Anticancer and anti-inflammatory activities of 1,8-naphthyridine-3-carboxamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.08.006

Several 1,8-naphthyridine-3-carboxamide derivatives (8-23) were synthesized and tested for in vitro cytotoxicity against eight cancer cell lines and a normal cell line. Compound 12 exhibited high cytotoxicity (IC₅₀ = 1.37 μM) in HBL-100 (breast) cell line while compounds 17 (IC₅₀ = 3.7 μM) and 22 (IC₅₀ = 3.0 μM) have shown high cytotoxicity in KB (oral) and SW-620 (colon) cell lines, respectively. The synthesized 1,8-naphthyridine-3-carboxamides were also evaluated for anti-inflammatory and myeloprotective activities, indicated by modulation in cytokine and chemokine levels secreted by dendritic cells.

Full text of DOI:10.1016/j.bmcl.2007.08.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6660–6664
Anticancer and anti-inflammatory activities of
1,8-naphthyridine-3-carboxamide derivatives
Sanjay K. Srivastava,a Manu Jaggi,b,* Anu T. Singh,b Alka Madan,b Nidhi Rani,a
Manupriya Vishnoi,b Shiv K. Agarwal,a,* Rama Mukherjeea,b and Anand C. Burmana,b
aDivision of Medicinal Chemistry, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad, 201 010 UP, India
bDivision of Preclinical Research & Developmental Therapeutics, Dabur Research Foundation, 22, Site IV,
Sahibabad, Ghaziabad, 201 010 UP, India
Received 16 April 2007; revised 20 July 2007; accepted 3 August 2007
Available online 11 August 2007
Abstract—Several 1,8-naphthyridine-3-carboxamide derivatives (8–23) were synthesized and tested for in vitro cytotoxicity against
eight cancer cell lines and a normal cell line. Compound 12 exhibited high cytotoxicity (IC50 = 1.37 lM) in HBL-100 (breast) cell line
while compounds 17 (IC50 = 3.7 lM) and 22 (IC50 = 3.0 lM) have shown high cytotoxicity in KB (oral) and SW-620 (colon) cell
lines, respectively. The synthesized 1,8-naphthyridine-3-carboxamides were also evaluated for anti-inflammatory and myeloprotec-
tive activities, indicated by modulation in cytokine and chemokine levels secreted by dendritic cells.
Ó 2007 Published by Elsevier Ltd.
Recently, quinolones and naphthyridine class of com-
pounds have been explored in cancer chemotherapy
and one molecule of naphthyridine class, namely SNS-
595, is presently in phase II clinical trial.1 SNS-595 acts
as a cell cycle modulator.1,2 However, a limited informa-
tion is available in the literature for the anticancer po-
tential of naphthyridine class of compounds. Most of
the chemical modifications were carried out at N-1, C-
5, C-6, and C-7 positions in 1,8-naphthyridines.1,3 The
C-3 position has not been well exploited. In order to
appreciate the actual utility of naphthyridines in cancer
chemotherapy and to understand structure–activity rela-
tionship, modification at C-3 in 1,8-naphthyridines is
required.
In addition, 1,8-naphthyridine class of molecules has
been reported to exhibit potent anti-inflammatory activ-
ity.4,5 1,8-Naphthyridine-3-carboxamide derivatives
were assessed for anti-inflammatory and myeloprotec-
tive activity using an in vitro screening assay based on
murine bone marrow derived dendritic cells (DCs).
The extent of modulation in pro-inflammatory cytokine
and chemokine levels was taken as an indicator of anti-
inflammatory and myeloprotective activity.
In the present paper, we have designed 1,8-naphthyri-
dine-3-carboxamides where cyclic as well as open amino
acids have been introduced at C-3 position. These amino
acids may provide interaction with receptors and may
lead to biological response. The propargyl group was
introduced at N-1 position due to its hydrogen bonding
capability. In order to have low molecular weight deriv-
atives, the pyridine ring of 1,8-naphthyridine has been
remained either unsubstituted or substituted with small
groups such as halo or methyl group. Herein, we report
the synthesis, cytotoxicity, anti-inflammatory, myelo-
protective activity, and structure–activity relationship
of 1,8-naphthyridine-3-carboxamides (8–23).
O
O
5
4
6
OH
3
1
N 7 N
8
N
S
H3CHN
H3CO
S
N
S
SNS-595
Synthesis of 1,8-naphthyridine-3-carboxamide deriva-
tives (8–22) has been described in Scheme 1. The appro-
priate nicotinic acid 1 was treated with CDI in dry THF
and the resulting imidazolide solution was reacted with
Keywords: Anticancer; Anti-inflammatory; Naphthyridine.
*
Corresponding authors. Tel.: +91 120 4378610; fax: +91 120
4376902; e-mail: jaggim@dabur.com
0960-894X/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.08.006

S. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6660–6664
6661
ethyl hydrogen malonate and methyl magnesium bro-
mide to give ketoester 2. Treatment of 2 with triethyl
orthoformate and acetic anhydride followed by the
addition of propargyl amine afforded the corresponding
ethyl nicotinoylacrylate 3. Compound 3 was cyclized by
K2CO3 in ethyl acetate to afford 1,8-naphthyridine-3-
carboxylate 4. The acidic hydrolysis of 4 provided the
corresponding acid derivative 5, which was converted
to carbonyl chloride derivative 6 with thionyl chloride.3
Compound 6 was reacted with suitable amino alcohol to
give compound 7. The 1-propargyl-1,8-naphthyridine-3-
carboxamide derivatives, 8 and 12, were prepared by
coupling of suitable derivative of 7 with 4R,5S-1-N-
(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazoli-
dine carboxylic acid6 in the presence of DCC and
DMAP.7 Using the same method as described for 8
and 12, compounds 9–11 and 13–20 were synthesized
from the reaction of 7 with commercially available
Boc-protected D-amino acids. The oxazolidine ring of
compound 12 was opened with 20% TFA/DCM to pro-
vide compound 21, which was then converted to 22 with
di-tert-butylpyrocarbonate. Compound 23 was prepared
in two steps starting from 7 as shown in Scheme 2. The
displacement of 7-Cl in compound 7 by 3-methylpiperi-
dine was performed by using K2CO3 as a base to
provide compound 7a, which was coupled with Boc-D-
proline, as described for 8–20, to give compound 23.
The 1,8-naphthyridine-3-carboxamide derivatives 8–20
are listed in Table 1.
(DU145), oral (KB), colon (SW-620), breast (HBL-
100), lung (A-549), pancreas (MiAPaCa2), and leukemia
(K562) cancers. Compounds 8–23 were also screened
against normal mouse fibroblast (NIH3T3) cell line to
evaluate their cancer cell specificity (safety index).8 The
cytotoxicity data are given in Table 2.
Except compound 10, none of the unsubstituted 1,8-
naphthyridine-3-carboxamides (8–11) have shown cyto-
toxicity. Compound 10 exhibited cytotoxicity in PA-1
and KB cell lines. The 7-chloro naphthyridines (12–16)
were found to be better than unsubstituted naphthyri-
dines (8–11). Compound 12, containing oxazolidine
ring, was found to be the most potent derivative as it
exhibited high cytotoxicity (IC50 = 1.37 lM) in HBL-
100 cell line and was also found to be active in SW-
620 and PA-1 cell lines. Compounds having proline
(13) and alanine (16) substituent showed cytotoxicity
in HBL-100 cell line. Compound 13 also showed cyto-
toxicity in MIAPaCa 2 cell line. Based on the above re-
sults, several analogs, related to active compounds 12,
13, and 16, were further designed and synthesized to im-
prove the cytotoxicity.
Upon replacing the 7-chloro group in compound 13 with
methyl group (compound 17), activity in HBL-100 cell
line was lost but was found to be most potent in KB cell
line (IC50 = 3.7 lM). It seems that nature ofgroup present
in pyridine ring in naphthyridine-3-carboxamides plays a
vital role in eliciting cytotoxicity profile. On the other
hand, by replacing propyl spacer with isopropyl (com-
pound 18) in compound 13, cytotoxicity profile has been
changed. Compound 18 was found to be more than 2-fold
less cytotoxic in HBL-100 than compound 13 but exhib-
ited activities in PA-1, KB, and SW-620 cell lines. It indi-
Results and discussion. The 1,8-naphthyridine-3-carbox-
amide derivatives (8–23) were tested for in vitro cytotox-
icity in eight tumor cell lines and IC50 values were
calculated in micromole (lM).8,9 The human tumor cell
lines used in the study were ovary (PA-1), prostate
O
O
O
O
O
1. (EtO)3CH, Ac2O
2. CH=C-CH2-NH2
1. CDI
OH
OEt
OEt
X
X
X
X
2. EtO2CCH2CO2H
3. MeMgBr
N
O
Cl
2
N
Cl
NH
N
1
Cl
3
K2CO3/EtOAc
O
O
O
H2N-Y-OH
Y
O
N
O
O
N
O
aq HCl
X
SOCl2
X
Cl
N
OH
X
OH
OEt
H
N
N
N
N
N
N
7
6
5
4
RCOOH,
DCC,
DMAP
O
N
O
O
Ph
O
O
O
20 % TFA/DCM
X = 7-Cl,
Y
N
O
NH2
N
O
R
X
H
H
OH
Cl
N
N
N
Ph
R
NBoc
Y = -(CH2)3- ; R =
S
8- 20
(Boc)2O/THF/NaHCO3
21
O
O
N
O
O
Ph
NHBoc
N
O
H
OH
Cl
N
22
Scheme 1.

6662
S. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6660–6664
O
N
O
O
Boc
N
N
OH
3-Methylpiperidine,
K2CO3, Acetone
,
HO
DCC, DMAP
H
N
N
7
X = 7-Cl
Y = -(CH2)3-
CH3
7a
O
O
O
Boc
N
N
O
H
N
N
N
CH3
23
Scheme 2.
propyl spacer with isopropyl (compound 19) in com-
pound 16, a similar cytotoxic profile was obtained except
in SW-620 cell line. Compound 19 was 2-fold less cyto-
toxic than 16 in HBL-100 but exhibited cytotoxicity in
PA-1 and KB cell lines. On the other hand, when oxazol-
idine ring in compound 12 was opened (compound 21),
activity was lost. Further, upon converting amine group
in compound 21 to its Boc derivative 22, activity HBL-
100 was lost but was found to be highly potent in SW-
620 cell lines (IC50 = 3.0 lM).
Table 1. 1,8-Naphthyridine-3-carboxamide derivatives (8–20)
Compound
X
Y
R
Ph
R
NBoc
8
H
–(CH2)3–
S
O
Boc
N
9
H
H
H
–(CH2)3–
–(CH2)3–
–(CH2)3–
10
11
–CH2–NHBoc
It reveals that 1,8-naphthyridine-3-carboxamides, in
general, exhibited cytotoxicity in oral, colon, and breast
cancers and the nature of group present in pyridine ring
and the spacer, in particular, determined cytotoxicity
profile. It was also interesting to note that 1,8-naph-
thyridine-3-carboxamides, in general, have shown good
safety index as well. However, compounds 12, 18, and
22 are under further biological evaluation.
NHBoc
H3C CH3
Ph
R
NBoc
12
7-Cl
–(CH2)3–
S
O
Boc
N
13
14
15
7-Cl
7-Cl
7-Cl
–(CH2)3–
–(CH2)3–
–(CH2)3–
Anti-inflammatory activity. 1,8-Naphthyridine-3-carbox-
amide derivatives (8–23) were able to downregulate the
levels of LPS stimulated TNF-a, IL-1b and IP-10 se-
creted by DCs that were identified to have potential
anti-inflammatory activity. The downregulation of cyto-
kine and chemokine levels by >25% was considered as
significant.10,11
–CH2–NHBoc
NHBoc
H3C CH3
NHBoc
CH3
16
17
18
19
20
7-Cl
–(CH2)3–
–(CH2)3–
Figure 1 shows the downregulation of a key pro-inflam-
matory cytokine TNF-a by selected molecules. Com-
pounds 12, 13, 14, and 22 exhibit >50% TNF-a
inhibition at 1 lg/ml, reflecting significant anti-inflam-
matory activity. Compound 13 shows a remarkable
downregulation of TNF-a activity even at 0.1 lg/ml.
Out of these few selected compounds, 13, 14, and 22
demonstrate a significant inhibition of IP-10 activity,
as shown in Figure 2. In addition, compounds 8, 12,
and 21 exhibited >50% IL-1b inhibitory activity (Fig. 3).
Boc
N
7-CH3
7-Cl
Boc
N
S
CH3
S
NHBoc
CH3
7-Cl
CH3
Boc
N
S
The downregulation of TNF-a, IL-1b, and IP-10 levels
by compounds 8, 12, 13, 14, 16, 21, and 22 suggests po-
tential anti-inflammatory activity.
7-CH3
CH3
cates that spacer has also played an important role in
determining cytotoxic profile. When the 7-chloro group
in compound 13 was replaced by 3-methylpiperidine
(compound 23), cytotoxicity was lost. As described in
the activity profile for compound 18, upon replacing the
1,8-Naphthyridine-3-carboxamides (8–23) with poten-
tial myeloprotective activity were identified by evaluat-
ing modulation in MIP-1-a, CCL-22, and TNF-a from
basal levels secreted by DCs, when incubated with these
molecules.12

S. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6660–6664
Table 2. In vitro cytotoxicity of 1,8-naphthyridine-3-carboxamide derivatives (8–23)
6663
Compound
IC50 (lM)
PA-1
(ovary)
DU-145
(prostate)
KB
(oral)
SW-620
(colon)
HBL-100
(breast)
A-549
(lung)
Miapaca
(pancreas)
K-562 (leukemia)
NIH3T3
(normal fibroblast)
10
12
13
16
17
18
19
22
9.0
6.0
>10
>10
>10
>10
>10
>10
>10
>10
7.0
>10
3.96
>10
>10
>10
3.3
>10
1.37
3.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
7.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8.4
>10
>10
>10
3.7
>10
>10
>10
5.2
5.4
>10
>10
>10
>10
>10
9.2
>10
8.4
>10
>10
>10
7.84
6.6
8.3
4.9
>10
>10
3.0
>10
>10
>10
Compound No.
Compound No.
14
8
12
13
14
16
21
22
0
13
16
-20
-40
300
250
-60
200
150
-80
1ug/ml
0.1ug/ml
100
50
0
-100
-120
1ug/ml
0.1ug/ml
-50
Figure 1. Anti-inflammatory activity of selected 1,8-naphthyridine-3-
carboxamide derivatives as a measure of TNF-a downregulation. %
change in TNF-a was calculated with reference to LPS stimulated
levels secreted by murine DCs.
Figure 4. Upregulation of MIP-1-a levels (% change calculated with
reference to basal levels secreted by DCs) by selected 1,8-naphthyri-
dine-3-carboxamide derivatives with potential myeloprotective
activity.
Compound No.
13
14
16
22
0
-20
Compound No.
-40
13
14
16
0
-20
-40
-60
-60
1ug/ml
-80
0.1ug/ml
-100
-120
1ug/ml
Figure 2. IP-10 downregulation (% change calculated with reference to
LPS stimulated levels secreted by DCs) by selected 1,8-naphthyridine-
3-carboxamide derivatives.
0.1ug/ml
Figure 5. CCL-22 downregulation (% change calculated with reference
to basal levels secreted by DCs) by selected 1,8-naphthyridine-3-
carboxamide derivatives.
Compound No.
8
12
21
0
-20
Compound No.
-40
13
14
16
-60
1ug/ml
0
-20
-40
-60
-80
-80
0.1ug/ml
-100
Figure 3. IL-1-b downregulation (% change calculated with reference
to LPS stimulated levels secreted by DCs) by selected 1,8-naphthyri-
dine-3-carboxamide derivatives.
1ug/ml
0.1ug/ml
Figure 4 demonstrates MIP-1-a upregulation by selected
compounds 13, 14, and 16, suggesting significant myelo-
protective activity. Also, compounds 13, 14, and 16 were
able to downregulate CCL-22 and TNF-a as shown in
Figures 5 and 6, respectively. Endogenous upregulation
of MIP-1-a under the influence of proposed 1,8-naph-
thyridine-3-carboxamide derivatives is also likely to ex-
ert similar myeloprotective effects.
Figure 6. TNF-a downregulation of selected 1,8-naphthyridine-3-
carboxamide derivatives with potential myeloprotective activity. %
change in TNF-a was calculated with reference to basal levels secreted
by DCs.
In addition, these molecules showing a downregula-
tion of TNF-a from basal levels (Fig. 6) were

6664
S. K. Srivastava et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6660–6664
1,8-naphthyridine-3-carboxamide derivatives (8–23) were
similarly incubated. The assay was terminated after 72 h
by adding 125 lg (25 lL) MTT to each well, then
incubating for three hours, and finally adding 50 lL of
10% SDS–0.01 N HCl to each well to lyse the cells and
dissolve formazan. After incubating for 1 h, the plate was
read spectrophotometrically at 540 mm and the cytotox-
icity percentage calculated using the following formula:
predicted to have lower tissue toxicity due to reduced
probability of TNF-a induced inflammation leading to
tissue damage.
References and notes
1. FDAnews Drug Pipeline Alert, March 21, 2006; Vol. 4,
No. 56.
cytotoxicity
percentage = (1 À X/R1) 100,
where
*
X = (absorbance of treated sample at 540 nm) À (absor-
bance of blank at 540 nm), R1 = absorbance of control
sample at 540 nm.
2. Tsuzuki, Y.; Tomita, K.; Shibamori, K.-i.; Sato, Y.;
Kashimoto, S.; Chiba, K. J. Med. Chem. 2004, 47, 2097.
3. Tomita, K.; Tsuzuki, Y.; Shibamori, K.-i.; Tashima, M.;
Kajikawa, F.; Sato, Y.; Kashimoto, S.; Chiba, K.; Hino,
K. J. Med. Chem. 2002, 45, 5564.
4. Grossi, G.; Di Braccio, M.; Roma, G.; Ballabeni, V.;
Tognolini, M.; Barocelli, E. Eur. J. Med. Chem. 2005, 40, 155.
5. Dianzani, C.; Collino, M.; Gallicchio, M.; Di Braccio, M.;
Roma, G.; Fantozzi, R. J. Inflamm. 2006, 3, 4.
doi:10.1186/1476-9255-3-4.
9. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
10. BMDC based ex-vivo septic shock assay to evaluate
potential anti-inflammatory activity: Primary DC cultures
were generated from femoral bone marrow of 8- to 12-
week-old C57BL/6 mice.11 Bone marrow progenitors
were cultured in RPMI-1640 supplemented with 10%
FBS and rmGMCSF (20ng/ml) at 37 °C, 5% CO2.
Immature DCs were stimulated with lipopolysaccharide
(LPS; 100 ng/ml) and incubated with the 1,8-naphthyri-
dine-3-carboxamide derivatives at various concentrations
ranging from 0.001 to 10 lg/ml, preferably between 0.1
and 1 lg/ml for 24 h. The TNF-a, IL-1-b-, and IP-10
secreted by DCs were measured in culture supernatants
by Enzyme Linked Immunosorbent Assays (R&D)
systems Inc., MN, USA. Percentage change in cytokine/
6. 4R,5S-N-(tert-Butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-
oxazolidine carboxylic acid was purchased from Dabur
Pharma Ltd, Kalyani, WB, India.
7. Compound 8: Rf 0.7 (5% MeOH/DCM); 1H NMR
(CDCl3, 300 MHz) d 9.76 (br s, 1H), 9.1 (s, 1H), 8.75–
8.66 (m, 2H), 7.43–7.39 (m, 1H), 7.25–7.19 (m, 5H), 5.21
(s, 2H), 5.01 (br s, 1H), 4.48 (d, 1H, J = 5.2 Hz), 4.26–4.24
(m, 2H), 3.45–3.43 (m, 2H), 2.46 (s, 1H), 1.96–1.92 (m,
2H), 1.70 (s, 3H), 1.63 (s, 3H) 1.18–1.05 (m, 9H); MS
(ES+) m/z (% relative intensity) 589 (M+H) (20), 611
(M+H+Na) (100).
chemokine = {(B À A)/A} 100, where B = concentration
*
of cytokine/chemokine (pg/ml) secreted by LPS stimulated
DCs when incubated with test molecule, A = concentration
of cytokine/chemokine (pg/ml) secreted by LPS stimulated
DCs alone.
8. Derivatives of 1,8-naphthyridine-3-carboxamide (8–23)
were screened for cytotoxic activity at the highest soluble
concentration of 10 lM and on four lower concentrations
on eight human tumors and one non-tumorous cell lines.
Briefly, a 3-day MTT in vitro cytotoxicity assay was
performed, which was based on the principle of uptake of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide), a tetrazolium salt, by the metabolically
active cells where it was metabolized by active mitochon-
dria into a blue colored formazan product that was read
spectrophotometrically.9 MTT was dissolved in phos-
phate-buffered saline with a pH of 7.4 to obtain a MTT
concentration of 5 mg/ml; the resulting mixture was
filtered through a 0.22-lm filter to sterilize and remove a
small amount of insoluble residue. For each type of tumor
and normal cell, 5000–10,000 cells were seeded in a 96-well
culture plate and incubated with various concentrations of
1,8-naphthyridine-3-carboxamide derivatives (8–23) in a
CO2 incubator for 72 h. Control cells not treated with
11. Lutz, M. B.; Kukutsch, N.; Ogilvie, A. L.; Rossner, S.;
Koch, F.; Romani, N.; Schuler, G. J. Immunol. Methods
1999, 223, 77.
12. BMDC based assay for myeloprotective activity: The
procedure employed to analyze changes in chemokine
(MIP-1-a, CCL-22) and cytokine (TNF-a) levels expressed
by DCs under the influence of 1,8-naphthyridine-3-car-
boxamide derivatives is described by below. Murine bone
marrow derived dendritic cells10 were incubated with test
compounds at 0.1 and 1 lg/ml. The amounts of MIP-1-a,
CCL-22, and TNF-a secreted by DCs after 24 h incuba-
tion were measured in the culture supernatants by
respective Enzyme-Linked Immunosorbent Assays (R&D
systems Inc., MN, USA). Percentage change in cytokine/
chemokine = {(B À A)/A} 100, where B = concentration
*
of cytokine/chemokine (pg/ml) secreted by DCs when
incubated with test molecule, A = concentration of cyto-
kine/chemokine (pg/ml) secreted by untreated DCs.