On the chemistry of cinnoline IV [1]. Synthesis and reactions of (4-aminocinnolin-3-yl)-aryl-methanones

Article abstract of DOI:10.1007/s007060170075

The synthesis of a series of disubstituted (4-aminocinnolin-3-yl)-aryl-methanones from aryl-hydrazonomalononitrile in a one-step procedure is described. Cyclocondensation of (4-amino-6,8-dimethyl-cinnolin-3-yl)-phenyl-methanone with malononitrile, diethylmalonate, and dimethylacetamide-dimethylacetal gave the corresponding pyrido[3,2-c]cinnoline derivatives. Treatment of (4-amino-6,8-dimethyl-cinnolin-3-yl)-phenyl-methanone with triethyl-orthoacetate under reflux readily afforded the corresponding imidoester which underwent cyclization to a pyrido[3,2-c]cinnoline derivative. This starting compound could also be annelated to the corresponding 1,2-dihydro-4-aryl-2-oxo-pyrido[3,2-c]cinnoline derivatives via the (4-acetamidocinnolin-3-yl)-aryl-methanones. Chemical and spectroscopic evidences for the structures of the new compounds are presented. The effect of three of the compounds against sixty cancer types was tested.

Full text of DOI:10.1007/s007060170075

Monatshefte fuÈr Chemie 132, 859±870 (2001)
On The Chemistry of Cinnoline IV [1]. Synthesis
and Reactions of (4-Aminocinnolin-3-yl)-aryl-
methanones
Atef M. Amer
Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt
Summary. The synthesis of a series of disubstituted (4-aminocinnolin-3-yl)-aryl-methanones from
aryl-hydrazonomalononitrile in a one-step procedure is described. Cyclocondensation of (4-amino-
6,8-dimethyl-cinnolin-3-yl)-phenyl-methanone with malononitrile, diethylmalonate, and dimethyla-
cetamide-dimethylacetal gave the corresponding pyrido[3,2-c]cinnoline derivatives. Treatment of
(4-amino-6,8-dimethyl-cinnolin-3-yl)-phenyl-methanone with triethyl-orthoacetate under re¯ux read-
ily afforded the corresponding imidoester which underwent cyclization to a pyrido[3,2-c]cinnoline
derivative. This starting compound could also be annelated to the corresponding 1,2-dihydro-4-aryl-2-
oxo-pyrido[3,2-c]cinnoline derivatives via the (4-acetamidocinnolin-3-yl)-aryl-methanones. Chemical
and spectroscopic evidences for the structures of the new compounds are presented. The effect of
three of the compounds against sixty cancer types was tested.
Keywords. Cycloadditions; Hydrazones; Cinnolines; Pyridines; Antitumor activity.
Introduction
Derivatives of cinnolines and their benzo and heterocyclic analogs exhibit
biological activity in various areas, including antihypertensive, antihrombotic, anti-
tumor, antisecretory [2±4], and bactericidal activities [5]. 4-Aminocinnolines have
recently become of importance due to their antibacterial, antihistaminic, and
insecticidal properties [6, 7]. Moreover, in recent years these derivatives have been
extensively utilized as intermediates for the synthesis of fused cinnolines of
potential biological activity [8, 9]. Some 4-amino-3-cinnolinecarboxylic acid deri-
vatives have been described as nonbenzodiazepine anxiolytic agents or benzodia-
zepine receptor antagonists [10, 11]. These pharmacological properties and interest
in the chemistry of cinnolines [11, 12] prompted the synthesis of a number of new
(4-aminocinnolin-3-yl)-p-tolyl-methanones as synthons for the preparation of new
pyrido[3,2-c]cinnolines with respect to a projected investigation of their utility as
pharmacological agents. Various biological activities, e.g. an interesting diuretic
effect [13], have been observed with derivatives of pyrido[2,3-d]pyridazines. The
method described provides a potential starting point for the synthesis of many new
pyrido[3,2-c]cinnoline derivatives.

860
A. M. Amer
Results and Discussion
The results of experiments designed to explore the scope and the limitations of the
Gewald procedure [14] as a synthetic method for the formation of (cinnolin-3-yl)-
aryl-methanones bearing various substituents 3a±q are reported. The aim of this
investigation is to study the effect of the substituent at the aryl group during the
cyclization of the aryl hydrazonomalononitriles 1a,h,o,q to obtain the new and
potentially useful substituted (cinnolin-3-yl)-aryl-methanones 3a±q. Therefore,
1a,h,o,q were synthesized in good yields by treatment of malononitrile with the
diazonium salts of the corresponding aniline derivatives (2,4-dimethylaniline, 2,5-
dimethylaniline, 2,3-dimethylaniline, and 4-bromo-3-methylaniline). The hydra-
1
zone structure was con®rmed by their H NMR spectra which included the broad
Scheme 1

(4-Aminocinnolin-3-yl)-aryl-methanones
861
signal of the NH group (exchangeable with D2O, 9.6 ppm). IR spectra (KBr) of 1
1
showed the characteristic absorption bands for NH groups at 3350 to 3198 cm
1
and for a cyano group at 2218 cm . The (4-aminocinnolin-3-yl)-phenyl-
methanones 3a and 3h were readily obtained via cyclization of 1 in presence of
AlCl3 in benzene under re¯ux conditions (Scheme 1).
Variation of the solvent during the cyclization of aryl-hydrazono-malononitriles
1 led to the new and useful substituted (4-aminocinnolin-3-yl)-aryl-methones 3a±q
in particularly excellent yields. Thus, toluene, ethyl benzene, o-xylene, m-xylene,
p-xylene, and 1,3,5-trimethylbenzene yielded under the same conditions via intra-
cyclization of 1a and 1h compounds 3b±g and 3i±q in excellent yields. However,
compounds 2a and 2h were not observed (Scheme 1). Furthermore, certain solvents
such as carbon disul®de, dichloromethane, carbon tetrachloride, pyridine, and
thiophene as well as solvent-free conditions did not lead to the cyclization of 1a,
irrespective of the temperature employed. In most of these cases, 1 could be
partially recovered.
The proposed mechanism involves an intramolecular Friedel-Crafts cyclization
gives 2a and 2h ®rst. Then, condensation of the cyano group at position 3 of 2 with
the nonpolar aromatic solvents mentioned above in presence of the Lewis acid
yields the corresponding ketones 3a±q in a Hoesch-type reaction [15].
Having available the novel ketones 3 as readily accessible starting materials,
synthesis of bi- and tricyclic systems containing a pyridazine subunit [16±17] was
attempted. Cyclocondensation of (4-amino-6,8-dimethylcinnolin-3-yl)-phenyl-
methanone 3a with malononitrile, diethylmalonate, and dimethylacetamide-di-
methylacetal gave the pyrido[3,2-c]cinnoline derivatives 4±6. Treatment of 3a with
triethyl-orthoacetate under re¯ux readily afforded the corresponding imidoester
which underwent cyclization to the pyrido[3,2-c]cinnoline derivative 7 in di-
methylformamide in presence of potassium carbonate at re¯ux temperature
(Scheme 2).
Scheme 2

862
A. M. Amer
Scheme 3
Treatment of the (4-aminocinnolin-3-yl)-p-tolyl-methanones 3a,i with acetic
anhydride under re¯ux readily afforded the 4-N-acetylated ketones 8a,b which
underwent cyclization to the pyrido[3,2-c]cinnoline derivatives 9a,b in dimethyl-
formamide in presence of potassium carbonate at re¯ux temperature. Interestingly,
9b was regioselectivity O-methylated in position 2 upon treatment with CH3I/KOH
to yield 10. The alternative N-position 1 seems shielded due to steric factors.
Nevertheless, this result is one of the rare exceptions [12] of the Kornblum rule
(Scheme 3).
Compounds 2a,h and 3a±q were tested for antimicrobial activity using the agar
diffusion method [18] against representatives of gram-positive bacteria (Bacillus
subtilis and Staphylococcus aureus) and gram-negative bacteria (E. coli and
3
Pseudomones aeruginosa). A concentration of 0.06 mg Á cm
showed good
inhibition zones of 10±15 mm, similar to those obtained with streptomycin.
The effects of 2a, 3b, and 3g were tested against several cancer lines (Table 1)
at a minmum of ®ve concentrations at 10-fold dilutions. A 48 hour continuous drug
exposure protocol was used, and a sulforhodamine B (SRB) protein assay was used
to estimate cell viability or growth. The results shown in Table 1 revealed that,
depending on the functional group on the cinnoline derivatives, the compounds
showed varying growth depression. Thus, the cyano group in 2a is less effective
compared to the carbonyl groups in compounds 3b and 3g.
Experimental
Melting points were measured on a Ko¯er hot stage microscope (Reichert, Vienna) and are
uncorrected. 1H and 13C NMR spectra were recorded on a Bruker DPX 200 spectrometer; chemical

(4-Aminocinnolin-3-yl)-aryl-methanones
863
Table 1. Effect of 2a, 3b, and 3g against several cancer lines
% Growth in presence of % Growth in presence of % Growth in presence of
2a
10logc (mg/cm3)
5.0
3a
10logc (mg/cm3)
5.0
3g
10logc (mg/cm3)
5.0
Panel/Cell line
4.0
4.0
4.0
Leukemia
MOLT-4
RPMI-8226
SR
112
105
113
103
92
108
94
4
11
4
83
64
70
33
29
18
102
115
Non-small cell lung cancer
A549/ATCC
HOP-62
99
110
106
84
91
97
86
67
89
90
98
74
83
80
82
85
14
12
38
18
2
76
67
44
73
75
76
5
63
17
32
2
HOP-92
NCI-H226
NCI-H23
NCI-H322M
Colon cancer
COLO 205
HCT-116
HT29
112
100
22
24
90
86
97
97
91
80
82
81
82
82
97
92
77
16
11
16
16
81
52
93
76
78
87
87
12
46
12
78
KM12
80
SW-620
105
CNS cancer
SF-268
126
97
117
91
104
93
6
19
5
98
90
86
28
18
7
SNB-19
U251
126
98
103
Melanoma
LOX IMVI
M14
103
108
136
86
97
95
100
96
2
19
61
16
83
4
80
69
76
72
72
89
69
67
61
75
24
95
22
18
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROVI
107
88
85
111
88
78
67
101
106
89
94
70
79
42
100
76
75
93
80
93
10
17
69
60
34
11
OVCAR-5
Renal cancer
786-0
96
111
92
91
95
80
85
97
91
13
22
4
61
79
74
59
10
ACHN
CAKI-1
Prostate cancer
PC-3
100
102
94
82
102
89
29
16
73
94
24
8
DU-145
Breast cancer
NCI/ADR-RES
105
116
101
93
85
78
89
73
20
29
25
12
90
70
13
17
23
37
MDA-MB-231/ATCC 101
MDA-MB-435
BT-549
107
88
68
78
103

864
A. M. Amer
shifts are given in ppm relative to internal TMS at 295 K. IR spectra were obtained on Biorad FT-IR-
45 instrument. All experiments were carried out with exclusion of moisture. For all newly
synthesized compounds satisfactory elemental analyses were obtained.
Preparation of aryl-hydrazonomalononitriles (1)
To a well stirred solution of 6.66 g malononitrile (100 mmol) in 130 cm3 EtOH and 20 cm3 H2O
containing 10 g CH3COONa, the diazonium salt (100 mmol) prepared in the usual way from the
corresponding aniline was added gradually with stirring during 20 min at 0±5ꢀC. The product was
®ltered, washed with H2O, dried, and recrystallized from the given solvent.
2,4-Dimethylphenyl-hydrazonomalononitrile (1a; C11H10N4)
Prepared from 2,4-dimethylaniline; crystallization from EtOH gave ®ne orange crystals (95%); m.p.:
1
96ꢀC; IR (KBr): ꢀꢀ ˆ 3350 br, 3199, 2920 br, 2218, 1610, 1532, 1480 cm 1; H NMR (CDCl3, ꢁ,
200 MHz): 2.33 (s, CH3), 2.36 (s, CH3), 7.03 (s, 1Har), 7.08 (d, 1Har), 7.38 (d, 1Har), 9.60 (br, NH)
ppm; 13C NMR (CDCl3, ꢁ, 50 MHz): 26.00 (CH3), 30.48 (CH3), 96.31 (CH), 117.00, 121.00, 125.98,
133.88, 138.21, 141.50, 144.90, 146.29 (aryl, 2CN) ppm.
2,5-Dimethylphenyl-hydrazonomalononitrile (1h; C11H10N4)
Prepared from 2,5-dimethylaniline; crystallization from MeOH gave ®ne yellow crystals (98%);
m.p.: 158±160ꢀC; IR (KBr): ꢀꢀ ˆ 3350 br, 3198, 2921, 2218, 1608, 1480 cm 1; 1H NMR (CDCl3, ꢁ,
200 MHz): 2.35 (s, CH3), 2.36 (s, CH3), 6.99 (d, J ˆ 7.7 Hz, 1Har), 7.08 (d, J ˆ 7.7 Hz, 1Har), 7.34 (s,
1Har), 9.65 (br, NH) ppm; 13C NMR (CDCl3, ꢁ, 50 MHz): 25.59 (CH3), 30.75 (CH3), 96.76 (CH),
117.62, 121.78, 126.26, 130.90, 137.05, 140.81, 146.80, 147.71 (aryl, 2CN) ppm.
2,3-Dimethylphenyl-hydrazonomalononitrile (1o; C11H10N4)
Prepared from 2,3-dimethylaniline; crystallization from EtOH gave ®ne yellow crystals (98%); m.p.:
1
198ꢀC; IR (KBr): ꢀꢀ ˆ 3350 br, 3198, 2920 br, 2218, 1608, 1474 cm
;
1H NMR (CDCl3, ꢁ,
200 MHz): 2.29 (s, CH3), 2.36 (s, CH3), 7.08 (d, J ˆ 7.4 Hz, 1Har), 7.17±7.25 (t, 1Har), 7.38 (d,
J ˆ 8.0 Hz, 1Har), 9.69 (br, NH) ppm; 13C NMR (CDCl3, ꢁ, 50 MHz): 12.27 (CH3), 20.28 (CH3),
87.21 (CH), 108.12, 112.18, 114.54, 123.16, 127.18, 128.33, 137.49, 138.26 (aryl, 2CN) ppm.
4-Bromo-3-methyl-phenyl-hydrazonomalononitrile (1q; C10H7BrN4)
Prepared from 4-bromo-3-methylaniline; crystallization from ethyl acetate gave ®ne yellow crystals
(80%); m.p.: 190ꢀC; IR (KBr): ꢀꢀ ˆ 3343 br, 3195, 2218, 1610, 1480, 1450 cm 1; 1H NMR (CDCl3,
ꢁ, 200 MHz): 2.44 (s, CH3), 6.99 (dd, J ˆ 8.6 Hz, J ˆ 2.4 Hz, 1Har), 7.21 (s, 1Har), 7.55 (d,
J ˆ 8.6 Hz, 1Har), 9.66 (br, NH) ppm; 13C NMR (CDCl3, ꢁ, 50 MHz): 32.72 (CH3), 96.91 (CH),
117.56, 121.48, 124.38, 127.49, 132.06, 143.25, 148.29, 149.71 (aryl, 2CN) ppm.
General procedure for the preparation of 4-aminocinnolin-3-carbonitrile and
(4-aminocinnolin-3-yl)-p-tolyl-methanone derivatives
A mixture of 10 mmol 1 and 2.50 g AlCl3 (0.04 mmol) in 150 cm3 of a nonpolar aromatic solvent
(benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, 1,3,5-trimethylbenzene) was heated
under re¯ux for 5 h with stirring and then allowed to cool to room temperature. The resulting product

(4-Aminocinnolin-3-yl)-aryl-methanones
865
was poured on 500 cm3 cold H2O and left for 2 h. The resulting solid product was collected by
®ltration, washed with petroleum ether (40±60ꢀ), and crystallized from the solvent given.
4-Amino-6,8-dimethylcinnolin-3-carbonitrile (2a; C11H10N4)
Prepared from 2,4-dimethylaniline; crystallization from petroleum ether gave greenish yellow
crystals (33%); m.p.: 189ꢀC; IR (KBr): ꢀꢀ ˆ 3459, 3319, 3199, 2919, 2217, 1641, 1571, 1532, 1502,
1
1465 cm 1; H NMR (CDCl3, ꢁ, 200 MHz): 2.57 (s, CH3), 2.95 (s, CH3), 5.54 (br, NH2), 7.47 (s,
1Har), 7.57 (s, 1Har) ppm.
4-Amino-5,8-dimethylcinnolin-3-carbonitrile (2h; C11H10N4)
Prepared from 2,5-dimethylaniline; crystallization from EtOH gave ®ne yellow crystals (35%); m.p.:
1
305±306ꢀC; IR (KBr): ꢀꢀ ˆ 3453, 3329 br, 3198, 2219, 1638, 1572, 1532, 1470 cm
;
1H NMR
(CDCl3, ꢁ, 200 MHz): 2.91 (s, CH3), 2.96 (s, CH3), 5.80 (br, NH2), 7.36 (d, J ˆ 7.3 Hz, 1Har), 7.58
(d, J ˆ 7.9 Hz, 1Har) ppm; 1H NMR (DMSO-d6, ꢁ, 200 MHz): 2.72 (s, CH3), 2.84 (s, CH3), 7.30 (br,
NH2), 7.38 (d, J ˆ 7.2 Hz, 1Har), 7.60 (d, J ˆ 7.2 Hz, 1Har) ppm; 13C NMR (DMSO-d6, ꢁ, 50 MHz):
28.03 (CH3), 33.00 (CH3), 124.10, 127.40, 138.85, 140.30, 141.41, 142.76, 157.14, 157.78 (aryl,
CN) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-phenyl-methanone (3a; C17H15N3O)
Prepared from 1a; crystallization from ethyl acetate gave cream white crystals (47%); m.p.:
> 330ꢀC; IR (KBr): ꢀꢀ ˆ 3340 br, 3131, 2974, 1631, 1606, 1566, 1526, 1474 cm 1; 1H NMR (CDCl3,
ꢁ, 200 MHz): 2.58 (s, CH3), 2.97 (s, CH3), 5.75 (br, NH), 7.45±7.56 (m, 5Har), 8.05 (d, J ˆ 8.0 Hz,
1
2Har) ppm; H NMR (CDCl3/DMSO-d6, ꢁ, 200 MHz): 2.48 (s, CH3), 2.75 (s, CH3), 7.46±7.49 (m,
3Har), 7.57 (s, 1Har), 7.84 (d, J ˆ 8.2 Hz, 2Har), 8.26 (s, 1Har), 9.42 (br, NH2) ppm; 13C NMR
(CDCl3, ꢁ, 50 MHz): 27.64 (CH3), 31.65 (CH3), 125.39, 126.15, 135.43, 137.35, 140.44, 141.09,
142.89, 144.52, 148.12, 148.54, 149.19, 154.48, 154.84 (aryl), 206.96 (C=O) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-p-tolyl-methanone (3b; C18H17N3O)
Prepared from 1a; crystallization from n-propanol gave yellowish white crystals (89%); m.p.: 336±
337ꢀC; IR (KBr): ꢀꢀ ˆ 3337 br, 3256, 3125, 2973, 2920, 1630, 1606, 1566, 1526, 1474, 1422, 1410,
1
1385 cm 1; H NMR (CDCl3, ꢁ, 200 MHz): 2.24 (s, CH3), 2.62 (s, CH3), 2.94 (s, CH3), 6.87 (d,
J ˆ 7.9 Hz, 2Har), 7.54 (s, 1Har), 7.56 (d, J ˆ 7.9 Hz, 2Har), 7.82 (s, 1Har), 10.10 (br, NH), 10.95 (br,
1
NH) ppm; H NMR (DMSO-d6, ꢁ, 200 MHz): 2.40 (s, CH3), 2.48 (s, CH3), 2.75 (s, CH3), 7.31 (d,
J ˆ 7.9 Hz, 2Har), 7.68 (s, 1Har), 7.79 (d, J ˆ 7.9 Hz, 2Har), 8.27 (s, 1Har), 9.19 (br, NH2) ppm; 13C
NMR (DMSO-d6, ꢁ, 50 MHz): 27.39 (CH3), 31.21 (CH3), 31.46 (CH3), 126.40, 129.70, 135.45,
138.39, 138.84, 140.71, 142.96, 144.44, 145.89, 149.12, 152.30, 155.61 (aryl), 206.00 (C=O) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-(4-ethylphenyl)-methanone (3c; C18H17N3O)
Prepared from 1a; crystallization from EtOH gave faint yellowish white crystals (83%); m.p.: 275±
1
276ꢀC; IR (KBr): ꢀꢀ ˆ 3344, 3131, 3032, 2932, 1630, 1610, 1560, 1528, 1474 cm
;
1H NMR
(DMSO-d6, ꢁ, 200 MHz): 1.19±1.27 (t, J ˆ 7.6 Hz, CH3), 2.49 (s, CH3), 2.69 (q, J ˆ 7.6 Hz, CH2),
2.75 (s, CH3), 7.36 (d, J ˆ 8.0 Hz, 2Har), 7.74 (s, 1Har), 7.83 (d, J ˆ 8.0 Hz, 2Har), 8.32 (s, 1Har), 9.35
1
(br, NH2) ppm; H NMR (CDCl3/DMSO-d6, ꢁ, 200 MHz): 1.17±1.25 (t, J ˆ 7.6 Hz, CH3), 2.48 (s,
CH3), 2.62±2.69 (q, J ˆ 7.6 Hz, CH2), 2.76 (s, CH3), 7.23 (d, J ˆ 8.0 Hz, 2Har), 7.55 (s, 1Har), 7.80
(d, J ˆ 8.0 Hz, 2Har), 8.23 (s, 1Har, 9.30 (br, NH2) ppm; 1H NMR (CDCl3, ꢁ, 200 MHz): 1.21±1.28 (t,

866
A. M. Amer
J ˆ 7.6 Hz, CH3), 2.58 (s, CH3), 2.61±2.68 (q, J ˆ 7.6 Hz, CH2), 2.96 (s, CH3), 7.22±7.27 (m, 3Har),
7.54 (s, 1Har), 7.80 (br, NH), 7.92 (d, J ˆ 8.0 Hz, 2Har) ppm; 13C NMR (DMSO-d6, ꢁ, 50 MHz):
25.46 (CH3), 27.32 (CH3), 31.44 (CH3), 38.28 (CH2), 126.78, 130.00, 135.43, 137.35, 140.78,
142.91, 145.64, 146.71, 149.39, 150.84, 157.49, 158.64 (aryl), 204.30 (C=O) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-(3,4-dimethylphenyl)-methanone (3d; C18H14N3O)
Prepared from 1a; crystallization from EtOH gave pale yellow crystals (69%); m.p.: 294±295ꢀC; IR
1
(KBr): ꢀꢀ ˆ 3310, 3228, 3030, 2920, 1635, 1590, 1550, 1530 cm
;
1H NMR (DMSO-d6, ꢁ,
200 MHz): 2.29 (s, CH3), 2.32 (s, CH3), 2.48 (s, CH3), 2.75 (s, CH3), 7.28 (d, J ˆ 7.8 Hz, Har), 7.64±
7.70 (m, 3Har), 8.32 (s, 1Har), 9.30 (br, NH2) ppm; 13C NMR (DMSO-d6, ꢁ, 200 MHz): 27.38 (CH3),
29.43 (CH3), 29.63 (CH3), 31.44 (CH3), 126.61, 129.89, 135.40, 138.34, 138.97, 141.39, 143.08,
143.36, 145.80, 146.38, 149.22, 151.30, 157.02 (aryl), 204.61 (C=O) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-(2,4-dimethylphenyl)-methanone (3e; C18H14N3O)
Prepared from 1a; crystallization from EtOH gave yellow crystals (77%); m.p.: 264ꢀC; IR (KBr):
1
ꢀꢀ ˆ 3563 br, 3380, 3030, 1637, 1600, 1570, 1520, 1416 cm 1; H NMR (DMSO-d6, ꢁ, 200 MHz):
2.05 (s, CH3), 2.31 (s, CH3), 2.48 (s, CH3), 2.63 (s, CH3), 7.07 (s, 3Har), 7.69 (s, 1Har), 8.31 (s, 1Har),
11.30 (br, NH2) ppm; 13C NMR (DMSO-d6, ꢁ, 50 MHz): 27.10 (CH3), 29.32 (CH3), 30.76 (CH3),
31.38 (CH3), 126.77, 130.01, 135.96, 137.33, 140.37, 140.62, 142.67, 143.89, 146.76, 146.22,
147.40, 147.86, 148.99, 158.29 (aryl), 188.16 (C=O) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-(2,5-dimethylphenyl)-methanone (3f; C18H14N3O)
Prepared from 1a; crystallization from EtOH gave pale yellowish white crystals (82%); m.p.: 287ꢀC;
1
IR (KBr): ꢀꢀ ˆ 3563 br, 3380, 3030, 1637, 1600, 1570, 1520, 1416 cm 1; H NMR (DMSO-d6, ꢁ,
200 MHz): 2.05 (s, CH3), 2.29 (s, CH3), 2.48 (s, CH3), 2.66 (s, CH3), 7.06 (s, 1Har), 7.15 (s, 2Har),
7.67 (s, 1Har), 8.30 (s, 1Har), 11.35 (br, NH2) ppm; 13C NMR (DMSO-d6, ꢁ, 50 MHz): 27.15 (CH3),
28.88 (CH3), 30.48 (CH3), 31.42 (CH3), 126.60, 129.78, 135.43, 137.64, 138.85, 139.71, 140.78,
142.80, 144.39, 146.14, 148.74, 150.59, 157.28 (aryl), 188.54 (C=O) ppm.
(4-Amino-6,8-dimethylcinnolin-3-yl)-(2,4,6-trimethylphenyl)-methanone (3g; C19H16N3O)
Prepared from 1a; crystallization from ethyl acetate gave pale brown crystals (75%); m.p.: 292ꢀC; IR
1
(KBr): ꢀꢀ ˆ 3563 br, 3380, 3030, 1637, 1600, 1570, 1520, 1416 cm
;
1H NMR (CDCl3, ꢁ,
200 MHz): 2.11 (s, 2CH3), 2.30 (s, CH3), 2.56 (s, CH3), 2.87 (s, CH3), 6.88 (s, 2Har), 7.26 (s, NH),
7.45 (s, 1Har), 7.57 (s, 1Har), 9.78 (br, NH) ppm; 1H NMR (DMSO-d6, ꢁ, 200 MHz): 1.98 (s, 2CH3),
2.27 (s, CH3), 2.47 (s, CH3), 2.71 (s, CH3), 6.86 (s, 2Har), 7.46 (s, 1Har), 8.06 (s, 1Har), 8.20 (br,
NH), 10.51 (br, NH) ppm; 13C NMR (DMSO-d6, ꢁ, 50 MHz): 27.38 (CH3), 29.47 (2CH3), 30.65
(CH3), 31.51 (CH3), 125.78, 128.78, 137.44, 143.15, 143.35, 146.03, 146.29, 147.60, 149.74,
153.31, 154.78 (aryl), 190.32 (C=O) ppm.
(4-Amino-5,8-dimethylcinnolin-3-yl)-phenyl-methanone (3h; C17H15N3O)
Prepared from 1h; crystallization from ethyl acetate gave yellow crystals (36%); m.p.: 160ꢀC, IR
1
(KBr): ꢀꢀ ˆ 3336 br, 3126, 2974, 1635, 1610, 1565, 1525, 1473 cm
;
1H NMR (CDCl3, ꢁ,
200 MHz): 2.91 (s, CH3), 2.96 (s, CH3), 5.86 (br, NH2), 7.34±7.48 (m, 4Har), 7.57 (d, J ˆ 7.6 Hz,
2Har), 7.94 (d, J ˆ 7.6 Hz, 1Har) ppm.

(4-Aminocinnolin-3-yl)-aryl-methanones
867
(4-Amino-5,8-dimethylcinnolin-3-yl)-p-tolyl-methanone (3i; C18H17N3O)
Prepared from 1h; crystallization from diethyl ether gave yellow crystals (83%); m.p.: 152±154ꢀC;
IR (KBr): ꢀꢀ ˆ 3337 br, 3251, 3130, 2973, 1632, 1574, 1530, 1425, 1385 cm 1; 1H NMR (CDCl3, ꢁ,
200 MHz): 2.44 (s, CH3), 2.93 (s, CH3), 3.00 (s, CH3), 7.30 (m, 3Har), 7.54 (d, J ˆ 7.29 Hz, 1Har),
1
7.92 (d, J ˆ 8.0 Hz, 2Har), 8.20 (br, NH2) ppm; H NMR (DMSO-d6, ꢁ, 200 MHz): 2.39 (s, CH3),
2.75 (s, CH3), 2.88 (s, CH3), 7.28 (d, J ˆ 8.0 Hz, 2Har), 7.36 (d, J ˆ 7.0 Hz, 1Har), 7.59 (d, J ˆ 7.0 Hz,
1Har), 7.72 (d, J ˆ 8.0 Hz, 2Har), 8.50 (br, NH2) ppm; 13C NMR (DMSO-d6, ꢁ, 50 MHz): 27.95
(CH3), 31.16 (CH3), 33.30 (CH3), 125.32, 128.19, 140.63, 141.24, 142.60, 143.06, 144.76, 146.70,
151.51, 157.12, 157.40 (aryl), 206.55 (C=O) ppm.
(4-Amino-5,8-dimethylcinnolin-3-yl)-(4-ethylphenyl)-methanone (3j; C18H17N3O)
Prepared from 1h; crystallization from MeOH gave faint yellow crystals (70%); m.p.: 195ꢀC; IR
1
(KBr): ꢀꢀ ˆ 3347, 3284, 3126, 2932, 1632, 1606, 1555, 1527, 1473 cm 1; H NMR (DMSO-d6, ꢁ,
200 MHz): 1.25 (t, J ˆ 7.6 Hz, CH3), 2.67 (q, J ˆ 7.6 Hz, CH2), 2.73 (s, CH3), 2.84 (s, CH3), 7.36 (m,
3Har), 7.60 (d, J ˆ 7.2 Hz, 1Har), 7.83 (d, J ˆ 8.0 Hz, 2Har), 9.22 (br, NH2) ppm.
(4-Amino-5,8-dimethylcinnolin-3-yl)-(3,4-dimethylphenyl)-methanone (3k; C18H14N3O)
Prepared from 1h; crystallization from CHCl3 gave red crystals (59%); m.p.: 225ꢀC; IR (KBr):
1
ꢀꢀ ˆ 3337, 3262, 3028, 2971, 1634, 1607, 1566, 1529, 1475 cm
;
1H NMR (DMSO-d6, ꢁ,
200 MHz): 2.29 (s, CH3), 2.32 (s, CH3), 2.44 (s, CH3), 2.48 (s, CH3), 6.94 (d, J ˆ 8.0 Hz, 1Har), 7.12
(d, J ˆ 7.5 Hz, 1Har), 7.57 (s, 1Har), 7.64 (s, 2Har), 10.00 (br, NH2) ppm; 1H NMR (CDCl3, ꢁ,
200 MHz): 2.28 (s, 2CH3), 2.85±2.87 (d, 2CH3), 7.17 (d, 2Har), 7.44 (d, 1Har), 7.72 (s, 2Har), 11.3
(br, NH) ppm.
(4-Amino-5,8-dimethylcinnolin-3-yl)-(2,4-dimethylphenyl)-methanone (3l; C18H14N3O)
Prepared from 1h; crystallization from EtOH gave orange yellow crystals (60%); m.p.: 218±219ꢀC;
1
IR (KBr): ꢀꢀ ˆ 3461 br, 3383, 3031, 1635, 1606, 1571, 1527, 1416 cm
;
1H NMR (CDCl3, ꢁ,
200 MHz): 2.37 (s, CH3), 2.39 (s, CH3), 2.45 (s, CH3), 2.57 (s, CH3), 7.12±7.16 (m, 3Har), 7.30±7.38
(m, 2Har), 11.27 (br, NH) ppm.
(4-Amino-5,8-dimethylcinnolin-3-yl)-(2,5-dimethylphenyl)-methanone (3m; C18H14N3O)
Prepared from 1h; crystallization from MeOH gave orange crystals (55%); m.p.: 235ꢀC; IR (KBr):
ꢀꢀ ˆ 3422, 3385, 3027, 1638, 1607, 1573, 1526, 1476 cm 1; 1H NMR (CDCl3, ꢁ, 200 MHz): 2.25 (s,
CH3), 2.32 (s, CH3), 2.86 (s, CH3), 2.95 (s, CH3), 7.11 (s, 2Har), 7.17 (s, 1Har), 7.23 (d, J ˆ 7.3 Hz,
1Har), 7.51 (d, J ˆ 7.3 Hz, 1Har), 11.30 (br, NH) ppm.
(4-Amino-5,8-dimethylcinnolin-3-yl)-(2,4,6-trimethylphenyl)-methanone (3n; C19H16N3O)
Prepared from 1h; crystallization from acetone gave orange crystals (72%); m.p.: 314ꢀC; IR (KBr):
ꢀꢀ ˆ 3553 br, 3377, 3031, 1637, 1610, 1568, 1525, 1422 cm 1; 1H NMR (CDCl3, ꢁ, 200 MHz): 2.11
(s, 2CH3), 2.30 (s, CH3), 2.56 (s, CH3), 2.87 (s, CH3), 6.88 (s, 2Har), 7.45 (s, 1Har), 7.57 (s, 1Har),
9.78 (br, NH) ppm; 1H NMR (DMSO-d6, ꢁ, 200 MHz): 1.98 (s, 2CH3), 2.27 (s, CH3), 2.47 (s, CH3),
2.71 (s, CH3), 6.86 (s, 2Har), 7.46 (s, 1Har), 8.06 (s, 1Har), 8.20 (br, NH), 10.51 (br, NH) ppm; 13C
NMR (DMSO-d6 ꢁ, 50 MHz): 27.38 (CH3), 29.47 (2CH3), 30.65 (CH3), 31.51 (CH3), 125.78,
128.78, 137.44, 143.15, 143.35, 146.03, 146.29, 147.60, 149.74, 153.31, 154.78 (aryl), 190.32 (C=O)
ppm.

868
A. M. Amer
(4-Amino-7,8-dimethylcinnolin-3-yl)-p-tolyl-methanone (3o; C18H17N3O)
Prepared from 1o; crystallization from MeOH gave buff crystals (80%); m.p.: 270ꢀC; IR (KBr):
1
ꢀꢀ ˆ 3345, 3284, 3127, 2971, 1633, 1567, 1526, 1474, 1385 cm
;
1H NMR (DMSO-d6, ꢁ,
200 MHz): 2.41 (s, CH3), 2.48 (s, CH3), 2.70 (s, CH3), 7.33 (d, J ˆ 8.0 Hz, 2Har), 7.64 (d, J ˆ 8.6 Hz,
1Har), 7.82 (d, J ˆ 8.0 Hz, 2Har), 8.41 (d, J ˆ 8.6 Hz, Har), 9.47 (br, NH2) ppm; 13C NMR (DMSO-d6,
ꢁ, 50 MHz): 13.40 (CH3), 20.78 (CH3), 21.64 (CH3), 115.37, 120.88, 128.93, 130.61, 131.14, 132.16,
133.07, 135.69, 142.28, 143.06, 143.84, 148.25 (aryl), 194.39 (C=O) ppm.
(4-Amino-6-bromo-7-methylcinnolin-3-yl)-(2,5-dimethylphenyl)-methanone (3p; C18H17BrN3O)
Prepared from 1q; crystallization from MeOH gave yellow crystals (32%); m.p.: 297ꢀC; IR (KBr):
1
ꢀꢀ ˆ 3445, 3259, 3125, 2965, 1637, 1612, 1587, 1526, 1431, 1387 cm
;
1H NMR (CDCl3, ꢁ,
200 MHz): 2.28 (s, CH3), 2.33 (s, CH3), 2.67 (s, CH3), 7.16 (s, 2Har), 7.23 (s, 1Har), 8.20 (s, 1Har),
8.26 (s, 1Har), 10.49±11.00 (br, NH2) ppm; 1H NMR (DMSO-d6, ꢁ, 200 MHz): 2.12 (s, CH3), 2.29 (s,
CH3), 2.58 (s, CH3), 7.11 (s, 1Har), 7.18 (s, 2Har), 8.21 (s, 1Har), 8.95 (s, 1Har), 9.50 (br, NH2) ppm;
13C NMR (DMSO-d6, ꢁ, 50 MHz): 29.06 (CH3), 30.48 (CH3), 32.94 (CH3), 125.43, 136.35, 138.24,
139.71, 139.91, 141.63, 143.27, 143.89, 150.83, 152.79, 154.22, 156.49 (aryl), 210.64 (C=O) ppm.
(4-Amino-6-bromo-5-methylcinnolin-3-yl)-2,5-dimethylphenyl-methanone (3q; C18H17BrN3O)
Prepared from 1q; crystallization from EtOH gave pale yellowish white crystals (24%); m.p.: 288ꢀC;
1
IR (KBr): ꢀꢀ ˆ 3371 br, 3277, 3130, 2970, 1635, 1610, 1582, 1526, 1431, 1387 cm
;
1H NMR
(CDCl3, ꢁ, 200 MHz): 2.17 (s, CH3), 2.20 (s, CH3), 2.57 (s, CH3), 7.00 (s, 2Har), 7.06 (s, 1Har), 7.59
(d, J ˆ 8.4 Hz, 1Har), 8.93 (d, J ˆ 8.4 Hz, 1Har), 10.13 (br, NH2) ppm; 13C NMR (CDCl3, ꢁ, 50 MHz):
29.32 (CH3), 30.36 (CH3), 31.86 (CH3), 125.11, 130.49, 133.99, 135.44, 139.21, 140.28, 141.05,
142.09, 143.06, 144.10, 146.94, 150.86, 157.36, 159.02 (aryl), 208.51 (C=O) ppm.
2-Amino-7,9-dimethyl-4-phenyl-pyrido[3,2-c]cinnoline-3-nitrile (4; C20H15N5)
A mixture of 0.28 g 3a (1 mmol), 0.07 g malononitrile (1 mmol), and 15 cm3 pyridine was re¯uxed
for 6 h. Excess pyridine was removed under reduced pressure, and the residue was poured on crushed
ice and HCl under vigorous stirring. The solid obtained was washed with H2O and crystallized from
EtOH to give pale yellow crystals (75%).
M.p.: > 300ꢀC; IR (KBr): ꢀꢀ ˆ 3422, 3345, 2926, 2218, 1617, 1554, 1492 cm
1
;
1H NMR
(CDCl3, ꢁ, 200 MHz): 2.70 (s, CH3), 3.06 (s, CH3), 5.71 (br, NH2), 7.52±7.57 (m, 5Har), 7.65 (s,
1Har), 8.34 (s, 1Har) ppm.
1,2-Dihydro-7,9-dimethyl-3-(ethylcarboxylate)-4-phenyl-2-oxo-pyrido[3,2-c]cinnoline
(5; C22H19N3O3)
A mixture of 0.28 g 3a (1 mmol) and 10 cm3 diethyl malonate was heated at 225ꢀC for 3 h. Excess
reagent was removed by distillation under reduced pressure, and the residue was washed with diethyl
ether and crystallized from toluene to give pale yellow crystals (55%).
M.p.: 220ꢀC; IR (KBr): ꢀꢀ ˆ 3320, 3085, 2927, 1723, 1655, 1582, 1489 cm 1; 1H NMR (DMSO-
d6, ꢁ, 200 MHz): 1.23 (t, J ˆ 7.5 Hz, CH3), 2.70 (s, CH3), 3.06 (s, CH3), 4.40 (AB system, J ˆ 7.5 Hz,
OCH2), 7.52±7.57 (m, 5Har), 7.65 (s, 1Har), 8.34 (s, 1Har), 12.94 (br, NH), ppm.
7,9-Dimethyl-4-phenyl-3-ethoxypyrido[3,2-c]cinnoline (6; C21H20N3O)
A mixture of 0.28 g 3a (1 mmol) and 15 cm3 triethyl orthoacetate was heated at 170ꢀC for 7 h. Excess
reagent was removed under reduced pressure, and the oily residue was dissolved in 15 cm3 dry DMF.

(4-Aminocinnolin-3-yl)-aryl-methanones
869
0.14 g K2CO3 were added, and the mixture was heated for 2 h. After concentration, the residue was
treated with 20 cm3 H2O. The precipitated solid was collected, washed with H2O, and crystallized
from toluene to give white crystals (62%).
M.p.: 293ꢀC; IR (KBr): ꢀꢀ ˆ 3092, 2937, 1612, 1562, 1491 cm
1
;
1H NMR (DMSO-d6, ꢁ,
200 MHz): 1.21 (t, J ˆ 7.5 Hz, CH3), 2.70 (s, CH3), 3.06 (s, CH3), 4.54 (q, J ˆ 7.5 Hz, OCH2), 7.20
(s, 1Har), 7.52±7.57 (m, 5Har), 7.65 (s, 1Har), 8.34 (s, 1Har) ppm.
7,9-Dimethyl-4-phenyl-2-dimethylaminopyrido[3,2-c]cinnoline (7; C21H21N4)
A mixture of 0.28 g 3a (1 mmol) and 10 cm3 dimethylacetamide dimethylacetal was heated at 150ꢀC
for 3 h. Excess reagent was removed under reduced pressure, and the oily residue was washed with
diethyl ether and crystallized from EtOH to give white crystals (72%).
M.p.: 318ꢀC; IR (KBr): ꢀꢀ ˆ 3092, 2937, 1612, 1562, 1491 cm 1; 1H NMR (CDCl3, ꢁ, 200 MHz):
2.70 (s, CH3), 3.06 (s, CH3), 3.52 (s, 2CH3), 7.08 (s, 1Har), 7.52±7.57 (m, 5Har), 7.65 (s, 1Har), 8.34
(s, 1Har) ppm.
Acetylation of (4-amino-dimethylcinnolin-3-yl)-aryl-methanones 3a,i; formation of
4-acetamidocinnolines 8a,b
A solution of 2.77 g ketones 3a and 3i (10 mmol) in 15 cm3 acetic anhydride was heated to boiling,
resulting a dark yellow solution. Within 5 min, needle-shaped crystals began to form. After cooling
in ice, the product was ®ltered off to give 4a,b. The crude products were used for the next step
without further puri®cation.
(4-Acetamido-5,8-dimethylcinnolin-3-yl)-phenyl-methanone (8a; C21H20N3O2)
Prepared from 3a; crystallization from EtOH/H2O gave grey crystals (85%); m.p.: 172ꢀC; IR (KBr):
ꢀꢀ ˆ 3453, 3123, 2958, 1710, 1688, 1626, 1579, 1490 cm 1; 1H NMR (CDCl3, ꢁ, 200 MHz): 2.33 (s,
COCH3), 2.62 (s, CH3), 3.09 (s, CH3), 7.48±7.67 (m, 5Har), 7.89 (d, J ˆ 7.2 Hz, 2Har) ppm.
(4-Acetamido-5,8-dimethylcinnolin-3-yl)-p-tolyl-methanone (8b; C22H22N3O2)
Prepared from 3i; crystallization from CH3COOH and H2O gave brownish red crystals (72%); m.p.:
1
160ꢀC; IR (KBr): ꢀꢀ ˆ 3446, 3130, 2932, 1712, 1690, 1625, 1580, 1490 cm 1; H NMR (CDCl3, ꢁ,
200 MHz): 2.08 (s, COCH3), 2.43 (s, CH3), 2.91 (s, CH3), 2.97 (s, CH3), 7.25±7.30 (m, 3Har), 7.52
(d, J ˆ 7.3 Hz, 1Har), 7.91 (d, J ˆ 8.2 Hz, 2Har) ppm.
Preparation of 1,2-dihydro-4-aryl-2-oxopyrido[3,2-c]cinnolines 9a,b
To a solution of the crude amide 8a or 8b (10 mmol) in 25 cm3 dry DMF, 1.4 g K2CO3 were added,
and the mixture was heated for 2 h. After concentration, the residue was treated with 30 cm3 H2O.
The pH was adjusted to 3±4 by addition of 2 N HCl, the precipitated solid was collected, washed
with H2O, and recrystallized from the given solvent to afford 9a,b.
1,2-Dihydro-7,9-dimethyl-4-phenyl-2-oxopyrido[3,2-c]cinnoline (9a; C19H15N3O)
Prepared from 8a; crystallization from DMF/H2O gave white crystals (82%); m.p.: 293ꢀC; IR (KBr):
ꢀꢀ ˆ 3400, 3144, 3082, 2926, 1656, 1552, 1491 cm 1; 1H NMR (CDCl3, ꢁ, 200 MHz): 2.70 (s, CH3),
3.06 (s, CH3), 7.05 (s, 1Har), 7.55±7.57 (m, 3Har), 7.65 (s, 1Har), 7.85±7.87 (m, 2Har), 8.34 (s, 1Har),
13.50 (br, NH) ppm.

870
A. M. Amer: (4-Aminocinnolin-3-yl)-aryl-methanones
1,2-Dihydro-7,10-dimethyl-4-(p-tolyl)-2-oxopyrido[3,2-c]cinnoline (9b; C20H17N3O)
Prepared from 8b; crystallization from EtOH gave white crystals (47%); m.p.: 202ꢀC; IR (KBr):
ꢀꢀ ˆ 3402, 3083, 2926, 1655, 1554, 1492 cm 1; 1H NMR (CDCl3, ꢁ, 200 MHz): 2.46 (s, CH3), 3.03
(s, CH3), 3.17 (s, CH3), 6.96 (s, 1Har), 7.33 (d, J ˆ 7.9 Hz, 2Har), 7.57 (d, J ˆ 7.3 Hz, 1Har), 7.66 (d,
J ˆ 8.0 Hz, 3Har), 11.70 (br, NH) ppm; 13C NMR (CDCl3, ꢁ, 50 MHz): 20.13 (CH3), 22.98 (CH3),
25.75 (CH3), 115.00, 123.39, 130.65, 130.79, 131.65, 133.39, 134.00, 134.81, 135.66, 135.79,
139.76, 141.13, 149.00, 156.80, (aryl), 163.75 (C=O) ppm.
7,10-Dimethyl-4-phenyl-2-methoxy-pyrido[3,2-c]cinnoline (10; C19H17N3O)
A mixture of 0.30 g 8b (1 mmol), 3 cm3 KOH (7%), and 0.30 cm3 CH3I was stirred overnight at room
temperature. The precipitate which separated was washed with diethyl ether and recrystallized from
EtOH to give red crystals (36%).
1
M.p.: > 300ꢀC; IR (KBr): ꢀꢀ ˆ 3080, 2933, 1682, 1625, 1550, 1492 cm 1; H NMR (CDCl3, ꢁ,
200 MHz): 2.43 (s, CH3), 3.09 (s, CH3), 3.37 (s, CH3), 4.24 (s, OCH3), 7.23 (s, 1Har), 7.35 (d,
J ˆ 7.9 Hz, 2Har), 7.62 (m, 2Har), 7.76 (d, J ˆ 7.9 Hz, 2Har) ppm.
References
[1] Amer AM, Attia IAG, El-Mobayad M, Asker S (2000) Polish J Chem 74: 681
[2] Cignarella G, Barlocco D, Pinna GA, Curzu MM (1994) Acta Chim Slov 41: 173
[3] Cignarella G, Barlocco D, Pinna GA, Loriga M, Curzu MM, Tofanetti O, Germini M, Cazzulani
P, Cavaletti E (1989) J Med Chem 32: 2277
[4] Lown JW, Morgan AR, Yen SF, Wang YH, Wilson WD (1985) Biochemistry 24: 4028
[5] Miyamoto K, Matsumoto J, Nakamura S (1990) Jap Patent 02 96 570; Chem Abstr (1990) 113:
97619
[6] Singerman GM (1973) Chem Heterocycl Compd 27: 1
[7] Glowka ML, Martynowski D, Napieraj A, Olczak A, Stanczak A, Ochocki Z, Lewgowd W
(1999) J Chem Crystallography 29: 687
[8] Nargund LVG, Nargund KS (1995) Arzneim Forsch 45: 1131
[9] ICI Americas Inc, JP 61.280.483 (1986) Chem Abstr (1988) 108: 131839
[10] Stanczak A, Kwapiszewski W, Szadoska A, Pakulska W (1994) Pharmazie 49: 406 and
references therein
[11] Resh JF, EP 328.282 (1989); Chem Abstr (1990) 113: 120786
[12] Amer AM, El-Bermaui MA, Ahmed AFS, Soliman SM (1999) Monatsh Chem 130: 1409
[13] Haider H, Heinisch G (1986) Synthesis 862 and references therein
[14] Gewald K, Calderon O, Schaefer H, Hain U (1984) Liebigs Ann Chem 7: 1390
[15] Spoerri PE, DuBois AS (1949) Organic Reactions, vol V, chapt. 9. Wiley, New York, pp 387±412
[16] Deeb A, El-Mobayad M, Essawy AN, El-Hamid AA, Amer AM (1990) Collect Czech Chem
Commun 55: 728, 2790
[17] El-Mobayad M, Essawy AN, El-Hamid AA, Amer AM (1991) Gazzetta Chimica Italiana 121:
209
[18] Collins CH (1964) Microbiological methods. Butterworth, London, p 9
Received November 27, 2000. Accepted December 4, 2000