Design and synthesis of new phthalazine-based derivatives as potential EGFR inhibitors for the treatment of hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bioorg.2018.12.039

Searching for new leads in the battle of cancer will never ends, we herein disclose the design and synthesis of new phthalazine derivatives and their in vitro and in vivo testing for their antiproliferative activity. Phthalazine was selected as a privilege moiety that is incorporated in a big number of anticancer drugs in clinical use or that are still under clinical or preclinical studies. We utilized the drug extension strategy to tailor the designed compounds to fit the EGFR hydrophobic sub pocket and cleft region. The designed phthalazine derivatives was synthesized by linking phthalazine moiety with 1,3,4-oxadiazole-thione and 1,2,4-triazole-thione. Alkylation and glycosylation of the new heterocyclic systems were successfully performed to be used in the drug extension. Coupling of some phthalazine derivatives with different amino acids was also performed to improve the drug selectivity. The synthesized compounds were tested for their antiproliferative activity against cancer cells both in vivo and in vitro. The in vitro activity against hepatocellular carcinoma (HepG2 cell line) ranged from 5.7 μg/mL to 43.4 μg/mL. Compounds 31a and 16 were the most active with an IC₅₀ 5.7 μg/mL and 7.09 μg/mL, respectively compared to the standard compound doxorubicin (4.0 μg/mL). In vivo, compounds 10 and 16 showed IC₅₀ values 7.25 μg/mL and 7.5 μg/mL, respectively compared to the standard compound cisplatin (IC₅₀ 9.0 μg/mL). In silico, testing of the phthalazine derivatives showed that they are good inhibitors for EGFR. The docking studies substantiated compounds 4, 10, 16 and 31a as new lead compounds and identified Arg841 as a key residue in the cleft region for binding stronger inhibitors.

Full text of DOI:10.1016/j.bioorg.2018.12.039

Accepted Manuscript
Design and synthesis of new phthalazine-based derivatives as potential EGFR
inhibitors for the treatment of hepatocellular carcinoma
Ahmed T.A. Boraei, Hanaa K. Ashour, El Sayed H. El Tamany, Nahla
Abdelmoaty, Abdullah I. El-Falouji, Mohamed S. Gomaa
PII:
S0045-2068(18)30970-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.039
YBIOO 2703
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 September 2018
26 November 2018
29 December 2018
Please cite this article as: A.T.A. Boraei, H.K. Ashour, E.S.H. El Tamany, N. Abdelmoaty, A.I. El-Falouji, M.S.
Gomaa, Design and synthesis of new phthalazine-based derivatives as potential EGFR inhibitors for the treatment
of hepatocellular carcinoma, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.039
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Design and synthesis of new phthalazine-based derivatives as
potential EGFR inhibitors for the treatment of hepatocellular
carcinoma
a*
a
a
a
Ahmed T. A. Boraei , Hanaa K. Ashour , El Sayed H. El Tamany , Nahla Abdelmoaty ,
b
c
Abdullah I. El-Falouji , Mohamed S. Gomaa
a
Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt
b
Institute of Biotechnology for Postgraduate Studies and Research, Suez Canal University,
Ismailia, Egypt
c
Chemistry Department, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal
University, Dammam, Kingdom of Saudi Arabia
Abstract:
Searching for new leads in the battle of cancer will never ends, we herein
disclose the design and synthesis of new phthalazine derivatives and their
in vitro and in vivo testing for their antiproliferative activity. Phthalazine
was selected as a privilege moiety that is incorporated in a big number of
anticancer drugs in clinical use or that are still under clinical or
preclinical studies. We utilized the drug extension strategy to tailor the
designed compounds to fit the EGFR hydrophobic sub pocket and cleft
region. The designed phthalazine derivatives was synthesized by linking
phthalazine moiety with 1,3,4-oxadiazole-thione and 1,2,4-triazole-
thione. Alkylation and glycosylation of the new heterocyclic systems
were successfully performed to be used in the drug extension. Coupling
1

of some phthalazine derivatives with different amino acids was also
performed to improve the drug selectivity.
The synthesized compounds were tested for their antiproliferative activity
against cancer cells both in vivo and in vitro. The in vitro activity against
hepatocellular carcinoma (HepG2 cell line) ranged from 5.7 µg/mL to
4
5
3.4 µg/mL. Compounds 31a and 16 were the most active with an IC50
.7 µg/mL and 7.1 µg/mL, respectively compared to the standard
compound doxorubicin (4.0 µg/mL). In vivo, compounds 10 and 16
showed IC values 7.25 μM and 7.5 μM, respectively compared to the
50
standard compound cisplatin (IC 9.0 μM). In silico, testing of the
50
phthalazine derivatives showed that they are good inhibitors for EGFR.
The docking studies substantiated compounds 4, 10, 16 and 31a as new
lead compounds and identified Arg841 as a key residue in the cleft region
for binding stronger inhibitors.
Corresponding Author Email Address: Ahmed_Tawfeek83@yahoo.com
Keywords: Anticancer activity; HepG2; phthalazines; alkylation; 1,3,4-
oxadiazolethione; 1,2,4-triazolethione
1. Introduction
Cancer is a main public health problem worldwide and is the second
leading cause of death in the United States.The continuous decline in
cancer death rates over the last 2 decades is due to the continuous effort
of researchers to overcome this disease [1].
2

It is well known that phthalazinone derivatives have great interest due to
their antidiabetic [2], vasorelaxant [3], antiallergic [4], PDE4 inhibitors
[5], vascular endothelical group factor receptor (VEGFR) tyrosine kinase
inhibitors for treatment of cancer [6,7], antiasthmatic [8] and herbicidal
agents [9]. Drug molecules like hydralazine [10,11], burdralazine [12,13],
ponalrest [14], azelastine [15,16], and zopolrestat [17]. Several
phthalazine derivatives were found to have antitumor [18-20],
anticovulsant [21,22], antihypertensive [23,24], antitrypanosomal [25],
antimicrobial [26], and anti-inflammatory activities [27,28]. Since, most
of drugs containing phthalazines are substituted at position 2 and 4 of
phathalazine scaffold; the present study is concerned with changing the
substituents at those two positions to find potent anticancer leads (Fig. 1.
Cl
O
H N
2
NH
OH
F
F
F
N
N
N
N
N
S
N
N
O
O
N
Hydralazine
Azelastine
Zopolrestat
Selected drugs
N
N
N
N
O
R
N
N
N
R
N
N
N
R
S
S
O
N
O
N
H
O
O
R1
O
O
Planned structures
3

Fig. 1. Structures of selected phthalazine containing drugs and planned
structures.
In continuation to our previous work [29], on the development of new
tyrosine kinase inhibitors for the treatment of cancer, which resulted on a
new indolyl-triazole lead, we hereby present the development of new lead
inhibitors for EGFR. The presence of more than one compound to choose
from would make future development and optimization easier in term of
pharmacokinetic properties and toxicity profile optimization. These
promising lead compounds will be further optimized or even hybridized
and tested in a hope to reach a final compound that would progress to
clinical trials and development. We designed compounds that would form
better interactions with residues at the active site than our previous lead
and have variable log P and pKa. We utilized ring variation, substituent
variation, chain extension, introduction of hydrogen-donors and hydrogen
acceptors techniques in our new lead search.
2. Results and discussion
2
.1. Chemistry
2
-(4-Benzyl-1-oxophthalazin-2(1H)-yl)acetic acid 2 was obtained from
the hydrolysis of ethyl 2-(4-benzyl-1-oxophthalazin-2(1H)-yl)acetate 1 in
alcoholic sodium hydroxide whereas, reaction of ester 1 with hydrazine
hydrate in ethanol afforded hydrazide 3. Refluxing hydrazide 3 with
4

carbon disulphide in ethanol containing aq. KOH then acidification led to
the formation of 5-(4-benzyl-1-oxo-1H-phthalazin-2-ylmethyl)-1,3,4-
oxadiazol-2-thione 4. Alkylation of oxadiazole 4 with allyl bromide and
benzyl bromide in the presence of potassium carbonate afforded S-allyl-
1
,3,4-oxadiazole 5 and S-benzyl-1,3,4-oxadiazole 6 respectively.
Whereas, glycosylation of the oxadiazole 4 with 2,3,4,6-tetra-O-acetyl-α-
D-glucopyranosyl bromide and 2,3,4,6-tetra-O-acetyl-α-D-
7
galactopyranosyl bromide 8 yielded a mixture of S-glycosylated 9,10 and
N-glycosylated analogs 11,12 (Scheme 1).
CH Ph
CH Ph
2
2
CH2Ph
N
N
N
N
N H H O
i) NaOH,EtOH
ii) HCl/H2O
2
4. 2
CONHNH2
N
N
CO Et
EtOH
2
O
CO2H
3
O
1
O
2
i. CS2, aq.KOH
EtOH
ii. Conc HCl/H O
2
O
R2
OAc
2
OAc
R
O
O
CH Ph
N
N
2
O
S
1
1
R
R
N
NH
AcO
OAc
N
N
N
AcO
OAc
Br
9,10
S
7,8
CH Ph
2
N
O
2 3
K CO ,
acetone
+
O
O
4
O
R-Br K2CO3, acetone
N
R2
OAc
S
O
N
N
N
1
R
CH Ph
2
AcO
CH Ph
2
OAc
11,12
N
N
N
N
R
S
O
1
2
7
,9,11: R = OAc, R = H
8,10,12: R = H, R = OAc
1
2
O
,6
5
5
6
: R = Allyl
: R = Benzyl
Scheme 1: Synthesis, alkylation, and glycosylation of phthalazino-1,3,4-
oxadiazolethione 4.
5

Stirring the hydrazide 3 with carbon disulphide in ethanol containing aq.
KOH gave potassium dithiocarbazate 13a which was separated and then
refluxed with hydrazine hydrate in water to afford 5-(4-benzyl-1-oxo-1H-
phthalazin-2-ylmethyl)-4-amino-1,2,4-triazole-3-thione 13 after cooling
and acidification. Reaction of hydrazide 3 with ammonium thiocyanate in
water containing hydrochloric acid led to the formation of 1-((4-benzyl-1-
oxo-1H-phthalazin-2-yl)acetyl)thiosemicarbazide 14 which failed to
cyclize to dihydro-1,2,4-triazole 15 upon treating with aq. KOH and
acidification. While, dihydro-1,2,4-triazole 15 was successfully obtained
from fusion of hydrazide 3 with thiourea. Stirring of hydrazide 3 with
phenyl isothiocyanate in absolute ethanol led to the formation of 1-((4-
benzyl-1-oxo-1H-phthalazin-2-yl)acetyl)-4-phenyl-thiosemicarbazide 16.
Cyclization of 16 was done by reflux in aq. KOH to the form 5-(4-benzyl-
1
-oxo-1H-phthalazin-2-ylmethyl)-4-phenyl-1,2,4-triazol-3-thione
17.
Benzylation of 1,2,4-triazole 17 with benzyl bromide in existence of
potassium carbonate afforded S-benzyl-4-phenyl-1,2,4-triazole 18
(Scheme 2).
6

CH Ph
2
CH Ph
2
N
N
N
N
Br
Ph
K CO ,
KOH
H O
O
CH Ph
2
CONHNHCSNHPh
2
3
H
S
acetone
2
N
N
N
N
N
O
N
O
Ph
N
16
N
Ph
SCH Ph
2
1
7
18
Ph-N=C=S,
EtOH
CH2Ph
CH Ph
CH Ph
2
2
N
N
NH SCN
N
N
O
H
N
NH
HN
CONHNH2
4
KOH / H O
2
N
N
HCl, H O
S
2
NH2
N
H
N
O
O
S
O
3
14
1
5
CS , aq. KOH
EtOH
NH CSNH , fusion
2
2
2
CH Ph
2
CH Ph
CH Ph
2
2
N
N
NH
N
NH
N
N
N
N
NH NH , H O
2 2 2
-
CONHNHCSS K+
NH2NH2, H2O
S
S
N
O
N
O
O
O
NH2
13a
13
4
Scheme 2: Synthesis of diverse 1,2,4-triazolo-phthalazines.
In addition, refluxing hydrazide 3 with α-D-glucose in ethanol containing
few drops of glacial acetic acid yielded 2-(4-benzyl-1-oxo-1H-phthalazin-
2
-yl)-N'-(β-D-glucopyranosyl)acetohydrazide 19. While, reaction of 3
with phthalic anhydride under the same conditions led to the formation of
-(4-benzyl-1-oxo-1H-phthalazin-2-yl)-N-(1,3-dioxoisoindolin-2-
2
yl)acetamide 20. Benzoylation of hydrazide 3 with benzoyl chloride was
highly dependent on the media used. The use of pyridene yielded N'-(2-
(4-benzyl-1-oxo-1H-phthalazin-2-yl)acetyl)benzohydrazide 21, whereas,
when done in ethanol gave 2-phenyl-5-(4-benzyl-1-oxo-1H-phthalazin-2-
ylmethyl)-1,3,4-oxadiazole 22. Treatment of 20 with phenyl hydrazine in
ethanol containing few drops of glacial acetic acid led to the formation of
N'-phenylacetohydrazide 23. Similarly hydrazinolysis of 20 at the same
conditions afforded the hydrazide 3. The reaction of hydrazide 3 with
ethyl acetoacetate in ethanol led to the formation of ethyl 3-(2-(2-(4-
7

benzyl-1-oxo-1H-phthalazin-2-yl)acetyl)hydrazono)butanoate
24.
(Scheme 3).
CH Ph
2
CH Ph
2
O
N
N
PhNHNH2
N
N
O
CONHNHPh
EtOH/AcOH
N
N
CH Ph
O
O
H
O
2
23
20
N
N
CONHNHCOPh
O
N H H O
2
4.
2
AcOH
O
21
O
EtOH/AcOH
O
CH Ph
2
O
N
N
CH Ph
Pyridene
2
H
H
C
N
N
OH
O
N
N
H
HO
H
H
D-glucose,
EtOH,AcOH
PhCOCl
CONHNH2
OH
OH
EtOH,3h
H
O
CH OH
3
2
CH Ph
2
CH COCH CO Et,
3
2
2
EtOH
N
N
N
N
Ph
O
OH
CH Ph
2
CH2Ph
H
O
O
O
N
N
N
N
CH3
HO
N
22
HO
CONHN
N
H
OH
O
CH CO Et
24
2
2
O
1
9
Scheme 3: Reactions of hydrazide 3 with D-glucose, phthalic anhydride,
benzoyl chloride, ethylacetoacetate and phenyl hydrazine.
Alkylation of 1,2,4-triazole 13 with ethyl chloroacetate and benzyl
chloride in the existence of potassium carbonate afforded S-alkylated-4-
amino-1,2,4-triazole 25 and 26 respectively. Triazolo-thiazole 27 was
obtained by fusion of 1,2,4-triazole 13 and urea. Condensation of 4-
amino-1,2,4-triazole 13 with aromatic aldehydes namely benzaldehyde
and salicylaldehyde in ethanol containing acetic acid afforded the
formation
of
5-(4-benzyl-1-oxo-1H-phthalazin-2-ylmethyl)-4-
(arylideneamino)-1,2,4-triazole-3-thione 28 and 29 respectively (Scheme
4
).
8

CH Ph
2
CH Ph
2
CH Ph
2
NH
N
N
N
NH
Ar'CHO, EtOH,
drops AcOH
N
N
N
R
S
N
R-X
N
N
N
N
S
N
K2C
O , EtOH
3
S
O
N
N
O
NH2
13
O
28,29
NH2
Ar'
25,26
NH CONH ,
2
2
fusion
Ar'
2
5 R = -CH COOC H
2 2 5
CH Ph
26 R = -CH Ph
2
8
9
2
2
HO
N
N
N
N
2
S
N
N
O
2
7
OH
Scheme 4: Alkylation and condensation of 4-amino-1,2,4-triazole 13.
Reaction of hydrazide 3 with NaNO /HCl at low temperature yielded the
2
azide 30 which was coupled with a number of amino acids esters (glycine,
L-leucine and L-serine) in the presence of triethyl amine to give coupled
products 31a-c respectively. Azide-coupling method was effectively
applied for coupling of azide 30 with benzyl amine, cyclohexylamine and
n-propylamine to produce the amides 32a-c (Scheme 5).
9

CH Ph
2
N
N
O
NH2
N
H
O
3
o
NaNO / HCl , -5 C
2
CH Ph
2
R
HClH N
CO CH
3
N
N
O
R'NH2
2
2
o
o
Et N, 0 C
Et3N, 0 C
3
N3
O
3
0
CH Ph
CH Ph
2
2
N
N
O
N
O
R
R'
N
OCH3
N
H
N
H
O
O
O
3
1a-c
32a-c
31
R
32
R'
a (Gly)
b ( leu)
c (ser)
a (benzyl)
-
H
-CH Ph
2
-CH CH(CH )
b ( cyclohexyl)
c (propyl)
2
3 2
-CH OH
-CH CH CH
3
2
2
2
Scheme (5): Coupling of azide 30 with amino acid esters and amines.
Significant analytical data used for structures confirmation
1
The H-NMR spectra of all compounds displayed a sharp singlet signal
around 4.30 ppm for -CH Ph directly attached to C-4 of phthalazine ring.
2
The methylene protons attached to N-2 of phthalazine (N-CH ) appeared
2
around 4.80 ppm as in 2 and 3 which revealed deshielding as a result of
the cyclization to appear around 5.60 ppm as in the rest of the
13
compounds. The aromatic protons appeared between 7.18-8.30 ppm. C-
10

NMR spectra revealed the methylene carbon of the -CH Ph group
2
(
attached to C-4) at 37.5ppm. While, NCH (attached to N-2) appeared
2
between 45.0 and 52.0 ppm. All the aromatic carbons were found
between 125.7 and 146.0 ppm. The carbonyl group of the phthalazinone
ring was observed around 158.0 ppm. The additional significant data
could be discussed as following:
1
The H-NMR spectrum of the acid 2 showed the carboxylic acid OH
signal(broad) at 13.04 ppm and the respective carbonyl group of the acid
appeared at 169.4 ppm. The NMRs of the hydrazide 3 showed the
hydarzino (NHNH ) group protons at 4.30 and 9.31 ppm and the carbonyl
2
group appeared at 166.4 ppm.
1
H-NMR spectrum of oxadiazolethione 4 revealed abroad signal at 14.60
13
ppm for the NH of the oxadiazolethione. C-NMR spectrum showed the
two oxadiazole signals (C-5Oxad.) at 154.8 and (C=SOxad.) at169.4 ppm.
1
Due to the allylation of oxadiazole 4, H-NMR spectrum of compound 5
showed SCH signal at 3.87. The olefinic methylene protons (CH=CH )
2
2
appeared as two doublet of doublets at 5.07 ppm for the cis proton with
coupling constant values JCis 9.0, JGem 0.9 Hz, while the trans proton
appeared at 5.26 ppm with coupling constant JTrans ≈ 16, JGem 1.2 Hz. The
13
olefinic CH (CH=CH ) appeared as multiplet at 5.92-5.98 ppm. C-NMR
2
spectrum showed a signal at 34.5 ppm corresponding to (SCH ). The
2
NMR spectrum of compound 6 showed a singlet signal for benzyl
methylene protons attached to sulfur (SCH Ph) at 4.46 ppm and the
2
1
13
corresponding methylene carbon at 35.8 ppm. H- and C-NMRs
unambiguously differentiated between S- and N-glycosides as following:
S-glycosides 9 and 10 showed the anomeric protons around 5.66 ppm.
Whereas, N-glycosides 11 and 12 displayed their anomeric protons
around 6.20. All anomeric protons revealed large coupling constant value
13
J > 9.3 Hz which confirm the β-configuration. Moreover, C-NMR of
1,2
11

glycosides 11 and 12 displayed C=S signal at 177.3 ppm which supports
the assignment. The structure of 4-amino-1,2,4-triazole 13 was confirmed
1
from H-NMR which showed two singlet signals at 5.63 ppm and 13.67
13
ppm which corresponding to NH and NH respectively. C-NMR
2
showed the two triazole carbon signals at 148.1 and 166.3 ppm due to C-
5Triazol and C=S respectively.
1
The structure of the thiosemicarbazide 14 was confirmed from H-NMR
which showed two singlet signals at 9.44 and 10.26 ppm for the two NH
13
groups (CONH-NH-). C-NMR spectrum revealed two signals at 166.6
and 182.0 ppm corresponding to carbonyl of amide and thiocarbonyl
1
(
C=S) groups. H-NMR spectrum of dihydro-1,2,4-triazole-3-thione 15
showed two singlet signals at 13.38 and 13.41 ppm due to two NH of
13
triazole ring. C-NMR spectrum presented two triazole carbon signals at
48.8 and 166.6 ppm.
1
The phthalazino-phenyl thiosemicarbazide 16 structure was elucidated
1
from H-NMR which displayed three NH signals (CONHNHCS-NHPh)
13
at 9.60, 9.81 and 10.46 ppm. C-NMR spectrum revealed two signals due
1
to C=O and (C=S) at 166.9 and 181.0 ppm respectively. H-NMR
spectrum of phthalazino-4-phenyl-1,2,4-triazole-3-thione 17 showed a
13
singlet signal at 13.91 ppm for NH group. C-NMR spectrum revealed
the two triazole carbon signals 148.3 ppm (C=NTriazol) and 168.1 ppm
(C=S). The NMRs spectra of S-benzyl-1,2,4-triazole 18 showed a singlet
signal for benzyl methylene protons attached to sulfur (SCH Ph) at 4.19
2
1
ppm and the corresponding methylene carbon appeared at 36.2 ppm. H-
NMR of the glucoside 19 (in DMSO-d + D O) showed multiplet signal
6
2
between 3.01-3.77 ppm corresponding to glucose six protons (H-2 , H-
Glu
3Glu, H-4Glu, H-5 Glu, H-6 and H-6΄ ). The anomeric proton appeared as
Glu
Glu
doublet at 3.85 ppm with coupling constant J 9.0 Hz. A doublet signal
2,3
was appeared between 5.04-5.29 ppm with coupling constant value
12

13
J 17.1 Hz corresponding to NCH group. C-NMR spectrum showed
Gem
2
1
the six glucose signals at 64.3, 64.9, 73.2, 73.8, 79.5 and 94.0 ppm. H-
NMR spectrum of compound 20 revealed a singlet signal at 11.08 ppm
13
due to NH group. C-NMR spectrum showed two signals at 164.8 and
1
1
66.8 ppm corresponding to three carbonyl carbons. H-NMR spectrum
of compound 21 showed two singlet signals at 10.31 and 10.48 ppm
13
corresponding to two NHs of (-CO-NHNHCOPh). C-NMR showed two
signals at 165.3 and 166.4 ppm for the two carbonyl groups. The structure
1
of 2-phenyl-1,3,4-oxadiazole 22 was deduced from H-NMR which
displayed NCH signal at 4.96 ppm and the corresponding carbon at 52.4
2
ppm.
1
H-NMR spectrum of the phenylacetohydrazide 23 showed the two NH
13
signals (-CONHNHPh) at 2.87 and 10.37 ppm. C-NMR revealed a
1
signal at 165.9 corresponding to carbonyl of amide group. H-NMR of
compound 24 displayed the two CH at 1.21 ppm and 1.97 ppm. The
3
methylene protons appeared as singlet at 3.37 ppm and quartet at 4.14
13
ppm. The NH signal appeared at 10.72 ppm. C-NMR spectrum revealed
three methylene carbons at 44.5, 52.8 and 60.9 ppm.
1
The structure of 25 was deduced from H-NMR which demonstrated
triplet and quartet signals at 1.17 and 4.09 ppm corresponding to the
13
methyl and methylene protons of the ethoxy group (OCH CH ). C-
2
3
NMR revealed the ethoxy carbons (OCH CH ) signals at 14.4 and 61.6
2
3
1
ppm. The carbonyl of the ester group was appeared at 168.9 ppm. H-
NMR of compound 26 showed the methylene protons of the new attached
benzyl group as singlet at 4.39 ppm and the corresponding carbon was
found at 34.7 ppm. NMRs of 27 displayed the spacer methylene protons
at 5.44 ppm and the corresponding methylene carbon at 44.8 ppm in
addition to a broad signal for hydroxyl group at 13.62 ppm. The structure
1
of 28 was elucidated from H-NMR which showed the disappearance of
13

NH signal and instead, a singlet signal was appeared 9.94 for
2
1
3
benzylidene group (N=CH). C-NMR revealed signal at 162.6 ppm for
1
C=S. The structure of 29 was elucidated from H-NMR which displayed
three singlet signals at 9.00, 10.29 and 13.94 ppm for benzylidene N=CH,
13
hydroxyl and NH groups respectively. C-NMR spectrum showed C=S
signal at 162.7 ppm.
The structures of the coupled amino acid esters were deduced from their
NMR spectra which demonstrated the methoxy protons (OCH ) as singlet
3
at 3.66 ppm while the corresponding carbon appeared around 52.2 ppm.
13
In addition, two signals appeared in C-NMR around 168.0 and 173.2
ppm for the carbonyl of ester and amide. Moreover, the characteristic
amino acid signals could be discussed as following:
1
H-NMR of Glycinated phthalizinone 31a showed the glycine CH as
2
13
singlet at 3.93 ppm and glycine NH at 8.55 ppm. C-NMRspectrum
1
revealed a signal at 41.1 attributable to glycine CH . H-NMR spectrum
2
of Leucinated phthalizinone 31b revealed leucine methyl protons at 0.90
13
ppm, CH and CH were observed around 1.58 and 1.70 ppm. C-NMR
2
spectrum revealed four signals at 21.9, 23.1, 24.7 and 50.9 ppm
corresponding to 2CH , CH, CH and NCH groups of leucine
3
2
1
respectively. H-NMR spectrum of serinated phthalizinone 31c showed
13
serine OH at 4.73 ppm, CH at 3.67 ppm and CH at 4.46 ppm. C-NMR
2
showed two signals at 55.3 and 61.9 ppm for NCH and CH OH. NMR of
2
compound 32a showed the methylene protons of the benzyl amine at 4.36
and the respective methylene carbon at 42.7 ppm. While, the NH of
amine appeared at 8.64 ppm. NMR spectrum of 32b revealed a multiplet
signal at 1.10-1.83 ppm corresponding to five methylene groups of the
cyclohexyl moiety whereas, the remaining CH was observed at 3.59 ppm.
The carbons of the cyclohexyl moiety appeared at 25.0, 25.7, 32.9 and
1
4
8.3 ppm. H-NMR spectrum of 32c displayed the propyl signals at 0.87,
14

13
1
.41, 3.11 ppm. C-NMRshowed the propyl signals at 11.8 (CH ), 22.8
3
(
CH ) and 53.6 ppm (NCH ).
2
2
2
.2. Anticancer Activity
The newly synthesized compounds were test for their anticancer activity
in vitro against human hepatocellular carcinoma cell line HepG2 with
comparison to the reference anticancer drug doxorubicin.
The method of Skehan et al., [30] was used to test the potential
cytotoxicity of the compounds. Different concentrations of the compound
under test (0, 5, 12.5, 25 and 50 µg/mL) were added to the cell line. After
o
4
8 hr of incubation at 37 C and in atmosphere of 5% CO , cells were
2
fixed, washed and stained with Sulfo-Rhodamine-B stain. Excess stain
was washed with acetic acid and attached stain was recovered withTris
EDTA buffer and color intensity was measured in an ELISA reader. The
relation between surviving fraction and compound concentration is
plotted to get the survival curve of tumor cell line after the specified
compound and determine the IC of the compound.
50
2.2.1. In vitro Anti-Tumor results
The synthesized phthalazine derivatives were biologically evaluated for
their antiproliferative activity against HepG2 cell lines where they
showed promising activity (Table 1). The activity ranged from 5.7-43.4
µg/mL. Compounds 31a and 16 were the most active with an IC of 5.7
50
µg/mL and 7.1 µg/mL respectively compared to the standard compound
doxorubicin (4.0 µg/mL). Compounds 4, 10 and 13 showed activity in the
tenth micromolar range; 15.8 µg/mL, 13.6 µg/mL, 16.2 µg/mL
respectively. All the remaining compounds exhibited lower but
comparable activity. The obtained biological results make this new
15

phthalazine series a good catch for further development and testing. Our
future directions will focus on biomedical assays to substantiate the
claimed activity against EGFR together with synthesizing more
derivatives for testing.
Table( 1 ) IC and binding affinities of the docked compounds in EGFR
50
calculated with autodock 4.3.
16

Compd.
Code
IC50
Binding
Affinity
kcal/mol)
(µg/mL)
(
Doxorubicin
4
4.0
15.8
13.6
28
-9.6
-8.6
1
1
1
1
1
1
1
2
2
2
2
2
0
2
3
4
6
7
8
4
6
7
8
9
-8.1
16.2
37.9
7.09
39.2
43.4
37.3
24.3
20.3
29.3
36
-9.8
-9.2
-10.8
-9.7
-10.2
-9.7
-11.0
-10.3
-10.2
-11.1
-9.3
3
1a
1b
5.7
3
29.3
43.4
41.4
31.3
-8.9
31c
32a
32c
-8.8
-9.6
-9.1
17

Figures (2 and 3) shows a dramatic decrease in tumor cell viability of
human liver carcinoma cell line HepG2 following 48 hr treatments.
Increasing the concentration of compounds 10 and 16 from 0.0 mM to 50
mM resulted in an increase in the percentage of cell inhibition as seen in
the figures. The results of the cell viability tests imply that these
compounds can be used to arrest the proliferation of tumor cells.
HepG2-16
HepG2-10
Survi
ving
Fracti
on
1
.2
1
1
0
.2
1
IC50=13.6µg/ml
0
0
0
0
.8
IC50=7.09 µg/ml
.8
.6
.4
.2
0.6
0.4
0.2
0
0
60
40
20
0
60
40
20
0
Conc; µg/ml
Conc; µg/ml
Fig. 2 and 3.The cytotoxic effect of compounds 10 and 16 on HepG2 data
represent mean ± S.E.
The antiproliferative activities of the tested compounds were comparable
to doxorubicin. The most active compounds were 4, 10, 16, and 31a
which give IC values 15.8 μM, 13.6 μM, 7.09 μM and 5.7 μM
50
respectively (Fig. 4, Table 1).
18

16
14
12
10
8
6
4
2
0
HEPG2
13.6
IC
5
/
0
*
7
.09
µ
4
compound 10
compound 16
doxorubicin
Fig. 4. IC values for compounds 10 and 16 against HepG2 (μM). IC
50
50
values are presented as the mean ± SD (standard error of the mean)
obtained from three independent experiments.
2.2.2. In vivo Anti-Tumor results
To determine the in vivo effect of 3-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosylsulfanyl)-5-(4-benzyl-1-oxo-1H-phthalazin-2-ylmethyl)-
1
4
,3,4-oxadiazole 10 and 1-((4-benzyl-1-oxo-1H-phthalazin-2-yl) acetyl)-
-phenyl-thiosemicarbazide 16 on tumor cell growth, Ehrlich Ascites
Carcinoma Cells (EACs) were treated with or without 10 and 16.
Cisplatin, which is one of the most effective metallo-anticancer agents,
was used as the reference drug in this study. The relationship between
surviving fraction and drug concentration was plotted to obtain the
survival curve of Ehrlich Ascites Carcinoma Cells (EACs). The response
parameter was the IC value, which corresponds to the concentration
50
required for 50 % inhibition of cell viability.
The treatment with 10 and 16 as shown in (Fig.5) produced a significant
reduction (P≤0.05) in tumor mass on days 14 post-inoculation as
compared with cisplatin or control. These compounds inhibit the growth
19

EAC with IC values of 7.25 and 7.5μM, respectively. These compounds
50
showed slight better cytotoxicity compared to cisplatin (IC = of 9.0 μM).
50
20
18
16
14
12
10
8
6
4
2
0
18
9.16
7.50
7.25
DMSO
cisplatin
compound 16
compound 10
Fig. 5. Statistical analysis of the compounds 10 and 16 treatment on solid
tumor mass.
3
. Molecular modeling
We utilized docking simulation studies to substantiate the proposed mode
of action of our synthesized compounds and to guide the structural
modification to optimize the compounds target interactions.
Study of crystal structures of the EGFR kinase bound to different
inhibitors revealed key residues involved in binding all the inhibitors
including Leu 718, Val 726, Leu 777, Leu 788, Thr 790, Leu 844, and
Asp 855.
The tested compounds were evaluated for their binding affinity to EGFR
and their binding energies were calculated using AutodockVina® (Table
1). Comparative docking with the targets crystallized ligands was
performed to further validate the docking results and substantiate the
compounds mode of action. The results were evaluated based on binding
affinity calculation together with cluster size determination and visually
20

through possible interaction with key residues at the active site. After
docking the cluster analysis of the obtained complexes was done. The
total number of best-scoring poses analyzed was 30.The procedure
successfully reproduced experimentally observed binding modes, and the
energy scores for these structurally related binders were in the range from
-8.1- -11.1 with the experimentally most active compounds had high but
not the highest binding energies. However, with respect to cluster size
compounds 16 and 31a had the largest cluster size which indicates that
these compounds are more likely to adopt this binding mode in biological
system.
Upon computational docking the inhibitors were found predominantly at
the crystallized inhibitor same position (Fig. 6). For the most active
compounds, docking poses for compounds 10, 16 were found to be very
close to the co-crystallized inhibitor, however compounds 4, 31a
occupied the same channel but with a slightly different pose and fewer
binding interactions. The reason behind that could be attributed to the
dimension of the molecule, being larger and more flexible in compounds
10 and 16 compared to compounds 4 and 31a.
Fig. 6. Compounds 16 red, 31a green, 4 brown, 10 purple, crystallized inhibitor blue,
docked in EGFR.
21

The common amino acids involved in binding all inhibitors include Lys
45, Cys 775, Leu 788, Leu 799, Asp 800, Arg 841 and Thr 854 (Fig. 7).
7
Fig. 7. Compounds 16 red, 31a green, 4 brown, 10 purple, docked in EGFR with key
residues involved in ligand binding.
The docking of the two most active compounds, 16 and 31a was
compared to identify any crucial binding requirements for optimum
activity. Although, the two compounds showed slightly different binding
poses, they are still binding some key residues in common. Compound 16
docked well in the EGFR active site with a docking pattern that allowed
possible hydrogen bonding between the two hydrazidenitrogens and Arg
841 and Asn 842 (Fig. 8). One of the phthalazine nitrogens is forming
hydrogen bonds with Thr854, and Asp855. Other residues holding the
inhibitor tightly in the active site through hydrophobic interactions
include Lys 721, Lys 745, Met 766, Leu 777, Leu 788, Thr 790, Leu 799,
Asp 800, Leu 844, Thr 854.
22

Fig. 8. Compound 16 docked in EGFR active site with key residues involved in ligand
binding; distances in angstrom is presented by red dotted lines for potential hydrogen
bonding.
Figure 9 showing the docking pose of compound 31a in EGFR active site
which similarly to compound 16 was able to bind Arg 841 with its ester
and amide oxygen through hydrogen bonding. Compound 31a established
several interactions with residues at the active site namely, Cys 797,
Asn842, Leu 844, Thr 854 and Asp 855. The cluster size determination
also agreed with the experimental antiproliferative activity.
23

Fig. 9. Compound 31a docked in EGFR active site with key residues involved in
ligand binding; distances in angstrom is presented by red dotted lines for potential
hydrogen bonding.
Both compounds 16 and 31a occupied the three main binding regions of
EGFR active site (Fig. 10)
Compound 16 (IC 7.09µg/mL) adopts a position like that of the well-
50
known inhibitor Gefitinib. The phthalazine ring lies in the pocket formed
by Thr790, Gln791, Leu792, and Met793. The benzyl substituent is
directed toward the hydrophobic pocket formed by Arg776, Leu777,
Arg855, and Phe856. The side chain is positioned at the cleft formed by
Ala840, and Arg841.
Compound 31a (IC 5.7µg/mL) shows slightly different pose with its side
50
chain slightly directed toward the cleft where it forms a strong hydrogen
bonding with its carboxylate and Arg841. The two most active
compounds identified Arg841 as an important residue in the cleft region
24

for binding of more potent inhibitors. Substitution on the side chain with
branched or bulky rings resulted in less active compounds due to steric
effect with residues in the cleft region. A chain length of 5-6 atoms
containing hydrogen donor or hydrogen acceptor atoms was found
optimum for this type of compounds for better binding with the cleft
region.
Possible future optimization would focus on the benzyl moiety for a
better binding with the hydrophobic pocket and better positioning of the
phthalazine ring in its binding pocket.
Fig. 10. Compound 16 and 31a docked in EGFR active site and showing interactions
with the three major binding pockets.
Molecular docking studies revealed that our synthesized compounds are
potential good inhibitors for EGFR, having similar binding mode to the
co-crystallized inhibitors. Compounds with a shorter side chain exhibited
slightly different conformations while those with longer side chains had
similar conformations to the co-crystallized inhibitors. The most active
compounds were able to set up stronger interactions with the active site
through hydrogen bonding especially with Arg 841.
25

4. Conclusion
In conclusion, a new heterocyclic systems of phthalazines attached to
,3,4-oxadiazoles, 1,2,4-triazoles, amino acid esters and amines were
1
synthesized and characterized using NMR, IR and elemental analysis.
The synthesized compounds showed good activity against hepatocellular
carcinoma both in vitro and in vivo. Molecular docking showed that the
synthesized phthalazine derivatives are good inhibitors for EGFR. We
were finally able to identify Compounds 4, 10, 16, 31a as new
phthalazine lead compounds added to our previous indole leads for EGFR
inhibition. These compounds will be further optimized and tested to get a
hit suitable for preclinical development.
5. Experimental Part
General procedures
Melting points were measured with a Buchi 510 melting point apparatus
in open capillaries and are uncorrected. Thin layer chromatography
(
TLC) was done on silica gel 60 F aluminium. The spots were detected
254
by UV lamp. Microanalysis was performed on Flash EA-1112 insrtument
1
at the microanalytical laboratory, Cairo University, Giza, Egypt. H-NMR
13
and C-NMR spectra were measured on Bruker spectrometer operating at
1
13
(
300 and 400 MHz for H and 75-100 MHz for C). The IR spectra were
recorded on Perkin Elmer FTIR spectrometer using KBr.
5
5
.1. Chemistry
.1.1. Synthesis of 2-(4-benzyl-1-oxo-1H-phthalazin-2-yl) acetic acid
(2)
To a solution of ester 1 (0.01 mol) in ethanol (95%, 25 mL), sodium
hydroxide (0.01 mol /0.5 mL H O) was added. The reaction mixture was
2
26

stirred at room temperature for 3 hrs. Then the mixture was acidified with
Conc. HCl, the formed precipitate was filtered off, washed with water,
dried and crystalized from methanol to give 2 as white crystals.
o
1
Yield: 92.3%. m.p. 196-198 C. H-NMR (DMSO-d , 300 MHz) δ 4.32 (s,
6
2
1
5
1
H, CH Ph ), 4.87 (s, 2 H, NCH COOH ), 7.16-8.29 (m, 9 H, ArH),
2
2
13
3.04 (brs, 1 H, OH ); C-NMR (DMSO-d , 75 MHz) δ 37.5 (CH Ph),
6
2
2.4 (NCH ), 125.9, 126.3, 126.5, 127.3, 128.3, 128.5, 128.8, 131.8,
2
33.5, 138.0 (12 Caromatic), 145.1 (C=NPhth.), 158.4 (C=OPhth.), 169.4
-1
(
C=O ). IR (KBr, ν cm ) 1625 (C=O ), 1750 (C=O ), 2500 - 3429
acid
Phth.
acid
(OHCarboxylic), EILR-MS: m/z (%) 65 (20.8), 91 (99.9), 235 (15.5), 250
+.
(
99.9), 294 (60 M ). Anal. Calcd for C H N O (294.10): C, 69.38; H,
17
14
2
3
4.79; N, 9.52; Found C, 69.52; H, 4.74; N, 9.56.
5.1.2. Synthesis of 2-(4-benzyl-1-oxo-1H-phthalazin-2-
yl)acetohydrazide (3)
A mixture of ethyl 2-(4-benzyl-1-oxophthalazin-2(1H)-yl) acetate (1)
0.01 mol) and hydrazine hydrate (0.04 mol) in ethanol (30 mL) was
(
refluxed for 4 hrs. On cooling, a solid product was formed, filtered off
and recrystallized from ethanol to give 3 as faint yellow crystals.
o
1
Yield: 80.51 %, m.p. 184-186 C; H-NMR (DMSO-d , 300 MHz) δ 4.30
6
(
s, 4 H, CH Ph, -NH ), 4.77 (s, 2 H, NCH -CO), 7.18-8.28 (m, 9 H,
2
2
2
13
ArH), 9.31 (s, 1 H, NH); C-NMR (DMSO-d , 75 MHz) δ 37.6 (CH Ph),
6
2
5
1
2.1 (NCH CO), 125.7, 126.3, 126.4, 127.6, 128.3, 128.5, 128.9, 131.5,
2
33.2, 138.0 (12Caromatic), 144.9 (C=N .), 158.5 (C=OPhth.), 166.4
Phth
-1
(
C=OHydrazide). IR (KBr, ν cm ) 1587 (C=OPhth.), 1658 (C=OHydrazide), 3290,
3
300 (NHNH ), EILR-MS: m/z (%) 65 (23), 91 (99.9), 249 (99.9), 277
2
+.
(
99.9), 293 (13.4), 308 (9.2 M ). Anal. Calcd forC H N O (308.13): C,
17
16
4
2
66.22; H, 5.23; N, 18.17; Found C, 66.32; H, 5.28; N, 18.12.
27

5.1.3. Synthesis 5-(4-benzyl-1-oxo-1H-phthalazin-2-ylmethyl)-1, 3, 4-
oxadiazol-2-thione (4)
To a solution of hydrazide 3 (0.01 mol) in ethanol (25 mL) containing
KOH (0.015 mol/0.5 mL H O), carbon disulphide (0.05 mol) was added
2
and the reaction mixture was refluxed for 3 hrs. The mixture was left to
cool to room temperature, and then acidified with Conc. HCl. The formed
precipitate was filtered off, washed with water dried and crystalized from
absolute ethanol to give 4 as white crystals.
o
1
Yield: 95.6 %, m.p. 242-244 C. H-NMR (DMSO-d , 300 MHz) δ 4.32
6
(
s, 2 H, CH Ph), 5.49 (s, 2 H, NCH ), 7.19-8.31 (m, 9 H, ArH), 14.60 (br,
2
2
13
1
H, NHOxad.); C-NMR (DMSO-d ,75 MHz) δ 37.5 (CH Ph), 45.2
6
2
(
NCH ), 125.8, 126.0, 126.3, 126.5, 127.1, 128.3, 128.5, 128.7, 128.8,
2
132.1, 133.8, 137.6 (12 Caromatic), 146.3 (C=NPhth.), 154.8 (C-5Oxad.), 158.2
-1
(
C=OPhth.), 169.4 (C=S). IR (KBr, ν cm ): 1633 (C=OPhth.), 3437 (NH).
EILR-MS: m/z (%) 65 (24.4), 91 (100), 249 (15.9), 277 (8.2), 350 (29.9
+.
M ). Anal. Calcd for C H N O S (350.08): C, 61.70; H, 4.03; N, 15.99;
18
14
4
2
S, 9.15; Found C, 61.73; H, 3.98; N, 16.02; S, 9.06.
5
1
.1.4. General procedure for preparation of compounds 5, 6 and (9-
2)
To a solution of oxadiazolethione 4 (0.01 mol) in acetone (20 mL),
potassium carbonate (0.015 mol) was added and stirred for 1 hr, then, the
appropriate alkyl or glycosyl bromide (7,8) (0.011 mol) was added. The
reaction mixture was stirred at room temperature overnight. The mixture
was filtered off and the solvent was removed. The products were purified
by crystallization from methanol in case of 5 and 6 or using column
chromatography (ethyl acetate/petroleum ether 4:7) in case of 9-12.
28

5
1
.1.4.1. 2-Allylsulfanyl-5-(4-benzyl-1-oxo-1H-phthalazin-2-ylmethyl)-
,3,4-oxadiazole (5)
o
1
Yield: 99.54 % as white crystals, m.p. 128 C. H-NMR (DMSO-d , 300
6
2
MHz) δ 3.87 (dd, 2 H, J7.8 Hz, J 0.9 Hz, SCH ), 4.31 ( s, 2 H, CH Ph),
2
2
2
5
.07 (dd, 1 H, J 9.0 Hz, J 0.9 Hz, CH=CHH), 5.26 (dd, 1 H, Jtrans≈
cis
2
1
6.0Hz, J 1.2 Hz, CH=CHH), 5.62 (s, 2 H, NCH ), 5.92-5.98 ( m, 1 H,
2
13
CH=CH ), 7.25-8.28 ( m, 9 H, ArH); C-NMR (DMSO-d , 75 MHz) δ
2
6
3
1
4.5 (SCH ), 37.4 (CH Ph), 45.1 (NCH ), 119.2, 126.0, 126.4, 126.5,
2
2
2
27.2, 128.3, 128.5, 128.7, 132.1, 132.4, 133.8, 137.7 (14 Caromatic
+olefinic),146.2 (C=NPhth.),158.2 (C=OPhth.),163.6,163.7 (C-2Oxad.,C-
-1
5Oxad.).IR (KBr, ν cm ) 1644 (C=OPhth.). EILR-MS: m/z (%) 65 (17.7),91
+
(
99.9),235 (26.4),249 (99.9),317 (99.9),349 (9.7),390 (99.9 M ).
Anal.Calcd For C H N O S (390.12): C, 64.60; H, 4.65; N, 14.35; S,
21
18
4
2
8.21 Found C, 64.85; H, 4.58; N, 14.44; S, 8.19.
5.1.4.2. 2-Benzylsulfanyl-5-(4-benzyl-1-oxo-1H-phthalazin-2-
ylmethyl)-1, 3,4-oxadiazole (6)
o
1
Yield: 94.69 % as white crystals, m.p. 94-97 C. H-NMR (DMSO-d , 300
6
MHz) δ 4.30 (s, 2 H, CH Ph), 4.46 (s, 2 H, SCH ), 5.62 (s, 2 H, NCH ),
2
2
2
1
3
7
3
1
.17-8.31 (m, 14 H, ArH); C-NMR (DMSO-d , 75 MHz) δ 35.8 (SCH ),
6
2
7.4 (CH Ph), 45.1 (NCH ), 126.0, 126.5, 127.2, 127.6, 128.3, 128.4,
2
2
28.5, 128.7, 128.8, 132.1, 133.8, 136.4, 137.7 (18 Caromatic), 146.2
(C=NPhth.), 158.2 (C=OPhth.), 163.62, 163.69 (C-5Oxad., C-2Oxad.). IR (KBr,
-1
ν cm ) 1652 (C=OPhth.).EILR-MS: m/z (%) 65 (29), 91 (99.9), 249 (99.9),
+
3
17 (65.7), 349 (14.9), 440 (99.9 M ). Anal. Calcd forC H N O S
25
20
4
2
(440.13): C, 68.16; H, 4.58; N, 12.72; S, 7.28 Found C, 68.11; H, 4.63;
N, 12.67; S, 7.32.
29