Synthesis, structure, and antibacterial activity of 4-imino-1,4-dihydrocinnoline-3-carboxylic acid and 4-oxo-1,4-dihydrocinnoline-3-carboxylic acid derivatives as isosteric analogues of quinolones

Article abstract of DOI:10.1002/ardp.200390000

Chemical modification of cinnoxacin was studied with the aim of improving its antibacterial activity and spectrum. A series of 4-imino-1,4-dihydrocinnoline-3-carboxylic acid derivatives was synthesized and their in vitro antibacterial activity was evaluated. These derivatives were designed as isosteric analogues of fluoroquinolones and are characterized by the presence of an imine group instead of an oxo group at the 4-position and a nitrogen atom in position 2. The crystal structure of one analogue determined by X-ray diffraction shows the dipolar form of the compound in the solid state. The in vitro antibacterial activity of the synthesized compounds against Gram-positive and Gram-negative bacteria was examined. The MIC of the most active compounds lies in the range of the first generation of quinolones such as nalidixic acid. The compounds with dichlorobenzyl substituent show enhanced activity against Gram-positive bacteria.

Full text of DOI:10.1002/ardp.200390000

18 Sta´nczak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 1, 18–30
Andrzej Sta´nczaka,
Zbigniew Ochockia,
Dariusz Martynowskib,
Marek Glówkab,
Synthesis, Structure, and Antibacterial Activity of
4-Imino-1,4-dihydrocinnoline-3-carboxylic Acid and
4-Oxo-1,4-dihydrocinnoline-3-carboxylic Acid
Derivatives as Isosteric Analogues of Quinolones
Ewa Nawrotc
a
Department of
Chemical modification of cinnoxacin was studied with the aim of improving its anti-
bacterial activity and spectrum. A series of 4-imino-1,4-dihydrocinnoline-3-car-
boxylic acid derivatives was synthesized and their in vitro antibacterial activity was
evaluated.These derivatives were designed as isosteric analogues of fluoroquino-
lones and are characterized by the presence of an imine group instead of an oxo
group at the 4-position and a nitrogen atom in position 2.The crystal structure of one
analogue determined by X-ray diffraction shows the dipolar form of the compound in
the solid state. The in vitro antibacterial activity of the synthesized compounds
against Gram-positive and Gram-negative bacteria was examined. The MIC of the
most active compounds lies in the range of the first generation of quinolones such as
nalidixic acid. The compounds with dichlorobenzyl substituent show enhanced
activity against Gram-positive bacteria.
Pharmaceutical Chemistry
and Drug Analysis,
Medical University of Lódz᝽,
Poland
b
Institute of General and
Ecological Chemistry,
Technical University of Lódz᝽,
Poland
c
Department of
Pharmaceutical Microbiology,
Medical University of Lódz᝽,
Poland
Key Words: Cinnolones; Quinolones; Antibacterial activity; DNA topoisomerase;
Crystal data
Received: February 27, 2002 [FP676]
good antibacterial activity mainly against Gram-negative
bacteria.We have been interested in the chemical modi-
Introduction
The quinolone class of antibacterial agents has rapidly
emerged as one of the most effective drugs in the treat-
ment of bacterial diseases. Quinolinones and naphthy-
ridinones were also found to inhibit human topoisomer-
ase II [1–5].The molecular target of these agents is DNA
gyrase, an enzyme that catalyses the introduction of
negative supercoils into circular duplex DNA.This proc-
ess is necessary for DNA replication in bacteria [6–9].Al-
though the structure-activity relationship (SAR) of fluoro-
quinolone has been extensively studied, no clear conclu-
sion has yet been reached. The most widely accepted
mechanism of action of quinolones is based on their in-
teraction with DNA bases via hydrogen bonds.Two mod-
els are usually described in the literature:
fication of cinnoline-3-carboxylic acids, in the hope of de-
veloping new antibacterial agents with improved activity.
As a part of a program designed to identify new DNA
gyrase inhibitors, modification of the cinnolone structure
was undertaken by implementation of the imine group in
position 4 (Figure 1).
– the model based on hydrogen bonding to DNA bases
and stacking with another quinolone molecule [6],
Figure 1
– an alternative model based on formation of Mg2+-qui-
nolone-DNA adducts and stacking with DNA bases
[7].
Also, X-ray studies of one of the resulting compounds
have been undertaken. Until now only three crystal
structures of 4-imine analogues of quinolones, all having
cinnoline ring system, have been determined [10, 11].
However, the compounds were either dihydrochlorides
(1-ethyl-7-methyl-1,4-dihydro-4-iminocinnoline-3-car-
boxylic acid) [10] or had a modified 3-carboxylic function,
as in 3-carbamoyl-1,4-dihydro-4-imino-1-methylcinno-
line [11], and the results of their structural studies could
Cinnoxacin, the chemical structure of which is character-
ized by a 4-oxocinnoline-3-carboxylic acid moiety, shows
Correspondence: Andrzej Sta´nczak, Department of Pharma-
ceutical Chemistry and Drug Analysis, Medical University
ul. Muszyn´ skiego 1, 90-151 Lódz᝽, Poland. Fax: +48 42 6779250,
e-mail: stanczak@pharm.am.lodz.pl.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/01/0018 $ 17.50+.50/0

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
4-Imino- and 4-Oxo-1,4-dihydrocinnoline-3-carboxylic 19
Figure 2
not be used directly for the discussion of structural simi-
larity with neutral quinolones. The present study has
been designed to investigate our hypothesis concerning
the possibility of formation of hydrogen bonding 4-imine-
analogues.Although the imine group is isosteric with the
carbonyl group, the former group may form hydrogen
bonds, both as a proton donor and as an acceptor (Fig-
ure 2).
hydrous potassium carbonate in dimethylformamide.
This gave a mixture of ethyl 1-alkyl-1,4-dihydro-4-oxo-
cinnolinecarboxylates and anhydro-bases of 2-alkyl-
3-ethoxycarbonyl-4-hydroxycinnolinium hydroxides.
Based on our earlier study, we have found that the ethyl
4-hydroxy- and 4-aminocinnoline-3-carboxylates 4 were
alkylated smoothly with benzyl bromides in the presence
of anhydrous potassium carbonate in acetonitrile [13].
Alkylation of the 4-hydroxycinnoline-3-carboxylic esters
4 was carried out with dimethyl sulphate, diethyl sul-
phate, benzyl bromide, allyl bromide, ethyl bromoace-
tate, and 2-bromoethanol. However, there were some
problems with alkylation of 4-aminocinnoline-3-carboxy-
lic acid esters 6 with “weak” alkylating agents such as
alkyl and cycloalkyl bromides. We have found that the
yield of alkylation depends strongly on the solubility of
esters 4 in the reaction medium, alkylating agent type,
and ease of formation of the imine form of 4-amino-3-cin-
nolinecarboxylic acid esters 4. In many cases the alkyla-
tion of esters failed. Structures of esters 6 were con-
firmed by IR1 and H NMR spectra. In the IR spectra of 6
the two bands in the range of carbonyl area at 1700 and
1645 cm–1 are different from signals of esters 4. There
was clear evidence of N1-alkylation when examining the
1H NMR.The signals of the ethyl group was noted as sec-
ond quartet (3.7–4.0 ppm) or singlet (5.8–6.2 ppm) for
benzyl CH2 protons. The singlet shift of the imine group
of esters 6 an exchangeable with D2O was about
9.6–9.9 ppm. Alkylation of the amides 1 by the same
method as used for esters 4 gave 1-alkyl-1,4-dihydro-4-
iminocinnoline-3-carboxamides and 1-alkyl-1,4-dihy-
dro-4-oxocinnoline-3-carboxamides 7. The esters 6
were converted into corresponding 1-alkyl-1,4-dihydro-
4-iminocinnoline-3-carboxylic acids hydrazides 9 and 1-
alkyl-1,4-dihydro-4-oxocinnoline-3-carboxylic acids hy-
drazides 9.The formation of hydrazides 9 was supported
by the disappearance of the 1H NMR band correspond-
ing to the OC2H5 group and the appearance two signals
NH of hydrazide protons at 4.0–6.1 ppm and 10.3–11.0
ppm exchangeable with D2O. The esters 6 were reacted
with hydroxylamine hydrochloride in the presence of
DBU in dimethylformamide to obtain corresponding
Appropriate analogues were synthesized on the basis of
molecular modelling.
Investigations, results, and discussion
Chemistry
The general synthetic routes employed to prepare 4-
aminocinnoline-3-carboxylic acid derivatives 2 and 4-hy-
droxycinnoline-3-carboxylic acids derivatives 3 are out-
lined in Scheme 1.The compounds 1 were prepared by
the intramolecular Friedel-Crafts reaction of correspond-
ing (arylhydrazono)(cyano)acetamides as described in
the reference [12]. Hydrolysis of the amides 1 with 80 %
sulphuric acid solution gave 4-aminocinnoline-3-carb-
oxylic acids 2. However, alkaline hydrolysis of appropri-
ate acids 2 or amides 1 led to 4-hydroxycinnoline-3-
carboxylic acids 3.The acids 2, 3 were converted into
corresponding ethyl 4-aminocinnoline-3-carboxylates
and ethyl 4-hydroxycinnoline-3-carboxylates 4 using
thionyl chloride and then ethyl alcohol according to the
method described in the literature [13].The esters 4 were
reacted with an excess of hydrazines in the presence of
DBU in dimethylformamide to give corresponding 4-
amino-3-cinnolinehydrazides and 4-hydroxy-3-cinno-
linehydrazides 5.
Alkylation of the 4-hydroxycinnolines derivatives was
examined extensively [14–16]. It was found that alkyla-
tion in protic solvents under basic conditions gave a mix-
ture of N-1 and N-2 alkylated products. Similar results
were reported by Miyamoto and Matsumoto [17], for
alkylation of ethyl 4-hydroxycinnolinecarboxylates with
alkyl iodide or dialkyl sulfate in the presence of an-

20 Stan´ czak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
4-Imino- and 4-Oxo-1,4-dihydrocinnoline-3-carboxylic 21
Table 1. Physical and analytical data of compounds (4 a–n).
Compd.
Y
R1
R2
R3
R4
Molecular formula
(weight)
Mp [°C]
Yield
[%]
4 a
4 b
4 c
4 d
4 e
4 f
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
OH
CH3
F
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
F
CH3
C11H11N3O2
(217.1)
C10H8FN3O2
(221.2)
C11H11N3O2
(217.1)
C12H13N3O2
(231.2)
C13H15N3O2
(245.3)
C11H10ClN3O2
(251.6)
C11H10FN3O2
(235.2)
C11H10FN3O2
(235.2)
C11H9Cl2N3O2
(286.1)
C11H9ClFN3O2
(269.7)
C12H12ClN3O2
(265.7)
C11H9ClN2O3
(252.6)
C11H9FN2O3
(236.2)
C11H9FN2O3
(236.2)
254–256
266–268
79.6
81.2
89.0
80.7
79.5
76.8
81.5
76.7
85.3
80.6
78.9
79.2
87.7
81.5
H
CH3
H
H
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
242–244 [13]
242–244 [13]
263–265
CH3
CH3
H
H
CH3
Cl
H
265–267 [13]
276–278
4 g
4 h
4 i
F
H
F
284–285
Cl
F
Cl
Cl
H
>330
4 j
>330
4 k
4 l
Cl
Cl
F
234–236
H
232–234 [13]
212–213
4 m
4 n
OH
H
OH
H
H
225–227
Table 2. Physical and analytical data of compounds (6 a–z).
Compd.
Z
R1
R2
R3
R5
Molecular formula
(weight)
Mp [°C]
Yield
[%]
6 a
6 b
6 c
NH
NH
NH
H
H
H
H
H
H
H
C18H17N3O2
(307.3)
215–217 [13]
207–208
74.8
76.9
82.3
F
C18H17FN3O2
(326.3)
CH3
C19H17Cl2N3O2
(390.3)
216–218
6 d
NH
CH3
CH3
H
C20H19Cl2N3O2
(404.3)
191–193
72.6

22 Stan´ czak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
Table 2. (continued).
Compd.
Z
R1
H
R2
R3
H
R5
Molecular formula
(weight)
Mp [°C]
Yield
[%]
6 e
NH
CH3
C19H17Cl2N3O2
(390.3)
224–226
80.4
6 f
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
CH3
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
CH3
C14H17N3O2
(259.3)
C13H15N3O2
(245.3)
C14H17N3O2
(259.3)
C13H14ClN3O2
(279.7)
C13H13Cl2N3O2
(314.2)
C13H14FN3O2
(263.2)
C13H13ClFN3O2
(297.7)
C17H16ClN3O2
(329.8)
>330
198–200
205–207
234–236
210–211
227–229
135–137
216–218
254–255
298–299
160–162 [13]
82.6
77.4
68.4
69.9
71.3
77.3
75.9
80.2
79.6
84.2
74.6
6 g
6 h
6 i
C2H5
H
CH3
Cl
Cl
H
C2H5
H
C2H5
6 j
Cl
F
C2H5
6 k
6 l
C2H5
F
Cl
H
C2H5
6 m
6 n
6 o
6 p
Cl
H
(CH2)2COOCH3
CH2COOC2H5
CH2COOC2H5
F
C15H16FN3O2
(289.3)
C15H15ClFN3O2
(323.8)
C18H15ClN2O3
(342.7)
F
Cl
H
Cl
6 r
O
O
H
CH3
H
H
H
C18H18N2O3
(322.4)
205–207
215–217
77.8
69.6
6 s
CH3
C19H16Cl2N2O3
(391.3)
6 t
O
H
CH3
H
C19H16Cl2N2O3
(391.3)
224–226
68.3
6 w
6 z
O
O
F
H
H
H
C2H5
C2H5
C13H13FN2O3
(264.3)
C14H16N2O3
(260.3)
187–189
216–217
80.3
78.9
H
CH3
1-alkyl-1,4-dihydro-4-iminocinnoline-3-hydroxamic acids
and 1-alkyl-1,4-dihydro-4-oxo-cinnoline-3-hydroxamic
acids 8.
with 20 % hydrochloric acid solution led to final 1-alkyl-
1,4-dihydro-4-iminocinnoline-3-carboxylic acids 10;
however, alkaline hydrolysis of the esters 6 gave the cor-
responding 1-alkyl-1,4-dihydro-4-oxocinnoline-3-car-
boxylic acids 10 j, 10 k, similarly to unsubstituted 4-ami-
nocinnoline-3-carboxylic acids as reported in a previous
The 1H NMR spectrum of the hydroxamic acids indicated
the presence of the singlet NH proton at 8.7–9.1 ppm ex-
changeable with D2O. The acidic hydrolysis of esters 6

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
4-Imino- and 4-Oxo-1,4-dihydrocinnoline-3-carboxylic 23
Table 3. Physical and analytical data of compounds (10 a–m).
Compd.
Z
R1
R2
R3
R5
Molecular formula
(weight)
Mp [°C]
Yield
[%]
10 a
10 b
10 c
10 d
NH
NH
NH
NH
F
H
H
H
H
H
C16H12FN3O2
(297.3)
240–242
265–266
263–265
279–280
60.4
48.6
51.3
60.2
H
Cl
H
CH3
H
C17H15N3O2
(293.3)
C16H12ClN3O2
(313.7)
CH3
C17H13Cl2N3O2
(362.2)
10 e
10 f
10 g
10 h
10 i
NH
NH
NH
NH
NH
NH
NH
O
H
H
H
H
H
H
H
H
H
H
H
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C11H11N3O2 · H2O
(235.2)
C12H13N3O2 · H2O
(249.3)
C12H13N3O2 · H2O
(249.3)
C13H15N3O2 · H2O
(263.3)
C11H10ClN3O2 · H2O
(269.7)
C11H10FN3O2 · H2O
(253.2)
C11H9ClFN3O2 · H2O
(287.7)
C11H9FN2O3
(236.2)
238–239 [12]
277–278
266–267
291–292
272–273
261–262
272–274
228–230
198–200
61.4
69.4
59.7
58.2
60.4
55.9
56.2
76.2
79.3
CH3
H
H
CH3
CH3
Cl
CH3
H
10 j
F
H
10 k
10 l
F
Cl
F
H
10 m
O
H
CH3
C12H12N2O3
(232.2)
Table 4. Physical and analytical data of compounds (5 a–w).
Compd.
Y
R1
R2
R3
R6
Molecular formula
(weight)
Mp [°C]
Yield
[%]
5 a
5 b
5 c
5 d
5 e
5 f
NH2
NH2
NH2
NH
CH3
CH3
F
H
H
H
H
H
H
H
H
C10H11N5O
(217.2)
C11H13N5O
(231.3)
C9H8FN5O
(221.2)
C9H8ClN5O
(237.6)
C10H10ClN5O
(251.7)
C9H7ClFN5O
(255.6)
279–281
>330
67.6
73.2
71.9
66.9
72.2
69.2
CH3
H
H
H
>330
Cl
H
H
>330
NH2
NH2
Cl
H
CH3
H
>330
F
Cl
>330

24 Stan´ czak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
Table 4. (continued).
Compd.
Z
R1
R2
R3
R5
Molecular formula
(weight)
Mp [°C]
Yield
[%]
5 g
5 h
5 i
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Cl
H
H
Cl
H
Cl
H
H
H
H
CH3
C10H10ClN5O
(251.7)
C10H10ClN5O
(251.7)
C10H10FN5O
(235.2)
C10H9ClFN5O
(269.7)
C11H12ClN5O
(265.7)
C11H10ClN5O2
(267.7)
C16H15N5O
(293.3)
>330
61.9
70.6
62.8
72.7
73.1
75.9
60.1
H
CH3
229–232
>330
F
H
CH3
5 j
F
H
CH3
245–246
220–222
218–220
276–278
5 k
5 l
Cl
Cl
CH3
CH3
H
CH3
COCH3
5 m
H
5 n
5 o
5 p
NH2
NH2
NH2
F
H
H
H
H
C15H12FN5O
(297.3)
235–237
248–250
245–247
61.2
57.8
60.9
Cl
Cl
H
C15H12ClN5O
(313.7)
CH3
C16H14ClN5O
(327.8)
5 r
5 s
5 t
OH
OH
OH
H
H
H
H
H
H
H
H
H
C9H8N4O2
(204.2)
C9H7ClN4O2
(238.6)
C15H12N4O2
(280.3)
>330
>330
59.9
61.4
57.9
Cl
H
289–291
5 w
OH
Cl
H
H
CH3
C10H9ClN4O2
(352.7)
>330
62.9
paper [14].In a typical example, the 1H NMR spectrum of
the acids 10 exhibited, besides the aromatic protons,
singlet or quartet for N1 methylene group and singlet of
NH protons at 10.5–11.2 ppm.We tried to substitute the
chlorine atom at 7-position by piperazine and 4-methyl-
piperazine because this is a basic substituent in 7 posi-
tion of most used flouoroquinolones. Unfortunately, at-
tempts to perform those replacements were unsuccess-
ful under a variety conditions. All the compounds were
characterized by physical constants, elemental analysis,
example because of the similarity of the spectral data in
corresponding groups of compounds. (The 1H NMR and
IR spectra of the remaining compounds are available on
request.) The physical properties of compounds 4–10
and the structures of their substituents are summarized
in Tables 1–6.
We have also analysed the optimised structures (PM3,
HyperChem, ChemPlus, Hypercube 1998) of com-
pounds 6 d, 10 and compared them with that of nalidixic
acid. Superimposition of the PM3 optimised conformers
of the individual compounds and nalidixic acid was per-
formed with the RMS fit procedure within ChemPlus 2.0.
As is usual in studies attempting to superimpose struc-
1
IR, and H NMR spectra.
Spectral data of the obtained compounds were consist-
ent with the assigned structure and are given for only one

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
4-Imino- and 4-Oxo-1,4-dihydrocinnoline-3-carboxylic 25
Table 5. Physical and analytical data of compounds (9 a–l).
Compd.
Z
R1
R2
R5
R6
Molecular formula
(weight)
Mp [°C]
Yield
[%]
9 a
9 b
NH
NH
CH3
CH3
H
H
C2H5
H
C12H15N5O
(245.3)
C18H19N5O
(321.4)
265–267
208–210
68.9
67.2
CH3
9 c
NH
CH3
H
H
C17H15Cl2N5O
(376.3)
257–259
75.7
9 d
9 e
NH
NH
CH3
CH3
H
C23H19Cl2N5O
(452.3)
278–279
267–269
65.6
59.8
CH3
CH3
CH3
C19H19Cl2N5O
(404.3)
9 f
NH
O
CH3
H
H
H
C18H17Cl2N5O
(390.3)
252–254
62.8
9 g
H
C16H14N4O2
(294.3)
185–187
175–177
79.8
68.9
9 h
9 i
O
O
Cl
Cl
H
H
CH3
C17H15ClN4O2
(342.8)
C22H17ClN4O2
(404.9)
213–215
61.8
9 j
O
O
Cl
H
H
H
C16H13ClN4O2
(328.8)
C18H18N4O2
(322.4)
220–222
173–175
71.7
62.5
9 k
CH3
C2H5
C2H5
9 l
O
H
CH3
CH3
C13H16N4O2
(260.3)
198–200
74.9
tures, we selected three pairs of atoms for the fitting pro-
cedure: N-1 atom of cinnoline ring, C-7 carbon atom of
cinnoline nucleus, and C atom of the carbonyl group at
3-position of cinnoline and analogous atoms of the struc-
ture of nalidixic acid. The three points mentioned were
selected as atom pairs for the fitting procedure. The
superimposition of nalidixic acid with cinnoline analog
10 g (RMS = 1.887 Å) is shown in Figure 3.However, the
fit of compound 6 d, which had the best antibacterial ac-
tivity, is worse than that of compounds with poor activity
(RMS = 5.139 Å).The superimposition of compound 6 d
with nalidixic acid is shown in Figure 4. It can be noted
that compounds with the best activity against Gram-
positive bacteria are inactive against Gram-negative
species Evidently, further refinement of the model is re-
quired to explain these facts.

26 Stan´ czak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
Table 6. Physical and analytical data of compounds (7 a–d, 8 a–d).
Compd.
Z
X
R1
R2
R5
Molecular formula
(weight)
Mp [°C]
Yield
[%]
7 a
7 b
7 c
7 d
NH
NH
NH
NH
H
H
H
H
H
F
H
C2H5
C2H5
C2H5
C11H12N4O
(216.2)
C11H10FClN4O
(268.7)
C11H11ClN4O
(250.7)
C17H16N4O
(292.3)
>330
>330
60.4
64.1
61.4
57.9
Cl
Cl
H
H
>330
CH3
287–289
8 a
8 b
NH
NH
OH
OH
H
H
H
C2H5
C11H12N4O2
(232.2)
C17H16N4O2
(308.3)
266–268
284–286
66.1
68.5
CH3
8 c
8 d
NH
O
OH
OH
F
H
H
C16H13FN4O2
(312.3)
238–240
202–204
69.2
75.8
Cl
C16H12ClN3O3
(313.7)
Figure 3.Superimposition of nalidixic acid with cinnoline
analog (10 g).
Figure 4. Superimposition of nalidixic acid with com-
pound (6 d).

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
4-Imino- and 4-Oxo-1,4-dihydrocinnoline-3-carboxylic 27
though formally classical isosteres of quinolones, can
not interact specifically with the same DNA base or in the
same fashion due to reversed functionality of a 4-imine
group in hydrogen bonds as compared with a 4-oxo one.
X-ray crystallography
The most surprising result of the X-ray study of 1-ben-
zyl-7-methyl-1,4-dihydro-4-iminocinnoline-3-carboxylic
acid 10 b was its existence in dipolar form in the crystal
state (Figure 5). It suggests that the same form may pre-
dominate in aqueous solution, especially in the case of
analogues containing a more basic substituent in posi-
tion 7, for example, 4-methylpiperazine.Other character-
istic features of the molecular structure of 10 b are:
Antibacterial activity
Antibacterial activity of 4-amino-3-cinnolinecarboxylic
acid derivatives was examined while indicating the mini-
mum inhibitory concentration (MIC) for representatives
of the Gram-positive (Staphylococcus aureus ATCC
6538P, Staphylococcus aureus ATCC 29213, Staphylo-
coccus aureus ATCC 25923, E. faecalis ATCC 29212,
Bacillus subtilis ATCC 6633), and Gram-negative
(Escherichia coli ATCC 35218, Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853) bacte-
rial strains. Nalidixic acid was used as a reference drug
of known antibacterial activity. The MIC of the chosen
compounds against Gram-positive and Gram-negative
bacteria are summarized in Table 7, which includes only
the compounds with MIC < 500 µg/mL. Surprisingly, the
most active compounds appeared to be ester 6 d and
methyl hydrazide 9 e, both with the same substituent at
N-1 position. In contrast to classical quinolones, these
– strong intramolecular hydrogen bonds between proto-
nated 4-imine group as a hydrogen bond donor and
3-carboxylate anion as an acceptor;
– partial localization of π electrons in the benzene ring
of the cinnoline system, shown by shortening of C-5–
C-6 and C-7–C-8 bonds to 1.361(3) and 1.376(3) Å,
respectively, as compared with the rest of the C–C
bonds, being 1.399–1.408 Å;
– stacking of the flat pseudo three ring structure of 10 b
in an antiparallel fashion (Figure 3).
Similar relative orientation of quinolones and its ana-
logues may be of great importance for their interactions
with DNA [18]. We are aware that 4-imine analogues,
Figure 5. Crystal structure of (10 b). Thermal elipsoids were drawn with 50 % probability with the XP program [19].
(a) General view of molecule, atom numbering system, and intramolecular hydrogen bond. (b) Arrangement of mole-
cules in the crystal state.

28 Stan´ czak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
Table 7. In vitro antibacterial activity of the compoundds.
Minimum inhibitory concentration (MIC) µg/mL
Compd.
Gram-positive organisms
Gram-negative organisms
S. aureus
ATCC
6538P
S. aureusa
ATCC
S. aureusa E. faecalisa B. subtilis E. colia E. coli Ps. aeruginosa
ATCC
25923
ATCC
29212
ATCC
6633
ATCC ATCC
35218 25922
ATCC
27853
29213
b
5 a
5 b
5 h
5 n
×
×
62
250
62
×
62
250
62
×
125
250
×
×
300
×
250
×
300
×
250
×
×
×
250
×
62
250
62
125
250
62
6 b
6 c
6 d
7 b
8 d
9 c
9 e
500
125
25
375
250
>500
50
500
125
25
500
125
25
×
250
50
500
125
100
187
250
300
50
×
×
×
187
125
300
×
×
×
×
93
125
300
×
×
×
×
×
>500
×
×
×
×
×
250
>500
50
250
300
50
500
500
50
9 h
9 j
10 j
250
500
×
>500
>50
250
250
×
×
>50
250
250
×
×
>50
250
250
×
×
>50
250
250
>400
500
6,2
×
×
100
250
6,2
×
×
×
×
×
×
200
250
<6,25
10 k
Nevigramon
>50
a
Multiple resistant bacteria.
No activity.
b
two compounds showed increased antibacterial activity
against Gram-positive bacterial strains and loss of activi-
ty against Gram-negative ones. Generally, among the
tested compounds, there can be seen rare antibacterial
activity, mainly against Gram-positive bacterial strains,
which is especially surprising for final acids 10, which are
very similar to typically applied quinolones.
NMR spectra of compounds were recorded on in DMSO-d6 on
a Varian Mercury 300 MHz spectrometer using tetramethyl-
silane as internal standard. The infrared spectra of the com-
pounds were recorded on a Mattson Infinity Series FTIR spec-
trophotometer. 1H NMR multiplicity data are denoted by s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br
(broad). Carbon, hydrogen, and nitrogen elemental analyses
were performed on a Perkin Elmer series II CHNS/O Analyzer
2400 and were within 0.4 % of theoretical values.
Chemistry
Acknowledgement
Synthesis of series 1, 2, 3
The authors gratefully acknowledge financial support
within the project 4 P05F 024 17 from the Polish State
Committee for Scientific Research.
Compounds 1, 2, 3 were synthesized as described in [12].
Synthesis of ethyl 4-aminocinnoline-3-carboxylates 4 and ethyl
4-hydroxycinnoline-3-carboxylates 4
Compounds 4 were synthesized as described in [13].
Experimental
Physical and analytical data for 4 are shown inTable 1.Example
of spectral data for 4 e: IR (KBr): νmax cm–1 = 3280–3000 (NH2),
1690 (C=O). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 1.3–1.6
(t, 3 H, CH3), 2.6 (d, 6 H, CH3-Ar), 3.7–4.0 (q, 2 H, CH2), 8.0 (s,
1 H, Ar-NH), 8.2 (s, 2 H, NH2), 8.3 (s, 1 H, Ar-H). 4 p: IR (KBr):
Apparatus
All chemicals and solvents used in this study were purchased
locally from Fluka and Aldrich and were used without further
purification.
ν
max cm–1 = 3200–3000 (OH), 1700 (C=O). 1H NMR (300 MHz,
Melting points were determined on an Electrothermal appara-
tus in open capillaries and have not been corrected. The 1H
DMSO-d6): δ (ppm) = 1.2–1.6 (t, 3 H, CH3), 4.1–4.6 (q, 2 H,
CH2), 7.8–8.0 (d, 2 H, Ar-H), 8.1–8.3 (d, 2 H, Ar-H + OH).

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
4-Imino- and 4-Oxo-1,4-dihydrocinnoline-3-carboxylic 29
Physical and analytical data for 9 are shown inTable 5.Example
of spectral data for 9 b: IR (KBr): νmax cm–1 = 3950–3780 (NH),
1690 (C=O). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 2.7 (s,
3 H, CH3-Ar), 3,8 (d, 3 H, N-CH3), 4.9 (bs, 1 H, NH-CH3), 5.9 (s,
2 H, CH2), 7.2–7.4 (m, 5 H, Ar-H), 7.8–7.9 (m, 2 H, Ar-H),
8.1–8.3 (m, 2 H, Ar-H + NH2), 10.8 (bs, 1 H, CO-NH).
Synthesis of ethyl 1-alkyl-1,4-dihydro-4-iminocinnoline-3-car-
boxylates 6 and ethyl 1-alkyl-1,4-dihydro-4-oxocinnoline-3-
carboxylates 6
To a stirred suspension of the appropriate esters 4 (10 mmol) in
CH3CN or DMF was added 15 mmol DBU or K2CO3 (for benzyl
derivatives) and the mixtures were refluxed for 0.5 h. After that
the alkylating agents (25 mmol) were added dropwise and the
mixtures were refluxed for 2 h before being evaporated down to
1/3 volume. The remaining solutions were rendered alkaline
with 10 % ammonia. The crude products were filtered off and
crystallized from a mixture of DMF and water.
Synthesis of 1-alkyl-1,4-dihydro-4-iminocinnoline-3-carboxylic
acid amides and 1-alkyl-1,4-dihydro-4-oxocinnoline-3-carb-
oxylic acid amides 7
Compounds 7 were synthesized as described above for com-
pound 6.
Physical and analytical data for 7 are shown inTable 6.Example
of spectral data for 7 b: IR (KBr): νmax cm–1 = 3250–31250 (NH),
1680 (C=O). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 1.4–1.5
(t, 3 H, CH3), 4.7–4.9 (q, 2 H, CH2), 8.0 (s, 1 H, Ar-H), 8.1 (s, 1 H,
Ar-H), 8.2–8.4 (bs, 2 H, CONH2), 10.3 (s, 1 H, NH).
Physical and analytical data for 6 are shown inTable 2.Example
of spectral data for 6 g: IR (KBr): νmax cm–1 = 3270–3000 (NH),
1700 (C=O). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 1.2–1.5
(dt, 6 H, CH3), 3.7–3.8 (q, 2 H, N-CH2), 4.3–4.4 (q, 2 H, –OCH2),
7.2–7.6 (m, 2 H, Ar-H), 8.0–8.2 (d, 1 H, Ar-H), 9.6 (s, 1 H, NH);
6 r: IR (KBr):νmax cm–1 = 1700, 1680 (C=O). 1H NMR (300 MHz,
DMSO-d6): δ (ppm) = 1.3–1.6 (t, 3 H, CH3), 4.2–4.5 (q, 2 H,
CH2), 5.9 (s, 2 H, CH2-Ar), 7.5–8.0 (m, 6 H, Ar-H), 8.3 (d, 2 H,
Ar-H).
Synthesis of 1-alkyl-1,4-dihydro-4-iminocinnoline-3-hydrox-
amic
acids
and
1-alkyl-1,4-dihydro-4-oxocinnoline-3-
hydroxamic acids 8
To a stirred solution of the appropriate esters 4 (10 mmol) in
DMF was added DBU (15 mmol) and the mixtures were reflux-
ed for 0.5 h.After that the hydroxylamine hydrochloride solution
25 mmol was added dropwise and mixtures were stirred for
20 h.The solvents were evaporated to 1/2 volume and acidified
with 30 % CH3COOH and filtered. The products were purified
by crystallization from mixture DMF and water with charcoal.
Synthesis of 1-alkyl-1,4-dihydro-4-iminocinnoline-3-carboxylic
acids 10
Compounds 10 a–k were synthesized as described in [13].
Physical and analytical data for 10 a–k are shown in Table 3.
Example of spectral data for 10 h: IR (KBr): νmax cm–1 = 3200–
3050 (OH, NH), 1670 (C=O). 1H NMR (300 MHz, DMSO-d6): δ
(ppm) = 1.4–1.5 (t, 3 H, CH3), 4.6–4.8 (q.2 H, CH2), 7.9–8.1 (m,
2 H, Ar-H), 8.3–8.4 (d, 1 H, Ar-H), 10.6 (s, 1 H, NH).
Physical and analytical data for 8 are shown inTable 6.Example
of spectral data for 8c: IR (KBr): νmax cm–1 = 3050–2860 (NH,
OH), 1660 (C=O). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 6.0
(s, 2 H, CH2), 7.2–7.4 (m, 5 H, Ar-H), 8.4-8.5 (d, 2 H, Ar-H),
8.9–9.0 (s, 1 H, NH), 10.5 (s, 1 H, NH).
Synthesis of 1-alkyl-1,4-dihydro-4-oxocinnoline-3-carboxylic
acids 10
X-ray structure determination
A stirred suspension of 5 mmol esters 6 and 2 M solution
(20 cm3) NaOH was refluxed for 2 h. After cooling the mixtures
were acidified with 30 % CH3COOH and filtered. The products
were purified by crystallization from CH3COOH with charcoal.
Physical and analytical data for 10 l–m are shown in Table 3.
X-ray data were collected on four-circle diffractometer KM-4 at
room temperature with Cu Kα. The structure was solved by di-
rect method and refined with SHELXL [19].
Crystal data and structure refinement details for 10 b.
Example of spectral data for 10 l: IR (KBr): νmax cm–1
=
2900–2400 (OH), 1675 (C=O). 1H NMR (300 MHz, DMSO-d6):
δ (ppm) = 1.4–1.5 (t, 3 H, CH3), 4.6–4.8 (q, 2 H, CH2), 7.9–8.0
(m, 1 H, Ar-H), 8.4–8.5 (dd, 1 H, Ar-H), 8.9–9.0 (m, 1 H, Ar-H).
Empirical formula
Crystal system and space group
Unit cell dimensions (A and °)
C17H15N3O2
monoclinic, P21/c
7.579(2)
14.808(3)
12.679(3)
90.10(3)
1456.8(6); 4
616
3–80
a
b
c
β
Synthesis of 4-aminocinnoline-3-carboxylic acid hydrazides 5
and 4-hydroxycinnoline-3-carboxylic acid hydrazides 5
Volume [A] and Z
F(000)
Theta range in data collection [°]
No. of reflections:
A solution of esters 4 or 6 (10 mmol) and DBU (15 mmol) in
DMF (50 cm3) was refluxed for 0.5 h. To the reaction mixtures
the corresponding hydrazines (10 mmol) were added and the
mixtures were heated under reflux for 2 h. After cooling water
(50 cm3) was added and the mixtures were left for 24 h.The sol-
ids were filtered off, washed with hot ethanol, and crystallized
from mixture DMF and water.
– collected 4019
– unique 2857
– observed 2254
Data/parameter ratio
Goodness of fit (on F2)
Final R (Rw)
10
1.017
0.048
Physical and analytical data for 5 are shown inTable 4.Example
of spectral data for 5 a: IR (KBr): νmax cm–1 = 3900–3550 (NH),
1690 (C=O). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 2.7 (s,
3 H, CH3-Ar), 4.5 (bs, 2 H, NH2), 7.8–7.9 (m, 2 H, Ar-H), 8.1–8.3
(m, 2 H, Ar-H + NH2), 10.5 (bs, 1 H, CONH).
Largest difference peak (e/Å3)
0.19
In vitro antibacterial activity
Synthesis of 1-alkyl-1,4-dihydro-4-iminocinnoline-3-carboxylic
acids hydrazides and 1-alkyl-1,4-dihydro-4-oxocinnoline-3-
carboxylic acid hydrazides 9
The minimal inhibitory concentrations (MICs) of the
studied compounds were determined according to the
method of serial twofold dilutions in agar, recommended
by NCCLS (National Committee for Clinical Laboratory
Compounds 9 were synthesized as described above for com-
pounds 5.

30 Stan´ czak et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 18–30
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