molecules
A icle
Ul asound Media ed One-Po , Th ee Componen
Syn hesis, Docking and ADME P edic ion o No el
5-Amino-2-(4-chlo ophenyl)-7-Subs i u ed
Phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)
py imidine-6-ca boni ile De i a i es as
An icance Agen s
Shailee V. Tiwa i 1, Julio A. Seijas 2, M. Pila Vazquez-Ta o 2, Anike P. Sa ka e 3,
Deepak K. Lokwani 3and Anna P a ima G. Nikalje 1,*
1Y.B. Cha an College o Pha macy, D . Ra iq Zaka ia Campus, Rauza Baug, Au angabad,
Maha ash a 431001, India; [email p o ec ed]
2Depa amen o de Química O gánica, Facul ad de Ciencias, Uni e sidad o San iago De Compos ela,
Al onso X el Sabio, Lugo 27002, Spain; [email p o ec ed] (J.A.S.); pila [email p o ec ed] (M.P.V.-T.)
3Depa men o Chemical Technology, D . Babasaheb Ambedka Ma a hwada Uni e si y, Au angabad,
Maha ash a 431004, India; [email p o ec ed] (A.P.S.); [email p o ec ed] (D.K.L.)
*Co espondence: [email p o ec ed]; Tel.: +91-916-892-9111
Academic Edi o : De ek J. McPhee
Recei ed: 28 May 2016; Accep ed: 5 July 2016; Published: 29 July 2016
Abs ac :
He ein, we epo an en i onmen ally iendly, apid, and con enien one-po ul asound-
p omo ed syn hesis o 5-amino-2-(4-chlo ophenyl)-7-subs i u ed phenyl-8,8a-dihyd o-7H-(1,3,4)
hiadiazolo(3,2-
α
)py imidine-6-ca boni ile de i a i es. The in- i o an icance ac i i ies o hese
compounds we e e alua ed agains ou human umo cell lines. Among all he syn hesized
de i a i es, compound
4i
, which has subs i uen 3-hyd oxy-4-me hoxyphenyl is ound o ha e
he highes GI
50
alue o 32.7
µ
M, 55.3
µ
M, 34.3
µ
M, 28.9
µ
M o MCF-7, K562, HeLa and PC-3 cance
cell lines espec i ely. A docking s udy o he newly syn hesized compounds we e pe o med, and
he esul s showed good binding mode in he ac i e si e o hymidyla e syn hase enzyme. ADME
p ope ies o syn hesized compounds we e also s udied and showed good d ug like p ope ies.
Keywo ds: 1,3,4- hiadiazolo(3,2-α)py imidine; ul asound-p omo ed syn hesis; ADME; docking
1. In oduc ion
Cance is a disease in which cells g ow and p oli e a e in an uncon olled manne . Cance
disease e okes a high le el o mo ali y ega dless o ecen ad ances in he de elopmen o clinically
au ho ized an icance agen s [
1
]. On he basis o sou ce and ac ion mechanisms, he an icance d ugs
a e classi ied as alkyla ing agen s, an ime aboli es, na u al p oduc s, ho mones and an agonis ic,
miscellaneous agen s. Many scien is s a e in ensi ely engaged in he de elopmen o new an icance
ac i e agen s ha e eal a selec i e cy o oxici y o cance cells o e no mal cells which is undoub edly
needed o ea he se e e cance disease mo e e icien ly and is also less oxic, since many o he
ma ke ed an icance d ugs a e oxic in na u e [2–4].
Fola e me abolism is conside ed as an impo an a ge o he de elopmen o new an icance agen s
due o i s ole in he biosyn hesis o nucleic acid p ecu so s [
5
,
6
]. The inhibi ion o ola e dependen
enzymes such as hymidyla e syn hase (TS), which ca alyzes he educ i e me hyla ion o deoxyu idyla e
(dUMP) o hymidyla e (dTMP) has also been ecognized as an in e es ing a ge o d ug disco e y [
7
,
8
].
Molecules 2016,21, 894; doi:10.3390/molecules21080894 www.mdpi.com/jou nal/molecules
Molecules 2016,21, 894 2 o 13
Inhibi ion o his enzyme leads o hymineless s a e, ende ing cells incapable o unde going accu a e
DNA eplica ion, ul ima ely esul ing in cell dea h. Classical, an i ola e inhibi o s o hymidyla e syn hase
o en su e om a numbe o po en ial disad an ages when used as an icance agen s. These include
impai ed up ake due o an al e a ion o he ac i e anspo sys em equi ed o cellula up ake, as well
as he o ma ion o long ac ing, non-e luxing polyglu ama es ia olypolyglu ama e syn he ase, which
a e esponsible o oxici y o no mal cells. Nonclassical an i ola es such as nola exed (AG337), AG331,
py ime hamine, ime hop im (TMP), pi i exim (PTX) and ime exa e (TMQ) do no equi e ola e
anspo sys ems bu en e cells ia passi e di usion. Recen ad ances in non-classical an i ola es ha e
been documen edin heli e a u e[
9
–
11
]. To o e come some o he disad an ages o classical hymidyla e
syn hase inhibi o s, ou eam has ied o syn hesize no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)py imidine-6-ca boni ile de i a i es.
Py imidine and i s de i a i es ha e been ecognized as impo an he e ocyclic compounds due o
hei a ie y o chemical and biological signi icance o medicinal chemis y [
12
–
15
]. Hyb idiza ion o wo
di e en bioac i e molecules wi h complemen a y pha macopho ic unc ions o en showed syne gis ic
e ec s [
16
,
17
]. Du ing ecen yea s, he e ha e been in ense in es iga ions o used hiadiazole and
py imidine sys ems. Li e a u e su ey e ealed ha (1,3,4) hiadiazolo(3,2-
α
)py imidine nucleus
is associa ed wi h di e se pha macodynamic and chemo he apeu ic ac i i ies [
18
,
19
], including
an imic obial [
19
–
21
] and an i umo ac i i ies [
18
,
20
], he bicidal, an i ungal, neu amidase inhibi o s.
1,3,4- hiadiazolo(3,2-
α
)py imidines ha e been used as key building blocks o he p epa a ion o a
a ie y o no el bioac i e agen s [
22
], he e o e, we hough i wo hwhile o explo e his coupled
he e ocyclic sys em o e alua ion o an icance ac i i y. The designing p o ocol o a a ge ed
compound is as shown in Figu e 1.
Molecules 2016, 21, 894 2 o 13
o unde going accu a e DNA eplica ion, ul ima ely esul ing in cell dea h. Classical, an i ola e
inhibi o s o hymidyla e syn hase o en su e om a numbe o po en ial disad an ages when used
as an icance agen s. These include impai ed up ake due o an al e a ion o he ac i e anspo sys em
equi ed o cellula up ake, as well as he o ma ion o long ac ing, non-e luxing polyglu ama es
ia olypolyglu ama e syn he ase, which a e esponsible o oxici y o no mal cells. Nonclassical
an i ola es such as nola exed (AG337), AG331, py ime hamine, ime hop im (TMP), pi i exim (PTX)
and ime exa e (TMQ) do no equi e ola e anspo sys ems bu en e cells ia passi e di usion.
Recen ad ances in non-classical an i ola es ha e been documen ed in he li e a u e [9–11]. To
o e come some o he disad an ages o classical hymidyla e syn hase inhibi o s, ou eam has ied o
syn hesize no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo
(3,2-α)py imidine-6-ca boni ile de i a i es.
Py imidine and i s de i a i es ha e been ecognized as impo an he e ocyclic compounds due
o hei a ie y o chemical and biological signi icance o medicinal chemis y [12–15]. Hyb idiza ion
o wo di e en bioac i e molecules wi h complemen a y pha macopho ic unc ions o en showed
syne gis ic e ec s [16,17]. Du ing ecen yea s, he e ha e been in ense in es iga ions o used
hiadiazole and py imidine sys ems. Li e a u e su ey e ealed ha (1,3,4) hiadiazolo(3,2-α)py imidine
nucleus is associa ed wi h di e se pha macodynamic and chemo he apeu ic ac i i ies [18,19], including
an imic obial [19–21] and an i umo ac i i ies [18,20], he bicidal, an i ungal, neu amidase inhibi o s.
1,3,4- hiadiazolo(3,2-α)py imidines ha e been used as key building blocks o he p epa a ion o a
a ie y o no el bioac i e agen s [22], he e o e, we hough i wo hwhile o explo e his coupled
he e ocyclic sys em o e alua ion o an icance ac i i y. The designing p o ocol o a a ge ed
compound is as shown in Figu e 1.
N
N
NH
O
O
F
O
OOH
OH
HN
N
H
F
O
O
Capeci ibine
5-Flu ou acil (5- FU) HN
NN
H
O
H2N
ONH
COOH
HOOC
HN
N
ON
S
HN
O
COOH
COOH
Ral i exed
Peme exed
HN
N
NH2
N
N
H2N
N
NH
O
COOH
COOH
Me ho exa e
HN
N
H2N
O
NH
O
COOH
HOOC
PDDF
N
N
N
S
NH2
CN
R
Cl
Syn hesized molecule
Figu e 1. Designing p o ocol o a ge ed molecule.
The con en ional mul is ep me hods o he p epa a ion o complex molecules in ol e la ge
syn he ic ope a ions, including ex ac ion and pu i ica ion p ocesses o each indi idual s ep, ha
lead o syn he ic ine iciency and he gene a ion o la ge amoun s o was e. The e o e, designing
mul icomponen eac ions (MCRs) in one po and he c ea ion o se e al bonds in a single ope a ion
a e he majo challenges o mode n o ganic chemis y.
The syn hesis o he e ocycles using oxic and haza dous chemicals which cause pollu ion has gi en
bi h o “G een Chemis y,” coined by Paul Anas as in 1998 [23]. Ul asound-p omo ed syn hesis is one
o he g een me hods o syn hesis which we ha e used in ou p esen esea ch wo k in o de o p e en
pollu ion. Ul asound-p omo ed syn hesis has a ious ad an ages o e con en ional syn he ic
echniques such as highly accele a ed eac ion a e, easonably good yields, simple open sys ems, e y
Figu e 1. Designing p o ocol o a ge ed molecule.
The con en ional mul is ep me hods o he p epa a ion o complex molecules in ol e la ge
syn he ic ope a ions, including ex ac ion and pu i ica ion p ocesses o each indi idual s ep, ha
lead o syn he ic ine iciency and he gene a ion o la ge amoun s o was e. The e o e, designing
mul icomponen eac ions (MCRs) in one po and he c ea ion o se e al bonds in a single ope a ion
a e he majo challenges o mode n o ganic chemis y.
The syn hesis o he e ocycles using oxic and haza dous chemicals which cause pollu ion has
gi en bi h o “G een Chemis y,” coined by Paul Anas as in 1998 [
23
]. Ul asound-p omo ed syn hesis
is one o he g een me hods o syn hesis which we ha e used in ou p esen esea ch wo k in o de o
p e en pollu ion. Ul asound-p omo ed syn hesis has a ious ad an ages o e con en ional syn he ic
Molecules 2016,21, 894 3 o 13
echniques such as highly accele a ed eac ion a e, easonably good yields, simple open sys ems, e y
low amoun o sol en s equi ed, eco iendly me hod, clean hea ing sys em, nea and clean syn he ic
p o ocol, cheape eagen s and less ex eme physical condi ions, con ol on eac ion pa ame e s, milde
eac ion condi ions.
The exis ing syn he ic me hodologies o (1,3,4) hiadiazolo(3,2-
α
)py imidine nucleus in a modula
ashion a e no s aigh o wa d and he syn he ic ou es in ol e mul iple s eps. Fo example,
1,3,4- hiadiazolo(3,2-
α
)py imidine-7-sul onamide de i a i es we e syn hesized om 5-aminol-3,4-
hiadiazole-2-sul onamide ia a wo s eps app oach [
24
]. Salimo e al. [
25
] p epa ed 2-b omo-7-me hyl-
5-oxo-5H-1,3,4- hiadiazolo(3,2-
α
)py imidine by wo s eps in ol ing he addi ion o 2-amino hiadiazole
de i a i es o e hyl ace oace a e, andem hyd olysis o he es e o he acid, and cycliza ion o
gi e he ing- used hiadiazolo(3,2-
α
)py imidines in PPA. Mos o hese a e mul is ep p o ocols,
which su e om gene a ion o by-p oduc s, low yields, and use o me al-con aining eagen s.
The e o e, i is qui e signi ican o de elop he di ec , e icien , and g een al e na i e app oaches o
ge he unc ionalized hiadiazolo(3,2-
α
)py imidine de i a i es om iewpoin o g een chemis y.
He ein we epo ed a new, simple p o ocol o an en i onmen iendly, apid and con enien
syn hesis, and an i umo ac i i y agains MCF-7, K562, HeLa, PC-3 cance cell lines, o no el
5-amino-2-(4-chlo ophenyl)-7-subs i u ed phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-
6-ca boni ile de i a i es ob ained in excellen yield h ough a one-po h ee componen condensa ion
eac ion o 5-(4-chlo ophenyl)-1,3,4- hiadiazol-2 amine, a oma ic aldehyde and malononi ile using
sodium hyd oxide as ca alys in ul asound. Mo eo e , docking s udies using hymidyla e syn hase
(TS) enzyme a e p esen ed in his pape as well.
2. Resul and Discussion
2.1. Chemis y
He ein we epo he one-po syn hesis o no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile de i a i es om h ee
componen eac ions o an 5-(4-chlo ophenyl)-1,3,4- hiadiazol-2 amine (
1
), a oma ic aldehydes (
3
) and
malononi ile (
2
) in p esence o NaOH unde e lux and ul asonic i adia ion as shown in Scheme 1.
To de e mine he op imal eac ion condi ions, he one po eac ions be ween 5-(4-chlo ophenyl)-
1,3,4- hiadiazol-2 amine (
1
), sui able aldehyde (
3
), malononi ile (
2
) we e ca ied ou using di e en
sol en s in he p esence o NaOH as a ca alys a di e en mole pe cen age as shown in Table 1, he
desi ed p oduc was no o med when H
2
O was chosen as sol en and ins ead ace oni ile, me hanol
and dime hyl o maide was chosen as sol en . The desi ed p oduc was o med in low yield unde
e lux and ul asonic i adia ion as shown in Table 1. The op imiza ion o eac ion condi ions o
1,3,4- hiadiazolo(3,2-
α
)py imidine skele on is as shown in Table 2. All he syn hesized compounds
we e cha ac e ized by 1H-NMR, 13C-NMR, mass spec oscopy and IR.
Molecules 2016, 21, 894 3 o 13
low amoun o sol en s equi ed, eco iendly me hod, clean hea ing sys em, nea and clean syn he ic
p o ocol, cheape eagen s and less ex eme physical condi ions, con ol on eac ion pa ame e s,
milde eac ion condi ions.
The exis ing syn he ic me hodologies o (1,3,4) hiadiazolo(3,2-α)py imidine nucleus in a modula
ashion a e no s aigh o wa d and he syn he ic ou es in ol e mul iple s eps. Fo example,
1,3,4- hiadiazolo(3,2-α)py imidine-7-sul onamide de i a i es we e syn hesized om 5-aminol-3,4-
hiadiazole-2-sul onamide ia a wo s eps app oach [24]. Salimo e al. [25] p epa ed 2-b omo-7-me hyl-
5-oxo-5H-1,3,4- hiadiazolo(3,2-α)py imidine by wo s eps in ol ing he addi ion o 2-amino hiadiazole
de i a i es o e hyl ace oace a e, andem hyd olysis o he es e o he acid, and cycliza ion o gi e
he ing- used hiadiazolo(3,2-α)py imidines in PPA. Mos o hese a e mul is ep p o ocols, which su e
om gene a ion o by-p oduc s, low yields, and use o me al-con aining eagen s. The e o e, i is qui e
signi ican o de elop he di ec , e icien , and g een al e na i e app oaches o ge he unc ionalized
hiadiazolo(3,2-α)py imidine de i a i es om iewpoin o g een chemis y. He ein we epo ed a new,
simple p o ocol o an en i onmen iendly, apid and con enien syn hesis, and an i umo ac i i y
agains MCF-7, K562, HeLa, PC-3 cance cell lines, o no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)py imidine-6-ca boni ile de i a i es ob ained in
excellen yield h ough a one-po h ee componen condensa ion eac ion o 5-(4-chlo ophenyl)-
1,3,4- hiadiazol-2 amine, a oma ic aldehyde and malononi ile using sodium hyd oxide as ca alys in
ul asound. Mo eo e , docking s udies using hymidyla e syn hase (TS) enzyme a e p esen ed in his
pape as well.
2. Resul and Discussion
2.1. Chemis y
He ein we epo he one-po syn hesis o no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)py imidine-6-ca boni ile de i a i es om h ee
componen eac ions o an 5-(4-chlo ophenyl)-1,3,4- hiadiazol-2 amine (1), a oma ic aldehydes (3) and
malononi ile (2) in p esence o NaOH unde e lux and ul asonic i adia ion as shown in Scheme 1.
To de e mine he op imal eac ion condi ions, he one po eac ions be ween 5-(4-chlo ophenyl)-
1,3,4- hiadiazol-2 amine (1), sui able aldehyde (3), malononi ile (2) we e ca ied ou using di e en
sol en s in he p esence o NaOH as a ca alys a di e en mole pe cen age as shown in Table 1, he
desi ed p oduc was no o med when H2O was chosen as sol en and ins ead ace oni ile, me hanol
and dime hyl o maide was chosen as sol en . The desi ed p oduc was o med in low yield unde
e lux and ul asonic i adia ion as shown in Table 1. The op imiza ion o eac ion condi ions o
1,3,4- hiadiazolo(3,2-α)py imidine skele on is as shown in Table 2. All he syn hesized compounds
we e cha ac e ized by 1H-NMR, 13C-NMR, mass spec oscopy and IR.
Scheme 1. One-Po , h ee componen syn hesis o no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)py imidine-6-ca boni ile de i a i es.
Scheme 1.
One-Po , h ee componen syn hesis o no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)py imidine-6-ca boni ile de i a i es.
Molecules 2016,21, 894 4 o 13
Table 1.
Op imiza ion o eac ion condi ions o no el 5-amino-2-(4-chlo ophenyl)-7-subs i u ed
phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile de i a i es using a ious
sol en and di e en mole pe cen age o NaOH.
En y Ca alys Amoun (% mol) Sol en Me hod A Con en ional Me hod B Ul asound
Time (h) Yield (%) Time (h) Yield (%)
1 No ca alys - E OH 9 - 2 -
2 NaOH 30 E OH 9 70 2 89
3 NaOH 20 E OH 9 70 2 89
4 NaOH 20 H2O 9 - 2 -
5 NaOH 20 MeOH 11 55 2.30 60
6 NaOH 20 CH3CN 11 40 2.30 50
7 NaOH 20 DMF 12 40 2.30 55
8 NaOH 10 E OH 13 60 2.45 65
9 NaOH 5 E OH 15 50 3.30 60
Table 2. Op imiza ion o eac ion condi ions o 1,3,4- hiadiazolo(3,2-α)py imidine skele on.
Compound RCon en ional Ul asound
Time (h) Yield (%) Time (h) Yield (%)
4a 4-chlo ophenyl 7 70 1 89
4b 2-chlo ophenyl 7.30 68 1.30 85
4c 3-chlo ophenyl 7.30 65 1.30 85
4d 4- lu ophenyl 7 62 1.30 80
4e 4-me hoxyphenyl 8 58 2 75
4 3,4,5-me hoxyphenyl 9 58 2 78
4g 3,4-me hoxyphenyl 9 55 2 75
4h Phenyl 7 60 1 80
4i 3-hyd oxy-4 me hoxyphenyl 9 60 2 82
4j u an-2-yl 8 45 1.30 65
2.2. In Vi o An icance Ac i i y
The a ge compounds (
4a
–
j
) we e e alua ed o hei an icance ac i i y agains MCF-7, K562,
HeLa and PC-3 cance cell lines. The GI
50
alues (concen a ion equi ed o G ow h inhibi ion o 50%)
o he syn hesized compounds we e de e mined using SRB assays me hod. The an icance e alua ion
esul s and GI
50
alues we e lis ed in Table 3and he well-known an icance d ug 5-FU was used as
posi i e con ol.
Table 3. In- i o an icance ac i i y.
Compound GI50 µM
MCF-7 K-562 HeLa PC-3
4a 88.5 47.9 56.2 38.9
4b 43.8 57.1 54.3 37.9
4c 55.0 60.1 55.7 38.4
4d 80.6 >100 58.1 30.2
4e 38.9 54.2 43.8 26.7
4 38.3 58.1 48.6 25.4
4g 34.8 54.3 47.9 25.3
4h 38.9 58.3 38.7 34.7
4i 32.7 55.3 34.3 28.9
4j 82.5 >100 60.9 55.3
5-FU 32.18 47.03 43.71 12.00
5-Flu ou acil (5-FU) was he s anda d d ug used. GI
50
is he concen a ion exhibi ing 50% inhibi ion o he
g ow h as compa ed o he g ow h o con ol. MCF-7 Human b eas cance , K-562 Human Leukemia cance ,
HeLa Human ce ical cance , PC-3 Human p os a e cance .
Molecules 2016,21, 894 5 o 13
The esul s indica ed ha he compounds,
4e
,
4
,
4g
and
4i
exhibi ed signi ican cell g ow h
inhibi ion compa ed o e e ence s anda d 5- luo ou acil agains MCF-7, K562, HeLa and PC-3 cance
cell lines. F om he an icance ac i i y esul s, i was obse ed ha compound
4i
, which has subs i uen
3-hyd oxy-4-me hoxyphenyl, is ound o ha e he highes GI
50
alues o 32.7
µ
M, 34.3
µ
M, 55.3
µ
M,
28.9
µ
M o MCF-7, HeLa, K562 and PC-3 cance cell lines espec i ely. Compound
4g
, which has
3,4-dime hoxyphenyl, is ound o ha e GI
50
alues o 34.8
µ
M, 54.3
µ
M, 47.9
µ
M, 25.3
µ
M o MCF-7,
K562, HeLa, and PC-3 cance cell lines espec i ely. All he syn hesized compounds we e less ac i e
owa ds PC-3 in compa ison o s anda d d ug 5-FU.
S uc u al ac i i y ela ionship (SAR) s udies o hese compounds demons a ed ha elec on
wi hd awing g oups such as luo o (
4d
), chlo o (
4a
,
4b
,
4c
) exhibi ed less ac i i y compa ed o elec on
dona ing, pola g oups. The an icance ac i i y o de i a i es bea ing an elec on wi hd awing g oup
such as chlo o g oup a o ho posi ion (
4b
) (43.8
µ
M, 57.1
µ
M, 54.3
µ
M, 37.9
µ
M o MCF-7, K-562,
HeLa and PC-3 cance cell lines espec i ely) exhibi ed mo e ac i i y han compound
4c
(chlo o g oup
a me a posi ion) (55.0
µ
M, 60.1
µ
M, 55.7
µ
M, 38.4
µ
M o MCF-7, K-562, HeLa and PC-3 cance cell
lines espec i ely) and compound
4a
(chlo o g oup a pa a posi ion) (88.5
µ
M, 47.9
µ
M, 56.2
µ
M,
38.9
µ
M o MCF-7, K-562, HeLa and PC-3 cance cell lines espec i ely). Replacemen o he phenyl
g oup in he pa en compound by u an ing in
4j
has shown dec eased ac i i y in compa ison o he
s anda d d ug 5-FU (82.5
µ
M, >100
µ
M, 60.9
µ
M, 55.30
µ
M, o MCF-7, K-562, HeLa and PC-3 cance
cell lines, espec i ely). F om SAR i can be conside ed ha compounds con aining elec on dona ing,
pola g oups such as
4e
,
4
,
4g
,
4i
ha e good an icance ac i i y in compa ison o elec on wi hd awing
g oups such as
4a
,
4b
,
4c
,
4d
. I is also clea ha he eplacemen o he phenyl ing wi h he u an ing
dec eases an icance ac i i y.
4i and 4g a e equipo en wi h he clinically used an icance d ug 5-FU agains MCF-7, HeLa cell
lines, hence hese compounds can be de eloped as an icance agen s in he u u e. Compound
4a
is
equipo en wi h he clinically used an icance d ug 5-FU agains K-562.
2.3. Molecula Docking
5-Fluo ou acil de i a i es and hei s uc u ally ela ed compounds like peme exed, Capeci ibine,
Ral i exed a e well known o inhibi hymidyla e syn hase. We decided o ca y ou a molecula
docking s udy on ou newly syn hesized 1,3,4- hiadiazolo(3,2-
α
)py imidine skele on de i a i es in o
he binding si e o hymidyla e syn hase (PDB ID: 1JU6) using 5-FU as a e e ence o docking esul s.
The docking esul s indica ed ha compounds we e held in he ac i e pocke by combina ion o a ious
hyd ogen and hyd ophobic in e ac ions wi h TS. The docking esul s e ealed ha he highes binding
compound o TS was
4i
wi h a G-Sco e o
´
7.17. The compound
4g
shows hyd ogen bonding wi h
ASN 112, ILE 108 and ARG 50 wi h a G-Sco e o
´
5.59 as shown in Figu e 2. Simila ly, he compound
4i
shows hyd ogen bonding wi h GLN 214, SER 214 and ASN 226 wi h a highes G-Sco e o
´
7.17 as
shown in Figu e 3. The compounds
4b
(
´
5.59),
4c
(
´
5.24),
4d
(
´
5.14),
4
(
´
5.14),
4h
(
´
5.38) and
4j (´4.98), all showed G-Sco e g ea e han 5-FU (´4.75).
On he basis o he an icance ac i i y and docking esul s, i was ound ha compounds
4g
,
4h
and
4i
had po en ial o inhibi hymidyla e syn hase. G-Sco e o all he syn hesized de i a i es is as
shown in Table 4.
Table 4. G-Sco e o all he syn hesized de i a i es.
Compound G-Sco e Compound G-Sco e
4a ´4.65 4g ´5.59
4b ´5.59 4h ´5.38
4c ´5.24 4i ´7.17
4d ´5.14 4j ´4.98
4e ´4.74 5-FU ´4.75
4 ´5.14
Molecules 2016,21, 894 6 o 13
Molecules 2016, 21, 894 6 o 13
Figu e 2. Docking pose o compound 4g in he ac i e si e o hymidyla e syn hase enzyme.
Figu e 3. Docking pose o compound 4i in he ac i e si e o hymidyla e syn hase enzyme.
2.4. In Silico ADME P edic ion
The p edic ion o he ADME pa ame e s p io o he expe imen al s udies is one o he mos
impo an aspec s o d ug disco e y and de elopmen o he d ug molecule. ADME s udies ha e
always played a c i ical ole in helping o op imize he pha macokine ic p ope ies o new d ugs,
he eby inc easing hei success a e. The analysis o Lipinski’s ule o i e was pe o med o indica e
whe he a chemical compound could be an o ally ac i e d ug in humans. I was obse ed ha he
compounds exhibi ed a good % abso p ion (% ABS) anging om 87.50% o 100% (Table 5).
The esul s o he p edic ion o ADME p ope ies a e depic ed in Table 5. All syn hesized
compounds 4 a–j had good abso p ion and we e ound o be non oxic.
Figu e 2. Docking pose o compound 4g in he ac i e si e o hymidyla e syn hase enzyme.
Molecules 2016, 21, 894 6 o 13
Figu e 2. Docking pose o compound 4g in he ac i e si e o hymidyla e syn hase enzyme.
Figu e 3. Docking pose o compound 4i in he ac i e si e o hymidyla e syn hase enzyme.
2.4. In Silico ADME P edic ion
The p edic ion o he ADME pa ame e s p io o he expe imen al s udies is one o he mos
impo an aspec s o d ug disco e y and de elopmen o he d ug molecule. ADME s udies ha e
always played a c i ical ole in helping o op imize he pha macokine ic p ope ies o new d ugs,
he eby inc easing hei success a e. The analysis o Lipinski’s ule o i e was pe o med o indica e
whe he a chemical compound could be an o ally ac i e d ug in humans. I was obse ed ha he
compounds exhibi ed a good % abso p ion (% ABS) anging om 87.50% o 100% (Table 5).
The esul s o he p edic ion o ADME p ope ies a e depic ed in Table 5. All syn hesized
compounds 4 a–j had good abso p ion and we e ound o be non oxic.
Figu e 3. Docking pose o compound 4i in he ac i e si e o hymidyla e syn hase enzyme.
2.4. In Silico ADME P edic ion
The p edic ion o he ADME pa ame e s p io o he expe imen al s udies is one o he mos
impo an aspec s o d ug disco e y and de elopmen o he d ug molecule. ADME s udies ha e
always played a c i ical ole in helping o op imize he pha macokine ic p ope ies o new d ugs,
he eby inc easing hei success a e. The analysis o Lipinski’s ule o i e was pe o med o indica e
whe he a chemical compound could be an o ally ac i e d ug in humans. I was obse ed ha he
compounds exhibi ed a good % abso p ion (% ABS) anging om 87.50% o 100% (Table 5).
The esul s o he p edic ion o ADME p ope ies a e depic ed in Table 5. All syn hesized
compounds 4a–jhad good abso p ion and we e ound o be non oxic.
Molecules 2016,21, 894 7 o 13
Table 5. Abso p ion, dis ibu ion, me abolism and exc e ion (ADME) esul s o syn hesized compounds.
Compound MW a
Pe cen age o
Abso p ion
(% ABS) b
logP o/w c
(–2.0 o 6.5) n-ON d(<10) n-OHNH e(<5) Pola Su ace A ea
(PSA) (7–200.0)
Numbe o
Viola ions o
Rule o 5 (<5)
Toxici y
4a 400 100 4.57 4 2 74.81 0 N
4b 400 100 4.42 4 2 74.87 0 N
4c 400 100 4.57 4 2 74.81 0 N
4d 383.8 100 4.30 4 2 74.81 0 N
4e 395.8 100 4.19 4.75 2 83.10 0 N
4 455.9 100 4.47 6.25 2 95.95 0 N
4g 425.8 100 4.36 5.5 2 88.02 0 N
4h 365.8 100 4.01 4 2 74.81 0 N
4i 411.8 87.50 3.46 5.5 3 104.7 0 N
4j 355.8 94.19 3.12 4.5 2 83.51 0 N
5-FU 130.0 66.30 ´0.89 3.5 2 87.56 0 N
a
Molecula weigh o he molecule;
b
Pe cen age human o al abso p ion (% ABS) (>80% is high, <25% is poo );
c
P edic ed oc anol-wa e pa i ion coe icien (logP o/w) (–2.0 o 6.5);
dn-ON numbe o hyd ogen bond accep o s <10; en-OHNH numbe o hyd ogen bonds dono s <5; Pola su ace a ea (PSA) (7.0–200.0); N: non- oxic.
Molecules 2016,21, 894 8 o 13
3. Ma e ials and Me hods
3.1. Gene al
All he eac ions we e pe o med in o en-d ied glasswa es. All eagen s and sol en s we e used
as ob ained om he supplie o ec ys allized/ edis illed unless o he wise no ed. The ul asound
sonica o (Sonics Vib a-cell, Modelno. VCX 500, New own, CT, USA) equipped wi h solid syn he ic
p obe, 13 mm in ip diame e , ope a ing a 20 kHz wi h a maximum powe ou pu o 500 W, was
used o syn hesis o inal i le compounds. The pu i y o he syn hesized compounds was moni o ed
by ascending hin laye ch oma og aphy (TLC) on silica gel-G (Me ck, Da ms ad , Ge many) coa ed
aluminum pla es, isualized by iodine apo and mel ing poin s we e de e mined in open capilla y
ubes. In a ed (IR) spec a we e eco ded on a PS 4000 FTIR (JASCO, Tokyo, Japan) using KB pelle s.
Elemen al analyses (C, H, and N) we e done wi h a FLASHEA 112 Shimadzu’ analyze (Mumbai,
Maha ash a, India) and all analyses we e consis en (wi hin 0.4%) wi h heo e ical alues. The
1
H-NMR and
13
C-NMR spec a o syn hesized compounds we e eco ded on B uke A ance II 400
NMR Spec ome e (Bille ica, MA, USA) a 400 MHz F equency in deu e a ed DMSO and CDCl
3
and
using TMS as in e nal s anda d (chemical shi
δ
in ppm). Mass spec a o some compounds we e
scanned on FTMS + p ESI ull mass (100.00–1500.00).
3.2. Gene al P ocedu e o he Syn hesis o 5-Amino-2-(4-chlo ophenyl)-7-Subs i u ed
Phenyl-8,8a-dihyd o-7H-(1,3,4) hiadiazolo(3,2-α)py imidine-6-ca boni ile De i a i es
Me hod A: A 25 mL ound bo om lask was cha ged wi h a mix u e o an 5-(4-chlo ophenyl)-
1,3,4- hiadiazol-2 amine (0.01 mol) (
1
), malononi ile (0.01 mol) (
2
), sui able aldehyde (0.01 mol) (
3
)
in e hanol (10–12 mL) and he ca alys NaOH (20% mmol) and he eac ion mix u e was e luxed .
A e comple ion o he eac ion (moni o ed by TLC), he mix u e was pou ed in o ice cold wa e . The
p oduc ob ained, was il e ed and d ied. The co esponding p oduc was ob ained in high pu i y a e
ec ys alliza ion o he c ude p oduc om e hanol. The au hen ici y o compounds was es ablished by
1H-NMR, 13C-NMR, IR and HRMS.
Me hod B: A 25 mL a beake was cha ged wi h a mix u e o an 5-(4-chlo ophenyl)-1,3,4-
hiadiazol-2 amine (0.01 mol) (
1
), malononi ile (0.01 mol) (
2
), sui able aldehyde (0.01 mol) (
3
) in
e hanol (10–12 mL) and he ca alys NaOH (20% mmol) and he eac ion mix u e was kep inside
an Ul asonica o acous ic chambe a 80
˝
C a 20%. A e comple ion o he eac ion (moni o ed by
TLC), he mix u e was pou ed in o ice cold wa e . The p oduc ob ained, was il e ed and d ied. The
co esponding p oduc was ob ained in high pu i y a e ec ys alliza ion o he c ude p oduc om
e hanol. The au hen ici y o compounds was es ablished by 1H-NMR, 13C-NMR, IR and HRMS.
5-Amino-2,7-bis(4-chlo ophenyl)-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4a
. M.P: 237–240
˝
C.
R alue: 0.28. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3100 (A oma ic C-H s e ching), 1623 (C=N), 740.55
(C-Cl o a oma ic ings),
1
H-NMR
δ
ppm: 10.00 (s, 2H, NH
2
), 8.00–7.02 (m, 8H wo a oma ic ings), 3.35
(s, 1H, A C, o py imidine ing),
13
C-NMR
δ
: 172.11 (C), 158.31 (C), 143.21 (C), 139.50 (C), 136.51 (C),
131.00 (C), 130.52 (CH), 129.87 (CH), 128.53 (CH), 128.23 (C), 127.93 (CH), 118.27 (C), 60.22 (C), 54.53
(C), m/z399 (100.0%), 401.01 (68.9%), 400.01 (22.7%) Molecula Fo mula: C
18
H
11
Cl
2
N
5
S. Elemen al
Analysis: Calcula ed: (C, H, Cl, N, S) 54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.04, 2.74, 17.68, 17.51, 8.00.
5-Amino-7-(2-chlo ophenyl)-2-(4-chlo ophenyl)7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4b
. M.P:
240–242
˝
C. R alue: 0.24. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3000 (A oma ic C-H s e ching), 1623
(C=N), 740.55 (C-Cl o a oma ic ings). 1H-NMR
δ
ppm: 9.98 (s, 2H, NH
2
), 8.00–7.09 (m, 8H wo
a oma ic ings), 3.35 (s, 1H, A C, o py imidine ing),
13
C-NMR
δ
ppm: 170.24 (C), 158.37 (C), 143.77
(C), 138.10 (C), 134.26 (C), 129.52 (CH), 129.00 (CH), 128.51 (CH), 128.03 (C), 127.99 (CH), 126.11 (CH),
117.59 (C), 60.00 (C), 55.02 (C), m/z399 (100.0%), 401.01 (68.9%), 400.01 (22.7%) Molecula Fo mula:
C
18
H
11
C
l2
N
5
S. Elemen al Analysis: Calcula ed: (C, H, Cl, N, S) 54.01, 2.77, 17.71, 17.50, 8.01 Found:
55.02, 2.76, 17.70, 17.52, 8.02.
Molecules 2016,21, 894 9 o 13
5-Amino-7-(3-chlo ophenyl)-2-(4-chlo ophenyl)7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4c
. M.P:
235–238
˝
C. R alue: 0.20. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3100 (A oma ic C-H s e ching), 162.3
(C=N), 740.55 (C-Cl o a oma ic ings), 1H-NMR
δ
ppm: 10.01 (s, 2H, NH
2
), 8.00–7.05 (m, 8H wo
a oma ic ings), 3.32 (s, 1H, A C, o py imidine ing),
13
C-NMR
δ
ppm: 172.21 (C), 158.20 (C), 155.00
(C), 142.50 (C), 137.69 (C), 136.17 (C), 134.09 (C), 130.03 (CH), 129.99 (CH), 129.02 (CH), 128.93 (CH),
128.37 (CH), 125.59 (CH), 123.95 (CH), 119.11 (CH), 117.57 (C), 59.97 (C), 52.00 (C), m/z399 (100.0%),
401.01 (68.9%), 400.01 (22.7%), Molecula Fo mula: C
18
H
11
C
l2
N
5
S. Elemen al Analysis: Calcula ed:
(C, H, Cl, N, S) 54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.01, 2.77, 17.67, 17.49, 8.03.
5-Amino-2-(4-chlo ophenyl)-7-(4- luo ophenyl)-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4d
. M.P:
239–240
˝
C. R alue: 0.37. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3000 (A oma ic C-H s e ching), 1623
(C=N), 740.55 (C-Cl o a oma ic ing), 1053 (C-F o a oma ic ings),
1
H-NMR
δ
ppm: 9.79 (s, 2H, NH
2
),
8.02–7.06 (m, 8H wo a oma ic ings), 3.33 (s, 1H, A C, o py imidine ing),
13
C-NMR
δ
ppm: 172.22
(C), 159.07 (C), 158.31 (C), 143.77 (C), 136.62 (C), 136.99 (C), 130.67 (CH), 129.52 (CH), 128.99 (C), 128.77
(CH), 117.54 (C), 115.34 (CH), 60.07 (C), 52.97 (C), m/z383.04 (100%), 385.04 (37%), 384.04 (22.7%),
Molecula Fo mula: C
18
H
11
ClFN
5
S. Elemen al Analysis: Calcula ed: (C, H, Cl, F, N, S) 56.33, 2.89, 9.24,
4.95, 18.25, 8.35 Found: 56.37, 2.90, 9.20, 4.91, 18.23, 8.36.
5-Amino-2-(4-chlo ophenyl)-7-(4-me hoxyphenyl)-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4e
.
M.P: 210–212
˝
C. R alue: 0.36. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
); 3000 (A oma ic C-H s e ching),
1623 (C=N), 1055 (C-OCH
3
), 740.55 (C-Cl o a oma ic ing),
1
H-NMR
δ
ppm: 9.99 (s, 2H, NH
2
), 8.02–6.79
(m, 8H wo a oma ic ings), 3.36 (s, 1H, A C, o py imidine ing), 3.56 (s, 3H, OCH
3
),
13
C-NMR
δ
ppm:
172.26 (C), 158.99 (C), 157.51 (C), 143.70 (C), 136.99 (C), 133.04 (C), 130.07 (CH), 129.55 (CH), 128.52
(CH), 128.00 (C), 117.37 (C), 114.26 (CH), 60.00 (C), 55.99 (CH
3
), 53.00 (C), m/z395.06 (100%), 397.06
(37.0%), 396.06 (23.8%), Molecula Fo mula: C
19
H
14
ClN
5
OS. Elemen al Analysis: Calcula ed: (C, H, Cl,
N, O, S) 57.65, 3.56, 8.96, 17.69, 4.04, 8.10 Found: 57.68, 3.59, 8.93, 17.67, 4.03, 8.11.
5-Amino-2-(4-chlo ophenyl)-7-(3,4,5- ime hoxyphenyl)-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4
. M.P: 220–222
˝
C. R alue: 0.26. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3100 (A oma ic C-H
s e ching), 1623 (C=N), 1059 (C-OCH
3
), 740.55 (C-Cl o a oma ic ing),
1
H-NMR
δ
ppm: 10.00 (s, 2H,
NH
2
), 8.02–6.79 (m, 6H wo a oma ic ings), 3.36 (s, 1H, A C, o py imidine ing), 3.56 (s, 9H, OCH
3
),
13
C-NMR
δ
ppm: 172.21 (C), 158.89 (C), 152.81 (C), 143.32 (C), 136.52 (C), 136.00 (C), 135.01 (C), 129.51
(CH), 128.52 (CH), 128.77 (C), 117.51 (C), 106.50 (CH), 61.00 (CH
3
), 60.52 (C), 56.00 (CH
3
), 53.00 (C),
m/z455.08 (100%), 457.08 (37.1%), 456.09 (23.7%), Molecula Fo mula: C
21
H
18
ClN
5
O
3
S. Elemen al
Analysis: Calcula ed: (C, H, Cl, N, O, S) 55.32, 3.98, 7.78, 15.36, 10.53, 7.03 Found: 55.37, 3.99, 7.77,
15.33, 10.52, 7.00.
5-Amino-2-(4-chlo ophenyl)-7-(3,4-dime hoxyphenyl)-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4g
.
M.P: 225–228
˝
C. R alue: 0.50. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3100 (A oma ic C-H s e ching),
1623 (C=N), 1623 (C=N), 1052 (C-OCH
3
), 740.55 (C-Cl o a oma ic ing),
1
H-NMR
δ
ppm: 10.02 (s, 2H,
NH
2
), 8.02–6.79 (m, 7H wo a oma ic ings), 3.36 (s, 1H, A C, o py imidine ing), 3.56 (s, 6H, OCH
3
),
13
C-NMR
δ
ppm: 172.07 (C), 158.00 (C), 149.07 (C), 146.72 (C), 143.54 (C), 136.58 (C), 134.51 (C), 129.59
(CH), 128.55 (CH), 128.70 (C), 122.01 (C), 117.56 (C), 114.99 (CH), 112.53 (CH), 59.11 (C), 56.09 (CH
3
),
53.00 (C), m/z425.07 (100.0%), 427.07 (37.0%), 426.07 (24.09%), Molecula Fo mula: C
20
H
16
ClN
5
O
2
S.
Elemen al Analysis: Calcula ed: (C, H, Cl, N, O, S) 56.40, 3.79, 8.32, 16.44, 7.51, 7.53 Found: 56.44, 3.82,
8.30, 16.00, 7.50, 7.52.
5-Amino-2-(4-chlo ophenyl)-7-phenyl-7H-(1,3,4) hiadiazolo(3,2-
α
)py imidine-6-ca boni ile
4h
. M.P: 218–220
˝
C.
R alue: 0.43. IR (KB )
υ
max cm
´1
: 3400 (C-NH
2
), 3000 (A oma ic C-H s e ching), 1623 (C=N), 740.55
(C-Cl o a oma ic ing),
1
H-NMR
δ
ppm: 10.00 (s, 2H, NH
2
), 8.02–7.27 (m, 9H wo a oma ic ings),
3.34 (s, 1H, A C, o py imidine ing),
13
C-NMR
δ
ppm: 172.12 (C), 158.37 (C), 143.77 (C), 141.00 (C),
136.50 (C), 129.55 (CH), 129.08 (CH), 128.61 (C), 128.52 (CH), 125.79 (CH), 125.60 (CH), 117.51 (C), 60.00
(C), 53.12 (C), m/z365.05 (100.0%), 367.05 (37.0%), 366.05 (22.7%), Molecula Fo mula: C
18
H
12
ClN
5
S.
