 94.70% (solvent system I); t_R=

8.33 min, 100% (solvent system II).

5-Bromo-8-hydroxy-N-(4-methoxybenzyl)-1,6-naphthyri-

dine-7-carboxamide (III-2-8). III-2-8 was prepared according to the

same procedure described for I-1, giving a white solid (yield: 65%). ¹H

NMR (300 MHz, CDCl₃) δ 9.19 (d, J = 4.2 Hz, 1H), 8.53 (d, J =

8.1 Hz, 1H), 8.12 (m, 1H), 7.72 (dd, J = 4.2, 7.8 Hz, 1H), 7.33 (d, J =

8.4 Hz, 2H), 6.91 (t, J = 8.7 Hz, 2H), 4.63 (d, J = 6.6 Hz, 2H), 3.80 (s,

3H). Anal. HPLC t_R= 22.47 min, 99.99% (solvent system I); t_R

=

5-Bromo-8-hydroxy-N-(4-(dimethylamino)benzyl)-1,6-naph-

thyridine-7-carboxamide (III-5-8). III-5-8 was prepared according

to the same procedure described for I-1, giving the desired crude

product as a brown solid (yield: 85%) that was used for the next step

directly.

5-Bromo-7-(4-(dimethylamino)benzylcarbamoyl)-1,6-naph-

thyridin-8-yl 4-Methylbenzenesulfonate (III-5-9). III-5-9 was

prepared according to the same procedure described for I-3, giving a

white solid (yield: 48%). ¹H NMR (300 MHz, CDCl₃): δ 9.03 (d, J =

4.5 Hz, 1H), 8.55 (d, J = 6.9 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.76

(m, 1H), 7.67 (dd, J = 4.5, 6.9 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.25

(m, 2H), 6.74 (d, J = 9 Hz, 2H), 4.49 (d, J = 5.7 Hz, 2H), 2.95 (s, 6H),

2.47 (s, 3H).

14.81 min, 99.81% (solvent system II).

5-Bromo-7-((4-methoxybenzyl)carbamoyl)-1,6-naphthyri-

din-8-yl 4-Methylbenzenesulfonate (III-2-9). III-2-9 was prepared

according to the same procedure described for I-3, giving a white solid

(yield: 62%). ¹H NMR (300 MHz, CDCl₃) δ 8.98 (dd, J = 1.5, 4.2 Hz,

1H), 8.52 (dd, J = 1.5, 8.7 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.87 (m,

1H), 7.65 (dd, J = 4.2, 8.4 Hz, 1H), 7.31−7.27 (m, 4H), 6.87 (d, J =

8.4 Hz, 2H), 4.52 (d, J = 5.7 Hz, 2H), 3.78 (s, 3H), 2.44 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(4-me-

thoxybenzyl)-1,6-naphthyridine-7-carboxamide (III-2). III-2

was prepared according to the same procedure described for I-18,

1

giving a yellow solid, yield 78%; mp 96−99 °C. H NMR (300 MHz,

CDCl₃): δ 9.64 (d, 1H, J = 8.1 Hz), 8.93 (dd, 1H, J = 1.2, 4.2 Hz),

8.40 (dd, 1H, J = 1.2, 8.4 Hz), 8.23 (m, 1H), 7.58 (dd, 1H, J = 4.2,

8.4 Hz), 7.30 (d, 2H, J = 8.4 Hz), 6.89 (d, 2H, J = 8.4 Hz), 4.95

(m, 1H), 4.56 (d, 2H, J = 6.3 Hz), 3.80 (s, 3H), 2.75 (m, 1H), 2.63

(m, 1H), 2.18 (m, 2H), 1.85 (m, 4H), 1.34 (m, 4H). EI-MS m/z: 483

(M)⁺, 485 (M + 2)⁺. Anal. HPLC t_R= 22.367 min, 97.28% (solvent

system I); t_R= 13.950 min, 97.37% (solvent system II).

N-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-bromo-8-hydroxy-1,6-

naphthyridine-7-carboxamide (III-3-8). III-3-8 was prepared

according to the same procedure described for I-1, giving a white

solid (yield: 77%). ¹H NMR (300 MHz, CDCl₃) δ 13.32 (s, 1H), 9.20

(dd, J = 1.5, 3.9 Hz, 1H), 8.54 (dd, J = 1.2, 8.4 Hz, 1H), 8.11 (m, 1H),

7.72 (dd, J = 4.2, 8.4 Hz, 1H), 6.89−6.79 (m, 3H), 5.97 (s, 2H), 4.60

(d, J = 6.3 Hz, 2H).

7-((Benzo[d][1,3]dioxol-5-ylmethyl)carbamoyl)-5-bromo-

1,6-naphthyridin-8-yl 4-Methylbenzenesulfonate (III-3-9). III-

3-9 was prepared according to the same procedure described for I-3,

giving a white solid (yield: 68%). ¹H NMR (300 MHz, CDCl₃) δ 9.01

(dd, J = 1.2, 4.2 Hz, 1H), 8.54 (dd, J = 1.5, 8.7 Hz, 1H), 7.91 (d, J =

8.1 Hz, 2H), 7.87 (m, 1H), 7.67 (dd, J = 4.2, 8.4 Hz, 1H), 7.33 (d, J =

8.1 Hz, 2H), 6.86−6.75 (m, 3H), 5.94 (s, 2H), 4.51 (d, J = 6.0 Hz,

2H), 2.46 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-N-(benzo[d][1,3]-

dioxol-5-ylmethyl)-5-bromo-1,6-naphthyridine-7-carboxa-

mide (III-3). III-3 was prepared according to the same procedure

described for I-18, giving a yellow solid, yield 43%; mp 131−134 °C.

¹H NMR (300 MHz, CDCl₃): δ 9.62 (d, 1H, J = 8.4 Hz), 8.93 (dd,

8-((1r,4r)-4-Aminocyclohexylamino)-N-(4-(dimethylamino)-

benzyl)-5-bromo-1,6-naphthyridine-7-carboxamide (III-5). III-

5 was prepared according to the same procedure described for I-18,

giving a yellow solid, yield 62%. ¹H NMR (300 MHz, CDCl₃): δ 9.72

(d, J = 8.1 Hz, 1H), 8.95 (d, 1H, J = 2.4 Hz), 8.39 (d, 1H, J = 8.7 Hz),

8.20 (t, J = 5.7 Hz, 1H), 7.56 (dd, 1H, J = 2.4, 8.7 Hz), 7.24−7.27 (m,

2H), 6.73 (d, 2H, J = 8.7 Hz), 5.03 (m, 1H), 4.52 (d, J = 5.7 Hz, 2H),

3.24 (m, 1H), 2.94 (s, 6H), 2.29 (br s, 4H), 1.77 (m, 2H), 1.47 (m, 2H).

MS-EI m/z: 496 (M)⁺, 498 (M + 2)⁺. Anal. Calcd for C₂₄H₂₉BrN₆O·1/

4CF₃COOH·3H₂O: C 50.74, H 6.13, N 14.49. Found: C 50.71, H 6.22,

N 14.22.

5-Bromo-8-hydroxy-N-(3-methoxybenzyl)-1,6-naphthyri-

dine-7-carboxamide (III-6-8). III-6-8 was prepared according to the

same procedure described for I-1, giving a white solid (yield: 65%). ¹H

NMR (300 MHz, CDCl₃) δ 9.14 (dd, J = 1.5, 4.2 Hz, 1H), 8.48 (dd,

J = 1.5, 8.4 Hz, 1H), 8.26 (m, 1H), 7.68 (dd, J = 4.2, 8.4 Hz, 1H), 7.23

(m, 2H), 6.84 (m, 2H), 4.64 (d, J = 6.0 Hz, 2H), 3.78 (s, 3H).

5-Bromo-7-((3-methoxybenzyl)carbamoyl)-1,6-naphthyri-

din-8-yl 4-Methylbenzenesulfonate (III-6-9). III-6-9 was prepared

according to the same procedure described for I-3, giving a white solid

(yield: 82%). ¹H NMR (300 MHz, CDCl₃) δ 8.97 (dd, J = 1.5, 4.2 Hz,

1H), 8.50 (dd, J = 1.8, 7.5 Hz, 1H), 7.94 (m, 1H), 7.88 (d, J = 8.4 Hz,

2H), 7.64 (dd, J = 4.2, 8.4 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.25

(m, 1H), 6.92 (m, 2H), 6.80 (m, 1H), 4.68 (d, J = 6.0 Hz, 2H), 3.78

(s, 3H), 2.42 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(3-me-

thoxybenzyl)-1,6-naphthyridine-7-carboxamide (III-6). III-6

was prepared according to the same procedure described for I-18,

1H, J = 1.2, 4.2 Hz), 8.40 (dd, 1H, J = 1.2, 8.4 Hz), 8.23 (m, 1H), 7.58

(dd, 1H, J = 4.2, 8.4 Hz), 6.87−6.76 (m, 3H), 5.94 (s, 2H), 4.95 (m,

1H), 4.53 (d, 2H, J = 6.0 Hz), 2.75 (m, 1H), 2.18 (m, 2H), 1.92 (m,

4H), 1.34 (m, 4H). EI-MS m/z: 401 (M)⁺, 403 (M + 2)⁺. Anal. HPLC

t_R= 14.43 min, 100% (solvent system I); t_R= 5.54 min, 100% (solvent

system II).

(S)-5-Bromo-N-(1-(4-ﬂuorophenyl)ethyl)-8-hydroxy-1,6-

naphthyridine-7-carboxamide (III-4-8). III-4-8 was prepared

according to the same procedure described for I-1, giving a white solid

1

giving a yellow solid, yield 44%; mp 84−87 °C. H NMR (300 MHz,

CDCl₃): δ 9.62 (d, 1H, J = 8.4 Hz), 8.92 (dd, 1H, J = 1.5, 4.2 Hz),

8.39 (dd, 1H, J = 1.5, 8.4 Hz), 8.31 (m, 1H), 7.56 (dd, 1H, J = 4.2,

8.4 Hz), 7.30 (d, 2H, J = 8.4 Hz), 6.89 (d, 2H, J = 8.4 Hz), 4.95 (m, 1H),

4.56 (d, 2H, J = 6.3 Hz), 3.80 (s, 3H), 2.75 (m, 1H), 2.63 (m, 1H),

2.18 (m, 2H), 1.85 (m, 4H), 1.34 (m, 4H). EI-MS m/z: 483 (M)⁺, 485

(M + 2)⁺. Anal. HPLC t_R= 5.71 min, 100% (solvent system I); t_R

=

8.80 min, 99.27% (solvent system II).

9505

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Article

5-Bromo-8-hydroxy-N-(4-phenoxybenzyl)-1,6-naphthyri-

dine-7-carboxamide (III-7-8). III-7-8 was prepared according to the

same procedure described for I-1, giving the crude product as a brown

solid (yield: 90%) that was used for the next step directly.

2.76 (m, 1H), 2.18 (m, 2H), 1.87 (m, 2H), 1.37 (m, 4H). ¹³C NMR

(126 MHz, CDCl₃) δ 167.96 (s), 150.83 (s), 144.97 (s), 144.80 (s),

136.50 (s), 134.56 (s), 134.03 (s), 133.68 (s), 129.98 (s), 129.34 (s),

127.29 (s), 126.79 (s), 124.64 (s), 124.57 (s), 123.51 (s), 53.16 (s),

50.20 (s), 40.42 (s), 32.79 (s), 32.69 (s). EI-MS m/z: 521 (M)⁺.

HR-EIMS calcd for C₂₂H₂₂N₅OCl₂Br, 521.0385; found, 521.0388.

Anal. HPLC t_R= 29.56 min, 94.27% (solvent system I); t_R= 23.65 min,

95.94% (solvent system II).

5-Bromo-7-(4-phenoxybenzylcarbamoyl)-1,6-naphthyridin-

8-yl 4-Methylbenzenesulfonate (III-7-9). III-7-9 was prepared

according to the same procedure described for I-3, giving a white solid

1

(yield: 20%). H NMR (300 MHz, CDCl₃): δ 9.03 (d, J = 4.2 Hz,

1H), 8.65 (d, J = 8.4 Hz, 1H), 7.93 (m, 3H), 7.68 (dd, J = 4.2, 8.4 Hz,

1H), 7.31−7.37 (m, 6H), 7.10 (t, J = 6 Hz, 1H), 6.98−7.03 (m, 4H),

4.60 (d, J = 6 Hz, 2H), 2.47 (s, 3H).

5-Bromo-N-(3-(triﬂuoromethyl)benzyl)-8-hydroxy-1,6-naph-

thyridine-7-carboxamide (III-10-8). III-10-8 was prepared accord-

ing to the same procedure described for I-1, giving a white solid (yield:

1

8-((1r,4r)-4-Aminocyclohexylamino)-5-bromo-N-(4-phenoxy-

benzyl)-1,6-naphthyridine-7-carboxamide (III-7). III-7 was

prepared according to the same procedure described for I-18, giving a

71%). H NMR (300 MHz, CDCl₃) δ 9.20 (d, J = 1.5, 4.2 Hz, 1H),

8.55 (d, J = 1.5, 7.8 Hz, 1H), 8.25 (m, 1H), 7.74 (dd, J = 4.5, 8.7 Hz,

1H), 7.64−7.44 (m, 4H), 4.76 (d, J = 6.6 Hz, 2H).

1

yellow solid, yield 61%. H NMR (300 MHz, CDCl₃): δ 9.65 (d, J =

5-Bromo-7-((3-(triﬂuoromethyl))carbamoyl)-1,6-naphthyri-

din-8-yl 4-Methylbenzenesulfonate (III-10-9). III-10-9 was

prepared according to the same procedure described for I-3, giving a

white solid (yield: 48%). ¹H NMR (300 MHz, CDCl₃) δ 9.02 (dd, J =

1.5, 4.2 Hz, 1H), 8.57 (dd, J = 1.5, 8.4 Hz, 1H), 8.06 (m, 1H), 7.92 (d,

J = 8.4 Hz, 2H), 7.69 (dd, J = 4.2, 8.4 Hz, 1H), 7.71−7.48 (m, 4H),

7.31 (d, J = 8.1 Hz, 2H), 4.70 (d, J = 6.6 Hz, 2H), 2.47 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(3-

(triﬂuoromethyl)benzyl)-1,6-naphthyridine-7-carboxamide

(III-10). III-10 was prepared according to the same procedure

described for I-18, giving a yellow solid, yield 74%; mp 94−96 °C. ¹H

NMR (300 MHz, CDCl₃): δ 9.59 (d, 1H, J = 8.1 Hz), 8.59 (s, 1H),

8.42 (m, 2H), 7.61−7.44 (m, 4H), 4.97 (m, 1H), 4.69 (d, 2H, J =

6.0 Hz), 2.78 (m, 1H), 2.17 (m, 2H), 2.00−1.83 (m, 4H), 1.43−

1.25 (m, 4H). EI-MS m/z: 521 (M)⁺, 523 (M + 2)⁺. Anal. HPLC t_R=

13.11 min, 100% (solvent system I); t_R= 9.94 min, 95.0% (solvent

system II).

5-Bromo-N-(furan-2-ylmethyl)-8-hydroxy-1,6-naphthyri-

dine-7-carboxamide (III-11-8). III-11-8 was prepared according to

the same procedure described for I-1, giving a white solid (yield: 78%).

¹H NMR (300 MHz, CDCl₃) δ 13.20 (brs, 1H), 9.19 (dd, J = 1.5,

4.2 Hz, 1H), 8.54 (dd, J = 1.5, 8.4 Hz, 1H), 8.13 (m, 1H), 7.73 (dd, J =

4.2, 8.4 Hz, 1H), 7.42 (m, 1H), 6.37 (m, 2H), 4.70 (d, J = 6.0 Hz, 2H).

EI-MS m/z: 347 (M)⁺, 349 (M + 2)⁺.

5-Bromo-7-((furan-2-ylmethyl)carbamoyl)-1,6-naphthyridin-

8-yl 4-Methylbenzenesulfonate (III-11-9). III-11-9 was prepared

according to the same procedure described for I-3, giving a white solid

(yield: 88%). ¹H NMR (300 MHz, CDCl₃) δ 9.03 (dd, J = 1.5, 4.2 Hz,

1H), 8.56 (dd, J = 1.5, 8.7 Hz, 1H), 7.91 (d, J = 8.1 Hz, 2H), 7.89 (m,

1H), 7.68 (dd, J = 4.2, 8.7 Hz, 1H), 7.40 (m, 1H), 7.32 (d, J = 8.1 Hz,

2H), 6.33 (m, 2H), 4.58 (d, J = 6.0 Hz, 2H), 2.47 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(furan-2-

ylmethyl)-1,6-naphthyridine-7-carboxamide (III-11). III-11 was

prepared according to the same procedure described for I-18, giving a

yellow solid, yield 46%; mp 96−98 °C. ¹H NMR (300 MHz, CDCl₃):

δ 9.56 (d, 1H, J = 8.1 Hz), 8.90 (dd, 1H, J = 1.5, 3.9 Hz), 8.37 (d, 1H,

J = 8.4 Hz), 8.23 (m, 1H), 7.54 (dd, 1H, J = 4.2, 8.4 Hz), 7.36 (s, 1H),

6.30 (m, 2H), 4.93 (m, 1H), 4.57 (d, 2H, J = 6.0 Hz), 2.71 (m, 1H),

2.18 (m, 2H), 1.92 (m, 4H), 1.42 (m, 4H). EI-MS m/z: 443 (M)⁺, 445

(M + 2)⁺. Anal. HPLC t_R= 5.91 min, 100% (solvent system I); t_R=

6.55 min, 100% (solvent system II).

7.8 Hz, 1H), 8.94 (d, J = 3.9 Hz, 1H), 8.42 (d, J = 8.7 Hz, 1H), 8.31

(t, J = 6 Hz, 1H), 7.59 (dd, J = 3.9, 8.7 Hz, 1H), 7.26−7.36 (m, 5H),

7.10 (t, J = 6.9 Hz, 1H), 6.98−7.03 (m, 3H), 4.99 (m, 1H), 4.61 (d, J =

6 Hz, 2H), 2.77 (m, 1H), 2.18 (d, J = 12.9 Hz, 2H), 1.91 (d, J = 12.3

Hz, 2H), 1.25−1.49 (m, 4H). MS-EI m/z: 545 (M)⁺, 547 (M + 2)⁺.

HRMS calcd for C₂₈H₂₈BrN₅O₂(M)⁺, 545.1426; found, 545.1427.

Anal. Calcd for C₂₈H₂₈BrN₅O₂: C 61.58, H 5.13, N 12.71. Found:

C 61.54, H 5.16, N 12.82.

5-Bromo-N-(4-chlorobenzyl)-8-hydroxy-1,6-naphthyridine-

7-carboxamide (III-8-8). III-8-8 was prepared according to the same

procedure described for I-1, giving a white solid (yield: 89%). ¹H

NMR (300 MHz, CDCl₃) δ 9.20 (d, J = 3.9 Hz, 1H), 8.54 (d, J = 9 Hz,

1H), 8.19 (m, 1H), 7.74 (dd, J = 4.2, 8.4 Hz, 1H), 7.35−7.23 (m, 8H),

4.67 (d, J = 6.3 Hz, 2H).

5-Bromo-7-((4-chlorobenzyl)carbamoyl)-1,6-naphthyridin-

8-yl 4-Methylbenzenesulfonate (III-8-9). III-8-9 was prepared

according to the same procedure described for I-3, giving a white solid

(yield: 38%). ¹H NMR (300 MHz, CDCl₃) δ 9.01 (dd, J = 1.5, 4.2 Hz,

1H), 8.56 (dd, J = 1.5, 8.7 Hz, 1H), 7.98 (m, 1H), 7.92 (d, J = 8.4 Hz,

2H), 7.68 (dd, J = 4.2, 8.7 Hz, 1H), 7.32−7.30 (m, 6H), 4.61 (d, J =

6.0 Hz, 2H), 2.46 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(4-chloro-

benzyl)-1,6-naphthyridine-7-carboxamide (III-8). III-8 was pre-

pared according to the same procedure described for I-18, giving a

1

yellow solid, yield 58.5%; mp 128−130 °C. H NMR (300 MHz,

CDCl₃): δ 9.60 (d, 1H, J = 8.1 Hz), 9.30 (dd, 1H, J = 1.5, 4.2 Hz),

8.40 (dd, 1H, J = 4.2, 8.7 Hz), 8.33 (m, 1H), 7.58 (dd, 1H, J = 4.2,

8.4 Hz), 7.30 (s, 4H), 4.96 (m, 1H), 4.59 (d, 2H, J = 6.0 Hz), 2.77

(m, 1H), 2.17 (m, 2H), 1.94 (m, 2H), 1.43−1.24 (m, 4H). ¹³C NMR

(126 MHz, CDCl₃) δ 167.83 (s), 150.97 (s), 150.64 (s), 144.90 (s),

144.58 (s), 137.01 (s), 136.54 (s), 133.15 (s), 128.98 (s), 128.93 (s),

128.82 (s), 126.71 (s), 124.89 (s), 124.59 (s), 124.46 (s), 123.69 (s),

52.57 (s), 50.38 (s), 42.35 (s), 32.34 (s), 30.28 (s). EI-MS m/z: 487

(M)⁺, 489 (M + 2)⁺. HR-EIMS calcd for C₂₂H₂₃N₅OClBr, 487.0775;

found, 487.0774. Anal. HPLC t_R= 30.39 min, 95.29% (solvent system I);

t_R= 20.43 min, 100% (solvent system II).

5-Bromo-N-(2,4-dichlorobenzyl)-8-hydroxy-1,6-naphthyri-

dine-7-carboxamide (III-9-8). III-9-8 was prepared according to

the same procedure described for I-1, giving a white solid (yield: 82%).

¹H NMR (300 MHz, CDCl₃) δ 9.20 (d, J = 4.2 Hz, 1H), 8.55 (d, J =

7.8 Hz, 1H), 8.27 (m, 1H), 7.75 (dd, J = 4.2, 8.4 Hz, 1H), 7.42 (m,

3H), 4.76 (d, J = 6.3 Hz, 2H).

5-Bromo-8-hydroxy-N-(thiophen-2-ylmethyl)-1,6-naphthyri-

dine-7-carboxamide (III-12-8). III-12-8 was prepared according to

the same procedure described for I-1, giving the desired crude product

as a brown solid (yield: 98%) that was carried onto the next step

directly.

5-Bromo-7-(thiophen-2-ylmethylcarbamoyl)-1,6-naphthyri-

din-8-yl 4-Methylbenzenesulfonate (III-12-9). III-12-9 was

prepared according to the same procedure described for I-3, giving a

white solid (yield: 48%). ¹H NMR (300 MHz, CDCl₃): δ 9.05 (d, J =

2.4 Hz, 1H), 8.57 (d, J = 8.7 Hz, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.68

(dd, J = 2.4, 8.7 Hz, 1H), 7.34 (d, J = 8.1 Hz, 2H), 7.25−7.27 (m, 1H),

7.07 (m, 1H), 6.99 (m, 1H), 4.77 (d, J = 4.5 Hz, 2H), 2.48 (s, 3H).

8-((1r,4r)-4-Aminocyclohexylamino)-5-bromo-N-(thiophen-

2-ylmethyl)-1,6-naphthyridine-7-carboxamide (III-12). III-12

was prepared according to the same procedure described for I-18,

giving a yellow solid, yield 43%. ¹H NMR (300 MHz, CDCl₃): δ 9.60

5-Bromo-7-((2,4-dichlorobenzyl)carbamoyl)-1,6-naphthyri-

din-8-yl 4-Methylbenzenesulfonate (III-9-9). III-9-9 was prepared

according to the same procedure described for I-3, giving a white solid

(yield: 44%). ¹H NMR (300 MHz, CDCl₃) δ 9.02 (dd, J = 1.5, 4.2 Hz,

1H), 8.57 (dd, J = 1.8, 8.7 Hz, 1H), 8.08 (m, 1H), 7.90 (d, J = 8.4 Hz,

2H), 7.68 (dd, J = 4.2, 8.7 Hz, 1H), 7.42 (m, 2H), 7.32 (d, J = 8.4 Hz,

2H), 7.24 (m, 1H), 4.67 (d, J = 6.3 Hz, 2H), 2.47 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-(2,4-di-

chlorobenzyl)-1,6-naphthyridine-7-carboxamide (III-9). III-9

was prepared according to the same procedure described for I-18,

giving a yellow solid, yield 80.3%; mp 72−74 °C. ¹H NMR (300 MHz,

CDCl₃): δ 9.55 (d, 1H, J = 7.8 Hz), 8.94 (dd, 1H, J = 1.5, 3.9 Hz),

8.41 (d, 1H, J = 7.2 Hz), 8.40 (m, 1H), 7.59 (dd, 1H, J = 1.5, 8.7 Hz),

7.37 (s, 2H), 7.23 (m, 1H), 4.96 (m, 1H), 4.67 (d, 2H, J = 6.0 Hz),

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(d, J = 7.8 Hz, 1H), 8.95 (d, J = 4.5 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H),

8.32 (t, J = 6 Hz, 1H), 7.59 (dd, J = 4.5, 8.4 Hz, 1H), 7.23−7.25 (m,

1H), 7.07 (m, 1H), 6.98 (m, 1H), 4.98 (m, 1H), 4.80 (d, J = 6 Hz,

2H), 2.81 (m, 1H), 2.19 (d, J = 11.4 Hz, 2H), 1.94 (d, J = 12.3 Hz,

2H), 1.31−1.49 (m, 4H). MS-EI m/z: 459 (M)⁺, 461 (M + 2)⁺. Anal.

Calcd for C₂₀H₂₂BrN₅O·1/2H₂O: C 51.68, H 5.08, N 14.70. Found: C

51.71, H 4.85, N 14.46.

5-Bromo-8-hydroxy-N-(pyridin-2-ylmethyl)-1,6-naphthyri-

dine-7-carboxamide (III-13-8). III-13-8 was prepared according to

the same procedure described for I-1, giving the crude product as a

brown solid (yield: 90%) that was used for the next step directly.

5-Bromo-7-(pyridin-2-ylmethylcarbamoyl)-1,6-naphthyri-

din-8-yl 4-Methylbenzenesulfonate (III-13-9). III-13-9 was

prepared according to the same procedure described for I-3, giving a

white solid (yield: 52%). ¹H NMR (300 MHz, CDCl₃): δ 9.05 (d, J =

4.2 Hz, 1H), 8.57−8.60 (m, 2H), 8.51 (t, J = 5.7 Hz, 1H), 7.90 (d, J =

8.1 Hz, 2H), 7.68 (m, 1H), 7.36 (d, 1H), 7.29 (m, 2H), 7.21 (m, 1H),

4.69 (d, J = 5.7 Hz, 2H), 2.43(s, 3H).

to the same procedure described for I-3, giving a white solid (overall

yield: 65%). ¹H NMR (300 MHz, CDCl₃) δ 9.36 (s, 1H), 9.14 (dd, J =

1.5, 4.2 Hz, 1H), 8.59 (dd, J = 1.8, 8.4 Hz, 1H), 7.88 (d, J = 8.1 Hz,

2H), 7.73 (dd, J = 4.2, 8.4 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.34 (m,

4H), 7.13 (m, 1H), 2.35 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-phenyl-

1,6-naphthyridine-7-carboxamide (III-15). III-15 was prepared

according to the same procedure described for I-18, giving a yellow

1

solid, yield 48%; mp 208−210 °C. H NMR (300 MHz, CD₃OD):

δ 10.12 (s, 1H), 9.02 (d, 1H, J = 4.2 Hz), 8.55 (d, 1H, J = 8.7 Hz),

7.77 (dd, 1H, J = 4.2, 8.7 Hz), 7.71 (m, 2H), 7.31 (m, 2H), 7.14

(m, 1H), 4.98 (m, 1H), 3.18 (m, 1H), 2.33 (m, 2H), 2.11 (m, 2H),

1.53 (m, 4H). EI-MS m/z: 439 (M)⁺, 441 (M + 2)⁺. Anal. HPLC t_R=

15.07 min, 97.83% (solvent system I); t_R= 9.25 min, 94.67% (solvent

system II).

5-Bromo-8-hydroxy-N-cyclohexyl-1,6-naphthyridine-7-car-

boxamide (III-16-8). III-16-8 was prepared according to the same

procedure described for I-1, giving the crude product as a brown solid

(yield: 75%) that was used for the next step directly.

5-Bromo-7-(cyclohexylcarbamoyl)-1,6-naphthyridin-8-yl 4-

Methylbenzenesulfonate (III-16-9). III-16-9 was prepared accord-

ing to the same procedure described for I-3, giving the desired crude

product as a brown solid (yield: 40%) that was carried onto the next

step directly.

8-((1r,4r)-4-Aminocyclohexylamino)-5-bromo-N-(pyridin-2-

ylmethyl)-1,6-naphthyridine-7-carboxamide (III-13). III-13 was

prepared according to the same procedure described for I-18, giving a

1

yellow solid, yield 45%. H NMR (300 MHz, CDCl₃): δ 9.6 (d, J =

8.4 Hz, 1H), 8.94 (d, J = 3.9 Hz, 1H), 8.73 (t, J = 6 Hz, 1H), 8.61 (d,

J = 4.8 Hz, 1H), 8.43 (d, J = 10.2 Hz, 1H), 7.68 (t, 1H), 7.58 (dd, J =

3.9, 10.2 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.2 (t, 2H), 4.95 (m, 1H),

4.77 (d, J = 6 Hz, 2H), 2.74 (m, 1H), 2.16 (d, J = 13.5 Hz, 2H), 1.9

(d, J = 12.6 Hz, 2H), 1.23−1.43 (m, 4H). MS-EI m/z: 454 (M)⁺, 456

(M + 2)⁺. Anal. Calcd for C₂₁H₂₃BrN₆O·1/2CF₃COOH·CH₃OH:

C 50.74, H 5.09, N 15.44. Found: C 50.81, H 4.95, N 15.29.

5-Bromo-8-hydroxy-N-phenethyl-1,6-naphthyridine-7-car-

boxamide (III-14-8). III-14-8 was prepared according to the same

procedure described for I-1, giving a white solid (yield: 77%).

¹H NMR (300 MHz, CDCl₃) δ 9.18 (dd, J = 1.5, 4.2 Hz, 1H), 8.53

(dd, J = 1.5, 8.4 Hz, 1H), 7.93 (m, 1H), 7.71 (dd, J = 4.2, 8.4 Hz, 1H),

7.73−7.18 (m, 10H), 3.75 (q, J = 6.9 Hz, 2H), 2.98 (m, 4H), 2.76 (m,

2H). EI-MS m/z: 371 (M)⁺, 373 (M + 2)⁺.

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-cyclohex-

yl-1,6-naphthyridine-7-carboxamide (III-16). III-16 was prepared

according to the same procedure described for I-18, giving a yellow

1

solid, yield 61%. H NMR (300 MHz, CDCl₃): δ 9.68 (d, 1H, J =

7.8 Hz), 8.93 (d, 1H, J = 2.7 Hz), 8.41 (d, 1H, J = 8.4 Hz), 7.87

(d, 1H, J = 8.4 Hz), 7.57 (dd, 1H, J = 3.9, 8.4 Hz), 4.92 (m, 1H), 3.87

(m, 1H), 2.73 (m, 1H), 2.16 (m, 2H), 2.03 (m, 2H), 1.90 (m, 2H),

1.81 (m, 3H), 1.45−1.26 (m, 9H). EI-MS m/z: 445 (M)⁺, 447

(M + 2)⁺. Anal. HPLC t_R= 15.46 min, 95.0% (solvent system I); t_R=

10.92 min, 97.8% (solvent system II).

Methyl 8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-1,6-

naphthyridine-7-carboxylate (III-17). Following the procedure

as compound I-18, tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate

(65 mg, 0.3 mmol), 10 (44 mg, 0.1 mmol), and triethylamine (30 mg,

0.3 mmol) gave methyl 5-bromo-8-(((1r,4r)-4-((tert-butoxycarbonyl)-

amino)cyclohexyl)amino)-1,6-naphthyridine-7-carboxylate as a yellow

solid, yield 70%. ¹H NMR (300 MHz, CDCl₃): δ 8.95 (dd, 1H, J = 1.5,

4.2 Hz), 8.88 (d, 1H, J = 7.5 Hz), 8.47 (dd, 1H, J = 1.5, 8.4 Hz), 7.64

(dd, 1H, J = 4.2, 8.4 Hz), 4.92 (m, 1H), 4.42 (m, 1H), 3.97 (s, 3H),

3.47 (m, 1H), 2.20 (m, 2H), 2.05 (m, 2H), 1.45 (s, 9H), 1.43−1.23

(m, 4H). EI-MS m/z: 478 (M)⁺, 480 (M + 2)⁺.

The resultant methyl 5-bromo-8-(((1r,4r)-4-((tert-butoxycarbonyl)-

amino)cyclohexyl)amino)-1,6-naphthyridine-7-carboxylate was depro-

tected in 20% TFA−DCM solution to give the target compound III-

17 as a yellow solid, yield 70%; mp 178−182 °C. ¹H NMR (300 MHz,

CD₃OD): δ 9.01 (dd, 1H, J = 1.5, 4.2 Hz), 8.49 (dd, 1H, J = 1.5,

8.4 Hz), 7.78 (dd, 1H, J = 4.2, 8.4 Hz), 4.94 (m, 1H), 3.93 (s, 3H),

3.19 (m, 1H), 2.31 (m, 2H), 2.13 (m, 2H), 1.68−1.39 (m, 4H). EI-MS

m/z: 378 (M)⁺, 380 (M + 2)⁺. Anal. HPLC t_R= 10.366 min, 100%

(solvent system I); t_R= 5.847 min, 100% (solvent system II).

Methyl 5-Bromo-8-(tosyloxy)-1,6-naphthyridine-7-carboxy-

late (10). The intermediate 10 was prepared according to the same

procedure described for I-3, giving a white solid (yield: 77%).

¹H NMR (300 MHz, CDCl₃) δ 9.06 (dd, J = 1.5, 4.2 Hz, 1H), 8.60

(dd, J = 1.5, 8.7 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.72 (dd, J = 4.2,

8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 3.83 (s, 3H), 2.47 (s, 3H).

EI-MS m/z: 436 (M)⁺.

5-Bromo-7-(phenethylcarbamoyl)-1,6-naphthyridin-8-yl 4-

Methylbenzenesulfonate (III-14-9). III-14-9 was prepared accord-

ing to the same procedure described for I-3, giving a white solid (yield:

1

86%). H NMR (300 MHz, CDCl₃) δ 8.98 (d, J = 3.6 Hz, 1H), 8.51

(d, J = 8.4 Hz, 1H), 7.90 (d, J = 7.8 Hz, 2H), 7.65 (dd, J = 4.2, 8.4 Hz,

1H), 7.64 (m, 1H), 7.35−7.21 (m, 7H), 3.63 (q, J = 6.9 Hz, 2H), 2.90

(t, J = 8.1 Hz, 2H), 2.44 (s, 3H).

8-(((1r,4r)-4-Aminocyclohexyl)amino)-5-bromo-N-pheneth-

yl-1,6-naphthyridine-7-carboxamide (III-14). III-14 was prepared

according to the same procedure described for I-18, giving a yellow

solid, yield 41%; mp 64−65 °C. ¹H NMR (300 MHz, CDCl₃): δ 9.60

(d, 1H, J = 8.4 Hz), 8.93 (d, 1H, J = 3.9 Hz), 8.40 (d, 1H, J = 8.4 Hz),

8.11 (m, 1H), 7.57 (dd, 1H, J = 4.2, 8.4 Hz), 7.36−7.24 (m, 5H), 4.93

(m, 1H), 3.65 (m, 2H), 2.95 (m, 2H), 2.75 (m, 1H), 2.18 (m, 2H),

1.94 (m, 2H), 1.42 (m, 4H). EI-MS m/z: 467 (M)⁺, 469 (M + 2)⁺.

Anal. HPLC t_R= 15.05 min, 97.78% (solvent system I); t_R= 8.30 min,

99.26% (solvent system II).

5-Bromo-8-hydroxy-N-phenyl-1,6-naphthyridine-7-carboxa-

mide (III-15-8). Compound 7 (150 mg, 0.735 mmol) was dissolved in

1N LiOH (2 mL) and MeOH (4 mL). The solution was heated to

reﬂux for 5 h. After cooling to rt, the pH of the reaction mixture was

adjusted to 3 with 1N HCl solution. The mixture was ﬁltered to give

crude product as a tan solid 0.116 g (yield: 81%). ¹H NMR (300 MHz,

CD₃OD): δ 9.21 (s, 1H), 8.57 (d, 1H, J = 7.8 Hz), 7.97 (s, 1H).

EI-MS m/z: 269 (M)⁺.

Triphosgene (150 mg, 0.735 mmol) was added to DMF solution of

5-bromo-8-hydroxy-1,6-naphthyridine-7-carboxylic acid (45 mg, 0.234

mmol) and DIPEA (0.16 mL, 0.398 mmol) at 0 °C. The solution was

stirred for 60 min at rt. And then excess aniline was added. After the

reaction was completed, the mixture was diluted with DCM. The

organic layer was washed with saturated NaCl solution. The organic

layer was dried over Na₂SO₄and evaporated to yield crude III-15-8,

which was used for the next step directly.

Methyl 5-Bromo-8-((4-ﬂuorobenzyl)amino)-1,6-naphthyri-

dine-7-carboxylate (11). Compound 11 was prepared according

to the same procedure described for I-18, giving a yellow solid, yield

1

61%. H NMR (300 MHz, CD₃OD): δ 9.15 (m, 1H), 8.93 (dd, 1H,

J = 1.5, 4.2 Hz), 8.48 (dd, 1H, J = 1.8, 8.4 Hz), 7.64 (dd, 1H, J = 4.2,

8.7 Hz), 7.37−7.32 (m, 2H), 7.02 (m, 2H), 5.30 (d, 2H, J = 6.0 Hz),

3.97 (s, 3H).

N-((1r,4r)-4-Aminocyclohexyl)-5-bromo-8-((4-ﬂuorobenzyl)-

amino)-1,6-naphthyridine-7-carboxamide (III-18). Compound

11 was dissolved in 1N LiOH (2 mL) and THF (2 mL). The solution

5-Bromo-7-(phenylcarbamoyl)-1,6-naphthyridin-8-yl-4-

methylbenzenesulfonate (III-15-9). III-15-9 was prepared according

9507

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Journal of Medicinal Chemistry

Article

was stirred under nitrogen at rt. After the reaction was completed, the

reaction mixture was concentrated. The residue was then partitioned

between H₂O and DCM. The pH of the H₂O phase was adjusted to

3 and was extracted with DCM 3 times. The DCM layer was dried

over Na₂SO₄and evaporated to give crude product as a tan solid, yield

92%. ¹H NMR (300 MHz, CDCl₃): δ 9.37 (m, 1H), 8.96 (dd, 1H, J =

1.5, 4.2 Hz), 8.48 (dd, 1H, J = 1.8, 8.4 Hz), 7.67 (dd, 1H, J = 4.5,

8.4 Hz), 7.35 (m, 2H), 6.99 (m, 2H), 5.42 (d, 2H, J = 5.7 Hz).

The resulting 5-bromo-8-((4-ﬂuorobenzyl)amino)-1,6-naphthyri-

dine-7-carboxylic acid (37.6 mg, 0.1 mmol), tert-butyl ((1r,4r)-4-

aminocyclohexyl)carbamate (33.6 mg, 0.15 mmol), EDCI (40 mg,

0.2 mmol), HOBt (27 mg, 0.2 mmol), and DIPEA (26 mg, 0.2 mmol)

were dissolved in dry DCM (15 mL), and the solution was stirred for

3 h at rt. After the reaction was completed, the mixture was diluted

with DCM. The organic layer was washed with saturated NaHCO₃

solution and saturated NaCl solution. Usual workup and puriﬁcation

by column chromatography on silica gel eluting with PE:EtOAc (4:1)

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1

gave a yellow solid (50 mg, yield 95%); mp 189−191 °C. H NMR

(300 MHz, CDCl₃): δ 8.90 (dd, 1H, J = 1.5, 3.9 Hz), 8.42 (dd, 1H, J =

1.5, 8.4 Hz), 7.81 (m, 1H), 7.57 (dd, 1H, J = 4.2, 8.4 Hz), 7.37

(m, 2H), 6.97 (m, 2H), 5.33 (s, 2H), 4.40 (m, 1H), 3.80 (m, 1H), 3.48

(m, 2H), 2.06 (m, 4H), 1.45 (s, 9H), 1.36 (m, 4H).

The resultant tert-butyl ((1r,4r)-4-(5-bromo-8-((4-ﬂuorobenzyl)-

amino)-1,6-naphthyridine-7-carboxamido)cyclohexyl)carbamate was

deprotected in 20% TFA−DCM solution to give the target compound

1

III-18 as a yellow solid, yield 95%; mp 210−213 °C. H NMR (300

MHz, DMSO-d₆): δ 9.07 (d, 1H, J = 3 Hz), 8.47 (d, 1H, J = 8.7 Hz),

8.25 (d, 1H, J = 7.8 Hz), 7.86 (dd, 1H, J = 4.2, 8.4 Hz), 7.40 (m, 2H),

7.15 (m, 2H), 5.26 (s, 2H), 3.72 (m, 2H), 2.99 (m, 2H), 2.73 (m, 1H),

1.93 (m, 4H), 1.48 (m, 4H). EI-MS m/z: 471 (M)⁺, 473 (M + 2)⁺.

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AUTHOR INFORMATION

Corresponding Author

*For Y.-Q.L.: phone, 86-21-50806876; fax, +86-21-50806876;

E-mail, yqlong@mail.shcnc.ac.cn. For N.N.: phone, 323-442-

2341; fax, 323-442-1390; E-mail: neamati@usc.edu.

■

Author Contributions

^∥These authors equally contributed to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This study was supported in part by the National Natural

Science Foundation of China nos. 81021062, 81072527, and

81123004 (Y-Q.L.), 81102483 (L-M. Y); the Key Scientiﬁc and

Technological Program of China (2012ZX10001-006) (Y.T.Z);

and the Sharon and William Cockrell Endowed Cancer Research

Fund (N.N.).

ABBREVIATIONS USED

■

AIDS, acquired immune deﬁciency syndrome; DCM,

dichloromethane; DEAD, diethyl azodicarboxylate; DIPEA,

diisopropylethylamine; DMF, dimethylformamide; DMPU,

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone; EDCI, 1-ethyl-

3-(3-dimethylaminopropyl) carbodiimide; EDTA, ethylenedia-

minetetraacetic acid; EGFR, epidermal growth factor receptor;

ErbB2, human epidermal growth factor receptor 2; HIV-1, human

immunodeﬁciency virus type 1; HOBt, hydroxybenzotriazole; IC₅₀,

half-maximal inhibitory concentration; IN, HIV-1 integrase; KDR,

kinase insert domain containing receptor; NBS, N-bromosucci-

nimide; SAR, structure−activity relationship; c-Src, cellular proto-

oncogene tyrosine kinase; TFA, triﬂuoroacetic acid; THF,

tetrahydrofuran

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Article Doi

Repositioning HIV-1 integrase inhibitors for cancer therapeutics: 1,6-naphthyridine-7-carboxamide as a promising scaffold with drug-like properties

DOI: 10.1021/jm300667v

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