Synthesis, dihydrofolate reductase inhibition, anti-proliferative testing, and saturation transfer difference1H-NMR study of some new 2-substituted-4,6-diaminopyrimidine derivatives

Article abstract of DOI:10.1248/cpb.60.70

A series of 2-substituted-4,6-diaminipyrimidine derivatives were synthesized and evaluated for their dihydrofolate reductase (DHFR) inhibitory activity. Saturation transfer difference (STD) ¹H-NMR experiments were used to probe the binding char

Full text of DOI:10.1248/cpb.60.70

—
70
Regular Article
Chem. Pharm. Bull. 60(1) 70 78 (2012)
Vol. 60, No. 1
Synthesis, Dihydrofolate Reductase Inhibition, Anti-proliferative Testing,
1
and Saturation Transfer Difference H-NMR Study of Some New
2-Substituted-4,6-diaminopyrimidine Derivatives
Shohreh Mohebbi,a Juan Manuel Falcón-Pérez,b Esperanza González,b Oscar Millet,c
,a,d
Jose Maria Mato,b and Farzad Kobarfard*
d
a Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences; Phyto-
b
chemistry Research Center, Shahid Beheshti University of Medical Sciences; Tehran 14155–6153, Iran: Metabolomics
c
Unit, CICbioGUNE, CIBERehd; Bizkaia Technology Park, Derio, Bizkaia 48160, Spain: and Structural Biology Unit,
CICbioGUNE; Bizkaia Technology Park, Derio, Bizkaia 48160, Spain.
Received August 24, 2011; accepted October 11, 2011; published online October 18, 2011
A series of 2-substituted-4,6-diaminipyrimidine derivatives were synthesized and evaluated for their di-
hydrofolate reductase (DHFR) inhibitory activity. Saturation transfer difference (STD) 1H-NMR experiments
were used to probe the binding characteristics of the compounds with human DHFR enzyme. The most po-
tent molecules, 12 and 15, in enzyme assay study showed the best results in STD experiments indicating their
intimate interaction with the receptor. The docking studies were followed to explain the structural basis for
the observed interaction between the ligands and DHFR. All the compounds were also assayed in vitro for
their growth inhibitory activity on MCF-7, HepG2, SKHep1, and Hela tumor cell lines. Compounds 16, 17,
and 22 demonstrated the most potent in vitro anti-proliferative activity among the others.
Key words diaminopyrimidine; dihydrofolate reductase inhibitory activity; synthesis
Interfering with folate metabolism in cancerous cells could
Similarly the DHFR inhibitor methotrexate (MTX) is a
provide a useful mean in cancer chemotherapy as a result of mainstay in single and combination cancer chemotherapy.
the inhibition of the biosynthesis of nucleic acid precursors.1,2) Several TS and DHFR inhibitors which are analogs of folate
The cofactor tetrahydrofolate (THF) is formed by have also shown antitumor activities in vitro and in vivo with
the reduced nicotinamide adenine dinucleotide phosphate some currently in clinical trials.7,8)
(NADPH)-dependent reduction of 7,8-dihydrofolate (7,8-DHF)
While the presence of 2 amino groups on positions 2 and
by the enzyme dihydrofolate reductase (DHFR) and serves as 4 of pteridine seems to be essential for DHFR inhibition, but
the principle component in folate metabolism as a carrier of there are several examples indicating the insignificant nature
one-carbon units in its various cofactor forms.3) Tetrahydro- of pteridine core. On the other hand, Yamini and Vijjulatha9)
folate (THF) picks up one carbon unit from L-serine and is showed that the replacement of pteridyl group by naphthalene
converted to coenzyme N5,N10-methylenetetrahydrofolic acid ring in methotrexate could still give molecules of great inter-
where CH2 group bridges N5 and N10 tetrahydrofolate.4) This est, indicating the not so significant role of nitrogen hetero-
coenzyme is utilized by thymidylate synthase (TS) for the atoms of pteridine core in methotrexate structure.
synthesis of deoxythymidylate monophosphate (dTMP) from
deoxyuridylate monophosphate (dUMP).
Replacement of pyrazine ring with benzene ring in the
structure of trimetrexate is another evidence for the insignifi-
Therefore thymidylate synthase (TS) coupled with dihydro- cant role of pyrazine ring in classical DHFR inhibitors.
folate reductase (DHFR) forms a crucial link responsible for
the synthesis of dTMP and hence DNA.
It is the aim of this study to make a group of 4,6-diamino-
pyrimidine derivatives with alkyl/arylalkylthio substituents on
Inhibitors of TS derived from substrate analogs such as position 2 (Fig. 2).
5-fluorouracil5) and from folate analogs such as raltitrexed6)
The alkyl/arylalkyl group (R) attached to sulfur atom
(N-[(5-{methyl[(2-methyl-4-oxo-1,4-dihydroquinazolin-6-yl) imparts various degrees of lipophilicity to the compounds
methyl]amino}-2-thienyl)carbonyl]-L-glutamic acid have found which could act in favor of increasing activity against DHFR
utility as clinically important antitumor agents. (Fig. 1).
enzyme of some cells such as Mycobacterium tuberculosis
Fig. 1. Chemical Structures of 5FU and Raltitrexed
*To whom correspondence should be addressed. e-mail: kobarfard@sbmu.ac.ir
© 2012 The Pharmaceutical Society of Japan

January 2012
71
Activities of the compounds were also evaluated using
clonogenic test against a selected group of tumor cell lines.
Chemistry The synthetic strategy to synthesize the inter-
—
mediate 3 and the final compounds 4 25 is depicted in Chart
1. 4,6-Diaminopyrimidine-2-thiol (3) was obtained as the key
intermediate by the reaction of thiourea and malononitrile in
absolute ethanol as solvent. Subsequent reaction of compound
(3) with various alkyl halides at room temperature afforded
Fig. 2. General Structures of 2-Alkylthio-4,6-diaminopyrimidines
10)
—
compounds 4 25 in good yields. Structure confirmation
of the synthesized intermediate and the final products was
performed using IR, NMR, mass spectrometry, and carbon,
which have more lipophilic characteristics in compared to the hydrogen, nitrogen, and sulfur (CHNS) elemental analysis.
same enzymes in human cells.
In order to investigate the DHFR inhibitory activity of the
synthesized compounds, enzyme inhibition assay was used
Result and Discussion
A group of 4,6-diaminopyrimidine bearing a thioether side
according to the protocol described in the manual of DHFR chain on the position 2 of pyrimidine ring were synthesized.
assay kit from Sigma-Aldrich company. The compounds were tested for their ability to inhibit human
The dynamic interactions of DHFR as the target protein DHFR and their potencies (IC50 values) were measured in
with the synthesized compounds as potential ligands were vitro. The results are presented in Table 1.
investigated using saturation transfer difference (STD) NMR
technique. The basis of the STD technique relies on the trans- (12µM), 25 (12µM), 20 (13µM), and 22 (14µM). The 2 most
ferred nuclear Overhauser effect (tr-NOE) phenomenon. potent compounds 15 and 12 both have o-substituted benzyl
The most potent compounds were 15 (9µM), 12 (11µM), 23
To further investigate the structural basis for the ligand-re- thioether on their position 2. Replacement of the substituent
ceptor binding, computer-assisted (in silico) experiments using on the benzyl group by an ortho-nitro group has been detri-
the X-ray crystal structure of human DHFR were conducted mental to the DHFR inhibitory activity. Changing the benzyl
for docking of the synthesized compounds into their presumed side chain with aliphatic or alicyclic groups both resulted in
receptor i.e. human DHFR.
decreased activity. However phenethyl and phenpropyl side
Reagents and conditions: (a) EtONa, reflux, 3h; (b) NaOH 0.1M, CH3OH, R-CH2-X (X=Cl, Br), rt, 18h10)
Chart 1

72
Vol. 60, No. 1
1
1
Fig. 3. (a) 500MHz H-NMR Spectrum of Compound 12 in Absence of Human DHFR; (b) 800MHz STD H-NMR Spectrum of Compound 12 in
Complex with Human DHFR Protein after Saturation of DHFR
1
1
Fig. 4. (a) 500MHz H-NMR Spectrum of Compound 15 in Absence of Human DHFR; (b) 800MHz STD H-NMR Spectrum of Compound 15 in
Complex with Human DHFR Protein after Saturation of DHFR

January 2012
73
1
1
Fig. 5. (a) 500MHz H-NMR Spectrum of Compound 11 in Absence of Human DHFR; (b) 800MHz STD H-NMR Spectrum of Compound 11 in
Complex with Human DHFR Protein after Saturation of DHFR
chains have been well tolerated indicating the possible role of study. The non-existence of any peak in difference spectrum
phenyl ring in ligand-receptor interaction. None of the com- indicated the lack of formation of ligand–receptor complex
1
pounds with fluorobenzyl side chains showed increased activi- for this compound. Figure 5a shows the H-NMR spectrum
ties against DHFR enzyme. The presence of a basic piperidine of compound 11 in absence of DHFR and Fig. 5b shows the
ring in the side chain has increased the enzyme inhibition STD NMR spectrum of compound 11 in complex with human
activity.
In order to examine the interaction of these ligands to
DHFR protein after saturation of DHFR.
To investigate the structural basis for the observed interac-
DHFR, saturation transfer difference (STD) NMR was used. tion between compound 12 and DHFR, a docking study was
STD is a fast and versatile method for investigating ligand- conducted using AutoDock 3.05 program. The binding mode
protein binding.11) It is based on magnetization transfer by of one of the most active compounds, 12, has been shown in
protein signal saturation and its relayed effect to the ligand. Fig. 6. It was found that the amine groups in positions 4 and
Saturation of a single protein resonance can result in a rapid 6 of pyrimidine ring were the main keys for hydrogen bond
spread of the saturation over the entire protein if spin dif- formation with =NH group of Arg 70 residue with distance:
fusion within the protein is efficient. During the saturation 2.27Å and –C=O group of Asn 64 with distance=2.36Å
period, progressive saturation will transfer from the protein respectively. This confirms the important role of diaminopy-
(DHFR in this study) to the ligand protons if the ligand binds rimidine moiety in ligand-receptor interaction. Furthermore,
’
to the target. If the ligand doesn t bind to the protein, the mag- the carbon atoms in positions 5 and 6 of the phenyl ring and
netization transfer will not occur and the difference spectrum the sulfur atom form hydrophobic contacts with the phenyl
thus obtained will not show any signal of the ligand.
Figure 3a shows the H-NMR spectrum of compound 12 respectively. Conducting a docking study for compound 15
ring of Phe 31 residue with distances: 3.92, 3.37, and 3.47Å,
1
in absence of DHFR while Figure 3b shows the STD NMR shows the same binding characteristics.
spectrum of compound 12 in complex with human DHFR
Anti-proliferative activity of the compounds were also
protein after saturation of DHFR. The appearance of aromatic evaluated using clonogenic method against 4 tumor cell lines
signals of compound 12 in STD NMR indicates the intimate MCF-7, HepG2, SK-Hep1, and HeLa (Table 1). Surprisingly
interaction of this compound with protein DHFR. Compound the most active DHFR inhibitors (12, 15) are not the most
15 demonstrated the same pattern in STD study. Figure 4 potent compounds against the 4 tumor cell lines. This could
shows 1H-NMR spectrum of this compound in absence of be due to the different distribution and accessibility of these
DHFR (Fig. 4a) and STD NMR spectrum after saturation of compounds to their target inside the cells. On the other hand,
the enzyme (Fig. 4b). Compound 11 which showed no inhibi- there are few compounds (16, 17) with fair anti-proliferative
tory activity against DHFR was also subjected to STD NMR activity against tumor cell lines but low DHFR inhibitory

74
Vol. 60, No. 1
Fig. 6. Stereoview of Compound 12 Docked into the Active Site of Human DHFR (PDB ID Code 1OHJ)12)
Fig. 7. General Structures of TS and DHFR Inhibitors
Fig. 8. Pemetrexed in Normal and Flipped Modes
activity. Other anti-proliferative mechanisms of action rather
than DHFR inhibition could be responsible for this phenom-
enon such as thymidylate synthase inhibition.
As shown in Fig. 7, folate analogs that inhibit TS gener-
ally contain a 2-amino-4-oxo or 2-methyl-4-oxo substitution
in their pyrimidine ring. For example PDDF(CB3717)13) and
raltitrexed.14)
In contrast, inhibitors of DHFR generally contain a 2,4-di-
amino substitution in the pyrimidine ring13,14) as typified by
methotrexate (MTX). Gangjee et al.15) have hypothesized that
Fig. 9. 4,6-Diaminopyrimidines in Normal and Flipped Modes
“
”
pemetrexed could bind to TS in a normal mode (Fig. 8)
which is similar to way that classic TS substrates or inhibitors
“
”
bind, or bind to DHFR in a flipped mode which is achieved
Experimental
°
by rotating the analogs in the normal binding mode by 180
about its NH2–C2 bond.
General All evaporations were carried out in vacuo with
°
a rotary evaporator. Melting points ( C) were determined
The alkylthio/arylalkylthio derivatives made in the present by capillary method on an electrothermal melting point ap-
“
”
study could also be speculates to mimic the Normal and paratus. Infrared spectra were recorded as thin films on KBr
“
”
Flipped forms of pemetrexed (Fig. 9). In their normal form, plates with νmax in inverse centimeters. Nuclear magnetic
they are similar to the normal form of pemetrexed and thus resonance spectra for proton (1H-NMR) were recorded on a
°
are expected to be DHFR inhibitors. By 180 rotating about Bruker DRX-Avance (500MHz) spectrometer to confirm the
C6–NH2 bond, the sulfur atom could mimic the oxo group of structure of synthesized compounds. Chemical shift values
TS inhibitors. Therefore it is expected that these compounds are expressed in ppm (parts per million) relative to tetra-
have both TS inhibitor as well.
methylsilane (TMS) as internal standard; s=singlet, d=dou-
blet, dd=double doublet, t=triplet, q=quartet, quin=quintet,
sex=sextet, m=multiplet, brs=broad singlet. J values are

January 2012
75
Table 1. Anti-proliferative Activity of Synthesized Compounds against 4 Human Tumor Cell Lines and Inhibitory Concentrations (IC50 in μM) of the
Compounds against Human DHFR
e)
EC50 (µM)
Human DHFR inhibition
Compound
(IC50f), μM)
MCF-7a)
>200
HepG2b)
SK-Hep1c)
HeLad)
4
>200
>200
>200
51
>200
>200
>200
62
>200
>200
160
110
>200
156
>200
147
138
151
117
93
21
18
5
>200
>200
90
6
22
7
24
8
150
84
93
61
>100
>100
23
9
81
65
10
>200
121
95
135
93
110
85
11
>100
11
12
56
60
13
90
49
55
>100
19
14
92
65
58
15
66
41
42
9
16
37
19
15
24
22
17
58
25
21
35
18
18
108
178
98
61
53
83
19
19
121
106
58
105
134
48
>200
>200
93
21
20
13
21
63
19
22
36
18
12
45
14
23
65
39
45
93
12
24
25
80
75
67
115
28
17
60
32
31
12
Methotrexate
1.6
25
10
0.9
0.022
a) Human breast adenocarcinoma cell line. b) Human hepatocellular carcinoma cell line. c) Human liver adenocarcinoma cell line. d) Human cervical carcinoma
cell line. e) EC50 value: the concentration, after which 50% of cells retain their clonogenic capacity and form colonies. f) IC50 value is the concentration of the com-
pound where the activity of the enzyme is reduced by half.
−
1
1
°
expressed in Hertz. The NMR experiments (STD) were per- 310 C (dec.); IR (KBr) νmax 3430, 3345, 1688, 1315cm ; H-
ΙΙΙ
formed on a Bruker Avance
generation (800MHz) spec- NMR (500MHz, DMSO-d6) δ: 5.05 (s, 1H, ArH), 6.48 (brs,
trometer. Thin layer chromatography (TLC) was performed 4H, NH2), 10.85 (brs, 1H); electrospray ionization (ESI)-
on Whatman Sil G/UV254 silica gel plates with fluorescent MS: Observed (M+H+)=143. Calcd for C4H6N4S=142.18.
indicator, and the spots were visualized under 254 and 366nm Anal. Found: C, 33.80; H, 4.25; N, 39.36; S, 22.59. Calcd for
illumination. Mass analyses were performed with an Agilent C4H6N4S: C, 33.79; H, 4.25; N, 39.40; S, 22.55%.
6400 Series equipped with an electrospray ionization source
2-(Methylthio)pyrimidine-4,6-diamine (4) Yield 85%,
—
°
(capillary voltage at 4000V, nebulizing gas temperature at mp 170 172 C; IR (KBr) νmax 3455, 3312, 3189, 1649, 1552,
−
300 C, nebulizing gas flow at 12L/min). All the compounds 1477, 1295, 935, 817cm ; 1H-NMR (500MHz, CDCl3) δ:
1
°
were analyzed for C, H, N, and S on a Costech model 4010 2.51(s, 3H, CH3), 4.57 (brs, 4H, NH2), 5.30 (s, 1H, ArH);
and agreed with the proposed structures within 0.4% of the ESI-MS: Observed (M+H+)=157. Calcd for C5H8N4S=156.21.
±
theoretical values.
Synthesis of 4,6-Diamino-pyrimidine-2-thiol (3)10) To C4H6N4S: C, 38.44; H, 5.16; N, 35.87; S, 20.53%.
a freshly prepared solution of sodium ethoxide (884mg, 2-(Ethylthio)pyrimidine-4,6-diamine (5) Yield 83%,
Anal. Found: C, 38.36; H, 5.15; N, 35.90; S, 20.59. Calcd for
—
°
13mmol) in absolute ethanol, were added thiourea (1) mp 142 144 C; IR (KBr) νmax 3522, 3414, 3229, 1658,
−
(13mmol) and malononitrile (2) (13mmol). The mixture was 1638, 1589, 1481, 1323, 1280, 991, 831, 620cm ; 1H-NMR
refluxed for 3h, and the precipitate was then filtered off. The (500MHz, CDCl3) δ: 1.90 (t, 3H, J=7.06Hz, CH3), 2.89 (q,
solid was dissolved in water (10mL), followed by adjusting pH 2H, J=7.30Hz, CH2), 4.86 (brs, 4H, NH2), 5.16 (s, 1H, ArH);
1
around 7 38, and the resulted precipitate was filtered to give ESI-MS: Observed (M+H+)=171. Calcd for C6H10N4S=170.24.
—
the pure product (3) as a white solid (75%).
Anal. Found: C, 42.28; H, 5.93; N, 32.93; S, 18.86. Calcd for
Representative Procedure for the Preparation of Com- C4H6N4S: C, 42.33; H, 5.92; N, 32.91; S, 18.84%.
10)
—
pounds (4 25)
To a solution of 4,6-diaminopyrimidine-
2-(Propyllthio)pyrimidine-4,6-diamine (6) Yield 81%,
—
°
2-thiol (3) (1.4mmol) in methanol, was added alkyl halide mp 140 142 C; IR (KBr) νmax 3458, 3330, 3163, 1651, 1477,
−
1
1
(3.5mmol) under basic conditions (NaOH 0.1M, 14mL). The 1312, 1235, 991, 819, 652cm ; H-NMR (500MHz, DMSO-
mixture was then stirred for 18h at room temperature. After d6) δ: 1.00 (t, 3H, J=7.34Hz, CH3), 1.70 (sex, 2H, J=7.32Hz,
removing the solvent under reduced pressure, the residue was S-CH2CH2), 3.02 (t, 2H, J=7.20Hz, S-CH2CH2), 4.70 (brs,
washed by water and the precipitate was collected as a solid. 4H, NH2), 5.26 (s, 1H, ArH); ESI-MS: Observed (M+H+)=185.
All compounds were obtained in acceptable purity and no fur- Calcd for C7H12N4S=184.26. Anal. Found: C, 45.60; H, 6.55;
ther purification was needed.
N, 30.43; S, 17.42. Calcd for C4H6N4S: C, 45.63; H, 6.56; N,
4,6-Diamino-pyrimidine-2-thiol (3) Yield 75%, mp 30.41; S, 17.40%.

76
Vol. 60, No. 1
—
°
2-(Butylthio)pyrimidine-4,6-diamine (7) Yield 76%, (13) Yield 75%, mp 115 117 C ; IR (KBr) νmax 3462,
−
mp 91 93 C; IR (KBr) νmax 3442, 3319, 3163, 2940, 1645, 3367, 3154, 1643, 1548, 1304, 814, 713, 641cm ; 1H-NMR
1
—
°
−
1
1
1578, 1471, 1315, 984, 932, 818, 647cm ; H-NMR (500MHz, (400MHz, DMSO-d6) δ: 2.38 (s, 3H, CH3), 4.21 (s, 2H,
DMSO-d6) δ: 0.97 (t, 3H, J=7.35Hz, CH3), 1.49 (sex, 2H, S-CH2), 5.16 (s, 1H, pyrimidine-CH), 6.14 (brs, 4H, NH2),
—
J=7.47Hz, S-CH2CH2CH2), 1.71 (quin, 2H, J=7.23Hz, S- 7.03 (d, 1H, J=6.8Hz, ArH), 7.15 7.21 (m, 3H, ArH); ESI-
CH2CH2), 3.10 (t, 2H, J=7.33Hz, S-CH2), 4.59 (brs, 4H, MS: Observed (M+H+)=247. Calcd for C12H14N4S=246.33.
NH2), 5.29 (s, 1H, ArH); ESI-MS: Observed (M+H+)=199. Anal. Found: C, 58.46; H, 5.75; N, 22.75; S, 13.04. Calcd for
Calcd for C8H14N4S=198.29. Anal. Found: C, 48.40; H, 7.11; C12H14N4S: C, 58.51; H, 5.73; N, 22.74; S, 13.02%.
N, 28.28; S, 16.21. Calcd for C4H6N4S: C, 48.46; H, 7.12; N,
28.26; S, 16.17%.
2-(2-Nitrobenzylthio)pyrimidine-4,6-diamine (14) Yield
—
°
73%, mp 164 166 C; IR (KBr) νmax 3498, 3457, 3390, 1600,
−
1
2-(Pentylthio)pyrimidine-4,6-diamine (8) Yield 74%, 1514, 1350, 959, 751, 704cm ; 1H-NMR (400MHz, DMSO-d6)
—
°
mp 92 94 C; IR (KBr) νmax 3500, 3372, 3188, 2947, 1614, δ: 4.52 (s, 2H, S-CH2), 5.15 (s, 1H, pyrimidine-CH), 6.16 (brs,
−
1585, 1468, 1316, 1249, 985, 823cm ; 1H-NMR (500MHz, 4H, NH2), 7.50 (t, 1H, J=8.2Hz, ArH), 7.65 (t, 1H, J=7.6Hz,
1
—
DMSO-d6) δ: 0.93 (t, 3H, J=7.21Hz, CH3), 1.36 1.47 (m, 4H, ArH), 7.89 (dd, 1H, J1 =7.8Hz, J2 =0.8Hz, ArH), 7.99 (dd, 1H,
S-CH2CH2CH2CH2), 1.72 (quin, 2H, J=7.47Hz, S-CH2CH2), J1 =8.2Hz, J2 =1.2Hz, ArH); ESI-MS: Observed (M+H+)=278.
3.09 (t, 2H, J=7.34Hz, S-CH2), 4.64 (brs, 4H, NH2), 5.28 Calcd for C11H11N5O2S=277.3. Anal. Found: C, 47.68; H, 4.01;
(s, 1H, ArH); ESI-MS: Observed (M+H+)=213. Calcd for N, 25.25; S, 11.54. Calcd for C11H11N5O2S: C, 47.64; H, 4.00;
C9H16N4S=212.32. Anal. Found: C, 50.94; H, 7.60; N, 26.37; N, 25.26; S, 11.56%.
S, 15.09. Calcd for C4H6N4S: C, 50.91; H, 7.60; N, 26.39; S,
15.10%.
2-(2-Chlorobenzylthio)pyrimidine- 4,6 -diamine
—
°
(15) Yield 75%, mp 146 148 C; IR (KBr) νmax 3475, 3354,
2-(Hexylthio)pyrimidine-4,6-diamine (9) Yield 68%, 3147, 1642, 1574, 1456, 1240, 1052, 980, 931, 812, 774, 748,
−
mp 80 82 C; IR (KBr) νmax 3487, 3191, 2938, 2857, 1644, 674cm ; 1H-NMR (500MHz, DMSO-d6) δ: 4.33 (s, 2H,
1
—
°
−
1
1566, 1464, 1242, 936, 805, 606cm ; 1H-NMR (500MHz, S-CH2), 5.16 (s, 1H, pyrimidine-CH), 6.16 (brs, 4H, NH2),
—
—
—
—
DMSO-d6) δ: 0.93 (t, 3H, J=6.74Hz, CH3), 1.33 1.35 (m, 7.25 7.28 (m, 2H, ArH), 77.43 7.45 (m, 1H, ArH), 7.67
4H, S-CH2CH2CH2CH2CH2), 1.46 (quin, 2H, J=7.46Hz, 7.70 (m, 1H, ArH); ESI-MS: Observed (M+H+)=267.6. Calcd
S-CH2CH2CH2), 1.72 (quin, 2H, J=7.45Hz, S-CH2CH2), for C11H11ClN4S=266.75. Anal. Found: C, 49.50; H, 4.15; N,
3.08 (t, 2H, J=7.35Hz,S-CH2), 4.61 (brs, 4H, NH2), 5.28 21.06; S, 11.96. Calcd for C11H11ClN4S: C, 49.53; H, 4.16; N,
(s, 1H, ArH); ESI-MS: Observed (M+H+)=227. Calcd for 21.00; S, 12.02%.
C10H18N4S=226.34. Anal. Found: C, 52.98; H, 8.03; N, 24.77;
S, 14.22. Calcd for C4H6N4S: C, 53.06; H, 8.02; N, 24.75; S, (16) Yield 77%, mp 147 149 C; IR (KBr) νmax 3499, 3462,
2-(2-Fluorobenzylthio)pyrimidine- 4,6 -diamine
—
°
−
1
14.17%.
3388, 3298, 3148, 1647, 1463, 1305, 1227, 978, 812, 760cm ;
2-(Cyclobutylmethylthio)pyrimidine-4,6-diamine 1H-NMR (500MHz, CDCl3) δ: 4.41 (s, 2H, S-CH2), 4.63
—
°
—
(10) Yield 70%, mp 107 109 C; IR (KBr) νmax 3484, 3358, (brs, 4H, NH2), 5.29 (s, 1H, pyrimidine-CH), 7.03 7.11
−
1
3154, 2979, 1633, 1580, 1470, 1316, 1250, 991, 942, 820cm ; (m, 2H, ArH), 7.23 (d, 1H, J=6.20Hz, ArH), 7.55 (t, 1H,
H-NMR (500MHz, DMSO-d6) δ: 1.65 1.69 (m, 2H, CH2), J=7.52Hz, ArH); ESI-MS: Observed (M+H+)=251. Calcd for
1
—
—
—
1.77 1.83 (m, 2H, CH2), 1.99 2.05 (m, 2H, CH2), 2.54 C11H11FN4S=250.3. Anal. Found: C, 52.75; H, 4.41; N, 22.40;
(d, 1H, J=7.50Hz, CH), 3.06 (d, 2H, J=7.53Hz, S-CH2), S, 12.83. Calcd for C11H11FN4S: C, 52.78; H, 4.43; N, 22.38; S,
5.17 (s, 1H, ArH), 6.03 (brs, 4H, NH2); ESI-MS: Observed 12.81%.
(M+H+)=211. Calcd for C9H14N4S=210.3. Anal. Found: C,
2-(3-Fluorobenzylthio)pyrimidine- 4,6 -diamine
—
°
51.35; H, 6.70; N, 26.67; S, 15.28. Calcd for C4H6N4S: C, (17) Yield 75%, mp 132 134 C; IR (KBr) νmax 3500, 3468,
51.40; H, 6.71; N, 26.64; S, 15.25%. 3372, 3307, 3159, 1648, 1610, 1547, 1463, 1311, 1141, 934,
2-(2-Morpholinoethylthio)pyrimidine-4,6-diamine 816, 799cm ; H-NMR (400MHz, DMSO-d6) δ: 4.25 (s, 2H,
−
1
1
°
(11) Yield 65%, mp 270 C (dec.); IR (KBr) νmax 3400, 3168, S-CH2), 5.17 (s, 1H, pyrimidine-CH), 6.18 (brs, 4H, NH2),
−
2832, 1654, 1583, 1305, 1121, 987, 874, 820cm ; 1H-NMR 7.01 7.06 (m, 1H, ArH), 7.25 7.35 (m, 3H, ArH); ESI-
1
—
—
(400MHz, DMSO-d6) δ: 2.42 2.44 (m, 4H, CH2), 3.07 (t, 2H, MS: Observed (M+H+)=251. Calcd for C11H11FN4S=250.3.
—
J=7.60Hz, S-CH2-CH2), 3.33 (s, 2H, S-CH2-CH2), 3.57 (t, 4H, Anal. Found: C, 52.75; H, 4.41; N, 22.40; S, 12.83. Calcd for
J=4.80Hz, CH2), 5.13 (s, 1H, ArH), 6.06 (brs, 4H, NH2); ESI- C11H11FN4S: C, 52.78; H, 4.43; N, 22.38; S, 12.81%.
MS: Observed (M+H+)=256. Calcd for C10H17N5OS=255.34.
2-(4 -Fluorobenzylthio)pyrimidine- 4,6 -diamine
—
°
Anal. Found: C, 47.00; H, 6.69; N, 27.46; S, 12.57. Calcd for (18) Yield 69%, mp 176 178 C; IR (KBr) νmax 3510, 3476,
C4H6N4S: C, 47.04; H, 6.71; N, 27.43; S, 12.56%.
−
1
3401, 3317, 3172, 1647, 1550, 1470, 1315, 1163, 849, 819cm ;
2-(2-Methylbenzylthio)pyrimidine- 4,6 -diamine 1H-NMR (500MHz, DMSO-d6) δ: 4.22 (s, 2H, S-CH2), 5.15
—
°
—
(12) Yield 69%, mp 165 167 C ; IR (KBr) νmax 3478, (s, 1H, pyrimidine-CH), 6.13 (brs, 4H, NH2), 7.07 7.12
—
3354, 3146, 1649, 1547, 1468, 1304, 1245, 981, 932, 815, (m, 2H, ArH), 7.44 7.46 (m, 2H, ArH); ESI-MS: Observed
−
782, 688cm ; 1H-NMR (500MHz, DMSO-d6) δ: 2.33 (s, (M+H+)=251. Calcd for C11H11FN4S=250.3. Anal. Found: C,
1
3H, CH3), 4.25 (s, 2H, S-CH2), 5.16 (s, 1H, pyrimidine-CH), 52.75; H, 4.41; N, 22.40; S, 12.83. Calcd for C11H11FN4S: C,
—
6.12 (brs, 4H, NH2), 7.09 7.17 (m, 3H, ArH), 7.40 (d, 1H, 52.78; H, 4.43; N, 22.38; S, 12.81%.
J=7.18Hz, ArH); ESI-MS: Observed (M+H+)=247. Calcd for
2-(3-Methoxybenzylthio)pyrimidine-4,6-diamine
C12H14N4S=246.33. Anal. Found: C, 58.46; H, 5.75; N, 22.75; (19) Yield 70%, mp 118 120 C; IR (KBr) νmax 3459, 3359,
—
°
−
1
S, 13.04. Calcd for C12H14N4S: C, 58.51; H, 5.73; N, 22.74; S, 3155, 1663, 1402, 1149, 1052, 980, 930, 882, 725, 687cm ;
13.02%.
1H-NMR (500MHz, DMSO-d6) δ: 3.37 (s, 3H, CH3), 4.22
2-(3-Methylbenzylthio)pyrimidine- 4,6 -diamine (s, 2H, S-CH2), 5.16 (s, 1H, pyrimidine-CH), 6.13 (brs, 4H,

January 2012
77
—
NH2), 6.78 (dd, 1H, J1 =8.17Hz, J2 =2.41Hz, ArH), 6.96 1.21 1.97 (m, 10H, piperidin-CH2, S-CH2-CH2), 2.16 (s, 3H,
—
—
—
(d, 1H, J=7.56Hz, ArH), 7.00 (s, 1H, ArH), 7.19 (t, 1H, CH3), 2.72 2.75 (m, 1H, piperidin-CH), 2.82 2.88 (m, 1H,
J=7.76Hz, ArH); ESI-MS: Observed (M+H+)=263. Calcd for S-CH2), 2.97 3.03 (m, 1H, S-CH2), 5.14 (s, 1H, ArH), 6.00
C12H14N4OS=262.33. Anal. Found: C, 54.91; H, 5.37; N, 21.38; (brs, 4H, NH2); ESI-MS: Observed (M+H+)=268. Calcd for
S, 12.23. Calcd for C12H14N4OS: C, 54.94; H, 5.38; N, 21.36; C12H21N5S=267.39. Anal. Found: C, 53.88; H, 7.91; N, 26.21;
S, 12.22%. S, 12.00. Calcd for C12H21N5S: C, 53.90; H, 7.92; N, 26.19; S,
2-(4 - Cyanobenzylthio)pyrimidine- 4,6 -diamine 11.99%.
—
°
(20) Yield 65%, mp 200 202 C; IR (KBr) νmax 3484,
Enzyme Inhibition Assay The DHFR enzyme assay is
3361, 3296, 3136, 2220, 1626, 1565, 1543, 1470, 1240, 976, a sensitive and specific method to screen DHFR inhibitors.
−
1
1
878, 644cm ; H-NMR (400MHz, DMSO-d6) δ: 4.29 (s, 2H, The assay is based on the ability of dihydrofolate reductase to
S-CH2), 5.15 (s, 1H, pyrimidine-CH), 6.18 (brs, 4H, NH2), catalyze the reversible NADPH-dependent reduction of dihy-
7.63 (d, 2H, J=8.4Hz, ArH), 7.75 (d, 2H, J=8.4Hz, ArH); drofolic acid to tertrahydrofolic acid.
ESI-MS: Observed (M+H+)=258. Calcd for C12H11N5S=257.31.
Anal. Found: C, 56.04; H, 4.32; N, 27.21; S, 12.43. Calcd for
DHFR
DHF + NADPH+ H+
THF + NADP+
C12H11N5S: C, 56.01; H, 4.31; N, 27.22; S, 12.46%.
The reduction progress is monitored by the decrease in ab-
2-(3-(Trifluoromethyl)benzylthio)pyrimidine-4,6-di- sorbance at 340nm that occurs when NADPH is converted to
amine (21) Yield 65%, mp 146 148 C; IR (KBr) νmax NADP+.
3497, 3467, 3395, 3305, 3189, 1648, 1553, 1465, 1336, 1245, In this study, DHFR enzyme kit from Sigma-Aldrich was
—
°
−
1
1
°
1173, 1121, 916, 813, 705cm ; H-NMR (400MHz, DMSO- used. The spectrophotometer was set at 340nm, 22 C, and
d6) δ: 4.34 (s, 2H, S-CH2), 5.18 (s, 1H, pyrimidine-CH), kinetic program (reading every 15s for 5min). To a solution
—
—
6.19 (brs, 4H, NH2), 7.50 7.58 (m, 2H, ArH), 7.75 7.79 of assay buffer (1X) and human DHFR, was added NADPH
(m, 2H, ArH); ESI-MS: Observed (M+H+)=301. Calcd for and mixed well. The test compounds were dissolved in di-
C12H11F3N4S=300.3. Anal. Found: C, 47.96; H, 3.68; N, 18.68; methyl sulfoxide (DMSO) and added to the mixture in differ-
S, 10.69. Calcd for C12H11F3N4S: C, 47.99; H, 3.69; N, 18.66; ent concentrations. The final concentration of DMSO in each
S, 10.68%. experiment was 0.4%. The changes in absorbance at 340nm
2-(Phenylethylthio)pyrimidine-4,6-diamine (22) Yield were followed using the test compounds and methotrexate (as
—
°
71%, mp 154 156 C; IR (KBr) νmax 3503, 3415, 3382, 3323, a positive control). The activity under these conditions was
−
1
3143, 1657, 1629, 1582, 1471, 1316, 1251, 992, 819, 777cm ; linear for 5min. Results were obtained as % inhibition of en-
1H-NMR (500MHz, DMSO-d6) δ: 2.89 (t, 2H, J=8.32Hz, zymatic activity calculated using the following formula:
S-CH2-CH2), 3.17 (t, 2H, J=8.35Hz, S-CH2-CH2), 5.16 (s,
ΔOD / min (test)
ΔOD / min (blank)


% Inhibition = 1−
×100
—
1H, pyrimidine-CH), 6.08 (brs, 4H, NH2), 7.20 7.22 (m,




—
1H, ArH), 7.29 7.31 (m, 4H, ArH); ESI-MS: Observed
(M+H+)=247. Calcd for C12H14N4S=246.33. Anal. Found: C,
The % inhibition values were plotted versus compounds
58.48; H, 5.74; N, 22.76; S, 13.02. Calcd for C12H14N4S: C, concentrations. The 50% inhibitory concentration (IC50) of
58.51; H, 5.73; N, 22.74; S, 13.02%. each compound was obtained using the GraphPad Prism 3
2-(3-Phenylpropylthio)pyrimidine- 4,6 -diamine program.
—
°
(23) Yield 65%, mp 118 120 C; IR (KBr) νmax 3473, 3354,
NMR Experiment (STD) Samples were prepared by
−
1
3140, 1638, 1589, 1466, 1286, 993, 827, 762cm ; 1H-NMR dissolving DHFR and a quantity of ligands (12, 15) to give
(400MHz, DMSO-d6) δ: 1.90 (quin, 2H, J=7.2Hz, S-CH2- a protein:ligand mole ratio of 1:100 in deuterated sodium
CH2-CH2), 2.69 (t, 2H, J=7.2Hz, S-CH2-CH2-CH2), 2.96 (t, phosphate buffer, pH 7.3. Prior to use, DHFR was dialyzed ex-
2H, J=7.2Hz, S-CH2-CH2-CH2), 5.14 (s, 1H, pyrimidine-CH), tensively and the ligand was dissolved into PBS buffer. All the
—
—
6.05 (brs, 4H, NH2), 7.16 7.22 (m, 3H, ArH), 7.27 7.31 NMR experiments were conducted on Bruker Avance DRX
(m, 2H, ArH); ESI-MS: Observed (M+H+)=261. Calcd for 800-MHz spectrometer using 5-mm inverse triple resonance
C13H16N4S=260.36. Anal. Found: C, 59.95; H, 6.20; N, 21.53; (15N/13C/1H) probes equipped with triple axis actively shielded
S, 12.32. Calcd for C13H16N4S: C, 59.97; H, 6.19; N, 21.52; S, gradient. STD spectra were collected with 16K data points to
12.32%.
2-((Naphthalen-1-yl)methylthio)pyrimidine-4,6-diamine
cover a sweep width of 10ppm.
The protein was saturated on its methyl signals around
—
°
(24) Yield 55%, mp 190 192 C; IR (KBr) νmax 3520, 3452, 0.9ppm at a frequency of 7000Hz and off-resonance at
−
1
3412, 3327, 3146, 1657, 1619, 1474, 1315, 1297, 800cm ; 20000Hz with a cascade of 40 selective Gaussian-shaped
1H-NMR (400MHz, DMSO-d6) δ: 4.78 (s, 2H, S-CH2), 5.23 pulses of 50ms duration with a 100µs delay between each
—
(s, 1H, pyrimidine-CH), 6.24 (brs, 4H, NH2), 7.41 7.53 pulse, resulting in a total saturation time of 2s.
(m, 3H, ArH), 7.68 (d, 1H, J=6.8Hz, ArH), 7.83 (d, 1H,
STD NMR spectra were recorded at 303K and 298K. The
J=8.00Hz, ArH), 7.94 (d, 1H, J=7.8Hz, ArH), 8.14 (d, 1H, watergate pulse train was used to suppress the H2O signal.
J=8.00Hz, ArH); ESI-MS: Observed (M+H+)=283. Calcd for The spectra were processed using Bruker xwinnmr. A cosine
C15H14N4S=282.36. Anal. Found: C, 63.83; H, 5.01; N, 19.82; function prior to zero-filling by a factor of 2 was applied
S, 11.34. Calcd for C15H14N4S: C, 63.80; H, 5.00; N, 19.84; S, before fourier transform. STD spectrum was obtained by sub-
11.36%. traction of saturated spectrum from the reference spectrum
2-(2-(1-Methylpiperidin-2-yl)ethylthio)pyrimidine-4,6- and STD intensity of individual signal was measured relative
—
°
diamine (25) Yield 65%, mp 165 167 C; IR (KBr) νmax to the corresponding signal intensity in the reference spec-
3468, 3416, 3302, 3159, 2937, 2788, 1630, 1562, 1460, 1297, trum.
−
1235, 972, 811cm ; 1H-NMR (500MHz, DMSO-d6) δ:
Docking Study The crystal structure of human DHFR
1

78
Vol. 60, No. 1
complexed with COP (N-(4-carboxy-4-{4-[(2,4-diamino- that 4,6-diaminopyrimidine derivatives synthesized in the pre-
pteridin-6-ylmethyl)-amino]-benzoylamino}-butyl)-phthalamic sent study which show acceptable DHFR inhibition and their
1
acid, and NADPH (PDB ID code 1OHJ) was downloaded binding to the enzyme which is confirmed by STD H-NMR
from protein data bank and the surrounding important resi- studies, could be concluded that 4,6-diaminopyrimidine could
dues such as Ile 7, Glu 30, Leu 22, Phe 31, Arg 28, Arg 32, be considered as new scaffold for design and synthesis of
Glu 35, Asn 64, Arg 70, Val 115, Tyr 121, Tyr 136 were rec- novel DHFR inhibitors. Lipophilic thioether side chains could
ognized and the native ligand was extracted to leave a cavity. help to improve the ligand-receptor binding in these group
Thereafter, the docking simulations were carried out with and of compounds. Anti-proliferative activity of the compounds
without cofactor NADPH and water molecules, to elucidate were also determined against tumor cell lines. Some of the
the role of NADPH and water molecules for the binding of compounds, despite their high IC50 against DHFR enzyme,
4,6-diamionpyrimidine derivatives. All compounds were showed good anti-proliferative activity which could be at-
drawn and optimized using Hyperchem 8.0.3 software. The tributed to the other possible mechanisms of anti-proliferative
protein and ligands were saved in pdbqs and pdbqt format activity rather than DHFR inhibition.
respectively. Docking was performed as in the AutoDock
3.05 manual and the results were visualized using AutoDock
Acknowledgement The authors would like to express
tools. Binding affinities were calculated and the highly ranked their thanks to the research deputy of Shahid Beheshti Univer-
compounds were selected and docking was repeated for these sity of Medical Sciences and CICbioGUNE Research Center
selected compounds to confirm their affinity.
for financial support of this research.
The grid maps representing the native ligand in the actual
docking target site were calculated. The grid box dimensions
were chosen to be sufficiently large to include all the previ-
ously mentioned residues.
References
“
”
1) Jackson R. C., Antifolate Drugs in Cancer Therapy, ed. by Jack-
—
man A. L., Humana Press, Totowa, 1999, pp. 1 12.
2) Berman E. M., Werbel L. M., J. Med. Chem., 34, 479 485 (1991).
—
Antiproliferative Activity (Clonogenic Assay) Four
cell lines consisting of HepG2 (human hepatocellular liver
carcinoma), MCF-7 (human breast adenocarcinoma), SKHep1
(human liver adenocarcinoma), and Hela (human cervical
carcinoma) which had been verified to be negative for my-
coplasma contamination were used in this experiment. To
apply the method of clonogenic assay,16) cells were plated in
6-well plates (200 cells/well) for 24h before treatment with
the test compounds to allow attachment of the cells to wells.
Seven different concentrations of each compound, methotrex-
ate (as reference), and DMSO 0.5% (applied solvent to dis-
solve the compound) were then added to the monolayer cells
“
”
3) MacKenzie R. E., Folates and Pterins, 2nd ed., Vol. 1, ed. by
Blakley R. L., Benkovic S. J., Wiley-Interscience, New York, 1984,
—
pp. 255 306.
“
”
4) Suckling C. J., Enzyme Chemistry, Chapman and Hall Ltd., New
York, 1984, p. 121.
“
”
5) Peters G. J., Kohne C. H., Antifolate Drugs in Cancer Therapy,
ed. by Jackson A. L., 1999, pp. 101 145.
6) Jackman A. L., Boyle F. T., Harrap K. R., Invest. New Drugs, 14,
—
—
305 316 (1996).
—
7) Takimoto C. H., Semin Oncol., S18-40 S18-51 (1997).
8) Jordell D. I., Bowman A., Rye R., Byrne B., Boddy A., Rafi I.,
Taylor G. A., Johnston A., Clendeninn N. J., Br. J. Cancer, 79,
—
915 920 (1999).
°
in triplicates. The plates were incubated for 10d at 37 C in
—
9) Yamini L., Vijjulatha M., E-Journal of Chemistry, 5, 263 270
atmosphere of 5% CO2. The media were removed after 10d
and the formed colonies were stained with a solution of 0.5%
crystal violet in ethanol for 10min and the number of colonies
containing more than 50 cells was counted under microscope.
The relation between the number of the colonies (as a percent-
(2008).
10) Crepaldi P., Cacciari B., Bonache M. C., Spalluto G., Varani K. P.
A., Borea P. A., Kügelgen I., Hoffmann K., Pugliano M., Razzari
C., Cattaneo M., Bioorg. Med. Chem., 17, 4612 4621 (2009).
11) Krishnan V. V. Current, Anal. Chem., 2005, 1 49 (2005).
—
—
age of the control containing DMSO 0.5%) and the concentra- 12) Cody V., Galitsky N., Luft J. R., Pangborn W., Rosowsky A., Blak-
—
ley R. L., Biochemistry, 36, 13897 13903 (1997).
tions of each compound was plotted to get survival curve of
the cell lines and EC50 values were calculated.
“
”
13) Rosowsky A., Progress in Medicinal Chemistry, ed. by Ellis G.
P., West G. B., Elsevier Science Publishers, Amsterdam, 1989, pp.
—
1
252.
Conclusion
14) Gangjee A., Elzein E., Kothare M., Vasudevan A., Curr. Pharm.
Des., 2, 263–280 (1996).
15) Gangjee A., Yu J., McGuire J. J., Cody V., Galitsky N., Kisliuk R.
A series of 4,6-diaminopyrimidine derivatives bearing
various alkyl side chains at position 2 of the ring via a sulfur
atom were tested for their DHFR inhibitory activity. The com-
pounds with the lowest IC50 values demonstrated high affinity
to human DHFR enzyme in STD studies. In light of the fact
—
L., Queener S. F., J. Med. Chem., 43, 3837 3851 (2000).
16) Franken N. A. P., Rodermond H. M., Stap J., Haveman J., van Bree
—
C., Nat. Protoc., 1, 2315 2319 (2006).