Identification of new potent phthalazine derivatives with VEGFR-2 and EGFR kinase inhibitory activity

Article abstract of DOI:10.1016/j.ejmech.2016.07.049

Efforts to develop new antitumor agents are now directed towards multitarget therapies that are believed to have high potency and low tendency to resistance compared to conventional drugs. Herein, we highlighted the synthesis and antitumor activity of five series of phthalazine-based compounds featuring a variety of bioactive chemical fragments at position 1 of the phthalazine nucleus. The antitumor activity of the target compounds was performed against fourteen cancer cell lines where all compounds were active in the nanomolar level. In addition, the mechanism of action of the target compounds was investigated through an enzymatic inhibitory assay against VEGFR-2 and EGFR kinases, revealing potent and preferential activity toward VEGFR-2. Binding mode of the most active compounds was studied using docking experiment.

Full text of DOI:10.1016/j.ejmech.2016.07.049

Accepted Manuscript
Identification of new potent phthalazine derivatives with VEGFR-2 and EGFR kinase
inhibitory activity
Kamilia M. Amin, Flora F. Barsoum, Fadi M. Awadallah, Nehal E. Mohamed
PII:
S0223-5234(16)30610-9
10.1016/j.ejmech.2016.07.049
EJMECH 8767
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 April 2016
Revised Date: 18 July 2016
Accepted Date: 20 July 2016
Please cite this article as: K.M. Amin, F.F. Barsoum, F.M. Awadallah, N.E. Mohamed, Identification of
new potent phthalazine derivatives with VEGFR-2 and EGFR kinase inhibitory activity, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.049.
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ACCEPTED MANUSCRIPT
Identification of new potent phthalazine derivatives with VEGFR-2 and
EGFR kinase inhibitory activity
R
HN
SO2NHR
6a-d
HN
5a-f
H
N
NHR
HN
S
?
7a-f
N
N
R
R2
R1
NHN
N
O
S
N
8a-f
NH
10a-f
General structures of the target compounds
The aniline derivatives 6a-d and the allylidene compounds 10a-f were the most active on
the panel of fourteen cell lines and against VEGFR-2 and EGFR kinases.

ACCEPTED MANUSCRIPT
Identification of new potent phthalazine derivatives with VEGFR-2 and
EGFR kinase inhibitory activity
Kamilia M. Amin, Flora F. Barsoum, Fadi M. Awadallah,* Nehal E.Mohamed.
Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University, Kasr El-Eini street, 11562, Cairo,
Egypt
Abstract
Efforts to develop new antitumor agents are now directed towards multitarget therapies that are
believed to have high potency and low tendency to resistance compared to conventional drugs.
Herein, we highlighted the synthesis and antitumor activity of five series of phthalazine-based
compounds featuring a variety of bioactive chemical fragments at position 1 of the phthalazine
nucleus. The antitumor activity of the target compounds was performed against fourteen cancer
cell lines where all compounds were active in the nanomolar level. In addition, the mechanism of
action of the target compounds was investigated through an enzymatic inhibitory assay against
VEGFR-2 and EGFR kinases, revealing potent and preferential activity toward VEGFR-2.
Binding mode of the most active compounds was studied using docking experiment.
Keywords: Phthalazine, cytotoxicity, VEGFR-2, EGFR, docking
1. Introduction
* Corresponding author: E-mail address: fadi2mae@gmail.com

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Cancer is a major worldwide health problem. Although there has been a progress in the treatment
and prevention of cancer, this disease remains the second major cause of death in the world.
Cancer chemotherapy has entered a new era of molecularly targeted therapeutics, which are
highly selective and not associated with the serious toxicities of conventional cytotoxic drugs [1].
The current strategy in developing new antitumor agents depends on multiple mechanistic
approach to avoid limited and sporadic responses [2,3]. Therefore, the present study aimed to
design new antitumor agents targeting multiple signal transduction pathways, namely; VEGFR-2
and EGFR, hoping to develop compounds with broad spectrum of activity and efficient control
of cancer cell growth and invasion. VEGFR regulates both vascular proliferation and
permeability. It is unique among pro-angiogenic factors because of its specificity for vascular
endothelium, its potency and it is expressed in most tumor cells [4]. On the other hand, EGFR
plays a key role in numerous processes that affect tumor growth and progression, including
proliferation, differentiation, angiogenesis, inhibition of apoptosis and invasiveness. EGFR has
been shown to be overexpressed in breast, colon, ovarian and pancreatic cancer cells [5,6].
In the course of identifying various chemical fragments that could serve as a scaffold for new
antitumor agents, the literature revealed that phthalazine ring was a privileged bioactive core
found in many agents of biological interest as antitumor [7-10], antihypertensive [11], anti-
inflammatory [12], anticonvulsant [13] and antimicrobial agents [14,15]. Potent antitumor
activity was demonstrated by many phthalazine-based compounds such as the anilino
phthalazines Vatalanib I and its congener II (Figure 1) [7,8,16,17]. Also, it should be pointed
out that the phthalazine ring is the isostere of the quinazoline ring that was frequently used as a
scaffold for many EGFR and VEGFR kinase inhibitors [18-22]. Based on these findings, and in
an attempt to discover new broad spectrum antitumor agents targeting multiple oncogenic
pathways, the phthalazine ring was selected as a nucleus for the new compounds. Moreover,
many bioactive fragments contributing to the antitumor activity such as sulfonamide, substituted
phenyl, thiosemicarbazide, thiazolidinone and allylidene moieties were attached to position 1 of
the phthalazine ring through an amino or hydrazino spacer. The antitumor activity of the target
compounds was assessed against fourteen human cancer cell lines, in addition to the
investigation of their inhibitory activity against VEGFR-2 and EGFR kinases.

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2. Results and discussion
2.1. Chemistry
The key intermediate compounds 1-chlorophthalazine 3 and 1-hydrazinylphthalazine 4 were
prepared according to the reported procedures (Scheme 1) [23-25]. The sulfonamide derivatives
5a-f were synthesized from the reaction of 1-chlorophthalazine 3 with the appropriate
sulfonamide in ethanol in the presence of potassium carbonate. 1H NMR spectra revealed
additional doublets in the aromatic region attributed to aromatic protons of the para-substituted
sulfonamido moiety. Deuterium exchangeable peak was also observed for the SO2NH-proton.
On the other hand, the substituted anilino derivatives 6a-d were obtained via reaction of 3 with
the appropriate aniline in ethanol (Scheme 1). 1H NMR spectra demonstrated peaks
corresponding to the aromatic protons of the anilino group, in addition to characteristic protons
of individual compounds such as the triplet-quartet pattern of the ethyl group of 6d and which
was also revealed in its 13C NMR spectrum as two peaks at 19.0 and 52.2 ppm. Reaction of the
chlorophthalazine 3 with thiosemicarbazide afforded the unsubstituted thiosemicarbazide
derivative 7a, while the synthesis of the other compounds in the series 7b-f was achieved
through the reaction of 1-hydrazinylphthalazine 4 with the appropriate isothiocyanate in ethanol.
Structures of the target compounds were evidenced by 1H NMR which showed characteristic
peaks such as the aliphatic protons in 7b, 7c and 7f. 13C NMR supported the carbon skeleton of
the synthesized compounds as in compound 7f which displayed a peak at 55.1 and 175.0 ppm
attributed to the methoxy and –C=S groups, respectively. The thiosemicarbazide derivatives 7a-f
underwent cyclocondensation with ethyl bromoacetate in ethanol in the presence of anhydrous
sodium acetate to give the hydrazonothiazolidinone derivatives 8a-f. Evidence of
cyclocondensation was deduced from 1H NMR spectra which revealed the CH2 protons of the
thiazolidinone ring around 3.80 ppm. The CH2 carbon was also observed in the 13C NMR spectra
around 32.0 ppm. Finally, the allylidene derivatives 10a-f were obtained from the reaction of 1-
hydrazinylphthalazine 4 with the appropriate propenones 9a-f in absolute ethanol and in the
presence of catalytic amount of glacial acetic acid (Scheme 2). The propenones 9a-f were
prepared through a Claisen-Schmidt condensation of the precursor aldehydes and acetophenones
according to the reported procedures [26-28]. The structures of compounds 10a-f were deduced
from their 1H NMR spectra which revealed the –CH=CH- protons as two doublets at 6.89-6.91
and 8.07-8.22 ppm, with coupling constants of 16.5-16.8 Hz, suggesting trans coupling.

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2.2. Antitumor activity study.
2.2.1. Cytotoxic activity
All synthesized compounds were tested for their cytotoxic activity against a panel of cancer cell
lines using sulforhodamine-B-stain (SRB) assay method [29]. The human cell lines used in the
assay were colon cancer cell lines: HT29 and colo205, ovarian carcinoma cells: Skov3 and
OVCAR3, cervical cancer cells: HeLa, glioblastoma line: SF268 and SF2956, human breast
cancer cells: MCF-7, melanoma: MDA-MB-435, prostatic cancer: DU145, kidney carcinoma:
UO31, lung carcinoma: H460 and leukemia cell lines: K562 and HL60. Doxorubicin was used as
positive control. The average IC50 values were calculated from at least three independent
experiments. Standard error of the mean (SEM) was within ± 10% of the values (Table 1).
Results obtained for the tested compounds revealed that all compounds were very promising as
antitumor agents with IC50 in the nanomolar level on all tested cancer cell lines. The most
susceptible cell lines were the ovarian SKOV-3 and glioblastoma SF295.
The most active derivatives were the aniline 6a-d and the allylidene derivatives 10a-f, followed
by the thiazolidinone derivatives 8a-f, sulfonamide derivatives 5a-f and finally the
thiosemicarbazides 7a-f. This order of activity was consistent in all cell lines.
Within the sulfonamide series 5a-f, an exceptional high activity was observed with the
dimethylpyrimidinyl derivative 5f which was 4-10 times more potent than other members of the
series. The N1-unsubstituted sulfonamide derivative 5a came second in activity. Other members
of the series, 5b-5d, interchanged position regarding their activity on the different panels of cell
lines. The aniline derivatives 6a-d showed promising activity; where the choice of electron
withdrawing groups e.g.-F, -CN, -COOH and -COOC2H5, especially the nitrile 6b and
ethylcarboxylate 6d derivatives was befitting to high potency. The thiosemicarbazide derivatives
7a-f were of lower potency compared to other series on most cell lines; except the ovarian
SKOV-3 and glioblastoma SF295 to which they retained high potency. The nature of substitution
showed no consistent relation with the cytotoxic activity. Structural modification of the
thiosemicarbazides 7a-f through rigidification into the corresponding cyclic thiazolidinone
derivatives 8a-f augmented the cytotoxic activity on most cancer cell lines. Finally, the diphenyl
allylidene derivatives 10a-f were by far the most active among all other compounds. In this

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series, it seemed that the nature of substitution on the phenyl rings did not show consistent effect
on the activity.
2.2.2. VEGFR-2 and EGFR kinase inhibitory activity.
VEGFR-2 and EGFR kinase inhibitory activity of the tested compounds was performed by
ELISA [30]. Experiments were run in triplicate and SEM was within ± 10% of the IC50 values
(Table 2). Same wise, IC50 of all compounds were in the nanomolar level but better inhibitory
activity was obtained against VEGFR-2 than EGFR. From the obtained results, it was observed
that there was a high degree of consistency between the in vitro cytotoxic activity on the cancer
cell lines and the enzyme inhibition assay. In accordance with this observation, the aniline
compounds 6a-d and the allylidene derivatives 10a-f revealed the highest inhibitory activity
against VEGFR-2 and EGFR kinases (IC50 ranged from 0.41 to 6.90 nM) compared to
Delphindin which was used as a reference standard (IC50=5.09 and 6.27 nM, respectively).
Within the sulfonamide series, the most active compound was the dimethylpyrimidinyl
derivative 5f with an exceptional inhibitory activity in the subnanomolar level against VEGFR-2
(IC50= 0.77 nM). The the thiazolyl, the pyridinyl and the pyrimidinyl derivatives 5b, 5d and 5e
were almost equipotent. The least in activity in this series were the unsubstituted and the
isoxazolyl derivatives 5a and 5c. The aniline derivatives 6a-d were the highest in potency
against both enzymes, the most active of which were the nitrile and the ethylcarboxylate
derivatives 6b and 6d, respectively. The thiosemicarbazide derivatives were of lower potency
compared to other series. The most active compounds in this series were the N-butyl and N-p-
methoxyphenyl derivatives, 7c and 7f, respectively. Also, it could be noticed that structure
rigidification of compounds 7a-f into the cyclic derivatives 8a-f resulted in remarkable increase
in VEGFR-2 inhibitory activity (IC50=1.20-4.40 nM) when compared to delphindin (IC50=5.09).
The order of activity in this series, 8a-f, was retained upon this structure rigidification. Finally,
the diphenyl allylidene derivatives 10a-f revealed potent inhibitory activity especially against
VEGFR-2 in the subnanomolar level (IC50= 0.41-0.88 nM). The most active compound in this
series was 10f which featured a –OCH3 on one phenyl ring and a –Cl group on the other.
2.3. Docking study.

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Docking study was performed for compounds 6b and 10a which showed the highest
activity in the enzyme binding assays in order to reveal the binding modes of these compounds in
the ATP-binding site of EGFR TK and VEGFR-2 TK. In the ATP-binding site of EGFR TK, the
most important amino acid in the interaction was Met769 where compound 6b interacted via a
H-bond through N2 of the phthalazine ring with a bonding distance of 3.18Å; in addition to
arene-H interaction with Val702 and other hydrophobic interactions (Figure 2). Similar
interactions were observed for compound 10a between N3 of the phthalazine ring and Met769
with a bonding distance of 3.25Å. Moreover, two arene-H interactions were observed with
Lys721 and Gly772, in addition to other hydrophobic interactions (Figure 3). Regarding the
docking of these compounds in the ATP-binding site of VEGFR-2 TK, compound 6b showed a
H-bond with the key amino acid Cys919 through N3 of the phthalazine ring with a bonding
distance of 3.12Å; in addition to arene-H interactions with Leu840, Val848 and Gly922 and
other hydrophobic interactions (Figure 4). Likewise, compound 10a interacted with Cys919 via
H-bond through N3 of the phthalazine ring with a bonding distance of 2.78Å. Multiple arene-H
interactions were observed with Leu840, Phe918, Leu1035 and Phe1047; in addition to other
hydrophobic interactions (Figure 5). To summarize, this docking study revealed that the
phthalazine nucleus, especially N3, was crucial in interacting with the key amino acids in the
binding site of both enzymes, in addition to participation of the fused benzo ring of phthalazine
in some arene-H interactions. Moreover, the aromatic substitution located at position 1 of the
phthalazine ring extended the interactions to other amino acids either through arene-H or
hydrophobic bonding.
3. Conclusion
Synthesis of five series of 1-substitutedphthalazine derivatives as potent broad spectrum
antitumor agents was described. Cytotoxic activity against fourteen different human cancer cell
lines as well as enzymatic inhibitory assays against VEGFR-2 and EGFR kinases, was carried
out. All series showed potent and broad spectrum of cytotoxic activity upon exposure to different
cell lines, where their IC50 values were in the nanomolar level. The kinase inhibitory activity
revealed potent inhibition of VEGFR-2 and relatively lower activity against EGFR. There was a
high degree of consistency in view of the activity of the tested compounds against the cell lines
and the two tested kinases, which suggested that targeting these two enzymes played the major
role in the cytotoxic effect of the new compounds. The aniline 6a-d and the allylidene 10a-f

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derivatives were the highest in activity and the most active compounds were 6b, 6d, 10a, 10d
and 10f. Study of the binding mode of two of the most active compounds, 6b and 10a, into the
ATP-binding site of EGFR TK and VEGFR-2 TK was performed using docking experiment;
where the ring nitrogens, mostly N3 of the phthalazine nucleus, was found to be crucial in
interacting with the key amino acids in the binding site.
Finally, it can be concluded that the constructed ring system, 1-substituted phthalazine
analogues, could be the basis for developing highly effective, broad spectrum and multitargeting
antitumor agents.
4. Experimental
4.1. Chemistry
Melting points were uncorrected and were carried out by the open capillary tube method using
Stuart SMP3 version 5 apparatus. Infrared spectra were done on Schimadzu IR spectrometer and
1
expressed in wave number (υ max) cm-1.
The H-NMR spectra were measured in chloroform
(CDCl3) or dimethylsulphoxide (DMSO-d6) on
a
Varian Mercury VX-300 MHz
spectrophotometer or Bruker AVANCE ӀӀӀ Nano Bay 400 MHz FT-NMR spectrophotometer.
Chemical shifts were recorded in δ as parts per million (ppm) downfield from tetramethylsilane
(TMS) as internal standard. 13C-NMR spectra were recorded at 75.45 or 100 MHz. Mass spectra
were performed on Hewlette Packard (HP)-5988 mass spectrometer at 70 eV at the Micro
Analytical Center, Faculty of Science, Cairo University. Elemental microanalyses were
performed at the Microanalytical Center, Cairo University and Al-Azhar University. TLC was
monitored on FLUKA silica gel TLC aluminum cards (0.2 mm thickness) with fluorescent
indicator 254 nm using chloroform/methanol (9:1) as eluent to follow the course of reactions and
to check the purity of products and visualized using UV lamp (Spectroline Model CM 10, USA).
Compounds 3, 4 [23-25], 9a, 9b, 9f [26], 9c, 9e [27], 9d [28] were prepared according to the
reported procedures.
4.1.1.. General procedure for the preparation of compounds (5a-f):
A mixture of 1-chlorophthalazine 3 (1 g, 6 mmol), the appropriate benzenesulfonamide (6
mmol) and anhydrous potassium carbonate (12 mmol) in ethanol (10 ml) was heated under reflux
for 24 h. The mixture was poured onto ice-cooled water and neutralized with concentrated

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hydrochloric acid. The separated solid was filtered off and crystallized from the appropriate
solvent.
4.1.1.1. 4-(Phthalazin-1-ylamino)benzenesulfonamide (5a):
M.p. 265-267 °C, yield: 79%. IR νmax cm-1: 3388, 3263 (NH2), 3062 (aromatic CH), 1325, 1152
(SO2). 1H NMR (DMSO-d6): 300 MHz, δ = 6.11 (d, 2H, J=8.7 Hz, CH-2+CH-6 phenyl),
7.15 (d, 2H, J=8.7 Hz, CH-3+CH-5 phenyl), 7.23 (s, 2H, SO2NH2, exchanged by D2O), 7.49 (t,
1H, J= 7.8 Hz, CH-6 phthalazine), 8.06 (t, 1H, J= 7.5 Hz, CH-7 phthalazine), 8.26(d, 1H, J= 7.8
Hz, CH-5 phthalazine), 8.69 (d, 1H, J=7.5 Hz, CH-8 phthalazine), 9.08 (s, 1H, CH-4
13
phthalazine), 9.55 (s, 1H, NH, exchanged by D2O). C-NMR (75 MHz) δ=109.4, 118.3, 119.5,
122.5, 123.0, 126.6, 129.1, 132.2, 134.6, 139.1 (aromatic Cs), 142.8 (C-4 phthalazine), 148.3 (C-
1 phenyl), 168.1 (C-1 phthalazine). MS m/z: 300 [M+] . Anal. Calcd. for C14H12N4O2S (300.34):
C, 55.99; H, 4.03; N, 18.66. Found: C, 56.12; H, 4.35; N, 18.89.
4.1.1.2. 4-(Phthalazin-1-ylamino)-N-(thiazol-2-yl)benzenesulfonamide (5b):
M.p. 278-280°C, yield: 88%. IR νmax cm-1: 3363 (NH), 3097 (aromatic CH), 1276,1141 (SO2).
1H NMR (DMSO-d6): 400 MHz, δ = 6.80 (d, 1H, J=4.5 Hz, CH-4 thiazole), 7.23 (d, 1H, J=4.5
Hz, CH-5 thiazole), 7.78 (d, 2H, J=8.6 Hz, CH-2+CH-6 phenyl), 7.98-8.12 (m, 5H, Ar-H), 8.58
(d, 1H, J=7.3 Hz, CH-8 phthalazine), 9.22 (s, 1H, CH-4 phthalazine), 9.52 (s, 1H, NH,
exchanged by D2O), 12.50 (s, 1H, SO2NH, exchanged by D2O). MS m/z: 383 [M+]. Anal. Calcd.
for C17H13N5O2S2 (383.44): C, 53.25; H, 3.42; N, 18.26. Found: C, 53.32; H, 3.48; N, 18.39.
4.1.1.3. N-(5-Methylisoxazol-3-yl)-4-(phthalazin-1-ylamino)benzenesulfonamide (5c):
M.p. 240-243°C, yield: 85%. IR νmax cm-1: 3388(NH), 3070 (aromatic CH), 2924 (Aliphatic CH),
1
1319,1161 (SO2). H NMR (DMSO-d6): 300 MHz, δ = 2.30 (s, 3H, CH3), 6.16 (s, 1H, CH
isoxazole), 7.86-8.17 (m, 7H, ArH+NH), 8.23 (d, 1H, J = 7.8 Hz, CH-5 phthalazine), 8.8 (d, 1H,
J=7.5 Hz, CH-8 phthalazine), 9.44 (s, 1H, CH-4 phthalazine),11.15 (s, 1H, SO2NH, exchanged
by D2O). 13C-NMR (75 MHz) δ=12.0 (CH3), 95.3 (C-4 isoxazole), 119.9, 121.0, 123.3, 127.8,
128.1, 128.7, 131.2, 133.7, 134.1 (aromatic Cs), 143.9 (C-4 phthalazine), 148.6 (C-1 phenyl),
152.7 (C-3 isoxazole), 157.5 (C-1 phthalazine), 170.1(C-5 isoxazole). MS m/z: 381 [M+]. Anal.

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Calcd. for C18H15N5O3S (381.41): C, 56.68; H, 3.96; N, 18.36. Found: C, 56.73; H, 3.91; N,
18.57.
4.1.1.4. 4-(Phthalazin-1-ylamino)-N-(pyridin-2-yl)benzenesulfonamide (5d):
M.p. 190-192°C, yield: 75%. IR νmax cm-1: 3414 (NH), 3070 (aromatic CH), 1334, 1149 (SO2).
1H NMR (DMSO-d6): 300 MHz, δ =6.53 (d, 2H, J=8.5 Hz, J=9 Hz, CH-2+CH-6 phenyl), 6.85
(t, 1H, J=7.5 Hz, CH-3 pyridine), 7.05 (d, 1H, J=7.8 Hz, CH-5 pyridine), 7.5 (d, 2H, J=8.7 Hz,
CH-3+CH-5 phenyl), 7.64 (t, 1H, J=7.8 Hz, CH-4 pyridine), 7.81 (t, 1H, J=7.8 Hz , CH-6
phthalazine), 7.90-8.15 (m, 2H, CH-7 phthalazine+CH-6 pyridine), 8.24 (d, 1H, J=7.6 Hz, CH-5
phthalazine), 8.60 (d, 1H, J=7.5 Hz, CH-8 phthalazine), 9.09 (s, 1H, CH-4 phthalazine), 10.04 (s,
2H, NH+SO2NH, exchanged by D2O). MS m/z: 377 [M+]. Anal. Calcd. for C19H15N5O2S
(377.42): C, 60.47; H, 4.01; N, 18.56. Found: C, 60.54; H, 4.11; N, 18.82.
4.1.1.5. 4-(Phthalazin-1-ylamino)-N-(pyrimidin-2-yl)benzenesulfonamide (5e):
M.p. 220-223°C, yield: 73%. IR νmax cm-1: 3356 (NH), 3035 (aromatic CH), 1327,1153 (SO2).
1H NMR (DMSO-d6): 400 MHz, δ =6.55 (d, 2H, J=8.6 Hz, CH-2+CH-6 phenyl), 7.00 (t, 1H,
J=4.7 Hz, CH-5 pyrimidine), 7.61 (d, 2H, J=8.5 Hz, CH-3+CH-5 phenyl), 8.02-8.36 (m, 2H,
CH-6+CH-7 phthalazine), 8.44 (d, 1H, J=7.5 Hz, CH-5 phthalazine), 8.47 (d, 2H, J=4.7 Hz, CH-
4+CH-6 pyrimidine), 8.55 (d, 1H, J=7.3 Hz, CH-8 phthalazine), 8.94 (s, 1H, CH-4 phthalazine),
11.27 (s, 2H, NH+SO2NH, exchanged by D2O). MS m/z: 378 [M+]. Anal. Calcd. for
C18H14N6O2S (378.41): C, 57.13; H, 3.73; N, 22.21. Found: C, 57.21; H, 3.82; N, 22.54.
4.1.1.6. N-(4,6-Dimethylpyrimidin-2-yl)-4-(phthalazin-1-ylamino)benzenesulfonamide (5f):
M.p. 256-260°C, yield: 75%. IR νmax cm-1: 3367 (NH), 3066 (aromatic CH), 2920 (aliphatic CH),
1315,1153 (SO2). 1H NMR (DMSO-d6): 300 MHz, δ =2.25 (s, 6H, 2CH3), 6.36 (d, 2H, J=8.1 Hz,
CH-2+CH-6 phenyl), 6.86 (s, 1H, CH-5 pyrimidine), 7.17 (d, 2H, J=8.7 Hz, CH-3+CH-5
phenyl), 7.61-8.12 (m, 2H, CH-6+CH-7 phthalazine), 8.19 (d, 1H, J=7.5 Hz, CH-5 phthalazine),
8.57 (d, 1H, J=7.5 Hz, CH-8 phthalazine), 9.03 (s, 1H, CH-4 phthalazine), 10.90 (br s, 2H,
NH+SO2NH, exchanged by D2O). 13C-NMR (75 MHz) δ=23.0 (2CH3), 111.8, 113.6, 122.2,
124.9, 126.5, 127.2, 127.7, 128.9, 130.2, 130.6, 131.0, 132.1, 134.2 (aromatic Cs), 142.7 (C-4
phthalazine), 144.5 (C-1 phenyl), 152.8 (C-4, C-6 pyrimidine), 156.6 (C-1 phthalazine), 167.2

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(C-2 pyrimidine). MS m/z: 406 [M+]. Anal. Calcd. for C20H18N6O2S (406.46): C, 59.10; H, 4.46;
N, 20.68. Found: C, 59.17; H, 4.52; N, 20.83.
4.1.2. General procedure for the preparation of compounds (6a-d):
An equimolar mixture of 1-chlorophthalazine 3 and the appropriate 4-substituted aniline (6
mmol) in ethanol (10 ml) was heated under reflux for 8 h. The mixture was left to cool and the
separated solid was filtered off and crystallized from the ethanol.
4.1.2.1. N-(4-Fluorophenyl)phthalazin-1-amine (6a):
1
M.p. 246-249°C, yield: 80%. IR νmax cm-1: 3446 (NH), 3068 (aromatic CH). H NMR (DMSO-
d6): 300 MHz, δ= 7.74 (d, 2H, J=8.9 Hz, CH-2+CH-6 phenyl), 7.92 (t, 2H, J=8.7 Hz CH-3+CH-
5 phenyl), 8.09 (t, 1H, J=7.4 Hz, CH-6 phthalazine), 8.21 (d, 1H, J=7.3 Hz, CH-7 phthalazine),
8.30 (d, 1H, J=7.3 Hz, CH-5 phthalazine), 8.56 (d, 1H, J=7.5 Hz, CH-8 phthalazine), 8.71 (s, 1H,
CH-4 phthalazine), 9.06 (s, 1H, NH, exchanged by D2O). 13C-NMR (DMSO-d6, 75 MHz)
δ=115.5, 123.0, 126.6, 127.8, 128.9, 130.3, 131.1, 134.3, 135.1 (aromatic Cs), 142.7 (C-4
phthalazine), 148.3 (C-1 phenyl), 162.1 (C-1 phthalazine). MS m/z: 239 [M+]. Anal. Calcd. for
C14H10FN3 (239.25): C, 70.28; H, 4.21; N, 17.56. Found: C, 70.41; H, 4.27; N, 17.68.
4.1.2.2. 4-(Phthalazin-1-ylamino)benzonitrile (6b):
1
M.p. 248-252°C, yield: 65%. IR νmax cm-1: 3263 (NH), 3041 (aromatic CH), 2220 (CN). H
NMR (DMSO-d6): 300 MHz, δ= 7.78 (d, 2H, J=9 Hz, CH-2+CH-6 phenyl), 7.98-8.11 (m, 3H,
CH-5, CH-6 and CH-7 phthalazine), 8.17 (d, 2H, J=9 Hz, CH-3+CH-5 phenyl), 8.58 (d, 1H,
J=7.6 Hz, CH-8 phthalazine), 9.26 (s, 1H, CH-4 phthalazine), 9.62 (s, 1H, NH exchanged by
D2O). MS m/z: 246 [M+]. Anal. Calcd. for C15H10N4 (246.27): C, 73.16; H, 4.09; N, 22.75.
Found: C, 73.42; H, 4.15; N, 22.94.
4.1.2.3. 4-(Phthalazin-1-ylamino)benzoic acid (6c):
M.p. 253-256°C, yield: 90%. IR νmax cm-1: 3444 (NH), 3363 (OH carboxylic), 3059 (aromatic
1
CH), 1685 (C=O). H NMR (DMSO-d6): 400 MHz, δ= 7.89 (d, 2H, J=8.7 Hz, CH-2+CH-6
phenyl), 7.90 (d, 2H, J=8.7 Hz, CH-3+CH-5 phenyl), 8.17 (t, 1H, J=7.3 Hz, CH-6 phthalazine),
8.25 (t, 1H, J=7.3 Hz, CH-7 phthalazine), 8.33 (d, 1H, J=7.5 Hz, CH-5 phthalazine), 9.00 (d, 1H,
J=7.6 Hz, CH-8 phthalazine), 9.54 (s, 1H, CH-4 phthalazine), 10.90 (s, 1H, NH exchanged with

ACCEPTED MANUSCRIPT
D2O), 12.80 (s, 1H, OH exchanged by D2O). MS m/z: 265 [M+]. Anal. Calcd. for C15H11N3O2
(265.27): C, 67.92; H, 4.18; N, 15.84. Found: C, 68.11; H, 4.24; N, 16.12.
4.1.2.4. Ethyl 4-(phthalazin-1-ylamino)benzoate (6d):
M.p. 288-290°C, yield: 67%. IR νmax cm-1: 3311 (NH), 3059 (aromatic CH), 2987 (aliphatic CH),
1693 (C=O). 1H NMR (DMSO-d6): 400 MHz, δ= 1.32 (t, 3H, J=7.1 Hz, CH3), 4.30 (q, 2H, J=7.1
Hz, CH2), 7.97 (d, 2H, J=8.6 Hz, CH-2+CH-6 phenyl), 8.03 (d, 2H, J=8.6 Hz, CH-3+CH-5
phenyl), 8.22 (t, 1H, J=7.5 Hz, CH-6 phthalazine), 8.28 (t, 1H, J=7.4 Hz, CH-7 phthalazine),
8.37 (d, 1H, J=7.5 Hz, CH-5 phthalazine), 9.04 (d, 1H, J=7.5 Hz, CH-8 phthalazine), 9.61 (s, 1H,
CH-4 phthalazine), 11.0 (s, 1H, NH exchanged by D2O). 13C-NMR (100 MHz) δ=19.0 (CH3),
52.23 (CH2), 113.1, 122.8, 123.7, 127.2, 129.2, 130.5, 131.3, 131.7, 132.9, 134.2, 135.5
(aromatic Cs), 142.2 (C-4 phthalazine), 148.0 (C-1 phenyl), 166.1 (C-1 phthalazine), 166.5
(C=O). MS m/z: 293 [M+]. Anal. Calcd. for C17H15N3O2 (293.33): C, 69.61; H, 5.15; N, 14.33.
Found: C, 69.74; H, 5.19; N, 14.45.
4.1.3. 2-(Phthalazin-1-yl)hydrazine-1-carbothioamide (7a):
An equimolar mixture of 1-chlorophthalazine 3 and thiosemicarbazide (6 mmol) in ethanol (10
ml) was heated under reflux for 8 h. The mixture was left to cool, the separated solid was filtered
off and crystallized from ethanol.
1
M.p. 225-228°C, yield: 60%. IR νmax cm-1: 3356 (NH), 3047 (aromatic CH), 1234 (C=S). H
NMR (DMSO-d6): 300 MHz, δ= 7.25 (s, 1H, NH phthalazine, exchanged by D2O), 8.00-8.12 (m,
2H, CH-6+CH-7 phthalazine), 8.18 (d, 1H, J=7.2 Hz, CH-5 phthalazine), 8.34 (d, 1H, J=7.8 Hz,
CH-8 phthalazine), 8.51 (s, 3H, NH-C=S-NH2, exchanged by D2O), 8.92 (s, 1H, CH-4
phthalazine). MS m/z: 219 [M+]. Anal. Calcd. for C9H9N5S (219.27): C, 49.30; H, 4.14; N,
31.94. Found: C, 49.41;H, 4.19;N, 32.25.
4.1.4. General procedure for the preparation of compounds (7b-f):
A mixture of equimolar amounts of 1-hydrazinylphthalazine 4 and the appropriate isothiocyanate
(5 mmol) in absolute ethanol (15 ml) was heated under reflux for 3 h. The reaction mixture was
filtered off while hot, the residue was separated and crystallized from methanol.
4.1.4.1. N-Ethyl-2-(phthalazin-1-yl)hydrazine-1-carbothioamide (7b):

ACCEPTED MANUSCRIPT
M.p. 288-290°C, yield: 85%. IR νmax cm-1: 3311 (NH), 3024 (aromatic CH), 2980 (Aliphatic
CH), 1136 (C=S). 1H NMR (CDCl3): 300 MHz, δ= 1.31 (t, 3H, J=7.2 Hz, CH2CH3), 3.78 (q, 2H,
J=6.9 Hz, CH2CH3), 7.27 (s, 1H, NH-C=S, exchanged by D2O), 7.47-7.64 (m, 3H, CH-5+CH-
6+CH-7 phthalazine), 7.85 (s, 1H, CH-4 phthalazine), 8.20 (d, 1H, J=7.8 Hz, CH-8 phthalazine),
9.49 (s, 1H, NH phthalazine, exchanged by D2O), 10.7 (s, 1H, NH-C=S, exchanged by D2O). MS
m/z: 247 [M+]. Anal. Calcd. for C11H13N5S (247.32): C, 53.42; H, 5.30; N, 28.32. Found: C,
53.63; H, 5.38; N, 28.51.
4.1.4.2. N-Butyl-2-(phthalazin-1-yl)hydrazine-1-carbothioamide (7c):
M.p. 168-173°C, yield: 87%. IR νmax cm-1: 3331 (NH), 3028 (aromatic CH), 2954 (Aliphatic
1
CH), 1138 (C=S). H NMR (CDCl3): 300 MHz, δ= 0.91 (t, 3H, J=7.5 Hz, CH2CH2CH2CH3),
1.34 (m, 2H, J=7.5 Hz, CH2CH2CH2CH3), 1.61 (m, 2H, J=7.8 Hz, CH2CH2 CH2CH3), 3.71 (t,
2H, J=7.2 Hz, CH2CH2CH2CH3), 7.26 (s, 1H, NH-C=S, exchanged by D2O), 7.38 (t, 2H, J=7.3
Hz, CH-6 phthalazine), 7.54-7.71 (m, 2H, CH-5+CH-7 phthalazine), 7.78 (s, 1H, CH-4
phthalazine), 8.07 (d, 1H, J=7.5 Hz, CH-8 phthalazine), 10.04 (s, 1H, NH phthalazine,
exchanged by D2O,), 11.07 (s, 1H, NH-C=S, exchanged by D2O). 13C-NMR (75 MHz) δ=14.0
(CH2CH2CH2CH3), 20.3 (CH2CH2CH2CH3), 31.7 (CH2CH2CH2CH3), 44.3 (CH2CH2CH2CH3),
123.7, 126.4, 126.7, 131.7, 131.9, 138.5, 139.2 (aromatic Cs), 176.2 (C=S). Anal. Calcd. for
C15H17N5S (275.37): C, 56.70; H, 6.22; N, 25.43. Found: C, 56.88; H, 6.41; N, 25.62.
4.1.4.3. N-Phenyl-2-(phthalazin-1-yl)hydrazine-1-carbothioamide (7d):
1
M.p. 284-286°C, yield: 70%. IR νmax cm-1: 3329 (NH), 3028 (aromatic CH), 1138 (C=S). H
NMR (DMSO-d6): 300 MHz, δ= 6.60 (d, 2H, J=8.5 Hz, Ar-H phenyl), 7.04 (t, 3H, J=8.5 Hz,
Ar-H phenyl), 8.02 (m, 3H, CH-6+CH-7 phthalazine+NH phthalazine, exchanged by D2O), 8.22
(d, 1H, J=7.2 Hz, CH-5 phthalazine), 8.37 (d, 1H, J=7.2 Hz, CH-8 phthalazine), 9.06 (s, 1H, CH-
4 phthalazine), 9.90 (s, 2H, 2NH-C=S exchanged by D2O). MS m/z: 295 [M+]. Anal. Calcd. for
C15H13N5S (295.36): C, 61.00; H, 4.44; N, 23.71. Found: C, 61.17; H, 4.48; N, 23.79.
4.1.4.4. N-(4-Chlorophenyl)-2-(phthalazin-1-yl)hydrazine-1-carbothioamide (7e):
1
M.p. above 300°C, yield: 90%. IR νmax cm-1: 3329 (NH), 3167 (aromatic CH), 1138 (C=S). H
NMR (DMSO-d6): 300 MHz, δ= 5.10 (s, 2H, NH- NH-C=S, exchanged by D2O), 6.53 (d, 2H,
J=9 Hz,CH-3+CH-5 phenyl), 6.99 (d, 2H, J=8.7 Hz, CH-2+CH-6 phenyl), 7.92 (m, 2H, CH-

ACCEPTED MANUSCRIPT
6+CH-7 phthalazine), 8.14 (d, 1H, J=7.6 Hz, CH-5 phthalazine), 8.29 (d, 1H, J=7.9 Hz, CH-8
phthalazine), 8.98 (s, 1H, CH-4 phthalazine), 14.6 (s, 1H, NH-phenyl, exchanged by D2O). Anal.
Calcd. for C15H12ClN5S (329.81): C, 54.63; H, 3.67; N, 10.75. Found: C, 54.81; H, 3.71; N,
10.89.
4.1.4.5. N-(4-Methoxyphenyl)-2-(phthalazin-1-yl)hydrazine-1-carbothioamide (7f):
M.p. above 300°C, yield: 84%. IR νmax cm-1: 3483 (NH), 3055 (aromatic CH), 2966 (aliphatic
CH), 1136 (C=S). 1H NMR (DMSO-d6): 300 MHz, δ= 3.76 (s, 3H, OCH3), 6.9 (d, 2H, J=8.3 Hz,
CH-3+CH-5 phenyl), 7.42 (d, 2H, J=8.5 Hz, CH-2+CH-6 phenyl), 7.6-7.96 (m, 4H, CH-5+CH-
6+CH-7 phthalazine+NH phthalazine), 8.58 (d,1H, J=7.7 Hz, CH-8 phthalazine), 9.80 (s, 1H,
CH-4 phthalazine), 10.25 (s, 1H, NH-C=S, exchanged by D2O), 11.63 (s, 1H, NH- phenyl,
exchanged by D2O). 13C-NMR (75 MHz) δ=55.1 (OCH3), 113.1, 124.4, 126.0, 126.2, 126.3,
127.2, 131.3, 131.6, 132.2, 135.4, 137.6 (aromatic Cs), 156.6 (C-1 phthalazine), 175.0 (C=S).
Anal. Calcd. for C16H15N5OS (325.39): C, 59.06; H, 4.65; N, 21.52. Found: C, 59.18; H, 4.73; N,
21.78.
4.1.5. General procedure for the preparation of compounds (8a-f):
A mixture of equimolar amounts of 7a-f and ethyl bromoacetate (5 mmol) in absolute ethanol
(15 ml) containing anhydrous sodium acetate (10 mmol) was heated under reflux for 2 h. The
reaction mixture was cooled and the separated crystals were filtered off and recrystallized from
benzene.
4.1.5.1. 2-(2-(Phthalazin-1-yl)hydrazono)thiazolidin-4-one (8a):
M.p. 272-275°C, yield: 64%. IR νmax cm-1: 3408 (NH), 3064 (aromatic CH), 2922 (aliphatic CH),
1718(C=O). 1H NMR (DMSO-d6): 300 MHz, δ= 3.85 (s, 2H, CH2thiazolidine), 5.05 (s, 1H, NH,
exchanged by D2O), 7.93 (t, 1H, J=7.8 Hz, CH-6 phthalazine), 8.07 (m,1H, J=7.8 Hz, CH-7
phthalazine), 8.21 (d, 1H, J=7.8 Hz, CH-5 phthalazine), 8.46 (s, 1H, CH-4 phthalazine), 8.57 (d,
1H, J=8.7, CH-8 phthalazine), 11.81 (s, 1H, NH thiazolidine, exchanged by D2O). MS m/z: 259
[M+]. Anal. Calcd. for C11H9N5OS (259.29): C, 50.96; H, 3.50; N, 27.01. Found: C, 51.08; H,
3.47; N, 27.23.
4.1.5.2. 3-Ethyl-2-(2-(phthalazin-1-yl)hydrazono)thiazolidin-4-one(8b):

ACCEPTED MANUSCRIPT
M.p. 218-220°C, yield: 80%. IR νmax cm-1: 3300 (NH), 3050 (aromatic CH), 2933 (aliphatic CH),
1708 (C=O). 1H NMR (CDCl3): 300 MHz, δ=1.30 (t, 3H, J=6.9 Hz, CH2CH3), 3.81 (s, 2H, CH2
thiazolidine), 3.89 (q, 2H, J=6.9 Hz, CH2CH3), 7.27 (t, 1H, J=7.8 Hz, CH-6 phthalazine), 7.51 (t,
1H, J=7.9 Hz, CH-7 phthalazine), 7.67 (d, 1H, J=7.8 Hz, CH-5 phthalazine), 7.84 (d, 1H, J=7.7
Hz, CH-8 phthalazine), 8.44 (s, 1H, CH-4 phthalazine), 10.04 (s, 1H, NH, exchanged by D2O).
13C-NMR (100 MHz) δ=12.4 (CH2-CH3), 32.6 (C-5 thiazolidine), 38.4 (CH2-CH3), 124.0, 126.0,
126.7, 128.3, 131.7, 131.8 (aromatic Cs), 138.0 (C-4 phthalazine), 146.3 (C-2 thiazolidine),
156.5 (C-1 phthalazine), 171.7 (C=O).MS m/z: 287 [M+]. Anal. Calcd. for C13H13N5OS (287.34):
C, 54.34; H, 4.56; N, 24.37. Found: C, 54.47; H, 4.54; N, 24.41.
4.1.5.3. 3-Butyl-2-(2-(phthalazin-1-yl)hydrazono)thiazolidin-4-one (8c):
M.p. 188-192°C, yield: 86%. IR νmax cm-1: 3277 (NH), 3050 (aromatic CH), 2958 (aliphatic CH),
1
1701 (C=O). H NMR (CDCl3): 300 MHz, δ=0.93 (t, 3H, J=7.2 Hz, CH3), 1.32 (m, 2H, J=7.2
Hz, CH2CH3), 1.66 (m, 2H, J=7.8 Hz, CH2CH2CH3), 3.76 (s, 2H, CH2 thiazolidine), 3.81 (t, 2H,
J=7.5 Hz, CH2CH2CH2CH3), 7.41 (t,1H, J=7.6 Hz, CH-6 phthalazine), 7.46-7.60 (m, 2H, CH-
5+CH-7 phthalazine), 7.70 (s, 1H, CH-4 phthalazine), 8.26 (d, 1H, CH-8 phthalazine), 9.90 (s,
1H, NH, exchanged by D2O). 13C-NMR (75 MHz) δ=13.6 (CH2CH2CH2CH3), 20.0
(CH2CH2CH2CH3), 29.1 (CH2CH2CH2CH3), 32.3 (C-5 thiazolidine), 43.1 (CH2CH2CH2CH3),
123.9, 125.9, 126.6, 126.8, 131.3, 131.6 (aromatic Cs), 137.7 (C-4 phthalazine), 146.0 (C-2
thiazolidine), 156.7 (C-1 phthalazine), 171.8 (C=O). MS m/z: 315 [M+]. Anal. Calcd. for
C15H17N5OS (315.40): C, 57.12; H, 5.43; N, 22.21. Found: C, 57.25; H, 5.52; N, 22.42.
4.1.5.4. 3-Phenyl-2-(2-(phthalazin-1-yl)hydrazono)thiazolidin-4-one (8d):
M.p. 247-250°C, yield: 75%. IR νmax cm-1: 3275 (NH), 3055 (aromatic CH), 2922 (aliphatic CH),
1
1716 (C=O). H NMR (DMSO-d6): 300 MHz, δ=4.06 (s, 2H, CH2 thiazolidine), 7.44 (d, 2H,
J=7.5 Hz, CH-2+CH-6 phenyl), 7.50 (d, 2H, J=7.9 Hz, CH-3+CH-5 phenyl), 7.88-8.03 (m, 3H,
CH-4 phenyl+CH-6+CH-7 phthalazine), 8.14 (d, 1H, J=7.8 Hz,CH-5 phthalazine), 8.29 (d, 1H,
J=7.8 Hz, CH-8 phthalazine), 8.98 (s, 1H, CH-4 phthalazine), 10.34 (s, 1H, NH, exchanged by
D2O). MS m/z: 335 [M+]. Anal. Calcd. for C17H13N5OS (335.39): C, 60.88; H, 3.91; N, 20.88.
Found: C, 60.97; H, 3.98; N, 20.97.
4.1.5.5. 3-(4-Chlorophenyl)-2-(2-(phthalazin-1-yl)hydrazono)thiazolidin-4-one (8e):

ACCEPTED MANUSCRIPT
1
M.p. 260-263°C, yield: 82%. IR νmax cm-1: 3263 (NH), 3059 (aromatic CH), 1718 (C=O). H
NMR (CDCl3): 300 MHz, δ= 3.96 (s, 2H, CH2 thiazolidine), 7.26-7.36 (m, 3H, Ar-H), 7.37-7.46
(m, 2H, Ar-H), 7.61-7.64 (m, 2H, Ar-H), 7.73 (s, 1H, CH-4 phthalazine), 8.31 (d, 1H, J=7.8 Hz,
CH-8 phthalazine), 9.81 (s, 1H, NH, exchanged by D2O). MS m/z: 369 [M+]. Anal. Calcd. for
C17H12ClN5OS (369.83): C, 55.21; H, 3.27; N, 18.94. Found: C, 55.29; H, 3.24; N, 19.06.
4.1.5.6. 3-(4-Methoxyphenyl)-2-(2-(phthalazin-1-yl)hydrazono)thiazolidin-4-one (8f):
M.p. 263-266°C, yield: 85%. IR νmax cm-1: 3275 (NH), 3014 (aromatic CH), 2964 (aliphatic CH),
1
1712 (C=O). H NMR (DMSO-d6): 300 MHz, δ=3.81 (s, 3H, OCH3), 4.02 (s, 2H, CH2
thiazolidine), 7.03 (d, 2H, J=9 Hz, CH-3+CH-5 phenyl), 7.33 (d, 2H, J=8.7 Hz, CH-2+CH-6
phenyl), 7.65-7.72 (m, 3H, CH-5+CH-6+CH-7 phthalazine), 7.93 (s, 1H, CH-4 phthalazine),
8.12 (d, 1H, J=7.8 Hz, CH-8 phthalazine), 10.37 (s, 1H, NH, exchanged by D2O). MS m/z: 365
[M+]. Anal. Calcd. for C18H15N5O2S (365.41): C, 59.17; H, 4.14; N, 19.14. Found: C, 59.34; H,
4.21; N, 19.4.
4.1.6. General procedure for the preparation of compounds (10a-f):
A mixture of equimolar amounts of 1-hydrazinylphthalazine 4 and the appropriate chalcone 9a-f
(4 mmol) in absolute ethanol (10 ml) containing glacial acetic acid (2.5 ml) was heated under
reflux for 30 h. Upon cooling, the separated solid was filtered off and crystallized from
acetonitrile.
4.1.6.1. 1-(2-(1,3-Diphenylallylidene)hydrazinyl)phthalazine (10a):
M.p. 170-172°C, yield: 82%. IR νmax cm-1: 3394 (NH), 3049 (aromatic CH), 2940 (aliphatic CH).
1H NMR (CDCl3): 300 MHz, δ=6.94 (d, 1H, J=16.8 Hz, N=C-CH=CH), 7.27-7.42 (m, 5H, Ar-
H), 7.47-7.59 (m, 5H, Ar-H), 7.68-7.72 (m, 3H, Ar-H), 7.85 (s, 1H, CH-4 phthalazine), 8.22 (d,
1H, J=16.8 Hz, N=C-CH=CH), 8.53 (d, 1H, J=7.7 Hz, CH-8 phthalazine), 10.70 (s, 1H, NH,
exchanged by D2O). MS m/z: 350 [M+]. Anal. Calcd. for C23H18N4 (350.43): C, 78.83; H, 5.18;
N, 15.99. Found: C, 78.97; H, 5.27; N, 16.04.
4.1.6.2. 1-(2-(1-(4-Chlorophenyl)-3-phenylallylidene)hydrazinyl)phthalazine (10b):
M.p. 188-192°C, yield: 65%. IR νmax cm-1: 3267 (NH), 3050 (aromatic CH), 2930 (aliphatic CH).
1H NMR (CDCl3): 300 MHz, δ=6.89 (d, 1H, J=16.5 Hz, N=C-CH=CH), 7.27-7.54 (m, 6H, Ar-

ACCEPTED MANUSCRIPT
H), 7.55-7.63 (m, 3H, Ar-H), 7.64-7.72 (m, 3H, Ar-H), 7.87 (d, 1H, J=7.6 Hz, CH-8
phthalazine), 8.17 (d, 1H, J=16.8 Hz, N=C-CH=CH), 8.52 (s, 1H, CH-4 phthalazine), 10.60 (s,
1H, NH, exchanged by D2O). MS m/z: 384 [M+]. Anal. Calcd. for C23H17ClN4 (384.87): C,
71.78; H, 4.45; N, 14.56. Found: C, 71.92; H, 4.49; N, 14.71.
4.1.6.3. 1-(2-(3-(4-Fluorophenyl)-1-phenylallylidene)hydrazinyl)phthalazine (10c):
M.p. 198-203°C, yield: 74%. IR νmax cm-1: 3298 (NH), 3035 (aromatic CH), 2925 (aliphatic CH).
1H NMR (CDCl3): 400 MHz, δ=6.91(d, 1H, J=16.6 Hz, N=C-CH=CH), 7.07 (t, 2H, J=8.3 Hz,
Ar-H), 7.48-7.53 (m, 3H, Ar-H), 7.53-7.58 (m, 3H, Ar-H), 7.67-7.74 (m, 4H, Ar-H), 7.80 (s, 1H,
CH-4 phthalazine), 8.17 (d, 1H, J=16.7 Hz, N=C-CH=CH), 8.56 (d, 1H, J=7.7 Hz, CH-8
phthalazine), 10.73 (s, 1H, NH, exchanged by D2O). 13C-NMR (100 MHz) δ=115.6, 115.8,
120.2, 120.3, 124.3, 126.1, 127.2, 127.4, 128.2, 128.9, 129.0, 129.4, 131.6, 132.0, 133.2, 133.24,
137.3 (aromatic Cs), 138.3 (N=C-CH=CH), 138.4 (C-4 phthalazine), 148.1 (C-1 allylidene),
160.4 (C-4 flourophenyl), 164.3 (C-1 phthalazine). Anal. Calcd. for C23H17FN4 (368.42): C,
74.98; H, 4.65; N, 15.21. Found: C, 75.12; H, 4.72; N, 15.34.
4.1.6.4. 1-(2-(3-(4-Chlorophenyl)-1-phenylallylidene)hydrazinyl)phthalazine (10d):
M.p. 220-224°C, yield: 75%. IR νmax cm-1: 3284 (NH), 3037 (aromatic CH), 2925 (aliphatic CH).
1H NMR (CDCl3): 400 MHz, δ=6.89 (d, 1H, J=16.6 Hz, N=C-CH=CH), 7.28-7.42 (m, 2H, Ar-
H), 7.5-7.56 (m, 5H, Ar-H), 7.68-7.88 (m, 5H, Ar-H), 8.21 (d, 1H, J=16.6 Hz, N=C-CH=CH),
8.54 (d, 1H, J=7.8 Hz, CH-8 phthalazine), 8.62 (s, 1H, CH-4 phthalazine), 10.67 (s, 1H, NH,
exchanged by D2O). 13C-NMR (100 MHz) δ=124.4, 126.1, 126.5, 127.1, 127.4, 128.1, 128.24,
128.28, 128.3, 128.6, 128.98, 129.4, 131.5, 131.6, 132.1, 134.4, 135.5, 137.2 (aromatic Cs),
138.2 (N=C-CH=CH), 138.4 (C-4 phthalazine), 148.2 (C-1 allylidene), 160.3 (C-1 phthalazine).
MS m/z: 384 [M+]. Anal. Calcd. for C23H17ClN4 (384.87): C, 71.78; H, 4.45; N, 14.56. Found: C,
71.89; H, 4.48; N, 14.68.
4.1.6.5. 1-(2-(3-(4-Fluorophenyl)-1-(p-tolyl)allylidene)hydrazinyl)phthalazine (10e):
M.p. 175-178°C, yield: 90%. IR νmax cm-1: 3267 (NH), 3051 (aromatic CH), 2918 (aliphatic CH).
1H NMR (CDCl3): 300 MHz, δ=2.45 (s, 3H, CH3), 6.9 (d, 1H, J=16.5 Hz, N=C-CH=CH), 7.05-
7.3 (m, 4H, Ar-H), 7.51-7.60 (m, 4H, Ar-H), 7.66-7.73 (m, 3H, Ar-H), 7.86 (d, 1H, J=7.7 Hz,
CH-8 phthalazine), 8.13 (d, 1H, J=16.5 Hz, N=C-CH=CH), 8.55 (s, 1H, CH-4 phthalazine),

ACCEPTED MANUSCRIPT
10.66 (s, 1H, NH, exchanged by D2O). 13C-NMR (100 MHz) δ=21.3 (CH3), 115.6, 120.4, 124.3,
126.0, 127.2, 127.4, 128.9, 129.0, 129.1, 129.3, 131.6, 132.0, 133.2, 133.29, 135.4, 137.3
(aromatic Cs), 138.2 (N=C-CH=CH), 139.0 (C-4 phthalazine), 147.9 (C-1 allylidene), 161.8 (C-4
flourophenyl), 164.2 (C-1 phthalazine).MS m/z: 382 [M+]. Anal. Calcd. for C24H19FN4 (382.16):
C, 75.37; H, 5.01; N, 14.65. Found: C, 75.44; H, 5.08; N, 14.73.
4.1.6.6.1-(1-(4-Chlorophenyl)-3-(4-methoxyphenyl)allylidene)hydrazinyl)phthalazine (10f):
M.p. 180-183°C, yield: 70%. IR νmax cm-1: 3327 (NH), 3051 (aromatic CH), 2953 (aliphatic CH).
1H NMR (CDCl3): 400 MHz, δ=3.86 (s, 3H, OCH3), 6.86 (d, 1H, J=16.6 Hz, N=C-CH=CH),
6.93-7.28 (m, 2H, Ar-H), 7.40-7.54 (m, 4H, Ar-H), 7.63-7.8 (m, 5H, Ar-H), 8.07 (d, 1H, J=16.6
Hz, N=C-CH=CH), 8.53 (d, 1H, J=7.7 Hz, CH-8 phthalazine), 8.99 (s, 1H, CH-4 phthalazine),
10.62 (s, 1H, NH, exchanged by D2O). 13C-NMR (100 MHz) δ=55.3 (OCH3), 114.2, 118.1,
120.4, 124.3, 126.1, 127.3, 127.7, 128.4, 128.9, 129.5, 130.7, 131.2, 131.6, 132.0, 134.8, 135.3,
137.0 (aromatic Cs), 138.1 (N=C-CH=CH), 138.4 (C-4 phthalazine), 148.0 (C-1 allylidene),
158.8 (C-4 methoxyphenyl ring), 160.4(C-1 phthalazine). MS m/z: 414 [M+]. Anal. Calcd. for
C24H19ClN4O (414.89): C, 69.48; H, 4.62; N, 13.5. Found: C, 69.61; H, 4.59; N, 13.59.
4.2. Antitumor activity study.
4.2.1. Cytotoxic activity.
Cytotoxic activity was measured in vitro using sulforhodamine-B-stain (SRB) assay method as
described by Skehan et al [29]. Cells seeded in 96-well plates were treated with the tested
compounds for 72 h. The medium was removed and the cells adhered to the plate were then fixed
with 10% trichloroacetic acid and stained with sulforhodamine-B. After three washings using 1%
acetum, sulforhodamine-B was dissolved in 100 µl buffer containing 10 mM Tris-base and the
optical density OD value was measured at 520 nm with a multiwell spectrophotometer
(VersaMax). The inhibitory activity percent on cell proliferation was calculated by using the
formula:
[(OD520 nm control cells - OD520 nm treated cells)/OD520 nm control cells ]* 100.
The average IC50 values were calculated from at least three independent tests. Standard error of
the mean (SEM) was within ± 10% of the values (Table 1).
4.2.2. VEGFR-2 and EGFR kinase inhibitory activity.

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The ELISA assay was performed in 96-well plates pre-coated with 20 ꢀg ml−1 poly (Glutamine,
Tyrosine) 4:1 (Sigma) as a substrate. In each well, 85 ꢀl of an 8 ꢀM ATP solution and 10 ꢀL of
the compound were added at varying concentrations. Delphinidin was used as a positive control
for VEGFR-2 and EGFR kinase, and 0.1% (v/v) DMSO was the negative control. Experiments at
each concentration were performed in triplicate. The reaction was initiated by adding 5 ꢀl of
VEGFR-2 or EGFR kinase. After incubation for 1 h at 37 °C, the plate was washed three times
with PBS containing 0.1% Tween 20 (T-PBS). Next, 100 ꢀl of anti-phosphotyrosine (PY99; 1:
500 dilution) antibody was added. After 1 h of incubation at room temperature, the plate was
washed three times. Goat anti-mouse IgG horseradish peroxidase (100 ꢀl; 1: 2000 dilution)
diluted in T-PBS containing 5 mg ml−1 BSA was added. The plate was reincubated at room
temperature for 1 h, and washed as before. Finally, 100 ꢀl of developing solution (0.03% H2O2, 2
mg ml−1 o-phenylenediamine in citrate buffer 0.1 M, pH 5.5) was added and incubated at room
temperature until color emerged. The reaction was terminated by the addition of 100 ꢀl of 2 M
sulfuric acid, A at 492 nm was measured using a multiwell spectrophotometer (VERSAmax™).
Experiments were run in triplicate and SEM was within ± 10% of the IC50 values (Table 2).
4.3. Docking study.
The molecular docking study was carried out on Molecular Operating Environment (MOE,
10.2008) software provided by chemical computing group, Canada. The program operated on
Windows 7 operating system installed on Intel core i5 2.27 MHz processor, 4.00 GB RAM. The
target compounds were built using the MOE builder interface and energy minimized using the
MMFF94X force field. The produced model was subjected to Systematic Conformational Search
where all items were set as default with RMS gradient of 0.01 kcal/mol and RMS distance of 0.1
Å. The X-ray crystallographic structures of EGRF TK and VEGFR-2 TK co-crystallized with
their ligands were downloaded from the protein data bank (PDB) code 1M17 and 4ASE,
respectively. The enzymes were prepared for docking as followed:
1- The co-docked ligand molecule was removed from the active site.
2- H atoms were added to the structure with their standard geometry.
3- MOE Alpha Site Finder was used for the active sites search in the enzyme structure and
dummy atoms were created from the obtained alpha spheres.

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4- Docking of the least energetic conformer for each compound in the active site of the ATP-
binding site of EGFR TK and VEGFR-2 TK was performed.
5- Docking protocol was verified by redocking of the co-crystallized ligand in the vicinity of the
active site of the enzyme with energy score= -10.4722 kCal/mol and RMSD= 1.53, for EGFR
TK and energy score= -35.483 kCal/mol and RMSD= 0.43 for VEGFR-2 TK.
Acknowledgements
The authors are grateful to Dr. Mohamed M. Abdulla, Saco Pharm, for his kind assistance in
performing the antitumor activity study. Also, thanks are extended to Ghada Fathy, assistant
lecturer, Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University for her
help in performing the docking study.
References
[1] L. Seymour, Cancer Treat. Rev., 25 (1999) 301-312.
[2] K. M. Amin, S. M. Abou-Seri, F. M. Awadallah, A. A. M. Eissa, G. S. Hassan, M. M.
Abdulla, Eur. J. Med. Chem. 90 (2015) 221-231.
[3] F. M. Awadallah, S. M. Abou-Seri, M. M. Abdulla, H. H. Georgey, Eur. J. Med. Chem. 94
(2015) 397-404.
[4] L. S. Rosen, Cancer control, 9 (2002) 36-44.
[5] M. Oliveira-Cunha, W. G. Newman, A. K. Siriwardena, Cancers (Basel), 3 (2011) 1513-
1526.
[6] P. Nair, Curr. Sci., 88 (2005) 890-898.
[7] E. N. Scott, G. Meinhardt, C. Jacques, D. Laurent, A. L. Thomas, Expert Opin. Investig.
Drugs, 16 (2007) 367-379.
[8] W. M. Eldehna, H. S. Ibrahim, H. A. Abdel-Aziz, N. N. Farrag, M. M. Youssef, Eur. J. Med.
Chem., 89 (2015) 549-560.
[9] J. Li, Y.-F. Zhao, X.-Y. Yuan, J.-X. Xu, P. Gong, Molecules, 11 (2006) 574-582.

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[10] X. Zhai, J. Li, L. He, S. Zheng, Y. B. Zhang, P. Gong, Chin. Chem. Lett., 19 (2008) 29-32.
[11] H. J. Knowles, Y.-M. Tian, D. R. Mole, A. L. Harris, Circ. Res., 95 (2004) 162-169.
[12] M. S. Abd Alla, M. I. Hegab, , N. A. Abo Taleb, , S. M. Hasabelnaby, A. Goudah, Eur. J.
Med. Chem., 45 (2010) 1267-1277.
[13] L. Zhang, L. P. Guan, X. Y. Sun, C. X. Wei, K. Y. Chai, Z. S. Quan, Chem. Biol. Drug
Des., 73 (2009) 313-319.
[14] Q.-R. Zhang, D.-Q. Xue, P. He, K.-P. Shao, P.-J. Chen, Y.-F. Gu, J.-L. Ren, L.-H. Shan, H.-
M. Liu, Bioorg. Med. Chem. Lett., 24 (2014) 1236-1238.
[15] M. A. El-Hashash, D. B. Guirguis, N. Abd El-Wahed, M. A. Kadhim, J. Chem. Eng.
Process Technol., 5 (2014) 1-6.
[16] S. P. Ivy, J. Y. Wick, B. M. Kaufman, Nat. Rev. Clin. Oncol., 6 (2009) 569-579.
[17] E. L. Piatnitski, M. A. Duncton, A. S. Kiselyov, R. Katoch-Rouse, D. Sherman, D. L.
Milligan, C. Balagtas, W. C. Wong, J. Kawakami, J. F. Doody, Bioorg. Med. Chem. Lett., 15
(2005) 4696-4698.
[18] K. Malinowsky, C. Wolff, S. Gündisch, D. Berg, K. Becker, J. Cancer, 2 (2011) 26-35.
[19] M. L. De Castro Barbosa, L. M. Lima, R. Tesch, C. M. R. Sant'Anna, F. Totzke, M. H.
Kubbutat, C. Schächtele, S. A. Laufer, E. J. Barreiro, Eur. J. Med. Chem., 71 (2014) 1-14.
[20] M. J. LaBonte, P. M. Wilson, W. Fazzone, J. Russell, S. G. Louie, A. El-Khoueiry, H.-J.
Lenz, R. D. Ladner, Cancer Res., 71 (2011) 3635-3648.
[21] A. Morabito, M. C. Piccirillo, F. Falasconi, G. De Feo, A. Del Giudice, J. Bryce, M. Di
Maio, E. De Maio, N. Normanno, F. Perrone, Oncologist, 14 (2009) 378-390.
[22] M. T. Conconi, G. Marzaro, L. Urbani, I. Zanusso, R. Di Liddo, I. Castagliuolo, P. Brun, F.
Tonus, A. Ferrarese, A. Guiotto, Eur. J. Med. Chem., 67 (2013) 373-383.
[23] Y. Chen, W. Hua, Spectrochim. Acta, 56 (2000) 1045-1049.
[24] D. J. Nelson, US 7,220,858 B2, 2007.
[25] F. M. Awadallah, W. I. El-Eraky, D. O. Saleh, Eur. J. Med. Chem., 52 (2012) 14-21.
[26] F. Hayat, A. Salahuddin, S. Umar, A. Azam, Eur. J. Med. Chem., 45 (2010) 4669-4675.

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[27] V. Kanagarajan, J. Thanusu, M. Gopalakrishnan, Eur. J. Med. Chem., 45 (2010) 1583-1589.
[28] H. L. Yadav, P. Gupta, R.Pawar, P. Singour, U. Patil, Med. Chem. Res., 20 (2011) 461-465.
[29] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H.
Bokesch, S. Kenney, M. R. Boyd, J. Natl. Cancer Inst., 82 (1990) 1107-1112.
[30] G. Bold, K. –H. Altmann, J. Frei, M. Lang, P.W. Manley, P. Traxler, B. Wietfeld, J.
Brüggen, E. Buchdunger, R. Cozens, J. Med. Chem., 43 (2000) 17174-17179.
Figure captions:
Figure 1. Examples of some phthalazine-based antitumor agents.
Figure 2. Docking of compound 6b into the ATP-binding site of EGFR kinase.
Figure 3. Docking of compound 10a into the ATP-binding site of VEGFR-2 kinase.
Figure 4. Docking of compound 6b into the ATP-binding site of EGFR kinase.
Figure 5. Docking of compound 10a into the ATP-binding site of VEGFR-2 kinase.
Scheme captions:
Scheme 1. Synthesis of compounds 4, 5a-f and 6a-d..
Scheme 2. Synthesis of compounds 7a-f. 8a-f and 10a-f.
Table captions:

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Table 1. IC50 (nM) of the tested compounds against the selected human cancer cell lines.
Table 2. IC50 (nM) of the tested compounds against VEGFR-2 and EGFR kinases.

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Table 1.
IC50 (nM )a
Colon
Ovarian
Cervical
OVCAR3 HeLa
Glioblastoma
Melanoma Breast Prostate Kidney Lung
Leukemia
Compd.
No
SKOV-
MDA-MB-
435
HT29 COLO205
SF268 SF295
MCF-7 DU145
UO31
H460 K562 HL60
3
12.00
34.00
23.00
38.00
29.00
3.30
23.00
56.00
43.00
44.00
43.00
7.90
0.80
7.70
6.70
0.90
0.80
0.08
0.06
0.02
0.06
0.04
0.80
0.70
0.90
0.60
0.50
0.80
0.80
0.90
4.50
14.00
43.00
54.00
83.00
72.00
3.40
12.00
33.00
45.00
24.00
25.00
34.00
7.60
5.30
6.50
4.40
8.40
7.60
0.56
0.43
0.32
0.44
0.33
9.30
0.40
6.30
9.50
8.30
7.20
2.40
3.50
4.60
18.00
36.00
27.00
36.00
45.00
3.40
37.00
55.00
46.00
53.00
64.00
7.70
14.00
23.00 17.00 14.00 18.00
12.00 39.00 54.00 77.00
33.00 28.00 23.00 68.00
44.00 43.00 34.00 86.00
23.00 33.00 43.00 67.00
5a
5b
5c
5d
5e
5f
15.00
13.00
26.00
14.00
3.50
24.00
35.00
54.00
45.00
7.80
3.40
4.70
2.00
5.60
1.90
0.80
8.80
7.70
0.70
8.60
4.90
6.70
2.60
7.80
5.50
9.50
6.40
5.50
5.30
6.60
2.10
4.50
5.40
2.80
4.50
4.00
3.50
4.30
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
1.20
2.50
4.30
1.50
3.30
2.00
2.30
2.10
2.20
5.60
3.30
3.92
5.40
5.40
4.50
2.10
1.30
3.40
5.40
2.70
3.40
1.00
2.40
3.20
47.00
59.00
30.00
40.00
30.00
20.00
49.00
58.00
44.00
55.00
66.00
66.00
77.00
78.00
67.00
55.00
43.00
22.00
74.00
95.00
54.00
6.00
37.00
48.00
49.00
39.00
20.00
38.00
57.00
65.00
22.00
17.00
66.00
75.00
57.00
76.00
86.00
6.00
52.00
83.00
67.00
74.00
95.00
88.00
56.00
65.00
33.00
36.00
25.00
79.00
47.00
59.00
60.00
89.00
98.00
0.30
65.00
54.00
56.00
45.00
34.00
25.00
67.00
86.00
86.00
33.00 32.00 23.00 94.00
24.00 23.00 54.00
36.00 20.00 74.00
4.00
6.00
56.00 16.00 76.00 95.00
68.00 27.00 84.00 84.00
47.00 39.00 94.00 74.00
95.00
73.00
46.00
55.00
54.00
25.00 19.00 85.00
54.00 28.00 76.00
75.00 36.00 44.00
4.00
3.00
0.30
8a
8b
8c
6.00
8.00

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47.00
54.00
46.00
1.10
2.30
4.40
1.40
2.40
1.00
0.36
32.00
45.00
34.00
2.40
5.70
7.70
3.50
6.80
2.20
0.52
7.90
6.80
5.40
0.01
0.07
0.07
0.05
0.07
0.01
0.05
44.00
53.00
45.00
3.30
5.40
2.30
4.50
4.50
3.40
0.47
56.00
42.00
34.00
1.40
3.30
4.45
2.60
3.40
1.20
1.20
7.00
6.00
8.00
3.20
6.30
7.80
4.30
6.50
2.10
0.09
4.60
1.30
2.40
0.33
0.53
0.57
0.42
0.55
0.14
0.23
44.00
35.00
44.00
3.00
5.60
3.40
3.00
5.40
2.00
1.30
77.00
6.00
8.00
2.20
3.60
7.90
3.40
5.60
1.10
0.04
97.00
6.00
97.00
1.30
4.60
7.60
4.50
5.60
1.20
0.07
63.00 30.00 57.00
75.00 17.00 46.00
84.00 25.00 35.00
3.00
4.00
0.40
4.70
7.40
9.00
7.50
8.60
5.66
0.40
8d
8e
8f
3.90
6.20
2.30
3.80
5.30
2.00
0.36
5.70
9.60
0.90
9.70
7.00
4.80
0.17
4.40
6.90
3.80
4.60
5.00
3.50
0.51
10a
10b
10c
10d
10e
10f
DOX
(µM)
a The results are the mean of three independent experiments and the SEM are within ± 10%.

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Table 2
.
IC50(nM)a
IC50(nM) a
Compd. No
Compd. No
VEGFR-2
9.80
8.60
9.70
7.80
8.50
0.77
0.67
0.43
0.68
0.54
7.50
7.30
4.50
6.20
EGFR
140.00
88.00
130.00
84.00
86.00
6.70
VEGFR-2
4.40
EGFR
51.00
47.00
23.00
45.00
42.00
36.00
5.60
5a
5b
5c
5d
5e
5f
8a
8b
3.60
8c
1.20
8d
3.50
8e
2.70
8f
2.40
6a
6b
6c
6d
7a
7b
7c
7d
7e
10a
10b
10c
10d
10e
10f
6.00
0.42
4.10
0.69
6.30
6.20
0.88
6.90
4.40
0.55
4.60
77.00
76.00
52.00
65.00
0.70
6.50
0.41
5.40
5.09
6.27
Delphinidin
6.10
5.70
64.00
53.00
7f
a The results are the mean of three independent experiments and the SEM are within ± 10%.

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F
O
F
Cl
Cl
HN
HN
N
N
N
N
N
H2N
II
O
I (Vatalanib)
Figure 1.
Figure 2.

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Figure 3.

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Figure 4.
Figure 5.