untitled


ISSN	215

Design,	
new	V600

Kamelia	M
Usama	Ma

1	Pharmaceutica
2	Pharmaceutica
3	Research	Unit,	

*	Corresponding
Tel.:	+20.02.383

	
	

	

ARTICLE	IN

DOI:	10.5155/e

Received:	22	Oc
Received	in	rev
Accepted:	05	De
Published	onlin
Printed:	31	Mar

KEYWORDS
V600EBRAF	
Melanoma	
Colon	cancer	
Ovarian	cancer	
Thyroid	cancer
Pyridopyrazino

	
1.	Introduct
	

Cancer	is
properties	 a
aggressive,	in
from	inter‐	a
can	 be	 orig
cytokines,	 g
generated	by

G	 protei
receptor	 tyr
triggers	of	in
[2].	 Rapidly
component	
carcinogenes
extracellular
translocated
biological	 pr
BRAF,	 and	 C
involved	 in	
cancer	 (35‐
(14%),	and	o

53‐2249	(Print)	

	

	

	

synthesis
0EBRAF	in

Mahmoud	Am
agdi	Ammar

al	Chemistry	Depar
al	Chemistry	Depar
	Saco	Pharma	Com

g	author	at:	Pharm
376751.	Fax:	+20.02

FORMATION	

	
eurjchem.7.1.19‐29

ctober	2015	
vised	form:	02	Dece
ecember	2015	
ne:	31	March	2016	
rch	2016	

S	

r	
one	

tion	

s	a	group	of	dis
are	 involved	 r
nvasive,	and	po
and	intra‐cellula
ginated	 from	
growth	 factor
y	oncogenes	[1]
ins,	 receptors	
rosine	 kinases	
ntracellular	sign
y	 growing	 fibr
of	 RTK	 that	
sis.	Phosphoryl
r	 signal‐regul
d	 to	nucleus	 to	
rocess	 [3].	 Thre
CRAF	 were	 kn
many	 cancer	 c
70%),	 colorec
ovarian	cancer	(

/	ISSN	2153‐225
ht

Europ

s,	antican
nhibitors	

min	1,	Ossam
r	2,*	and	Moh

rtment,	Faculty	of	P
rtment,	Faculty	of	P
mpany,	6th	of	Octob

aceutical	Chemistry
2.38334379.	E‐mai

9.1346	

ember	2015	

seases	in	which
related	 to	 the	
ossibility	of	met
ar	communicat
false	 messen

rs	 or	 false	 s
].	
tyrosine	 kinas
(NRTK)	 are	 co
nal	transduction
rosarcoma	 (RA
included	 the

lation	of	RAF	re
ated	 kinase	
trigger	 cell	pro
ee	 isoforms	 of	
own	 [4].	 BRAF
cells	 as	 melan
tal	 cancer	 (5‐
(30%)	[5‐11].	

European	Journ

Europ
57	(Online)		20
ttp://dx.doi.org

pean	Jo
Journal	web

cer	evalu
derived	fr

ma	Metwally
hamed	Most

Pharmacy,	Cairo	Un
Pharmacy,	Ahram	C
er	City,	Egypt	

ry	Department,	Fac
l	address:	usama_a

	 	

	 	

	 ABSTRACT
	 Design	and	sy
described.	Th
(LOXIMVI),	ov
and	 colon	 ce
compounds	w
activity	again
V600EBRAF	wa
tested	 compo
kinase	inhibit
6b	were	poten
melanoma,	ov
with	different
potent	 V600EB
cancer	types	s

Cite	this:	Eur.

h,	three	characte
cancer	 cells:	
tastasis	[1].	It	r
tion	disorders,	w
gers	 by	 horm
signal	 transdu

ses	 (RTK)	 and
onsidered	 impo
n	of	cell	prolifer
AF)	 is	 conside
	 key	 mediato
esults	in	activat
(ERK)	 that	

oliferation	and	
RAF	 kinases;	 A
F	 mutation	 is	 h
oma	 (66%),	 th
‐20%),	 liver	 c

al	of	Chemistry	7

	

pean	Journal	of	C
016	Atlanta	Pub
/10.5155/eurjch

	

ournal	
bpage:	www.

	

ation	and
rom	pyrid

y	El‐Badry	2,
tafa	Abdalla	

niversity,	Cairo,	115
Canadian	University

culty	of	Pharmacy,	A
acu@hotmail.com		(

ynthesis	of	some
he	cytotoxic	activ
varian	cell	 line	
ell	 lines	 (HT29	
were	 active	 and	
st	all	tested	cell	
s	performed	for
ounds	 was	 estab
tion	assay	and	m
nt	inhibitors	for
varian	and	thyro
t	substituents	at
RAF	 inhibitors	
such	as	melanom

.	J.	Chem.	2016,	

eristic	
being	
results	
which	
mones,	
uction	

d	 non‐
ortant	
ration	
red	 a	
ors	 of	
tion	of	
was	
other	
ARAF,	
highly	
hyroid	
cancer	

mut
sub
loop
dur
to	 t
ren
gro
crit

cate
whi
con
targ

targ
only
pha
SB5
in	 v
pro

7	(1)	(2016)	19‐

Chemistry	
lishing	House	LL
hem.7.1.19‐29.1

of	Che
.eurjchem.co

d	molecul
dopyrazin

,	Doaa	Ezzat
3	

562,	Egypt	
ty,	Giza,	12566,	Egy

Ahram	Canadian	U
(U.M.	Ammar).	

e	new	pyridopyra
vities	of	 the	syn
(OVCAR3),	 thyr
and	 HCT116)	
compound	 3d	w
lines.	In	additio
r	all	synthesized
blished	 with	 act
molecular	dockin
r	V600EBRAF	kinas
oid	cancer.	The	n
t	C‐3	or	 fused	w
and	 exhibited	
ma,	ovarian,	thyr

7(1),	19‐29	

The	 most	 com
tations	 is	 V600E
bstitution	 (val	
p)	 which	 is	 ad
ring	BRAF	activ
the	 phosphory
dering	 V600EBRA
wth	 [5,6,12,13
tical	therapeutic
BRAF	 kinase	
egories;	 type	 I
ich	 bind	 to	
nformation.	Typ
get	that	enzyme
Biarylurea	 de
get	V600EBRAF	in
y.	 It	 has	 no	 si
ase	 II	 clinical	
590885,	is	cons
vitro,	 but	 had	p
ofiles	in	vivo	[20
	

‐29	

LC	‐	All	rights	re
1346	

emistry
m	

ar	dockin
none	

t	Abdel	Rahm

ypt	

University,	Giza,	125

azinone	derivati
nthesized	compo
oid	 cell	 lines	 (C
were	 investigat
was	 the	most	 a
on,	in	vitro	kinas
d	compounds.	Fu
tive	 site	 of	 V600E
g	revealed	that,	
se	enzyme	invol
newly	synthesize
with	 triazine	het
promising	 cyto
roid	and	colon	ca

mmon	 mutatio
BRAF.	 V600EBRA
600	 is	 replace
djacent	 to	 pho
vation.	The	pola
ylated	 serine	 in
AF	 active	 and	
3].	 Therefore,	 B
c	target	in	diffe
enzyme	 inhibi
I	 inhibitors,	 su
the	 BRAF	 kin
pe	II	 inhibitors,
e	in	its	inactive	
rivative,	 sorafe
n	melanoma	an
ignificant	 resp
trials	 [15‐19].	
sidered	a	poten
poor	 pharmaco
0].		

served	‐	Printed

y	

ng	of		

man	1,		

566,	Egypt.		

ives	as	anti‐prol
ounds	against	m
CAL62,	FTC133,	
ted.	 Results	 rev
ctive	 one.	 It	 exh
se	assay	against	
urthermore,	mol
EBRAF	 kinase	 do
compounds	1,	3
lved	in	number	o
ed	pyridopyrazin
terocycle	at	C‐3	
otoxic	 activities	
ancer.	

n	 in	 BRAF	 am
AF	 is	 a	 single	
ed	 with	 glu	 in	
osphorylation	 t
arity	of	glutamic
n	 activation	 lo
resulted	 in	 un
BRAF	 kinase	 is
rent	types	of	ca
itors	 are	 class
uch	 as	 SB5908
nase	 enzyme	
	such	as	sorafe
conformation.	
enib	 was	 the	 f
nd	exhibited	in	
onse	 in	 neithe
Triarylimidaz
t	type	I	inhibito
okinetic	 and	 ph

d	in	the	USA	

	

	

	

iferative	agents	
melanoma	cell	 lin
BCPAP	and	ML
vealed	 that	 mo
hibited	 promisin
both	WTBRAF	an
lecular	docking	
omain.	 Results	
3d,	e,	h,	i,	5d,	e	an
of	cancer	types	a
nones	substitute
and	C‐4	afforde
against	 differe

mong	 95	 BRAF
base	 missense
the	 activation
target	 ser	 599
c	acid	is	similar
op	 that	 led	 to
ncontrolled	 cell
s	 considered	 a
ancer	[14].	
sified	 into	 two
885	 (Figure	 1),
in	 its	 active

enib	(Figure	1),

first	 drug	 that
vitro	inhibition
er	 phase	 I	 nor
zole	 derivative,
or	of	V600EBRAF
harmcodynamic

is
ne
1)
ost
ng
nd
of
of
nd
as
ed
ed
nt

F	
e	
n	
9	
r	
o	
l	
a	

o	
,	
e	
,	

t	
n	
r	
,	
F	
c	



20	 Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	
	

	

	

SB590885	 Sorafenib	
	 	

S
N
H

F

O O

N

S

N

N NH2

F

F

	 	
Dabrafenib	 Vemurafenib	

	

	
RAF265	 CCT23905	

Figure	1.	BRAF	kinase	inhibitors.	
	
	
However,	 derivatives	 of	 pyrimidine,	 azaindole	 and	

benzimidazole	 such	 as	 darafenib,	 vemurafenib	 and	 RAF265,	
respectively,	 were	 involved	 in	 clinical	 trials	 as	 V600EBRAF	
kinase	inhibitors	[21]	(Figure	1).	

Pyridopyrazinone	 derivatives	 had	 considerable	 thera‐
peutic	 activities	 in	 the	 treatment	 of	 different	 cancer	 types	 as	
melanoma,	 thyroid,	 colon,	 and	 ovarian	 cancer	 through	 their	
selective	 inhibition	 of	 V600EBRAF	 [21].	 Pyridopyrazinone	
derivatives	 were	 considered	 type	 II	 inhibitors	 of	 V600EBRAF,	
both	 in	 vitro	 and	 in	 vivo.	 Pyridopyrazinone	 activity	 was	
exhibited	through	selective	binding	with	Cys	532	backbone	of	
hinge	 region	 of	 V600EBRAF	 by	 hydrogen	 bonding	 [14,22].	
CCT23905	 was	 the	 first	 derivative	 of	 pyridopyrazinones	
described	for	inhibiting	activity	towards	oncogenic	BRAF	[14].	

Accordingly,	new	pyridopyrazinones	were	designed	 to	be	
substituted	 with	 different	 substituents	 at	 C‐3	 or	 fused	 with	
another	 heterocycle	 at	 C‐3	 and	 C‐4	 to	 afford	 an	 additional	
binding	sites	that	affect	mode	of	interaction.	These	compounds	
were	 evaluated	 for	 their	 cytotoxic	 activity	 against	 different	
cancer	 cell	 lines.	 In	 addition,	 in	 vitro	 kinase	 assay	 was	
performed	 to	 evaluate	 their	 BRAF	 inhibition	 activities.	
Moreover,	a	molecular	docking	study	was	performed	to	assign	
the	binding	mode	with	active	site	of	BRAF	enzyme	model.	
	
2.	Experimental	
	
2.1.	Chemistry	
	

Melting	 points	 were	 determined	 by	 open	 capillary	 tube	
method	using	Gallen	Kamp	melting	point	apparatus	MFB‐595‐
010M	(Gallen	Kamp,	London,	England)	and	were	uncorrected.	
Microanalysis	 was	 carried	 out	 at	 The	 Regional	 Center	 for	
Mycology	 and	Biotechnology,	 Al‐Azhar	 University.	 IR	 Spectra	
were	recorded	as	potassium	bromide	discs	on	Schimadzu	FT‐
IR	 8400S	 spectrophotometer	 (Shimadzu,	 Kyoto,	 Japan)	 and	
expressed	in	wavenumber	(ν)	cm‐1.	The	1H	NMR	spectra	were	
recorded	 on	 a	 Bruker	 NMR	 spectrometer	 at	 400	 MHz	 and	
Varian	 Mercury	 VX‐300	 NMR	 spectrometer	 at	 300	 MHz.	
Chemical	 Shifts	 are	 quoted	 in	 δ	 as	 parts	 per	 million	 (ppm)	
downfield	from	tetramethylsilane	(TMS)	as	internal	standard.	
Mass	 spectra	 were	 recorded	 using	 Shimadzu	 Gas	 Chroma‐

tograph	Mass	spectrometer	QP	1000	Ex	(Shimadzu).	TLC	was	
carried	out	using	Art.	DC‐Plastikfolien,	Kieselgel	60F254	sheets	
(Merck,	 Darmstadt,	 Germany),	 the	 developing	 solvent	 was	
chloroform/methanol	(9:1,	v:v)	and	the	spots	were	visualized	
at	 366	 and	 254	 nm	 by	 UV	 Vilber	 Lourmat	 77202	 (Vilber,	
Marne	La	Vallee,	France).		
	
2.1.1.	Synthesis	of	3‐methylpyrido[2,3‐b]pyrazin‐2(1H)one	
(1)	
	

Ethyl	pyruvate	(2.90	g,	2.77	mL,	0.025	mol)	was	added	to	a	
solution	of	2,3‐diaminopyridine	(2.75	g,	0.025	mol)	in	ethanol	
(30	mL)	 and	 refluxed	 for	 10	h.	Reaction	mixture	was	 cooled,	
filtered,	 washed	 with	 ethanol	 (5	 mL)	 and	 dried.	 The	 crude	
product	was	crystalized	from	ethanol	(Scheme	1).	Yield:	59%.	
M.p.:	 278‐280	 °C.	 FT‐IR	 (KBr,	,	 cm‐1):	 3186	 (NH),	 3018	 (CH	
Ar),	 2943,	 2839	 (CH	 Aliph),	 1685	 (C=O),	 1641,	 1631,	 1597,	
1559,	1546,	1527	(C=N,	NH,	C=C).	 1H	NMR	(300	MHz,	DMSO‐
d6,	 δ,	 ppm):	 2.41	 (s,	 3H,	 CH3),	 7.48	 (d,	 J=	 8.4	Hz,	 1H,	H‐8Ar),	
7.67	(t,	1H,	H‐7Ar),	8.11	(d,	J=	7.6	Hz,	1H,	H‐6Ar),	12.44	(s,	1H,	
NH,	exchanged	with	D2O).	Anal.	calcd.	for	C8H7N3O:	C,	59.62;	H,	
4.38;	N,	26.07.	Found:	C,	59.90;	H,	4.14;	N,	26.73%.	
	
2.1.2.	Synthesis	of	3‐bromomethylpyrido[2,3‐b]pyrazin‐2	
(1H)one	(2)	
	

Compound	 1	 (3.22	 g,	 0.02	 mol)	 was	 dissolved	 in	 glacial	
acetic	 acid	 (10	 mL)	 and	 anhydrous	 sodium	 acetate	 (1.64	 g,	
0.02	mol)	 was	 added.	 Bromine	 (3.04	 g,	 0.99	mL,	 0.019	mol)	
was	 added	 dropwise	 and	 the	 mixture	 was	 heated	 on	 steam	
bath	 for	 30	 min.	 then	 cooled.	 The	 formed	 precipitate	 was	
filtered,	washed	with	glacial	acetic	acid	(5	mL)	and	dried.	The	
crude	product	was	crystalized	from	ethanol	(Scheme	1).	Yield:	
43%.	M.p.:	237‐239	°C.	FT‐IR	(KBr,	,	cm‐1):	3414	(NH),	3055	
(CH	Ar),	2980,	2883	(CH	Aliph),	1670	(C=O),	1647,	1616,	1558,	
1544,	 1508	 (C=N,	 NH,	 C=C).	 1H	 NMR	 (300	MHz,	 DMSO‐d6,	 δ,	
ppm):	3.10	(s,	2H,	CH2),	7.40	(d,	J	=	7.4	Hz,	1H,	H‐8Ar),	7.61	(t,	
1H,	H‐7Ar),	 8.35	 (d,	 J	=	6.1	Hz,	1H,	H‐6Ar),	 11.50	 (s,	 1H,	NH,	
exchanged	with	 D2O).	MS	 (EI,	m/z	 (%)):	 240	 (M+,	 0.53),	 242	
(M++2,	0.67).	Anal.	 calcd.	 for	C8H6BrN3O:	C,	 40.03;	H,	2.52;	N,	
17.50.	Found:	C,	40.13;	H,	2.47;	N,	17.68%.	



Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	 21	
 

	

N N

H
N O

CH3N NH2

NH2

O

C2H5O O

CH3 N N

H
N O

Br

N N

H
N O

N
R'R

N N

H
N O

H
N
NH2

N N

H
N O

HN
N

RN N

H
N O

NH
NR

N N

H
N O

H
N
R

R'

+

1 2

3a-j

4

5a-e6a-d

7a-c

3a R,R'= C2H5

3b NRR'=

3c NRR'=

3d R= H, R'= C6H5
3e R= H, R'= 2-CH3C6H4
3f R= H, R'= 3-CH3C6H4
3g R= H, R'= 4-CH3C6H4
3h R= H, R'= 4-ClC6H4
3i R= H, R'= 4-BrC6H4
3j R= C6H5, R'= C6H5

N

N O

N

O

O

N

O

O

N

O

O

7a ,7c,7b

5a R= H, R'= C6H5
5b R= H, R'= 4-ClC6H4
5c R= H, R'= 4-NO2C6H4
5d R= H, R'= 4-CH3OC6H4
5e R= CH3, R'= C6H5

6a R= C6H5
6b R= 4-ClC6H4
6c R= 4-NO2C6H4
6d R= 4-CH3OC6H4

(i) (ii)

(iii)
(iv)

(v)

(vi)

(vii)

	
	
Reagents	and	conditions:	(i)	Ethanol,	reflux,	10	h.	(ii)	Bromine,	glacial	acetic	acid,	anhydrous	sodium	acetate,	reflux,	30	min.	(iii)	Appropriate	amine,	ethanol,	
sodium	iodide,	reflux,	10	h.	(iv)	Hydrazine	hydrate,	ethanol,	reflux,	9	h.	(v)	Appropriate	aldehyde	or	ketone,	glacial	acetic	acid,	reflux,	8	h.	(vi)	Bromine,	glacial	
acetic	acid,	sodium	acetate,	room	temp.,	3	h.	(vii)	Appropriate	cyclic	acid	anhydride,	glacial	acetic	acid,	reflux,	6	h.	
	

Scheme	1
	
	
2.1.3.	General	procedure	for	synthesis	of	3‐substituted	
methylpyrido[2,3‐b]pyrazin‐2(1H)ones	(3a‐j)	
	

Appropriate	amine	(0.02	mol)	was	added	 to	a	 solution	of	
compound	2	 (2.4	 g,	 0.01	mol)	 in	 ethanol	 (30	mL)	 containing	
sodium	 iodide	 (0.15	g,	0.001	mol)	 and	refluxed	 for	10	h.	The	
reaction	 mixture	 was	 cooled,	 formed	 precipitate	 filtered,	
washed	with	ethanol	(5	mL)	and	dried.	The	crude	product	was	
crystalized	from	ethanol	(Scheme	1).	

3‐(N,N‐Diethylamino)methylpyrido[2,3‐b]pyrazin‐2(1H)one	
(3a):	Yield:	81%.	M.p.:	262‐263	°C.	FT‐IR	(KBr,	,	cm‐1):	3238	
(NH),	3051	(CH	Ar),	2924,	2852	(CH	Aliph),	1672	(C=O),	1637,	
1624,	 1612,	 1593,	 1508	 (C=N,	 NH,	 C=C).	 1H	NMR	 (300	MHz,	
DMSO‐d6,	 δ,	 ppm):	 1.41	 (t,	 6H,	 2xCH2CH3),	 2.29	 (q,	 4H,	 2x	
CH2CH3),	2.43	(s,	2H,	CH2),	7.11	(d,	J	=	7.4	Hz,	1H,	H‐8Ar),	7.30	
(t,	1H,	H‐7Ar),	8.21	(d,	J	=	6.4	Hz,	1H,	H‐6Ar),	10.80	(s,	1H,	NH,	
exchanged	with	D2O).	 Anal.	 calcd.	 for	 C12H16N4O:	 C,	 62.05;	H,	
6.94;	N,	24.12.	Found:	C,	62.18;	H,	6.97;	N,	24.13%.	

3‐(Piperidin‐1‐yl)methylpyrido[2,3‐b]pyrazin‐2(1H)one	
(3b):	Yield:	80%.	M.p.:	277‐279	°C.	FT‐IR	(KBr,	,	cm‐1):	3387	
(NH),	3066	(CH	Ar),	2964,	2866	(CH	Aliph),	1700	(C=O),	1649,	
1612,	 1591,	 1508,	 1500	 (C=N,	 NH,	 C=C).	 1H	NMR	 (300	MHz,	
DMSO‐d6,	 δ,	 ppm):	 2.06‐2.32	 (m,	 10H,	 piperidine	H),	 2.51	 (s,	
2H,	 CH2),	 7.31	 (d,	 J	=	8.0	Hz,	 1H,	H‐8Ar),	 7.53	 (t,	 1H,	H‐7Ar),	
8.22	 (d,	 J	 =	 6.6	 Hz,	 1H,	 H‐6Ar),	 11.13	 (s,	 1H,	 NH,	 exchanged	
with	 D2O).	 Anal.	 calcd.	 for	 C13H16N4O:	 C,	 63.91;	 H,	 6.60;	 N,	
22.93.	Found:	C,	63.98;	H,	6.64;	N,	23.10%.	

3‐(Morpholin‐4‐yl)methylpyrido[2,3‐b]pyrazin‐2(1H)one	
(3c):	Yield:	74%.	M.p.:	234‐235	°C.	FT‐IR	(KBr,	,	 cm‐1):	3394	
(NH),	3059	(CH	Ar),	2920,	2840	(CH	Aliph),	1674	(C=O),	1640,	
1618,	 1593,	 1580,	 1540	 (C=N,	 NH,	 C=C).	 1H	NMR	 (300	MHz,	
DMSO‐d6,	δ,	ppm):	2.04	(t,	4H,	morpholine	H),	2.41	(s,	2H,	CH2),	
3.30	(t,	4H,	morpholine	H),	7.33	(d,	J	=	8.0	Hz,	1H,	H‐8Ar),	7.75	

(t,	1H,	H‐7Ar),	8.12	(d,	J	=	7.4	Hz,	1H,	H‐6Ar),	10.89	(s,	1H,	NH,	
exchanged	with	D2O).	Anal.	 calcd.	 for	C12H14N4O2:	C,	58.53;	H,	
5.73;	N,	22.75.	Found:	C,	58.61;	H,	5.71;	N,	22.92%.	

3‐Phenylaminomethylpyrido[2,3‐b]pyrazin‐2(1H)one	(3d):	
Yield:	60%.	M.p.:	248‐250	°C.	FT‐IR	(KBr,	,	cm‐1):	3404,	3385	
(2NH),	 3049	 (CH	 Ar),	 2926,	 2852	 (CH	 Aliph),	 1672	 (C=O),	
1593,	 1560,	 1521,	 1508	 (C=N,	 NH,	 C=C).	 1H	NMR	 (400	MHz,	
DMSO‐d6,	δ,	ppm):	3.10	(s,	2H,	CH2),	6.52‐7.10	(m,	5H,	H‐2’,	3’,	
4’,	5’,	6’	Ar),	7.21	(d,	J	=	9.6	Hz,	1H,	H‐8Ar),	7.50	(t,	1H,	H‐7Ar),	
8.17	 (d,	 J	=	 8.2	 Hz,	 1H,	 H‐6Ar),	 10.62	 (s,	 1H,	 NH,	 exchanged	
with	D2O),	11.51	(s,	1H,	NH,	exchanged	with	D2O).	Anal.	calcd.	
for	C14H12N4O:	C,	66.65;	H,	4.79;	N,	22.21.	Found:	C,	66.83;	H,	
4.74;	N,	22.37%.	

3‐(2‐Methylphenyl)aminomethylpyrido[2,3‐b]pyrazin‐2(1H)	
one	 (3e):	 Yield:	 64%.	M.p.:	 296‐298	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	
3414,	3385	(2NH),	3049	(CH	Ar),	2924,	2854	(CH	Aliph),	1672	
(C=O),	1649,	1622,	1593,	1570,	1521	(C=N,	NH,	C=C).	1H	NMR	
(400	MHz,	 DMSO‐d6,	 δ,	 ppm):	 2.13	 (s,	 3H,	 CH3),	 3.11	 (s,	 2H,	
CH2),	6.30‐7.00	(m,	4H,	H‐3’,	4’,	5’,	6’	Ar),	7.21	(d,	J	=	7.2	Hz,	1H,	
H‐8Ar),	 7.50	 (t,	 1H,	 H‐7Ar),	 8.16	 (d,	 J	 =	 8.4	 Hz,	 1H,	 H‐6Ar),	
10.80	 (s,	 1H,	 NH,	 exchanged	 with	 D2O),	 11.44	 (s,	 1H,	 NH,	
exchanged	with	D2O).	 Anal.	 calcd.	 for	 C15H14N4O:	 C,	 67.65;	H,	
5.30;	N,	21.04.	Found:	C,	67.82;	H,	5.32;	N,	21.08%.	

3‐(3‐Methylphenyl)aminomethylpyrido[2,3‐b]pyrazin‐2(1H)	
one	 (3f):	 Yield:	 57%.	 M.p.:	 258‐260	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	
3414,	3388	(2NH),	3057	(CH	Ar),	2922,	2852	(CH	Aliph),	1674	
(C=O),	1622,	1593,	1558,	1518	(C=N,	NH,	C=C).	1H	NMR	(400	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 2.15	 (s,	 3H,	 CH3),	 3.32	 (s,	 2H,	 CH2),	
6.12‐6.99	(m,	4H,	H‐2’,	4’,	5’,	6’	Ar),	7.20	(d,	J	=	6.3	Hz,	1H,	H‐
8Ar),	7.50	(t,	1H,	H‐7Ar),	8.19	(d,	J	=	8.3	Hz,	1H,	H‐6Ar),	10.81	
(s,	 1H,	 NH,	 exchanged	 with	 D2O),	 11.50	 (s,	 1H,	 H	 NH,	
exchanged	with	D2O).	 Anal.	 calcd.	 for	 C15H14N4O:	 C,	 67.65;	H,	
5.30;	N,	21.04.	Found:	C,	67.84;	H,	5.34;	N,	21.06%.	



22	 Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	
	

3‐(4‐Methylphenyl)aminomethylpyrido[2,3‐b]pyrazin‐2(1H)	
one	 (3g):	 Yield:	 73%.	M.p.:	 284‐286	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	
3369,	3350	(2NH),	3051	(CH	Ar),	2926,	2856	(CH	Aliph),	1672	
(C=O),	1612,	1591,	1544,	1514	(C=N,	NH,	C=C).	 1H	NMR	(300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 2.10	 (s,	 3H,	 CH3),	 2.41	 (s,	 2H,	 CH2),	
6.30‐6.83	(m,	4H,	H‐2’,	3’,	5’,	6’	Ar),	7.22	(d,	J	=	6.7	Hz,	1H,	H‐
8Ar),	7.54	(t,	1H,	H‐7Ar),	8.17	(d,	J	=	7.0	Hz,	1H,	H‐6Ar),	10.09	
(s,	1H,	NH,	exchanged	with	D2O),	11.51	(s,	1H,	NH,	exchanged	
with	 D2O).	 Anal.	 calcd.	 for	 C15H14N4O:	 C,	 67.65;	 H,	 5.30;	 N,	
21.04.	Found:	C,	67.80;	H,	5.30;	N,	21.18%.	

3‐(4‐Chlorophenyl)aminomethylpyrido[2,	3‐b]pyrazin‐2(1H)	
one	(3h):	Yield:	66%.	M.p.:	>300	°C.	FT‐IR	(KBr,	,	cm‐1):	3421,	
3385	 (2NH),	 3093	 (CH	 Ar),	 2924,	 2852	 (CH	 Aliph),	 1674	
(C=O),	1647,	1624,	1593,	1570	(C=N,	NH,	C=C).	 1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	3.31	(s,	2H,	CH2),	6.42‐7.12	(m,	4H,	H‐
2’,	3’,	5’,	6’	Ar),	7.21	(d,	 J	=	6.3	Hz,	1H,	H‐8Ar),	7.59	(t,	1H,	H‐
7Ar),	 8.11	 (d,	 J	 =	 6.2	 Hz,	 1H,	 H‐6Ar),	 11.00	 (s,	 1H,	 NH,	
exchanged	with	D2O),	11.59	(s,	1H,	NH,	exchanged	with	D2O).	
Anal.	calcd.	for	C14H11ClN4O:	C,	58.65;	H,	3.87;	N,	19.54.	Found:	
C,	58.78;	H,	3.92;	N,	19.73%.	

3‐(4‐Bromophenyl)aminomethylpyrido[2,3‐b]pyrazin‐2(1H)	
one	(3i):	Yield:	79%.	M.p.:	>300	°C.	FT‐IR	(KBr,	,	cm‐1):	3400,	
3388	 (2NH),	 3101	 (CH	 Ar),	 2924,	 2897	 (CH	 Aliph),	 1664	
(C=O),	1649,	1618,	1593	 (C=N,	NH,	C=C).	 1H	NMR	 (400	MHz,	
DMSO‐d6,	δ,	ppm):	3.11	(s,	2H,	CH2),	6.36‐7.20	(m,	4H,	H‐2’,	3’,	
5’,	 6’	Ar),	 7.41	 (d,	 J	=	8.1	Hz,	1H,	H‐8Ar),	 7.69	 (t,	 1H,	H‐7Ar),	
8.22	 (d,	 J	 =	 6.0	 Hz,	 1H,	 H‐6Ar),	 11.02	 (s,	 1H,	 NH,	 exchanged	
with	D2O),	11.61	(s,	1H,	NH,	exchanged	with	D2O).	Anal.	calcd.	
for	C14H11BrN4O:	C,	50.77;	H,	3.35;	N,	16.92.	Found:	C,	50.91;	H,	
3.37;	N,	16.98%.	

3‐(N,N‐Diphenylamino)methylpyrido[2,	 3‐b]pyrazin‐2(1H)	
one	 (3j):	 Yield:	 46%.	 M.p.:	 292‐293	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	
3410	(NH),	3057	(CH	Ar),	2924,	2852	(CH	Aliph),	1662	(C=O),	
1649,	1610,	1591,	 1570,	 1508	 (C=N,	NH,	 C=C).	 1H	NMR	 (400	
MHz,	DMSO‐d6,	δ,	ppm):	3.32	(s,	2H,	CH2),	6.50‐7.11	(m,	10H,	
Ar	H),	7.21	(d,	J	=	7.2	Hz,	1H,	H‐8Ar),	7.55	(t,	1H,	H‐7Ar),	8.15	
(d,	 J	=	7.0	Hz,	 1H,	H‐6	Ar),	 11.44	 (s,	 1H,	NH,	 exchanged	with	
D2O).	 Anal.	 calcd.	 for	 C20H16N4O:	 C,	 73.15;	 H,	 4.91;	 N,	 17.06.	
Found:	C,	73.28;	H,	4.97;	N,	17.22%.	
	
2.1.4.	Synthesis	of	3‐Hydrazinomethylpyrido[2,3‐b]pyrazin‐
2(1H)one	(4)	
	

Hydrazine	hydrate	(7.00	g,	6.86	mL,	0.14	mol)	was	added	
to	a	solution	of	compound	2	(2.40	g,	0.01	mol)	in	ethanol	(20	
mL)	and	refluxed	 for	9	h.	The	mixture	was	cooled	 in	 ice	bath	
for	 10	 min.	 and	 formed	 precipitate	 was	 filtered.	 The	
precipitate	 was	 washed	 with	 ethanol	 (5	mL)	 and	 dried.	 The	
crude	product	was	crystallized	from	ethanol	(Scheme	1).	Yield:	
73%.	M.p.:	243‐245	°C.	FT‐IR	(KBr,	,	cm‐1):	3369,	3329	(NH2,	
2NH),	3066	(CH	Ar),	2924,	2852	(CH	Aliph),	1670	(C=O),	1612,	
1591,	1570,	1508	(C=N,	NH,	C=C).	 1H	NMR	(400	MHz,	DMSO‐
d6,	 δ,	 ppm):	2.51	 (s,	2H,	CH2),	7.21	 (d,	 J	=	6.2	Hz,	1H,	H‐8Ar),	
7.50	(t,	1H,	H‐7Ar),	8.22	(d,	J	=	8.2	Hz,	1H,	H‐6Ar),	10.72	(s,	1H,	
NH,	 exchanged	with	D2O),	 11.22	 (s,	 2H,	NH2,	 exchanged	with	
D2O),	 11.50	 (s,	 1H,	NH,	 exchanged	with	D2O).	Anal.	 calcd.	 for	
C8H9N5O:	C,	50.26;	H,	4.74;	N,	36.63.	Found:	C,	50.41;	H,	4.73;	
N,	37.01%.	
	
2.1.5.	General	procedure	for	synthesis	of	3‐(2‐(un)substitu‐
ted	benzylidene)hydrazinomethylpyrido[2,3‐b]pyrazin‐
2(1H)‐ones	(5a‐e)	
	

Appropriate	aromatic	aldehyde	or	ketone	 (0.01	mol)	was	
added	to	a	solution	of	compound	4	(1.91	g,	0.01	mol)	in	glacial	
acetic	 acid	 (10	 mL)	 and	 refluxed	 for	 8	 h.	 The	 mixture	 was	
allowed	 to	 cool	 then	 poured	 onto	 crushed	 ice	 (30	 g).	 The	
precipitate	was	filtered,	washed	with	water	(5	mL)	and	dried.	
The	crude	product	was	crystallized	from	ethanol	(Scheme	1).	

3‐(2‐Benzylidene)hydrazinomethylpyrido[2,	 3‐b]pyrazin‐2	
(1H)‐one	(5a):	Yield:	62%.	M.p.:	234‐236	°C.	FT‐IR	(KBr,	,	cm‐

1):	 3398,	 3364	 (2NH),	 3061	 (CH	 Ar),	 2901,	 2829	 (CH	 Aliph),	
1676	(C=O),	1624,	1600,	1583,	1544,	1508	(C=N,	NH,	C=C).	1H	
NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	2.32	(s,	2H,	CH2),	7.21	(d,	J	
=	7.2	Hz,	1H,	H‐8	Ar),	7.31‐7.42	(m,	3H,	H‐3’,4’,5’	Ar),	7.45	(t,	
1H,	 H‐7	 Ar),	 7.62	 (d,	 J	 =	 6.7	 Hz,	 2H,	 H‐2’,6’	 Ar),	 8.11	 (s,	 1H,	
N=CH),	 8.22	 (d,	 J	 =	 6.2	 Hz,	 1H,	 H‐6Ar),	 11.32	 (s,	 1H,	 NH,	
exchanged	with	D2O),	11.54	(s,	1H,	NH,	exchanged	with	D2O).	
Anal.	calcd.	for	C15H13N5O:	C,	64.51;	H,	4.69;	N,	25.07.	Found:	C,	
64.67;	H,	4.73;	N,	25.19%.	

3‐[2‐(4‐Chlorobenzylidene)]hydrazinomethylpyrido[2,	 3‐b]	
pyrazin‐2(1H)‐one	 (5b):	 Yield:	 69%.	 M.p.:	 237‐239	 °C.	 FT‐IR	
(KBr,	,	 cm‐1):	 3421,	 3387	 (2NH),	 3012	 (CH	 Ar),	 2920,	 2837	
(CH	Aliph),	 1683	 (C=O),	1616,	1593,	1558,	1541,	1521	 (C=N,	
NH,	 C=C).	 1H	NMR	 (400	MHz,	DMSO‐d6,	 δ,	 ppm):	 2.42	 (s,	 2H,	
CH2),	7.22	(d,	J	=	7.3	Hz,	1H,	H‐8	Ar),	7.34	(d,	J	=	6.1	Hz,	2H,	H‐
3’,5’	Ar),	7.46	(t,	1H,	H‐7Ar),	7.59	(d,	J	=	7.0	Hz,	2H,	H‐2’,6’	Ar),	
8.11	(s,	1H,	N=CH),	8.22	(d,	J	=	6.3	Hz,	1H,	H‐6	Ar),	11.30	(s,	1H,	
NH,	 exchanged	 with	 D2O),	 11.51	 (s,	 1H,	 NH,	 exchanged	with	
D2O).	MS	(EI,	m/z	(%)):	313	(M+,	2.63),	315	(M++2,	2.77).	Anal.	
calcd.	 for	 C15H12ClN5O:	 C,	 57.42;	 H,	 3.86;	 N,	 22.32.	 Found:	 C,	
57.52;	H,	3.91;	N,	22.48%.	

3‐[2‐(4‐Nitrobenzylidene)]hydrazinomethylpyrido[2,	3‐b]	
pyrazin‐2(1H)‐one	 (5c):	 Yield:	 70%.	 M.p.:	 238‐240	 °C.	 FT‐IR	
(KBr,	,	 cm‐1):	 3444,	 3365	 (2NH),	 3021	 (CH	 Ar),	 2926,	 2850	
(CH	 Aliph),	 1677	 (C=O),	 1653,	 1614,	 1597,	 1558	 (C=N,	 NH,	
C=C),	1519,	1344	(NO2).	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	
2.33	(s,	2H,	CH2),	7.21	(d,	J	=	6.4	Hz,	1H,	H‐8Ar),	7.66	(t,	1H,	H‐
7Ar),	7.91‐8.10	(m,	4H,	H‐2’,	3’,	5’,	6’	Ar),	8.19	(s,	1H,	N=CH),	
8.23	 (d,	 J	=	 7.2	 Hz,	 1H,	 H‐6Ar),	 11.32	 (s,	 1H,	 NH,	 exchanged	
with	D2O),	11.50	(s,	1H,	NH,	exchanged	with	D2O).	Anal.	calcd.	
for	C15H12N6O3:	C,	55.55;	H,	3.73;	N,	25.91.	Found:	C,	55.74;	H,	
3.74;	N,	26.07%.	

3‐[2‐(4‐Methoxybenzylidene)]hydrazinomethylpyrido[2,	 3‐
b]pyrazin‐2(1H)‐one	(5d):	Yield:	63%.	M.p.:	256‐258	°C.	FT‐IR	
(KBr,	,	 cm‐1):	 3417,	 3392	 (2NH),	 3039	 (CH	 Ar),	 2933,	 2837	
(CH	Aliph),	 1670	 (C=O),	1598,	1575,	1558,	1541,	1508	 (C=N,	
NH,	 C=C).	 1H	NMR	 (400	MHz,	DMSO‐d6,	 δ,	 ppm):	 2.12	 (s,	 2H,	
CH2),	3.52	(s,	3H,	OCH3),	6.76	(d,	J	=	8.2	Hz,	2H,	H‐3’,5’	Ar),	7.21	
(d,	J	=	7.1	Hz,	1H,	H‐8	Ar),	7.43	(t,	1H,	H‐7	Ar),	7.66	(d,	J	=	7.4	
Hz,	2H,	H‐2’,6’	Ar),	8.10	(s,	1H,	N=CH),	8.22	(d,	J	=	7.6	Hz,	1H,	
H‐6Ar),	 11.30	 (s,	 1H,	NH,	 exchanged	with	D2O),	 11.45	 (s,	 1H,	
NH,	exchanged	with	D2O).	Anal.	calcd.	for	C16H15N5O2:	C,	62.13;	
H,	4.89;	N,	22.64.	Found:	C,	62.26;	H,	4.87;	N,	22.72%.	

3‐(2‐Methyl‐2‐phenyl)hydrazinomethylpyrido[2,	3‐b]pyrazin	
‐2(1H)‐one	 (5e):	 Yield:	 69%.	M.p.:	 230‐233	 °C.	 FT‐IR	 (KBr,	,	
cm‐1):	3446,	3363	(2NH),	3032	(CH	Ar),	2929,	2856	(CH	Aliph),	
1670	(C=O),	1645,	1616,	1570,	1558,	1521,	(C=N,	NH,	C=C).	1H	
NMR	(400	MHz,	DMSO‐d6,	 δ,	ppm):	1.42	 (s,	3H,	CH3),	2.50	 (s,	
2H,	CH2),	7.22	(d,	 J	=	6.2	Hz,	1H,	H‐8Ar),	7.30‐7.43	(m,	3H,	H‐
3’,4’,5’	Ar),	7.49	(t,	1H,	H‐7Ar),	7.63	(d,	 J	=	8.1	Hz,	2H,	H‐2’,6’	
Ar),	8.22	(d,	J	=	7.2	Hz,	1H,	H‐6Ar),	11.31	(s,	1H,	NH,	exchanged	
with	D2O),	11.53	(s,	1H,	NH,	exchanged	with	D2O).	Anal.	calcd.	
for	C16H15N5O:	C,	65.52;	H,	5.15;	N,	23.88.	Found:	C,	65.59;	H,	
5.12;	N,	23.98%.	
	
2.1.6.	General	procedure	for	synthesis	of	4‐(un)substituted	
phenyl‐2,5,10,11‐tetrahydropyrido[2,3‐g]pyrazino[4,3‐e]	
1,2,4‐triazin‐11‐ones	(6a‐d)	
	

Bromine	 (1.60	g,	 0.53	mL,	0.01	mol)	 in	glacial	 acetic	acid	
(0.6	mL)	was	 added	dropwise	 to	 solution	 of	 compound	5a‐d	
(0.01	 mol)	 in	 glacial	 acetic	 acid	 (9	 mL)	 containing	 sodium	
acetate	 (2.46	 g,	 0.03	mol)	 at	 room	 temperature.	 The	mixture	
was	stirred	for	3	h	at	room	temperature	then	poured	onto	cold	
water	 (50	mL).	 The	 formed	 precipitate	was	 filtered,	 washed	
with	 water	 (10	 mL)	 and	 dried.	 The	 crude	 product	 was	
crystallized	from	acetic	acid	(Scheme	1).	



Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	 23	
 

4‐Phenyl‐2,5,10,11‐tetrahydropyrido[2,	3‐g]pyrazino[4,	3‐e]	
1,2,4‐triazin‐11‐one	 (6a):	 Yield:	 80%.	M.p.:	 219‐221	 °C.	 FT‐IR	
(KBr,	,	 cm‐1):	 3365,	3355	 (2NH),	3035	 (CH	Ar),	 1760	 (C=O),	
1608,	1558,	1541,	 1521,	 1508	 (C=N,	NH,	 C=C).	 1H	NMR	 (400	
MHz,	DMSO‐d6,	δ,	ppm):	6.01	(s,	1H,	H‐1	Ar),	6.99	(t,	1H,	H‐8	
Ar),	7.01	(d,	J	=	6.4	Hz,	1H,	H‐9	Ar),	7.29‐7.41	(m,	3H,	H‐3’,4’,5’	
Ar),	7.63	(d,	J	=	7.2	Hz,	2H,	H‐2’,6’	Ar),	7.91	(d,	J	=	6.0	Hz,	1H,	H‐
7	 Ar),11.29	 (s,	 1H,	 NH,	 ,	 exchanged	with	 D2O),	 11.52	 (s,	 1H,	
exchanged	with	D2O).	 Anal.	 calcd.	 for	 C15H11N5O:	 C,	 64.97;	H,	
4.00;	N,	25.26.	Found:	C,	65.13;	H,	4.06;	N,	25.49%.	

4‐(4‐Chlorophenyl)‐2,	 5,	 10,	 11‐tetrahydropyrido[2,	 3‐g]	
pyrazino[4,3‐e]1,2,4‐triazin‐11‐one	(6b):	Yield:	75%.	M.p.:	222‐
225	°C.	FT‐IR	(KBr,	,	cm‐1):	3390,	3385	(2NH),	3032	(CH	Ar),	
1680	(C=O),	1635,	1608,	1593,	1570,	1558	(C=N,	NH,	C=C).	1H	
NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	5.99	(s,	1H,	H‐1	Ar),	6.86	(t,	
1H,	H‐8	Ar),	7.02	(d,	J	=	8.1	Hz,	1H,	H‐9	Ar),	7.33	(d,	J	=	7.0	Hz,	
2H,	H‐3’,5’	Ar),	7.62	(d,	 J	=	6.5	Hz,	2H,	H‐2’,6’	Ar),	8.22	(d,	 J	=	
6.3	 Hz,	 1H,	 H‐7	 Ar),	 11.31	 (s,	 1H,	 NH,	 exchanged	with	 D2O),	
11.53	 (s,	 1H,	 NH,	 exchanged	 with	 D2O).	 Anal.	 calcd.	 for	
C15H10ClN5O:	 C,	 57.79;	 H,	 3.23;	 N,	 22.47.	 Found:	 C,	 57.96;	 H,	
3.19;	N,	22.63%.	

4‐(4‐Nitrophenyl)‐2,	 5,	 10,	 11‐tetrahydropyrido[2,	 3‐g]	
pyrazino[4,3‐e]1,2,4‐triazin‐11‐one	(6c):	Yield:	86%.	M.p.:	222‐
224	°C.	FT‐IR	(KBr,	,	cm‐1):	3410,	3393	(2NH),	3041	(CH	Ar),	
1674	 (C=O),	 1654,	 1635,	 1600,	 1568,	 1558	 (C=N,	 NH,	 C=C),	
1521,	1346	(NO2).	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	6.21	
(s,	1H,	H‐1	Ar),	6.62	(t,	1H,	H‐8	Ar),	6.84	(d,	J	=	7.2	Hz,	1H,	H‐9	
Ar),	7.56	(d,	J	=	8.1	Hz,	1H,	H‐7Ar),	7.91‐8.10	(m,	4H,	H‐2’,	3’,	5’,	
6’	Ar),	11.33	(s,	1H,	NH,	exchanged	with	D2O),	11.50	(s,	1H,	NH,	
exchanged	with	D2O).	Anal.	 calcd.	 for	C15H10N6O3:	C,	55.90;	H,	
3.13;	N,	26.08.	Found:	C,	56.03;	H,	3.17;	N,	26.32%.	

4‐(4‐Methoxyphenyl)‐2,	5,	10,	11‐tetrahydropyrido[2,3‐g]	
pyrazino[4,3‐e]1,2,4‐triazin‐11‐one	(6d):	Yield:	66%.	M.p.:	229‐
231	°C.	FT‐IR	(KBr,	,	cm‐1):	3415,	3388	(2NH),	3020	(CH	Ar),	
2933,	2841	 (CH	Aliph),	 1674	 (C=O),	1633,	1597,	1575,	1558,	
1508	 (C=N,	NH,	 C=C).	 1H	NMR	 (300	MHz,	DMSO‐d6,	 δ,	 ppm):	
3.72	 (s,	 3H,	OCH3),	 5.82	 (s,	 1H,	H‐1	Ar),	 6.54	 (t,	 1H,	H‐8	Ar),	
6.96	(d,	J	=	6.0	Hz,	1H,	H‐9	Ar),	7.03‐7.40	(m,	4H,	H‐2’,	3’,	5’,	6’	
Ar),	 7.67	 (d,	 J	 =	 7.3	 Hz,	 1H,	 H‐7	 Ar),	 11.33	 (s,	 1H,	 NH,	
exchanged	with	D2O),	11.51	(s,	1H,	NH,	exchanged	with	D2O).		

	
2.1.7.	General	procedure	for	synthesis	of	3‐(2,5‐dioxopyrrol	
(or	pyroliden)‐1‐yl)aminomethylpyrido[2,3‐b]pyrazin‐2	
(1H)ones	(7a‐c)	
	

Appropriate	cyclic	acid	anhydride	(0.005	mol)	was	added	
to	 solution	 of	 hydrazine	 compound	4	 (0.96	 g,	 0.005	mol)	 in	
glacial	acetic	acid	(10	mL)	and	refluxed	for	6	h.	After	cooling,	
the	mixture	was	poured	onto	crushed	 ice	 (30	g).	The	 formed	
precipitate	was	filtered,	washed	with	water	(5	mL)	and	dried.	
The	crude	product	was	crystalized	from	ethanol	(Scheme	1).	

3‐(2,	 5‐Dioxopyrroliden‐1‐yl)aminomethylpyrido[2,	 3‐b]	
pyrazin‐2(1H)one	 (7a):	 Yield:	 86%.	 M.p.:	 215‐217	 °C.	 FT‐IR	
(KBr,	,	 cm‐1):	 3410,	 3396	 (2NH),	 3026	 (CH	 Ar),	 2962,	 2848	
(CH	Aliph),	1668	(3C=O),	1635,	1602,	1589,	1558,	1521	(C=N,	
NH,	 C=C).	 1H	NMR	 (300	MHz,	DMSO‐d6,	 δ,	 ppm):	 2.56	 (s,	 2H,	
CH2),	3.11	(t,	4H,	CH2‐CH2),	7.22	(d,	J	=	6.1	Hz,	1H,	H‐8Ar),	7.58	
(t,	1H,	H‐7Ar),	8.18	(d,	J	=	6.4	Hz,	1H,	H‐6Ar),	11.23	(s,	1H,	NH,	
exchanged	with	D2O),	11.44	(s,	1H,	NH,	exchanged	with	D2O).	
Anal.	calcd.	for	C12H11N5O3:	C,	52.75;	H,	4.06;	N,	25.63.	Found:	
C,	52.84;	H,	4.11;	N,	25.89%.	

3‐(2,5‐Dioxo‐2,5‐dihydro‐1H‐pyrrol‐1‐yl)aminomethylpyrido	
[2,3‐b]pyrazin‐2(1H)one	 (7b):	 Yield:	 85%.	 M.p.:	 212‐215	 °C.	
FT‐IR	 (KBr,	,	 cm‐1):	 3421,	 3398	 (2NH),	 3030	 (CH	Ar),	 2964,	
2848	(CH	Aliph),	1668	(3C=O),	1635,	1602,	1589,	1558,	1521	
(C=N,	NH,	C=C).	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	2.55	(s,	
2H,	CH2),	5.11	(d,	J	=	8.7	Hz,	2H,	CH=CH),	7.21	(d,	J	=	6.3	Hz,	1H,	
H‐8Ar),	 7.50	 (t,	 1H,	 H‐7Ar),	 8.01	 (d,	 J	 =	 7.7	 Hz,	 1H,	 H‐6Ar),	
11.33	 (s,	 1H,	 NH,	 exchanged	 with	 D2O),	 11.49	 (s,	 1H,	 NH,	

exchanged	with	D2O).	 Anal.	 calcd.	 for	 C12H9N5O3:	 C,	 53.14;	H,	
3.34;	N,	25.82.	Found:	C,	53.22;	H,	3.31;	N,	26.04%.	

3‐(2,	5‐Dioxo‐2,	5‐dihydro‐1H‐benzo[c]pyrrol‐1‐yl)amino	
methylpyrido[2,3‐b]pyrazin‐2(1H)one	 (7c):	 Yield:	 86%.	 M.p.:	
218‐221	°C.	FT‐IR	(KBr,	,	cm‐1):	3446,	3395	(2NH),	3010	(CH	
Ar),	 2966,	 2854	 (CH	Aliph),	 1683	 (3C=O),	 1637,	 1602,	 1587,	
1568,	1541,	1521	(C=N,	NH,	C=C).	 1H	NMR	(400	MHz,	DMSO‐
d6,	 δ,	ppm):	2.70	 (s,	2H,	CH2),	7.22	 (d,	 J	=	6.2	Hz,	1H,	H‐8Ar),	
7.46	(t,	1H,	H‐7Ar),	7.66‐8.01	(m,	4H,	H‐3’,	4’,	5’,	6’	Ar),	8.26	(d,	
J	=	8.0	Hz,	1H,	H‐6	Ar),	11.33	(s,	1H,	NH,	exchanged	with	D2O),	
11.55	 (s,	 1H,	 NH,	 exchanged	 with	 D2O).	 Anal.	 calcd.	 for	
C16H11N5O3:	 C,	 59.81;	 H,	 3.45;	 N,	 21.80.	 Found:	 C,	 59.97;	 H,	
3.52;	N,	22.04%.	
	
2.2.	Biological	activity	
	
2.2.1.	Antitumor	screening	
	

The	cytotoxicity	of	the	newly	synthesized	compounds	was	
performed	 against	 different	 cancer	 cell	 lines,	 melanoma	 cell	
line	 (LOXIMVI),	 ovarian	 cell	 line	 (OVCAR3),	 thyroid	 cell	 lines	
(CAL62,	FTC133,	BCPAP	and	ML1)	and	colon	cell	 lines	(HT29	
and	HCT116),	with	the	MTT	assay	according	to	the	Mosmann’s	
method	[23].	The	cells	used	in	cytotoxicity	assay	were	cultured	
in	 RPMI	 1640	 medium	 supplemented	 with	 10%	 fetal	 calf	
serum.	 Cells	 suspended	 in	 the	 medium	 (2×104/mL)	 were	
plated	in	96‐well	culture	plates	and	incubated	at	37°C	in	a	5%	
CO2	incubator.	After	12	h,	the	test	sample	(2	μL)	was	added	to	
the	cells	 (2×104)	 in	96‐well	plates	and	cultured	at	37°C	 for	3	
days.	 The	 cultured	 cells	 were	 mixed	 with	 20	 μL	 of	 MTT	
solution	 and	 incubated	 for	 4	h	 at	 37°C.	The	 supernatant	was	
carefully	 removed	 from	each	well	 and	100	μL	of	DMSO	were	
added	 to	 each	 well	 to	 dissolve	 the	 formazan	 crystals	 which	
were	 formed	 by	 the	 cellular	 reduction	 of	 MTT.	 After	 mixing	
with	 a	 mechanical	 plate	 mixer,	 the	 absorbance	 of	 each	 well	
was	measured	by	a	microplate	reader	using	a	test	wavelength	
of	 570	nm	 [24].	 The	 results	were	 expressed	 as	 the	 IC50	 [23],	
which	inducing	a	50%	inhibition	of	cell	growth	of	treated	cells	
when	 compared	 to	 the	 growth	 of	 control	 cells.	 Each	
experiment	was	performed	at	least	3	times.	The	concentration	
ranges	 used	 were	 0.00001,	 0.00005,	 0.0001,	 0.0005,	 0.001,	
0.005,	 0.01,	 0.05,	 0.1,	 0.5,	 1,	 5	 and	10	μM.	There	was	 a	 good	
reproducibility	 between	 replicate	wells	with	 standard	 errors	
below	10%.		
	
2.2.2.	In	vitro	kinase	assay	
	
2.2.2.1.	Protein	expression	and	purification	
	

Human	BRAF	kinase	domain,	BRAF‐KD	(residues	433‐726)	
with	 an	 N‐terminal	 purification	 tag	 (MDRGSH6GS),	 and	 full‐
length	mouse	p50cdc37	were	cloned	into	a	pFastBac	Dual	vector.	
BRAF	 wild‐type	 and	 V600E	 mutant	 kinase	 domains	 were	
expressed	 and	 purified.	 Briefly,	 Sf9	 cells	 infected	 with	 the	
BRAF	 kinase	 domain	 harboring	 baculo	 virus	 were	 re‐
suspended	 in	 sonication	 buffer	 for	 sonication,	 and	 the	 lysate	
was	 cleared	 by	 high‐speed	 centrifugation.	 Equilibrated	 Talon	
resin	was	added	into	the	cleared	lysate;	the	resin	was	washed	
with	 10	 column	 volumes	 of	 wash	 buffer	 (25	mM	 Tris	 (pH	 =	
8.0),	 250	mM	NaCl,	 5	mM	 imidazole,	 and	 10%	 glycerol)	 and	
then	eluted	with	addition	of	buffer	(25	mM	Tris	(pH	=	7.0),	250	
mM	NaCl,	 160	mM	 imidazole,	 and	 10%	 glycerol).	 The	 eluant	
from	 the	 Talon	 resin	 was	 diluted	 3‐fold	 and	 loaded	 on	 a	 SP	
resin	 column.	 SP	 resin	was	 extensively	washed	 (25	mM	Tris	
(pH	 =	 8.0),	 50	 mM	 NaCl,	 1	 mM	 dithiothreitol,	 and	 10%	
glycerol)	 and	eluted	with	high‐salt	buffer	 (25	mM	Tris	 (pH	=	
8.0),	500	mM	NaCl,	1	mM	dithiothreitol,	1	mM	EDTA,	and	10%	
glycerol).	 Recombinantly	 expressed	λ	phosphatase	 was	 then	
added	 to	 the	 SP	 eluant	 and	 incubated	 for	 2	 h	 at	 room	
temperature	followed	by	7‐fold	dilution	in	buffer	(25	mM	Tris	
(pH	 =	 8.0),	 1	 mM	 dithiothreitol,	 1	 mM	 EDTA,	 and	 10%	



24	 Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	
	
glycerol).	This	dilution	was	reloaded	on	an	SP	resin	column	to	
remove	 the	λ	phosphatase.	 The	 final	 SP	 eluant	 was	
concentrated	 and	 loaded	 onto	 a	 Superdex	 200	 gel	 filtration	
column	equilibrated	in	buffer	(25	mM	Tris	(pH	=	8.0),	300	mM	
NaCl,	1	mM	dithiothreitol,	1	mM	EDTA,	and	5%	glycerol).	Gel	
filtration	 fractions	 were	 concentrated	 with	 a	 final	 glycerol	
concentration	of	15%	to	a	protein	concentration	of	1.5	mg/mL.	
BRAF‐KD	protein	was	immediately	used	for	crystallization.	

GST‐MEK‐His	 protein	 was	 overexpressed	 at	 37	 °C	
in	Escherichia	coli	BL21	(Gold)	cells	(Invitrogen)	in	LB	medium	
until	 the	 OD	 value	 reached	 0.4‐0.6.	 Isopropyl	 1‐thio‐β‐D‐
galactopyrano‐side	 (IPTG,	 0.5	 mM)	 was	 then	 added	 to	 the	
culture,	which	was	grown	at	15	°C	for	an	additional	16	h.	The	
cell	pellet	was	re‐suspended	and	sonicated	in	sonication	buffer	
(20	 mM	 HEPES	 (4‐(2‐hydroxyethyl)‐1‐piperazineethane	
sulfonic	 acid))	 (pH	=	 7.5),	 500	mM	NaCl,	 10	mM	2‐mercapto	
ethanol	 (BME),	10	mM	imidazole,	0.1	mg/mL	phenylmethane	
sulfonyl	 fluoride	 (PMSF),	 and	 5%	 glycerol).	 The	 lysate	 was	
cleared	by	high	speed	centrifugation	before	it	was	loaded	onto	
a	 Ni‐NTA	 resin	 column	 pre‐equilibrated	 in	 sonication	 buffer.	
The	protein‐bound	Ni‐NTA	resin	was	then	extensively	washed	
with	20	column	volumes	of	wash	buffer,	and	the	GST‐MEK‐His	
protein	was	eluted	with	an	imidazole	gradient	from	10	to	150	
mM	 in	wash	buffer.	Fractions	 corresponding	 to	GST‐MEK‐His	
were	pooled	and	applied	to	a	Superdex	200	column	equilibra‐
ted	in	buffer	(20	mM	HEPES	(pH	=	7.5),	150	mM	NaCl,	10	mM	
BME,	 and	 5%	 glycerol).	 Gel	 filtration	 fractions	 were	 pooled	
and	 concentrated	 to	 10	 mg/mL	 before	 being	 flash‐frozen	 in	
liquid	nitrogen	and	stored	at	‐80	°C	until	use	[25].	
	
2.2.2.2.	In	vitro	ELISA‐based	kinase	assay	

	
Recombinantly	 expressed	 GST	 MEK‐His,	 diluted	 in	 TTBS	

buffer	 (20	 mM	 Tris	 (pH	 =	 7.5),	 150	 mM	 NaCl,	 and	 0.05%	
Tween	20)	to	50	μg/mL	in	a	volume	of	100	μL,	was	bound	to	
the	 wells	 of	 a	 96‐well	 glutathione‐coated	 plate	 (Pierce	
Biotechnology).	 One	 microliter	 of	 compound	 (as	 racemic	
mixtures)	 with	 2	 serial	 dilutions	 in	 a	 100%	 DMSO	 stock	
solution	was	added	to	a	mixture	of	50	μL	of	a	buffer	containing	
50	mM	HEPES	 (pH	=	7.0)	with	0.7	pmol	of	 V600EBRAF	kinase.	
This	 mixture	 was	 incubated	 at	 room	 temperature	 for	 1	 h	
before	 it	 was	 added	 to	 the	 GST‐MEK‐His‐bound	wells	 of	 the	
96‐well	 plate.	 An	 additional	 50	 μL	 of	 phosphorylation	 buffer	
(50	mM	HEPES	 (pH	=	7.0),	200	mM	NaCl,	 10	mM	MgCl2,	 and	
200	μM	ATP)	was	added	to	the	well	mixture	to	start	the	kinase	
reaction	 at	 37	 °C	 for	 30	min.	 with	 intermittent	 shaking.	 The	
kinase	reaction	was	stopped	by	extensive	washing	with	TTBS	
buffer,	 and	 a	 1:5000	 dilution	 of	 anti‐phospho‐MEK1	
(Ser218/222)/MEK2	 (Ser222/226)	 monoclonal	 antibody	
(Millipore)	 in	 TTBS	 buffer	 was	 subsequently	 added	 to	 the	
wells	and	incubated	for	1	h	with	shaking.	Goat	anti‐rabbit	IgG	
(H+L)‐HRP	 conjugate	 (Bio‐Rad	 Laboratories)	 in	 a	 1:5000	
dilution	 was	 added	 to	 the	 wells	 for	 incubation	 at	 room	
temperature	with	shaking.	Finally,	the	Super	Signal	ELISA	Pico	
chemiluminescent	 substrate	 (Pierce	 Biotechnology)	 was	
added	to	the	wells.	The	luminescence	signal	was	recorded	with	
a	 luminescence	 filter	 using	 a	 Wallac	 1420	 luminometer	
(PerkinElmer)	[25].	

High	 throughput	 inhibitor	 screening	 was	 performed.	
Assays	were	conducted	 in	glutathione	coated	384‐well	plates	
and	 followed	 the	 procedures	 essentially	 as	 described	 above	
but	using	a	50	μL	reaction	volume	instead	of	a	100	μL	reaction	
volume	 to	 fit	 384‐well	 plate	 format.	 Specifically,	 GST‐MEK	
protein	diluted	 in	TTBS	 to	50	μg/mL	was	dispensed	 into	 the	
wells	of	the	glutathione	coated	384‐well	plate	to	a	final	volume	
of	 50	 μL/well	 using	 a	 Matrix	 Wellmate	 Dispanser	 with	 a	
microplate	 stacker	 (Thermo	 Scientific).	 Each	 plate	 was	
agitated	 using	 an	 orbital	 shaker	 at	 2500	 rpm	 for	 1	min.	 and	
incubated	at	room	temperature	for	1	h.	Plates	were	aspirated	
and	 washed	 once	 using	 a	 wash	 program	 with	 vigorous	
agitation	using	an	automated	microplate	washer	 (Biotek).	3.5	

pico	moles	of	V600EBRAF	kinase	domain	diluted	in	25	μL	of	50	
mM	 HEPES	 (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic	
acid))	 pH	 =	 7.5	 buffer	was	 added	 into	 each	well	 of	 the	 plate	
using	 the	Matrix	Wellmate	Dispanser,	 and	25	nl	of	 individual	
compound	 (10	mM	 in	100%	DMSO)	was	 transferred	 into	 the	
solution	using	a	Cybi‐Well	pin‐transfer	station	(Cybio).	Plates	
were	agitated	using	an	orbital	shaker	for	1	min	and	incubated	
at	room	temperature	for	1	h.	25	μL	of	phosphorylation	buffer	
was	 then	 added	 into	 the	 wells	 to	 start	 the	 kinase	 reaction	
(controls	were	tested	to	ensure	the	precise	timing	for	the	start	
of	 the	 kinase	 reaction).	 Kinase	 reactions	 were	 conducted	 at	
room	 temperature	 for	30	min	and	stopped	by	washing	using	
the	 microplate	 washer.	 A	 1:5000	 dilution	 of	 Anti‐phospho‐
MEK1	 (Ser218/222)/MEK2	 (Ser222/226)	 monoclonal	
antibody	 (Millipore)	 in	 TTBS	 buffer	 was	 subsequently	
dispensed	 into	 the	 wells	 to	 a	 final	 volume	 of	 50	 μl	 and	
incubated	for	1	h	with	shaking.	Goat	anti‐rabbit	IgG	(H+L)‐HRP	
conjugate	(BioRad	Laboratories)	in	a	1:5000	dilution	was	then	
dispensed	into	the	wells	to	a	final	volume	of	50	μL	to	incubate	
at	room	temperature	with	agitation.	Finally,	50	μL	of	the	Super	
Signal	 ELISA	 Pico	 chemiluminescent	 substrate	 (Pierce	
Biotechnology)	was	 dispensed	 into	 the	wells	 to	 generate	 the	
chemiluminescence	signal,	which	was	detected	using	a	700	nm	
luminescence	 filter	 by	 an	 Envision	 chemiluminescence	
detector	 (PerkinElmer).	 A	 total	 of	 31976	 compounds	 were	
screened	in	duplicate	including	libraries	of	a	diversity	oriented	
synthesis	(DOS),	commercially	available	drug‐like	compounds,	
bioactive	 compounds,	 natural	 products,	 compounds	 collected	
from	 academic	 organic	 synthesis	 laboratories	 and	 a	
ChemBridge	Kinase	inhibitor	biased	library.	Compounds	were	
ranked	 based	 on	 a	 composite	 Z‐score	 of	 both	 duplicates	 and	
the	 top	 100	 compounds	 were	 cherry‐picked	 from	 the	
compound	 plates	 at	 the	 Broad	 Institute	 and	 they	 were	 re‐
analyzed	 by	 the	 same	 assay	 to	 confirm	 their	 inhibitory	
activities.	According	to	the	results,	the	top	23	compounds	that	
were	deemed	to	have	drug‐like	properties	were	confirmed	by	
reordering	 the	 compounds	 from	 their	 source	 vendors	 and	
confirming	their	inhibitor	activities	[25].	
	
2.2.2.3.	IC50	value	determination	
	

For	 IC50	 calculations	of	 the	 related	quinolol	 and	naphthol	
inhibitors,	 the	 same	 assay	 described	 above	 was	 used	 at	
different	inhibitor	concentrations	to	generate	a	sigmoidal	dose	
response	 curve	 using	 V600EBRAF	 or	 WTBRAF	 protein.	 All	 dose	
response	 measurements	 were	 carried	 out	 in	 duplicate	 or	
triplicate	and	IC50	values	were	derived	from	fitting	the	data	to	
a	sigmoidal	dose	response	curve	with	a	four‐parameter	logistic	
model	using	Graph	Pad	Prism	[26].	

IC50	values	 were	 calculated	 using	 GraphPad	 Prism	
software	 (GraphPad	 Software,	 La	 Jolla,	 CA,	 USA).	 Data	 are	
reported	 as	 means±SEM	 and	 significance	 was	 calculated	 by	
Student's	 t‐test	 using	 SPSS	 software	 (SPSS	 Inc.,	 Paris,	
France).	The	 concentrations	 range	 used	 were	 0.01,	 0.05,	 0.1,	
0.5	1,	5	and	10	µM.		
	
2.3.	Molecular	docking	
	
2.3.1.	Docking	procedure	
	

Docking	 studies	 of	 all	 the	 synthesized	 compounds	 were	
performed	by	Molecular	 Operating	 Environment	 (MOE)	 [27].	
The	 program	 operated	 under	 “Window	 7”	 operating	 system	
installed	on	an	Dell	Pentium	 IV	PC	with	a	2.8	MHz	processor	
and	 512	 RAM.	 All	 minimizations	 were	 performed	 with	 MOE	
until	a	RMSD	gradient	of	0.05	Kcal/mol.Å	with	MMFF94	force	
field	 and	 the	 partial	 charges	 were	 automatically	 calculated.	
The	score	 function,	dock	 function	 (S,	Kcal/mol)	developed	by	
MOE	 program	 was	 used	 for	 the	 evaluation	 of	 the	 binding	
affinity	of	the	ligand.	
	



Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	 25	
 

2.3.2.	Preparation	of	the	target	V600EBRAF	kinase	protein	
	

The	X‐ray	 crystal	 structure	 of	 the	 enzyme	with	PLX3203,	
N‐{2,4‐difluoro‐3‐[(5‐pyridin‐3‐yl‐1H‐pyrrolo[2,3‐b]pyridin‐3‐
yl)carbonyl]phenyl}ethanesulfonamide	 (PDB	 code:	 4FK3)	
[28]	was	obtained	from	the	protein	data	bank	(www.rcsb.org)	
in	PDB	format.	The	enzyme	was	prepared	for	docking	studies.	
(i)	3D	protonation	for	the	amino	acid	side	chain	and	ligand.	(ii)	
Deleting	all	water	of	crystallization	away	from	the	active	site.	
(iii)	Isolation	of	the	active	site,	fixation	to	be	dealt	with	as	rigid	
structure	and	recognition	of	 the	amino	acids.	 (iv)	Creation	of	
dummies	around	the	active	site.	(v)	Studying	the	 interactions	
of	the	ligand	with	the	amino	acids	of	the	active	site.	
	
2.3.3.	Preparation	of	compounds	for	docking	
	

The	 3D	 structures	 of	 the	 synthesized	 compounds	 in	
addition	 to	 sorafenib	 (standard	 used	 for	 enzyme	 inhibition	
assay)	 were	 built	 using	 MOE	 and	 subjected	 to	 the	 following	
procedure:	 (i)	 3D	 protonation	 of	 the	 structures.	 (ii)	 Running	
conformational	 analysis	 using	 systemic	 search.	 (iii)	 Selecting	
the	least	energetic	conformer.	(iv)	Applying	the	same	docking	
protocol	used	with	ligand	
	
2.3.4.	Docking	running	

	
Prior	 to	 the	 docking	 of	 the	pyridopyrazinone	 derivatives,	

redocking	of	 the	PLX3203	bound	 in	 the	 V600EBRAF	active	 site	
was	performed	to	validate	the	docking	protocol.	The	generated	
most	stable	conformer	of	each	compound	was	virtually	docked	
into	 the	 predefined	 active	 site	 of	 V600EBRAF.	 The	 developed	
docked	models	were	energetically	minimized	and	then	used	to	
predict	 the	 interaction	 of	 the	 ligand	with	 the	 amino	 acids	 in	
the	active	site	of	the	enzyme.	

	
3.	Results	and	discussion		

	
3.1.	Chemistry	
	

The	 target	 compounds	1,	2,	3a‐j,	4,	5a‐e,	6a‐d,	 and	7a‐c	
were	 synthesized	 as	 depicted	 in	 Scheme	 1.	 Condensation	 of	
2,3‐diaminopyridine	with	 ethyl	 pyruvate	 in	 ethanol	 provided	
methylpyridopyrazinone,	 1.	 IR	 spectrum	 showed	 a	 band	 at	
3186	 cm‐1	 assigned	 to	 NH	 group	 and	 a	 band	 at	 1685	 cm‐

1attributed	to	C=O	group.	1H	NMR	showed	a	singlet	peak	at	δ	
2.41	 ppm	 assigned	 to	 CH3	 protons	 in	 addition	 to	 doublet,	
triplet	 and	 doublet	 at	 δ7.48,	 7.67,	 8.11	 ppm,	 respectively,	
corresponding	to	pyridine	protons.	Bromomethyl	compound	2	
was	 achieved	 via	bromination	 of	 compound	1	with	 bromine.	
Use	of	bromine	in	presence	of	sodium	acetate	in	glacial	acetic	
acid	 gave	 higher	 yield	 than	 bromination	 with	 N‐bromo‐
succinamide	 [29].	 1H	 NMR	 spectrum	 revealed	 disappearance	
of	methyl	protons	singlet	peak	at	δ	2.41	ppm	and	appearance	
of	 singlet	 peak	 at	 δ	 3.10	 ppm	 assigned	 to	 CH2	 protons.	Mass	
spectrum	 showed	 both	 M+	 and	 M++2	 at	 240	 and	 242	 m/z,	
respectively.	

Refluxing	 bromomethyl	 compound	 2	 with	 different	
primary	or	secondary	amines	 in	ethanol	afforded	compounds	
3a‐j.	Catalytic	amount	of	sodium	iodide	was	used	to	accelerate	
the	reaction	[30].	IR	spectra	showed	a	band	at	3421‐3350	cm‐1	
corresponding	to	additional	NH	group	for	compounds	3d‐i.	In	
addition,	1H	NMR	spectra	showed	added	aliphatic	protons	at	δ	
1.41‐3.30	 ppm	 for	 compounds	 3a‐c	 or	 additional	 aromatic	
protons	at	δ	6.12‐7.20	ppm	for	compounds	3d‐j	and	additional	
singlet	 peak	 at	 δ	 10.09‐11.61	 ppm	 exchanged	 with	 D2O	
corresponding	to	NH	proton	for	compounds	3d‐i.	

Refluxing	 bromomethyl	 compound	 2	 with	 hydrazine	
hydrate	 in	 ethanol	 afforded	 compound	 4.	 1H	 NMR	 spectrum	
showed	additional	two	singlet	peaks	at	δ	10.72	and	11.22	ppm	
exchanged	 with	 D2O	 corresponding	 to	 added	 NH	 and	 NH2	
protons,	 respectively.	 Compounds	 5a‐e	 were	 prepared	 via	

reaction	of	hydrazino	compound	4	with	different	appropriate	
aldehyde	 or	 ketone	 in	 glacial	 acetic	 acid.	 1H	 NMR	 spectra	
showed	 additional	 aromatic	 protons	 at	 δ	 6.76‐8.10	 ppm	 in	
addition	 to	 signal	 at	 δ	 8.10‐8.19	 ppm	 corresponding	 to	
benzylidene	proton	for	compounds	5a‐d.	Adopting	cyclization	
with	 the	 aid	 of	 bromine	 and	 sodium	 acetate	 in	 glacial	 acetic	
acid	 at	 room	 temperature	 [31,32]	 for	 compounds	 5a‐d	
afforded	compounds	6a‐d.	1H	NMR	spectra	showed	additional	
singlet	 signal	 at	 δ	 5.82‐6.21	 ppm	 corresponding	 triazine	
proton.	

Refluxing	 compound	 4	 with	 different	 cyclic	 acid	
anhydrides	in	glacial	acetic	acid	provided	compounds	7a‐c.	1H	
NMR	 spectra	 showed	 additional	 triplet	 signal	 at	 δ	 3.11	 ppm	
assigned	 to	 CH2‐CH2	 protons	 for	 compound	 7a	 or	 a	 doublet	
signal	 at	 δ	 5.11	 ppm	 corresponding	 to	 CH=CH	 protons	 for	
compound	7b	or	additional	multiplet	signal	at	δ	7.66‐8.11	ppm	
attributed	to	added	aromatic	protons	for	compound	7c.	
	

3.2.	Biological	activity	
	

3.2.1.	Antitumor	screening	
	

Cell	 lines	 were	 provided	 by	 American	 Type	 Culture	
Collection	 (Rockville,	 MD).	 These	 cells	were	 grown	 in	 RPMI‐
1640;	all	supplemented	with	10%	heat	inactivated	fetal	bovine	
serum	 (FBS),	 2	 mM	 L‐glutamine,	 and	 1%	 penicillin‐
streptomycin.	DMEM	was	also	supplemented	with	0.01	mg/mL	
insulin	and	1	mM	sodium	pyruvate.	Cells	were	incubated	in	a	
5%	CO2	humidified	incubator	at	37	°C	and	passaged	bi‐weekly.	

All	 the	synthesized	compounds	were	subjected	to	 in	vitro	
tumor	 growth	 inhibitory	 activity	 against	 eight	 different	 cell	
lines	 of	 human	 cancer	 cells.	 The	 cytotoxicity	 of	 the	 newly	
synthesized	 compounds	 against	 cancer	 cell	 lines	 in	 vitro	was	
performed	 with	 the	 MTT	 assay	 according	 to	 the	 Mosmann’s	
method	[23].	The	MTT	assay	is	based	on	the	reduction	of	the	
soluble	 3‐(4,5‐methyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazo‐
lium	 bromide	 (MTT)	 into	 a	 blue‐purple	 formazan	 product,	
mainly	by	mitochondrial	 reductase	activity	 inside	 living	cells.	
Results	were	expressed	in	term	of	IC50	[μM]	(Table	1).	

All	newly	synthesized	compounds	showed	various	activity	
against	 different	 cell	 lines	 compared	 with	 sorafenib.	 Com‐
pound	7c	showed	good	activity	among	the	tested	compounds	
against	 melanoma	 (LOXIMVI)	 cell	 line	 (IC50	 =	 0.0045	 μM)	
compared	 to	 sorafenib	 (IC50	 =	 0.0034	 μM).	 However,	
compounds	3c,	d,	 j	 were	more	 active	 than	 sorafenib	 against	
ovarian	 cell	 line	 (OVCAR3)	 as	 they	 exhibited	 lower	 IC50	
(0.00456‐0.043	μM)	 than	 that	of	 sorafenib	 (0.12	μM).	On	 the	
other	 hand,	 compounds	 3b‐f,	 5a,	 6b‐d	 and	 7a‐c	 exhibited	
higher	 activity	 than	 sorafenib	 (IC50	 0.17	 μM)	 against	 thyroid	
cell	line	(CAL62)	as	they	showed	lower	IC50	(0.003‐0.094	μM)	
while	 compounds	1	 and	3j	 showed	 comparable	 activity	with	
sorafenib	(IC50	=	0.12	and	0.15	μM,	respectively).	Compounds	
3g,	j	and	5a	showed	higher	activity	than	sorafenib	(IC50	0.006	
μM)	against	thyroid	cell	line	(FTC133)	as	they	exhibited	lower	
IC50	 (0.0032‐0.0043	 μM)	 while	 compound	 3h	 showed	
comparable	 activity	 with	 sorafenib	 at	 this	 cell	 line	 (0.0056	
μM).	In	addition,	compounds	3f	and	7b	were	more	active	than	
sorafenib	 against	 thyroid	 cell	 line	 (BCPAP)	 as	 they	 exhibited	
lower	 IC50	(0.08054	and	0.064	μM,	respectively)	compared	to	
IC50	of	sorafenib	(0.087	μM)	while	compounds	3d,	e,	6d	and	7c	
showed	 comparable	 activity	 with	 sorafenib	 against	 this	 cell	
line	 as	 they	 exhibited	 IC50	 (0.085‐0.09	 μM).	 Moreover,	
compounds	1,	2,	3a‐d,	4,	5a,b,	6b	and	7b,c	were	more	active	
than	sorafenib	against	thyroid	cell	line	(ML1)	as	revealed	from	
lower	IC50	(0.10‐0.58	μM)	compared	to	IC50	of	sorafenib	(0.61	
μM)	while	 compounds	3e,j	 showed	 comparable	 activity	with	
sorafenib	against	this	cell	line	as	they	exhibited	IC50	(0.60	and	
0.64	 μM,	 respectively).	 On	 the	 other	 hand,	 compounds	 3d‐i	
exhibited	promising	activity	against	colon	cell	lines	HT29	and	
HCT116	 than	 sorafenib	 as	 they	 exhibited	 much	 lower	 IC50	
(0.00032‐0.00078	μM)	compared	to	IC50	of	sorafenib	(0.73	and	
0.18	μM,	respectively).		



26	 Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	
	
	
Table	1.	In	vitro	growth	inhibitory	activity	(IC50,	μM)	against	melanoma,	thyroid,	and	ovarian	cell	lines.	
Compound	 Melanoma	 Ovarian	 Thyroid Colon	

LOXIMVI	 OVCAR3	 CAL62 FTC133 BCPAP	 ML1 HT29	 HCT116
1	 1.45	 1.34	 0.12 0.21 0.13 0.17 1.23	 *	
2	 2.67	 2.35	 0.32 0.26 0.24 0.26 5.43	 7.66
3a	 4.54	 8.76	 0.45 0.53 0.54 0.35 *	 *	
3b	 6.34	 3.56	 0.09	 0.68	 0.67	 0.28	 8.78	 4.56	
3c	 5.40	 0.00456	 0.0065	 0.98	 0.89	 0.39	 0.98	 5.44	
3d	 0.023	 0.043	 0.0095 0.09 0.09 0.40 0.00034	 0.00032
3e	 6.43	 9.39	 0.075	 0.09	 0.09	 0.60	 0.00056	 0.00056	
3f	 12.45	 12.56	 0.087	 0.0098	 0.08054	 0.79	 0.00032	 0.00078	
3g	 34.43	 34.56	 0.43 0.0043 0.56 0.88 0.00035	 0.00065
3h	 0.13	 23.56	 0.57 0.0056 0.45 0.95 0.00043	 0.00047
3i	 0.43	 5.67	 0.63 0.0075 0.11 0.74 0.00047	 0.00065
3j	 0.35	 0.034	 0.15 0.0032 0.24 0.64 *	 *	
4	 0.45	 0.30	 0.34 0.08 0.32 0.55 *	 *	
5a	 0.45	 0.234	 0.003 0.0043 0.57 0.46 *	 7.50
5b	 0.45	 0.45	 0.436 0.034 0.68 0.57 0.098	 7.60
5c	 2.45	 0.45	 0.576	 0.053	 0.65	 0.68	 *	 55.70	
5d	 5.78	 5.49	 0.605	 0.072	 0.34	 0.70	 *	 32.60	
5e	 9.28	 10.21	 0.905	 0.098	 0.24	 0.80	 0.433	 12.70	
6a	 3.29	 10.11	 0.874 0.0086 0.56 0.90 23.56	 *	
6b	 5.69	 23.56	 0.094 0.99 0.78 0.10 3.67	 *	
6c	 4.89	 3.45	 0.063 0.54 0.75 0.94 *	 7.80
6d	 9.08	 6.034	 0.033 0.37 0.086 0.75 *	 23.60
7a	 8.18	 5.88	 0.082 0.65 0.096 0.76 *	 14.37
7b	 0.065	 2.12	 0.082 0.47 0.064 0.58 *	 16.54
7c	 0.0045	 7.13	 0.011 0.38 0.085 0.57 *	 17.67
Sorafenib	 0.0034	 0.12	 0.17	 0.006	 0.087	 0.61	 0.73	 0.18	
*	Inactive.	
	
Table	2.	IC50	(95%	confidence	interval	[nM])	of	tested	compounds	for	both	WTBRAF	and	V600EBRAF.	
Compound	 IC50	(95%	Confidence	Interval)WTBRAF(nM)	 IC50	(95%	Confidence	Interval)V600EBRAF	(nM)	
1	 15.5	 0.23
2	 24.4	 1.23
3a	 36.5	 0.45
3b	 47.6	 0.45
3c	 58.5	 1.45	
3d	 84.6	 0.44	
3e	 73.5	 0.47	
3f	 67.6	 3.45	
3g	 58.7	 3.45
3h	 69.9	 0.54
3i	 78.8	 0.65
3j	 78.0	 6.78
4	 67.0	 5.45
5a	 46.6	 0.56
5b	 55.7	 7.45	
5c	 44.5	 0.34	
5d	 55.4	 0.56	
5e	 66.5	 0.34
6a	 77.6	 0.31	
6b	 88.7	 0.28
6c	 67.5	 3.45
6d	 56.6	 7.65
7a	 45.4	 3.27
7b	 56.5	 4.30
7c	 45.5	 4.00
Sorafenib	 37.8	 0.48
	

	
Furthermore,	compounds	5b,e	showed	good	activity	(IC50	

=	 0.098	 and	 0.433	 μM,	 respectively)	 against	 colon	 cell	 lines	
HT29	(sorafenib	IC50	0.73	μM).	All	tested	compounds	exhibited	
high	 activity	 against	 thyroid	 (CAL62,	 FTC133,	 BCPAP	 and	
ML1)	 cell	 lines.	 Compound	 3d	 exhibited	 potential	 activity	
against	all	tested	cell	lines.	
	

3.2.2.	In	vitro	kinase	assay	
	

The	 in	 vitro	 kinase	 assay	 of	 the	 synthesized	 compounds	
was	investigated	against	both	WTBRAF	(BRAF	kinase	wild	type)	
and	V600EBRAF	(mutant	BRAF	kinase)	(Table	2).	All	compounds	
were	 highly	 active	 inhibitors	 for	 V600EBRAF	 (0.23‐7.65	 nM)	
compared	 with	moderate	 activity	 against	 WTBRAF	 (15.5‐88.7	
nM).	

Compounds	 1,	 3a,b,d,	 5c,e,	 and	 6a,b	 were	 more	 active	
than	 sorafenib	 as	 they	 exhibited	 lower	 IC50	 (0.23‐0.45	 nM)	
than	that	of	sorafenib	(0.48	nM).	Compounds	3e,h,i,	and	5a,d	
had	comparable	activity	with	sorafenib	(0.47‐0.65	nM).	On	the	

other	hand,	 compounds	2,	3c,f,g,j,	4,	5b,	6c,d	 and	7a‐c	were	
less	active	than	sorafenib	(1.23‐7.65	nM).	
	

3.3.	Molecular	docking	
	

BRAF	 is	 isoform	 of	 RAF	 protein	 kinase	 (Rapidly	
Accelerated	 Fibrosarcoma),	 that	 was	 known	 at	 1988.	 BRAF	
proteins	are	encoded	by	RAF	oncogenes	which	are	 located	 in	
chromosome	 7q32.	 BRAF	 is	 a	 serine/threonine	 specific	
protein	kinase	that	has	long	been	viewed	as	key	players	in	the	
MAPK	 pathway.	 Under	 normal	 circumstances,	 BRAF	 is	
activated	in	a	RAS	small	G‐protein	dependent	manner.	It	then	
phosphorylates	and	activates	the	protein	kinase	MEK	and	ERK	
orderly,	 regulating	gene	expression	and	controlling	how	cells	
respond	to	extracellular	signals.	

BRAF	 kinase	 domain	 consists	 of	 two	 lobes,	 small	 N‐
terminal	lobe	and	large	C‐terminal	lobe.	The	small	N‐terminal	
lobe	 has	 antiparallel	 β‐sheet	 structure	 which	 anchors	 and	
orients	ATP	molecule.	 The	 large	 C‐terminal	 lobe	 is	mainly	 α‐
helical	which	binds	to	MEK	as	a	protein	substrate.		



Moreove
phosphate‐b
these	 two	 lo
inhibitors	[3

In	 each	
segment	is	lo
mations.	In	t
αC‐helix	rota
On	 the	 othe
terminal	 lob
sequences	an
In	the	inactiv
the	phenylal
(DFG	Asp‐ou
the	phenylal
chains	expos

Most	 BR
segment	 (P‐
active	 state	
mutation	(va
approximate
as	V600EBRAF

The	 X‐ra
(native	 ligan
[2,3‐b]pyridi
code:	 4FK3)
with	 binding
kinase.	 The	
rearrangeme
movement	 o
hydrophobic
movement	re
to	ATP	bindi
which	 the	 a
bonding	 inte
which	is	nece
binding	site	a

The	 bind
energy	score
indicates	goo
cation	intera
The	 results	
kinase	 prot
moieties	and
and	PLX3203

	

	
Fig

	
Analysis	
(i)	 The	

reproduced	b
root	 mean	 s
(‐19.2870	 k
protocol	 wa
forming	two

er,	 it	 contain
binding	loop).	T
obes	 that	 wer
3].	
lobe	 (N	 and	
ocated	which	h
the	small	N‐term
ates	and	makes	
er	 hand,	 this	 a
be	 begins	 with	
nd	adjusted	to	
ve	conformatio
anine	side	chai
ut	conformation
anine	side	chai
sed	into	the	bin
RAF	 kinase	 m
‐loop).	 These	 m
rather	 than	 t

al	600	is	replace
ely	 90%	of	 kno
F	kinase	[33].	
ay	 crystal	 stru
nd),	 N‐{2,4‐difl
in‐3‐yl)carbony
	 [28]	 shows	 th
g	 to	 the	 DFG‐o
allosteric	 bind
ent	 of	 the	 a
of	 a	 phenylala
c	 pocket	 and	
esults	in	a	conf
ing	and	also	cre
allosteric	 kinas
eractions	with	
essary	for	tight
and	is	present	i
ding	 affinity	 o
e	 (S,	Kcal/mol)
od	affinity.	Hyd
action	were	also
of	 docking	 stud
tein	 active	 sit
d	hydrogen	bon
3	(Figure	2).	

gure	2.	2D	interac

of	the	docking	
PLX3203‐(V60

by	the	docking	
square	 deviatio
kcal/mol,	 Table
as	 valid.	 PLX32
	hydrogen	bon

Amin	et	al

ns	 a	 P‐loop	
The	catalytic	sit
e	 occupied	 by

C‐terminal	 lo
has	both	active	
minal	lobe,	a	se
part	of	the	acti
activation	 segm
a	 DFG	 (Asp/P
make	part	of	t
on	of	this	segme
n	exposed	away
n),	while	in	the	
n	rotates	out	an
ding	sit	[33].	

mutations	 occu
mutations	 keep
the	 inactive	 st
ed	by	glu	in	the
own	BRAF	mut

ucture	 of	 BRAF
luoro‐3‐[(5‐pyr
yl]phenyl}ethan
hat	 it	 exists	 m
out	 allosteric	 p
ding	 site	 is	 m
activation	 loop
anine	 side	 ch
into	 the	 ATP
formation	that	i
eates	a	large	hy
se	 inhibitors	 b
Cys532	 in	 the	
t	binding	of	the	
in	most	BRAF	in
of	 the	 ligand	 w
	 (Table	3).	 Low
drogen	bond,	ar
o	used	to	assess
dy;	 dock	 score
te	 amino	 acid
nd	length	for	ea

ction	of	PLX3203w

results	reveale
00EBRAF)	 comp
procedure	as	d
on,	 RMSD	 (1.21
e	 3),	 achieved
203	 nearly	 fits
ding	interactio

l.	/	European	Jou

(glycine‐rich	
te	is	located	bet
y	 most	 BRAF	 k

bes),	 a	 polype
and	inactive	co
egment	designa
ive	ATP‐	bindin
ment	 in	 the	 lar
Phe/Gly)	 amino
the	ATP‐bindin
ent	in	the	large
y	from	the	activ
active	conform
nd	the	aspartat

r	 in	 the	 activ
p	 the	 P‐loop	 i
tate.	 The	 Val6
	P‐loop)	accoun
ations	which	 k

F	 with	 its	 PLX
ridin‐3‐yl‐1H‐py
nesulfonamide(
mainly	 in	 dimer
pocket	 of	 V600E
ade	 accessible
p	 and	 subse
ain	 out	 of	 a	
P	 binding	 site.
is	mutually	exc
ydrophobic	poc
bind.	 In	 additio
ATP	binding	 p
inhibitor	in	the
nhibitors	[34].
was	 evaluated
w	dock	 score	e
rene	arene	and	
s	the	binding	m
,	 involved	 V600E
d	 interacting	 l
ach	active	comp

ith	V600EBRAF.	

ed	that:	
plex	 was	 pre
demonstrated	b
173)	 and	 dock	
d	 that	 the	 do
s	 in	 the	 activ
ns	with	Cys	53

urnal	of	Chemistr
 

ATP‐
tween	
kinase	

eptide	
onfor‐
ted	as	
ng	site.	
rge	 C‐
o	 acid	
g	site.	
e	lobe,	
ve	site	
mation,	
te	side	

vation	
in	 the	
00Glu	
nts	for	
known	

X3203	
yrrolo	
(PDB	
r	 form	
EBRAF	
	 by	 a	
equent	
large	
.	 This	
clusive	
cket	to	
on,	 H‐
pocket	
e	ATP‐

d	 with	
energy	
arene	

models.	
EBRAF	
ligand	
pound	

ecisely	
by	low	
score				
ocking	
e	 site	
32	and	

Phe
531

enz
fits	
and

ran
one
cati
com
acti
‐14
583
invo

‐19
com
to	
Mor
‐17
com
Kca

Kca
with
inte
Lys

nM)
with
one
seco
and
to	 a
and
	
4.	C

syn
acti
line

ry	7 (1)	(2016)	1

e	595,	three	are
1	and	one	arene
(ii)	 The	 docki
zyme	 inhibition
in	the	active	si
d	one	arene	aren
	

Figure	3.	
	
(iii)	The	dockin
ge	‐10.6210	to
e	 or	 two	 hydro
ion	interactions
mpounds	 3j	 an
ive	 site	 altho
.6555	respectiv
3,	 Asp	 594	 an
olved	in	these	i
(iv)	 The	 docki
.0422	 Kcal/mo
mpounds	5a‐e	s
‐21.0419	 Kcal/
reover,	the	doc
.4771	Kcal/mo
mpounds	7a‐c	s
al/mol	and	IC50
(v)	 Compoun
al/mol)	and	bin
h	 Asp	 594	 an
eraction	with	Tr
	483	(Figure	4)
	

Figure	4.	2D
	
Compound	1,	
)	while	gave	a	d
h	the	active	sit
e	 arene	 arene	
ond	most	 activ
d	showed	a	doc
active	 site	with
d	one	arene	cati

Conclusion	
	
A	 number	 of

nthesized.	 Most
ivity	towards	th
es.	Compound	3

9‐29

ene	arene	inter
e	cation	interact
ing	 score	 for	 s
n	 assay)	 was	 ‐
ite	forming	one
ne	interaction	w

2D	interaction	of	s

ng	score	of	test
	 ‐21.0419	Kcal
ogen	 bonds	 an
s	with	 the	enzy
nd	 7a	 showed	
ugh	 binding	
vely.	Lys	483,	T
nd	 Phe	 595	 a
nteractions.	
ing	 scores	 of	 c
ol)	 with	 IC50	
showed	docking
/mol	 while	 th
cking	scores	of	c
ol),	with	IC50	(0.
showed	docking
3.27‐4.30	nM.
d	 5dgave	 bes
nds	with	2	hydr
nd	 Lys	 483	 re
rp	531	and	one
).	

D	interaction	of	com

most	 active	 co
docking	score	o
e	with	one	hyd
interaction	 wi

ve	 compound	 e
cking	score	of	‐1
h	 one	 arene	 ate
ion	interaction	w

f	 new	 pyridop
t	 of	 these	 com
hyroid	cell	lines
3d	was	the	most

actions	with	Ph
tion	with	Lys	48
sorafenib	 (stan
23.4155	 Kcal/
	hydrogen	bond
with	Phe	583	(F

sorafenib	with	V600

ted	compounds
/mol.	All	comp
nd/	 or	 arene	 a
yme	active	site	
no	 binding	 in
scores	 were	
Trp	531,	Cys	53
re	 the	 amino	

compounds	3a‐
(0.44‐6.78	 nM
g	scores	in	the	r
eir	 IC50	 were	
compounds	6a‐
.28	to	7.65	nM)
g	scores	‐14.65

st	 docking	 sc
rogen	bonds	(3.
espectively,	 on
e	arene	cation	in

mpound	5d	with	V

ompound	gave	
of	‐11.3281	Kca
drogen	bond	wi
ith	 Phe	 583.	 C
exhibited	 good	
16.0553	Kcal/m
ene	 interaction
with	Phe	583.	

pyrazinone	 der
mpounds	 have
s	rather	than	re
t	active	one.		

27

he	583	and	Trp
83	(Figure	2).
ndard	 used	 for
/mol.	 Sorafenib
d	with	Asp	594
Figure	3).	

0EBRAF.

s	were	all	in	the
pounds	showed
arene	 or	 arene
residues.	Only

nteraction	 with
‐15.8120	 and
2,	Gly	534,	Phe
acids	 residue

‐j	 (‐10.6210	 to
).	 In	 addition,
range	‐13.3412
0.34‐7.45	 nM.
‐d	(‐14.6422	to
).	Furthermore,
55	to	‐18.1910

core	 (‐21.0419
.12	and	3.37	Å)
e	 arene	 arene
nteraction	with

V600EBRAF.

best	 IC50	 (0.23
al/mol	and	bind
th	Cys	532	and
Compound	 6b,
IC50	 (0.28	nM)
mol	which	bind
n	with	 Lys	 483

rivatives	 were
e	 a	 significant
est	of	other	cell

p	

r	
b	
4	

e	
d	
e	
y	
h	
d																	
e	
e	

o						
,	
2	
.	
o	
,	
0	

9	
)	
e	
h	

3	
d	
d	
,	
)	
d	
3	

e	
t	
l	



28	 Amin	et	al.	/	European	Journal	of	Chemistry	7	(1)	(2016)	19‐29	
	

	
Table	3.	Docking	results.	
Compound	 Energy	score	

S	(Kcal/mol)	
Amino	acids	
interaction

Interacting	
groups

Hydrogen	bond		
length	(Å)	

PLX3203	 ‐19.2870	 Lys	483	(arene	cation) Phenyl	ring 		
Trp	531	(arene	arene)	 N	Pyridine	 		
Cys	532	 N	Pyridine	 3.1	
Phe	583	(arene	arene)	 N	Pyridine	 		
Phe	583	(arene	arene)	 N	Pyrrole	 		
Phe	595 SO2 2.99	

Sorafenib	 ‐23.4155	 Phe	583	(arene	arene) Phenyl	ring 		
Asp	594 C=O 2.6	

1	 ‐11.3281	 Cys	532 N	Pyridine 3.02	
Phe	583	(arene	arene) Pyridine	ring 		

2	 ‐13.0101	 Cys	532 N	Pyridine 3.05	
Phe	583	(arene	arene) Pyridine	ring 		

3a	 ‐13.2439	 Cys	532	 C=O	 2.69	
Phe	583	(arene	arene)	 Pyridine	ring	 		

3b	 ‐10.6210	 Lys	483	(arene	cation) Pyridine	ring 		
Phe	583	(arene	cation)	 Piperidine	ring	 		
Asp	594 N	Pyridine 2.98	

3c	 ‐11.6002	 Cys	532 C=O 3.13	
Phe	583	(arene	arene) Pyridine	ring 		

3d	 ‐17.5978	 Cys	532 NH	Piperazine 2.27	
Cys	532 C=O 2.69	

3e	 ‐17.7071	 Lys	483	(arene	cation) Phenyl	ring 		
3f	 ‐16.0602	 Cys	532	 NH	Piperazine	 1.83	

Cys	532 C=O 3.22	
3g	 ‐18.4843	 Cys	532	 NH	Piperazine	 2.2	

Cys	532	 C=O	 2.61	
3h	 ‐18.2255	 Cys	532	 NH	Piperazine	 2.26	

Cys	532 C=O 2.56	
3i	 ‐19.0422	 Cys	532 NH	Piperazine 2.24	

Cys	532 C=O 2.58	
3j	 ‐15.8120	 	‐	 ‐ 	‐	
4	 ‐12.0174	 Cys	532 C=O 2.57	

Gly	534 NH2 1.94	
Phe	583	(arene	arene)	 Pyridine	ring	 		

5a	 ‐16.5954	 Lys	483	(arene	cation)	 Phenyl	ring	 		
Cys	532	 NH	Piperazine	 1.83	
Cys	532 C=O 2.52	

5b	 ‐16.7560	 Lys	483	(arene	cation)	 Pyridine	ring	 		
Asp	594 N	Pyridine 2.63	

5c	 ‐13.3412	 Cys	532 NH	Piperazine 1.86	
Cys	532 C=O 2.57	

5d	 ‐21.0419	 Lys	483	(arene	cation) Phenyl	ring 		
Lys	483 OCH3 3.37	
Trp	531	(arene	arene) Pyridine	ring 		
Asp	594	 OCH3	 3.12	

5e	 ‐18.4503	 Lys	483	(arene	cation) Pyridine	ring 		
Asp	594	 N	Pyridine	 2.64	

6a	 ‐14.6422	 Cys	532	 C=O	 2.53	
Phe	583	(arene	arene)	 Pyridine	ring	 		

6b	 ‐16.0553	 Lys	483	(arene	cation) Phenyl	ring 		
Phe	583	(arene	arene) Pyridine	ring 		

6c	 ‐15.7831	 Cys	532 NH	Piperazine 1.58	
6d	 ‐17.4771	 Lys	483 OCH3 2.99	
7a	 ‐14.6555	 	‐	 ‐ 	‐	
7b	 ‐17.4101	 Lys	483	(arene	cation) Pyridine	ring 		

Asp	594	 N	Pyridine	 2.65	
7c	 ‐	18.1910	 Lys	483	(arene	cation)	 Pyridine	 		

Asp	594 N	Pyridine 2.66	
	
	

It	exhibited	promising	activity	against	all	 tested	cell	 lines.	
In	 addition,	 the	 in	 vitro	 kinase	assay	 revealed	 that	 the	 tested	
compounds	were	more	 active	 towards	 V600EBRAF	 rather	 than	
WTBRAF	enzyme.	The	docking	study	revealed	binding	of	most	
compounds	with	Cys	 532	 at	 the	 active	 site.	 Finally,	 based	 on	
both	 in	 vitro	 and	 docking	 studies,	 introduction	 of	
unsubstituted,	o‐CH3,	p‐Cl,	p‐Br	phenyl	amino	group	increased	
activity	 of	 V600EBRAF	 inhibitors	 (compounds	 3d,e,h,i).	 Also	
Shiff’s	 compounds	 with	 unsubstituted	 benzylidine	 increased	
activity,	 while	 p‐substituted	 benzylidine	 ring	 with	 electron	
withdrawing	 group	 decreased	 activity	 of	 these	 inhibitors	
(compounds	5d,e).	In	case	of	tricyclic	derivatives;	substitution	
with	 Cl	 at	 position	 4	 increased	 activity	 (compound	 6b).	
Although	 the	 docking	 study	 showed	 non‐significant	
correlation	 with	 results	 of	 enzyme	 inhibition	 assay,	 most	 of	
docked	compounds	shared	some	of	binding	 interactions	with	
V600EBRAF	 active	 sites	 similar	 to	 those	 of	 native	 ligand	

(PLX3203).	 This	 suggests	 that	 these	 compounds	 might	
possibly	act	as	V600EBRAF	inhibitors.	
	
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