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European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	

	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2017	Atlanta	Publishing	House	LLC	‐	All	rights	reserved	‐	Printed	in	the	USA	

http://dx.doi.org/10.5155/eurjchem.8.4.391-399.1652 

	
	

	

	

European	Journal	of	Chemistry	
Journal	webpage:	www.eurjchem.com	 	

	 	 	

Synthesis	and	biological	evaluation	of	phthalimide	dithiocarbamate	and	
dithioate	derivatives	as	anti‐proliferative	and	anti‐angiogenic	agents‐I	

Magdy	Zahran	1,	Hussein	Agwa	1,2,*,	Amany	Osman	1,	Sherif	Hammad	2,3,	Bishoy	El‐Aarag	4,		
Nasser	Ismail	5,	Tarek	Salem	6	and	Amira	Gamal‐Eldeen	7	

1	Chemistry	Department,	Faculty	of	Science,	Menoufia	University,	Shebin	El‐Koom,	32511,	Egypt	
2	Research	and	Development	Department,	Pharco	B	International	Company	for	Pharmaceutical	Industries,	Borg	El‐Arab,	Alexandria,	21934,	Egypt	
3	Department	of	Pharmaceutical	Chemistry,	Faculty	of	Pharmacy,	Helwan	University,	Cairo,	11599,	Egypt	
4	Biochemistry	Division,	Chemistry	Department,	Faculty	of	Science,	Menoufia	University,	Shebin	El‐Koom,	32511,	Egypt	
5	Department	of	Pharmaceutical	Chemistry,	Faculty	of	Pharmacy,	Ain‐Shams	University,	Cairo,	11599,	Egypt	
6	Molecular	Biology	Department,	Genetic	Engineering	and	Biotechnology	Research	Institute	(GEBRI),	Sadat	City	University,	Sadat	City,	32897,	Egypt	
7	Cancer	Biology	and	Genetics	Laboratory,	Centre	of	Excellence	for	Advanced	Sciences,	National	Research	Centre,	Dokki,	Cairo,	11599,	Egypt	

*	Corresponding	author	at:	Chemistry	Department,	Faculty	of	Science,	Menoufia	University,	Shebin	El‐Koom,	32511,	Egypt.		
Tel.:	+2.034.626448.	Fax:	+2.034.626026.	E‐mail	address:	husseinagwa@gmail.com	(H.	Agwa).	

	
	

	 	

	 	 	

ARTICLE	INFORMATION	 	 ABSTRACT	

	
DOI: 10.5155/eurjchem.8.4.391-399.1652	

Received:	15	September	2017	
Received	in	revised	form:	23	October	2017	
Accepted:	25	October	2017	
Published	online:	31	December	2017	
Printed:	31	December	2017

 

	 A	 facile	synthesis	of	new	phthalimide	dithiocarbamate	and	dithioate	analogs	8a‐j,	9a‐e	and
9g‐j	 were	 achieved	 by	 the	 reaction	 of	N‐chloromethyl	 and	N‐bromoethylphthalimide	 with
carbon	disulfide	 (CS2)	and	various	amines.	The	 structures	of	 the	 synthesized	analogs	were
elucidated	 by	 spectroscopic	 methods,	 including	 IR,	 1H	 NMR	 and	 13C	 NMR,	 and	 ESI‐HRMS
techniques.	 The	 antiproliferative	 activity	 of	 the	 newly	 synthesized	 compounds	 was	 also
evaluated	 against	 various	 human	 cancer	 cell	 lines.	 The	 compound	 9e	 and	 9i	 exhibited	 the
highest	activity	against	human	breast	adenocarcinoma	MCF‐7	and	hepatocellular	carcinoma
HepG2	 cells.	 Compound	 8f	 showed	 better	 antiproliferative	 effect	 against	 colon	 carcinoma
HCT‐116	 and	 cervical	 carcinoma HeLa	 compared	 to	 thalidomide.	 The	 binding	 affinity	 to
vascular	 endothelial	 growth	 factor	 receptor	 (VEGFR)	 of	 some	 compounds	was	 assessed	 in
addition	 to	 molecular	 docking	 study.	 Compounds	 9e	 and	 9i	 showed	 high	 docking	 score
values	and	they	significantly	declined	the	concentration	of	VEGFR.	

KEYWORDS	
Cytotoxicity	
Phthalimide	
Thalidomide	
Dithiocarbamate	
Antitumor	activity	
Molecular	Docking	

Cite	this: Eur.	J.	Chem.	2017,	8(4),	391‐399

	
1.	Introduction	
	

Thalidomide	(TH,	Figure	1),	was	originally	launched	in	the	
1950s	as	sedative	and	antiemetic	drug	and	was	subsequently	
withdrawn	from	the	market	in	1960s	owing	to	its	teratogenic	
properties	 [1‐3].	 Recently,	 TH	 has	 attracted	 a	 considerable	
attention	 due	 to	 its	 pronounced	 antiangiogenic	 activity.	 TH	
has	been	re‐approved	by	FDA	in	2006	and	re‐purposed	in	the	
treatment	 of	 various	 hematological	 malignancies	 such	 as	
multiple	myeloma	and	solid	 tumors	such	as	 colon,	brain,	 and	
prostate	cancers	[4].		

TH	 is	 considered	 to	 be	 a	 soft	 drug	 yielding	 biologically	
active	 metabolites	 upon	 biotransformation.	 Among	 these	
metabolites,	phthalimide	species	represents	a	pharmacophoric	
group	which	might	be	the	reason	for	their	biological	activities	
[5].	 Based	 on	 these	 results	 and	 assumption,	 new	 series	 of	
phthalimide	 derivatives	 were	 synthesized	 and	 evaluated	 as	
potential	antitumor	drug	candidates	[6,7].	Recently,	our	group	

reported	 the	 potency	 of	 the	 thalidomide	 dithiocarbamate	
analogs	as	novel	antitumor	derivatives	[8‐14].		

	

N

O

O

NH

O

O

Phthalimide Piperidine-2,6-dione

	
	

Figure	1.	Chemical	structure	of	thalidomide	(TH).
	
On	the	other	hand,	dithiocarbamic	acid	esters	have	drawn	

a	 great	 attention	 due	 to	 their	 cancer	 chemopreventive	 and	
antitumor	activities	[15,16].		

	



392	 Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	
	

	

N

O

O

N

O

O S

Amine

S

N

O

O

S

Amine

S

(CH2)n

n=1, compounds 8a-j
n=2, compounds 9a-e, 9g-j

N

O

O

NH

O

O

Molecular conjugation
with dithiocarbamate

Molecular simplification

by removal of piperidinedione

Reservation of the phthalimide 
pharmacophore

Thalidomide
(Prototype)

(Current work)

Thalidomide 
dithiocarbamate analogs

(Previous work) 	
	

Figure	2.	Rational	design	of	the	targeted	synthesized	compounds.
	
	
Some	 molecules	 that	 incorporated	 dithiocarbamic	 acid	

ester	 functionality,	 as	 a	 linker	 between	 different	 pharma‐
cophores,	showed	significant	anticancer	activities,	such	as	TH	
derivatives	 [8‐14],	 chromones	 [17],	 quinazolinones	 [18]	 and	
naphthalimide	derivatives	[19].	

Our	research	interest	is	still	existing	in	development	of	TH	
analogs	and	potential	antitumor	compounds	[8‐14,20‐22].	So,	
we	 herein	 describe	 the	 design,	 synthesis	 and	 biological	
evaluation	of	novel	dithiocarbamate	analogs	connected	either	
through	methylene	 or	 ethylene	 bridges	 to	 phthalimide	 phar‐
macophoric	core.	The	 insertion	of	one	or	 two	carbon	spacers	
to	 connect	 the	 N‐terminus	 of	 phthalimide	 with	 dithiocarba‐
mate	 groups	was	 executed	 to	 explore	 the	 influence	 of	 exclu‐
ding	 the	 2,6‐piperidinedione	moiety	 from	TH	 from	 one	 side	
and	the	impact	of	the	linker	length	on	antitumor	activity	from	
the	 other	 side	 (Figure	 2).	 Moreover,	 the	 newly	 synthesized	
phthalimide	 derivatives	 were	 evaluated	 for	 their	 growth	
inhibitory	 effects	 against	 various	 cancer	 cell	 lines.	Molecular	
docking	 studies	 as	 well	 as	 their	 binding	 affinity	 on	 VEGFR	
were	also	investigated.	
	
2.	Experimental	
	
2.1.	Instrumentation	
	

The	 progress	 of	 all	 reactions	 and	 synthesized	 product	
were	 monitored	 via	 analytical	 silica	 gel	 thin	 layer	 chroma‐
tography	 (TLC)	 plates	 60	 F254	 which	 were	 purchased	 from	
Merck	 and	 spots	 were	 located	 by	 UV	 light.	 1H	 and	 13C	 NMR	
spectra	 for	 all	 synthesized	 compounds	 were	 recorded	 on	 a	
Bruker	 400	 MHz	 spectrometer	 for	 1H	 and	 100	 MHz	 for	 13C,	
with	TMS	as	an	internal	standard	for	1H	NMR,	chemical	shifts	
are	 reported	 in	 parts	 per	 million	 (ppm)	 relative	 to	 the	
respective	deuterated	solvent	peak	CDCl3	(δ	7.27	ppm),	DMSO‐
d6	 (δ	2.50	ppm)	 for	 1H	and	CDCl3	 (δ	77.00	ppm),	DMSO‐d6	 (δ	
39.51	ppm)	 for	 13C	NMR.	MALDI	mass	spectra	of	 synthesized	
compounds	 were	 recorded	 on	 JEOL	 JMS‐700N	 for	 electron	
ionization	or	on	JEOL	JMS‐T100TD	for	electro‐spray	ionization	
using	 α‐cyano‐4‐hydroxycinnamic	 acid	 (CHCA)	 as	 a	 matrix	
(m/z	 189.17)	 (Nagasaki	 University,	 Japan).	 Electro‐spray	
ionization	 high	 resolution	 mass	 spectra	 (ESI‐HRMS)	 were	
performed	on	PE	SCIEX	API	Q‐Star	Pulsar	Mass	Spectrometer.	
For	accurate	ion	mass	determinations,	the	[MH+]	or	[MNa+]	ion	

was	 peak	 matched	 by	 calibration	 with	 NaI	 (University	 of	
Southern	 Denmark,	 Denmark).	 Infrared	 (IR)	 spectra	 were	
recorded	(KBr)	on	a	Pye‐Unicam	Sp‐883	Perkins‐Elmer	spect‐
rometer,	Micro‐analytical	Laboratory,	Faculty	of	Science,	Cairo	
University.	Melting	 points	were	 recorded	 on	 Stuart	 scientific	
melting	 point	 apparatus.	 All	 chemicals	 and	 solvents	 were	
purchased	 from	 E.	 Merck	 (Darmstadt,	 Germany)	 and	 Sigma‐
Aldrich.	
	
2.2.	Synthesis	
	
2.2.1.	General	procedure	for	the	synthesis	of	phthalimide	
dithiocarbamate	and	dithioate	analogs	(8a‐j,	9a‐e,	9g‐j)	
	

A	 solution	 of	 carbon	 disulfide	 (CS2)	 (1	mL)	 and	 different	
amines	 (1	 equivalent)	 in	 acetonitrile	 (CH3CN)	 (5	 mL)	 was	
stirred	 for	 30	 min	 at	 room	 temperature.	 N‐chloromethyl	
phthalimide	 (3)	 or	 N‐bromoethylphthalimide	 (7)	 (1	 equiva‐
lent)	was	added	to	the	resulting	mixture	and	the	stirring	was	
continued	 for	 48	 h.	 The	 reaction	 was	 monitored	 using	 TLC.	
After	 completion	 the	 reaction,	 the	 solvent	 removed	 in	 vacuo,	
the	residue	co‐evaporated	twice	with	dichloromethane	and	the	
products	 obtained	 were	 crystallized	 from	 ethanol	 (20	 mL)	
(Scheme	1).	

(1,	3‐Dioxoisoindolin‐2‐yl)methyl	methylcarbamodithioate	
(8a):	Color:	White	crystals.	Yield:	72%.	M.p.:	135‐137	°C.	FT‐IR	
(KBr,	 ,	 cm‐1):	 3466	 (NH	 broad),	 3056	 (Ar	 CH),	 2997,	 2930	
(CH2),	1772,	1715	(2	CO	sharp),	1072	(CS	sharp).	1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	3.03	 (d,	3H,	 J	=	2.7	Hz,	CH3),	5.45	(s,	
2H,	 NCH2S),	 7.86‐7.93	 (m,	 4H,	 Harom),	 10.03	 (s,	 1H,	 NH).	 13C	
NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	30.71	(CH3),	42.20	(NCH2S),	
123.44,	131.00,	134.87	(Aryl),	167.00	(2	×	C=O),	193.00	(C=S).	
HRMS	 (ESI,	 m/z)	 calcd.	 for	 C11H10N2O2S2Na+	 [M+Na]+:	
289.0080, Found:	289.0076.	

(1,	 3‐Dioxoisoindolin‐2‐yl)methyl	 propylcarbamodithioate	
(8b):	Color:	Pale	yellow	crystals.	Yield:	65%.	M.p.:	144‐146	°C.	
FT‐IR	 (KBr,	 ,	 cm‐1):	 3293	 (NH	 broad),	 3062	 (ArCH),	 2961,	
2918	 (CH2),	 1762,	 1710	 (2	 CO	 sharp),	 1072	 (CS	 sharp).	 1H	
NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	0.85‐0.90	(t,	3H,	CH3),	1.52‐
1.63	 (m,	2H,	CH3CH2),	 3.31‐3.56	 (m,	2H,	CH3CH2CH2),	 5.45	 (s,	
2H,	 NCH2S),	 7.85‐7.93	 (m,	 4H,	 Harom),	 10.04	 (s,	 1H,	 NH).	 13C	
NMR	(100	MHz,	CDCl3,	δ,	ppm):	11.15	(CH3),	20.98	 (CH3CH2),	
40.33		(NCH2S),		48.86		(CH3CH2CH2),		123.62,		131.16,		134.46		



Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	 393	
 

	 	

NH

O

O
1

(i) N

O

O

Cl N

O

O

S
R

S

8a-j

O

O

O
4

(iii) (v)
N

O

O

OH

N

O

O
OH

2

5

3

N

O

O
X

N

O

O

9a-e, 9g-j

S
S

R

(v)

6 X = Cl

7 X = Br

(ii)

(iv)

(iii)

	
 
Reagents	and	conditions:	(i)	Formaldehyde	(37%	solution	in	water),	reflux,	1	h;	(ii)	a)	KOH,	EtOH,	b)	BrCH2CH2Br;	(iii)	 thionyl	chloride,	DMF,	0	°C,	1	h;	(iv)	
Ethanolamine,	EtOH,	reflux;	(v)	RCS2H,	CH3CN,	room	temperature,	48	h.	
	

Scheme	1
	
	
(aryl),	167.16	(2	×	C=O),	192.29	(C=S).	HRMS	(ESI,	m/z)	calcd.	
for	C13H14N2O2S2Na+	[M+Na]+:	317.0390,	Found:	317.0389.	

(1,3‐Dioxoisoindolin‐2‐yl)methyl	 bis(2‐hydroxyethyl)carba	
modithioate	(8c):	Color:	White	crystals.	Yield:	77%.	M.p.:	100‐
102	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	 3374	 (OH	 broad),	 2932,	 2879	
(CH2),	1773,	1718	(2	CO	sharp),	1071	(CS	sharp).	1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	3.58‐3.77	(m,	4H,	CH2NCH2),	3.83‐3.87	
(m,	2H,	CH2OH),	4.07‐4.11	 (m,	2H,	CH2OH),	4.86‐4.90	 (m,	1H,	
OH),	4.96‐5.02	(m,	1H,	OH),	5.42	(s,	2H,	NCH2S),	7.83‐7.93	(m,	
4H,	Harom).	13C	NMR	(100	MHz,	CDCl3,	δ,	ppm):	44.09	(NCH2S),	
57.69,	 58.87	 (2	 ×	 CH2N),	 60.53	 (CH2OH),	 123.63,	 131.82,	
134.30	(aryl),	166.92	(2	×	C=O),	195.60	(C=S).	HRMS	(MALDI,	
m/z)	 calcd.	 for	 C14H17N2O4S2+	 [M+H]+:	 341.0630,	 Found:	
341.0624.	

(1,	3‐Dioxoisoindolin‐2‐yl)methyl	diethylcarbamodithioate	
(8d):	 Color:	 Colorless	 crystals.	 Yield:	 65%.	M.p.:	 128‐130	 °C.	
FT‐IR	 (KBr,	 ,	 cm‐1):	 2971,	 2932	 (CH2),	 1773,	 1718	 (2	 CO	
sharp),	 1064	 (CS	 sharp).	 1H	 NMR	 (400	MHz,	 CDCl3,	 δ,	 ppm):	
1.23‐1.28	 (m,	6H,	2	×	CH3),	3.65	 (q,	2H,	 J	=	12	Hz,	CH2NCH2),	
4.01	(q,	2H,	J	=	12	Hz,	CH2NCH2),	5.63	(s,	2H,	NCH2S),	7.72‐7.75	
(m,	 2H,	 Harom),	 7.85‐7.88	 (m,	 2H,	 Harom).	 13C	 NMR	 (100	MHz,	
CDCl3,	δ,	ppm):	11.50	(CH3),	12.57	(CH3),	43.83	(NCH2S),	46.87,	
49.34	(2	×	CH2NCH2),	123.60,	131.93,	134.25	(aryl),	166.88	(2	
×C=O),	 192.27	 (C=S).	 HRMS	 (ESI,	m/z)	 calcd.	 for	 C14H16N2O2	
S2Na+	[M+Na]+	:	331.0551,	Found:	331.0545.	

(1,	3‐Dioxoisoindolin‐2‐yl)methyl	cyclohexylcarbamodithio	
ate	 (8e):	 Color:	White	 crystals.	 Yield:	 60%.	M.p.:	 185‐187	 °C.	
FT‐IR	(KBr,	,	cm‐1):	3283	(NH	broad),	2934,	2853	(CH2),	1767,	
1712	(2	CO	sharp),	1075	(CS	sharp).	1H	NMR	(400	MHz,	CDCl3,	
δ,	ppm):	1.19‐1.84	(m,	10H,	cyclohex.	C2,3,4,5,6‐H),	2.11‐2.20	(m,	
1H,	 cyclohex.	 C1‐H),	 5.13	 (s,	 2H,	 NCH2S),	 7.75‐7.80	 (m,	 2H,	
Harom),	7.89‐7.92	 (m,	2H,	Harom),	8.30	 (br	 s,	 1H,	NH).	 13C	NMR	
(100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 23.75	 (cyclohex.	 C4),	 24.61	
(cyclohex.	C3),	25.04	(cyclohex.	C5),	30.31	(cyclohex.	C2),	30.88	
(cyclohex.	 C6),	 41.08	 (NCH2S),	 55.95	 (cyclohex.	 C1),	 123.45,	
131.39,	134.88	 (aryl),	166.70	 (2	×	C=O),	191.26	 (C=S).	HRMS	

(MALDI,	 m/z)	 calcd.	 for	 C16H19N2O2S2+	 [M+H]+	 :	 335.0888,	
Found:	335.0883.	

(1,	 3‐Dioxoisoindolin‐2‐yl)methyl	 2‐(piperidin‐1‐yl)ethylcar	
bamodithioate	(8f):	Color:	Yellow	solid.	Yield:	79%.	M.p.:	143‐
146	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	 3438	 (NH	 broad),	 2929	 (CH2),	
1772,	1718	(2	CO	sharp),	1071	(CS	sharp).	1H	NMR	(400	MHz,	
DMSO‐d6,	 δ,	 ppm):	 1.74‐1.78	 (m,	 6H,	 pip.	 C3,4,5‐H),	 2.48‐2.49	
(m,	4H,	pip.	C2,6,‐H),	3.25‐3.27	(m,	2H,	CH2N)	3.40‐3.45	(m,	2H,	
NHCH2),	5.47	(s,	2H,	NCH2S),	7.86‐7.93	(m,	4H,	Harom),	10.51	(s,	
1H,	NH).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	21.20	(pip.	C4),	
22.36	 (pip.	 C3,	 C5),	 40.99	 (NHCH2),	 41.36	 (NCH2S),	 52.29	
(CH2N),	 53.46	 (pip.	 C2,	 C6),	 123.59,	 131.50,	 135.05	 (aryl),	
166.83	 (2	 ×	 C=O),	 194.62	 (C=S).	 HRMS	 (ESI,	m/z)	 calcd.	 for	
C17H22N3O2S2+	[M+H]+	:	364.1153,	Found:	364.1148.	

(1,	3‐Dioxoisoindolin‐2‐yl)methyl	morpholinocarbamodithio	
ate	(8g):	Color:	White	solid.	Yield:	92%.	M.p.:	198‐200	°C.	FT‐
IR	(KBr,	,	cm‐1):	3435	(NH	broad),	3098	(Ar	CH),	2968,	2836	
(CH2),	1773,	1724	(2	CO	sharp),	1039	(CS	sharp).	1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	2.79	(m,	4H,	morph.	C3,5‐H),	3.66	(m,	
4H,	 morph.	 C2,6‐H),	 5.27	 (s,	 2H,	 NCH2S),	 7.85‐7.92	 (m,	 4H,	
Harom),	 9.67	 (br	 s,	 1H,	 NH).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	
ppm):	40.08	(NCH2S),	53.82	(morph.	C3,	C5),	65.64	(morph.	C2,	
C6),	 123.36,	 131.45,	 134.78	 (aryl),	 166.76	 (2	 ×	 C=O),	 196.33	
(C=S).	 HRMS	 (ESI,	 m/z)	 calcd.	 for	 C14H16N3O3S2+	 [M+H]+	 :	
338.0633,	Found:	338.0628.	

(1,	3‐Dioxoisoindolin‐2‐yl)methyl	4‐methylpiperazin‐1‐ylcar	
bamodithioate	 (8h):	 Color:	 White	 crystals.	 Yield:	 72%.	 M.p.:	
142‐144	 °C.	FT‐IR	 (KBr,	,	 cm‐1):	 3477	 (NH	broad),	3088	 (Ar	
CH),	 2925,	 2888	 (CH2),	 1773,	 1721	 (2	 CO	 sharp),	 1072	 (CS	
sharp).	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	2.17	(s,	3H,	CH3),	
2.19‐2.24	(m,	4H,	pip.	C3,5‐H),	2.79	(brs,	4H,	pip.	C2,6‐H),	5.27	(s,	
2H,	 NCH2S),	 7.84‐7.92	 (m,	 4H,	 Harom),	 11.12	 (s,	 1H,	 NH).	 13C	
NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	40.06	(CH3),	45.20	(NCH2S),	
53.07	 (pip.	C3,	 C5),	 53.87	 (pip.	 C2,	 C6),	 123.34,	131.45,	134.75	
(aryl),	166.76	(2	×	C=O),	196.31	(C=S).	HRMS	(ESI,	m/z)	calcd.	
for	C15H19N4O2S2+[M+H]+:	351.0949,	Found:	351.0942.	



394	 Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	
	

(1,	3‐Dioxoisoindolin‐2‐yl)methyl	piperidine‐1‐carbodithio	
ate	(8i):	Color:	Colorless	crystals.	Yield:	63%.	M.p.:	129‐130	°C.	
FT‐IR	 (KBr,	 ,	 cm‐1):	 2931,	 2855	 (CH2),	 1776,	 1715	 (2	 CO	
sharp),	1071	(CS	sharp).	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	
1.57‐1.64	(m,	6H,	pip.	C3,4,5‐H),	3.82	(br	s,	2H,	pip.	C2‐H),	4.19	
(br	 s,	 2H,	 pip.	 C6‐H),	 5.43	 (s,	 2H,	 NCH2S),	 7.84‐7.93	 (m,	 4H,	
Harom).	 13C	 NMR	 (100	 MHz,	 CDCl3,	 δ,	 ppm):	 24.17	 (pip.	 C4),	
25.99	(pip.	C3),	26.10	(pip.	C5),	43.85	(NCH2S),	52.62	(pip.	C2),	
52.90	(pip.	C6),	123.58,	131.92,	134.24	(aryl),	166.86	(2×C=O),	
192.16	 (C=S).	 HRMS	 (MALDI,	 m/z)	 calcd.	 for	 C15H17N2O2S2+	
[M+H]+:	321.0731,	Found:	321.0722.	

(1,	 3‐Dioxoisoindolin‐2‐yl)methyl	morpholine‐4‐carbodithio	
ate	(8j):	Color:	White	solid.	Yield:	88%.	M.p.:	179‐181	°C.	FT‐IR	
(KBr,	,	cm‐1):	2905,	2855	(CH2),	1771,	1720	(2	CO),	1105	(CS	
sharp).	 1H	 NMR	 (400	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 3.10‐3.13	 (m,	
2H,	morph.	C3‐H),	3.65	(m,	2H,	morph.	C5‐H),	3.75‐3.78	(m,	2H,	
morph.	 C2‐H),	 4.27‐4.30	 (m,	 2H,	 morph.	 C6‐H),	 5.46	 (s,	 2H,	
NCH2S),	7.87‐7.93	(m,	4H,	Harom).	13C	NMR	(100	MHz,	DMSO‐d6,	
δ,	ppm):	43.22	(NCH2S),	63.52	(morph.	C3,	C5),	66.10	(morph.	
C2,	C6),	123.47,	131.39,	134.91	(aryl),	166.60	(2	×	C=O),	192.54	
(C=S).	 HRMS	 (ESI,	m/z)	 calcd.	 for	 C14H14N2O3S2Na+	 [M+Na]+:	
345.0344,	Found:	345.0338.	

2‐(1,3	‐Dioxoisoindolin‐2‐yl)ethyl	methylcarbamodithioate	
(9a):	Color:	White	crystals.	Yield:	75%.	M.p.:	106‐108	°C.	FT‐IR	
(KBr,	 ,	 cm‐1):	 3319	 (NH	 broad),	 3019	 (Ar	 CH),	 2930	 (CH2),	
1766,	1705	(2	CO	sharp),	1080	(CS	sharp).	1H	NMR	(400	MHz,	
DMSO‐d6,	 δ,	 ppm):	 2.98‐2.99	 (m,	 3H,	 CH3),	 3.49‐3.52	 (m,	 2H,	
CH2S),	3.84‐3.88	(m,	2H,	NCH2),	7.82‐7.88	(m,	4H,	Harom),	9.93	
(s,	1H,	NH).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	32.44	(CH3),	
33.76	 (NCH2),	 36.86	 (CH2S),	 123.26,	 131.52,	 134.67	 (aryl),	
167.66	 (2	×	C=O),	 195.33	ppm	 (C=S).	HRMS	 (ESI,	m/z)	 calcd.	
for	C12H12N2O2S2Na+	[M+Na]+:	303.0238,	Found:	303.0232.	

2‐(1,	 3‐Dioxoisoindolin‐2‐yl)ethyl	 propylcarbamodithioate	
(9b):	Color:	White	crystals.	Yield:	61%.	M.p.:	120‐122	°C.	FT‐IR	
(KBr,	,	cm‐1):	3311	(NH	broad),	2965,	2933,	2871	(CH2),	1769,	
1706	(2	CO	sharp),	1072	(CS	sharp).	1H	NMR	(400	MHz,	DMSO‐
d6,	 δ,	 ppm):	 0.80‐0.85	 (m,	 3H,	 CH3),	 1.45‐1.57	 (m,	 2H,	 CH2),	
3.44‐3.60	(m,	4H,	NHCH2,	CH2S),	3.84‐3.94	(m,	2H,	NCH2),	7.81‐
7.87	 (m,	 4H,	 Harom),	 9.96	 (s,	 1H,	 NH).	 13C	 NMR	 (100	 MHz,	
DMSO‐d6,	δ,	ppm):	11.34	(CH3),	20.82	(CH3CH2),	32.27	(NCH2),	
36.93	 (CH2S),	 48.51(NHCH2),	 123.07,	 131.55,	 134.44	 (aryl),	
167.65	 (2	 ×	 C=O),	 194.70	 (C=S).	 HRMS	 (ESI,	m/z)	 calcd.	 for	
C14H16N2O2S2Na+	[M+Na]+:	331.0551,	Found:	331.0545.	

2‐(1,	 3‐Dioxoisoindolin‐2‐yl)ethyl	 bis(2‐hydroxyethyl)carba	
modithioate	 (9c):	 Color:	White	 solid.	 Yield:	 82%.	M.p.:	 93‐95	
°C.	 FT‐IR	 (KBr,	,	 cm‐1):	 3371	 (OH	broad),	 2955,	 2857	 (CH2),	
1768,	1704	(2	CO	sharp),	1073	(CS	sharp).	1H	NMR	(400	MHz,	
DMSO‐d6,	 δ,	 ppm):	 3.55‐3.64	 (m,	 6H,	 CH2NCH2,	 CH2S),	 3.83‐
3.91	(m,	4H,	2	×	CH2OH),	4.06	(t,	2H,	 J	=	6	Hz,	NCH2),	4.81	(t,	
1H,	J	=	6	Hz,	OH),	4.96	(t,	1H,	J	=	6	Hz,	OH),	7.81‐7.85	(m,	4H,	
Harom).	13C	NMR	(100	MHz,	CDCl3,	δ,	ppm):	34.99	(NCH2),	36.39	
(CH2S),	 57.79	 (CH2NCH2),	 59.19	 (CH2NCH2),	 60.01	 (CH2OH),	
60.33	 (CH2OH),	 123.34,	 131.86,	 134.18	 (aryl),	 168.45	 (2×	
C=O),	 196.63	 (C=S).	 HRMS	 (ESI,	 m/z)	 calcd.	 for	 C15H18N2O4	
S2Na+	[M+Na]+:	377.0606,	Found:	377.0600.	

2‐(1,	3‐Dioxoisoindolin‐2‐yl)ethyl	diethylcarbamodithioate	
(9d):	Color:	 Colorless	 crystals.	 Yield:	 83%.	M.p.:	 115‐117	 °C.	
FT‐IR	 (KBr,	 ,	 cm‐1):	 2968,	 2936	 (CH2),	 1769,	 1707	 (2	 CO	
sharp),	 1082	 (CS	 sharp).	 1H	 NMR	 (400	MHz,	 CDCl3,	 δ,	 ppm):	
1.21‐1.26	 (m,	6H,	2	×	CH3),	 3.66‐3.69	 (m,	4H,	2	×	CH2),	 3.99‐
4.06	 (m,	4H,	 CH2S,	NCH2),	 7.70‐7.73	 (m,	2H,	Harom),	 7.81‐7.86	
(m,	2H,	Harom).	13C	NMR	(100	MHz,	CDCl3,	δ,	ppm):	11.47	(CH3),	
12.44	 (CH3),	 34.81	 (NCH2),	 37.00	 (CH2S),	 46.79	 (CH2NCH2),	
49.65	 (CH2NCH2),	 123.23,	 132.06,	 133.90	 (aryl),	 168.04	 (2×	
C=O),	 193.92	 (C=S).	 HRMS	 (MALDI,	m/z)	 calcd.	 for	 C15H19N2	

O2S2+	[M+H]+:	323.0888,	Found:	323.0880.	
2‐(1,	3‐Dioxoisoindolin‐2‐yl)ethyl	cyclohexylcarbamodithio	

ate	(9e):	Color:	White	solid.	Yield:	61%.	M.p.:	114‐115	°C.	FT‐
IR	 (KBr,	,	 cm‐1):	 3317	 (NH	 broad),	 2937,	 2852	 (CH2),	 1760,	
1717	(2	CO	sharp),	1088	(CS	sharp).	1H	NMR	(400	MHz,	DMSO‐

d6,	δ,	ppm):	1.09‐1.27	(m,	6H,	cyclohex.	C3,4,5‐H),	1.55‐1.83	(m,	
5H,	cyclohex.	C1,2,6‐H),	3.52	(t,	2H,	J	=	8	Hz,	CH2S),	3.87	(t,	2H,	J	
=	8	Hz,	NCH2),	7.82‐7.88	(m,	4H,	Harom),	9.86	(d,	1H,	 J	=	8	Hz,	
NH).	 13C	NMR	 (100	MHz,	DMSO‐d6,	 δ,	 ppm):	 24.56	 (cyclohex.	
C3,	 C5),	 25.01	 (cyclohex.	 C4),	 30.84	 (cyclohex.	 C2,	 C6),	 32.14	
(NCH2),	 36.90	 (CH2S),	 55.84	 (cyclohex.	 C1),	 123.03,	 131.53,	
134.39	 (aryl),	 167.59	 (2	 ×	 C=O),	 193.21	 (C=S).	 HRMS	 (ESI,	
m/z)	 calcd.	 for	 C17H21N2O2S2+	 [M+H]+:	 349.1044,	 Found:	
349.1046.	

2‐(1,	3‐Dioxoisoindolin‐2‐yl)ethyl	morpholinocarbamodithio	
ate	(9g):	Color:	White	solid.	Yield:	78%.	M.p.:	208‐210	°C.	FT‐
IR	 (KBr,	,	 cm‐1):	 3208	 (NH	 broad),	 2961,	 2917	 (CH2),	 1765,	
1704	(2	CO	sharp),	1035	(CS	sharp).	1H	NMR	(400	MHz,	DMSO‐
d6,	 δ,	 ppm):	 2.75	 (m,	 4H,	 morph.	 C3,5‐H),	 3.09‐3.11	 (m,	 2H,	
morph.	 C2‐H),	 3.39‐3.43	 (m,	 2H,	 CH2S),	 3.64‐3.76	 (m,	 2H,	
morph.	 C6‐H),	 3.85‐3.89	 (m,	 2H,	 NCH2),	 7.81‐7.88	 (m,	 4H,	
Harom),	 10.99	 (s,	 1H,	 NH).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	
ppm):	 30.61	 (NCH2),	 36.85	 (CH2S),	 42.86	 (morph.	 C3),	 53.69	
(morph.	 C5),	 63.26	 (morph.	 C2),	 65.66	 (morph.	 C6),	 123.00,	
131.58,	134.40	 (aryl),	167.69	 (2	×	C=O),	198.21	(C=S).	HRMS	
(ESI,	m/z)	 calcd.	 for	 C15H18N3O3S2+	 [M+H]+:	 352.0790,	 Found:	
352.0784.	

2‐(1,	3‐Dioxoisoindolin‐2‐yl)ethyl	4‐methylpiperazin‐1‐ylcar	
bamodithioate	(9h):	Color:	White	solid.	Yield:	64%.	M.p.:	119‐
121	 °C.	 FT‐IR	 (KBr,	 ,	 cm‐1):	 3462	 (NH	 broad),	 2941,	 2811	
(CH2),	1775,	1724	(2	CO	sharp),	1040	(CS	sharp).	1H	NMR	(400	
MHz,	DMSO‐d6,	 δ,	 ppm):	 2.18	 (s,	 3H,	 CH3),	 2.33	 (brs,	 4H,	 pip.	
C3,5‐H),	 3.59‐3.63	 (m,	 2H,	 CH2S),	 3.86‐3.91	 (m,	 4H,	 pip.	 C2‐H,	
NCH2),	4.17	(brs,	2H,	pip.	C6‐H),	7.82‐7.88	(m,	4H,	Harom),	10.85	
(s,	 1H,	 NH).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 34.25	
(NCH2),	36.63	(CH2S),	45.04	(NCH3),	53.84	(pip.	C3,	C5,	C2,	C6),	
123.07,	131.52,	134.44	(aryl),	167.62	(2	×	C=O),	193.91	(C=S).	
HRMS	 (ESI,	m/z)	 calcd.	 for	 C16H21N4O2S2+	 [M+H]+:	 365.1106,	
Found:	365.1095.	

2‐(1,	3‐Dioxoisoindolin‐2‐yl)ethyl	piperidine‐1‐carbodithio	
ate	(9i):	Color:	Colorless	crystals.	Yield:	85%.	M.p.:	170‐172	°C.	
FT‐IR	 (KBr,	 ,	 cm‐1):	 2932,	 2854	 (CH2),	 1767,	 1707	 (2	 CO	
sharp),	1082	(CS	sharp).	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	
1.51‐1.65	 (m,	 6H,	 pip.	 C3,4,5‐H),	 2.99‐3.03	 (m,	 2H,	 pip.	 C2‐H),	
3.58	(m,	2H,	CH2S),	3.84	(m,	3H,	pip.	C6‐H,	NCH2),	4.15	(brs,	1H,	
pip.	C6‐H),	7.81‐7.87	(m,	4H,	Harom).	13C	NMR	(100	MHz,	DMSO‐
d6,	 δ,	 ppm):	 21.61	 (pip.	 C4),	 22.16	 (pip.	 C3),	 23.54	 (pip.	 C5),	
34.30	(NCH2),	37.55	(CH2S),	43.71	(pip.	C2,	C6),	123.06,	131.55,	
134.46	 (aryl),	 167.69	 (2	 ×	 C=O),	 192.52	 (C=S).	 HRMS	 (ESI,	
m/z)	 calcd.	 for	 C16H18N2O2S2Na+	 [M+Na]+:	 357.0707,	 Found:	
357.0700.	

2‐(1,	 3‐Dioxoisoindolin‐2‐yl)ethyl	morpholine‐4‐carbodithio	
ate	(9j)	[23]:	Color:	White	solid.	Yield:	84%.	M.p.:	176‐178	°C.	
FT‐IR	 (KBr,	 ,	 cm‐1):	 2849	 (CH2),	 1714,	 1417	 (2	 CO	 sharp),	
1104	(CS	sharp).	1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	3.69‐3.75	
(m,	6H,	morph.	C3,5‐H,	CH2S),	3.90‐4.08	(m,	6H,	morph.	C2,6‐H,	
NCH2),	 7.69‐7.73	 (m,	 2H,	 Harom),	 7.83‐7.87	 (m,	 2H,	Harom).	 13C	
NMR	 (100	MHz,	 CDCl3,	 δ,	 ppm):	 34.81	 (NCH2),	 36.80	 (CH2S),	
51.12	 (morph.	 C3,	 C5),	 66.19	 (morph.	 C2,	 C6),	 123.29,	 132.00,	
134.00	(aryl),	168.04	(2	×	C=O),	195.86	(C=S).	HRMS	(MALDI,	
m/z)	 calcd.	 for	 C15H17N2O3S2+	 [M+H]+:	 337.0681,	 Found:	
337.0694.	
	
2.3.	Biological	evaluation	
	
2.3.1.	Antiproliferation	assay	
	

Human	breast	adenocarcinoma	MCF‐7	cells,	hepatocellular	
carcinoma	 HepG2	 cells,	 colon	 carcinoma	 HCT‐116	 cells,	
cervical	 carcinoma	 HeLa	 cells	 and	 alveolar	 adenocarcinoma	
A549	 cells	 were	 purchased	 from	 American	 Type	 Culture	
Collection	 (ATCC,	 Rockville,	 MD,	 USA).	 HepG2,	 MCF‐7,	 A549	
and	HeLa	cells	were	routinely	cultured	in	Dulbecco’s	Modified	
Eagle’s	Medium	(DMEM),	while	HCT‐116	cells	were	cultured	in	
McCoy’s	 medium.	 Media	 were	 supplemented	 with	 2	 mM	 L‐
glutamine,	 100	 units/mL	 penicillin	 G	 sodium,	 250	 ng/mL	



Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	 395	
 

amphotericin	 B,	 100	 units/mL	 streptomycin	 sulphate,	 and	
10%	fetal	bovine	serum	(FBS).	Cells	were	maintained	in	37	°C	
humidified	 air	 containing	 5%	 CO2	 at	 sub‐confluence.	 Cells	
were	 harvested	 when	 confluence	 had	 reached	 75%	 using	
trypsin/EDTA.	 All	 cell	 culture	 materials	 were	 obtained	 from	
Gibco®/Invitro	 gen,	 USA.	 All	 chemicals	 were	 obtained	 from	
Sigma/Aldrich,	USA,	except	the	mentioned.		

The	 antiproliferative	 activity	 of	 phthalimide	 derivatives	
against	 Hep‐G2,	 MCF‐7,	 HCT‐116,	 A549,	 and	 HeLa	 cells	 was	
estimated	 by	 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetra	
zolium	bromide	(MTT)	assay	[24].	Briefly,	cells	were	seeded	in	
96‐well	plates	at	5×104	cells/well	in	the	corresponding	media	
supplemented	with	 culture	materials	mentioned	 above.	 After	
24	 h	 of	 culture,	 phthalimide	 derivatives	 were	 individually	
added	 in	 triplicate	 in	 a	 range	 of	 12.5‐1000	 µM,	 and	 the	 cells	
were	further	cultured	for	24	h.	The	cells	were	then	exposed	to	
MTT	(5	mg/mL	in	PBS)	at	a	final	concentration	of	1	mg/mL	in	
culture	 for	 4	 h.	 Formazan	 crystals	 formed	 during	 the	 incu‐
bation	 period	 were	 dissolved	 overnight	 at	 37	 °C	 by	 adding	
10%	 SDS	 containing	 0.02	 N	 HCl.	 The	 absorbance	 was	 then	
measured	at	570	nm	(FLUOstar	OPTIMA;	BMG	Labtech	GmbH,	
Offenburg,	 Germany).	 The	 half	 maximal	 growth	 inhibitory	
concentration	 (IC50	 values)	 was	 calculated	 from	 the	 dose‐
dependent	curve	equation	of	each	compound.	The	experiment	
was	 repeated	 three	 times.	 Thalidomide	was	 used	 as	 drug	 of	
reference	(TH).	
	
2.3.2.	Molecular	docking	study	
	

Molecular	modeling	 study	was	 performed	 using	 Accelrys	
Discovery	 Studio	 2.5	 operating	 system	 (Accelrys	 Inc.,	 San	
Diego,	CA,	USA),	at	Faculty	of	Pharmacy,	Ain‐Shams	University,	
Cairo,	Egypt.	Molecules	were	built	within	Docking	Study	(DS)	
and	 conformational	 models	 for	 each	 compound	 were	
generated	automatically.	Docking	study	involved	the	following	
steps:	The	docking	analysis	was	carried	out	on	VEGFR	enzyme.	
The	 3D	 protein	 structure	 of	 VEGFR	 enzyme	 co‐crystallized	
with	lead	compound	(Code;	2OH4),	was	downloaded	from	the	
Protein	Data	Bank	of	the	Research	Collaboration	for	Structural	
Bioinformatics	 (RCSB)	 website	 www.rcsb.org.	 The	 binding	
pocket	 was	 prepared	 for	 docking	 by	 cleaning	 the	 protein,	
adding	 the	 missing	 hydrogen	 and	 side	 chains	 and	 energy	
minimization	according	to	DS	protocol.	The	binding	pocket	of	
the	complexed	lead	compound	with	the	connected	amino	acid	
residues	was	 identified	 at	 sphere	 of	 radius	 =	 12	 Å	 and	 then	
was	 used	 in	 docking	 of	 test	 compounds	 using	 Genetic	
Optimization	 for	 Ligand	 Docking	 (GOLD)	 module.	 After	 that	
the	GOLD	score	 fitness	 of	 the	best	 fitted	 conformation	of	 the	
docked	molecule	were	recorded.	
	
2.3.3.	Estimation	of	compound’s	binding	affinity	to	VEGFR	
	

The	 direct	 binding	 of	 pure	 compounds	 to	 VEGFR	 protein	
were	 tested	 in	 cell‐free	 ELISA	 to	 discover	 the	possible	 direct	
compound/receptor	binding	that	may	block	the	receptor.	The	
assay	 was	 mainly	 examining	 the	 possible	 binding	 between	
each	compound	to	 the	antigenic	determinant	sequence	 in	 the	
VEGFR	protein	 that	can	prevent	 its	 further	binding	 to	VEGFR	
antibodies.	 In	 brief,	 each	 pure	 compound	 (10	 mg/mL)	 was	
incubated	with	 a	 fixed	 concentration	 of	 recombinant	 human	
VEGFR	(0.5	ng/mL,	Abcam,	USA)	at	37	°C	for	1h	under	orbital	
shaking	 in	 non‐coated	 microplates.	 After	 incubation,	 the	
compound/receptor	mixture	was	transferred	to	ELISA‐micro‐
plates	 that	 were	 previously	 coated	 with	 VEGFR	 monoclonal	
antibodies	and	the	level	of	free	VEGFR	protein	was	quantified	
by	sandwich	ELISA	as	previously	mentioned	[25].	
	
2.3.4.	Statistical	analysis		
	

Statistical	 analyses	 of	 data	 were	 performed	 using	 the	
Statistical	 Package	 for	 the	 Social	 Sciences	 (SPSS)	 program	

Version	11	and	one	way	ANOVA	test	followed	by	Tuckey’s	Post	
Hoc	 test.	 Data	 were	 expressed	 as	 mean±standard	 division	
(SD).	 The	 results	 were	 considered	 to	 be	 significant	 when	 p	
value	is	less	than	0.05.	
	
3.	Results	and	discussion	
	
3.1.	Chemistry	
	

Merging	the	bridgeheads	of	different	dithiocarbamate	and	
dithioate	 moieties	 to	 phthalimide	 is	 the	 core	 of	 our	 rational	
design	 of	 a	 series	 of	 novel	 dithiocarbamate	 phthalimide	
hybrids	 (8a‐j,	9a‐e,	9g‐j).	 The	 novel	 phthalimide	 derivatives	
were	 synthesized	 according	 to	 the	 procedures	 depicted	 in	
Scheme	 1.	 Phthalimide	 (1)	 with	 its	 (NH)	 acidic	 hydrogen	
underwent	 reaction	 with	 formaldehyde	 affording	 the	 inter‐
mediate	 hydroxymethyl	 derivative	 2	 that	 was	 subjected	 to	
chlorination	 using	 thionyl	 chloride	 to	 furnish	 the	 chloro‐
methylene	 derivative	 3	with	 perfect	 yields	 according	 to	 the	
reported	 procedure	 [26].	 Similarly,	N‐chloroethylene	 phthal‐
imide	 derivative	 could	 be	 prepared	 by	 condensation	 of	
phthalic	 anhydride	 (4)	 with	 ethanolamine	 to	 construct	 the	
hydroxyethyl	derivative	5	 that	is	to	be	subsequently	halogen‐
nated	to	afford	chloroethyl	derivative	6	[27‐29].	Alternatively,	
N‐bromoethylene	phthalimide	derivative	could	be	prepared	by	
the	 reaction	 of	 phthalimide	 1	 with	 potassium	 hydroxide	 to	
afford	 potassium	 phthalimide	 which	 reacted	 with	 ethylene	
dibromide	to	give	bromoethyl	derivative	7	[30].	In	the	current	
study,	 the	commercially	available	bromoethylene	counterpart	
7	 has	 been	utilized	 for	 the	 synthesis	 of	 the	 ethylene	 bridged	
analogs.	 N‐Chloromethylphthalimide	 (3)	 and	 N‐bromoethyl‐
phthalimide	 (7)	were	allowed	 to	 react	with	CS2	and	different	
amines	under	stirring	for	48	h	at	room	temperature	in	CH3CN	
to	 afford	 novel	 phthalimide	 dithiocarbamate	 and	 dithioate	
derivatives	 (8a‐j,	9a‐e,	9g‐j).	Phthalimide	derivative	9f	 failed	
to	 proceed	 properly	 and	 no	 indication	 for	 its	 formation	was	
achieved.	 The	 structures	 of	 amines	 applied	 in	 this	 reaction	
protocol	 are	 given	 in	Table	1.	 The	 chemical	 structures	 of	 the	
synthesized	 compounds	 were	 confirmed	 by	 IR,	 1H	 NMR,	 13C	
NMR,	and	Electrospray	ionization	high	resolution	mass	spectra	
(ESI‐HRMS).	 The	 IR	 spectra	 of	 phthalimide	 dithiocarbamate	
derivatives	 8a‐j,	 9a‐e	 and	 9g‐j	 supported	 the	 expected	
structures	 and	showed	absorption	bands	 in	 the	 region	1704‐
1776	cm‐1	as	 two	peaks	owing	 to	asymmetric	and	symmetric	
stretching	vibrations	of	the	two	carbonyls	of	phthalimide.	The	
IR	 spectra	 of	 phthalimide	 dithiocarbamate	 and	 dithioate	
derivatives	 8a‐j,	 9a‐e	 and	 9g‐j	 showed	 absorption	 bands	 in	
the	 1035‐1088	 cm‐1	 region	 resulting	 from	 C=S	 function.	 1H	
NMR	 spectra	 of	 phthalimide	 dithiocarbamate	 and	 dithioate	
derivatives	 8a‐j	 showed	 singlets	 at	 δ	 5.13‐5.63	 ppm	 corres‐
ponding	to	the	methylene	protons	(N‐CH2‐S).	13C	NMR	spectra	
of	phthalimide	dithiocarbamate	and	dithioate	derivatives	8a‐j	
showed	 signals	 at	 δ	 40.08‐45.20	 ppm	 corresponding	 to	 the	
methylene	 carbon	 (N‐CH2‐S).	 On	 the	 other	 hand,	 1H	 NMR	
spectra	 of	phthalimide	dithiocarbamate	 and	dithioate	deriva‐
tives	 9a‐e	 and	 9g‐j	 showed	 triplet	 at	 δ	 3.44‐3.75	 ppm	
corresponding	 to	 the	 methylene	 proton	 (CH2S),	 triplet	 at	 δ	
3.84‐4.08	ppm	corresponding	to	(N‐CH2).	These	two	triplets	in	
some	of	synthesized	compounds	appeared	as	multiplet	due	to	
the	overlap	with	protons	for	the	derivatized	amine	side	chain.	
Similarly,	13C	NMR	spectra	of	phthalimide	dithiocarbamate	and	
dithioate	derivatives	9a‐e	and	9g‐j	showed	signals	at	δ	30.61‐
34.99	 ppm	 corresponding	 to	 (N‐CH2),	 signals	 at	 36.39‐37.35	
ppm	corresponding	to	(CH2S).	In	addition,	13C	NMR	spectra	of	
all	compounds	showed	signals	at	δ	191.26‐198.21	ppm	corres‐
ponding	to	(C=S).	ESI‐HR	mass	spectra	of	phthalimide	dithio‐
carbamate	 and	 dithioate	 derivatives	 8a‐j,	 9a‐e	 and	 9g‐j,	
respectively,	 displayed	molecular	 ions	 (M +),	which	 confirmed	
their	 molecular	 weights.	 All	 the	 analytical	 data	 were	 in	
complete	 accordance	with	 the	proposed	 structures.	Reagents	
and	analytical	data	are	presented	in	Experimental	part.		



396	 Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	
	
	
Table	1.	Appropriate	amine	and	corresponding	phthalimide	dithiocarbamate	and	dithioate	derivatives.	
Amine Product Structure Product Structure 

 
8a	

 

9a

	

 
8b	

N

O

O
S

HN

S

CH3 	

9b 

N

O

O NH
S

CH3

S

 
8c	

	

9c 

N

O

O N
S

OH

OH

S

 
8d	 9d

	

 

8e	

	

9e

	

 

8f	 9f Not	proceed	

 

8g	

	

9g

	
	

 

8h	

 

9	h

 

8i	

N

O

O
S

N

S

9i

	

 

8j	 9ja

 

a	Derivative	9j	was	previously	reported	for	another	biological	target	but	not	characterized	[23].	
	
	
3.2.	Biological	evaluation	
	
3.2.1.	Effect	of	phthalimide	derivatives	on	proliferation	of	
human	cancer	cells		
	

The	 antiproliferative	 effects	 of	 the	 newly	 synthesized	
phthalimide	 derivatives	 were	 tested	 against	 MCF‐7,	 HepG2,	

HCT‐116,	 cervical	 carcinoma HeLa,	 A549	 cells	 by	 using	MTT	
assay.	 Phthalimide	 derivatives	 were	 dissolved	 in	 dimethyl‐
sulphoxide	 (DMSO),	 and	 then	 diluted	 1000	 times	 for	 the	
assays.	 The	 solubility	 and	 absence	 of	 crystals	 as	 well	 as	
precipitation	were	analyzed	under	microscope	after	dilution	of	
compounds/DMSO	in	their	corresponding	culture	media.		

	



Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	 397	
 

	 	
Table	2.	Antiproliferative	activity	of	phthalimide	derivatives	on	different	human	cancer	cells	*.	
Compounds	 MCF‐7	 HepG2 HCT‐116 HeLa A549	
TH	 131.9	 207.3 361.8 227.3 144.1	
8c	 514.6	 236.37	 741.3	 846.5	 552.4	
8e	 534.4	 412.5	 486.1	 555.1	 362.2	
8f	 608.5	 207.2	 156.9	 179.1	 553.6	
8g	 424.4	 342.5	 273.6	 312.4	 223.8	
8h	 493.3	 256.4 539.0 615.6 401.7	
8i	 482.8	 411.6 373.3 426.4 278.2	
9c	 501.4	 269.9 425.7 486.1 317.2	
9e	 66.9	 87.1 742.9 348.4 116.9	
9i	 132.0	 151.9 273.2 426.1 178.1	
9j	 603.2	 299.3 616.8 704.4 459.6	
*	Proliferation	assay	was	determined	by	MTT	assay	and	obtained	from	at	least	three	independent	experiments. Cells	were	treated	for	24	h	with	phthalimide	
derivatives	 in	 concentration	 range	 12.5‐1000	µM.	Data	 are	 presented	 as	 IC50	 (µM),	which	were	 calculated	 from	 the	 exponential	 equation	 of	 dose/viability	
%response.	Thalidomide	was	used	as	drug	of	reference	(TH).	

	
Table	3.	Docking	score	for	each	compound	in	the	test	set	with	active	site	of	VEGFR. 
Compounds	 	 GOLD	score	(‐kcal/mol)	
TH		 44.45
8c	 50.80
8e	 51.37
8f	 49.87
8g	 50.98	
8h	 50.44	
8i	 46.63
9c	 49.15	
9e	 54.40	
9i	 51.70
9j	 45.98
		
	

(a)	 (b)	
	

Figure	3.	a)	Alignment	of	bioactive	conformer	of	lead	from	Pdb	and	docked	conformer,	b)	2D	binding	mode	of	lead	compound.	

	
	
Compounds	 8a,	 8b,	 8d,	 8j,	 9a,	 9b,	 9d,	 9g	 and	 9h	 that	

precipitated	in	cell	culture	media	were	omitted	from	biological	
evaluation	experiments.		

As	 shown	 in	Table	2,	 our	 results	 indicated	 that	 in	MCF‐7	
cells,	 compound	 9e	 possessed	 the	 highest	 antiproliferative	
activity	 (IC50	 =	 66.9	 µM),	 whereas,	 derivative	 9i	 showed	
similar	antiproliferative	effect	with	TH	(IC50	about	132	µM).	In	
HepG2	 cells,	 derivatives	 9e	 and	 9i	 exhibited	 the	 maximum	
antiproliferative	 effect	 with	 IC50	 =	 87.1	 µM	 and	 151.9	 µM,	
respectively,	compared	to	TH	 (IC50	=	207.3	µM).	Moreover,	 in	
lung	 A549	 cells,	 all	 of	 the	 tested	 derivatives	 showed	 non‐
antiproliferative	activity	with	IC50	>	200	µM,	except	derivatives	
9e	 and	 9i	 which	 revealed	 antiproliferative	 activity	 (IC50	 =	
116.9	 and	 178.1	 µM,	 respectively)	 compared	 to	 TH	 (IC50	 =	
144.1	µM).	Furthermore,	 in	both	colon	HCT‐116	and	cervical 
HeLa,	 only	 derivative	8f	 showed	 antiproliferative	 effect	with	
IC50	=	156.9	and	179.1	µM,	respectively	 than	TH	 (IC50	=	361.8	
and	227.3	µM,	respectively).	
	
3.2.2.	Molecular	docking	study		
	

It	 was	 reported	 that	 TH	 and	 its	 analogs	 have	 VEGFR	
inhibitory	 activities	 [31‐33].	 Molecular	 docking	 investigated	
the	affinity	of	 the	designed	compounds	 into	 the	active	site	of	
VEGFR	enzyme	(PDP:	2OH4)	[34].	Both	the	binding	modes	and	
the	docking	scores	of	 the	designed	target	compounds	compa‐
red	to	the	lead	compound	were	evaluated.	The	selection	of	pdb	
2OH4	 was	 based	 on	 similarity	 in	 structure	 between	 the	
designed	compounds	and	the	selected	lead	which	proposed	to	
have	the	same	pharmacophoric	features	(Figure	3).		

Interactive	 docking	 using	 GOLD	protocol	was	 carried	 out	
for	all	the	conformers	of	each	compound	of	the	test	set	(8c,	8e,	
8f,	8g,	8h,	8i,	9c,	9e,	9i,	9j	and	TH)	to	the	selected	active	site	
after	energy	minimization.	The	small	RMSD	values	proved	the	
validity	 of	 the	 used	 docking	 processes	 [35].	 Each	 docked	
compound	was	assigned	a	score	according	to	its	binding	mode	
onto	the	binding	site	[36]	that	predicted	binding	energies	and	
the	corresponding	experimental	values	as	outlined	in	Table	3.	

The	lead	binding	mode	involved	shows	that	NH	attached	to	
benzimidazole	 nucleus	 forms	H‐bond	with	 the	 Cys917	 in	 the	
hinge	 region.	 On	 the	 other	 hand,	 the	 benzimidazole	 nucleus	
forms	 hydrophobic	 interaction	 with	 Phe916	 in	 the	 hinge	
region.	 Furthermore,	 the	 extended	 chains	 are	 solvated	 and	
occupied	in	the	hydrophobic	pocket	[37,38].	Also,	the	binding	
mode	of	ATP	was	matched	with	the	reported	results	 [39].	All	
interaction	 behaviors,	 as	 obtained	 from	 the	 docking	 studies,	
were	 found	 to	 be	 in	 accordance	 with	 the	 reported	 binding	
mode	of	the	VEGFR	active	site	[37,38].	The	active	compounds	
elicited	 the	 same	 conformation	 of	 the	 lead	 compound	 with	
bioisosteric	 replacement	 of	 benzimidazole	 nucleus	 by	 isoin‐
dole	nucleus	and	hence	acquired	the	same	binding	mode	of	the	
lead.	 Derivatives	 9e	 and	 9i,	 which	 showed	 similar	 binding	
mode	to	the	lead	compound	with	high	docking	score	values	(‐
54.4	 and	 ‐51.7	 kcal/mol),	 revealed	 the	 highest	 inhibitory	
activity	of	64.8	and	60.5%,	respectively	(Figures	4	and	5).	The	
carbonyl	group	in	the	active	hits	(9e	and	9i)	conserved	the	H‐
bond	 interaction	 with	 CYS917	 in	 the	 hinge	 region	 and	 the	
extended	chain	occupied	the	hydrophobic	pocket	and	solvated	
to	form	extra	hydrogen	bonding	(Figures	4	and	5).		

	



398	 Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	
	

	 	

(a) (b)
	

Figure	4.	a)	2D	and	b)	3D	interaction	diagram	of	compound	9e	in	the	active	site	of	VEGFR.	

	

	(a)	 	(b)	

Figure	5.	a)	2D	and	b)	3D	alignment	of	compound	9i	having	fitting	score	=	‐51.7	Kcal/mol	and	biological	activity	=	60.5%.	

	

	
	

Figure	6.	Alignment	of	compound	9e	(with	grey	color)	and	the	lead	compound	(with	purple	color)	in	the	active	site	of	VEGFR.	
	

	
Compared	to	lead	compound	(potent	and	selective	VEGFR	

inhibitor),	docking	behavior	with	derivative	9e	exhibited	that	
our	 constructed	 analog	 was	 perfectly	 aligned	 with	 the	 lead	
compound	 taking	 the	 same	 orientation	 of	 the	 features	 inside	
the	active	site	(Figure	6).	These	observations	support	the	attai‐
ned	 structure	 activity	 relationship	 (SAR)	 assumptions	 due	 to	
enzymatic	inhibitory	activity	data.	
	
3.2.3.	Assessment	of	phthalimide	derivatives	on	VEGFR	
concentration	
	

The	 present	 study	 explored	 the	 affinity	 of	 the	 prepared	
derivatives	to	directly	bind	the	VEGFR	protein	molecule	in	the	
antigenic	 sequence	 that	may	 lead	 to	a	 change	 in	 its	ability	 to	
bind	 VEGF.	 As	 shown	 in	 Figure	 7,	 the	 results	 demonstrated	
that	 the	 incubation	 of	 recombinant	 human	 VEGFR	 with	 the	
tested	derivatives	resulted	in	a	variable	range	of	 inhibition	in	
the	VEGFR	detection	by	 its	 corresponding	antibodies.	 In	des‐
cending	order,	derivatives	9e	>	9i	>	TH	were	the	most	potent	
inhibitors	of	VEGFR	detection,	among	all	of	 the	tested	deriva‐
tives,	at	percentage	of	inhibition	>	50%.	

Finally,	preliminary	biological	screening	indicated	that	the	
nature	 of	 dithiocarbamate	 and	 dithioate	moieties	 introduced	
at	the	phthalimide	nitrogen	atom	play	an	important	role	in	the	

potency	of	phthalimide	derivatives	9e	and	9i	toward	different	
biological	 targets.	 The	 outcome	 results	 of	 phthalimide	
derivatives	8f,	9e	and	9i	revealed	the	superior	relative	activity	
of	cyclohexylamine	and	piperidine	side	chains	which	supports	
our	previously	reported	data	using	the	same	dithiocarbamate	
and	dithioate	moieties	with	thalidomide	[8,11].	Moreover,	the	
introduction	 of	 alkyl	 linker	 in	 between	 phthalimide	 and	
dithiocarbamate	 moieties	 in	 derivatives	 9e	 and	 9i	 added	 a	
value	 in	 improving	 their	 biological	 activities.	 These	 findings	
are	 in	 agreement	with	 the	previously	 reported	 investigations	
which	 stated	 that	 alkyl	 linkers	 could	 modulate	 the	
physicochemical	 properties	 and	 thus	 improve	 the	 biological	
potency	[40‐42].	
	
4.	Conclusion	
	

Herein;	we	report	 the	 synthesis	and	biological	evaluation	
of	 phthalimide	 moiety	 with	 different	 dithiocarbamate	 and	
dithioate	side	chains.	The	improvement	of	biological	activities	
compared	 to	 thalidomide	 depends	 on	 the	 nature	 of	 dithio‐
carbamate	and	dithioate	groups	attached	to	phthalimide	core	
and	 the	 presence	 of	 alkyl	 linker	 in	 between.	 Phthalimide	
derivatives	9e	and	9i	showed	remarkable	antitumor	activities.		

	



Zahran	et	al.	/	European	Journal	of	Chemistry	8	(4)	(2017)	391‐399	 399	
 

	

	
	
Figure	7.	Effect	of	phthalimide	derivatives	on	VEGFR	concentration.	The	concentration	of	VEGFR	was	measured	after	the	incubation	of	each	tested	phthalimide	
derivative	(10	µg/mL)	with	a	fixed	concentration	of	the	standard	recombinant	human	VEGFR	(0.5	ng/mL)	using	ELISA

	
	
The	active	hits	exhibited	the	highest	inhibitory	activity	on	

VEGFR	enzyme	which	was	consistent	with	biological	 findings	
and	 consequently	 lead	 to	 inhibition	 of	 tumor	 growth.	 These	
results	 suggest	 that	 piperidine	 and	 cyclohexylamine	 deriva‐
tives	 have	 potential	 promising	 activities.	 Future	 directions	
may	 include	 insertion	 of	 some	 other	 linkers	 of	 different	
lengths	and	functionalities	in	between	phthalimide	and	dithio‐
carbamate	moieties	for	further	improvement	the	activity.	
	
Acknowledgement		
	

This	 research	was	 supported	 by	 Science	 and	 Technology	
Development	 Fund	 (STDF)	 through	 Project	 No.	 3627.	 The	
corresponding	 author	 and	 all	 the	 co‐authors	 appreciate	 the	
Egyptian	fund	for	supporting	this	research.	We	appreciate	our	
thank	 to	 Prof.	 Erik	 Pedersen	 from	 department	 of	 Physics,	
Chemistry	 and	 Pharmacy,	 Nucleic	 Acid	 Center,	 University	 of	
Southern	 Denmark,	 Odense,	 Denmark	 for	 the	 scientific	
supporting	this	research.	
	
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