Synthesis and anti-intestinal nematode activity of variously substituted benzonaphthyridine derivatives

Article abstract of DOI:10.3390/molecules16021593

A series of benzonaphthyridine derivatives bearing the C=N linkage moiety were designed and synthesized. The structures of all the newly synthesized compounds were identified by elemental analysis, 1H-NMR, 13C-NMR and MS. Their anti-intestinal nematode ac

Full text of DOI:10.3390/molecules16021593

Molecules 2011, 16, 1593-1602; doi:10.3390/molecules16021593
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Anti-Intestinal Nematode Activity of Variously
Substituted Benzonaphthyridine Derivatives
Li-Ping Duan, Ai-Dan Wen, Ning-Bo Wu, Yi Tao and Hao-Bing Zhang *
Key Laboratory of Parasitology and Vector Biology at National Institute of Parasitic Diseases, Chinese
Center for Disease Control and Prevention, WHO Collaborating Centre for Malaria, Schistosomiasis,
and Filariasis, National Institute of Parasitic Diseases, Shanghai 200025, China
* Author to whom correspondence should be addressed; E-Mail: zhanghaobing@hotmail.com.
Received: 21 December 2010; in revised form: 18 January 2011 / Accepted: 25 January 2011 /
Published: 14 February 2011
Abstract: A series of benzonaphthyridine derivatives bearing the C=N linkage moiety
were designed and synthesized. The structures of all the newly synthesized compounds
1
were identified by elemental analysis, H-NMR, 13C-NMR and MS. Their anti-intestinal
nematode activities against Nippostrongylus brazilliensis were evaluated in vivo by an oral
route in male rats. Among these compounds, at concentrations of 10 mg/kg of rat, the
compound
7-chloro-2-methoxy-10-(4-(4′-(1H-indol-5′-yl)methylene)aminophenyl)-
amino-benzo[b][1,5] naphthyridine (4n) produced the highest activity, with 80.2%
deparasitization. These compounds may find usefulness in the discovery and development
of new anti-intestinal drugs.
Keywords: benzonaphthyridine; C=N linkage; Nippostrongylus brazilliensis
1. Introduction
Intestinal parasitic nematodes or roundworms infect over 1 billion people in tropical countries.
According to a 2005 report by the World Health Organization (WHO), approximately 0.807–1.221
billion humans have ascariasis, 604–795 million have trichuriasis, and 576–740 million have
hookworm infections worldwide [1]. Despite their harmfulness, few drugs for dealing with them exist
[2-4]. Tribendimidine has good single-dose efficacy against some roundworm parasites [5], and in
studies [6-8] tribendimidine showed high efficacy against Nippostrongylus braziliensis, Necator

Molecules 2011, 16
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americanus, Ancylostoma caninum, and Toxocara canis. Furthermore, tribendimidine can continue to
be effective in areas where albendazole has become less so due to resistance. This has been attributed
to the presence of a toxophoric C=N linkage [5]. In our research group, we have been interested in
studying the design, synthesis, and biological activity of compounds, especially heterocyclic
compounds [9-14]. During the last 30 years a large number of derivatives belonging to the general
benzonaphthyridine class have been prepared and evaluated extensively as antimalarial,
antileishmanial and antitrypanosomal compounds [15-19]. This inspired us to assume that
benzo[b][1,5]-naphthyridine derivatives incorporating a Schiff base might have some improved or
different biological activities. In the present work, we describe the synthesis and results of some
preliminary in vivo biological tests of 15 different novel benzonaphthyridine derivatives bearing a
C=N linkage moiety.
2. Results and Discussion
2.1. Synthesis and Characterization of substituted benzo-naphthyridine derivatives (4a-4o)
The synthetic route to the target compounds 4a-4o is shown in Scheme 1. Benzo[b][1,5]-
naphthyridine derivative 1 had already been described [20].
Scheme 1. Synthesis of compounds 4a-4o.
NO2
Cl
N
O
O2N
NH2
Cl
N
NH
N
N
O
1
Cl
2
N
CH R
NH2
SnCl2, HCl
NH
N
NH
N
N
O
N
O
Cl
Cl
4a-4m
Br
3
OH
Cl
F
b
c
d
a
e
OH
H3CO
OCH3
OCH3
R=
HO
i
j
f
g
h
Cl
N
HN
N
Cl
N
k
l
m
m
n
Direct nucleophilic substitution of chlorine at the 10 position of the benzo[b][1,5]-naphthyridine
ring by p-nitroaniline afforded desired chloride 2 in good yield. The subsequent reduction of 2 using
SnCl2 as reducing agent led to the key intermediate 3. Then 3 and different aromatic aldehydes were

Molecules 2011, 16
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mixed in boiling ethanol affording the target compounds 4a–4o, which were recrystallized twice from
ethanol to give satisfactory yields of 35–50%, respectively. Compounds 4a–4o were characterized by
elemental analysis, 1H-NMR, 13C-NMR and MS. All results were in full agreement with the proposed
structures. For example, the 1H-NMR spectrum of compound 4b showed a singlet at 4.18 ppm (OCH3),
singlet at 8.45 ppm (NH) and a singlet at around δ = 8.40 due to one proton of the Schiff base. This
13
was also confirmed by C-NMR spectrum, which showed C=N signals at 160.71 ppm, moreover, the
13C-NMR spectrum showed δ 53.90(OCH3), 159.73, 154.89, 148.65, 148.02, 146.56, 143.71, 142.92,
142.50, 142.20, 140.49, 136.62, 135.11, 129.95, 129.09, 128.87, 127.76, 127.71, 126.60, 124.51,
122.58, 122.18, 119.42, 118.59，115.76 (Ar-C), all consistent with its proposed structure. The
elemental analyses results were in good agreement with those calculated for the suggested formula.
Further confirmation for the structure of compound 4b was obtained by MS, which showed the
expected [M+1]+ ion at 457.27. Additionally, the melting points are sharp, suggesting the purity of
these compounds.
2.2. Anti-intestinal nematode activity
From Table 1, we can see that some of compounds showed significant anti-intestinal nematode
activity in a two-day in vivo test in rats. At concentrations of 10 mg/kg of rat, compound 4n produced
the highest activity against Nippostrongylus brazilliensis, with 80.3% deparasitization.
Table 1. Effect of benzo[b][1,5] naphthyridine derivatives in treatment of rats infected
with NbL3 for 11 days (4a-4o).
Worm recovery
Compound Dose(mg/kg)
Number of rats
Expelled
245
114
75
Total
248
543
297
322
237
426
269
326
287
306
370
310
189
197
268
248
Reduction (%)
98.8
Albendazole
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4a
4b
4c
4d
4e
4f
21.0
25.2
74
23.0
48
20.2
100
75
23.4
27.9
4g
4h
4i
68
20.8
69
24.0
82
26.8
4j
89
24.0
4k
4l
79
25.4
95
50.2
4m
4n
4o
101
215
176
51.2
80.2
70.9
Expelled: number of expelled worms; Total: total worm number; Reduction: worm burden reduction.
To investigate the role of substituent effects on activity, a variety of substituents could be
introduced on the phenyl ring. The F, OCH3, OH, Cl and Br-substituted derivatives all have almost the

Molecules 2011, 16
1596
same activity as 4a. Furthermore, disubstituted derivatives also appeared to have similar potency as the
monsubstituted phenyl analogues. For instance, the replacement of the 4′-F in 4′ by a hydroxyl group
led to only slightly less potency, and compound 4f inhibited Nippostrongylus brazilliensis with a value
similar to that of the 2,4-disubstituted analogue 4j. In contrast, derivatives 4g and 4l were slightly less
active than the corresponding isomers 4h and 4m. On the other hand, replacement of the phenyl ring
by a pyridyl ring, indole group, or quinoline group resulted in increased effects, suggesting a
preference for hetero-aromatic rings in maximizing activity. This indicated that indole group was the
most important group involved in this activity. The quinoline group also seems to influence this
activity, while the pyridyl group appeared to give only a small contribution. Because compound 4n
displayed anti-intestinal nematode potency that was comparable to albendazole (10 mg/kg), further
anti-intestinal nematode activity assayd were carried out for compound 4n. It was found that at
concentrations
of
30 mg/kg of rat, 4n produced the highest activity against Nippostrongylus brazilliensis with 98.8%
effectiveness, which implies further possibilities for the development of new anti-intestinal drugs.
3. Experimental
3.1. Materials and reagents
All solvents and other reagents were of high purity (Aldrich and Sigma) and were used without
further purification. Elemental analysis was performed on a PE-2400 Elemental Analyzer, the C, H
1
and N analysis were repeated twice. H-NMR and 13C-NMR spectra were obtained in CDCl3 with
TMS as internal standard on a Bruker AM-400 spectrometer. Chemical shifts were reported as ppm.
Mass spectra were measured on a HP 1100 LC-MS spectrometer. Melting points were determined by
an BÜCHI melting point B-540 apparatus and are uncorrected.
3.2. Synthesis of 7-chloro-2-methoxy-10-aminobenzo[b][1,5]naphthyridine (2)
A solution obtained dissolving equimolar amounts of the starting compound 7-chloro-2-methoxy-
10-chloro-benzo[b][1,5]naphthyridine (1) and p-nitroaniline in absolute ethanol (150 mL) was refluxed
for 2.5–3 h until the starting materials disappeared (TLC, ethyl acetate/n-hexane 1:4). On standing and
cooling a red-orange precipitate formed which was filtered off and dried (70–80% yields). Purification
was accomplished by repeated crystallization from ethanol. LC-MS: [M+1]+ 381.17.
3.3. Synthesis of 7-chloro-2-methoxy-10-(4-aminophenyl)-aminobenzo[b][1,5] naphthyridine (3)
Compound 2 (3.80 g, 0.01 mol) was added to a previously prepared solution of glacial acetic acid
(40 mL) containing SnCl2 (15.3 g, 0.08 mmol) and aerated with hydrogen chloride. The reaction
mixture was stirred for 1 h. The precipitated solid was collected by filtration and washed with water.
Crystallization from acetic acid gave 3 as red–orange needles: 3.46 g (98.8%); LC-MS: [M+1]+
351.29.

Molecules 2011, 16
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3.4. General procedure for the synthesis of Schiff bases 4a-4o
To a mixture of 3 (0.005 mol) and substituted aromatic aldehyde (0.005 mol) in absolute ethanol
(40 mL) was added few drops of glacial acetic acid. Then the mixture was refluxed on water bath for
5–6 h. The excess of solvent was distilled off, poured onto ice cold water. The separated solid was
filtered, washed and recrystallized from ethanol.
7-Chloro-2-methoxy-10–(4-benzylideneaminophenyl)-aminobenzo[b][1,5]naphthyridine (4a). Yield
35%, m.p. 180.2–181.3 °C. 1H-NMR δ: 8.41 (s, 1H, Ar-NH), 8.37(s, 1H, CH=N), 8.24–6.82 (m, 14H,
Ar-H), 4.16 (s, 3H,OCH3); 13C-NMR δ: 160.20, 159.62, 154.70, 149.00, 148.12, 146.45, 143.71,
142.00, 140.50, 137.63, 131.65, 130.86, 130.78, 129.24, 124.78, 123.16, 123.10, 122.17, 122.16,
121.76, 119.28, 119.16, 115.78, 114.44, 113.15, 53.69; Anal. Calcd. for C26
71.15, H 4.36, N 12.77; found C 71.16, H 4.38, N 12.77.
H
19 Cl N O (439.82): C
4
7-Chloro-2-methoxy-10–(4-(4′-fluorobenzylidene)aminophenyl)-aminobenzo[b][1,5]naphthyridine
1
(4b). Yield 40%, m.p. 191.6–191.8 °C. H-NMR δ: 8.45 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N),
13
8.29–7.02 (m, 13H, Ar-H), 4.18 (s, 3H,OCH3); C-NMR δ: 160.71, 159.73, 154.89, 148.65, 148.02,
146.56, 143.71, 142.92, 142.50, 142.20, 140.49, 136.62, 135.11, 129.95, 129.09, 128.87, 127.76,
127.71, 126.60, 124.51, 122.58, 122.18, 119.42, 118.59, 115.76, 53.90; Anal. Calcd. for C26
O (457.27): C 68.39, H 4.00, N 12.07; found C 68.39, H 4.00, N 12.07.
H18 ClF
N4
7-Chloro-2-methoxy-10–(4-(4′-chlorobenzylidene)aminophenyl)-aminobenzo[b][1,5]naphthyridine
1
(4c). Yield 42%, m.p. 193.4–194.1 °C. H-NMR δ: 8.45 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N),
13
8.25–6.98 (m, 13H, Ar-H), 4.16 (s, 3H,OCH3); C-NMR δ: 160.83, 159.80, 154.70, 149.79, 148.32,
144.40, 143.87, 142.21, 140.36, 138.06, 132.56, 131.45, 129.65, 126.09, 124.87, 123.76, 122.67,
122.48, 121.69, 119.20, 119.17, 117.09, 116.47, 115.20, 53.80 ; Anal. Calcd. for C26
(474.25): C 65.97, H 3.83, N 14.98; found C 65.99, H 3.83, N 14.98.
H
18 Cl2 N O
4
7-Chloro-2-methoxy-10–(4-(4′-bromobenzylidene)aminophenyl)-aminobenzo[b][1,5]naphthyridine
1
(4d). Yield 37%, m.p. 188.6–191.0 °C. H-NMR δ: 8.47 (s, 1H, Ar-NH), 8.40(s, 1H, CH=N),
13
8.25–7.00 (m, 13H, Ar-H), 4.17 (s, 3H,OCH3); C-NMR δ: 160.83, 159.60, 149.71, 148.38, 144.84,
143.89, 142.41, 140.40, 138.96, 132.66, 131.95, 129.75, 126.49, 125.67, 124.86, 123.69, 123.68,
120.69, 119.29, 119.20, 118.89, 117.57, 116.27, 115.48, 53.90 ; Anal. Calcd. for C26
(519.21): C 60.31, H 3.50, N 10.82; found C 60.31, H 3.51, N 10.85.
H
18 BrCl N O
4
7-Chloro-2-methoxy-10–(4-(4′-hydroxylbenzylidene)aminophenyl)-amino-benzo[b][1,5] naphthyridine
1
(4e). Yield 39%, m.p. 201.3–201.5 °C. H-NMR δ: 8.44 (s, 1H, Ar-NH), 8.20 (s, 1H, CH=N),
13
8.28–6.84 (m, 13H, Ar-H), 5.50 (s, 1H,OH), 4.18 (s, 3H,OCH3); C-NMR δ: 160.83, 160.21, 159.74,
149.68, 148.45, 144.91, 143.56, 142.35, 138.95, 132.45, 130.68, 129.87, 127.49, 125.86, 123.89,
123.70, 123.45, 120.69, 119.34, 119.08, 118.54, 116.60, 116.27, 115.50, 114.56, 59.62 ; Anal. Calcd.
for C26
H
19ClN O2 (455.28): C 64.43, H 4.05, N 11.58; found C 64.43, H 4.05, N 11.58.
4

Molecules 2011, 16
1598
7-Chloro-2-methoxy-10–(4-(4′-methoxylbenzylidene)aminophenyl)-aminobenzo[b][1,5] naphthyridine
1
(4f). Yield 45%, m.p. 170.8–171.1 °C. H-NMR δ: 8.87 (s, 1H, Ar-NH), 8.40(s, 1H, CH=N),
8.20–6.68 (m, 13H, Ar-H), 4.18 (s, 3H,OCH3), 3.82 (s, 3H,OCH3); 13C-NMR δ: 160.73, 159.23,
149.91, 148.98, 145.82, 143.90, 142.53, 138.96, 132.70, 131.90, 129.86, 127.31, 126.77, 125.77,
123.54, 123.25, 121.99, 120.29, 119.78, 118.96, 116.78, 116.20, 106.48, 103.76, 54.60, 53.82; Anal.
Calcd. for C27
H
21ClN O2 (419.18): C 69.15, H 4.51, N 11.95; found C 69.16, H 4.51, N 11.96.
4
7-Chloro-2-methoxy-10–(4-(2′,4′-dimethylbenzylidene)aminophenyl)aminobenzo[b][1,5] naphthyrid-
ine (4g). Yield 46%, m.p. 161.1–161.9 °C. 1H-NMR δ: 8.80 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N),
8.39–6.78 (m, 12H, Ar-H), 4.18 (s, 3H,OCH3), 2.55(s, 3H, CH3), 2.35 (s, 3H, CH3); 13C-NMR δ:
160.61, 158.34, 149.11, 148.74, 143.12, 142.87, 141.49, 140.81, 140.48, 138.58, 135.18, 131.88,
131.48, 125.77, 128.86, 128.41, 127.91, 127.26, 126.75, 124.28, 122.79, 122.15, 119.37, 115.46,
53.79, 21.57,19.47; Anal. Calcd. for C28
H 5.34, N 11.43.
H
23ClN O (467.29): C 69.10, H 5.34, N 11.49; found C 69.18,
4
7-Chloro-2-methoxy-10–(4-(2′,3′-dimethylbenzylidene)aminophenyl)-aminobenzo[b][1,5] naphthyrid-
ine (4h). Yield 48%, m.p. 125.1–125.7 °C. 1H-NMR δ: 8.45 (s, 1H, Ar-NH), 8.40(s, 1H, CH=N), 8.20–
7.01 (m, 12H, Ar-H), 4.18 (s, 3H,OCH3), 2.34(s, 6H, CH3); 13C-NMR δ: 160.62, 158.34, 149.11,
148.74, 143.10, 142.80, 141.48, 140.80, 140.47, 138.56, 135.08, 131.78, 131.48, 125.77, 128.86,
128.41, 127.90, 127.26, 126.70, 124.28, 122.78, 122.05, 119.37, 115.46, 53.89, 21.47,19.37; Anal.
Calcd. for C28
H
23ClN O (467.28): C 71.87, H 4.62, N 11.68; found C 71.87, H 4.62, N 11.68.
4
7-Chloro-2-methoxy-10–(4-(2′,4′-dihydroxylbenzylidene)aminophenyl)-aminobenzo[b][1,5]naphthyr-
1
idine (4i). Yield 44%, m.p. 232.1–232.9 °C. H-NMR δ: 8.45 (s, 1H, Ar-NH), 8.40(s, 1H, CH=N),
8.20–7.01 (m, 12H, Ar-H), 4.18 (s, 3H,OCH3), 1.44(s, 2H, OH); 13C-NMR δ: 160.63, 158.38, 149.16,
148.77, 143.13, 142.80, 141.48, 140.83, 140.49, 138.58, 135.68, 131.80, 131.56, 125.79, 128.89,
128.43, 127.92, 127.36, 126.78, 124.59, 122.78, 122.16, 119.39, 115.48, 53.91; Anal. Calcd. for
C26
H
19ClN O3 (471.27): C 66.07, H 3.76, N 11.50; found C 66.07, H 3.76, N 11.50.
4
7-Chloro-2-methoxy-10–(4-(2′,4′-methoxylbenzylidene)aminophenyl)-aminobenzo[b][1,5] naphthyr-
1
idine (4j).Yield 45%, m.p. 160.2–161.8 °C. H-NMR δ: 8.89 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N),
13
8.20–6.55 (m, 12H, Ar-H), 4.18 (s, 3H,OCH3), 3.83 (s, 3H,OCH3), 3.82 (s, 3H,OCH3); C-NMR δ:
160.73, 159.23, 149.91, 148.98, 145.82, 143.90, 142.53, 138.96, 132.70, 131.90, 129.86, 127.31,
126.77, 125.77, 123.54, 123.25, 121.99, 120.29, 119.78, 118.96, 116.78, 116.20, 115.48, 114.76,
54.60, 53.82, 53.80; Anal. Calcd. for C28
66.21, H 5.19, N 10.52.
H
23Cl N O3 (499.29): C 66.21, H 5.19, N 10.52; found C
4
7-Chloro-2-methoxy-10–(4-(2′,4′-dichlorobenzylidene)aminophenyl)-aminobenzo[b][1,5] naphthyr-
1
idine (4k). Yield 39%, m.p. 220.1–221.2 °C. H-NMR δ: 8.59 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N),
13
8.20–6.99 (m, 12H, Ar-H), 4.18 (s, 3H,OCH3); C-NMR δ: 160.63, 159.13, 149.00, 148.88, 145.83,
143.91, 142.59, 138.99, 132.72, 131.91, 129.88, 127.32, 126.79, 125.79, 123.56, 123.35, 121.99,

Molecules 2011, 16
1599
120.39, 119.79, 118.98, 116.79, 116.21, 115.49, 114.78, 53.90; Anal. Calcd. for C26
(507.21): C 60.65, H 3.70, N 10.69; found C 60.90, H 3.53, N 10.87.
H
17Cl3 N O
4
7-Chloro-2-methoxy-10–(4-(2′-pyridylidene)aminophenyl)-amino-benzo[b][1,5] naphthyridine (4l).
Yield 48%, m.p. 227.2–228.7 °C. 1H-NMR δ: 8.75 (s, 1H, Ar-NH), 8.41(s, 1H, CH=N), 8.80–7.05 (m,
13H, Ar-H), 4.18 (s, 3H,OCH3); 13C-NMR δ: 160.69, 159.23, 149.09, 148.78, 145.85, 143.92, 142.59,
138.99, 132.76, 131.93, 129.88, 127.35, 126.69, 125.81, 123.66, 123.36, 121.98, 120.49, 119.89,
118.45, 116.80, 116.20, 115.38, 114.78, 53.91; Anal. Calcd. for C25
3.79, N 14.40; found C 62.70, H 3.94, N 14.42.
H
18ClN O (440.26): C 62.76, H
5
7-Chloro-2-methoxy-10–(4-(3′-pyridylidene)aminophenyl)-aminobenzo[b][1,5]-naphthyridine (4m).
Yield 41%, m.p. 190.1–190.2 °C. 1H-NMR δ: 8.76 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N), 8.91–7.09 (m,
13H, Ar-H), 4.18 (s, 3H,OCH3); 13C-NMR δ: 160.67, 157.97, 148.73, 147.91, 142.91, 141.22, 140.55,
135.07, 130.75, 130.68, 129.88, 127.32, 126.79, 125.79, 123.56, 123.35, 121.99, 120.39, 119.79,
118.98, 116.79, 116.21, 115.49, 114.78, 53.90; Anal. Calcd. for C25
4.12, N 15.92; found C 68.28, H 4.14, N 15.94.
H
18Cl N O (440.26): C 68.26, H
5
7-Chloro-2-methoxy-10–(4-(4′-(1H-indol-5′-yl)methylene)aminophenyl)-aminobenzo[b][1,5]-naph-
1
thyridine (4n). Yield 45%, m.p. 226.8–227.7 °C. H-NMR δ: 8.68 (s, 1H, Ar-NH), 8.40 (s, 1H,
CH=N), 8.45–7.10 (m, 14H, Ar-H), 6.61 (s, 1H, Pyrrol-NH), 4.18 (s, 3H,OCH3); 13C-NMR δ: 160.60,
157.98, 148.76, 147.87, 142.96, 141.24, 140.55, 140.33, 139.80, 136.31, 135.76, 130.69, 130.65,
129.89, 127.36, 126.80, 125.78, 123.58, 123.38, 122.01, 120.39, 119.82, 118.98, 116.79, 116.21,
115.47, 114.96, 53.78; Anal. Calcd. for C28
69.33, H 4.31, N 14.23.
H
20Cl N O (478.30): C 69.33, H 4.31, N 14.23; found C
5
7-Chloro-2-methoxy-10–(4-(4′-(quinoline-2′-yl)methylene)aminophenyl)-aminobenzo[b][1,5]naph-
thyridine (4o). Yield 44%, m.p. 219.7–220.9 °C. 1H-NMR δ: 9.24 (s, 1H, Ar-NH), 8.40 (s, 1H, CH=N),
13
9.05–7.15 (m, 15H, Ar-H), 4.18 (s, 3H,OCH3); C-NMR δ: 160.67, 159.21,158.45, 158.21, 157.99,
148.84, 147.68, 142.91, 141.20, 140.59, 135.14, 130.36, 130.23, 129.89, 127.33, 126.76, 125.78,
123.56, 123.35, 121.99, 120.39, 119.87, 118.98, 116.82, 116.71, 115.49, 115.41, 53.92; Anal. Calcd.
for C29
H
20ClN O (490.32): C 70.98, H 4.17, N 14.04; found C 71.01, H 4.18, N 14.12.
5
3.5. Biological assays
All analogues were tested against N. brazilliensis to evaluate their anti-intestinal nematode
activities. These compounds were tested on 15 groups of rats, each containing five rats. Eighty male
rats, weighing 70–80 g, were provided by the Shanghai Animal Center (Chinese Academy of
Sciences). Rats were kept at the animal facility of our institute. Animals were acclimatized for 1 week
before infection, and they had free access to water and food. The life cycle of N. braziliensis were
maintained in our laboratory. Egg-positive feces were obtained from N. braziliensis-infected rats at
12–20 days post-infection, and then used for the cultivation of N. braziliensis third stage infective
larvae (NbL3) as described previously [21]. For the in vivo test, rats were each infected subcutaneously

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with 300 NaL3, all infected rats were treated with compounds 11 days post-infection. For oral
administration, compounds were suspended or dissolved in 7% Tween 80 and 3% alcohol, and the
concentrations of each drug were 10 mg/kg. The reference compound was albendazole, and water
containing 3% alcohol and 7% Triton X-80 was used as a negative control. The percentage
deparasitization was calculated using the following formula:
n/N × 100
where N = average number of total worms found from small intestine, large intestine and feces in the
control animals and n = average number of worms found from large intestine and feces in the control
animals. Evaluations were based on a percentage scale of 0–100, in which 100 was total kill and 0 was
no activity.
4. Conclusions
In summary, various types of substituted benzonaphthyridine derivatives were synthesized and their
biological activities towards the N. brazilliensis were demonstrated. Among these compounds, 4n
produced the highest activity against N. brazilliensis, with 80.2% deparasitization. The present work
suggested that 4n may be a useful lead compound for anti-intestinal nematode medicine development.
Further studies of the structure-activity relationships around the designed compounds are underway.
Acknowledgements
The authors wish to acknowledge that this project is supported by National Institute for Diseases
(2010A102) and Shanghai Health Bureau (2009Y109).
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Sample Availability: Samples of the compounds 4a-4o are available from the authors.
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