4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.bmc.2014.05.028

Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC ₅₀ values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

Full text of DOI:10.1016/j.bmc.2014.05.028

Accepted Manuscript
4-Quinolone-3-carboxylic Acids as Cell-permeable Inhibitors of Protein Tyro-
sine Phosphatase 1B
Ying Zhi, Li-Xin Gao, Yi Jin, Chun-Lan Tang, Jing-Ya Li, Jia Li, Ya-Qiu Long
PII:
S0968-0896(14)00377-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.028
BMC 11589
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 April 2014
6 May 2014
7 May 2014
Please cite this article as: Zhi, Y., Gao, L-X., Jin, Y., Tang, C-L., Li, J-Y., Li, J., Long, Y-Q., 4-Quinolone-3-
carboxylic Acids as Cell-permeable Inhibitors of Protein Tyrosine Phosphatase 1B, Bioorganic & Medicinal
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.028
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4-Quinolone-3-carboxylic Acids as Cell-permeable Inhibitors of
Protein Tyrosine Phosphatase 1B
Ying Zhi,a Li-Xin Gao,b Yi Jin,c Chun-Lan Tang,b Jing-Ya Li,b Jia Li,b,* Ya-Qiu
Longa,*
aCAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
bThe National Center for Drug Screening, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences,189 Guoshoujin Road, Shanghai 201203, China
cSchool of Chemical Science and Technology, Yunnan University, Kunming 650091,
China
∗To whom correspondence should be addressed. Ya-Qiu Long, Phone:
86-21-50806876; Fax: +86-21-50806876; Email: yqlong@mail.shcnc.ac.cn and Jia Li,
Phone: 86-21-50801552; E-mail: jli@mail.shcnc.ac.cn
1

Abstract: Protein tyrosine phosphatase 1B is a negative regulator in the insulin and
leptin signaling pathways, and has emerged as an attractive target for the treatment of
type 2 diabetes and obesity. However, the essential pharmacophore of charged
phosphotyrosine or its mimetic confer low selectivity and poor cell permeability.
Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a
drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time
as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic
groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent
PTP1B inhibitors with low micromolar IC50 values. These quinolone-3-carboxylate
based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP’s.
Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated
with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin
signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that
the quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B
active site in the WPD loop closed conformation. Taken together, our study shows that
the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological
properties over previously described PTB1B inhibitors and warrant further preclinical
studies.
Key words: protein tyrosine phosphatase 1B inhibitor; quinolone-3-carboxylic acid;
bidentate ligand; bioisostere; insulin receptor signaling
2

Introduction
The rapidly increased incidence of type 2 diabetes mellitus (T2DM) and obesity
in the population worldwide has fueled an intensified search for new therapeutic
treatment options.1, 2 Insulin resistance is a major pathophysiological factor in the
development of T2DM, occurring mainly in muscles, adipose tissues, and liver,
leading to reduced glucose uptake and utilization and increased glucose production,
respectively.3, 4 The protein tyrosine phosphatase 1B (PTP1B) is a crucial negative
6
regulator in signal transduction for both insulin and leptin pathways,5,
by
dephosphorylating and inactivating the insulin receptor (IR),7 insulin receptor
substrates (IRS)8 as well as Janus kinase 2 (JAK2).9, 10 Two independent studies of
PTP1B knockout mice have revealed phenotypes of enhanced insulin sensitivity,
improved glycemic control, and resistance to high fat diet induced obesity.
Interestingly, the PTP1B deficient mice still have normal development and
12
longevity.11,
Furthermore, treatment of diabetic mice with PTP1B antisense
oligonucleotides reduced the expression level of the enzyme and subsequently
normalized blood glucose and improved insulin sensitivity.13 Therefore, PTP1B has
emerged as a promising and a validated therapeutic target to effectively treat T2DM
and obesity.14, 15 Accordingly, small molecule inhibitors of this enzyme have been
pursued extensively by both industry and academia.16-18 However, only two small
molecule PTP1B inhibitors entered clinical trials.18, 19 Poor bioavailability and low
selectivity of negatively charged phosphotyrosine mimetics are the major concerns of
the many previously disclosed PTP1B inhibitors.20 More significantly, because of the
structural homologies in PTPs, designing selective PTP1B inhibitors has remained
elusive.
Previously, we identified aryl diketoacid chemotype as a new class of PTP1B
noncompetitive inhibitors that target the enzyme inactive conformation, conferring
excellent selectivity and cellular efficacy.21 Our compounds are distinctly different
from most reported PTP1B inhibitors that are active site-directed competitive
18
inhibitors.16,
Crystal structures of ligand-bound PTP1B reveal that the aryl
diketoacid derivatives bind to the PTP1B active site. Remarkably, they bind to the
3

catalytically inactive, WPD loop open conformation, thus providing a unique
opportunity for creating active site-directed PTP1B inhibitors with improved
pharmacological properties. Because some diketoacids are biologically labile, we
decided to develop novel bioavailable PTP1B inhibitors using bioisostere replacement
strategy. 4-Quinolone-3-carboxylic acid has been reported as an effective bioisostere
of the diketoacid pharmacophore in the HIV-1 integrase inhibitor design.22, 23 Also,
this scaffold is present in various bioactive compounds and several approved drugs.24
Therefore, starting from the aryl diketoacid PTP1B inhibitor LZP-25,21 we introduced
for the first time the 4-quinolone-3-carboxylic acid scaffold into the design of novel
PTP1B inhibitors for enhanced potency and properties. As depicted in Scheme 1, the
substitution on the quinolone motif was optimized to generate bidentate inhibitors,
affording a new class of selective and cell-permeable PTP1B inhibitors bearing the
4-quinolone-3-carboxyl motif.
Scheme 1. The design of 4-quinolone-3-carboxylic acid-based PTP1B inhibitors with
investigations on the substitution effect and the conjugation pattern of the bidentate
inhibitor
Inhibitor Design
Previously, we showed that the hydrophobic group was essential for the PTP1B
inhibition by the aryl diketoacid lead compound LZP-25.21 Therefore, the new series
of quinolones were designed bearing the same hydrophobic moiety. Our initial SAR
study on the 4-quinolone-3-carboxylate-based PTP1B inhibitors was focused on the
appropriate installation of a hydrophobic moiety and the neutralization of the
4

carboxylic acid for a better bioavailability. Thus, the substituent effect of the
quinolone motif on the PTP1B inhibitory activity was investigated, with respect to the
substituents on the phenyl ring, the 3-carboxyl derivatization and 1-N substituent
(Scheme 1, compounds 1a-k and 2a-i).
Inspired by the previous finding that the dimerization of the aryl diketoacid
through an amide linkage remarkably improved the PTP1B inhibitory activity
(LZP-40, IC50 = 2.8 µM vs. LZP-25, IC50 = 20 µM),21 we further explored
3-carboxylquinolone-containing bidentate PTP1B inhibitors. To achieve an optimal
conjugation and thus gain higher potency and selectivity, three types of bifunctional
molecules were designed by conjugating a salicylic acid or an aryl diketoacid
functionality with the 4-quinolone-3-carboxylic acid at N-1, C-3 or C-6 position
(Scheme 1, compounds 3a-h). For the PTP1B enzyme, a secondary non-catalytic
arylphosphate-binding site adjacent to the active site was also identified representing
an important structural feature of this phosphatase.25 The salicylic acid moiety was
reported to facilitate the interaction with the second binding site of PTP1B pocket.26
Taken together, two classes of novel quinolone-3-carboxy-based PTP1B inhibitors, i.e.
substituted monomers and bidentate ligands, were designed and synthesized as shown
in Scheme 1.
Synthesis
The variously substituted quinolone-3-carboxylic acid derivatives (1a-k, 2a-i)
were synthesized according to a known procedure22 based on Grohe-Heitzer
reaction,27 as depicted in Scheme 2. Starting from the substituted benzoic acid, the
acid chloride was condensed with ethyl 3-(dimethylamino)acrylate to produce the
enaminoester 4. After the substituent exchange at N-1 position from dimethyl to
benzyl or cyclopropyl group, the intramolecular SNAr reaction of the halo substituted
phenylenamine 5 with potassium carbonate gave the key intermediate quinolone
(6a-f). Further substituents on the phenyl ring was introduced by Negishi coupling of
6-iodide quinolone with aryl zinc bromides (6g-i). Hydrolysis of these
quinolone-3-carboxylates (6a-i) with HCl (6N) in MeOH at reflux produced the target
5

compounds (1a-e, 1g-j). Hydrogenolysis catalyzed by Pd-C converted the 6-nitro
substituted quinolone-3-carboxylic acid (1e) into the 6-amino analog (1f).
Nucleophilic substitution of the 6-fluoro substituted quinolone-3-carboxylic acid 1a
by piperazine in NMP under microwave irradiation followed by reductive amination
with benzaldehyde delivered compound 1k. Further coupling of these substituted
quinolone-3-caroxylic acids with various amines by BOP and Et3N readily generated
the 3-amidation analogs 2c-i. It was worth noting that the 3-esterification products
2a-b were obtained as the side-products of the Negishi coupling of 6-iodide
quinolone-3-ester 6b with the zinc benzyl bromide and zinc 4-fluorobenzyl bromide,
respectively.
Scheme 2. Synthesis of substituted 4-quinolone-3-carboxylic acid derivativesa
O
O
O
O
O
a
b
d
c
OEt
OEt
OH
R1
R1
R1
F
X
X
HN R3
N
5a
5b
5c
5d
5e
5f
X= F, Cl
R1=5-F, R3= c-Pr
R1=5-I, R3= c-Pr
R1= 5-F, R3= Bn
R1=5-I, R3= Bn
R1= 5-NO2, R3= 4-F-Bn
R1=4-F, R3= Bn
4a
4b
4c
4d
4e
R1= 5-F, X = F
R1= 5-I, X = F
R1= 5-NO2, X = F
R1= 4-F, X = F
R1= 5-I, X = Cl
O
O
O
O
O
O
j
R2
e
OH
OEt
R1
R1
R1
N
N
N
R3
R3
R3
1a
1b
1c
1d
1e
1f
R1= 6-F, R3= c-Pr
R1= 6-I, R3= c-Pr
R1= 6-F, R3= Bn
R3:
6a
6b
6c
6d
6e
6f
R1= 6-F, R3= c-Pr
R1= 6-I, R3= c-Pr
R1= 6-F, R3= Bn
R1= 6-I, R3= Bn
R1= 6-NO2, R3= 4-F-Bn
R1= 7-F, R3= Bn
2a
R1= 6-I, R2= OBn
R1= 6-(4-F-Bn), R2= O-(4-F
R1= 6-I, R2= NHBn
R1= 6-I, R2= NH-(4-F-Bn)
R1= 6-F, R2= NHBn
R1= 7-F, R2= NHBn
2b
2c
2d
2e
2f
R1= 6-I, R3= Bn
R1= 6-NO2, R3= 4-F-Bn
R1= 6-NH2, R3= 4-F-Bn
R1= 7-F, R3= Bn
g
1g
1h
1i
h,i
f
f
R= Bn, R3= c-Pr
R= 4-F-Bn, R3= c-Pr
R= Bn, R3= Bn
6g
6h
R1= 6-Bn, R3= c-Pr
1j
R3:
R1= 6-(4-F-Bn), R3= c-Pr
R1= 6-Bn, R3= Bn
6i
2g
2h
2i
R1= 6-I, R2= NHBn
R1= 6-F, R2= NHBn
R1= 6-Bn, R2=OBn
Bn
R3= c-Pr
N
1k
R1=6-
N
aReagents and conditions: (a) SOCl2, reflux; (b) ethyl 3-(dimethylamino)acrylate, Et3N, toluene,
reflux; (c) cyclopropylamine or benzylamine, Et2O/EtOH (1/3, v/v), rt; (d) K2CO3, DMF, 100°C;
(e) 6N HCl, MeOH, reflux; (f) i. Zinc dust, benzyl bromide or 4-fluorobenzyl iodide, TMSCl,
BrCH2CH2Br, THF, 65°C, 2.5h; ii. Pd2(dba)3, tri(2-furyl)phosphine, THF, 65°C; (g) H2, Pd/C (5%),
MeOH, rt; (h) piperazine, NMP, 200°C, 0.5 hr, microwave irradiation; (i) aldehyde, NaBH(OAc)3,
6

AcOH, DCE, rt, 6 h; (j) amine, BOP, Et3N, DCM, rt (compounds 2a and 2b were isolated during
the preparation of 6h and 6i under Negishi coupling reaction conditions).
Three classes of quinolone-3-carboxylic acid based bidentate inhibitors were
designed with N-1, C-3 and C-6 as the attaching points using salicylic acid and aryl
diketoacid as the second binding groups (3a-h). As shown in Scheme 3, the synthesis
of N1-salicylic acid conjugated quinolone-3-carboxylates was commenced with
5-methylsalicylic acid, which was converted to the amino component 9 through
bromination and amination. Then the nucleophilic attack of the enaminoester 4b by
the aminomethyl substituted salicylic ester 9 gave the quinolone-salicylic conjugate
precursor 10. The subsequent cyclization and hydrolysis/amidation furnished the
target compounds 3a-c.
Scheme 3. Synthetic route toward the N1-salicylic acid conjugated quinolone PTP1B
inhibitorsa
aReagents and conditions: (a) CH3I, K2CO3, acetone, reflux; (b) Perbenzoic acid, NBS, CCl4,
reflux; (c) NH3 (7 N in MeOH), 70°C, 2 hr; (d) 4e, Et2O / EtOH (1 / 3, v / v), rt; (e) K2CO3, DMF,
100°C; (f) 6N HCl, MeOH, reflux; (g) BBr3, DCM, rt; (h) Benzylamine, Bop, Et3N, DCM, rt.
Similarly, the attachment of an aryl diketoacid moiety on the N-1 site was
7

realized by the introduction of an aminomethyl substituted acetophenone, which was
further transformed into the diketoacid functionality (3d-e). As described in Scheme 4,
3-acetylbenzonitrile served as the starting material and underwent the ketal protection
and cyano reduction reactions, affording the amino component (13) for the N-1
substitution of the enaminoester 4e.
Scheme 4. Synthetic route toward the N1-aryl diketoacid conjugated
quinolone-3-carboxylate PTP1B inhibitorsa
O
N
O
O
OEt
OEt
EtO
EtO
I
CN
CN
CH2NH2
OEt
a
b
c
OEt
OEt
12
14
13
O
N
O
O
O
O
N
O
I
I
I
OH
g
d
e, f
OH
OEt
O
OH
N
O
OH
O
OEt
OH
3e
O
3d
O
15
aReagents and conditions: (a) p-toluenesulfonic acid, triethoxy methane, EtOH, rt; (b) Lithium
aluminum hydride, anhydrous THF, 0°C -rt; (c) i. 4b, Et2O/EtOH (1/3, v/v), rt; ii. K2CO3, DMF,
100°C; (d) formic acid, rt; (e) 6N HCl, MeOH, reflux; (f) (EtOOC)2, NaOMe, anhydrous THF,
50°C, 30 min; (g) 1N NaOH, 1N HCl, THF / EtOH (1 / 1, v / v), rt.
The C-3 conjugated bifunctional molecules (3f-g) were synthesized through a
3-oxypropylamino linker (Scheme 5). Starting from commercially available methyl
2,6-dihydroxybenzoate, etherification with 3-bromopropyl carbamate provided the
alkylamino substituted salicylate (16). Then, amidation of various
quinolone-3-carboxylic acids with the salicylic acid containing propylamine (17)
furnished the C3-salicylate conjugated quinolone-3-carboxylic acid derivatives (3f-g).
We also tried the incorporation of the salicylate moiety into the C-6 position of
the quinolone. An aldehyde group was introduced into the salicylate moiety as a
handle (20) to join the quinolone motif by a reductive amination reaction, producing a
C6-piperazino substituted quinolone-3-carboxylic acid (3h).
8

Scheme 5. Synthetic route toward the C-3/6 salicylic acid conjugated quinolone
PTP1B inhibitorsa
aReagents and conditions: (a) tert-Butyl (3-bromopropyl)carbamate, K2CO3, DMF, 80°C; (b) TFA,
DCM, rt; (c) 1g or 1e, Bop, Et3N, DCM, rt; (d) benzyl bromide, K2CO3, anhydrous CH3CN, 60°C;
(e) 3-bromopropan-1-ol, K2CO3, anhydrous CH3CN, 80°C; (f) Dess-Martin periodinane,
anhydrous DCM, rt;(g) 1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic
acid, NaBH(OAc)3, AcOH, DCE, rt.
Results and Discussion
4-Quinolone-3-carboxylates with optimal hydrophobic substitution displayed
effective inhibition against PTP1B enzyme. This is the first attempt to develop the
4-quinolone-3-carboxylic acid motif as an isostere of aryl diketoacid and as a new
phosphotyrosine surrogate to improve cell permeability and PTP1B inhibition.
Initially, substitution with respect to the electronic and hydrophobic effect on the
phenyl ring and N1-position were investigated. Typical substituents such as halogen,
nitro, amino and phenyl group on the C6/7 position in combination with a
hydrophobic group on N-1 site were assessed (Table 1). Several new
quinolone-3-carboxylate compounds exhibited effective PTP1B inhibition. As
expected, the introduction of a hydrophobic group into the quinolone-3-carboxylate
motif was beneficial for the PTP1B binding. An optimal combination of a bulky
benzyl group on the phenyl ring and a small cyclopropyl group on the N-1 position
resulted in low micromolar inhibitory activity against the PTP1B enzyme (Table 1, 1h,
1i, 1k). Notably, the planar hydrophobic group installed on the phenyl ring was
superior to the N-1 position, as N1-benzyl substituted analogs displayed weak PTP1B
9

inhibition regardless of the substituents on the phenyl ring (Table 1, 1c-g). However,
double benzyl substitutions on C-6 and N-1 positions attenuated the inhibitory activity
(1j). More significantly, the introduction of a hydrophobic group such as benzyl or
4-fluorobenzyl into the 3-carboxylic acid site by amidation or esterification generated
neutral potent PTP1B inhibitors (Table 2, 2a-b, 2g), which might confer better cell
permeability because of decreased charge. Interestingly, the C3-benzylamino
substituent greatly boosted the PTP1B inhibitory activity of the inert N1-benzyl
substituted quinolone (2g vs 1d). This suggests a suitable combination of the
hydrophobic groups on the polar 4-quinolone-3-carboxylic acid motif advances the
PTP1B binding. As shown in Tables 1 and 2, an optimal combination includes an
aromatic group at C-6/C-3 and a small alkyl group on N-1, or double aromatic
substitutions at N-1/C-6 and C-3.
Table 1. Investigation on the phenyl substitution and the N1-substitutiona
PTP1B inhibition
% at 20 µg/mL IC50 (µg/mL)b
Compd.
R1
R3
1a
1b
1c
1d
1e
1f
6-F
6-I
c-Pr
c-Pr
Bn
0.8
3.5
-
-
6-F
15.1
3.1
-
6-I
Bn
-
6-NO2
6-NH2
7-F
4-F-Bn
4-F-Bn
Bn
36.2
12.4
0.7
-
-
-
1g
1h
1i
6-Bn
6-(4-F-Bn)
6-Bn
c-Pr
c-Pr
Bn
87.5
71.1
15.1
7.5±1.0
12.3±0.9
-
1j
c-Pr
62.3
9.2±1.2
1k
6-
10

aAssay details were described in the Experimental section. b The pNPP competitive assay. IC50
values were determined by regression analysis and expressed as means SD of three independent
replications.
Table 2. Investigation on the phenyl substitution and the carboxyl-derivatizationa
O
O
O
O
R2
R2
R1
R1
N
N
2a-f
2g-i
PTP1B inhibition
% at 20 µg/mL IC50 (µg/mL)b
Compd.
R1
R2
6-I
OBn
80.5
73.0
6.1
11.4±1.7
2a
2b
2c
2d
2e
2f
6-(4-F-Bn)
6-I
O-(4-F-Bn)
NHBn
14.0±2.8
-
3.3
-
6-I
NH-(4-F-Bn)
NHBn
0.2
-
6-F
7.4
-
7-F
NHBn
61.8
41.4
0.1
13.9±2.5
6-I
NHBn
2g
2h
2i
6-F
NHBn
-
6-Bn
OBn
-
b
aAssay details were described in the Experimental section. The pNPP competitive assay. IC50
values were determined by regression analysis and expressed as means SD of three independent
replications.
Optimal
conjugation
of
the
quinolone-3-carboxylate
with
aryl
diketoacid/salicylic acid enhanced the PTP1B inhibitory activity. We showed that
the 4-quinolone-3-carboxylic acid, with suitable modifications, can serve as a new
motif for effective PTP1B inhibition. Further structural optimization was turned to the
bidentate ligand exploration to gain an improved PTP1B potency and selectivity over
other PTPs. Taken into account our preliminary SAR study and synthetic accessibility,
we chose 6-iodo-4-quinolone-3-carboxylic acid as a template to join the second
binding moiety (Table 2, compounds 2a and 2g). In order to achieve an optimal
11

conjugation of the two functional motifs, we designed three types of bidentate
inhibitors by employing the aryl diketoacid and salicylic acid as the second binding
groups and incorporated them into the quinolone scaffold at the N-1, C-3 and C-6
positions, respectively (Table 3).
Most of these bidentate inhibitors exhibited improved PTP1B inhibitory activity
compared to the corresponding quinolone-3-carboxylic acid counterpart. As shown in
Table 3, the incorporation of a salicylic acid into the quinolone-3-carboxylic scaffold
facilitated the enzyme inhibition (3b vs 1d, 3f vs. 1l, 3g vs 1j), resulting in potent
PTP1B inhibition with low micromolar IC50 values (Table 3, 3b, IC50 = 8.1 µg/mL; 3f,
IC50 = 9.4 µg/mL; 3g, IC50 = 6.5 µg/mL). As a comparison, the low potency of the
methoxyl-substituted analogs 3a and 3c suggested that the free hydroxyl group of the
salicylic acid moiety was critical for the PTP1B inhibitory activity. For the aryl
diketoacid as the second binding group, its conjugate with the quinolone motif at N-1
position afforded the most potent PTP1B inhibitors among the quinolone series herein
(Table 3, 3d, IC50 = 3.12 µg/mL; 3e, IC50 = 2.40 µg/mL). Our previous study showed
that only the free aryl diketo acid exhibited PTP1B inhibitory activity and no effect
was observed with the ester form. However, compound 3d in which the aryl diketo
acid was esterified still displayed a comparable activity with 3e, probably due to the
presence of 4-quinolone-3-carboxylic acid, which might function in a different
inhibitory mechanism.
Table 3. The PTP1B inhibitory activity of the designed bidentate PTP1B inhibitors
bearing the quinolone core structurea
O
O
R2
3a-h
R1
N
R3
Compd.
R1
R2
R3
IC50 (µg/mL) b
6-I
OH
> 20
3a
12

6-I
6-I
6-I
6-I
7-F
OH
NHBn
OH
8.1 0.7
> 20
3b
3c
3d
3e
3f
3.1 0.2
2.4 0.2
9.4 1.5
6.5 1.4
OH
Bn
6-NO2
6-
4-F-Bn
3g
OH
c-Pr
45.2 8.5
3h
b
aAssay details were described in the Experimental section. The pNPP competitive assay. IC50
values were determined by regression analysis and expressed as means SD of three independent
replications.
Kinetic analysis indicated that 4-quinolone-3-carboxylate acted as a competitive
PTP1B inhibitor. The effect of the inhibitor on the PTP1B catalyzed reaction was
studied with the substrate p-nitrophenyl phosphate (pNPP) in the presence and
absence of different concentrations of 3d. The kinetic parameters are summarized in
Table 4. As the concentration of the inhibitor increased, the Km value of PTP1B
increased accordingly while the Vmax value kept constant, demonstrating a
competitive inhibition pattern with a Ki value of 1.33 µM.
Table 4. The kinetic parameters of PTP1B enzyme in the pNPP hydrolysis in the
presence of different concentrations of the inhibitor 3da.
[Inhibitor] (µM)
Km (mM)
1.21
Vmax (mOD/Min)
72.82
0.00
2.00
4.00
6.00
2.62
72.71
3.58
72.69
5.43
13
71.64

8.00
7.40
68.65
a Km is the Michaelis constant, Vmax is the maximum rate and were calculated from the Michaelis-Menten
equation.28
Further Lineweaver-Burk plot, as shown in Figure 1, confirmed the competitive
inhibition mechanism by the quinolone-4-carboxylate based PTP1B inhibitors. This
mode of inhibition was contrary to that of the aryl diketoacid-based PTP1B inhibitors,
which were active site-directed non-competitive inhibitors by targeting an inactive
conformation of the enzyme.21
Figure 1. The effect of 3d on PTP1B-catalyzed pNPP hydrolysis. The experiment was
performed at 25 °C and pH 7.0. The inhibitor 3d concentrations were 0 (◆), 2.0 (■),
4.0 (▲), 6.0 (ꢀ) and 8.0 (ꢁ) µM, respectively.
The quinolone-3-carboxylate displayed selective inhibition against PTP1B.
Several active compounds with representative structures (1c, 1d, 2g, 3b, 3d, 3e and 3g)
were selected to test against a panel of PTPs including cytosolic PTPs, TCPTP, the
closest homologue of PTP1B (74% sequence identity in their catalytic domains),29
SHP1, SHP2 and the receptor-like PTPs, LAR. As shown in Table 5, all these
inhibitors demonstrated a moderate to excellent PTP1B selectivity over other PTPs.
For monomer quinolones 1c, 1d and 2g, no inhibition against other PTPs was
14

observed at 20 µg/mL, indicating that 4-quinolone-3-carboxylic acid motif was
selective for PTP1B. The bidentate inhibitors 3b and 3g bearing salicylic acid moiety
exhibited a better potency and higher selectivity for PTP1B. The most potent aryl
diketoacid-quinolone conjugates 3d and 3e displayed a selective inhibition of PTP1B
over TCPTP by a factor of 2-fold, and at least 10-fold over SHP2 and LAR.
Table 5. The inhibitory activity of select quinolone-based PTP1b inhibitors against a
panel of PTPs, IC50 (µg/mL) a
Compd.
PTP1B
TCPTP
SHP1
SHP2
LAR
7.5 1.0
12.3 0.9
13.9 2.5
8.1 0.7
3.1 0.2
2.4 0.2
6.5 1.34
NAb
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1h
1i
NA
NA
2g
3b
3d
3e
3g
NA
NA
6.3 0.7
4.9 0.7
NA
6.8 1.1
5.1 0.4
NA
a The pNPP assay. IC50 values were determined by regression analysis and expressed as means SD of three
replications. b Inhibition rate < 50% at 20 µg/ mL concentration.
The quinolone-3-carboxylate exhibited distinct cellular efficacy. Previous studies
have illustrated that PTP1B negatively regulates the insulin activated signaling
pathways by catalyzing the dephosphorylation of the insulin receptor β (IRβ) subunit.
Thus, inhibition of PTP1B activity should enhance insulin signaling. To determine the
cellular efficacy of 3d, we assessed its effect on insulin signaling in CHO/hIR cells.
As shown in Figure 2, 3d markedly increased insulin stimulated IRβ phosphorylation
at 90 µM concentration while there was no significant effect at the lower
concentrations.
15

Figure 2. Effect of 3d on insulin-mediated IRβ phosphorylation in CHO/hIR cells.
The quinolone-3-carboxylates bind to PTP1B in the active site interacting with
the WPD-loop closed conformation, as predicted by molecular modeling. Finally,
we tried to interpret the binding mode of the quinolone-3-carboxylate motif by the
molecular modeling. Based on the X-ray cocrystal structure of the PTP1B inhibitor
bound to the active site of the enzyme in the WPD loop open conformation (PDB:
3QKP),30 and in WPD loop closed conformation (PDB: 1PXH),31 we predicted the
binding mode of the lead inhibitor 3d in the PTP1B domain using Autodock
(Autodock.Scripps.edu).
When docking 3d into the active pocket of the enzyme in both conformations,
we observed that this compound binding to WPD loop closed conformation was more
stable (Figure 3A). The quinolone ring resided well to position the carboxyl oxygen in
hydrogen bonding distance to Ala217, Arg221, Ile219 and Gly220 residues, and the
phenyl ring of the quinolone formed a π-π stacking interaction with the Try46 residue.
However, when ligand 3d binding to WPD loop open conformation (Figure 3B), the
carboxyl oxygen of quinolone only formed two hydrogen bonds with Ala217 and
Arg221 residues, and no π-π stacking interaction existed. Further calculation of the
binding free energy of two ligand-protein complexes confirmed that this compound
preferred binding the WPD-closed conformation to the WPD-open conformation, i.e.
△G-WPD-open (-43.623kcal/mol) > △G-WPD-close (-68.453kcal/mol). The binding
mode established by the molecular modeling was consistent with the kinetic
experiment of 3d showing a competitive PTP1B inhibition.
16

A) WPD loop closed, ∆G = -68.45 kcal/mol
B) WPD loop open, ∆G = -43.623kcal/mol
Figure 3. A proposed binding mode of 3d to the PTP1B enzyme, based on the X-ray
co-crystal structure of PTP1B in complex with a small molecule inhibitor in WPD
loop closed (3A)31 or WPD loop open state (3B).30
Conclusions
By
employing
bioisostere
replacement
strategy,
we
introduced
4-quinolone-3-carboxylic acid motif into the aryl diketoacid chemotype for the design
and discovery of novel PTP1B inhibitors. Efficient synthesis was established to
provide variously substituted quinolone-3-carboxylic acid derivatives for an optimal
template exploration. The SAR study revealed that hydrophobic group was favored at
C6-position of the quinolone ring and the steric effect at N1-position interfered with
the binding. By introducing salicylic acid or aryl diketoacid as a second binding group
17

into the quinolone motif, several bidentate inhibitors were obtained with improved
inhibitory activity and good selectivity. The C3-aryl diketoacid conjugated quinolones
(3d and 3e) displayed the highest PTP1B inhibitory activity with low micromolar IC50
values, acting as a competitive inhibitor. More significantly, 3d possesses distinct
cellular efficacy in inhibiting IRβ phosphorylation in CHO/hIR cells. In summary, we
identified for the first time the 4-quinolone-3-carboxylic acid as a novel surrogate of
phosphotyrosine leading to the design of potent PTP1B inhibitors with excellent
selectivity and cell permeability.
Experimental Section
General Synthetic Methods.
All Reagents and solvents were obtained from commercial sources in analytical
grade and used without further purification. Melting points were determined by SGW
X-4B; 1H NMR spectra were recorded on a Varian Mercury-400 or 300 MHz and 13C
NMR spectra were recorded on a Varian Mercury-125 MHz. Mass specta were
recorded using a Finnigan MAT-95 instrument for EI and Finnigan LCQ Deca for
ESI.
General procedure for the preparation of the key intermediate 6: the acid
chlorides of commercially available substituted benzoic acids were coupled with ethyl
3-(dimethylamino)acrylate to produce the acrylates 4. Substitution with
cyclopropylamine or benzylamine and subsequent cyclization with potassium
carbonate gave quinolone-3-carboxylate 6.
Ethyl 2-(2,5-difluorobenzoyl)-3-(dimethylamino)acrylate (4a). A mixture of
2,5-difluorobenzoic acid (1.58 g, 10 mmol) and thionyl chloride (7 mL) was refluxed
for 3 hrs. The excess thionyl chloride was removed by distillation under reduced
pressure to give an oil residue which was then dissolved in dry toluene (25 mL). To
this solution was added the mixture of (E)-ethyl 3-(dimethylamino)acrylate (1.43 mL,
10 mmol) and dry Et3N (2.1 mL, 10 mmol). The resulting solution was heated at
90 °C for 2 hrs. After cooling and filtering, the solvent was evaporated to dryness and
the residue was purified by silica gel column chromatography eluting with PE /
18

EtOAc (10 / 1) to give 4a (2.83 g, 100%) as a yellow solid. 1H NMR (300 MHz,
CDCl3) δ 7.76 (s, 1H), 7.63 (d, J = 7.2 Hz, 1H), 6.89 (td, J = 8.3, 2.4 Hz, 1H), 6.81 –
6.70 (m, 1H), 3.99 (q, J = 7.1 Hz, 2H), 3.28 (s, 3H), 2.91 (s, 3H), 0.95 (t, J = 7.1 Hz,
3H).
Ethyl 1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (6a). To a
stirred solution of 4a (283 mg, 1 mmol) in EtOH (3 mL) and Et2O (1 mL) was added
cyclopropylamine (93 mg, 1.63mmol) dropwise. After stirring at room temperature
for 15 min, the mixture was evaporated to dryness to give compound 5a as a white
solid, which was used directly for the next step. The crude product 5a was dissolved
in dry DMF (2 mL) and was added K2CO3 (248 mg, 1.8 mmol). The reaction mixture
was heated at 100 °C for 40 min, then cooled to room temperature and poured into
ice-water. The precipitate was filtered off, washed with water, and dried to give 6a
( 248 mg, 90%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.95 (s, 1H), 8.49 (dd,
J = 8.9, 3.0 Hz, 1H), 8.29 (dd, J = 9.3, 4.3 Hz, 1H), 7.18 (ddd, J = 9.3, 7.5, 3.0 Hz,
1H), 4.75 (q, J = 7.1 Hz, 2H), 3.88 – 3.79 (m, 1H), 1.77 (t, J = 7.1 Hz, 3H), 1.74 –
1.66 (m, 2H), 1.54 – 1.47 (m, 2H).
Ethyl 2-(2-chloro-5-iodobenzoyl)-3-(dimethylamino)acrylate (4b). Analogous to
the preparation of 4a, 2-chloro-5-iodobenzoic acid (2.81 g, 10 mmol) was treated with
thionyl chloride (7 mL) followed by the addition of (E)-ethyl
3-(dimethylamino)acrylate (1.43 mL, 10 mmol) to give 4b (4.0 g, 100%) as a yellow
solid. 1H NMR (300 MHz, CDCl3) δ 7.84 (s, 1H), 7.67 (s, 1H), 7.60 – 7.54 (m, 1H),
7.06 (d, J = 8.4 Hz, 1H), 3.92 (q, J = 7.1 Hz, 2H), 3.33 (s, 3H), 2.97 (s, 3H), 0.88 (t, J
= 7.1 Hz, 3H).
Ethyl
1-cyclopropyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate
(6b).
Analogous to the preparation of 6a, 4b (407 mg, 1.0 mmol) was treated with
cyclopropylamine (93 mg, 1.63 mmol) followed by cyclization to afford 352 mg of 6b
(92%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 2.1 Hz, 1H), 8.59 (s,
1H), 7.96 (dd, J = 8.9, 2.1 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H),
3.44 (tt, J = 7.1, 3.9 Hz, 1H), 1.41 (t, J = 7.1 Hz, 3H), 1.36 – 1.30 (m, 2H), 1.15 –
1.10 (m, 2H).
19

Ethyl
1-benzyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate
(6c).
According to the same procedure described for 6a, 4a (283 mg, 1.0 mmol) was treated
with benzylamine (0.18 mL, 1.63 mmol) followed by cyclization to afford 310 mg
1
(95%) of 6c as a white solid. H NMR (300 MHz, CDCl3) δ 8.61 (s, 1H), 8.21 – 8.16
(m, 1H), 7.42 – 7.32 (m, 3H), 7.32 – 7.27 (m, 2H), 7.18 – 7.12 (m, 2H), 5.40 (s, 2H),
4.41 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H).
Ethyl 1-benzyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate (6d). According
to the same procedure described for 6a, 4b (407 mg, 1.0 mmol) was treated with
benzylamine (0.18 mL, 1.63mmol) followed by cyclization to afford 390 mg (90%) of
1
6d as a white solid. H NMR (300 MHz, CDCl3) δ 8.84 (d, J = 2.1 Hz, 1H), 8.59 (s,
1H), 7.79 (dd, J = 8.9, 2.2 Hz, 1H), 7.41 – 7.32 (m, 3H), 7.14 (dd, J = 7.2, 2.2 Hz, 2H),
7.06 (d, J = 8.9 Hz, 1H), 5.36 (s, 2H), 4.41 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz,
3H).
Ethyl 1-(4-fluorobenzyl)-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (6e).
1
6e was prepared in a similar fashion as described for 6a, yellow solid, 84%. H NMR
(300 MHz, CDCl3) δ 8.26 – 8.10 (m, 2H), 7.50 (d, J = 8.7 Hz, 1H), 7.33 – 7.27 (m,
2H), 7.20 – 7.04 (m, 3H), 5.67 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz,
3H).
Ethyl 1-benzyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (6f). 6f was
1
prepared in a similar fashion as described for 6a, a white solid, 95%. H NMR (400
MHz, CDCl3) δ 8.59 (s, 1H), 8.54 (dd, J = 9.0, 6.4 Hz, 1H), 7.42 – 7.33 (m, 3H), 7.17
(dd, J = 7.9, 1.5 Hz, 2H), 7.13 – 7.07 (m, 1H), 7.00 – 6.95 (m, 1H), 5.34 (s, 2H), 4.41
(q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H).
Ethyl 6-benzyl-1-cyclopropyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (6g).
Zinc powder (195 mg, 3.0 mmol) was placed in a three-neck flask. Anhydrous THF
(1.0 mL) was added. Then TMSCl (4 µL, 0.03 mmol) and 1,2-dibromomethane (20
µL, 0.015 mmol) were added sequentially. The mixture was stirred at rt for 10 min.
Benzyl bromide (0.18 mL, 1.5 mmol) was added slowly to keep the solvent gently
refluxing. After addition, the mixture was stirred at 65°C for 2.5 hr. Compound 6b
(383 mg, 1.0 mmol), Pd2(dba)3 (23 mg, 0.04 mmol) and trifurylphosphine (19 mg,
20

0.08 mmol) were placed in a two-neck flask, evacuated and refilled with N2. Then
anhydrous THF (3.0 mL) was added. The solution was stirred at 65°C for reflux. The
zinc bromide mixture was added dropwise to the solution, then stirred at this
temperature for 3 hrs. Saturated NH4Cl solution was added and extracted with ethyl
acetate (20 mL). The organic phase was combined, washed with brine and dried over
anhydrous Na2SO4.The solvent was evaporated to give a residue which was purified
1
by SGC to give 240 mg of 6g as a white solid (69%). H NMR (300 MHz, CDCl3) δ
8.57 (s, 1H), 8.38 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.53 – 7.48 (m, 1H),
7.32 – 7.26 (m, 2H), 7.23 – 7.17 (m, 3H), 4.39 (q, J = 7.1 Hz, 2H), 4.11 (s, 2H), 3.48
– 3.39 (m, 1H), 1.41 (t, J = 7.1 Hz, 3H), 1.30 – 1.25 (m, 2H), 1.15 – 1.08 (m, 2H);
ESI m/z: 348.1 (M+H) +.
Ethyl 1-cyclopropyl-6-(4-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate
(6h). According to the same procedure described for 6g, 6b (383 mg, 1.0 mmol) was
treated with 4-fluorobenzyl zinc bromide (190 mg, 1.5 mmol), giving 157 mg (43%)
of 6h as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 8.33 (d, J = 2.0 Hz,
1H), 8.02 (d, J = 8.8 Hz, 1H), 7.63 (dd, J = 8.7, 2.0 Hz, 1H), 7.18 (dd, J = 10.4, 3.8
Hz, 2H), 7.03 – 6.92 (m, 2H), 4.41 (q, J = 7.1 Hz, 2H), 4.12 (s, 2H), 3.60 – 3.49 (m,
1H), 1.42 (t, J = 7.1 Hz, 3H), 1.31 – 1.24 (m, 2H), 1.17 – 1.12 (m, 2H).
Ethyl 1,6-dibenzyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (6i). According to
the same procedure described for 6g, 6d (433 mg, 1.0 mmol) was treated with benzyl
1
zinc bromide (0.18 mL, 1.5 mmol) to afford 203 mg (51%) of 6i as a white solid. H
NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 8.41 (d, J = 2.1 Hz, 1H), 7.41 – 7.28 (m, 5H),
7.26 – 7.23 (m, 2H), 7.22 – 7.19 (m, 1H), 7.16 (ddd, J = 9.5, 5.8, 1.8 Hz, 4H), 5.36 (s,
2H), 4.40 (q, J = 7.1 Hz, 2H), 4.05 (s, 2H), 1.42 (t, J = 7.1 Hz, 3H).
1-Cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1a). To the
solution of 6a (230 mg, 0.88 mmol) in MeOH (3.0 mL) was added HCl (6N, 3.0 mL).
The solution was stirred at reflux for 2 hr. The solvent was evaporated to give a white
solid. Then the solid was washed with 20 mL EtOH and dried in the oven, affording
1a as a white solid (187 mg, 86%). Mp: 272-273°C; 1H NMR (300 MHz, CDCl3) δ
14.62 (s, 1H), 8.88 (s, 1H), 8.15 – 8.12 (m, 2H), 7.64 – 7.57 (m, 1H), 3.68 – 3.58 (m,
21

1H), 1.48 – 1.39 (m, 2H), 1.27 – 1.22 (m, 2H); EI-MS m/z: 247 (M+); HRMS (EI)
(M+): calcd for C13H10FNO3 247.0661, found 247.0653.
1-Cyclopropyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(1b).
According to the same procedure described for 1a, 6b (188 mg, 0.49 mmol) was
treated with HCl (6N, 3.0 mL) to afford 151 mg (85%) of 1b as a white solid. Mp:
1
243-245°C; H NMR (300 MHz, CDCl3) δ 8.88 (s, 1H), 8.85 – 8.84 (m, 1H), 8.13 –
8.09 (m, 1H), 7.84 (d, J = 9.0 Hz, 1H), 3.62 – 3.56 (m, 1H), 1.46 – 1.39 (m, 2H), 1.26
– 1.17 (m, 2H); ESI-MS m/z: 355.98 (M+H)+; HRMS (ESI) (M+H)+: calcd for
C13H11O3NI 355.9778, found 355.9789.
1-Benzyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1c). According
to the same procedure described for 1a, 6c (98 mg, 0.3 mmol) was treated with HCl
(6N, 1.2 mL) to afford 71 mg (80%) of 1c as a white solid. Mp: 237-239°C; 1H NMR
(300 MHz, CDCl3) δ 8.90 (s, 1H), 8.20 (dd, J = 8.4, 2.7 Hz, 1H), 7.57 – 7.37 (m, 5H),
7.15 (dd, J = 7.3, 2.3 Hz, 2H), 5.52 (s, 2H); ESI-MS m/z: 320.07 (M+Na)+; HRMS
(ESI) (M+Na)+: calcd for C17H12O3NFNa320.0693, found 320.0702.
1-Benzyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1d). According to
the same procedure described for 1a, 6d (150 mg, 0.35 mmol) was treated with HCl
(6N, 2.0 mL) to afford 126 mg (90%) of 1d as a white solid. Mp: 207-209°C; 1H
NMR (400 MHz, DMSO-d6) δ 14.86 (s, 1H), 9.30 (s, 1H), 8.64 (d, J = 2.0 Hz, 1H),
8.15 (dd, J = 9.0, 2.0 Hz, 1H), 7.65 (d, J = 9.1 Hz, 1H), 7.40 – 7.22 (m, 5H), 5.86 (s,
2H); ESI-MS m/z: 405.99 (M+H)+; HRMS (ESI) (M+H)+: calcd for C17H13O3NI
405.9935, found 405.9933.
1-(4-Fluorobenzyl)-6-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1e).
According to the same procedure described for 1a, 6e (200 mg, 0.54 mmol) was
treated with HCl (6N, 4.0 mL) to afford 124 mg (67%) of 1e as a light yellow solid.
Mp: 227-228°C; 1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 9.01 (d, J = 2.0 Hz,
1H), 8.58 (d, J = 9.3 Hz, 1H), 8.07 (d, J = 9.2 Hz, 1H), 7.41 – 7.38 (m, 2H), 7.20 (t, J
= 8.6 Hz, 2H), 5.91 (s, 2H); ESI-MS m/z: 343.07 (M+H)+; HRMS (ESI) (M+H)+:
calcd for C17H12O5N2F 343.0725, found 343.0725.
6-Amino-1-(4-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1f).
22

The reaction mixture of 1e (162 mg, 0.5 mmol) and Pd/C (5%, 8 mg) in anhydrous
MeOH was stirred under the atmosphere of H2 for 6 hr at rt. The solution was filtered
through a pad of Celite and the filtrate was collected. The solvent was evaporated to
give a crude product which was purified by Prep-HPLC, affording 118 mg (80%) of
1f as a yellow solid. Mp: 261-263°C; 1H NMR (300 MHz, CDCl3) δ 9.36 (d, J = 2.6
Hz, 1H), 8.98 (s, 1H), 8.50 (dd, J = 9.3, 2.5 Hz, 1H), 7.66 (d, J = 9.7 Hz, 1H), 7.24 –
7.05 (m, 4H), 5.57 (s, 2H); EI-MS m/z: found 312 (M+); HRMS (EI) (M)+: calcd for
C17H13FN2O3 312.0983, found 312.0991.
Benzyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1g). According to
the same procedure described for 1a, 6f (100 mg, 0.31 mmol) was treated with HCl
1
(6N, 1.2 mL) to afford 87 mg (95%) of 1g as a white solid. Mp: 227-230°C; H NMR
(300 MHz, CDCl3) δ 9.93 (s, 1H), 8.92 (s, 1H), 8.55 – 8.46 (m, 1H), 7.36 (d, J = 6.8
Hz, 3H), 7.16 (s, 3H), 7.05 (d, J = 8.2 Hz, 1H), 5.40 (s, 2H), 3.05 – 2.93 (m, 1H), 0.90
– 0.80 (m, 2H), 0.71 – 0.63 (m, 2H); EI-MS m/z: 297 (M+); HRMS (EI) (M)+: calcd
for C13H12FNO3 297.0802, found 297.0794.
6-Benzyl-1-cyclopropyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(1h).
According to the same procedure described for1a, 6g (70 mg, 0.2 mmol) was treated
with HCl (6N, 1.0 mL) to afford 58 mg (90%) of 1h as a white solid. Mp: 193-195°C;
1H NMR (300 MHz, CDCl3) δ 8.86 (s, 1H), 8.39 (d, J = 2.2 Hz, 1H), 8.00 (d, J = 8.8
Hz, 1H), 7.66 (dd, J = 8.8, 2.2 Hz, 1H), 7.35 – 7.28 (m, 2H), 7.26 – 7.18 (m, 3H),
4.16 (s, 2H), 3.64 – 3.52 (m, 1H), 1.43 – 1.34 (m, 2H), 1.19 (dt, J = 11.0, 5.4 Hz, 2H);
13C NMR (125 MHz, CDCl3) δ 178.6, 167.1, 147.8, 140.1, 139.8, 139.6, 135.0, 128.9,
128.9, 126.6, 126.3, 126.0, 117.5, 108.6, 41.4, 35.4, 8.2; EI-MS m/z: 319 (M+);
HRMS (EI) (M+) calcd for C20H17NO3 319.1208, found 319.1207.
Cyclopropyl-6-(4-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(1i). According to the same procedure described for 1a, 6h (53 mg, 0.15 mmol) was
treated with HCl (6N, 1.0 mL) to afford 46 mg of 1i (92%) as a white solid. Mp:
213-216°C; 1H NMR (400 MHz, CDCl3) δ 14.90 (s, 1H), 8.86 (s, 1H), 8.35 (d, J = 2.0
Hz, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.64 (dd, J = 8.7, 2.0 Hz, 1H), 7.16 (dd, J = 10.4,
3.8 Hz, 2H), 7.04 – 6.95 (m, 2H), 4.13 (s, 2H), 3.63 – 3.53 (m, 1H), 1.39 (q, J = 6.5
23

13
Hz, 2H), 1.22 – 1.18 (m, 2H); C NMR (125 MHz, CDCl3) δ 178.5, 167.0, 161.6 (d,
J = 245.1 Hz), 147.8, 139.9, 139.6, 135.4, 134.8, 130.4 (J = 7.5 Hz), 126.2, 126.0,
117.6, 115.6 (d, J = 21.3 Hz), 108.7, 40.6, 35.3, 8.2; EI-MS m/z: 337 (M+); HRMS (EI)
(M+) calcd for C20H16NO3F 337.1114, found 337.1111.
1,6-Dibenzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1j). According to the
same procedure described for 1a, 6i (102 mg, 0.26 mmol) was treated with HCl (6N,
1.5 mL) to afford 81 mg of 1j (85%) as a white solid. Mp: 199-201°C; 1H NMR (400
MHz, CDCl3) δ 14.96 (s, 1H), 8.87 (s, 1H), 8.41 (d, J = 1.8 Hz, 1H), 7.51 (dd, J = 8.8,
2.0 Hz, 1H), 7.43 (d, J = 8.9 Hz, 1H), 7.40 – 7.32 (m, 3H), 7.32 – 7.27 (m, 2H), 7.24
– 7.07 (m, 5H), 5.48 (s, 2H), 4.11 (s, 2H); ESI-MS m/z: 370.2 (M+H)+; HRMS (ESI)
(M+Na)+: calcd for C24H19NO3Na 392.1263, found 392.1255.
6-(4-Benzylpiperazin-1-yl)-1-cyclopropyl-4-oxo-1,4-dihydroquinoline-3-carboxyli
c acid (1k). A solution of compound 1a (247 mg, 1.0 mmol) and piperazine (258 mg,
3.0 mmol) in NMP (0.5 mL) was stirred under microwave heating at 200°C for 0.5 hr,
then cooled to rt. After evaporating the solvent, the residue was diluted with EtOH
and filtered, washed with EtOH, giving a yellow solid which was used directly in the
next step. To the solution of the solid obtained above (313 mg, 1.0 mmol) in DCE (5.0
mL) were added AcOH (86 µL, 1.5 mmol), NaBH(OAc)3 (509 mg, 2.4 mmol) and
benzaldehyde (0.15 mL, 1.5 mmol) at rt. The mixture was stirred for 6 hr at rt, then
diluted with DCM (20 mL). Washed with saturated NaHCO3 (5.0 mL) solution, brine
and dried over anhydrous Na2SO4. Concentrated, and the residue was purified by SGC
to give 174 mg (43%) of 1k as a light yellow solid. Mp: 223-225°C; 1H NMR (400
MHz, CDCl3) δ 15.24 (s, 1H), 8.73 (s, 1H), 7.97 (d, J = 9.4 Hz, 1H), 7.76 (d, J = 2.9
Hz, 1H), 7.46 (dd, J = 9.4, 2.9 Hz, 1H), 7.35 (d, J = 2.0 Hz, 2H), 7.30 (dd, J = 5.8, 2.9
Hz, 1H), 7.19 (t, J = 7.9 Hz, 2H), 6.86 (dd, J = 7.9, 4.9 Hz, 2H), 3.60 – 3.55 (m, 1H),
3.37 (d, J = 2.4 Hz, 4H), 2.85 (s, 2H), 2.70 – 2.62 (m, 4H), 1.35 (q, J = 6.8 Hz, 2H),
1.21 – 1.14 (m, 2H); EI-MS m/z: 403 (M+); HRMS (EI) (M)+ calcd for C24H25N3O3
403.1896, found 403.1890.
Benzyl 1-cyclopropyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate (2a).
According to the same procedure described for 6g, 6b (376 mg, 1.0 mmol) was
24

treated with benzyl zinc bromide (0.18 mL, 1.5 mmol), producing 25 mg (5.6%) of 2a
1
as a white solid. Mp: 154-156°C; H NMR (300 MHz, CDCl3) δ 8.80 (d, J = 2.1 Hz,
1H), 8.59 (s, 1H), 7.96 (dd, J = 9.0, 2.0 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 7.55 – 7.49
(m, 2H), 7.42 – 7.29 (m, 3H), 5.39 (s, 2H), 3.43 (ddd, J = 10.7, 7.2, 3.9 Hz, 1H), 1.35
– 1.27 (m, 2H), 1.11 (qd, J = 5.7, 2.8 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 172.7,
165.0, 148.9, 141.0, 140.0, 136.3, 129.7, 128.6, 128.0, 128.0, 123.5, 118.6, 111.0,
90.1, 66.4, 34.6, 8.2; ESI-MS m/z: 468.2 (M+Na)+; HRMS (ESI) (M+H)+: calcd for
C20H16NO3I 468.0073, found 468.0084.
4-Fluorobenzyl-1-cyclopropyl-6-(4-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-c
arboxylate (2b). According to the same procedure described for 6g, 6b (383 mg, 1.0
mmol) was treated with 4-fluorobenzyl zinc bromide (190 mg, 1.5 mmol) to afford
149 mg (32%) of 2b as a white solid. Mp: 136-138°C; 1H NMR (300 MHz, CDCl3) δ
8.59 (s, 1H), 8.39 – 8.35 (m, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.53 – 7.49 (m, 3H), 7.18
– 7.12 (m, 2H), 7.05 (t, J = 8.8 Hz, 2H), 6.96 (t, J = 8.9 Hz, 2H), 5.36 (s, 2H), 4.07 (s,
2H), 3.48 – 3.40 (m, 1H), 1.30 (d, J = 9.0 Hz, 2H), 1.14 – 1.08 (m, 2H); 13C NMR
(125 MHz, CDCl3) δ 174.3, 165.8, 162.5 (d, J = 243.8 Hz), 161.5 (d, J = 240.0 Hz),
148.5, 139.0, 138.6, 136.0, 133.3, 132.2, 130.3 (d, J = 7.9 Hz), 129.8 (d, J = 8.2 Hz),
128.6, 127.2, 116.9, 115.5 (d, J = 5.0 Hz), 115.3 (d, J = 5.0 Hz), 110.5, 65.9, 40.6,
34.5, 8.1; ESI-MS m/z: 446.3 (M+H)+; HRMS (ESI) (M+Na)+: calcd for
C27H21NO3NaF2 468.1387, found 468.1381.
Benzyl 1,6-dibenzyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (2i). According to
the same procedure described for 6g, 6d (433 mg, 1.0 mmol) was treated with benzyl
zinc bromide (0.18 mL, 1.5 mmol) to afford 106 mg (23%) of 2i as a white solid. Mp:
147-149°C; 1H NMR (300 MHz, CDCl3) δ 8.58 (s, 1H), 8.43 (s, 1H), 7.55 – 7.50 (m,
2H), 7.42 – 7.29 (m, 7H), 7.24 (s, 2H), 7.22 – 7.10 (m, 6H), 5.41 (s, 2H), 5.34 (s, 2H),
4.05 (s, 2H); ESI-MS m/z: 460.4 (M+H)+; HRMS (ESI) (M+Na)+: calcd for
C31H25NO3Na 482.1732, found 482.1722.
N-benzyl-1-cyclopropyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxamide (2c).
To a solution of 1b (75 mg, 0.21 mmol) in DCM (2mL) was added Bop (111 mg, 0.25
mmol) and Et3N (73 ul, 0.53 mmol) at rt, and the reaction mixture was stirred for 15
25

min. Then benzylamine (27 mg, 0.25 mmol) was added and the reaction mixture was
stirred for 2 hrs. The reaction mixture was dissolved in DCM (15 mL) and washed
with water. The organic phase was combined, washed with brine and dried over
anhydrous Na2SO4. The residue was purified by SGC to give 74 mg (80%) of 2c as a
white solid. Mp: 169-171°C; 1H NMR (300 MHz, CDCl3) δ 10.24 (t, J = 6.3 Hz, 1H),
8.93 (s, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.00 (dd, J = 8.9, 2.0 Hz, 1H), 7.74 (d, J = 8.9
Hz, 1H), 7.35 (dt, J = 15.0, 7.4 Hz, 4H), 7.23 (d, J = 7.2 Hz, 1H), 4.67 (d, J = 5.8 Hz,
2H), 3.54 – 3.45 (m, 1H), 1.39 – 1.30 (m, 2H), 1.20 – 1.12 (m, 2H); EI-MS m/z: 444
(M+); HRMS (EI) (M)+: calcd for C20H17IN2O2 444.0331, found 444.0329.
1-Cyclopropyl-N-(4-fluorobenzyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxa
mide (2d). According to the same procedure described for 2c, 1b (107 mg, 0.3 mmol)
was treated with 4-fluorobenzylamine (46 mg, 0.36 mmol) to afford 115 mg (83%) of
2d as a white solid. Mp: 178-180°C; 1H NMR (300 MHz, CDCl3) δ 10.24 (t, J = 6.1
Hz, 1H), 8.91 (s, 1H), 8.78 (d, J = 1.8 Hz, 1H), 8.00 (dd, J = 8.9, 1.9 Hz, 1H), 7.74 (d,
J = 8.9 Hz, 1H), 7.33 (dd, J = 8.1, 5.6 Hz, 2H), 7.00 (t, J = 8.6 Hz, 2H), 4.62 (d, J =
5.7 Hz, 2H), 3.55 – 3.44 (m, 1H), 1.34 (q, J = 6.9 Hz, 2H), 1.21 – 1.11 (m, 2H);
EI-MS m/z: 462 (M+); HRMS (EI) (M)+: calcd for C20H16F I N2O2 462.0257, found
462.0249.
N-Benzyl-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (2e).
According to the same procedure described for 2c, 1a (25 mg, 0.1 mmol) was treated
with benzylamine (13 mg, 0.12 mmol) to afford 30 mg (90%) of 2e as a white solid.
Mp: 161-163°C; 1H NMR (300 MHz, CD3OD) δ 10.55 (t, J = 7.4 Hz, 1H), 8.90 (s,
1H), 8.30 (dd, J = 9.4, 4.4 Hz, 1H), 8.01 (dd, J = 9.0, 3.1 Hz, 1H), 7.67 (ddd, J = 9.4,
7.8, 3.1 Hz, 1H), 7.40 – 7.22 (m, 4H), 4.64 (d, J = 5.8 Hz, 2H), 3.79– 3.72 (m, 1H),
1.42 – 1.32 (m, 2H), 1.23 – 1.15 (m, 2H); ESI-MS m/z: 359.2 (M+Na) +; HRMS (ESI)
(M+Na)+: calcd for C20H19N2O2NaF359.1171, found 359.117.
N-Benzyl-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (2f).
According to the same procedure described for 2c, 1g (50 mg, 0.2 mmol) was treated
with benzylamine (26 mg, 0.24 mmol) to afford 58 mg (87%) of 2f as a white solid.
Mp: 165-167°C; 1H NMR (300 MHz, CDCl3) δ 10.29 (t, J = 6.2 Hz, 1H), 8.93 (s, 1H),
26

8.49 (dd, J = 9.0, 6.3 Hz, 1H), 7.65 (dd, J = 10.3, 2.3 Hz, 1H), 7.41 – 7.30 (m, 4H),
7.24 – 7.18 (m, 1H), 4.67 (d, J = 5.8 Hz, 2H), 3.52 – 3.43 (m, 1H), 1.41 – 1.33 (m,
2H), 1.18 (dt, J = 3.9, 3.0 Hz, 2H); EI-MS m/z: 336 (M+); HRMS (EI) (M)+: calcd for
C20H17F N2O2 336.1276, found 336.1275.
N,1-Dibenzyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxamide (2g). According
to the same procedure described for 2c, 1d (81 mg, 0.2 mmol) was treated with
benzylamine (26 mg, 0.24 mmol) to afford 89 mg (90%) of 2g as a white solid. Mp:
171-174°C; 1H NMR (300 MHz, CDCl3) δ 10.33 – 10.27 (m, 1H), 8.95 (s, 1H), 8.84
(d, J = 2.1 Hz, 1H), 7.84 (dd, J = 8.9, 2.1 Hz, 1H), 7.43 – 7.27 (m, 8H), 7.18 – 7.12
(m, 3H), 5.44 (s, 2H), 4.70 (d, J = 5.8 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 175.4,
164.5, 148.8, 141.3, 138.6, 136.4, 133.8, 129.4, 128.8, 128.6, 127.6, 127.1, 125.0,
118.6, 112.7, 111.0, 100.9, 89.9, 57.8, 43.2; ESI-MS m/z: 337.1 (M+H)+; HRMS (ESI)
(M+H)+ calcd for C24H19N2O2I 495.0570, found 495.0549.
N,1-Dibenzyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide
(2h).
According to the same procedure described for 2c, 1c (75 mg, 0.25 mmol) was treated
with benzylamine (33 mg, 0.3 mmol) to afford 65 mg (63%) of 2h as a white solid.
Mp: 166-168°C; 1H NMR (300 MHz, CDCl3) δ 10.34 (t, J = 5.5 Hz, 1H), 8.97 (s, 1H),
8.15 (dd, J = 8.9, 2.9 Hz, 1H), 7.48 – 7.27 (m, 10H), 7.18 – 7.12 (m, 2H), 5.47 (s, 2H),
4.70 (d, J = 5.8 Hz, 2H); ESI-MS m/z: 397.2 (M+H)+; HRMS (ESI) (M+Na)+: calcd
for C24H19N2O2NaF 409.1328, found 409.1334.
Methyl
2-methoxy-5-methylbenzoate
(7).
To
a
solution
of
2-hydroxy-5-methylbenzoic acid (761 mg, 5 mmol) and K2CO3 (2.07 g, 15 mmol) in
anhydrous acetone (15 mL) was added CH3I (0.78 mL, 12.5 mmol) dropwise at rt.
The reaction mixture was stirred at reflux overnight, then dissolved in DCM (15 mL)
and washed with water. The organic phase was combined, washed with brine and
dried over anhydrous Na2SO4. The residue was purified by SGC to give 687 mg (77%)
1
of 7 as a colorless oil. H NMR (300 MHz, CDCl3) δ 7.60 (dd, J = 2.5, 0.9 Hz, 1H),
7.28 – 7.23 (m, 1H), 6.87 (d, J = 8.5 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 2.30 (s, 3H).
Methyl 5-(bromomethyl)-2-methoxybenzoate (8). To a solution of 7 (728 mg, 3.82
mmol) and perbenzoic acid (20 mg, 0.076 mmol) in CCl4 (10 mL) was added
27

N-bromosuccinimide (680 mg, 3.82 mmol). The reaction mixture was stirred at reflux
until the starting compound was totally consumed. The solvent was evaporated to give
a residue, which was dissolved in DCM (50 mL) and washed with water. The organic
phase was combined, washed with brine and dried over anhydrous Na2SO4. The
residue was purified by SGC to give 840 mg (85%) of 8 as a white solid. 1H NMR
(300 MHz, CDCl3) δ 7.84 (d, J = 2.4 Hz, 1H), 7.51 (dd, J = 8.6, 2.5 Hz, 1H), 6.95 (d,
J = 8.6 Hz, 1H), 4.48 (s, 2H), 3.91 (s, 3H), 3.89 (s, 3H); ESI-MS m/z: 259.1 (M+H)+.
Methyl 5-(aminomethyl)-2-methoxybenzoate (9). A solution of 8 (130 mg, 0.5
mmol) in NH3 (12.5 mL, 7 N in MeOH) was stirred at 70°C for 2 hrs. Then the
solvent was evaporated to give a residue which was purified by SGC, affording 90 mg
1
(98%) of 9 as an off yellow solid. H NMR (300 MHz, CDCl3) δ 7.73 (d, J = 2.4 Hz,
1H), 7.41 (dd, J = 8.5, 2.4 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 5.27 (s, 2H), 3.91 – 3.84
(m, 6H), 3.81 (s, 2H); ESI-MS m/z: 196.0 (M+H)+.
(Z)-Methyl5-(((2-(2-chloro-5-iodobenzoyl)-3-ethoxy-3-oxoprop-1-en-1-yl)amino)
methyl)-2-methoxybenzoate (10). A stirred solution of 4b (283mg, 1 mmol) in EtOH
(3 mL) and Et2O (1 mL) was added dropwise a solution of 9 (0.18, 1.63mmol) in
EtOH (0.5 mL). The reaction mixture was stirred at rt for 15 min, then dissolved in
DCM (15 mL) and washed with water. The organic phase was washed with brine and
dried over anhydrous Na2SO4. The residue was purified by SGC to give 373 mg (67%)
of 10 as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 11.19 (t, J = 8.1 Hz, 1H),
8.32- 8.18 (m, 1H), 7.78 – 7.72 (m, 1H), 7.60 – 7.52 (m, 1H), 7.49 (d, J = 2.1 Hz, 1H),
7.44 – 7.38 (m, 1H), 7.09 – 7.00 (m, 2H), 4.60 – 4.54 (m, 2H), 3.98 (q, J = 7.1 Hz, 2H),
3.93 (s, 3H), 3.91 (s, 3H), 0.94 (t, J = 7.1 Hz, 3H).
Ethyl
6-iodo-1-(4-methoxy-3-(methoxycarbonyl)benzyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylate (11). To a solution of 10 (373 mg, 0.67 mmol) in dry DMF (2 mL) was
added K2CO3 (248 mg, 1.8 mmol). The mixture was heated at 100°C for 40 min. After
cooling, the reaction mixture was poured into ice-water. The precipitated solid was
filtered off, washed with water, and dried to give 331 mg (95%) of 11 as a white solid.
1H NMR (300 MHz, CDCl3) δ 8.83 (d, J = 2.2 Hz, 1H), 8.57 (s, 1H), 7.80 (dd, J = 8.9,
28

2.2 Hz, 1H), 7.67 (d, J = 2.5 Hz, 1H), 7.17 (dd, J = 8.7, 2.5 Hz, 1H), 7.04 (d, J = 9.0
Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 5.31 (s, 2H), 4.41 (q, J = 7.2 Hz, 2H), 3.91 – 3.87
(m, 6H), 1.42 (t, J = 7.2 Hz, 3H).
6-Iodo-1-(4-methoxy-3-(methoxycarbonyl)benzyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (3a). According to the same procedure described for 1a, 11 (331 mg,
0.64 mmol) was treated with HCl (6N, 5.0 mL) to afford 282 mg (90%) of 3a as a
white solid. Mp: 244-246°C; 1H NMR (400 MHz, CDCl3) δ 14.57 (s, 1H), 8.89 (s,
2H), 7.97 (dd, J = 9.0, 2.1 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.25 – 7.17 (m, 2H), 6.97
(d, J = 8.7 Hz, 1H), 5.43 (s, 2H), 3.893 (s, 3H), 3.892 (s, 3H); ESI-MS m/z: 494.0
(M+H)+; HRMS (ESI) (M+Na)+: calcd for C20H16NO6NaI 515.9920, found 515.9924.
1-(4-Hydroxy-3-(methoxycarbonyl)benzyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (3b). To a stirred solution of 3a (34 mg, 0.07 mmol) in DCM (5 mL)
was added BBr3 (0.07 mL, 0.28 mmol) in an ice bath. The mixture was stirred at rt for
6 hrs. Saturated NaHCO3 solution (1 mL) was added to quench the reaction. The
mixture was then distributed between DCM (20 mL) and H2O (5 mL). The organic
phase was combined, washed with brine and dried over anhydrous Na2SO4. The
residue was purified by Prep-HPLC (CH3CN / H2O (0.5% TFA) = 50 / 50) to give 32
1
mg (95%) of 3b as a white solid. Mp: 240-242°C; H NMR (300 MHz, CDCl3) δ
10.86 (s, 1H), 8.91 – 8.85 (m, 2H), 7.98 (dd, J = 8.5, 2.2 Hz, 1H), 7.65 – 7.62 (m, 1H),
7.32 – 7.27 (m, 2H), 7.02 (d, J = 8.7 Hz, 1H), 5.40 (s, 2H), 3.94 (s, 3H); ESI-MS m/z:
480.2 (M+H)+; HRMS (ESI) (M+H)+: calcd for C19H14NO6I 479.9944, found
479.9952.
Methyl-5-((3-(benzylcarbamoyl)-6-iodo-4-oxoquinolin-1(4H)-yl)methyl)-2-metho
xybenzoate (3c). According to the same procedure described for 2c, 3a (123 mg, 0.25
mmol) was treated with benzylamine (33 mg, 0.3 mmol) to afford 52 mg (35%) of 3c
as a white solid. Mp: 215-216°C; 1H NMR (300 MHz, CDCl3) δ 10.28 (t, J = 6.1 Hz,
1H), 8.94 (s, 1H), 8.84 (d, J = 2.1 Hz, 1H), 7.85 (dd, J = 8.6, 2.4 Hz, 1H), 7.68 (d, J =
2.4 Hz, 1H), 7.43 – 7.27 (m, 5H), 7.19 – 7.10 (m, 2H), 6.93 (d, J = 8.7 Hz, 1H), 5.38
(s, 2H), 4.70 (d, J = 5.8 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 3H); ESI-MS m/z: 605.1
(M+Na)+; HRMS (ESI) (M+Na)+: calcd for C27H23N2O5NaI 605.0549, found
29