Synthesis and evaluation of anti-proliferative activity of 1,4-disubstituted phthalazines

Article abstract of DOI:10.1007/s00044-011-9874-z

A series of new phthalazine derivatives 3a-i and 4a-c were synthesized via the reaction of 1-chlorophthalazine derivative 2 with either N-substituted-piperazines, primary or their secondary amines. The structure of the synthesized, new compounds were characterized by spectral data. The anti-proliferative activity on human breast cancer cell line MCF-7 of the synthesized compounds was determined. The results showed that six of the test compounds (3a, 3g, 3i, and 4a-c) displayed potent cytotoxic activity ranging from 1.4 to 2.3 μmol.

Full text of DOI:10.1007/s00044-011-9874-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3288–3293
DOI 10.1007/s00044-011-9874-z
ORIGINAL RESEARCH
Synthesis and evaluation of anti-proliferative activity
of 1,4-disubstituted phthalazines
•
Khaled A. M. Abouzid Nadia A. Khalil
•
Eman M. Ahmed
Received: 8 August 2011 / Accepted: 3 November 2011 / Published online: 16 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of new phthalazine derivatives 3a–i and
4a–c were synthesized via the reaction of 1-chlorophthal-
azine derivative 2 with either N-substituted-piperazines,
primary or their secondary amines. The structure of the
synthesized, new compounds were characterized by spec-
tral data. The anti-proliferative activity on human breast
cancer cell line MCF-7 of the synthesized compounds was
determined. The results showed that six of the test com-
pounds (3a, 3g, 3i, and 4a–c) displayed potent cytotoxic
activity ranging from 1.4 to 2.3 lmol.
unique mechanisms of action have been developed con-
tinuously, many of them have not been therapeutically
useful due to low tumor selectivity and harsh side effects
(Chari, 2008). These facts prompted us to design and
develop novel potent and selective anti-breast cancer
agents.
1,4-Disubstituted phthalazines have received a consid-
erable attention as antitumor agents in the past few years
(Menear et al., 2008a; Menear et al., 2008b; Miller-Moslin
et al., 2009; Vasiliou et al., 2009). A successful example is
Vatalanib (PTK-787), (Fig. 1), which is VEGFR (vascular
endothelial growth factor receptor) inhibitor and is cur-
rently in Phase III clinical trials for metastatic colorectal
cancer (Scott et al., 2007). Growing interest in the potential
of 1,4-disubstituted phthalazines as anticancer agents led to
the synthesis of phthalazine derivatives with N-arylpiper-
azine side chains (Zhang et al., 2010). The piperazinyl
moiety, a small and rigid heterocyclic backbone, has been
an attractive pharmacological scaffold present in several
antitumor drugs (Zhang et al., 2010; Hennequin et al.,
2006; Pollard and Mortimore, 2009). Among them, certain
compounds, II, displayed excellent selectivity against
MDA-MB-231 cell line (Zhang et al., 2010). Furthermore,
the 1,4-disubstituted phthalazine derivatives III has shown
more potent cytotoxicity than cisplatin (Xin et al., 2008)
(Fig. 1).
Keywords Breast cancer ꢀ Anti-proliferative activity ꢀ
Phthalazine ꢀ Phthalazinone
Introduction
Breast cancer is the most common form of cancer and the
second most frequent cause of cancer death among women
(Wang et al., 2004; Albrand and Terret, 2008). Regardless
of the use of surgical treatment and irradiation, chemo-
therapy still remains an important option for the treatment
of solid cancers. Chemotherapeutic drugs should prefer-
entially target tumor cells without harming normal cells or
tissues. However, although new cytotoxic agents with
In an effort to discover and develop potent cytotoxic
agent, we synthesized a series of novel 1,4-disubstituted
phthalazine derivatives bearing piperazinyl, primary, or
secondary amino functions in position (1) of phthalazine
pharmacophore and a 4-bromophenyl group in the (4)
position. In addition, we screened their cytotoxic activities
against MCF-7 cancer cell line in vitro as potent anti-breast
cancer agents. Some of these compounds showed promis-
ing cytotoxic effect (Fig. 1).
K. A. M. Abouzid (&)
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Ain Shams University, Cairo, 11566, Egypt
e-mail: abouzid@yahoo.com
N. A. Khalil ꢀ E. M. Ahmed
Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo, Egypt
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Med Chem Res (2012) 21:3288–3293
3289
Fig. 1 Examples of 1,4-
R
Cl
disubstituted phthalazine lead
compounds with anti-cancer
activity, compounds I, II, and
III, and the synthesized target
compounds 3a–i and 4a–c
HN
HN
N
N
F
N
N
F
S
I
N
R= F or Cl
II
Vatalanib (PTK-787)
R
O
N
NHR2
XRR1
H
N
N
N
N
N
N
N
N
Br
Br
N
III
3a-k
4a-c
Results and discussion
d 2.49–3.63 ppm and d 3.32–3.68 ppm. Compound 3f
showed in its HNMR spectrum a sharp singlet signal due
1
1
to four equivalent benzylic protons at 3.66 ppm. HNMR
Chemistry
spectra of 4b exhibited two triplets of two CH2 protons of
thiazoline at 3.62 and 4.59 ppm. All the other aromatic and
aliphatic protons were observed at the expected regions.
The synthetic pathways leading to the 1,4-disubstituted-
phthalazine derivatives 3a–i and 4a–c are outlined in
Scheme 1. 4-(4-Bromophenyl)phthalazinone (1) was syn-
thesized by cyclization of 2-(4-bromobenzoyl)benzoic acid
with hydrazine hydrate according to a reported method
Yamaguchi et al., 1993). Compound 1 was treated with
phosphorus oxychloride to give 1-chlorophthalazine
derivative 2 (Colotta et al., 1994). Reacting 2 with
N-substituted-piperazines, secondary amines or 2-mercap-
tobenzothiazole in n-butanol afforded 3a–i. Furthermore,
reaction of 1-chlorophthalazine derivative 2 with different
primary amines in n-butanol, yielded 4a–c. The structures
of all the newly synthesized compounds were elucidated
using IR, 1HNMR, MS, and element analyses. 1HNMR
spectra of the compounds 3a–e displayed two characteristic
signals corresponding to piperazine protons in the range of
Biological evaluation
In vitro antitumor activity for compounds 3a–i and 4a–
c was determined by measurement of their cytotoxic
properties against MCF-7 (human breast adenocarcinoma
cell line) by SRB assay using doxorubicin as a standard.
The results were expressed as IC50 and summarized in
Table 1. The IC50 values are the average of three inde-
pendent experiments.
As shown in Table 1 and Fig. 2, most of the prepared
compounds showed good-to-excellent cytotoxic activities
against MCF-7 cancer cell line. In general, the cytotoxic
activities of compounds 4a–c were more active than
123

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Med Chem Res (2012) 21:3288–3293
XRR1
Scheme 1 Synthesis of 1-(4-
bromophenyl)phthalazine-4-
amine derivatives
Cl
O
N
POCl3/ reflux
XHRR1
N
N
NH
N
N
Br
Br
Br
3
2
1
N
CH3
N
N
N
NH2R2R3
N
N
H3CO
N
NHR2
N
OCH3
N
N
C2H5O
N
N
CH2
N
NHR2
CH2
Cl
Br
HN
N
O
4
Cl
S
N
N
N
H
Cl
HN
S
N
S
N
3a–i series. Six of the tested compounds 3a, 3g, 3i, and
4a–c demonstrated higher cytotoxic activity than the
reference drug doxorubicin. Preliminary structure–activity
relationship (SAR) showed that among the substituted-
piperazine derivatives, 3a–e, N-methylpiperazine, 3a,
exhibited the most potent antiproliferative activity
(IC50 = 1.5 lM). On the other hand, the unsubstituted
and ortho-substituted N-phenylpiperazine derivatives
showed more potent effect than the para–substituted
derivative. On the other hand, replacement of piperazine
with morpholine or piperidine reduced the potency
drastically. Furthermore, the cytotoxicity of the
S-substituted compound 3i was found to be twice more
potent than the N,N-dibenzyl derivative 3f. Moving our
attention back to the compounds, 4a–c, the pharmaco-
logical results indicated that the phthalazine derivatives
that contain NH (compounds 4a–c) displayed excellent
cytotoxicity compared to phthalazines attached to tertiary
amino functions, (compounds 3a–h).
Conclusion
In summary, we have synthesized a series of 1,4-dis-
ubstitutedphthalazine derivatives, and evaluated their
cytotoxic activities against MCF-7 (human breast adeno-
carcinoma cell line). Most of the prepared compounds
displayed good to excellent cytotoxic activity, in particular,
six compounds (3a, 3g, 3i, and 4a–c) with IC50 values
ranging from 1.4 to 2.3 lM. From the structure–activity
relationships (SARs), we may conclude that the introduc-
tion of the primary amines to the 4-substitutedphthalazine
scaffold plays an important role in enhancing antitumor
activities of these compounds.
Experimental
Melting points were determined on Griffin apparatus and
the values given are uncorrected. IR spectra were
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Med Chem Res (2012) 21:3288–3293
3291
1-(4-Substitutedpiperazin-1-yl) 4-(4-
Table 1 In vitro cytotoxic activity of the test compounds against
human breast cancer cell line (MCF-7)
methoxyphenyl)phthalazines (3a–i), (4a–c)
Compound
IC50 (lM)a,b
To a solution of 1-chloro-4-(4-bromophenyl)phthalazine
(2) (0.318 gm, 0.001 mol) in n-butanol (20 ml), the
appropriate N-substituted-piperazine or amine (0.001 mol)
was added, and the mixture was heated under reflux for
5 h. The reaction mixture was filtered while hot, the filtrate
was evaporated to half its volume, and cooled. The sepa-
rated solid was filtered, washed by sodium carbonate
solution, and crystallized from isopropanol.
Doxorubicin
2.97
1.5
3a
3b
3c
3d
3e
3f
9.7
8.6
17.5
4.77
4.8
3g
3h
3i
1.9
9.1
1-(4-Bromophenyl)-4-(4-methylpiperazin-1-yl)phthalazine
(3a)
2.3
4a
4b
4c
a
1.7
Yield 65%; mp 150–151°C; IR (t cm-1): 3074 (C–H
aromatic), 1605 (C=N); HNMR (DMSO–d6): 2.30 (s, 3H,
1.4
1
1.7
N–CH3), 2.63–2.65 (br m, 4H, piperazine), 3.45–3.48 (br
m, 4H, piperazine), 7.61 (dd, 2H, Ar–H), 7.70 (dd, 2H, Ar–
H), 7.79–8.40 (m, 4H, Ar–H); MS (EI) m/z (% rel. Int.): 99
(10.14), 283 (11.63), 285 (7.53), 382 (M ¯l?, 0.31), 384
(M ? 2¯l?, 0.30). Anal. Calcd for C19H19BrN4: C, 59.54;
H, 5.00; N, 14.62. Found: C, 59.30; H, 5.20; N, 14.55.
IC50 dose of the compound which inhibit tumor cell proliferation by
50%
b
Values are means of three experiments
determined on Shimadzu IR 435 spectrophotometer (KBr,
1
cm-1). H-NMR spectra were carried out using a Varian
Gemini 200 MHz Spectrophotometer and Varian Mercury-
300 (300 MHz) Spectrophotometer using TMS as internal
standard. Chemical shift values are recorded in ppm on d
scale, Microanalytical Center, Cairo University, Egypt.
Mass spectra were recorded on a GCMP-QP1000 EX Mass
spectrometer, Microanalytical Center, Cairo University,
Egypt. Elemental analyses were carried out at the Micro-
analytical Center, Cairo University, Egypt. Progress of the
reactions was monitored using TLC sheets precoated with
UV fluorescent silica gel Merck 60F 254 using acetone/
benzene (1: 9) and were visualized using UV lamp.
All chemicals were obtained from Aldrich, Fluka, or
Merck chemicals.
1-(4-Bromophenyl)-4-(4-phenylylpiperazin-1-
yl)phthalazine (3b)
Yield 70%; mp 169–170°C; IR (t cm-1): 3051 (C–H
aromatic), 1597 (C=N); HNMR (DMSO–d6): 3.40–3.45
1
(br m, 4H, piperazine), 3.60–3.62 (br m, 4H, piperazine),
6.79–8.28 (m, 13H, Ar–H); MS (EI) m/z (% rel. Int.): 161
(3.34), 162 (37.77), 283 (0.21), 285 (0.33), 311 (0.09), 444
(M¯l?, 0.00). Anal. Calcd for C24H21BrN4: C, 64.73; H,
4.75; N, 12.58. Found: C, 64.50; H, 4.80; N, 12.60.
1-(4-Bromophenyl)-4-(4-(2-methoxyphenyl)piperazin-1-
yl)phthalazine (3c)
4-(Bromophenyl)phthalazin-1(2H)-one
(Yamaguchi
Yield 72%; mp 110–111°C; IR (t cm-1): 3051 (C–H
aromatic), 1597 (C=N); HNMR (DMSO–d6): 3.30–3.32
(br m, 4H, piperazine), 3.36 (s, 3H, OCH3), 3.42–3.45 (br
et al., 1993) and 1-chloro-4-(4-methoxyphenyl) phthal-
azine (Colotta et al., 1994) were prepared according to
reported procedures.
1
Fig. 2 IC50 values of test
compounds 3a–i, 4a–c and
doxorubicin against MCF-7
IC50
20
18
16
14
12
10
8
IC50
6
4
2
0
Dox
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
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Med Chem Res (2012) 21:3288–3293
1-(4-Bromophenyl)-4-(piperidin-1-yl)phthalazine (3h)
Yield 70%; mp 152–153°C; IR (t cm-1): 3050 (C–H
m, 4H, piperazine), 7.60 (dd, 2H, Ar–H), 7.76 (dd, 2H, Ar–
H), 7.79–8.27 (m, 8H, Ar–H); MS (EI) m/z (% rel. Int.):
443 (9.79), 476 (M ? 2 l?, 24.39). Anal. Calcd for
¯
1
aromatic), 1605 (C=N); HNMR (DMSO–d6): 1.67–1.69
C25H23BrN4O: C, 63.16; H, 4.88; N, 11.79. Found: C,
63.30; H, 4.75; N, 11.66.
(m, 2H, CH2 piperidinyl), 1.80–1.85 (m, 4H, 2CH2 pipe-
ridinyl), 3.39–3.41 (m, 4H, 2CH2 piperidinyl), 7.62 (dd,
2H, Ar–H), 7.78 (dd, 2H, Ar–H), 7.87–8.13 (m, 4H, Ar–H);
MS (EI) m/z (% rel. Int.): 84 (100), 283 (11.13), 285
(31.96), 367 (M¯l?, 25.96), 369 (M ? 2¯l?, 24.37). Anal.
Calcd for C19H18BrN3: C, 61.97; H, 4.93; N, 11.41. Found:
C, 61.59; H, 4.80; N, 11.60.
1-(4-Bromophenyl)-4-(4-(4-methoxyphenyl)piperazin-1-
yl)phthalazine (3d)
Yield 75%; mp 183–184°C; IR (t cm-1): 3070 (C–H
aromatic), 1616 (C=N); HNMR (DMSO–d6): 3.60–3.63
1
(br m, 4H, piperazine), 3.66–3.68 (br m, 4H, piperazine),
3.71 (s, 3H, OCH3), 6.86 (dd, 2H, Ar–H), 6.99 (dd, 2H, Ar–
H), 7.64 (dd, 2H, Ar–H), 7.80 (dd, 2H, Ar–H), 7.93–8.25
(m, 4H, Ar–H); MS (EI) m/z (% rel. Int.): 163 (100), 191
(2.38), 283 (7.23), 2.85 (7.07), 311 (1.85), 313 (11.91), 474
(M l?, 6.37), 476 (M ? 2 l?, 6.18). Anal. Calcd for
C25H23BrN4O: C, 63.16; H, 4.88; N, 11.79. Found: C,
63.40; H, 4.65; N, 11.70.
1-(4-Bromophenyl)-4-[(benzothioazol-2-
yl)thio]phthalazine (3i)
Yield 74%; mp 115–116°C; IR (t cm-1): 3082 (C–H
aromatic), 1581 (C=N); 1HNMR (DMSO–d6): 7.57 (dd,
2H, Ar–H), 7.63 (dd, 2H, Ar–H), 7.76–8.27 (m, 8H, Ar–H).
MS (EI) m/z (% rel. Int.): 166 (8.1), 283 (2.63), 285 (3.15),
449 (M¯l?, 0.01), 451 (M ? 2¯l?, 0.01). Anal. Calcd for
C21H12BrN3S2: C, 56.00; H, 2.69; N, 9.33. Found: C,
56.10; H, 2.75; N, 9.50.
¯
¯
1-(4-Bromophenyl)-4-(4-(2-ethoxyphenyl)piperazin-1-
yl)phthalazine (3e)
Yield 60%; mp 112–113°C; IR (t cm-1): 3050 (C–H
aromatic), 1581 (C=N); HNMR (DMSO–d6): 2.49–2.52
1-(4-Bromophenyl)-4-(2,6-dichloroanilino-1-yl)
phthalazine (4a)
1
(br m, 4H, piperazine), 2.90 (t, 3H, OCH2CH3), 3.32–3.35
(br m, 4H, piperazine), 4.65 (q, 2H, OCH2CH3), 7.60 (dd,
2H, Ar–H), 7.76 (dd, 2H, Ar–H), 7.79-8.27 (m, 8H, Ar–H);
MS (EI) m/z (% rel. Int.): 176 (100), 177 (65.84), 312
Yield 75%; mp 125–126°C; IR (t cm-1): 3082 (C–H
aromatic), 1581 (C=N); 1HNMR (DMSO–d6): 7.56 (dd,
2H, Ar–H), 7.63 (dd, 2H, Ar–H), 7.93–8.26 (m, 7H, Ar–H);
MS (EI) m/z (% rel. Int.): 283 (2.58), 285 (3.08), 298
(6.67%), 300 (100), 443 (M ¯l?, 0.00). Anal. Calcd for
C20H12BrCl2N3: C, 53.96; H, 2.72; N, 9.44. Found: C,
53.70; H, 2.85; N, 9.50.
(39.82), 314 (35.57), 488(M ¯l?, 2.09), 490 (M ? 2 ¯l?
,
6.06). Anal. Calcd for C26H25BrN4O: C, 63.81; H, 5.15; N,
11.45. Found: C, 63.70; H, 5.50; N,11.50.
1-(4-Bromophenyl)-4-(N,N-dibenzylamino)phthalazine (3f)
Yield 64%; mp 100–101°C; IR (t cm-1): 3040 (C–H
aromatic), 1581 (C=N); HNMR (DMSO–d6): 3.66 (s, 4H,
1-(4-Bromophenyl)-4-(4,5-dihydrothiazol-2-
yl)aminophthalazine (4b)
1
1-(4-bromophenyl)-4-(4,5-dihydrothiazol-2-yl) aminoph-
thalazine Yield 70%; mp 114–115°C; IR (t cm-1), 3082
(C–H aromatic), 1581 (C=N), HNMR (DMSO–d6): 3.62
2CH2), 7.29–8.26 (m, 18H, Ar–H); MS (EI) m/z (% rel.
Int.): 283 (2.46), 285 (2.95), 300 (100), 302 (94.83), 479
¯
1
(M l?, 0.01), 481 (M ? 2 l?, 0.01). Anal. Calcd for
¯
(t, 2H, CH2 thiazoline), 4.59 (t, 2H, CH2 thiazoline), 7.59
(dd, 2H, Ar–H), 7.62 (dd, 2H, Ar–H), 7.92–8.25 (m, 4H,
Ar–H); MS (EI) m/z (% rel. Int.): 102 (8.22), 285 (19.75),
384 (M¯l?, 35.75), 386 (M ? 2¯l?, 30.47). Anal. Calcd for
C17H13BrN4S: C, 53.00; H, 3.40; N, 14.54. Found: C,
52.90; H, 3.75; N, 14.50.
C28H22BrN3: C, 70.00; H, 4.62; N, 8.75. Found: C, 69.90;
H, 4.50; N, 8.65.
1-(4-Bromophenyl)-4-(morpholin-1-yl)phthalazine (3g)
Yield 75%; mp 139–140°C; IR (t cm-1): 3050 (C–H
aromatic), 1605 (C=N); HNMR (DMSO–d6): 3.44–3.45
1
1-(4-Bromophenyl)4-(2-chloropyridin-3-
yl)aminophthalazine (4c)
(m, 4H, 2CH2–N), 3.88–3.90 (m, 4H, 2CH2–O), 7.62 (dd,
2H, Ar–H), 7.79 (dd, 2H, Ar–H), 7.89–8.22 (m, 4H, Ar–H);
MS (EI) m/z (% rel. Int.): 86 (100), 283 (19.77), 285
¯
Yield 73%; mp 138–139°C; IR (t cm-1): 3050 (C–H
aromatic), 1605 (C=N); 1HNMR (DMSO-d6): 7.67 (dd, 2H,
Ar–H), 7.82 (dd, 2H, Ar–H), 8.03-8.38 (m, 7H, Ar–H); MS
(23.46), 369 (M l?, 55.38), 371 (M ? 2 l?, 44.54). Anal.
¯
Calcd for C18H16BrN3O: C, 58.39; H, 4.36; N, 11.35.
Found: C, 58.50; H, 4.20; N, 11.50.
123

Med Chem Res (2012) 21:3288–3293
3293
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410 (M¯l?, 0.12), 412 (M ? 2¯l?, 0.13). Anal. Calcd for
C19H12BrClN4: C, 55.43; H, 2.94; N, 13.61. Found: C,
55.60; H, 2.75; N, 13.50.
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Emax model.
ꢀ
ꢁ
m
½Dꢃ
% Cell viability ¼ ð100 ꢁ RÞ ꢂ 1ꢁꢁꢁ
þ R
m
Kdm þ ½Dꢃ
where R is the residual unaffected fraction (the resistance
fraction), [D] is the drug concentration used, Kd is the drug
concentration that produces a 50% reduction of the maxi-
mum inhibition rate, and m is a Hill-type coefficient. IC50
was defined as the drug concentration required to reduce
fluorescence to 50% of that of the control (i.e., Kd = IC50
when R = 0 and Emax = 100 - R).
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Acknowledgment The authors are grateful to Dr. Ashraf Bahi Abd
El-Naim, Professor of Pharmacology, Faculty of Pharmacy, Ain
Shams University, for carrying out anti-proliferative activity.
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