Synthesis, molecular modelling and anticancer evaluation of new pyrrolo[1,2-b]pyridazine and pyrrolo[2,1-a]phthalazine derivatives

Article abstract of DOI:10.1080/14756366.2018.1550085

Two new series of heterocyclic derivatives with potential anticancer activity, in which a pyrrolo[1,2-b]pyridazine or a pyrrolo[2,1-a]phthalazine moiety was introduced in place of the 3′-hydroxy-4′-methoxyphenyl ring of phenstatin have been synthesised and their structure-activity relationship (SAR) was studied. Fourteen of the new compounds were evaluated for their in vitro cytotoxic activity by National Cancer Institute (NCI) against 60 human tumour cell lines panel. The best five compounds in terms of in vitro growth inhibition were screened in the second stage five dose-response studies, three of them showing a very good antiproliferative activity with GI₅₀<100 nM on several cell lines including colon, ovarian, renal, prostate, brain and breast cancer, melanoma and leukemia. Docking experiments on the biologically active compounds showed a good compatibility with the colchicine binding site of tubulin.

Full text of DOI:10.1080/14756366.2018.1550085

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, molecular modelling and anticancer
evaluation of new pyrrolo[1,2-b]pyridazine and
pyrrolo[2,1-a]phthalazine derivatives
Lacramioara Popovici, Roxana-Maria Amarandi, Ionel I. Mangalagiu, Violeta
Mangalagiu & Ramona Danac
To cite this article: Lacramioara Popovici, Roxana-Maria Amarandi, Ionel I. Mangalagiu, Violeta
Mangalagiu & Ramona Danac (2019) Synthesis, molecular modelling and anticancer evaluation of
new pyrrolo[1,2-b]pyridazine and pyrrolo[2,1-a]phthalazine derivatives, Journal of Enzyme Inhibition
and Medicinal Chemistry, 34:1, 230-243, DOI: 10.1080/14756366.2018.1550085
To link to this article: https://doi.org/10.1080/14756366.2018.1550085
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 230–243
https://doi.org/10.1080/14756366.2018.1550085
RESEARCH PAPER
Synthesis, molecular modelling and anticancer evaluation of new
pyrrolo[1,2-b]pyridazine and pyrrolo[2,1-a]phthalazine derivatives
Lacramioara Popovicia, Roxana-Maria Amarandia , Ionel I. Mangalagiua , Violeta Mangalagiub and
Ramona Danaca
b
aFaculty of Chemistry, Alexandru Ioan Cuza University of Iasi, Iasi, Romania; CERNESIM Research Centre, Alexandru Ioan Cuza University of Iasi,
Iasi, Romania
ABSTRACT
ARTICLE HISTORY
Received 23 October 2018
Revised 14 November 2018
Accepted 14 November 2018
Two new series of heterocyclic derivatives with potential anticancer activity, in which a pyrrolo[1,2-b]
pyridazine or a pyrrolo[2,1-a]phthalazine moiety was introduced in place of the 30-hydroxy-40-methoxy-
phenyl ring of phenstatin have been synthesised and their structure-activity relationship (SAR) was
studied. Fourteen of the new compounds were evaluated for their in vitro cytotoxic activity by National
Cancer Institute (NCI) against 60 human tumour cell lines panel. The best five compounds in terms of in
vitro growth inhibition were screened in the second stage five dose-response studies, three of them show-
ing a very good antiproliferative activity with GI50<100nM on several cell lines including colon, ovarian,
renal, prostate, brain and breast cancer, melanoma and leukemia. Docking experiments on the biologically
active compounds showed a good compatibility with the colchicine binding site of tubulin.
KEYWORDS
Anticancer; phenstatin;
pyrrolo[1,2-b]pyridazine;
pyrrolo[2,1-a]phthalazine;
3 þ 2 dipolar cycloaddition;
docking; N-heterocycles
Introduction
being reported to have antimicrobial25,26
,
antifungal25 or
anticancer effects27,28
,
or to act as acyl CoA:diacylglycerol
Considerable efforts have been focussed in the past decades, on
the design and development of new antiproliferative drugs with
improved efficiency, limited toxicity, cost-effectiveness, which are
acyltransferase (DGAT1) inhibitors24, JAK inhibitors29, HER-2
tyrosine kinase inhibitors30, IRAK4 inhibitors31, or MEK inhibitors32.
The replacement of one of the substituted phenyl ring of
phenstatin with pyrrolo-fused heterocycles has been a major focus
in rational drug design in the recent years, as there are several
reported biological active phenstatin analogues containing an
indole ring5,19, an indolizine ring33, or a pyrrolo[2,3-d]pyrimidine
ring34. However, to our knowledge, there are no reported
analogues of phenstatin with pyrrolo[1,2-b]pyridazine or pyrrolo
[2,1-a]phthalazine scaffolds, respectively.
synchronously less prone to develop multidrug resistance1–3
.
Among the variety of targets used in this huge anticancer fight,
tubulin targeting appears to be a key focus in cancer treatment,
the research in this field remaining very active in past years4–7
.
After the success of Colchicine8, combretastatin A-49, vincristine or
vinblastine10 as anticancer drugs acting by inhibiting tubulin poly-
merisation, research efforts focused on developing new colchicine
binding site inhibitors with improved pharmacological profiles4,11
.
With the aim of exploring new potential antitumour scaffolds,
One of the simplest known structures synthesised and tested
as an anticancer agent in the past years is phenstatin12,13 which
stand as one of the most potent tubulin polymerisation inhibitors
by binding to the colchicine site of the tubulin and thus,
interfering with the equilibrium dynamics associated with the cell
division14,15. Because of its biological properties and structural
simplicity, phenstatin continues to be a lead compound for
rational design in anticancer therapy, the recent literature being
the target compounds described in this paper possess
a
pyrrolo[1,2-b]pyridazin-7-yl or pyrrolo[2,1-a]phthalazin-3-yl
a
moiety in place of the 30-hydroxy-40-methoxyphenyl ring of phen-
statin. In order to establish structure-activity relationships (SARs),
we extended our structural modifications by introducing different
substituents at position 2 of the pyrrolo[1,2-b]pyridazine unit,
including methyl or 4-substituted phenyl rings (4-chlorophenyl,
4-bromophenyl or p-tolyl). At the same time, the trimethoxyphenyl
ring of phenstatin was replaced either by 3,5-dihydroxyphenyl,
3,4-dimethoxyphenyl or 4-bromophenyl (Figure 1).
plentiful of such phenstatin analogues16–18
.
Pyrrolo-fused derivatives comprise a class of biologically active
heterocyclic compounds which can serve as promising scaffolds
for the development of anticancer, antimicrobial, antiviral, anti-
malarial, antitubercular, anti-inflammatory, and enzyme inhibiting
drugs19. Among the fused pyrrolo-heterocyclic compounds,
pyrrolo[1,2-b]pyridazines and its condensed pyrrolo[2,1-a]phthala-
zine system are compounds well known for their strong lumines-
Materials and methods
Chemistry
All commercially available reagents and solvents employed were
cence20,21 and photochromic properties22, and at the same time used without further purification. Melting points were recorded
are promising in the field of drug design23,24, some derivatives on an A. Kruss Optronic Melting Point Meter KSPI and are
€
CONTACT Ramona Danac
rdanac@uaic.ro
Faculty of Chemistry, Alexandru Ioan Cuza University of Iasi; Violeta Mangalagiu
ionelm64@yahoo.com
CERNESIM Research Centre, Alexandru Ioan Cuza University of Iasi, 11 Carol I, Iasi 700506, Romania.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
231
A
O
N
N
O
N
N
O
MeO
MeO
OH
R1
R2
R1
R2
OMe
COOEt
COOEt
OMe
Phenstatin
R3
target compounds
R3
target compounds
Figure 1. The structures of phenstatin and the target compounds.
uncorrected. Proton and carbon nuclear magnetic resonance 2H, H10, H14), 8.76 (t, J ¼ 6.0 Hz, 1H, H4), 8.90 (t, J ¼ 6.0 Hz, 1H,
(dH, dC) spectra were recorded on a DRX-500 Bruker or a Bruker H5), 9.74 (d, J ¼ 3.5 Hz, 1H, H3), 9.74 (d, J ¼ 5.0 Hz, 1H, H6).
Avance 400 DRX spectrometers. The following abbreviations were 13 C NMR (125 MHz DMSO-d6) d 56.4 (2 ꢂ OMe), 60.4 (OMe), 70.3
(C7), 106.2 (C10, C14), 136.0 (C5), 137.5 (C4), 143.3 (C12), 151.9
(C6), 153.1 (C11, C13), 154.7 (C3), 189.3 (C8). Anal. calcd. for
C15H17BrN2O4: C, 48.80; H, 4.64; N, 7.59%. Found: C, 48.79; H, 4.55;
N, 7.62%.
used to designate chemical shift multiplicities: s: singlet, d:
doublet, t: triplet, q: quartet, m: multiplet, bs: broad singlet, as:
apparent singlet. All chemical shifts are quoted on the d-scale in
ppm. Coupling constants are given in Hz. IR spectra were
recorded on a FTIR Shimadzu or Jasco 660 plus FTIR spectropho-
tometer. Analyses indicated by the symbols of the elements or
functions were within 0.4% of the theoretical values. Thin layer
chromatography (TLC) was carried out on Merck silica gel 60F254
plates. Visualisation of the plates was achieved using a UV lamp
(kmax ¼ 254 or 365 nm).
1–(2-Oxo-2–(3,5-dimethoxyphenyl)ethyl)pyridazin-1-ium bromide
7b. Beige powder, Yield: 60%; m.p. 196–199 ꢀC; IR (KBr, cmꢁ1):
1684, 1586, 1438, 1334, 1209, 1157, 1058. 1H NMR (500 MHz
DMSO-d6): d 3.85 (s, 6H, 2 ꢂ OMe), 6.76 (s, 2H, H7), 6.94 (t,
J ¼ 2.0 Hz, 1H, H12), 7.21 (d, J ¼ 2.5 Hz, 2H, H10, H14), 8.75 (dd,
J ¼ 8.0; 4.5 Hz, 1H, H4), 8.88 (m, 1H, H5), 9.74 (d, J ¼ 5.5 Hz, 1H,
H3), 9.90 (d, J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6): d
55.8 (2 ꢂ OMe), 70.4 (C7), 106.3 (C10, C14), 106.5 (C12), 135.2 (C9),
136.0 (C5), 137.6 (C4), 151.9 (C6), 154.7 (C3), 160.9 (C11, C13),
190.2 (C8). Anal. calcd. for C14H15BrN2O3: C, 49.57; H, 4.46; N,
8.26%. Found: C, 49.59; H, 4.42; N, 8.32%.
Compounds 7d, 7h, 7l, 8d, 10a, and 10d were previously
reported35–39
.
General procedure for the synthesis of monoquaternary salts 7
and 10
1–(2-Oxo-2–(3,4-dimethoxyphenyl)ethyl)pyridazin-1-ium bromide
1 mmol of heterocycle (pyridazine 1, 3-methylpyridazine 2, 3–(4- 7c. Beige solid, Yield: 60%; m.p. 123–124 ꢀC; IR (KBr, cmꢁ1): 2979,
chlorophenyl)pyridazine) 3, 3–(4-bromophenyl)pyridazine 4, 3-(p- 1677, 1586, 1518, 1294, 1205, 1168. 1H NMR (500 MHz DMSO-d6):
d 3.85 (s, 3H, OMe), 3.90 (s, 3H, OMe), 6.79 (s, 2H, H7), 7.22 (d,
J ¼ 8.5 Hz, 1H, H13), 7.53 (d, J ¼ 2.0 Hz, 1H, H10), 7.81 (dd, J ¼ 8.5;
2.0 Hz, 1H, H14), 8.77 (m, 1H, H4), 8.91 (m, 1H, H5), 9.75 (dd,
J ¼ 5.0; 1.0 Hz, 1H, H3), 9.97 (d, J ¼ 6.0 Hz, 1H, H6). 13 C NMR
(125 MHz DMSO-d6): d 55.8 (OMe), 56.1 (OMe), 70.1 (C7), 110.4
(C10), 111.3 (C14), 123.7 (C13), 126.0 (C9), 136.0 (C5), 137.5 (C4),
151.9 (C6), 154.7 (C3), 148.9 (C11), 154.5 (C12), 188.7 (C8). Anal.
calcd. for C14H15BrN2O3: C, 49.57; H, 4.46; N, 8.26%. Found: C,
49.55; H, 4.44; N, 8.30%.
1–(2-Oxo-2–(4-bromophenyl)ethyl)pyridazin-1-ium bromide 7d.
White solid, Yield: 66%; m.p. 235–237 ꢀC; IR (KBr, cmꢁ1): 3017,
2976, 1695, 1580, 1439, 1394, 1229, 976, 822. 1H NMR (400 MHz
DMSO-d6): d 6.75 (s, 2H, H7), 7.90 (d, J ¼ 8.4 Hz, 2H, H11, H13),
8.03 (d, J ¼ 8.4 Hz, 2H, H10, H14), 8.78 (m, 1H, H4), 8.92 (m, 1H,
H5), 9.76 (dd, J ¼ 6.0; 0.8 Hz, 1H, H3), 9.99 (d, J ¼ 6.0 Hz, 1H, H6).
13 C NMR (100 MHz DMSO-d6): d 70.2 (C7), 129.1 (C12), 130.4 (C10,
C14), 132.2 (C11, C13), 135.4 (C9), 136.0 (C5), 137.5 (C4), 151.8
(C6), 154.7 (C3), 189.8 (C8). Anal. calcd. for C12H10Br2N2O: C, 40.26;
H, 2.82; N, 7.82%. Found: C, 40.25; H, 2.79; N, 7.84%.
3-Methyl-1–(2-oxo-2–(3,4,5-trimethoxyphenyl)ethyl)pyridazin-1-ium
bromide 7e. Liquid, Yield: 50%; IR (cmꢁ1): 3069, 2943, 1687, 1586,
1417, 1332, 1126, 1050, 921. 1H NMR (500 MHz DMSO-d6) d 2.81
(s, 3H, Me), 3.80 (s, 3H, OMe), 3.89 (s, 6H, 2 ꢂ OMe), 6.76 (s, 2H,
H7), 7.38 (s, 2H, H10, H14), 8.65 (d, J ¼ 8.5 Hz, 1H, H4), 8.77 (dd,
J ¼ 8.5; 6.0 Hz, 1H, H5), 9.80 (d, J ¼ 6.0 Hz, 1H, H6). 13 C NMR
(125 MHz DMSO-d6): d 21.6 (Me), 56.4 (2 ꢂ OMe), 60.4 (OMe), 70.1
(C7), 106.2 (C10, C14), 128.5 (C9), 135.0 (C5), 138.4 (C4), 143.3
(C12), 149.7 (C6), 153.1 (2 x C11), 164.9 (C3), 189.3 (C8).
tolyl)pyridazine 5 or phthalazine 9 was dissolved in 7 ml acetone
(for compounds 1–5) or acetonitrile for compound 9. Then
1.1 mmol of reactive halide (2-bromo-1–(3,4,5-trimethoxyphenyl)
ethanone 6a, 2-bromo-1–(3,5-dimethoxyphenyl) ethanone 6 b, 2-
bromo-1–(3,4-dimethoxyphenyl) ethanone 6c or 2-bromo-1–(4-
bromophenyl) ethanone 6d) was added and the resulted mixture
was stirred overnight at room temperature (to obtain compounds
7) or reflux (for synthesis of compounds 10). The reaction mixture
was cooled and the formed precipitate was filtered and washed
with diethyl ether to give the desired product which was used in
the next reaction without any further purification. In case of salts
7e–g, the resulting salts have not crystallised; for these com-
pounds, the solvent was removed under vacuum, and the result-
ing liquid was used in the next step.
General procedure for preparation of compounds 8a–t and 11a–d
The cycloimmonium salt (7a–d or 10a–d) (1 mmol) and ethyl pro-
piolate (1.1 mmol) were added to 10 ml of anhydrous acetone and
the obtained suspension was stirred at room temperature. Then, a
solution of triethylamine (TEA) (3 mmol, 3 equiv.) in anhydrous
acetone (3 ml) was added drop-wise over 1 h (magnetic stirring)
and the resulting mixture was then stirred overnight at room
temperature. Water (10 ml) was added and the formed solid was
collected by filtration to give a powder which was washed with
5 ml methanol. The product was crystallised from dichlorome-
thane/methanol (1:1, v/v).
1–(2-Oxo-2–(3,4,5-trimethoxyphenyl)ethyl)pyridazin-1-ium bromide
7a. Beige powder, Yield: 61%; m.p. 152–153 ꢀC; IR (KBr, cmꢁ1):
3-Methyl-1–(2-oxo-2–(3,5-dimethoxyphenyl)ethyl)pyridazin-1-ium
1
1672, 1583, 1416, 1319, 1165, 1128. H NMR (500 MHz DMSO-d6) d bromide 7f. Liquid, Yield: 50%; IR (cmꢁ1): 3069, 2943, 1687, 1586,
3.80 (s, 3H, OMe), 3.90 (s, 6H, 2 ꢂ OMe), 6.82 (s, 2H, H7), 7.39 (s, 1417, 1332, 1164, 1050, 921. 1H NMR (500 MHz DMSO-d6): d 2.90

232
L. POPOVICI ET AL.
(s, 3H, Me), 3.84 (s, 6H, 2 ꢂ OMe), 6.63 (s, 2H, H7), 6.90 110.5 (C10), 111.3 (C13), 123.7 (C14), 126.0 (C9), 129.7 (C16, C20,
(t, J ¼ 2.0 Hz, 1H, H12), 7.20 (d, J ¼ 2.5 Hz, 2H, H10, H14), 8.67 (d, C17, C19), 130.6 (C15), 134.7 (C4), 136.3 (C5), 137.8 (C18), 148.9
J ¼ 8.5 Hz, 1H, H4), 8.78 (dd, J ¼ 8.5; 5.5 Hz, 1H, H5), 9.87 (d, (C11), 150.1 (C6), 154.6 (C12); 160.1 (C3), 188.4 (C8). Anal. calcd.
J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6) d 21.6 (Me), 55.9 for C20H18BrClN2O3: C, 53.41; H, 4.03; N, 6.23%. Found: C, 53.44; H,
(2 ꢂ OMe), 70.3 (C7), 106.3 (C10, C14), 106.6 (C12); 135.0 (C9), 3.99; N, 6.27%.
3–(4-Chlorophenyl)-1–(2-oxo-2–(4-bromophenyl)ethyl)pyridazin-1-
ium bromide 7l. Yellow solid, Yield: 75%; m.p. 200–202 ꢀC; IR (KBr,
135.2 (C5), 138.4 (C4), 149.6 (C6), 160.8 (C11, C13), 164.8 (C3),
190.2 (C8).
3-Methyl-1–(2-oxo-2–(3,4-dimethoxyphenyl)ethyl)pyridazin-1-ium cmꢁ1): 3055, 2992, 1690, 1587, 1447, 1395, 1333, 1233, 1096, 986,
bromide 7g. Liquid; Yield: 51%; IR (cmꢁ1): 2969, 1682, 1586, 1517, 824. 1H NMR (500 MHz DMSO-d6) d 6.86 (s, 2H, H7), 7.76 (d,
1270, 1019, 806. 1H NMR (DMSO-d6, 500 MHz) d 2.80 (s, 3H, Me), J ¼ 6.5 Hz, 2H, H17, H19), 7.90 (d, J ¼ 6.0 Hz, 2H, H11, H13), 8.04
3.84 (s, 3H, OMe), 3.84 (s, 3H, OMe), 6.78 (s, 2H, H7), 7.18 (d, (d, J ¼ 6.0 Hz, 2H, H10, H14), 8.25 (d, J ¼ 6.5 Hz, 2H, H16, H20),
J ¼ 8.5 Hz, 1H, H12), 7.51 (d, J ¼ 2.0 Hz, 1H, H10), 7.79 (dd, J ¼ 8.5; 8.96 (bs, 1H, H5), 9.33 (d, J ¼ 7.5 Hz, 1H, H4), 9.93 (bs, 1H, H6). 13 C
2.0 Hz, 1H, H14), 8.68 (1H, d, J ¼ 8.5 Hz, H4), 8.80 (1H, dd, J ¼ 8.5; NMR (125 MHz DMSO-d6) d 70.5 (C7), 129.2 (C12), 129.7 (C16, C20),
5.5 Hz, H5), 9.93 (d, J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO- 129.8 (C17, C19), 130.4 (C11, C13), 130.6 (C15), 132.3 (C10, C14),
d6): d ¼ 21.7 (Me), 55.8 (OMe), 56.0 (OMe), 70.0 (C7), 110.5 (C10), 132.4 (C9), 134.8 (C4), 136.4 (C5), 137.8 (C18), 150.1 (C6), 160.1
111.4 (C14), 123.8 (C13), 126.1 (C9), 135.0 (C5), 138.3 (C4), 149.7 (C6), (C3), 189.7 (C8). Anal. calcd. for C18H13Br2ClN2O: C, 46.14; H, 2.80;
148.9 (C11), 154.5 (C12), 164.8 (C3), 188.7 (C8).
N, 5.98%. Found: C, 46.14; H, 2.77; N, 6.01%.
3-Methyl-1–(2-oxo-2–(4-bromophenyl)ethyl)pyridazin-1-ium brom-
3–(4-Bromophenyl)-1–(2-oxo-2–(3,4,5-trimethoxyphenyl)ethyl)pyri-
ide 7h. Beige solid, Yield: 70%; m.p. 216–218 ꢀC; IR (KBr, cmꢁ1): dazin-1-ium bromide 7m. Brown solid, Yield: 75%; m.p. 185–187 ꢀC;
3013, 2976, 1692, 1586, 1468, 1231, 1072, 988, 828. 1H NMR IR (KBr, cmꢁ1): 2930, 1680, 1585, 1456, 1340, 1132. 1H NMR
(500 MHz DMSO-d6) d 2.81 (s, 3H, Me), 6.74 (s, 2H, H7), 7.89 (d, (500 MHz DMSO-d6) d 3.82 (s, 3H, OMe), 3.92 (s, 6H, 2 ꢂ OMe), 6.90
J ¼ 8.5 Hz, 2H, H11, H13), 8.02 (d, J ¼ 8.5 Hz, 2H, H10, H14), 8.66 (s, 2H, H7), 7.43 (s, 2H, H10, H14), 7.91 (d, J ¼ 8.0 Hz, 2H, H17,
(d, J ¼ 8.5 Hz, 1H, H4), 8.78 (dd, J ¼ 8.5; 6.0 Hz, 1H, H5), 9.82 (d, H19), 8.18 (d, J ¼ 8.0 Hz, 2H, H16, H20), 8.97 (dd, J ¼ 9.0; 4.5 Hz,
J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6) d 21.6 (Me), 70.0 1H, H5), 9.33 (d, J ¼ 9.0 Hz, 1H, H4), 9.92 (d, J ¼ 4.5 Hz, 1H, H6).
(C7), 129.1 (C12); 130.4 (C10, C14), 132.3 (C11, C13), 132.4 (C9), 13 C NMR (125 MHz DMSO-d6) d 56.4 (2 ꢂ OMe), 60.4 (OMe), 70.6
135.0 (C5), 138.4 (C4), 149.7 (C6), 164.8 (C3), 189.8 (C8). Anal. (C7), 106.2 (C10, C14), 126.8 (C18), 128.5 (C9), 129.9 (C16, C20),
calcd. for C13H12Br2N2O: C, 41.97; H, 3.25; N, 7.53%. Found: C, 130.9 (C15), 132.6 (C17, C19), 134.7 (C4), 136.4 (C5), 143.3 (C12),
42.00; H, 3.18; N, 7.55%.
150.1 (C6), 153.0 (C11, C13), 160.3 (C3), 189.2 (C8). Anal. calcd. for
3–(4-Chlorophenyl)-1–(2-oxo-2–(3,4,5-trimethoxyphenyl)ethyl)pyr- C21H20Br2N2O4: C, 48.12; H, 3.85; N, 5.34%. Found: C, 48.15; H,
idazin-1-ium bromide 7i. Beige solid, Yield 74%; m.p. 186–188 ꢀC;
3.79; N, 5.37%.
IR (KBr, cmꢁ1): 3059, 2932, 1682, 1599, 1450, 1343, 1132. 1H NMR
3–(4-Bromophenyl)-1–(2-oxo-2–(3,5-dimethoxyphenyl)ethyl)pyrida-
(500 MHz DMSO-d6) d 3.81 (s, 3H, OMe), 3.90 (s, 6H, 2 ꢂ OMe), 6.91 zin-1-ium bromide 7n. Brown solid, Yield: 73%; m.p. 217–220 ꢀC; IR
(s, 2H, H7), 7.42 (s, 2H, H10, H14), 7.76 (d, J ¼ 7.5 Hz, 2H, H17, (KBr, cmꢁ1): 3078, 2926, 1695, 1591, 1452, 1385, 1103, 1074. 1H
H19), 8.25 (d, J ¼ 8.0 Hz, 2H, H16, H20), 8.96 (dd, J ¼ 9.0; 4.5 Hz, NMR (500 MHz DMSO-d6) d 3.85 (s, 6H, 2 ꢂ OMe), 6.82 (s, 2H, H7),
1H, H5), 9.33 (d, J ¼ 9.0 Hz, 1H, H4), 9.93 (d, J ¼ 4.5 Hz, 1H, H6). 7.23 (s, 2H, H10, H14), 7.91 (d, J ¼ 8.0 Hz, 2H, H17, H19), 8.17 (d,
13 C NMR (125 MHz DMSO-d6) d 56.3 (2 ꢂ OMe), 60.3 (OMe), 70.5 J ¼ 8.0 Hz, 2H, H16, H20), 8.95 (dd, J ¼ 9.0; 4.5 Hz, 1H, H5), 9.30 (d,
(C7), 106.2 (C10, C14), 128.4 (C9), 129.6 (C16, C20, C17, C19), 130.5 J ¼ 9.0 Hz, 1H, H4), 9.86 (d, J ¼ 4.5 Hz, 1H, H6). 13 C NMR (125 MHz
(C15), 134.7 (C4), 136.3 (C5), 137.7 (C18), 143.2 (C12), 150.0 (C6), DMSO-d6) d 56.7 (2 ꢂ OMe), 70.7 (C7), 106.6 (C12), 107.1 (C10,
153.0 (C11, C13), 159.9 (C3), 189.1 (C8). Anal. calcd. for C14), 127.7 (C18), 128.9 (C16, C20), 130.8 (C15), 132.7 (C17, C19),
C21H20BrClN2O4: C, 52.57; H, 4.20; N, 5.84%. Found: C, 52.55; H, 134.8 (C4), 135.2 (C9), 136.3 (C5), 150.8 (C6), 160.3 (C3), 160.9 (C11,
4.18; N, 5.87%.
C13), 189.9 (C8). Anal. calcd. for C20H18Br2N2O3: C, 48.61; H, 3.67;
N, 5.67%. Found: C, 48.65; H, 3.66; N, 5.67%.
3–(4-Chlorophenyl)-1–(2-oxo-2–(3,5-dimethoxyphenyl)ethyl)pyrida-
zin-1-ium bromide 7j. Beige solid, Yield: 75%; m.p. 220–222 ꢀC; IR
3–(4-Bromophenyl)-1–(2-oxo-2–(3,4-dimethoxyphenyl)ethyl)pyrida-
(KBr, cmꢁ1): 3034, 2980, 1697, 1599, 1456, 1356, 1204, 1153, 841. zin-1-ium bromide 7o. Brown solid, Yield: 72%; m.p. 210–212 ꢀC; IR
1H NMR (500 MHz DMSO-d6) d 3.86 (s, 6H, 2 ꢂ OMe), 6.83 (s, 2H, (KBr, cmꢁ1): 3049, 2975, 1697, 1596, 1453, 1358, 1261, 1207. 1H
H7), 6.95 (as, 1H, H12), 7.24 (as, 2H, H10, H14), 7.77 (d, J ¼ 8.5 Hz, NMR (500 MHz DMSO-d6) d 3.86 (s, 3H, 2 OMe), 3.91 (s, 3H, 2
2H, H17, H19), 8.25 (d, J ¼ 8.5 Hz, 2H, H16, H20), 8.95 (dd, J ¼ 9.0; OMe), 6.80 (s, 2H, H7), 7.23 (d, J ¼ 8.5 Hz, 1H, H13), 7.55 (d,
5.5 Hz, 1H, H5), 9.31 (d, J ¼ 9.0 Hz, 1H, H4), 9.87 (d, J ¼ 5.5 Hz, 1H, J ¼ 2.0 Hz, 1H, H10), 7.83 (dd, J ¼ 8.5; 2.0 Hz, 1H, H14), 7.91 (d,
H6). 13 C NMR (125 MHz DMSO-d6) d 55.8 (2 ꢂ OMe), 70.7 (C7), J ¼ 8.5 Hz, 2H, H17, H19), 8.17 (d, J ¼ 8.5 Hz, 2H, H16, H20), 8.95
106.3 (C10, C14), 106.6 (C12); 129.7 (C16, C20), 129.8 (C17, C19), (dd, J ¼ 9.0; 5.5 Hz, 1H, H5), 9.31 (d, J ¼ 9.0 Hz, 1H, H4), 9.87 (d,
130.6 (C15), 134.8 (C4), 135.2 (C9), 136.4 (C5), 137.8 (C18), 150.1 J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6) d 55.8 (OMe),
(C6), 160.2 (C3), 160.9 (C11, C13); 190.0 (C8). Anal. calcd. for 56.1 (OMe), 70.4 (C7), 110.4 (C10), 111.3 (C13), 123.7 (C14), 126.0
C20H18BrClN2O3: C, 53.41; H, 4.03; N, 6.23%. Found: C, 53.45; H, (C9), 126.9 (C18), 129.9 (C16, C20), 131.0 (C15), 132.7 (C17, C19),
4.00; N, 6.27%.
134.7 (C4), 136.4 (C5), 148.9 (C11), 150.1 (C6), 154.6 (C12); 160.3
3–(4-Chlorophenyl)-1–(2-oxo-2–(3,4-dimethoxyphenyl)ethyl)pyrida- (C3), 188.5 (C8). Anal. calcd. for C20H18Br2N2O3: C, 48.61; H, 3.67; N,
zin-1-ium bromide 7k. Beige solid, Yield 77%; m.p. 144–146 ꢀC; IR
5.67%. Found: C, 48.62; H, 3.64; N, 5.68%.
(KBr, cmꢁ1): 3055, 2936, 1680, 1597, 1518, 1452, 1333, 1271, 1157,
3–(4-Bromophenyl)-1–(2-oxo-2–(4-bromophenyl)ethyl)pyridazin-1-
1
1092, 1015. H NMR (500 MHz DMSO-d6) d 3.86 (s, 3H, OMe), 3.91 ium bromide 7p. Brown solid, Yield: 80%; m.p. 206–208 ꢀC; IR (KBr,
(s, 3H, OMe), 6.79 (s, 2H, H7), 7.23 (d, J ¼ 8.0 Hz, 1H, H13), 7.55 (s, cmꢁ1): 3055, 1690, 1587, 1447, 1387, 1333, 1233, 1076, 986, 827.
1H, H10), 7.76 (d, J ¼ 7.5 Hz, 2H, H17, H19), 7.82 (d, J ¼ 8.0 Hz, 1H, 1H NMR (500 MHz DMSO-d6) d 6.84 (s, 2H, H7), 7.90 (d, J ¼ 8.5 Hz,
H14), 8.24 (d, J ¼ 7.5 Hz, 2H, H16, H20), 8.94 (dd, J ¼ 8.5; 5.5 Hz, 2H, H11, H13), 7.91 (d, J ¼ 8.0 Hz, 2H, H17, H19), 8.04 (d,
1H, H5), 9.30 (d, J ¼ 8.5 Hz, 1H, H4), 9.87 (d, J ¼ 4.5 Hz, 1H, H6). J ¼ 8.0 Hz, 2H, H16, H20), 8.17 (d, J ¼ 8.5 Hz, 2H, H10, H14), 8.95
13 C NMR (125 MHz DMSO-d6) d 55.8 (OMe), 56.0 (OMe), 70.3 (C7), (dd, J ¼ 8.0; 5.5 Hz, 1H, H5), 9.32 (d, J ¼ 8.0 Hz, 1H, H4), 9.89 (d,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
233
J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6) d 70.5 (C7), 126.9 J ¼ 9.0; 4.5 Hz, 1H, H3), 7.17 (s, 2H, H12, H16), 7.77 (s, 1H, H6), 8.53
(C18), 129.2 (C12), 129.9 (C10, C14), 130.4 (C16, C20), 131.0 (C15), (as, 1H, H4), 8.67 (d, J ¼ 9.0 Hz, 1H,H2). 13 C NMR (125 MHz CDCl3)
132.2 (C11, C13), 132.4 (C9), 132.6 (C17, C19), 134.8 (C4), 136.4 d 14.7 (CH3), 55.5 (2 ꢂ OMe), 60.6 (CH2), 61.2 (OMe), 105.5 (C5),
(C5), 150.1 (C6), 160.3 (C3), 189.6 (C8). Anal. calcd. for 107.4 (C12, C16), 117.6 (C3), 124.4 (C6), 126.8 (C7), 128.2 (C2),
C18H13Br3N2O: C, 42.14; H, 2.55; N, 5.46%. Found: C, 42.12; H, 2.54; 133.5 (C8), 134.2 (C14), 142.2 (C11), 144.3 (C4), 153.2 (C13, C15),
N, 5.48%.
1–(2-Oxo-2–(3,4,5-trimethoxyphenyl)ethyl)-3-(p-tolyl)pyridazin-1-ium
bromide 7q. Beige solid, Yield: 78%; m.p. 208–210 ꢀC; IR (KBr, cmꢁ1):
163.7 (COO), 183.6 (C10). Anal. calcd. for C20H20N2O6: C, 62.49; H,
5.24; N, 7.29%. Found: C, 64.59; H, 5.15; N, 7.32%.
Ethyl 7–(3,5-dimethoxybenzoyl)pyrrolo[1,2-b]pyridazine-5-carboxyl-
3040, 2990, 2928, 1688, 1587, 1454, 1343, 1132, 990. 1H NMR ate 8b. Yellow powder, Yield: 57%; m.p. 178–180 ꢀC; IR (KBr,
(500 MHz DMSO-d6) d 2.43 (s, 3H, Me), 3.81 (s, 3H, OMe), 3.91 (s, 6H, cmꢁ1): 1685, 1631, 1587, 1479, 1358, 1225, 1047 (C-O); 1H NMR
2 ꢂ OMe), 6.87 (s, 2H, H7), 7.42 (s, 2H, H10, H14), 7.48 (d, J ¼ 8.0 Hz, (500 MHz CDCl3) d 1.40 (t, J ¼ 7.0 Hz, 3H, CH3), 3.85 (s, 6H,
2H, H17, H19), 8.13 (d, J ¼ 8.0 Hz, 2H, H16, H20), 8.92 (dd, J ¼ 9.0; 2 ꢂ OMe), 4.39 (q, J ¼ 7.0 Hz, 2H, CH2), 6.68 (s, 1H, H14), 7.01 (d,
5.5 Hz, 1H, H5), 9.28 (d, J ¼ 9.0 Hz, 1H, H4), 9.86 (d, J ¼ 5.5 Hz, 1H, J ¼ 1.5 Hz, 2H, H12, H16), 7.15 (dd, J ¼ 9.5; 4.5 Hz, 1H, H3), 7.78 (s,
H6). 13 C NMR (125 MHz DMSO-d6) d 21.1 (Me), 56.4 (2 ꢂ OMe), 60.4 1H, H6), 8.54 (d, J ¼ 3.0 Hz, 1H, H4), 8.67 (d, J ¼ 9.0 Hz, 1H, H2).
(OMe), 70.6 (C7), 106.2 (C10, C14), 127.8 (C16, C20), 128.5 (C9), 128.9 13 C NMR (125 MHz CDCl3) d 14.6 (CH3), 55.8 (2 ꢂ OMe), 60.6 (CH2),
(C15), 130.2 (C17, C19), 134.3 (C4), 136.2 (C5), 143.2 (C18), 143.3 104.7 (C14), 105.5 (C5), 107.6 (C12, C16), 117.8 (C3), 125.2 (C6),
(C12), 149.6 (C6), 153.1 (C11, C13), 161.1 (C3), 189.3 (C8). Anal. calcd. 126.7 (C7), 128.2 (C2), 133.7 (C8), 141.1 (C11), 144.4 (C4), 160.8
for C22H23BrN2O4: C, 57.53; H, 5.05; N, 6.10%. Found: C, 57.55; H, (C13, C15), 163.7 (COO), 184.1 (C10). Anal. calcd. for C19H18N2O5:
5.01; N, 6.13%.
C, 64.40; H, 5.12; N, 7.91%. Found: C, 64.52; H, 5.05; N, 7.99%.
1–(2-Oxo-2–(3,5-dimethoxyphenyl)ethyl)-3-(p-tolyl)pyridazin-1-ium
Ethyl 7–(3,4-dimethoxybenzoyl)pyrrolo[1,2-b]pyridazine-5-carboxyl-
bromide 7r. Beige solid, Yield: 75%; m.p. 180–182 ꢀC; IR (KBr, ate 8c. Beige solid, Yield: 50%; m.p. 123–124 ꢀC; IR (KBr, cmꢁ1):
cmꢁ1): 3042, 2974, 1695, 1591, 1456, 1202, 1155, 808. 1H NMR 1704, 1629, 1580, 1371, 1132, 1023; 1H NMR (500 MHz CDCl3) d
(500 MHz DMSO-d6) d 2.43 (s, 3H, Me), 3.86 (s, 6H, 2 ꢂ OMe), 6.82 1.41 (t, J ¼ 7.0 Hz, 3H, CH3), 3.96 (s, 3H, OMe), 3.98 (s, 3H, OMe),
(s, 2H, H7), 6.94 (t, J ¼ 2.5 Hz, 1H, H12), 7.24 (d, J ¼ 2.5 Hz, 2H, H10, 4.39 (q, J ¼ 7.0 Hz, 2H, CH2), 6.95 (d, 1H, J ¼ 8.0 Hz, H15), 7.12 (dd,
H14), 7.48 (d, J ¼ 8.5 Hz, 2H, H17, H19), 8.13 (d, J ¼ 8.5 Hz, 2H, J ¼ 9.5; 4.5 Hz, 1H, H3), 7.55 (d, J ¼ 1.5 Hz, 1H, H12), 7.56 (dd,
H16, H20), 8.90 (dd, J ¼ 9.0; 5.5 Hz, 1H, H5), 9.27 (d, J ¼ 9.0 Hz, 1H, J ¼ 8.0; 1.5 Hz, 1H, H16), 7.75 (s, 1H, H6), 8.50 (dd, J ¼ 4.0; 2.0 Hz,
H4), 9.84 (d, J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6) d 1H, H4), 8.65 (dd, J ¼ 9.0; 2.0 Hz, 1H, H2). 13 C NMR (125 MHz
21.1 (Me), 55.8 (2 ꢂ OMe), 70.7 (C7), 106.3 (C10, C14), 106.6 (C12); CDCl3) d 14.7 (CH3), 56.2 (OMe), 56.3 (OMe), 60.5 (CH2), 105.2 (C5),
127.8 (C16, C20), 128.9 (C15), 130.2 (C17, C19), 134.3 (C4), 135.2 110.1 (C12), 112.0 (C15), 117.3 (C3), 123.9 (C16), 124.8 (C6), 126.9
(C9), 136.1 (C5), 143.2 (C18), 149.6 (C6), 160.9 (C11, C13); 161.1 (C7), 128.2 (C2), 131.7 (C11), 133.2 (C8), 144.2 (C4), 149.2 (C13),
(C3), 190.1 (C8). Anal. calcd. for C21H21BrN2O3: C, 58.75; H, 4.93; N, 153.2 (C14), 163.8 (COO), 184.4 (C10). Anal. calcd. for C19H18N2O5:
6.53%. Found: C, 58.77; H, 4.89; N, 6.55%.
C, 64.40; H, 5.12; N, 7.91%. Found: C, 64.44; H, 5.07; N, 7.97%.
Ethyl 7–(4-bromobenzoyl)pyrrolo[1,2-b]pyridazine-5-carboxylate
1–(2-Oxo-2–(3,4-dimethoxyphenyl)ethyl)-3-(p-tolyl)pyridazin-1-ium
bromide 7s. Beige solid, Yield: 75%; m.p. 150–151 ꢀC; IR (KBr, 8d. Yellow solid, Yield: 57%; m.p. 133–135 ꢀC; IR (KBr, cmꢁ1): 1707,
cmꢁ1): 3009, 2962, 1679, 1590, 1518, 1455, 1268, 1160, 1024. 1H 1638, 1468, 1235, 1190, 1096; 1H NMR (500 MHz CDCl3) d 1.40 (t,
NMR (500 MHz DMSO-d6) d 2.42 (s, 3H, Me), 3.86 (s, 3H, OMe), 3.91 J ¼ 7.0 Hz, 3H, CH3), 4.38 (q, J ¼ 7.0 Hz, 2H, CH2), 7.17 (dd, J ¼ 9.0;
(s, 3H, OMe), 6.79 (s, 2H, H7), 6.94 (d, J ¼ 8.5 Hz, 1H, H13), 7.55 (d, 4.5 Hz, 1H, H3), 7.65 (d, J ¼ 8.0 Hz, 2H, H13, H15), 7.72 (s, 1H, H6),
J ¼ 1.5 Hz, 2H, H10), 7.48 (d, J ¼ 8.0 Hz, 2H, H17, H19), 7.83 (dd, 7.77 (d, J ¼ 8.0 Hz, 2H, H12, H16), 8.54 (dd, J ¼ 4.0; 1.5 Hz, 1H, H4),
J ¼ 8.5; 1.5 Hz, 1H, H14), 8.13 (d, J ¼ 8.0 Hz, 2H, H16, H20), 8.90 8.67 (dd, J ¼ 9.5; 4.0 Hz, 1H, H2). 13 C NMR (125 MHz CDCl3) d 14.6
(dd, J ¼ 8.5; 5.5 Hz, 1H, H5), 9.27 (d, J ¼ 9.0 Hz, 1H, H4), 9.83 (d, (CH3), 60.7 (CH2), 105.7 (C5), 118.0 (C3), 125.0 (C6), 126.4 (C7),
J ¼ 5.5 Hz, 1H, H6). 13 C NMR (125 MHz DMSO-d6) d 21.1 (Me), 55.8 127.4 (C14), 128.2 (C2), 131.2 (C12, C16), 131.9 (C13, C15), 133.8
(OMe), 56.1 (OMe), 70.3 (C7), 110.4 (C10), 111.3 (C13); 123.7 (C14), (C8), 137.9 (C11), 144.5 (C4), 163.6 (COO), 183.3 (C10). Anal. calcd.
126.1 (C9), 127.9 (C16, C20), 128.9 (C15), 130.3 (C17, C19), 134.3 for C17H13BrN2O3: C, 54.71; H, 3.51; N, 7.51%. Found: C, 54.74; H,
(C4), 136.1 (C5), 143.3 (C18), 148.9 (C11), 149.7 (C6), 154.5 (C12), 3.47; N, 7.57%.
Ethyl
2-methyl-7–(3,4,5-trimethoxybenzoyl)pyrrolo[1,2-b]pyrida-
161.1 (C3), 188.6 (C8). Anal. calcd. for C21H21BrN2O3: C, 58.75; H,
4.93; N, 6.53%. Found: C, 58.78; H, 4.90; N, 6.56%.
zine-5-carboxylate 8e. Beige solid; Yield: 47%; m.p. 91–93 ꢀC; IR
1–(2-Oxo-2–(4-bromophenyl)ethyl)-3-(p-tolyl)pyridazin-1-ium brom- (KBr, cmꢁ1): 2978,1695, 1656, 1586, 1470, 1368, 1233, 1124, 754;
ide 7t. Yellow solid, Yield: 77%; m.p. 208–210 ꢀC; IR (KBr, cmꢁ1): 1H NMR (500 MHz CDCl3) d 1.40 (t, J ¼ 7.0 Hz, 3H, CH3), 2.64 (s,
1
3022, 2988, 1694, 1584, 1452, 1219, 984. H NMR (500 MHz DMSO- 3H,Me), 3.90 (s, 6H, 2 ꢂ OMe), 3.95 (s, 3H, OMe), 4.38 (q, J ¼ 7.0 Hz,
d6) d 2.42 (s, 3H, Me), 6.84 (s, 2H, H7), 7.47 (d, J ¼ 8.0 Hz, 2H, H11, 2H, CH2), 7.02 (d, J ¼ 9.0 Hz, 1H, H3), 7.18 (s, 2H, H12, H16), 7.68 (s,
H13), 7.91 (d, J ¼ 8.5 Hz, 2H, H17, H19), 8.05 (d, J ¼ 8.5 Hz, 2H, H16, 1H, H6), 8.52 (d, J ¼ 9.0 Hz, 1H, H4). 13 C NMR (125 MHz CDCl3) d
H20), 8.13 (d, J ¼ 8.0 Hz, 2H, H10, H14), 8.91 (dd, J ¼ 9.0; 6.0 Hz, 1H, 14.7 (CH3), 22.4 (Me), 56.5 (2 ꢂ OMe), 60.5 (CH2), 61.2 (OMe), 105.1
H5), 9.28 (d, J ¼ 9.0 Hz, 1H, H4), 9.86 (d, J ¼ 6.0 Hz, 1H, H6). 13 C (C5), 107.5 (C12, C16), 119.7 (C3), 123.8 (C6), 126.6 (C7), 127.6
NMR (125 MHz DMSO-d6) d 21.1 (Me), 70.5 (C7), 127.9 (C16, C20), (C11), 132.3 (C8), 134.3 (C14), 142.2 (C4), 153.1 (C13, C15), 153.4
128.9 (C15), 129.2 (C12), 130.2 (C17, C19), 130.4 (C11, C13), 132.3 (C2), 163.9 (COO), 183.5 (C10). Anal. calcd. for C21H22N2O6: C,
(C10, C14), 132.5 (C9), 134.3 (C4), 136.1 (C5), 143.2 (C18), 149.7 (C6), 63.31; H, 5.57; N, 7.03%. Found: C, 63.29; H, 5.55; N, 7.00%.
Ethyl 2-methyl-7–(3,5-dimethoxybenzoyl)pyrrolo[1,2-b]pyridazine-
5-carboxylate 8f. Beige solid, Yield: 45%; m.p. 96–98 ꢀC; IR (KBr,
161.0 (C3), 189.7 (C8). Anal. calcd. for C19H16Br2N2O: C, 50.92; H,
3.60; N, 6.25%. Found: C, 50.93; H, 3.56; N, 6.26%.
Ethyl 7–(3,4,5-trimethoxybenzoyl)pyrrolo[1,2-b]pyridazine-5-carb- cmꢁ1): 2937, 1681, 1654, 1592, 1436, 1361, 1232, 1158, 754; 1H
oxylate 8a. Beige powder, Yield: 55%; m.p. 188–190 ꢀC; IR (KBr, NMR (500 MHz CDCl3) d 1.39 (t, J ¼ 7.0 Hz, 3H, CH3), 2.65 (s,
cmꢁ1): 1703, 1635, 1584, 1474, 1323, 1217, 1128, 1051; 1H NMR 3H,Me), 3.84 (s, 6H, 2 ꢂ OMe), 4.37 (q, J ¼ 7.0 Hz, 2H, CH2), 6.68 (s,
(500 MHz CDCl3) d 1.41 (t, J ¼ 7.0 Hz, 3H, CH3), 3.90 (s, 6H, 1H, H14), 7.02 (overlapped signals, 3H, H3, H12, H16), 7.69 (s, 1H,
2 ꢂ OMe), 3.96 (s, 3H, OMe), 4.40 (q, J ¼ 7.0 Hz, 2H, CH2), 7.15 (dd, H6), 8.52 (d, J ¼ 9.5 Hz, 1H, H4). 13 C NMR (125 MHz CDCl3) d 14.6

234
L. POPOVICI ET AL.
(CH3), 22.4 (Me), 55.8 (2 ꢂ OMe), 60.5 (CH2), 104.6 (C14), 105.2 (C5), 2H, CH2), 7.47 (d, J ¼ 8.5 Hz, 2H, H19, H21), 7.59 (d, J ¼ 9.5 Hz, H4),
107.6 (C12, C16), 119.9 (C3), 124.6 (C6), 126.6 (C7), 127.6 (C11), 7.67 (d, J ¼ 8.0 Hz, 2H, H12, H16), 7.76 (s, 1H, H6), 7.78 (d,
132.5 (C8), 141.2 (C4), 153.5 (C2), 160.7 (C13, C15), 163.8 (COO), J ¼ 8.0 Hz, 2H, H13, H15), 7.96 (d, J ¼ 8.5 Hz, 2H, H18, H22), 8.70
184.1 (C10). Anal. calcd. for C20H20N2O5: C, 65.21; H, 5.47; N, (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d 14.7 (CH3), 60.7
7.60%. Found: C, 65.18; H, 5.45; N, 7.63%.
(CH2), 105.9 (C5), 116.1 (C4), 124.8 (C6), 126.9 (C14), 127.4 (C7),
128.4 (C18, C22), 128.5 (C3), 129.5 (C19, C21), 132.4 (C8), 133.5
Ethyl 2-methyl-7–(4-bromobenzoyl)pyrrolo[1,2-b]pyridazine-5-carb-
oxylate 8h. Beige solid, Yield: 42%; m.p. 145–147 ˚C; IR (KBr, (C17), 131.1 (C13, C15), 131.9 (C12, C16), 137.0 (C20), 138.0 (C11),
cmꢁ1): 2990, 1701, 1645, 1547, 1462, 1362, 1261, 1229, 1188, 151.3 (C2), 163.6 (COO), 183.5 (C10). Anal. calcd. for
1
1098, 754; H NMR (500 MHz CDCl3) d 1.40 (t, J ¼ 7.0 Hz, 3H, CH3),
C23H16BrClN2O3: C, 57.11; H, 3.33; N, 5.79%. Found: C, 57.10; H,
2.65 (s, 3H, Me), 4.38 (q, J ¼ 7.0 Hz, 2H, CH2), 7.04 (d, J ¼ 9.5 Hz, 3.29; N, 5.85%.
1H, H3), 7.63 (s, 1H, H6), 7.65 (d, J ¼ 8.0 Hz, 2H, H13, H15), 7.77 (d,
Ethyl 2–(4-bromophenyl)-7–(3,4,5-trimethoxybenzoyl)pyrrolo[1,2-
J ¼ 8.0 Hz, 2H, H12, H16), 8.54 (d, J ¼ 9.0 Hz, 1H, H4). 13 C NMR b]pyridazine-5-carboxylate 8m. Beige solid, Yield: 50%; m.p.
(125 MHz CDCl3) d 14.6 (CH3), 22.4 (Me), 60.6 (CH2), 105.4 (C5), 238–240 ˚C; IR (KBr, cmꢁ1): 2984, 2930, 1697, 1657, 1586, 1503,
1
120.1 (C3), 124.4 (C6), 126.3 (C7), 127.3 (C14), 127.6 (C4), 131.2 1458, 1314, 1234, 1169, 1128, 808, 752; H NMR (500 MHz CDCl3) d
(C12, C16), 131.8 (C13, C15), 132.6 (C8), 138.1 (C11), 153.7 (C2), 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 3.90 (s, 6H, 2 ꢂ OMe), 3.97 (s, 3H,
163.7 (COO), 183.3 (C10). Anal. calcd. for C18H15BrN2O3: C, 55.83; OMe), 4.42 (q, J ¼ 7.0 Hz, 2H, CH2), 7.20 (s, 2H, H12, H16), 7.58 (d,
H, 3.90; N, 7.23%. Found: C, 55.85; H, 3.87; N, 7.26%.
J ¼ 9.0 Hz, H4), 7.63 (d, J ¼ 8.5 Hz, 2H, H19, H21), 7.80 (s, 1H, H6),
Ethyl 2–(4-chlorophenyl)-7–(3,4,5-trimethoxybenzoyl)pyrrolo[1,2- 7.94 (d, J ¼ 8.5 Hz, 2H, H18, H22), 8.69 (d, J ¼ 9.0 Hz, 1H, H3). 13 C
b]pyridazine-5-carboxylate 8i. Beige solid, Yield: 41%; m.p. 230–232 NMR (125 MHz CDCl3) d 14.7 (CH3), 56.5 (2 ꢂ OMe), 60.6 (CH2), 61.2
˚C; IR (KBr, cmꢁ1): 2984, 1697, 1657, 1583, 1503, 1460, 1314, 1234, (OMe), 105.6 (C5), 107.4 (C12, C16), 115.7 (C4), 124.4 (C6), 125.3
1
1169, 1130, 808; H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H, (C20), 127.1 (C7), 128.5 (C3), 128.7 (C18, C22), 132.5 (C19, C21),
CH3), 3.90 (s, 6H, 2 ꢂ OMe), 3.97 (s, 3H, OMe), 4.42 (q, J ¼ 7.0 Hz, 132.2 (C11), 134.1 (C17), 134.2 (C8), 142.3 (C14), 151.2 (C2), 153.2
2H, CH2), 7.20 (s, 2H, H12, H16), 7.47 (d, J ¼ 8.5 Hz, 2H, H19, H21), (C13, C15), 163.7 (COO), 183.6 (C10). Anal. calcd. for C26H23BrN2O6:
7.47 (d, J ¼ 9.0 Hz, 1H, H4), 7.79 (s, 1H, H6), 8.01 (d, J ¼ 8.5 Hz, 2H, C, 57.90; H, 4.30; N, 5.19%. Found: C, 57.95; H, 4.27; N, 5.24%.
H18, H22), 8.69 (d, J ¼ 9.0 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d
Ethyl
2–(4-bromophenyl)-7–(3,5-dimethoxybenzoyl)pyrrolo[1,2-
14.7 (CH3), 56.5 (2 ꢂ OMe), 60.6 (CH2), 61.2 (OMe), 105.6 (C5), 107.3 b]pyridazine-5-carboxylate 8n. Beige solid, Yield: 51%; m.p.
(C12, C16), 115.8 (C4), 124.4 (C6), 127.1 (C7), 128.4 (C3, C18, C22), 193–195 ˚C; IR (KBr, cmꢁ1): 2982, 1695, 1659, 1591, 1458, 1298,
1
129.5 (C19, C21), 132.2 (C11), 133.6 (C17), 134.2 (C8), 136.9 (C20), 1155, 1096, 808; H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H,
142.3 (C14), 151.1 (C2), 153.2 (C13, C15), 163.7 (COO), 183.6 (C10). CH3), 3.85 (s, 6H, 2 ꢂ OMe), 4.40 (q, J ¼ 7.0 Hz, 2H, CH2), 6.71 (t,
Anal. calcd. for C26H23ClN2O6: C, 63.10; H, 4.68; N, 5.66%. Found: C, J ¼ 2.5 Hz, 1H, H14), 7.05 (d, J ¼ 2.5 Hz, 2H, H12, H16), 7.58 (d,
63.15; H, 4.67; N, 5.69%.
J ¼ 9.5 Hz, H4), 7.63 (d, J ¼ 8.5 Hz, 2H, H19, H21), 7.81 (s, 1H, H6),
Ethyl
2–(4-chlorophenyl)-7–(3,5-dimethoxybenzoyl)pyrrolo[1,2- 7.94 (d, J ¼ 8.5 Hz, 2H, H18, H22), 8.70 (d, J ¼ 9.5 Hz, 1H, H3). 13 C
b]pyridazine-5-carboxylate 8j. Beige solid, Yield: 41%; m.p. 193–195 NMR (125 MHz CDCl3) d 14.7 (CH3), 55.8 (2 ꢂ OMe), 60.6 (CH2),
˚C; IR (KBr, cmꢁ1): 2949, 1694, 1659, 1599, 1458, 1298, 1094, 810; 104.8 (C14), 105.7 (C5), 107.5 (C12, C16), 115.9 (C4), 125.0 (C6),
1H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 3.85 (s, 6H, 125.2 (C20), 127.1 (C7), 128.4 (C3), 128.7 (C18, C22), 132.4 (C19,
2 ꢂ OMe), 4.40 (q, J ¼ 7.0 Hz, 2H, CH2), 6.71 (bs, 1H, H14), 7.05 (d, C21, C8), 134.1 (C17), 141.1 (C11), 151.2 (C2), 160.9 (C13, C15),
J ¼ 2.0 Hz, 2H, H12, H16), 7.47 (d, J ¼ 8.5 Hz, 2H, H19, H21), 7.59 163.7 (COO), 184.2 (C10). Anal. calcd. for C25H21BrN2O5: C, 58.95;
(d, J ¼ 9.5 Hz, 1H4), 7.81 (s, 1H, H6), 8.01 (d, J ¼ 8.5 Hz, 2H, H18, H, 4.16; N, 5.50%. Found: C, 58.94; H, 4.09; N, 5.55%.
H22), 8.70 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d 14.7
Ethyl
2–(4-bromophenyl)-7–(3,4-dimethoxybenzoyl)pyrrolo[1,2-
(CH3), 55.8 (2 ꢂ OMe), 60.6 (CH2), 104.8 (C14), 105.7 (C5), 107.5 b]pyridazine-5-carboxylate 8o. Beige solid, Yield: 52%; m.p.
(C12, C16), 115.9 (C4), 125.0 (C6), 127.1 (C7), 128.4 (C18, C22), 166–167 ˚C; IR (KBr, cmꢁ1): 2975, 2929, 1719, 1680, 1592, 1458,
128.5 (C3), 129.5 (C19, C21), 132.4 (C8), 133.7 (C17), 136.8 (C20), 1269, 1236, 1147, 1087; 1H NMR (500 MHz CDCl3) d 1.42 (t,
141.1 (C11), 151.2 (C2), 160.9 (C13, C15), 163.7 (COO), 184.2 (C10). J ¼ 7.0 Hz, 3H, CH3), 3.97 (s, 3H, OMe), 3.99 (s, 3H, OMe), 4.41 (q,
Anal.calcd. for C25H21ClN2O5: C, 64.59; H, 4.55; N, 6.03%. Found: C, J ¼ 7.0 Hz, 2H, CH2), 6.95 (d, J ¼ 7.5 Hz, 1H, H15), 7.57–7.62 (m, 5H,
64.64; H, 4.50; N, 6.10%.
H12, H16, H4, H19, H21), 7.76 (s, 1H, H6), 7.93 (d, J ¼ 8.0 Hz, 2H,
Ethyl
2–(4-chlorophenyl)-7–(3,4-dimethoxybenzoyl)pyrrolo[1,2- H18, H22), 8.67 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d
b]pyridazine-5-carboxylate 8k. Beige solid, Yield: 42%; m.p. 161–163 14.7 (CH3), 56.2 (OMe), 56.3 (OMe), 60.6 (CH2), 105.4 (C5), 110.1
˚C; IR (KBr, cmꢁ1): 2982, 1696, 1649, 1595, 1462, 1269, 1234, 1090, (C15), 111.8 (C12), 115.5 (C4), 124.1 (C6), 124.8 (C16), 125.1 (C20),
806; 1H NMR (500 MHz CDCl3) d 1.43 (t, J ¼ 7.0 Hz, 3H, CH3), 3.97 127.3 (C7), 128.4 (C3), 128.7 (C18, C22), 131.7 (C11), 132.4 (C19,
(s, 3H, OMe), 3.99 (s, 3H, OMe), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 6.95 C21), 132.0 (C8), 134.2 (C17), 149.3 (C13), 151.1 (C2), 153.2 (C14),
(d, J ¼ 8.0 Hz, 1H, H15), 7.46 (d, J ¼ 8.5 Hz, 2H, H19, H21), 7.56 (m, 163.8 (COO), 183.4 (C10). Anal. calcd. for C25H21BrN2O5: C, 58.95;
3H, H12, H16, H4), 7.76 (s, 1H, H6), 8.00 (d, J ¼ 8.5 Hz, 2H, H18, H, 4.16; N, 5.50%. Found: C, 58.97; H, 4.10; N, 5.53%.
H22), 8.68 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d 14.8
Ethyl 2–(4-bromophenyl)-7–(4-bromobenzoyl)pyrrolo[1,2-b]pyrida-
zine-5-carboxylate 8p. White solid, Yield: 45%; m.p. 171–173 ˚C; IR
(CH3), 56.3 (OMe), 56.4 (OMe), 60.7 (CH2), 105.4 (C5), 110.2 (C15),
112.0 (C12), 115.7 (C4), 124.2 (C6), 124.9 (C16), 127.4 (C7), 128.6 (KBr, cmꢁ1): 3051, 2988, 1694, 1657, 1458, 1242, 1209, 1094, 1072,
(C3, C18, C22), 129.5 (C19, C21), 131.9 (C11), 132.1 (C17), 133.8 806, 748; 1H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3),
4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 7.63 (d, J ¼ 8.5 Hz, 2H, H19, H21), 7.59
(C8), 136.9 (C20), 151.1 (C2), 149.4 (C13), 153.4 (C14), 163.9 (COO),
183.5 (C10). Anal. calcd. for C25H21ClN2O5: C, 64.59; H, 4.55; N, (d, J ¼ 9.5 Hz, H4), 7.67 (d, J ¼ 8.0 Hz, 2H, H12, H16), 7.76 (s, 1H,
H6), 7.77 (d, J ¼ 8.0 Hz, 2H, H13, H15), 7.88 (d, J ¼ 8.5 Hz, 2H, H18,
6.03%. Found: C, 64.60; H, 4.49; N, 6.07%.
Ethyl 2–(4-chlorophenyl)-7–(4-bromobenzoyl)pyrrolo[1,2-b]pyrida- H22), 8.70 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d 14.6
zine-5-carboxylate 8l. White solid; Yield: 45%; m.p. 163–165 ˚C; IR (CH3), 60.7 (CH2), 105.9 (C5), 116.0 (C4), 124.8 (C6), 125.3 (C20),
(KBr, cmꢁ1) 3051, 2988, 1694, 1458, 1242, 1207, 1090, 808; H N,R 126.9 (C14), 127.4 (C7), 128.5 (C3), 128.7 (C18, C22), 131.1 (C19,
1
(500 MHz CDCl3) d 1.43 (t, J ¼ 7.0 Hz, 3H, CH3), 4.41 (q, J ¼ 7.0 Hz, C21), 131.9 (C13, C15), 132.4 (C8), 132.5 (C12, C16), 133.9 (C17),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
235
138.0 (C11), 151.4 (C2), 163.6 (COO), 183.5 (C10). Anal. calcd. for 3.80 (s, 3H, OMe), 3.91 (s, 6H, 2 ꢂ OMe), 6.87 (s, 2H, H11), 7.44 (s,
C23H16Br2N2O3: C, 52.30; H, 3.05; N, 5.30%. Found: C, 52.30; H, 2H, H14, H18), 8.50 (t, J ¼ 7.5 Hz, 1H, H6), 8.62 (t, J ¼ 8.0 Hz, H5),
3.00; N, 5.32%.
Ethyl
8.69 (d, J ¼ 8.0 Hz, 1H, H4), 8.76 (d, J ¼ 8.0 Hz, 1H, H7), 10.24 (s,
1H, H3), 10.77 (s, 1H, H8). 13 C NMR (125 MHz DMSO-d6) d 56.4
2-(p-tolyl)-7–(3,4,5-trimethoxybenzoyl)pyrrolo[1,2-b]pyrida-
zine-5-carboxylate 8q. White solid, Yield: 40%; m.p. 198–200 ˚C; IR (2 ꢂ OMe), 60.4 (OMe), 69.0 (C11), 106.3 (C14, C18), 127.3 (C10),
(KBr, cmꢁ1): 3020, 2978, 2943, 1697, 1657, 1586, 1503, 1460, 1333,
127.5 (C9), 128.6 (C13), 128.7 (C4), 130.8 (C7), 136.6 (C6), 139.9
1234, 1130, 1130, 806, 750; 1H NMR (500 MHz CDCl3) d 1.42 (t,
(C5), 143.3 (C16), 153.1 (C15, C17), 153.5 (C8), 154.9 (C3), 189.6
J ¼ 7.0 Hz, 3H, CH3), 2.41 (s, 3H, Me), 3.89 (s, 6H, 2 ꢂ OMe), 3.97 (s, (C12). Anal. calcd. for C19H19BrN2O4: C, 54.43; H, 4.57; N, 6.68%.
3H, OMe), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 7.20 (s, 2H, H12, H16), 7.29
Found: C, 54.47; H, 4.54; N, 6.71%.
(d, J ¼ 8.5 Hz, 2H, H19, H21), 7.60 (d, J ¼ 9.5 Hz, 1H4), 7.78 (s, 1H,
2–(2-Oxo-2–(3,5-dimethoxyphenyl)ethyl)phthalazin-2-ium bromide
H6), 7.94 (d, J ¼ 8.5 Hz, 2H, H18, H22), 8.66 (d, J ¼ 9.5 Hz, 1H, H3). 10b. Brown solid, Yield: 76%; m.p. 178–180 ˚C; IR (KBr, cmꢁ1):
13 C NMR (125 MHz CDCl3) d 14.7 (CH3), 21.5 (Me), 56.5 (2 ꢂ OMe), 2976, 1703, 1589, 1319, 1011. 1H NMR (500 MHz DMSO-d6) d 3.86
60.5 (CH2), 61.2 (OMe), 105.4 (C5), 107.3 (C12, C16), 116.3 (C4), (s, 6H, 2 ꢂ Me), 6.79 (s, 2H, H11), 6.93 (t, J ¼ 2.0 Hz, 1H, H16), 7.26
124.2 (C6), 127.0 (C18, C22), 127.1 (C7), 128.1 (C3), 129.9 (C19, C21), (d, J ¼ 2.0 Hz, 2H, H14, H18), 8.51 (t, J ¼ 8.0 Hz, 1H, H6), 8.61 (t,
132.2 (C11), 132.3 (C17), 134.3 (C8), 140.9 (C20), 142.1 (C14), 152.2 J ¼ 8.0 Hz, H5), 8.67 (d, J ¼ 8.0 Hz, 1H, H4), 8.75 (d, J ¼ 8.0 Hz, 1H,
(C2), 153.1 (C13, C15), 163.8 (COO), 183.7 (C10). Anal. calcd. for H7), 10.21 (s, 1H, H3), 10.69 (s, 1H, H8). 13 C NMR (125 MHz DMSO-
C27H26N2O6: C, 68.34; H, 5.52; N, 5.90%. Found: C, 68.35; H, 5.47; d6) d 55.8 (2 ꢂ OMe), 69.1 (C11), 106.3 (C14, C18), 106.6 (C16),
N, 5.94%.
Ethyl 2-(p-tolyl)-7–(3,5-dimethoxybenzoyl)pyrrolo[1,2-b]pyridazine-
127.3 (C10), 127.5 (C9), 128.6 (C4), 130.8 (C7), 135.3 (C13), 136.6
(C6), 139.9 (C5), 153.6 (C8), 154.9 (C3), 160.9 (C15, C17), 190.5
5-carboxylate 8r. Beige solid, Yield: 40%; m.p. 142–144 ˚C; IR (KBr, (C12). Anal. calcd. for C18H17BrN2O3: C, 55.54; H, 4.40; N, 7.20%.
cmꢁ1): 2999, 2918, 1686, 1649, 1593, 1452, 1302, 1236, 1159,
Found: C, 55.55; H, 4.37; N, 7.22%.
1053, 804, 754; 1H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H,
2–(2-Oxo-2–(3,4-dimethoxyphenyl)ethyl)phthalazin-2-ium bromide
CH3), 2.41 (s, 3H, Me), 3.84 (s, 6H, 2 ꢂ OMe), 4.40 (q, J ¼ 7.0 Hz, 2H, 10c. Brown solid, Yield: 74%; m.p. 224–226 ˚C; IR (KBr, cmꢁ1):
CH2), 6.97 (bs, 1H, H14), 7.05 (bs, 2H, H12, H16), 7.29 (d, J ¼ 8.0 Hz, 3017, 2974, 1701, 1589, 1313, 1204, 1011. 1H NMR (500 MHz
2H, H19, H21), 7.60 (d, J ¼ 9.5 Hz, H4), 7.79 (s, 1H, H6), 7.95 (d, DMSO-d6) d 3.86 (s, 3H, OMe), 3.92 (s, 3H, OMe), 6.74 (s, 2H, H11),
J ¼ 8.5 Hz, 2H, H18, H22), 8.65 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR 6.98 (d, J ¼ 8.0 Hz, 1H, H17), 7.47 (overlapped signals, 2H, H14,
(125 MHz CDCl3) d 14.7 (CH3), 21.5 (Me), 55.8 (2 ꢂ OMe), 60.5 H18), 8.47 (t, J ¼ 8.0 Hz, 1H, H6), 8.60 (t, J ¼ 8.0 Hz, H5), 8.68 (d,
(CH2), 104.7 (C14), 105.4 (C5), 107.4 (C12, C16), 116.4 (C4), 124.9 J ¼ 8.0 Hz, 1H, H4), 8.76 (d, J ¼ 8.0 Hz, 1H, H7), 10.25 (s, 1H, H3),
(C6), 127.0 (C7), 127.1 (C18, C22), 128.0 (C3), 129.9 (C19, C21), 10.72 (s, 1H, H8). 13 C NMR (125 MHz DMSO-d6) d 55.8 (OMe), 56.1
132.3 (C8), 132.5 (C17), 140.8 (C20), 141.3 (C11), 152.3 (C2), 160.8 (OMe), 68.9 (C11), 110.5 (C14), 111.4 (C18), 123.8 (C17), 126.2
(C13, C15), 163.8 (COO), 184.2 (C10). Anal. calcd. for C26H24N2O5: (C13), 127.5 (C9), 128.7 (C4), 130.9 (C7), 136.6 (C6), 139.9 (C5),
C, 70.26; H, 5.44; N, 6.30%. Found: C, 70.29; H, 5.39; N, 6.33%.
148.9 (C15), 153.5 (C8), 154.9 (C3), 154.5 (C16), 188.9 (C12). Anal.
calcd. for C18H17BrN2O3: C, 55.54; H, 4.40; N, 7.20%. Found: C,
Ethyl 2-(p-tolyl)-7–(3,4-dimethoxybenzoyl)pyrrolo[1,2-b]pyridazine-
5-carboxylate 8s. Beige solid, Yield: 40%; m.p. 150–151 ˚C; IR (KBr, 55.55; H, 4.37; N, 7.22%.
cmꢁ1): 3088, 2974, 2929, 1721, 1681, 1614, 1514, 1457, 1272, 1148,
2–(2-Oxo-2–(4-bromophenyl)ethyl)phthalazin-2-ium bromide 10d.
1
1088; H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 3.96 White solid, Yield: 76%; m.p. 222–224 ˚C; IR (KBr, cmꢁ1): 3013,
1
(s, 3H, OMe), 3.98 (s, 3H, OMe), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 6.95 (d, 1684, 1613, 1580, 1352. H NMR (500 MHz DMSO-d6) d 6.83 (s, 2H,
J ¼ 8.0 Hz, 1H, H15), 7.28 (d, J ¼ 8.0 Hz, 2H, H19, H21), 7.59 (m, 3H, H11) 7.89 (d, J ¼ 8.5 Hz, 2H, H15, H17), 8.08 (d, J ¼ 8.5 Hz, 2H, H14,
H12, H16, H4), 7.74 (s, 1H, H6), 7.94 (d, J ¼ 8.0 Hz, 2H, H18, H22), H18), 8.48 (t, J ¼ 8.0 Hz, 1H, H6), 8.61 (t, J ¼ 8.0 Hz, H5), 8.68 (d,
8.65 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d 14.7 (CH3), J ¼ 8.0 Hz, 1H, H4), 8.75 (d, J ¼ 8.0 Hz, 1H, H7), 10.23 (s, 1H, H3),
56.2 (OMe), 56.3 (OMe), 60.5 (CH2), 105.0 (C5), 110.0 (C15), 111.8 10.76 (s, 1H, H8). 13 C NMR (125 MHz DMSO-d6) d 68.8 (C11), 127.3
(C12), 116.1 (C4), 123.9 (C6), 124.7 (C16), 127.1 (C18, C22), 127.2 (C10), 127.5 (C9), 128.6 (C4), 129.2 (C16), 130.5 (C14, C18), 130.8
(C7), 128.0 (C3), 129.9 (C19, C21), 131.9 (C11), 132.1 (C17), 132.4 (C7), 132.3 (C15, C17), 132.5 (C13), 136.6 (C6), 139.9 (C5), 153.5
(C8), 140.7 (C20), 149.2 (C13), 152.1 (C2), 153.1 (C14), 164.0 (COO), (C8), 154.9 (C3), 190.1 (C12). Anal. calcd. for C16H12Br2N2O: C,
183.5 (C10). Anal. calcd. for C26H24N2O5: C, 70.26; H, 5.44; N, 6.30%. 47.09; H, 2.96; N, 6.86%. Found: C, 48.11; H, 2.94; N, 6.88%.
Found: C, 70.30; H, 5.40; N, 6.35%.
Ethyl 3–(3,4,5-trimethoxybenzoyl)pyrrolo[2,1-a]phthalazine-1-carb-
Ethyl
2-(p-tolyl)-7–(4-bromobenzoyl)pyrrolo[1,2-b]pyridazine-5- oxylate 11a. White solid, Yield: 40%; m.p. 235–237 ˚C; IR (KBr,
carboxylate 8t. Yellow solid, Yield: 40%; m.p. 160–162 ˚C; IR (KBr, cmꢁ1): 3041, 2933, 2835, 1711, 1650, 1583, 1261, 1172, 1130,
cmꢁ1): 3072, 2974, 1697, 1620, 1503, 1452, 1219, 1211, 1082, 816; 1042, 785; H NMR (500 MHz CDCl3) d 1.41 (t, J ¼ 7.0 Hz, 3H, CH3),
1
1H NMR (500 MHz CDCl3) d 1.42 (t, J ¼ 7.0 Hz, 3H, CH3), 2.42 (s, 3H, 3.90 (s, 6H, 2 ꢂ OMe), 3.90 (s, 3H, OMe), 4.41 (q, J ¼ 7.0 Hz, 2H,
Me), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 7.29 (d, J ¼ 8.5 Hz, 2H, H19, H21), CH2), 7.24 (s, 2H, H16, H20), 7.73 (s, 1H, H2), 7.76 (d, J ¼ 7.5 Hz, 1H,
7.62 (d, J ¼ 9.5 Hz, H4), 7.67 (d, J ¼ 7.5 Hz, 2H, H12, H16), 7.75 (s, H7), 7.90 (overlapped signals, 2H, H8, H6), 8.75 (s, 1H, H5), 9.84 (d,
1H, H6), 7.76 (d, J ¼ 8.0 Hz, 2H, H13, H15), 7.89 (d, J ¼ 8.0 Hz, 2H, J ¼ 8.0 Hz, 1H, H9). 13 C NMR (125 MHz CDCl3) d 14.6 (CH3), 56.5
H18, H22), 8.67 (d, J ¼ 9.5 Hz, 1H, H3). 13 C NMR (125 MHz CDCl3) d (2 ꢂ OMe), 60.9 (CH2), 61.2 (OMe), 107.6 (C16, C20), 108.3 (C1),
14.7 (CH3), 21.6 (Me), 60.6 (CH2), 105.7 (C5), 116.5 (C4), 124.6 (C6), 122.3 (C11), 124.2 (C2), 127.0; 127.1 (C12, C3), 127.6 (C9), 127.7
127.0 (C14), 127.1 (C18, C22), 127.2 (C7), 128.1 (C3), 130.0 (C19, (C6), 129.8 (C7), 130.2 (C13), 133.1 (C8), 134.1 (C15), 142.3 (C18),
C21), 132.2 (C17), 132.5 (C8), 131.1 (C13, C15), 131.9 (C12, C16), 146.5 (C5), 153.1 (C17, C19), 164.4 (COO), 183.8 (C14). Anal. calcd.
138.0 (C11), 141.0 (C20), 152.4 (C2), 163.7 (COO), 183.6 (C10). Anal. for C24H22N2O6: C, 66.35; H, 5.10; N, 6.45%. Found: C, 66.58; H,
calcd. for C24H19BrN2O3: C, 62.22; H, 4.13; N, 6.05%. Found: C, 5.05; N, 6.48%.
62.25; H, 4.05; N, 6.08%.
Ethyl
3–(3,5-dimethoxybenzoyl)pyrrolo[2,1-a]phthalazine-1-carb-
2–(2-Oxo-2–(3,4,5-trimethoxyphenyl)ethyl)phthalazin-2-ium brom- oxylate 11b. White solid, Yield: 41%; m.p. 220–222 ˚C; IR (KBr,
ide 10a. Brown solid, Yield: 77%; m.p. 150–152 ˚C; IR (KBr, cmꢁ1): cmꢁ1): 3040, 2969, 2835, 1707, 1649, 1595, 1458, 1383, 1173, 1096,
2976, 1670, 1584, 1339, 1125, 764. 1H NMR (500 MHz DMSO-d6) d 756; 1H NMR (500 MHz CDCl3) d 1.41 (t, J ¼ 7.0 Hz, 3H, CH3), 3.85

236
L. POPOVICI ET AL.
(s, 6H, 2 ꢂ OMe), 4.41 (q, J ¼ 7.0 Hz, 2H, CH2), 6.70 (bs, 1H, H18),
7.08 (d, J ¼ 2.0 Hz, 2H, H16, H20), 7.74 (overlapped signals, 2H, H2,
H7), 7.90 (overlapped signals, 2H, H8, H6), 8.76 (s, 1H, H5), 9.83 (d,
J ¼ 8.5 Hz, 1H, H9). 13 C NMR (125 MHz CDCl3) d 14.6 (CH3), 55.8
(2 ꢂ OMe), 60.9 (CH2), 105.0 (C18), 107.8 (C16, C20), 108.4 (C1),
122.3 (C11), 124.8 (C2), 127.0; 127.1 (C12, C3), 127.6 (C9), 127.7 (C6),
129.8 (C7), 130.4 (C13), 133.0 (C8), 141.1 (C15), 146.6 (C5), 160.7
(C17, C19), 164.4 (COO), 184.3 (C14). Anal. calcd. for C23H20N2O5: C,
68.31; H, 4.98; N, 6.93%. Found: C, 68.34; H, 4.95; N, 6.96%.
Results and discussion
Chemistry
The chosen method for the assembly of pyrrolo[1,2-b]pyridazine
moieties relied on 1,3-dipolar cycloaddition of different pyridazi-
nium ylides to ethyl propiolate.
First, pyridazines 1–5 (Scheme 1) were used for the synthesis
of their monoquaternary salts with 2-bromoacetophenones 6a–d.
While compound 6d is commercially available, compounds 6a–c
were synthesised using reported procedures46. The quaternisation
reactions were carried out at room temperature (r.t.) in a minimal
amount of acetone, leading to the formation of salts 7a–t
(Scheme 1).
As shown in Scheme 2, ethyl propiolate was reacted with the
corresponding pyridazinium ylides 70a–t (in situ generated in basic
medium from salts 7a–t) to give the intermediate dihydropyr-
rolo[1,2-b]pyridazines 80a–t, which in turn underwent oxidative
dehydrogenation under atmospheric conditions, yielding the final
compounds 8a–t in moderate yields (40–52%) (Scheme 2).
For the synthesis of compounds 11, in a similar manner, phtha-
lazine was first reacted with 2-bromoacetophenones 6a–d, to give
monoquaternary salts 10a–d (Scheme 3). Phthalazinium salts
10a–d furnished pyrrolo[2,1-a]phthalazines 11a–d when treated
with triethylamine and ethyl propiolate in acetone at room
temperature (Scheme 3).
Ethyl 3–(3,4-dimethoxybenzoyl)pyrrolo[2,1-a]phthalazine-1-carb-
oxylate 11c. White solid, Yield: 41%; m.p. 226–228 ˚C; IR (KBr,
cmꢁ1): 3101, 2959, 2932, 1713, 1645, 1466, 1412, 1263, 1177, 1024,
1
764; H NMR (500 MHz CDCl3) d 1.41 (t, J ¼ 7.0 Hz, 3H, CH3), 3.96 (s,
3H, OMe), 3.97 (s, 3H, OMe), 4.40 (q, J ¼ 7.0 Hz, 2H, CH2), 6.94 (d,
J ¼ 8.0 Hz, 1H, H19), 7.58 (overlapped signals, 2H, H16, H20), 7.68
(s, 1H, H2), 7.73 (t, J ¼ 7.5 Hz, 1H, H7), 7.88 (overlapped signals, 2H,
H8, H6), 8.71 (s, 1H, H5), 9.82 (d, J ¼ 8.0 Hz, 1H, H9). 13 C NMR
(125 MHz CDCl3) d 14.6 (CH3), 56.22 (OMe), 56.23 (OMe), 60.8 (CH2),
108.1 (C1), 110.1 (C16), 112.1 (C19), 122.2 (C11), 123.5 (C2), 125.1
(C20), 127.1 (C3), 127.3 (C12), 127.5 (C9), 127.7 (C6), 129.7 (C7),
129.9 (C13), 131.7 (C15), 133.0 (C8), 146.4 (C5), 149.2 (C17), 153.3
(C18), 164.5 (COO), 183.8 (C14). Anal. calcd. for C23H20N2O5: C,
68.31; H, 4.98; N, 6.93%. Found: C, 68.32; H, 4.97; N, 6.94%.
Ethyl 3–(4-bromobenzoyl)pyrrolo[2,1-a]phthalazine-1-carboxylate
11d 35. White solid, Yield: 51%; m.p. 180–182 ˚C; IR (KBr, cmꢁ1):
2980, 1722, 1653, 1587, 1464, 1379, 1242; 1H NMR (500 MHz
CDCl3) d 1.38 (t, J ¼ 7.0 Hz, 3H, CH3), 4.37 (q, J ¼ 7.0 Hz, 2H, CH2),
7.62 (d, J ¼ 8.5 Hz, 2H, H17, H19), 7.65 (s, 1H, H2), 7.74 (t,
J ¼ 8.0 Hz, 1H, H7), 7.78 (d, J ¼ 8.5 Hz, 2H, H16, H20), 7.87 (over-
lapped signals, 2H, H8, H6), 8.73 (s, 1H, H5), 9.80 (d, J ¼ 8.5 Hz, 1H,
H9). 13 C NMR (125 MHz CDCl3) d 14.5 (CH3), 60.9 (CH2), 108.5 (C1),
122.3 (C11), 124.7 (C2), 126.7 (C12); 126.8 (C18), 127.5 (C3), 127.6
(C9), 127.7 (C6), 129.9 (C7), 130.5 (C13), 131.3 (C16, C20), 131.7
(C17, C19), 133.1 (C8), 137.9 (C15), 146.6 (C5), 164.2 (COO), 183.5
(C14). Anal. calcd. for C21H15BrN2O3: C, 59.59; H, 3.57; N, 6.62%.
Found: C, 59.62; H, 3.55; N, 6.64%.
Biological activity
Fourteen of the synthesised compounds (8a, b, d, e, f, h, i, j, k,
n, q, and 11a–c) were selected by the National Cancer Institute
(NCI) for screening against a panel of 60 human tumour cell lines
at a single dose of 10 μM43, the representative results for the
active compounds being summarised in Table 1.
Pyrrolo[2,1-b]pyridazines 8a, 8b, 8e, 8f, and pyrrolo[2,1-
a]phthalazine 11b showed a very good growth inhibition effect
on almost all 60 cell lines, the best results being registered on leu-
kemia HL-60 (TB) cell, colon cancer COLO205 cell, melanoma
MDA-MB-435 cell, ovarian cancer cell OVCAR-3, and renal cancer
A498 cell. Compound 8a also showed a moderate cytotoxic effect,
notably on melanoma MDA-MB-435 cells (57% cytotoxic). Mild to
moderate cytotoxic effects were also observed for compounds 8b,
8e, 8f, and 11b against several cell types. Interestingly, the substi-
tution of pyrrolo[2,1-b]pyridazine heterocycle at position 2 with a
4-substituted phenyl group resulted in the loss of the activity,
compounds 8i–j, 8k, 8n, and 8q showing almost no inhibition
effect against the 60 cell tested lines (data not shown). In contrast,
2-methylpyrrolo[2,1-b]pyridazines 8e–f showed similar activity to
unsubstituted compounds 8a–b. Another interesting aspect is that
substitution of the 3,4,5-trimethoxyphenyl ring of with a 3,5-dime-
thoxyphenyl one, did not diminish the inhibitory activity. In fact,
Molecular modelling
Flexible docking experiments were carried out in Autodock Vina40
,
using a 18x22x22 Å3 grid box centered on the colchicine binding
site of the a,b-tubulin heterodimer crystal structure (PDB: 1SA0)41.
The 3 D structures of the compounds were constructed in
Avogadro v1.2.042 and were subjected to 10,000 steepest descent
steps of energy minimisation in the MMFF94 force field. One hun-
dred poses were generated for each ligand, and the best-ranked
models were chosen for further visual inspection in order to
assess the consistency of the generated docking solutions relative
to the docking poses of known inhibitor colchicine. Molecular pyrrolo[2,1-a]phthalazine compound 11b showed better growth
inhibitory properties when compared with 11a. Substitution of
the 3,4,5-trimethoxyphenyl ring with 3,4-dimethoxyphenyl or 4-
bromophenyl also caused a reduction in biological activity, with
the exception of compound 8d, which maintained moderate GI%
values on most tested cell lines, although lower than 3,4,5-trime-
thoxyphenyl-substituted analogues 8a–b.
graphics and visual analyses were performed in The PyMOL
€
Molecular Graphics System, Version 1.8.2. (Schrodinger, LLC). Logp
values were calculated using the ChemAxon/Chemicalize server
(www.chemicalize.com).
The most active compounds 8a, 8b, 8e, 8f, and 11b were
selected for the second stage five dose-response studies42–44
selected results being presented in Table 2.
All five tested compounds confirmed the preliminary results by
displaying good antiproliferative properties. The best candidate, 2-
methyl-pyrrolo[2,1-b]pyridazine 8e exhibited GI50 values <100 nM
Cell proliferation assay
The compounds were tested against a panel of 60 human cancer
cell lines at the National Cancer Institute, Rockville, MD. The cyto-
toxicity experiments were realised using a 48 h exposure protocol
using sulphorhodamine B assay43–45
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
237
4
3
A
1
2
. A= H
. A= Me
5
Br
A
N2
O
6
3
4
5
1 N
. A= C6H4Cl(p)
. A= C6H4Br(p)
. A= C6H4Me(p)
Br
R3
N
N
O
acetone
7
+
8
9
14
1
2
3
R1
10
11
6a
6b
6c
. R = R = R = OMe;
1- 5
1
3
2
R2
13
. R = R = OMe, R = H;
R1
R3
12
1
2
3
. R = R = OMe, R = H;
6a-d
R2
1
3
2
6d
. R = R = H, R = Br;
7a-t
1
2
3
1
2
3
k
l
m
n
o
p
q
r
s
. A= C6H4Cl(p), R = R = OMe, R = H;
a
b
c
d
e.
f
g
h
i
j
. A= H, R = R = R = OMe;
1
3
2
1
3
2
. A= C6H4Cl(p), R = R = H, R = Br;
. A=H, R = R = OMe, R = H;
1
2
3
1
2
3
. A= C6H4Br(p), R = R = R = OMe;
. A= H, R = R = OMe, R = H;
1
3
2
1
3
2
. A= C6H4Br(p), R = R = OMe, R = H;
. A= H, R = R = H, R = Br;
1
2
3
1
2
3
. A= C6H4Br(p), R = R = OMe, R = H;
A= Me, R = R = R = OMe;
1
3
2
1
3
2
. A= C6H4Br(p), R = R = H, R = Br;
. A=Me, R = R = OMe, R = H;
1
2
3
1
2
3
. A= C6H4Me(p), R = R = R = OMe;
. A= Me, R = R = OMe, R = H;
. A= Me, R = R = H, R = Br;
. A= C6H4Cl(p), R = R = R = OMe;
. A= C6H4Cl(p), R = R = OMe, R = H;
1
3
2
1
3
2
. A= C6H4Me(p), R = R = OMe, R = H;
1
2
3
1
2
3
. A= C6H4Me(p), R = R = OMe, R = H;
1
3
2
1
3
2
t
. A= C6H4Me(p), R = R = H, R = Br;
Scheme 1. Synthesis of pyridazin-1-ium quaternary salts 7a–t.
A
A
A
Br
N
A
O
N
N
N
N
N
N
Et3N
R3
R2
O
N
O
COOEt
O
acetone
R1
EtOOC
R1
R3
R1
R3
8'a-t
R1
R3
7'a-t
R2
R2
R2
-2[H]
7a-t
1
2
3
a
b
c
d
e.
f
h
i
j
. A= H, R = R = R = OMe;
1
N
A
1
3
2
2
O
l
m
n
o
p
q
r
s
. A= C6H4Cl(p), R = R = H, R = Br;
1
3
2
. A=H, R = R = OMe, R = H;
9
1
2
3
11
R3
15
. A= C6H4Br(p), R = R = R = OMe;
7
1
2
3
3
N
. A= H, R = R = OMe, R = H;
10
16
1
3
2
. A= C6H4Br(p), R = R = OMe, R = H;
1
3
2
14
. A= H, R = R = H, R = Br;
8
4
6
1
2
3
12
R2
. A= C6H4Br(p), R = R = OMe, R = H;
1
2
3
5
13
A= Me, R = R = R = OMe;
1
3
2
R1
. A= C6H4Br(p), R = R = H, R = Br;
1
3
2
EtOOC
. A=Me, R = R = OMe, R = H;
1
2
3
. A= C6H4Me(p), R = R = R = OMe;
. A= C6H4Me(p), R = R = OMe, R = H;
. A= C6H4Me(p), R = R = OMe, R = H;
. A= C6H4Me(p), R = R = H, R = Br;
1
3
2
8a-t
. A= Me, R = R = H, R = Br;
. A= C6H4Cl(p), R = R = R = OMe;
. A= C6H4Cl(p), R = R = OMe, R = H;
. A= C6H4Cl(p), R = R = OMe, R = H;
1
3
2
1
2
3
1
2
3
1
3
2
1
3
2
t
1
2
3
k
Scheme 2. Synthesis of pyrrolo[1,2-b]pyridazines 8a–t.
in thirteen cell lines, notably on melanoma MDA-MB-435 cell
Notably, compound 8e showed a very good cytostatic activ-
(GI50 ¼ 25.6 nM), leukemia SR cell (GI50 ¼ 48.1 nM) and breast cancer ity on melanoma MDA-MB-435 cell with a total growth inhib-
MCF7 cell (GI50 ¼ 48.1 nM). Compound 8e showed better GI50 val- ition level of effect (TGI) of 76.3 nM and leukemia HL-60(TB)
ues against melanoma MDA-MB-435, SK-MEL-5, and UACC-62 and (TGI ¼ 588 nM), whereas compounds 8a and 8f showed the
colon cancer HCT-15, KM12 and SW-620 cell than Doxorubicin best cytostatic activity on melanoma MDA-MB-435 cell with a
(NSC: 123127 code), the NCI standard drug for this type of analysis. TGI of 117 nM and 420 nM, respectively. Significant cytotoxic
Interestingly, even if it exhibits an overall inhibitory activity activity was exhibited only by compound 8a on MDA-MB-435
lower than the 2-methyl substituted compound 8e, compound 8a melanoma cell with
a
lethal concentration (LC50
)
value
shows an excellent inhibitory activity on melanoma MDA-MB-435 of 438 nM.
cell (GI50 ¼ 31.4 nM), leukemia SR cell (GI50 ¼ 46.1 nM) and renal
Although pyrrolo[2,1-a]phthalazine 11b displayed the best
cancer A498 (GI50 ¼ 46.6 nM). Also, compound 8f displayed very mean growth inhibitory effect in preliminary single dose evalu-
good activity against melanoma MDA-MB-435 cell (GI50 ¼ 45.6 nM). ation (Table 1), it did not exhibit GI50 values under the 100 nm

238
L. POPOVICI ET AL.
5
4
6
7
9
3
10
4
Br
5
O
N2
N 10
N
O
8
20
19
1 N
11
6
8
3
COOEt
Et3N
R3
R2
Br
R3
15
14
13
N
N
O
acetone
reflux
11
7
+
12
9
12
16
2
1
18
13
18
rt
17R1
14
R1
EtOOC
9
17
R2
11a-d
R1 15
R3
1
2
3
16
a
b
c
d
. R = R = R = OMe;
. R = R = OMe, R = H;
. R = R = OMe, R = H;
. R = R = H, R = Br;
R2
10a-d
6a-d
1
3
2
1
2
3
1
3
2
Scheme 3. Synthesis of pyrrolo[2,1-a]phthalazines 11a–d from phthalazine via quaternary phthalazinum salts 10a–d.
Table 1. Results of the in vitro growth inhibition (GI %) caused by compounds 8a, b, d, e, f, h and 11a-c against human cancer cell lines in the single-dose assaya.
Compound
Cell line
8a
8b
8d
8e
8f
8h
11a
11b
11c
Cell type
Leukemia
GI (%) (10ꢁ5M)a
CCRF-CEM
K-562
SR
89
89
76
78
23
79
72
47
31
51
27
23
0
84
29
54
30
40
60
48
26
22
31
35
9
45
88
39
55
37
22
50
27
24
83
90
77
86
90
82
4
6
30
77
77
71
38
18
45
10
49
60
40
49
41
70
64
60
27
13
21
19
34
50
93
30
22
66
53
36
17
22
16
26
21
12
3
77
94
11
11
27
18
31
21
20
10
0
16
0
19
23
0
1
4
13
4
14
14
4
6
1
1
3
34
2
12
7
12
0
18
9
10
15
14
5
24
2
82
68
18
11
23
22
23
21
0
34
1
22
8
27
6
8
1
6
21
28
11
0
0
7
19
10
0
95
HL-60(TB)
MOLT-4
RPMI-8226
A549/ATCC
HOP-62
NCI-H460
NCI-H522
COLO205
HCT-116
HCT-15
HT-29
SW-620
KM12
SF-295
SF-539
100b (33)
100b (22)
100b (24)
82
100b (17)
100b (8)
81
79
76
68
67
71
65
69
85
70
88
73
52
71
67
82
57
93
97
81
96
70
98
92
84
82
73
49
90
Non-small
Cell lung cancer
90
89
65
75
96
100b (11)
Colon cancer
100b (25)
100b (13)
86
100b (39)
85
100b (11)
88
76
96
66
75
68
96
82
70
76
96
82
77
78
93
81
72
72
80
76
75
75
92
73
72
59
85
52
81
69
79
97
41
72
97
87
79
76
75
48
77
51
66
22
78
79
CNS cancer
Melanoma
86
91
100b (12)
89
SNB-75
U251
LOX IMVI
M14
66
85
57
100b (10)
100b (13)
60
51
89
90
84
96
58
96
100b (16)
99
MDA-MB-435
UACC-62
SK-MEL-2
SK-MEL-5
OVCAR-3
NCI/ADR-RES
SK-OV-3
OVCAR-8
OVCAR-4
A498
ACHN
RXF393
TK-10
MCF7
MDA-MB-468
100b (57)
100b (16)
53
70
42
74
79
74
99
93
80
62
38
84
69
99
81
94
70
43
85
74
81
Ovarian cancer
Renal cancer
100b (15)
100b (13)
97
73
70
7
81
62
25
10
19
7
66
38
100b (18)
100b (5)
48
100b (3)
47
64
36
75
73
15
25
100b (2)
40
84
98
71
1
100b (4)
46
86
35
79
70
75
78
21
20
12
8
24
16
100b (9)
84
Breast cancer
80
51
22
33
6
70
1
45
6
100b (12)
78
63
69
79
57
68
54
Prostate cancer
PC-3
DU-145
64
63
76
The most active compounds are highlighted in bold.
aData obtained from NCI’s in vitro 60 cell one dose screening at 10ꢁ5M concentration.
bCytotoxic effect; lethality percent is represented in brackets.
threshold, as was the case for the more simple pyrrolo[1,2- Molecular modelling
b]pyridazines. Therefore, the introduction of a bulkier hetero-
Because both computational and biological models of 3,4,5-
trimethoxyphenyl-containing phenstatin analogues supported the
hypothesis that the antiproliferative effects of these compounds
are induced by inhibiting tubulin polymerisation5,7,33,47,48, docking
cycle, such as pyrrolo[2,1-a]phthalazine, in place of the 30-
hydroxy-40-methoxyphenyl ring of phenstatin is less favourable
in terms of antiproliferative activity than pyrrolo[1,2-b]
pyridazine.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
239
Table 2. Results of the 5-dose in vitro human cancer cell growth inhibitiona for compounds 8a–b, e–f and 11b and compared with standard drug Doxorubicin.
Compound !
Cell line #
8a
8b
8e
8f
11b
Doxorubicinc
Cell type
Leukemia
GI50 (nM)b
CCRF-CEM
HL-60(TB)
K-562
261
228
90.6
443
246
46.1
487
363
312
343
193
276
171
208
216
155
733
180
276
769
211
402
247
176
31.4
385
269
176
145
200
426
395
46.6
301
185
166
333
94.6
236
437
n.d.c
281
2510
1380
538
2630
1820
573
11100
10200
2580
346
797
n.d.
587
403
212
160
n.d.
396
n.d.
48.1
223
398
133
171
n.d.
164
84.4
348
248
n.d.
527
n.d.
75.9
767
691
365
303
n.d.
331
280
401
254
280
676
311
349
752
384
549
n.d.
251
45.6
494
276
477
289
308
878
523
388
n.d.
239
317
906
313
284
1990
501
297
n.d.
820
n.d.
n.d.
n.d.
442
n.d.
n.d.
494
236
n.d.
455
484
384
351
483
1590
483
1060
1570
471
730
1070
485
188
n.d.
623
692
341
476
n.d.
n.d.
n.d.
n.d.
1070
839
n.d.
410
1840
1180
n.d.
403
79
126
200
32
79
25
63
63
16
32
MOLT-4
RPMI-8226
SR
Non-small
Cell Lung cancer
A549/ATCC
HOP-62
NCI-H460
NCI-H522
COLO205
HCT-116
HCT-15
HT-29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-5
UACC-62
OVCAR-3
NCI/ADR-RES
SK-OV-3
786-0
Colon cancer
CNS cancer
Melanoma
200
79
6310
126
251
100
100
100
126
40
133
n.d.
518
57.7
68.7
550
65.7
130
420
n.d.
331
n.d.
136
25.6
495
58.5
61.5
64.9
123
546
335
76.2
n.d.
116
93.9
391
48.1
190
878
>100000
26500
2100
1850
4260
399
2000
>100000
394
221
738
508
523
402
463
4900
11400
n.d.
2976
1640
7450
3960
1310
1990
1220
17200
1110
63
40
126
159
251
159
79
159
398
7943
200
126
100
1000
100
316
100
40
Ovarian cancer
Renal cancer
A498
CAKI-1
RXF 393
PC-3
DU-145
MCF7
HS 578T
BT-549
T-47D
MDA-MB-468
Prostate cancer
Breast cancer
316
251
63
66.8
50
The most active compounds are highlighted in bold.
aData obtained from NCI’s in vitro 60 cell 5-dose screening43–45
.
bGI50 – the molar concentration of tested compound causing 50% growth inhibition of tumor cells. Determined at five concentration levels (100, 10, 1.0, 0.1
and 0.01 lM).
cGI50 data for Doxorubicin tested at a highest concentration of 100 lM were obtained from NCI database: https://dtp.cancer.gov/dtpstandard/dwindex/index.jsp.
n.d.: Not determined.
experiments were performed on the colchicine binding site of adopted a conformation in which the p-bromo substituted phenyl
the a,b-tubulin heterodimer (PDB:1SA0), in order to evaluate the was accommodated more deeply in the colchicine binding pocket,
shape and electrostatic complementarity between ligands and resulting in a shift in the position of the central heterocyclic moi-
the a,b-tubulin heterodimer interface, which could account for the ety towards the center of the colchicine binding site, which led to
observed antiproliferative effects.
the disruption of the hydrogen bond with bCys241 (Figure 2(d)).
Compounds 8a and 8b displayed similar docking con-
Overall, the docking experiments suggest that the removal of
formations grouped into two distinct clusters, both having the the 4-methoxy group does not influence the accommodation of
trimethoxyphenyl subunit overlapping with the one in the the ligand in the binding pocket, in agreement with the biological
co-crystallised DAMA-colchicine ligand (Figure 2(a)), and interact- data, while the introduction of a bromine atom as substituent can
ing with the protein through hydrogen bonding with bCys241. induce a different binding conformation which leads to the dis-
The ligands are further stabilised in the binding pocket through ruption of the hydrogen bond between the ligand and bCys241,
hydrophobic interactions with bLeu242, bLeu248, bAla250, which could account for the reduced antiproliferative activity of
bLeu252, bLeu255, and bVal238. The diazine moiety either compound 8d.
extended on top of the binding pocket, with the ester functional
The 2-methyl-substituted analogues of 8a and 8b (8e and 8f)
group orienting towards the dimer interface (Figure 2(b,c)), or was were accommodated in a similar fashion to that of parent com-
flipped at about 180ꢀ, to have the ester group roughly overlap- pounds (Figure 3(e,f)), suggesting that the introduction of a
ping with the third colchicine ring in the crystal structure. methyl substituent does not influence the binding preferences of
Interestingly, the 4-bromo-substituted compound 8d, which dis- the compounds in the colchicine binding site, in agreement with
played a less pronounced biological activity than 8a and 8b, the biological data in terms of antiproliferative activity. Compound

240
L. POPOVICI ET AL.
Figure 2. Structure and docking of diazines in the tubulin binding site: (a) DAMA-colchicine, (b) 8a, (c) 8b, (d) 8d, (e) 8e, (f) 8f; the a,b-tubulin heterodimer is
represented as ribbons; amino acids in the binding site are represented as sticks.
Figure 3. Structure and docking of diazines in the tubulin binding site: (a) 8i, (b) 8j, (c) 8k, (d) 8n; the a,b-tubulin heterodimer is represented as ribbons; amino acids
in the binding site are represented as sticks.
8h, which displayed a marked reduction in biological activity
Interestingly, 2-(p-halogeno-phenyl)-substituted compounds 8i,
when compared to parent compound 8d, did not form the two 8j, 8k, and 8n, which showed a marked decrease in growth inhib-
expected well-defined clusters of conformations, but rather had a ition activity when compared to unsubstituted analogues, were com-
broad range of unrelated docking poses, the most energetically patible with the colchicine binding site, and were accommodated
favourable being similar to the second cluster of compound 8d.
in a similar fashion to their unsubstituted or 2-methyl-substituted

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
241
Table 3. Theoretical logp values of biologically tested compounds.
Compound
logp
Compound
logp
Compound
logp
8a
8b
8d
8e
8f
2.77
2.93
4.01
2.90
3.06
8h
8i
8j
8k
8n
4.14
5.41
5.56
5.56
5.73
8q
5.32
3.76
3.92
3.92
11a
11b
11c
Values were calculated using the ChemAxon/Chemicalize server (www.chemicalize.com).
Figure 4. Structure and docking of diazines in the tubulin binding site: (a) 11a, (b) 11b, (c) 11c; the a,b-tubulin heterodimer is represented as ribbons; amino acids in
the binding site are represented as sticks.
analogues with biological activity to the a,b-tubulin heterodimer of a more bulkier substituent is completely detrimental for the
(Figures 2 and 3). A closer inspection of the basic physicochemical growth inhibitory properties, despite the fact that docking studies
properties of these four compounds reveals, however, a violation of showed a good compatibility with the colchicine binding site of
Lipinski’s rule of five in terms of logp values49 (Table 3), which could tubulin. The lack of proliferative activity in the case of the bulkier
account for the loss in antiproliferative efficacy in spite of apparent 2–(4-X-phenyl)-pyrrolo[1,2-b]pyridazines could be explained by
activity at the colchicine binding site50.
the suboptimal lipophilicity and solubility of these compounds.
However, further assaying in terms of tubulin polymerisation is
needed in order to confirm the proposed antiproliferative mechan-
ism of action of the newly synthetised compounds. Compound
8f could serve as a useful lead compound for further structural
optimisation in the development of new anticancer agents.
Docking of pyrrolo[2,1-a]phthalazines 11a–c revealed a single
cluster of conformations for each compound, similar to the second
cluster obtained for pyrrolo[1,2-b]pyridazines 8a, 8b, 8e, and 8f,
in which the heterocyclic subunit is oriented as to have the ester
group roughly overlapping with the third colchicine ring in
the crystal structure (Figure 4). The methoxyphenyl subunit is
stabilised by a hydrogen bond interaction with bCys241, similar to
the case of pyrrolo[1,2-b]pyridazine analogues. Notably, com-
pound 11b adopts a conformation slightly deeper in the hydro-
phobic pocket, which induces a rotation of the heterocyclic core
and facilitates a hydrophobic interaction with bLeu248, which is
unique among the three docked pyrrolo[2,1-a]phthalazines. A
tighter hydrophobic interaction between 11b and the protein
could account for the pronounced antiproliferative activity exerted
by 11b among the three tested pyrrolo[2,1-a]phthalazines.
However, for all compounds, complementary tubulin polymer-
isation assays are needed in order to confirm the proposed
molecular mechanism.
Acknowledgements
The authors acknowledge to National Cancer Institute (NCI) for
biological evaluation of compounds on their 60- cell panel:
the testing was performed by the Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis (the URL to
the Program’s website: http://dtp.cancer.gov/). We also thank
CERNESIM Research Centre from Alexandru Ioan Cuza University
of Iasi, for the NMR experiments.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Conclusion
The authors are thankful for financial support to Romanian
Ministry of Research and Innovation, Program 1- Development
of the national R & D system, Subprogram 1.2 - Institutional
performance - RDI excellence financing projects, Grant no. 34PFE
In summary, five of the newly synthesised pyrrolo[1,2-b]pyridazine
and pyrrolo[2,1-a]phthalazine phenstatin analogues showed in vitro
antiproliferative activity, the most potent being compounds 8f
with GI50 values <100 nM on thirteen cell lines including colon,
ovarian, renal, prostate, brain and breast cancer, melanoma and
leukemia. Notably, compound 8a showed a very good antiprolifer-
ative effect on melanoma MDA-MB-435 cell, renal cancer A498,
and leukemia SR cell. The substitution of position 2 of pyrrolo[1,2-
b]pyridazine with a methyl group generally appears to increase the
ORCID
Roxana-Maria Amarandi
http://orcid.org/0000-0002-9937-6539
http://orcid.org/0000-0002-4632-5076
http://orcid.org/0000-0003-4370-5353
Ionel I. Mangalagiu
antiproliferative potency of the compounds, while the introduction Ramona Danac

242
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