Pyridazine and phthalazine derivatives with potential antimicrobial activity

Article abstract of DOI:10.1002/jhet.5570440528

(Chemical Equation Presented) Fifteen new pyridazine and phthalazine derivatives were prepared (in good to excellent yields) and tested in vitro as antimicrobial compounds. All the compounds have proved to have a remarkable activity against Gram positive

Full text of DOI:10.1002/jhet.5570440528

Sep-Oct 2007
1149
Pyridazine and Phthalazine Derivatives with
Potential Antimicrobial Activity
Roxana M. Butnariu,1 Maria D. Caprosu,1 Vasilichia Bejan,1 Margareta Ungureanu,2
Antonia Poiata,2 Cristina Tuchilus,2 Margareta Florescu3 and Ionel I. Mangalagiu1*
1 “Al. I. Cuza” University of Iasi, Organic and Biochemistry Department, Bd. Carol 11, 700506 Iasi, Romania.
e-mail: ionelm@uaic.ro
2 University of Medicine and Pharmacy “Gr.T.Popa” Iasi, Str. Universitatii 16, 700115 Iasi, Romania.
3 Academy of Economical Sciences, Str. Piata Romana 6, Sector 1, 010374 Bucuresti, Romania. e-mail: icefaceus@yahoo.com
Received May 9, 2006
R
R
Z-dipolarophiles
CH3
N
N
N
N
O
O
C
H
Z
Z
CH3
Fifteen new pyridazine and phthalazine derivatives were prepared (in good to excellent yields) and
tested in vitro as antimicrobial compounds. All the compounds have proved to have a remarkable activity
against Gram positive germs, the results on Sarciria Luteea being spectacular. Correlation structure -
biological activity have been done. Stereo- and region- chemistry involved in these reactions are discussed.
J. Heterocyclic Chem., 44, 1149 (2007).
Considering the pyridazine – acetophenone skeleton (2)
as the pharmacophoric group for the activity [4,9], in this
work we aimed to obtain new diazine heterocycles with
antimicrobial activity, having in mind three structural
modifications: introduction of a pyrrolo (I) and/or a
benzene (II) ring, and a substituient (R). In equal
measure, we were interested in the chemistry of
cycloaddition reactions.
In order to synthetise the desired pyridazine and phthal-
azine derivatives, first we obtained the corresponding
cycloimmonium salt (3) which in alkaline medium (Et3N)
generated the ylide (4) in situ. N-Phenylmaleimide
(NPMI), as a symmetrical cyclic Z-alkene, reacts with
ylides (4) giving the cycloadduct (5).
INTRODUCTION
The literature describes a large variety of pyridazine
and phthalazine compounds (1) with different
biological activities: anticancer [1,2], antituberculosis
[3], antimicrobial [4-6], antihypertensive [7-9],
platelent aggregation inhibitor [9,10], etc. A facile way
to obtain diazine derivatives uses cycloimmonium
ylides as reactive species [11-15]. The reaction
pathway involves, in the most frequent cases, a
Huisgen 3+2 dipolar cycloaddition of ylides to
dipolarophiles (activated alkenes and alkynes). The
problems of stereo- and regiochemistry involved in
these types of cycloadditions are discussed.
The Huisgen 3+2 cycloaddition occurs with high
stereospecificity and no formation of other isomers was
observed. The reaction of ylides (4) with dimethyl acetyl-
endicarboxylate (DMAD) leads to pyrrolopyridazine (8a)
and dihydropyrrolophthalazines (8b,c). As to the
mechanism, the formation of compounds (8b,c) could be
explained by prototropic rearrangement of (7b,c) while
R
X
N
N
N
O
II
N
I
1
CH3
2
N
N
N
N
N
Br
N
CH
1
CH
CH2
7
O
9
6
C
COC6H4Me-4
COC6H4Me-4
N
N
3.a
4.a
BrCH2COC6H4Me-4
12
R
N
R
4
R
R
Me
N
N
N
Br
N
N
N
N
O
CH
CH2
CH
8
1
9
C
COC6H4Me-4
COC6H4Me-4
3.b. R= H
c. R= Me
11
4.b. R= H
c. R= Me
14
Me

1150
R. M. Butnariu, M. D. Caprosu, V. Bejan, M. Ungureanu, A. Poiata, C. Tuchilus,
M. Florescu and I. I. Mangalagiu
Vol 44
formation of (8a) could be explained by oxidative
dehydrogenation of (7a).
The in vitro antibacterial and antifungal activities of the
newly obtained diazine compounds were tested. The tests
were performed using the diffusiometric method with
rustless steel cylinders based on the diffusion of the tested
substances on the gelose surface (for bacteria) and
Sabouraud environment (for fungus Candida albicans).
The cylinders were maintained for 24 h at 34 °C for
bacteria and at 37 °C for Candida. The tested substances
were previously dissolved in dimethylformamide (DMF)
5% (v/v). A control solvent sample was also included.
The inhibition diameter zone, in mm, of development of
microbial strain was measured. A compound is considered
active when the difference between the inhibition
diameter zone of compound and witness is up to 2 mm (3-
4 mm moderate active and up to 5 very active). The
results are listed in Table 1.
The reaction of ylides (4) with non-symmetrical
dipolarophiles such as acrylonitrile and ethyl propiolate
involves additional regiochemical problems [11,12] since
alternative addition of the dipole (ylide) to the
dipolarophile has often been found. No matter what
conditions we used (different solvents, temperature, time),
the reaction of ylides (4) with acrylonitrile and ethyl
propiolate leads to a single isomer, the tetrahydropyrrolo-
diazine adduct (6) and the pyrrolo- diazine adduct (10),
respectively. This means that one bond is formed between
the ylide carbon and the non-substituted carbon atom of
the dipolarophile and the second bond between the
positively charged carbon of the ylide and the substituted
carbon of the dipolarophile. This is in accordance with the
Me
14
Me
Me
15
15
Me
12
8b
C
8
12
O
N
O
N
H
10
C
H
H
3a
12
H
R
N
H
9
11
H
C
Ha
H4
O
N
H
N
O
O
H
8a
6
N
H
1
N
H
R
C
3
H
H
N
7
O
Ha
3
9a
11a
9
11b
1
9b
9
H
N
2
Hb
16
N
15
11
16
1
6
10b
1
Hb
4a
O
O
5
CN
4
10
5.b. R= H
CN
5.a
6.a
c. R= Me
6.b. R= H
c. R= Me
O
C6H5
N
CN
4a-c
O
MeOOC
COOMe
COOEt
COC6H4Me-4
COOMe
COC6H4Me-4
COOMe
COC6H4Me-4
COOEt
R
N
COC6H4Me-4
N
N
R
N
N
N
N
N
COOMe
COOMe
7.a
COOEt
7.b. R= H
c. R= Me
9
Me
15
15
12
Me
Me
12
O
O
9
12
12
O
Me
C
9
C
1
O
C
3
3
11
11
11
R
N
H
N
6
6
1
R
N
C
N
N
11
N
6
N
H
COOMe
COOMe
N
6
2a
1
1
10b
10b
10
5
4
COOMe
COOEt
5
10
COOMe
1a
4
COOMe
10.b. R= H
c. R= Me
8.a
10.a
8.b. R= H
c. R= Me
usual electronic effects in dipolarophiles. Because a single
regioisomer is obtained, the reaction is regiospecific being
under charge control.
The comparative analysis of the data leads to the
conclusions that both pyridazine and phthalazine
derivatives have excellent antimicrobial activity (non
selective) against Gram positive germs. The results on
Sarciria Luteea are of particular note. However,
pyridazine derivatives are, in most case, more active
compared with the corresponding phthalazine, which
means that introduction of a benzene ring (II) on the
The structure of the new compounds was proved by
elemental and spectral analysis. The following spectral
1
methods were used: IR, H NMR, 13C NMR, and two-
dimensional experiments 2D-COSY, 2D-HETCOR
(HMQC), long range 2D-HETCOR (HMBC).

Sep-Oct 2007
Pyridazine and Phthalazine Derivatives with Potential Antimicrobial Activity
1151
Table 1
Results of in vitro antibacterial and antifungal activities for some diazine derivatives described in the text.
Strainꢂ
Product
ꢃ
Staphylococcus
aureus Oxford
Bacillus
subtillis
Pseudomonas
aeruginosa
Candida
albicans
Sarciria Luteea
Escherichia coli
Witness
(3a)
3b)
3c)
5a)
5b)
5c)
6a)
6b
6c
8a
8b
8c
12
26
24
24
13
12
14
25
24
24
25
23
22
22
22
21
10
52
51
51
48
42
43
35
31
31
37
32
34
25
22
21
14
28
27
28
22
14
13
26
15
14
14
14
15
24
14
15
17
18
18
17
17
17
17
17
18
17
17
18
17
17
18
18
14
14
14
15
14
14
14
14
15
14
14
14
14
14
15
14
16
16
17
16
16
17
17
16
17
16
16
16
17
16
17
17
10a
10b
10c
of alkenes, then washed thoroughly three times with water (30
ml), the chloroform layer was dried on sodium sulfate, filtered
and evaporated. The residue was crystallized from an
appropriate solvent.
pyridazine moiety it is not beneficial for antimicrobial
activity. The influence of pyrrolo (I) moiety and
substituient (R) seems to be negligible.
8-(4-Methylbenzoyl)-10-phenyl-8,8a,11a,11b-tetrahydro-
pyrrolo[3',4':3,4]pyrrolo[2,1-a]phthalazine-9,11-dione (5b).
This compound was obtained as white crystals (ethanol), 0.73 g
(84%), mp 232-234°C; ir: 2952 (C-H), 1772, 1721 (C=O imide),
1692 (CO keto), 1603, 1565, 1498, 1449 (C=C, C=N); 1H nmr
(deuteriochloroform): ꢀ 8.02-8.00 (d, 2H, H13, J=8.0), 7.52 (s,
1H, H5), 7.49-7.31 (m, 9H, H1, H2, H3, H4, 2H14, 2H18, H19),
7.02-7.00 (d, J=7.2, 2H, H17), 5.93 (s, 1H, H8), 4.69-4.67 (d,
1H, H11b, J=8.4), 4.18-4.16 (d, 1H, H8a, J=8.0), 3.74-3.70 (dd,
1H, H11a, J=8.0, 8.4), 2.51 (s, 3H, CH3); 13C nmr: ꢀ 193.43
(C8b, keto), 177.48 (C9, keto imide), 174.87 (C11, keto imide),
144.89 (C15), 140.61 (C5), 135.06 (C16), 132.39 (C12), 131.23
(C2), 129.50 (C13), 129.31 (C18), 129.26 (C3), 129.12 (C14),
129.01 (C1), 128.24 (C1a), 128.21 (C19), 126.60 (C17), 126.23
(C4), 123.70 (C4a), 76.42 (C8), 59.67 (C11b), 51.43 (C11a),
46.90 (C8a), 21.91 (CH3).
3-(4-Methylbenzoyl)-1,2,3,10b-tetrahydro-pyrrolo[2,1-a]-
phthalazine-1-carbonitrile (6b). This compound was obtained
as white crystals (methanol), 0.42 g (67%), mp 172-173°C; ir:
3051 (C-H), 2951 (C-H), 2232 (CN), 1694 (CO keto), 1597,
1561, 1492, 1451 (C=C, C=N); 1H nmr (deuteriochloroform): ꢀ
8.08-8.06 (d, 2H, H13, J=8.0), 7.46 (s, 1H, H6), 7.43-7.36 (m,
2H, H9, H10), 7.29-7.25 (m, 3H, 2H14, 1H8), 7.12-7.10 (dd,
1H, H7, J=8.00, 1.6), 5.73-5.70 (dd, 1H, H3, J=2.8, 8.4), 4.51-
4,50 (d, 1H, H10b, J=7.2), 3.38-3.33 (8 lines: dxdxd, 1H, H1),
2.93-2.87 (8 lines: dxdxd, 1H, H2b, J=22.8, 2.8, 4.4), 2.37-2.30
(8 lines: dxdxd, 1H, H2a, J=22.8, 4.4, 8.4), 2.49 (s, 3H, CH3);
13C nmr: ꢀ 195.58 (C=O), 144.90 (C15), 139.87 (C6), 132.34
(C12), 131.25 (C10), 129.56 (C13), 129.41 (C9), 129.12 (C14),
128.35 (C10a), 126.66 (C7), 126.40 (C8), 124.36 (CN), 120.33
(C6a), 70.32 (C3), 59.10 (C10b), 35.53 (C1), 29.14 (C2), 21.75
(CH3).
EXPERIMENTAL
1H NMR and 13C NMR spectra were acquired on a Bruker
Avance 400 DRX spectrometer at 400 MHz. Chemical shifts are
given in ppm (ꢀ-scale), coupling constants (J) in Hz. IR spectra
(ꢁ, cm-1) were recorded with a JASCO V-570 spectrometer in
KBr. Melting points were determined on a MELTEMP II
apparatus and are uncorrected. The microanalyses were in
satisfactory agreement with the calculated values: C, ±15; H,
±10; N, ±30.
General Procedure for Synthesis of Salts (3a-c). A solution
of 2-bromo-4'-methylacetophenone (2.13 g, 10 mmol, dissolved
in 15 ml of anhydrous benzene) and diazine compound (10
mmol, dissolved in 10 ml of anhydrous benzene), was stirred
for 3 h at room temperature to give the corresponding
cycloimmonium salt (3). The obtained salts were collected by
filtration and dried in vacuo. No purification required.
2-[2-(4-Methylphenyl)-2-oxoethyl]phthalazinium bromide
(3b). This compound was obtained as white crystals, 3.36 g
(98%), mp 112-115°C; ir: 3051(CH), 2986(CH), 1694(C=O),
1
1610, 1572, 1494, 1462 (C=C, C=N); H nmr (DMSO-d6): ꢀ
11.49 (s, 1H, H1), 9.90 (s, 1H, H4), 8.77-8.74 (d, 1H, H8,
J=8.4), 8.60-8.58 (d, 1H, H5, J=8.0), 8.37-8.35 (t, 1H, H6, J=7.2,
8.0), 8.27-8.25 (t, 1H, H7, J=7.2, 8.4), 7.95-7.93 (d, 2H, H12,
J=8.0), 7.29-7.27 (d, 2H, H13, J=8.0), 6.91 (s, 2H, CH2), 2.39 (s,
3H, CH3); 13C nmr: ꢀ 188.96 (C=O),154.12 (C4), 153.50 (C1),
146.53 (C14), 139.73 (C6), 136.53 (C7), 131.40 (C8), 130.87
(C11), 129.93 (C13), 128.76 (C12), 128.31 (C5), 127.81 (C4a),
127.55 (C8a), 69.15 (CH2), 21.91 (CH3).
General Procedure to obtain [3 + 2] cycloadducts (5 - 10).
The corresponding cycloimmonium salt (3) (2.0 mmol) was
suspended in 10 ml of chloroform. A solution of an activated
alkene or alkyne (2.2 mmol) and triethylamine (0.31 g, 3.0
mmol) in the same solvent (5 ml) was then added. The solution
was refluxed for 2 h in the case of alkynes and for 3 h in the case
7-(4-Methylbenzoyl)H pyrrolo[2,1-a]pyridazine-5,6-dicar-
boxylic acid dimethyl ester (8a). This compound was obtained
as yellow crystals (ethanol), 0.1 g (12%), mp 176-178°C; ir:
3071 (C-H), 2954 (C-H), 1739 (C=O ester-1), 1708 (C=O ester-

1152
R. M. Butnariu, M. D. Caprosu, V. Bejan, M. Ungureanu, A. Poiata, C. Tuchilus,
M. Florescu and I. I. Mangalagiu
Vol 44
2), 1635 (C=O keto), 1604, 1537, 1504, 1452 (C=C, C=N),
1247, 1105 (C-O-C); 1H nmr (deuteriochloroform): ꢀ 8.62-8.59
(dd , 1H, H4, J=9.2, J=1.6), 8.32-8.31 (dd, 1H, H2, J=4.4,
J=1.6), 7.72-7.70 (d, 2H, H10, J=8.4), 7.26-7.24 (d, 2H, H11,
J=8.4), 7.07-7.04 (dd, 1H, H3, J=4.4, J=9.2), 3.92 (s, 3H,
CO2CH3-5), 3.59 (s, 3H, CO2CH3-6), 2.42 (s, 3H, CH3). 13C nmr:
ꢀ 184.26 (C=O), 163.24 (C7), 144.27 (C2), 143.22 (C12),
136.57 (C9), 129.62 (C11), 129.15 (C10), 128.34 (C4), 117.49
(C3), 103.21 (C5), 101.37 (C6), 53.69 (CO2CH3), 51.37
(CO2CH3), 21.98 (CH3)
3-(4-Methylbenzoyl)-1,10b-dihydropyrrolo[2,1-a]phthala-
zine-1,2-dicarboxylic acid dimethyl ester (8b). This compound
was obtained as yellow crystals (methanol), 0.46 g (58%), mp
179-180°C; ir: 3051 (C-H), 2958 (C-H), 1745 (C=O ester-1),
1720 (C=O ester-2), 1671 (C=O keto), 1598, 1557, 1496, 1454
(C=C, C=N), 1226, 1123 (C-O-C); 1H nmr (deuteriochloroform):
ꢀ 8.42-40 (d, 2H, H13, J=8.0), 7.52-7.49 (t, 1H, H8, J=7.2, 8.4),
7.43 (s, 1H, H6), 7.41-7.37 (m, 2H, H9, H10), 7.31-7.29 (d, 2H,
H14, J=8.0), 7.27-7.22 (d, 1H, H7, J=8.4), 5.29-5.25 (d, 1H,
H10b, J=13.2), 4.51-4.47 (d, 1H, H1, J=13.2), 3.91 (s, CO2CH3-
2), 3.50 (s, CO2CH3-1), 2.42 (s, 3H, CH3); 13C nmr: ꢀ 187.92
(C=O), 166.71 (COO-1), 164.45 (COO-2), 153.23 (C3), 145.66
(C6), 142.17 (C15), 132.62 (C12), 131.71 (C10), 130.91 (C10a),
129.71 (C14), 129.48 (C13), 128.99 (C9), 125.78 (C7), 124.89
Acknowledgements. To CNCSIS Bucuresti, grant no.1155/
2006 and to Rounanian Ministry of Research and Education-
BIOTECH program, grant no. 49/2005, for financial support.
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(C6a), 123.53 (C8), 101.51 (C2),
61.75 (C10b), 53.02
(CO2CH3-2), 52.36 (C1), 51.36 (CO2CH3-1), 21.92 (CH3).
3-(4-Methylbenzoyl)-pyrrolo[2,1-a]phthalazine-1-carbox-
ylic acid ethyl ester (10b). This compound was obtained as
white crystals (ethanol), 0.29 g (42%), mp 189-190°C; ir: 3058
(C-H), 2956 (C-H), 1697 (C=O ester), 1668 (C=O keto), 1596,
1
1562, 1498, 1458 (C=C, C=N), 1228, 1125 (C-O-C); H nmr
(deuteriochloroform): ꢀ 9.85-9.83 (d, 1H, H10, J=8.0), 8.74 (s,
1H, H6), 7.92-7.87 (m, 4H, 2H13, H9, H8), 7.76-7.72 (d, 1H,
H7, J=8.0), 7.70 (s, 1H, H2), 7.33-7.31 (d, 2H, H14, J=8), 4.44-
4.38 (q, 2H, CO2CH2, J=7.2), 2.47 (s, 3H, CH3), 1.43-1.39 (t,
3H, CO2CH2CH3, J=7.2); 13C nmr: ꢀ 184.48 (C=O), 164.31
(C=O est), 146.34 (C6), 143.28 (C15), 136.33 (C12), 134.27
(C3), 132.89 (C9), 132.12 (C10b), 130.02 (C13), 129.59 (C8),
129.07 (C14), 127.56 (C10), 127.29 (C7), 126.92 (C10a), 124.12
(C2), 122.15 (C6a), 108.15 (C1), 60.71 (CO2CH2), 21.70 (CH3),
14.46 (CO2CH2CH3).
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