Synthesis and cytotoxic activity of some new 2,6-substituted purines

Article abstract of DOI:10.3390/molecules16075840

A seriesof twenty four acyclic unsaturated 2,6-substututed purines 5a-20b were synthesized. These compounds were evaluated for cytotoxic activity against NCI-60 DTP human tumor cell line screen at 10?Mconcentration. N ₉-[(Z)-4′-chloro-2′-butenyl-1′-yl]-2, 6-dichloropurine(5a), N₉-[4′-chloro-2′-butynyl-1′- yl]-2,6-dichloropurine(10a), N₉-[(E)-2′,3′-dibromo- 4′-chloro-2′-butenyl-1′-yl]-6-methoxypurine(14)and N ₉-[4′-chloro-2′-butynyl-1′-yl]-6-(4-methoxyphenyl) -purine(19)exhibited highly potent cytotoxic activity with GI₅₀ values in the 1-5 μM range for most human tumor cell lines. Other compounds exhibited moderate activity.

Full text of DOI:10.3390/molecules16075840

Molecules 2011, 16, 5840-5860; doi:10.3390/molecules16075840
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Cytotoxic Activity of Some New
2,6-Substituted Purines
Nageswara Rao Kode and Shashikant Phadtare *
Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana,
1 Drexel Drive, New Orleans, LA 70125, USA
* Author to whom correspondence should be addressed; E-Mail: sphadtar@xula.edu;
Tel.: +1-504-520-5378; Fax: +1-504-520-7954.
Received: 20 June 2011 / Accepted: 29 June 2011 / Published: 11 July 2011
Abstract: A seriesof twenty four acyclic unsaturated 2,6-substututed purines 5a-20b were
synthesized. These compounds were evaluated for cytotoxic activity against NCI-60 DTP
human tumor cell line screen at 10µMconcentration. N9-[(Z)-4'-chloro-2'-butenyl-1'-yl]-
2,6-dichloropurine(5a), N9-[4'-chloro-2'-butynyl-1'-yl]-2,6-dichloropurine(10a), N9-[(E)-
2',3'-dibromo-4'-chloro-2'-butenyl-1'-yl]-6-methoxypurine(14)and N9-[4'-chloro-2'-butynyl-
1'-yl]-6-(4-methoxyphenyl)-purine(19)exhibited highly potent cytotoxic activity with GI50
values in the 1–5 µM range for most human tumor cell lines. Other compounds exhibited
moderate activity.
Keywords: Suzuki-Miyaura cross coupling reaction; pyridiniumtribromide bromination;
2,6-substituted purines; cytotoxic activity
1. Introduction
According to WHO report on cancer about 7.6 million people died in the year 2005 and the number
is expected to raise to 9 million by the year 2015 and 11.5 million by 2030 [1]. Hence, development of
new potent and selective anticancer agents has become one of most intensely pursued goals in drug
development around the world. Neplanocin A, (1, Figure 1) is considered a carbocyclic analogue of a
natural nucleoside and has shown potent antitumor and antiviral properties [2-4]. As a part of our
research program on the synthesis of anti-cancer agents, we have synthesized some aromatic
neplanocin-A analogues like 3a-3b, 4a-c [5-7].

Molecules 2011, 16
5841
The N9-hydroxymethyl analogues of adenine, guanine and 2,6-diaminopurine related to 3a-3b did
not exhibit any anticancer activity, however, their N9-chloromethyl arylpurine intermediates, related to
4a-4c (Figure 1), were found to be potent in vitro growth inhibitors of several human tumor cell lines.
These results prompted us to consider purines with an unsaturated N9-linker that has been terminated
with a chloromethyl group.
Figure 1. Neplanocin A and aromatic neplanocin-A analogues.
NH2
O
7
N
6
5
N
N
HN
N
1
8
2
N
9
O
N
N
3
4
NH2
H
O
H
O
c c ov r
A
l
i
2
y
H
O
H
O
e anoc n
N
l
i
A 1
p
R2
R2
N
N
N
N
N
N
N
a
R1
N
R1
H
O
l
C
=
=
4
R1 H R
NH2
=
l
C
,
a
3
=
=
=
NH2
2
R1 H R
3b R1 NH2
,
2
=
4b R1 NH2
R
,
=
OH
2
R
,
2
c
4
=
=
R1 R2 l
C
In the synthesis of anti-cancer purine compounds, many times either the purine base is modified or
the sugar moiety is modified or replaced with a non-sugar linker or sometimes all these changes have
been done by researchers simultaneously in an attempt to make an active compound. We have
followed a very similar path in the present work.
Purine base selection: 2,6-Dichloropurine is selected where the chlorines are expected to serve as
powerful electron withdrawing centers on the purine ring. 2-Chloro-6-methoxypurine is expected to
serve a double role, with electron withdrawing and electron donating centers on the purine ring. The 6-
methoxy group was selected for its electron donating nature to the purine ring. 6-(4-Methoxy)phenyl-
and 6-(4-fluoro)phenyl-substituted purines were selected to significantly alter the purine base
properties and to improve the lipophilicity. It is interesting to note that purines with those substitution
patterns were also reported to elicit wide range of anti-viral and anti-cancer activities. 6-
Methoxypurine arabinoside was reported as a potent inhibitor of Varicella-Zoster virus [8]. 6-Methoxy
group-containing Nelarabine and the 2-chloro group-containing compound Clofarabine elicit anti-
cancer activities [9]. Further, 6-(4-methoxphenyl)purine and 6-(4-fluorophenyl)purine ribonucleosides
were reported to elicit significant cytostatic activity [10].
Linker selection: We chose linkers like cis-1,4-dichlorobutene, trans-1,4-dichlorobutene and 1,4-
dichlorobutyne. All these linkers are acyclic five carbon length open chain liners analogous to the
linker of acyclovir, with some degree of unsaturation. All these liners are common for each of the
above purine bases selected, like 2,6-dichloropurine, 6-methoxyurine, 2-chloro-6-methoxypurine, 6-(4-
methoxyphenyl) purine and 6-(4-fluorophenyl)purine. Reaction of each of these purine bases with

Molecules 2011, 16
5842
cis-a 1,4-dichlorobutene linker furnishes N9 substituted purines with methylchloromethyl-cis-butene
units, e.g., compounds 5a, 6, 7, 15a and 16a. Reaction with the trans-1,4-dichlorobutene linker
furnishes N9 substituted purines with methylchloromethyl-trans-butene units, e.g., compounds 8a, 17
and 18a. Reactionwith1,4-dichlorobutyne is expected to furnish N9 substituted purines with
methylchloro-methyl-butyne moieties, e.g. compounds 9a, 10a, 11, 12, 19 and 20a. Compounds 13
and 14 represent vinylic dibromides, a new class of purines, which were also synthesized in this work
to assess their cytotoxic activity. This plan gives an opportunity for us to assess the cytotoxicity for a
group of purine compounds (Figure 2) and to understand how the activity is changing for a given
linker with a change on the substitution pattern on the purine ring.
Figure 2. Purines with acyclic unsaturated linkers.
R2
R2
N
N
N
N
N
N
N
H
H
H
R1
N
R1
H
l
l
C
a
8
= =
R1 R2 Cl
a
=
=
=
R1 R2 l
C
5
6
7
C
=
OCH3
=
OC
R1 H R
,
2
=
R1
l R
H
C
,
2
3
R2
R2
N
N
N
N
r
B
N
N
R1
N
R1
N
=
=
=
l
C
l
C
a
9
=
H R2 Cl
,
r
B
R1
=
=
=
=
13 R1 H R
14 R1 H R
,
l
C
H
OC
,
2
2
a
=
10 R1 R2 l
C
3
=
OC
11 R1 H R
H
,
2
3
=
=
OC
12 R1
R
l R
H
C
,
2
3
R
13
12
14
15
11
R
10
6
5
7
N
N
1
N
8
N
N
N
2'
2
4'
9
N
3
4
H
l
C
N
N
N
H
l
1'
N
3'
H
H
=
19
20
R
H
OC
3
a
a
=
=
N
R
15
16
R
R
H
OC
F
3
=
R F
C
a
l
C
=
17
H
OC
3
a
18
=
F
R
Here, we have focused primarily on the synthesis of N9 substituted purines because they were found
to be more active when compared to N7 isomers at our laboratory. Furthermore the N7 isomers are
expected to be minor products in the synthesis.

Molecules 2011, 16
5843
2. Results and Discussion
2.1. Chemistry
The N9-alkylated compounds 5a-20b were prepared by the direct alkylation approach on the
appropriately substituted purine bases in presence of K2CO3 in dimethyl formamide (DMF) medium
(Schemes 1 and 2). A 1-3 fold excess of the alkylating agent and anhydrous potassium carbonate were
employed for one equivalent of the purine base taken to isolate N9-purine isomers as the major product
in moderate to good yields.
Scheme 1. Synthesis of N9-alkylated compounds.
R2
R2
N
N
N
i
N
N
H
N
H
R1
N
R1
N
:
:
=
=
=
l R
OC
2
22 R1
H
C
,
:
=
=
,
=
21 R1 R2
l
=
C
3
=
24 R1 H R
,
H
OC
:
23 R1 H R
l
C
2
3
2
R2
N
R2
N
N
N
ii iii
,
N
H
N
H
R1
N
R1
N
H
l
a
+
5
R2
N
:
=
=
21 R1 R2 l
C
N
N
C
N
H
R1
H
5b
N
R1
N
N
N
R2
v
,
ii i
v
ii i
,
a +
10
a +
8
10b
8b
21
22
24
v
ii
ii
,
ii iii
,
12
11
7
6
v
ii iii
,
,
ea en s an con ons:
R
t
d
diti
g
=
a
re ux
fl
rs.
18 h
,
i
N
H
OC
H
H
C
3O
,
,
3
=
=
-
rs.
ii
iii
K
C
DMF RT 1 24 h
2CO3/
s-
,
-
c
oro- - u ene
i 1 4 di hl 2 b t
,
v = rans-
-
c
oro- - u ene
i
T
1 4 di hl 2 b t
,
v =
-
c
oro- - u ne
1 4 di hl 2 b t
,
y

Molecules 2011, 16
Scheme 2. Synthesis of 6-(4'-methoxyphenyl) and 6-(4'-fluorophenyl)-purines.
5844
R
l
C
l
C
N
N
v
i
N
i
N
ii
N
N
N
N
iii
N
H
N
N
N
23
25
O
=
=
26 R
27 R
H
OC
3
F
O
R
R
R
R
+
N
N
N
N
N
N
N
N
v v
,
i
N
N
N
H
N
N
N
H
C C
H H
N
N
H
l
:
:
=
=
28 R
29 R
H
3
OC
F
a:
15
=
=
R
R
H
OC
3
:
=
=
15b R
H
OC
F
3
a:
16
C
F
:
16b R
v v
ii
,
v v
iii
,
28
29
19
20
17
v v
,
v v
,
ii
iii
a +
20b
a +
18
18b
ea en s an con ons:
R
t
d
diti
g
=
=
-
rs.
l RT 24 65 h
,
i
PPT DHP
H
2C 2
S
C
,
,
,
a
N
ii
Pd PPh
K
2CO3/ 2CO3
,
)
(
3 4
OC
rs.
8 24 h
,
o une
-
-
T l
DME 80 100
/
,
= -
-
-
- -
-
O
(
iii
4
H Ph B
OC
H
R 4 F Ph B
H
)
2
O
(
2 O
)
3
v = ce c or e-
rs.
H 24 h
3O
,
-
i
A
K
C
t l hl id
H
C
y
v
v
v
v
=
=
=
rs.
DMF RT 1 24 h
2CO3
,
-
,
,
s-
c oro- - u ene
2 b t
i
i 1 4 Di hl
,
rans- - c oro- - u ene
T
1 4 di hl 2 b t
ii
,
=
- c oro- - u ne
iii 1 4 di hl
2 b t
,
y
A ten equivalent excess is not required as previously reported [12]. Minor dimeric products (Figure 3)
have been isolated whenever formed during the synthesis for each linker. Increasing the reaction time
and the molar ratio of the potassium carbonate favors the higher yields of the dimeric products. The
UV maxima for N7 isomers were 10–15 nm higher (275–320 nm) than the N9-isomers
(265–310 nm) [13].
Minor modifications were made to the Suzuki-Miyaura cross coupling procedure [10,14]. Reaction
of appropriate phenylboronic acid with 9-(tetrahydropyran-2-yl)-6-chloropurine under Suzuki-Miyaura
cross coupling methodology afforded 6-(4'-methoxyphenyl), 6-(4'-fluorophenyl)-purines (Scheme 2).
The reported procedure of pyridiniumtribromide bromination of the acetyleneic compounds [11] was
adopted with minor modifications to furnish the vinylicdibromides13, 14 (scheme 3).

Molecules 2011, 16
5845
Figure 3. Dimers.
R2
R2
N
R2
N
N
N
H
H
N
N
N
N
N
N
R1
N
N
R1
N
N
R1
C C
H H
R1
N
N
R2
=
=
=
8b R1 R2 Cl
=
=
l
C
5b R1 R2
= -
18b R1 H R 4 F Ph
,
2
=
= -
4 H Ph
OC
15b R1 H R
,
2
3
=
= -
16b R1 H R 4 F Ph
,
2
F
F
13
13''
14''
12
11
14
15
12''
11''
15''
10
5
10''
R2
R2
6
6''
7
7
''
5''
N
N
N
1
2
N
1''
2''
N
N
N
N
N
N
N
8
8
''
N
9
9''
N
3
N
4
4''
R1
N
N
R1
C C
3''
C C
1' 2' 3' 4'
=
=
Cl
=
l
C
9b R1 H R
,
2
20b
=
10b R1 R2
Scheme 3. Synthesis of the vinylicdibromides13, 14.
R2
R2
r
n um r rom e
P idi i id
t ib
y
N
N
N
N
N
N
r
B
H
l
C
2C 2
R1
N
R1
N
l
C
l
C
r
B
a:
:
=
=
Cl
R1 H R
2
:
:
=
=
=
=
9
,
13 R1 H R
l
C
,
2
2
=
=
11 R1 H R
OC
2
H
,
14 R1 H R
H
3
OC
,
3
Acetyl chloride-mediated THP protection and deprotection of hydroxyl functional groups on a wide
range of aliphatic and aromatic systems has been reported [15]. We have extended the concept to
6-chloropurine and found acetyl chloride to be a versatile clean inexpensive deprotective agent in
methanol medium for the THP removal. No side products are found on the procedure we described,
although Dowex 50W X 8 works [10]. During the protection of the NH of 6-chloropurine, acetyl
chloride was found to form one side product, possibly an N-acetyl derivative, still the % yield was
about 70 after column purification. The pyridinium-p-toluenesulfonate (PPTS) catalyzed THP
protection of 6-chloropurine was found to be relatively very clean, no major side products formed and
the yield was about 90%. Hence we chose PPTS catalyst for the THP protection of 6-chloropurine.
Reaction of sodium methoxide with 6-chloropurine and 2,6-dichloropurine in methanol medium
furnished 6-methoxypurine and 2-chloro-6-methoxypurine.respectively. Further reaction of these
purine bases with various alkenyl and akynyl linkers as explained above furnished the target
1
13
compounds 5a-20b. The structures of all the purines 5a-20b were confirmed by H-NMR, C-NMR,
LC-MS and satisfactory elemental (C, H, N) analysis within ± 0.4% of theoretical values.

Molecules 2011, 16
5846
2.2. Cytotoxicity
Compounds 5a, 5b,6, 7, 8a, 8b, 9a, 9b, 10a, 10b, 11, 12, 13, 14, 15a, 15b, 16a, 16b, 17, 19, 20 and
20b were initially tested at 10 µM concentration (Table 1).
Table 1.Cytotoxicity Data.
Cell Line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
5a
5b
8b
10a
10b
14
17
19
–45
–71
–63
–69
–53
–76
–73
–70
–25
–55
–61
–48
18
–76
–70
–67
–63
–76
–76
–62
–58
–13
1.0
–39
47
63
65
67
--
19
49
58
5.0
35
--
–3.0
17
8
MOLT-4
RPMI-8226
SR
81
19
–17
–14
–57
–53
–25
8.0
52
--
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
–76
–91
–72
–93
–74
–78
–97
–63
–85
–7.0
–68
23
72
87
94
–13
94
95
95
84
28
–77
–72
–82
–92
–81
–70
–100
–66
–89
9.0
–39
62
9.0
114
16
81
60
115
77
9.0
57
59
92
68
49
108
99
HOP-62
HOP-92
–34
–59
9.0
21
–88
6.0
39
--
38
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
--
89
15
64
47
79
100
67
4.0
–77
13
–41
–72
–71
49
–65
–87
–80
–79
–61
–86
–87
–41
--
63
--
–77
–85
–100
–90
–70
–73
–89
–27
--
–82
90
87
91
46
61
85
85
61
80
85
52
50
94
73
49
–71
–44
–66
–69
–52
33
50
48
79
45
–35
–66
–44
–45
–45
–82
32
HT-29
–57
–6
KM-12
SW-620
–74
CNS Cancer
SF-268
–83
–92
–90
–89
–92
–72
–2.0
–33
–53
10
62
60
47
75
65
57
–74
–84
–93
–99
–97
–96
–38
6.0
–66
12
12
97
12
90
47
–72
88
63
95
92
72
94
29
SF-295
109
76
SF-539
SNB-19
88
SNB-75
–26
–56
8.0
–76
128
64
U251
Melanoma
LOX IMVI
MALME-3M
M14
–83
–73
–85
–90
–77
–96
–80
–72
–33
–67
24
34
–89
–70
–93
–86
–76
–98
–83
–75
8.0
4.0
65
–87
–7
54
62
71
58
69
98
–32
20
70
40
53
87
111
101
98
–25
4
MDA-MB-435
SK-MEL-2
SK-MEL-28
96
52
–19
99
–26

Molecules 2011, 16
5847
Table 1. Cont.
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-Res
SK-OV-3
Renal cancer
786-0
–91
–90
–96
–82
--
69
--
–96
–67
--
54
86
81
77
62
59
51
–91
–92
–41
57
–73
79
–12
–96
–97
–86
–85
–83
–56
–98
–82
–99
–89
–9.0
–57
30
18
–93
–93
–88
–86
–84
–57
–99
–75
–91
–71
–3.0
–35
60
12
94
46
53
51
97
65
56
86
61
–94
–47
62
78
95
53
112
8.0
99
114
67
1
–40
148
77
33
103
88
111
–92
–92
–89
–95
--
–9.0
–18
–94
–97
--
93
90
90
–60
--
–99
–89
–99
–88
--
8.0
--
8.0
95
15
96
70
11
51
57
70
68
59
71
8.0
84
86
60
36
56
53
62
30
46
70
–87
A498
ACHN
–53
–96
3.0
--
CAKI-1
RXF 393
SN12C
–84
–86
–95
–11
–45
--
96
80
6.0
–87
–93
–98
TK-10
–32
--
UO-31
Prostate cancer
PC-3
–93
–99
–58
–98
67
44
–98
–72
–87
46
37
84
82
57
88
DU-145
–100
Breast cancer
MCF-7
–49
–90
–43
–94
–54
–72
–78
–68
–42
--
38
96
–52
–89
–44
–93
–62
–61
–23
–53
--
0.0
42
24
70
1.0
17
40
36
70
107
50
53
21
MDA-MB-231/ATCC
HS578T
89
46
131
54
BT549
–33
38
--
T-47D
–44
--
–21
--
68
MDA-MB-468
–4.0
* Where the number 100 = control growth, 0 = 100% inhibition, –100 = total cell kill. Compounds
5, 6, 7a, 10, 11, 14a-b, 15a-b, 17a-b, 19a-b were not active at 10 µM concentration. * Growth %
for one dose testing at 10 µM concentration.
Out of these compounds 6, 7, 8a, 11, 12, 15a, 15b, 16a, 16b, 20a, 20b exhibited growth %in the
range of 70–100 plus and hence may be considered inactive. Table 1 summarizes the single dose
10 µM test results for the active compounds. Compounds 5a, 5b, 8b, 10a, 10b and14 elicited
significant cytotoxicity on almost all the cell lines such as leukemia, non-small cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer. Under
the same single dose testing, compound 17 elicited cytotoxicity to MDA-MB-468 breast cancer cell
line while compound 19 elicited cytotoxicity to LOXIMVI melanoma and UO-32 renal cancer cell
lines. Table2 represent the five dose testing results GI50 and LC50for compounds 5a, 10a, 14 and 19.
Compound 5a was found very active with GI50 values 1–2 µM for almost all the cell lines. Compound
10a was also found very active with GI50values under 2 µM for many cell lines. For leukemia
HL-60TB and melanoma UACC-62 the GI50 values are 3.5 and 3.7 µM respectively. Compound 14

Molecules 2011, 16
5848
displayed impressive activity, with GI50 values of 2–3 µM for leukemia, melanoma, renal cancer and
breast cancer. It also elicited significant activity on non-small cell lung cancer, colon cancer and CNS
cancer. Compound 19 exhibited striking activity, with GI50 values of 2–4 µM for breast cancer and
2–8 µM for leukemia, colon, renal and prostate cell lines. Replacing the chlorine at 6-position in
compound 5a with a methoxy group results in compound 11. Similarly replacing6-chlorine in
compound 5a with a methoxy group results in compound 12. These changes resulted in a total loss of
cytotoxicity. When the N-9 cis-butene stereochemistry in 5a is changed to a trans form as in 8a also
resulted in the loss of activity, although the corresponding dimer 8b with a trans stereochemistry
elicited good cytotoxicity.
On the 6-phenyl substituted compounds, only 19 elicited good activity and all other compounds
were inactive. The reported procedure [11] was employed to transform the triple bond compounds 9a
and 11 in to the corresponding vinylicdibromides13 and 14(Scheme2).Indeed one of the vinylic
dibromide with a 6-methoxy substituent,14, was found very active for leukemia, melanoma, renal
cancer, breast cancer (GI 50 value 2–3 µM) and significant activity on all other cancers. The other
vinylic dibromide with a 6-chlorine group 13 did not elicit any cytotoxicity. Compounds 18a and 18b
were not tested.
Table 2. GI50 and LC50 data. Units: µM.
Compound
5a
10a
14
19
Cell Line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
GI 50
LC 50
GI 50
LC 50
GI 50
LC 50
GI 50
LC 50
0.19
0.33
0.23
0.23
0.64
--
0.78
>100
>100
>100
>100
--
0.21
3.50
0.22
0.50
0.23
--
0.93
4.89
4.89
>100
>100
--
2.73
2.03
3.29
2.91
3.19
2.73
>100
>100
>100
>100
>100
>100
3.84
3.06
8.06
2.68
3.46
5.24
>100
>100
>100
>100
>100
>100
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
0.37
1.54
0.19
0.20
1.30
0.22
1.64
0.20
1.01
>100
7.18
7.18
3.41
5.10
5.10
5.68
0.67
7.51
0.54
2.01
0.23
0.73
1.61
0.19
1.63
0.19
2.41
>100
8.83
0.94
5.70
5.70
0.84
5.59
0.78
29.0
10.80
16.30
3.90
4.08
2.82
3.16
12.40
--
62.50
55.10
37.50
45.30
30.70
39.40
50.20
--
7.40
12.80
1.79
2.75
4.45
7.83
12.50
3.27
3.09
>100
68.70
61.10
49.80
>100
50.50
52.30
45.50
55.10
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
0.88
5.27
0.20
0.20
0.18
0.17
0.26
0.22
0.20
0.69
0.71
0.71
0.65
>100
0.80
0.73
0.19
0.27
0.20
0.18
0.23
0.36
0.22
0.67
0.72
0.76
0.73
>100
3.73
3.73
1.73
17.20
1.73
1.84
1.63
3.54
--
5.83
56.90
7.05
9.39
8.41
37.60
--
3.51
17.60
3.50
4.06
7.12
4.84
2.99
41.60
57.20
41.30
54.90
71.70
45.20
42.40
HT-29
KM-12
SW-620

Molecules 2011, 16
5849
Table 2. Cont.
CNS Cancer
SF-268
0.20
1.29
0.12
0.91
0.18
0.18
0.62
6.97
0.53
4.72
0.68
0.57
0.19
1.60
1.03
0.57
0.20
0.18
0.67
0.70
4.92
4.46
0.73
0.62
1.96
10.60
2.17
20.30
51.60
28.00
49.40
29.60
7.62
1.66
2.35
6.48
4.87
1.64
1.96
10.90
40.30
52.70
44.20
18.20
>100
SF-295
SF-539
SNB-19
12.10
2.01
SNB-75
U251
1.74
Melanoma
LOX IMVI
MALME-3M
M14
0.17
0.22
0.42
0.21
--
0.62
1.32
6.76
1.71
--
0.17
0.22
0.39
0.32
--
0.65
2.67
6.01
6.14
--
2.79
1.49
1.82
2.94
--
31.30
6.89
1.77
2.58
4.55
2.71
--
7.01
60.10
45.90
42.80
--
9.43
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-Res
SK-OV-3
Renal Cancer
786-0
39.20
--
0.20
0.28
0.24
0.22
0.62
3.53
1.34
0.83
0.20
0.28
0.21
3.66
0.66
3.82
0.90
4.33
2.62
2.19
0.78
2.65
30.00
18.90
21.00
34.60
9.87
3.16
5.74
3.32
47.50
36.20
64.70
42.30
0.21
0.20
0.39
0.20
0.24
0.21
1.69
0.79
0.59
4.52
0.72
2.80
2.80
5.64
0.21
0.19
0.41
0.40
0.25
0.24
1.74
0.74
0.61
>100
0.95
4.51
0.90
5.72
1.77
1.77
1.61
3.83
3.01
6.12
14.80
18.20
5.86
--
--
1.93
2.66
3.25
11.60
2.72
10.50
22.30
36.20
37.30
>100
>100
49.00
5.67
39.20
28.50
44.20
52.90
0.45
1.80
0.55
0.19
--
6.08
6.35
4.32
0.74
--
0.75
1.80
1.52
0.20
--
6.66
6.64
5.43
0.75
--
3.38
--
33.90
--
1.86
2.15
3.63
1.47
2.29
2.26
7.29
2.43
8.33
47.50
42.10
6.56
A498
ACHN
2.14
1.87
1.64
2.44
2.54
2.41
13.50
8.00
5.69
31.00
26.90
32.50
CAKI-1
RXF 393
SN12C
25.20
29.00
67.50
48.90
0.26
1.82
0.21
1.75
6.34
0.95
0.37
1.56
0.23
6.16
5.98
2.22
TK-10
UO-31
Prostate Cancer
PC-3
0.18
0.20
0.62
0.62
0.20
0.18
0.663
0.59
9.12
5.31
55.90
42.70
6.82
4.74
74.90
45.40
DU-145
Breast Cancer
MCF-7
0.16
0.18
0.82
0.62
0.16
0.18
0.77
0.70
1.43
2.40
6.54
2.48
2.32
44.10
39.20
MDA-MB-
231/ATCC
HS 578T
BT549
26.10
0.26
0.21
0.23
0.21
>100
0.74
0.74
0.74
0.28
0.50
0.23
0.21
>100
4.21
0.48
>100
--
--
--
3.80
2.98
2.89
2.37
>100
38.80
56.80
44.80
--
T-47D
2.37
1.56
78.50
6.91
MDA-MB-468

Molecules 2011, 16
5850
2.3. Pharmacology
A total of 60 human cell lines, derived from nine cancer types (leukemia, lung, colon, brain,
melanoma, ovarian, renal, prostate, breast) formed the basis of this NCI-60 DTP human tumor cell line
screen [16,17].The tumor cells were cultured in RPMI1640 medium supplemented with 5% fetal
bovine serum and 2 mM L-glutamine. The tumor cells were inoculated in to 96-well microtiter plates,
100 µL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of
the individual cell lines [16-19].After this cell inoculation, the microtiter plates are incubated at 37 °C,
5% CO2, 95% air and 100% relative humidity for 24 h. Two plates of each cell line are fixed in situ
with TCA to represent a measure of the cell population (T0)before adding the target compounds. The
target compounds are dissolved in DMSO and diluted in the test medium to obtain the desired
concentration. 100 µL of each of the test compound solution is now added to the above appropriate cell
line microtiter wells and incubated for 48 h at 37 °C, 5% CO2, 95% air and 100% relative humidity. A
sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The cytotoxic
effects were evaluated and the assay results and dose response parameters calculated as previously
described [17,20-22]. At the present time at NCI the target compounds were tested initially at a single
dose at 10 µM concentration and those promising target compounds are further tested at five dose
testing concentrations 0.01, 0.1, 1.0, 10, 100 µM.
Concentration parameters GI50, TGI and LC50: The NCI re-named the IC50 as GI50. GI50 value
represents the concentration of the target compound that causes 50% growth inhibition, that is derived
from the formula 100 × (T – T0) / (C – T0) = 50, where T is the optical density of the target compound
after 48 h exposure. T0 is the optical density at time 0 and C is the control optical density. TGI
represents the concentration of the target compound where 100 × (T – T0) / (C – T0) = 0 and it is the
cytostatic effect. LC50 is the concentration of the target compound where 100 × (T – T0) / T0 = –50.
LC50 also signifies the cytostatic effect and the control optical density is not used in the calculation.
3. Experimental
3.1. General
Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich Chemical
Co. Melting points were determined on an Electrothermal MEL-TEMP apparatus and are uncorrected.
13
1H-NMR and C-NMR spectra were recorded in DMSO-d6 on a Bruker 500 MHz instrument and the
chemical shift (δ) values are reported in parts per million (ppm)relative to TMS. A Thermo Scientific
LTQ Linear Trap LC/MS/MS system was used for mass spectrometry. UV spectra were recorded on a
Beckman-Coulter DU-800 spectrophotometer. Analytical TLC was carried out on Sigma-Aldrich (cat
# Z122785-25EA), 0.2 mm percolated silica gel polyester sheets with UV indicator. Elemental analysis
was carried out by M-H-W Laboratories, Phoenix, AZ. Analysis of C, H, N were within ± 0.4% of
theoretical values. The carbon numbering was shown for representative monomer 20a and for one
representative dimer 20b. All others were referred similarly on the 13C-NMR assignments.

Molecules 2011, 16
5851
3.2. General Procedure-A for N9-[(Z)-4'-chloro-2'-butenyl-1'-yl]-6-methoxypurine (6)
Synthesis of 6-methoxypurine: To a stirred suspension of 6-chloropurine (5.0 g, 32 mmol) in anhydrous
methanol (240 mL) was slowly added 30 W% sodium methoxide in methanol (17.3 g,
320 mmol) at room temperature and then the reaction mixture was refluxed for 18 h. It was cooled to
room temperature, neutralized with glacial acetic acid to pH 7.5–8.0 and then evaporated in a rotary
evaporator to remove the solvent. The residue was treated with cold water (5 °C) (100 mL), the
resulting solid was filtered, thoroughly washed with DI water. The product was crystallized from
1
methanol as brownish white solid (3.5 g), 72% yield. H-NMR: δ 13.39 (1H, br s, NH), 8.50 (1H, s,
H-8), 4.10 (3H, s, OCH3).
To a suspension of 6-methoxypurine (1.51 g, 10 mmol), anhydrous potassium carbonate (2.1 g,
15 mmol) in DMF (50 mL) at room temperature, was added cis-1,4-dichloro-2-butene (1.25 g,
10 mmol) and the contents stirred at room temperature for 5 h. The reaction mixture was filtered to
remove the potassium carbonate that was also washed with DMF (25 mL). The filtrate and washings
combined and evaporated under vacuum. The residue was chromatographed on a column of silica gel
and the product was eluted with ethyl acetate: hexane 1:1 v/v. Evaporation of the homogeneous
fractions resulted in a residue that was further crystallized from ethyl acetate-hexane as cream white
rosettes, (1.3 g), 54% yield, m.p. 104–106 °C.1H-NMR: δ 8.54 (1H, s, H-2), 8.36 (1H, s, H-8),
5.89–5.84 (2H, m, HC=CH), 5.4 (2H, s, N-CH2), 4.10 (3H, s,OCH3), 2.71 (2H, m, CH2Cl), 1.10 (3H, t,
J = 7.5 Hz, OCH3). 13C-NMR: δ 160.22 (C-6), 151.76 (C-4), 151.49 (C-2), 129.66 (C-3'), 128.11
(C-2'), 120.51 (C-5), 53.84 (OCH3), 40.01 (C-1'), 39.85 (C-4'). Anal. Calcd. forC10H11N4OCl: C 50.32,
H 4.65, N 23.47; Found: C 50.20, H 4.74, N 23.55.
N9-[(Z)-4'-Chloro-2'-butenyl-1'-yl]-2,6-dichloropurine (5a). A suspension of 2,6-dichloropurine (2.0 g,
11 mmol), anhydrous potassium carbonate (4.6 g, 34 mmol), 1,4-dichloro-2-butyne (2.1 g, 17 mmol)
in anhydrous DMF was stirred at r.t. for 6 h. The reaction has been worked-up and purified on a
column of silica gel as described in the general procedure-A , to give a major product 5a and a minor
dimeric product 5b. Compound 5a was isolated as a cream white solid (1.1g), 46% yield, m.p.
1
65–67 °C. H-NMR: δ 8.67 (1H, s, H-8), 5.93–5.91 (2H, m, HC=CH), 5.10–5.09 (2H, m, N-CH2),
13
4.54–4.52 (2H, m,CH2Cl). C-NMR: δ 153.23 (C-2), 150.95 (C-6), 149.59 (C-4), 148.0 (C-8), 130.44
(C-5), 130.24 (C-3'), 127.16 (C-2'), 40.53 (C-1'), 39.13 (C-4'). LC-MS (m/z): 277 [M+1]+, 100%. Anal.
Calcd. forC9H7N4Cl3: C 38.95, H 2.54, N 20.19; Found: C 39.84, H 2.45, N 20.25.
N9,N9'' -bis[(Z)-2'-Butenyl-1',4'-diyl]-2,6-dichloropurine (5b). Compound 5b was isolated in the above
1
reaction as a brownish white solid (0.45g), 15% yield, m.p. 226–228 °C. H-NMR: δ 8.65(2H, H-8,
H-8''), 5.97 (m, 2H, HC=CH), 4.97 (4H, m, 2 × N-CH2). 13C-NMR: δ 153.33 (C-2, C-2''), 150.93 (C-6,
C-6''), 149.60 (C-4, C-4''), 148.17 (C-8, C-8''), 130.52 (C-5, C-5''), 127.69 (C-2', C-3'), 40.95 (C-1',
C-4'). LC-MS (m/z): 431 [M+1]+, 100%. Anal. Calcd. forC14H8N8Cl4: C 39.10, H 1.88, N 26.05;
Found: C 39.0, H 2.0, N 25.95.

Molecules 2011, 16
5852
3.3. N9-[(Z)-4'-Chloro-2'-butenyl-1'-yl]-2-chloro-6-methoxypurine (7)
Synthesis of 2-chloro-6-methoxypurine: 2,6-dichloropurine (5.0 g, 27 mmol) was reacted with sodium
methoxide (30 wt.% 14.6 g, 270 mmol) in anhydrous methanol (250 mL) under reflux for 16 h. The
reaction has been worked-up as described above under general procedure-A The crude product was
1
crystallized from methanol as snow white solid (4.0g), 82% yield. H-NMR: δ 13.52 (1H, br s, NH),
8.42 (1H, s, H-8), 4.1 (3H, s, OCH3).
2-Chloro-6-methoxypurine (1.9 g, 10 mmol), anhydrous potassium carbonate (2.1 g, 15 mmol),
cis- 1,4-dichloro-2-butene (1.5 g, 12mmol) were stirred in DMF (50 mL) at room temperature for 4 h.
The crude product was isolated as described under the general procedure-A. The product was
chromatographed on a column of silica gel, eluent ethyl acetate/hexane 1:1 v/v. Cream white solid
1
(1.8g) 64% yield, m.p. 136–138 °C. H-NMR: δ8.38 (1H, s, H-8), 5.9–5.83 (2H, m, HC=CH), 5.02
13
(2H, d, J = 5.5 Hz, N-CH2), 4.5 (2H, d, J = 5.5 Hz, CH2Cl), 4.1 (3H, s, OCH3). C-NMR: δ 160.73
(C-6), 152.94 (C-2), 151.32 (C-4), 144.04 (C-8), 129.96 (C-3'), 127.64 (C-2'), 119.77 (C-5), 54.87
(OCH3), 40.05 (C-1'), 39.15(C-4'). Anal. Calcd. for C10H10N4OCl2: C 43.93, H 3.69, N 20.51; Found:
C 43.85, H 3.75, N 20.45.
N9-[(E)-4'-Chloro-2'-butenyl-1'-yl]-2,6-dichloropurine (8a). A suspension of 2,6-dichloropurine (2.0 g,
11 mmol), anhydrous potassium carbonate (4.6 g, 34 mmol), trans-1,4-dichloro-2-butene (2.15 g,
17 mmol) in anhydrous DMF was stirred at r.t. for 10 h. The reaction was worked-up as described in
the general procedure-A. Chromatography of the resulting crude product on a column of silica gel
yielded one major product 8a and a minor dimeric product 8b.Compound 8a was isolated as a cream
1
white solid (1.2 g), 40% yield, m.p. 70–72 °C. H-NMR: δ 8.78 (1H, H-8), 6.42 (1H, m, HC=CH),
5.80 (1H, m, HC=CH), 5.0 (2H, d, J = 5.5 Hz, N-CH2), 4.21 (2H, m, CH2Cl). LC-MS (m/z): 277
[M+1]+, 100%. Anal. Calcd. for C9H7N4Cl3: C 38.95, H 2.54, N 20.19; Found C 38.82, H 2.65,
N 20.25.
N9,N9''-bis[(E)-2'-Butenyl-1',4'-diyl]-2,6-dichloropurine (8b). Pale brown solid (0.6g), 13% yield, m.p.
1
240 °C, decomposes. H-NMR: δ 8.77 (2H, s, H-8), 5.97 (2H, m, HC=CH), 4.96 (4H, 2 × N-CH2).
LC-MS (m/z): 431 [M+1]+,100%. Anal. Calcd. for C14H8N8Cl4: C 39.10, H 1.88, N 26.05; Found: C
39.20, H 2.0, N 26.15.
N9-[4'-Chloro-2'-butynyl-1'-yl]-6-chloropurine (9a). A suspension of 6-chloropurine (2.5 g, 16 mmol),
anhydrous potassium carbonate (3.5 g, 25 mmol) and 1,4-dichlorobutyne (3.0 g, 24 mmol) in DMF
(150 mL) was stirred at room temperature for 3.0 h. The crude product was isolated as described in the
general procedure-A. It was chromatographed on a column of silica gel using ethyl acetate/hexane 1:1
1
(v/v)as eluents to furnish 9a as a brownish white solid, 2.3 g, 59% yield, m.p. 92–94 °C. H-NMR: δ
8.83 (1H, s, H-2), 8.77 (1H, s, H-8), 5.32 (2H, t, J = 2.0 Hz, N-CH2), 4.49 (2H, t, J = 2.0 Hz, CH2Cl).
13C-NMR: δ 151.79 (C-2), 151.35 (C-6), 149.23 (C-4), 146.74 (C-8), 130.72 (C-5), 80.76 (C-3'), 79.71
(C-2'), 33.43 (C-1'), 30.47 (C-4'). Anal. Calcd. for C9H6N4Cl2: C 44.84, H 2.51, N 23.24; Found: C
44.75, H 2.64, N 23.15. From the above column, the ethyl acetate/hexane 2:1 v/v and 100% ethyl

Molecules 2011, 16
5853
acetate eluents furnished the minor dimeric product N9,N9'' -bis[2'-butynyl-1',4'-diyl]-6-chloropurine
1
(9b) as a pale brown solid. 0.7 g, 12% yield, m.p. 220 °C decomposes. H-NMR: δ 8.95 (2H, s, H-2,
H-2, H-2''), 8.75 (2H, s, H-8, H-8''), 5.24 (4H, 2 × N-CH2). 13C-NMR: δ 151.9 (C-2, C-2''), 151.5
(C-6,C-6''), 149.4 (C-4, C-4''), 146.9 (C-8, C-8''), 130.8 (C-5, C-5''), 80.5 (C-2', C-3'), 40.9
(C-1',C-4'). Anal. Calcd. for C14H8N8Cl2: C 46.82, H 2.25, N 31.20; Found: C 46.92, H 2.14, N 31.25.
N9-[4'-Chloro-2'-butynyl-1'-yl]-2,6-dichloropurine (10a). A suspension of 2,6-dichloropurine (1.9 g,
10 mmol), anhydrous K2CO3 (4.2 g, 30 mmol) and 1,4-dichloro-2-butyne (1.85 g, 15 mmol) was
stirred in DMF at r.t. for 8 h under argon. The reaction has been worked-up as described in the general
procedure-A. The resulting product was chromatographed on a column of silica gel tofurnish 10a as
the major product and 10b as a minor dimeric product.Compound 10a cream white solid (1.4 g) 50%
1
yield, m.p. 80–82 °C. H-NMR: δ 8.79 (1H, s, H-8), 5.29 (2H, t, J = 2.0 Hz, N-CH2), 4.50 (2H, t,
13
J = 2.0 Hz, CH2Cl). C-NMR: δ 152.87 (C-2), 151.23 (C-6), 149.91 (C-4), 147.66 (C-8), 130.41 (C-
5), 81.11 (C-3'), 79.32 (C-2'), 33.66 (C-1'), 30.46 (C-4')). LC-MS (m/z): 275 [M+1]+, 100%.Anal.
Calcd. for C9H5N4Cl3: C 39.23, H 1.83, N 20.33; Found: C 39.10, H 1.70, N 20.15.
N9, N9'' -bis[2'-Butynyl-1',4'-diyl]-2,6-dichloropurine (10b). It was isolated as a brownish white solid
1
(0.6g), 14% yield, m.p. 210 °C decomposes. H-NMR: δ 8.78 (2H, s, H-8, H-8''), 5.25 (4H, s, 2 ×
N-CH2). LC-MS (m/z): 429 [M+1]+, 100%. Anal. Calcd. for C14H6N8Cl4: C 39.28, H 1.41, N 26.18;
Found: C 39.12, H 1.55, N 26.25.
N9-[4'-Chloro-2'-butynyl-1'-yl]-6-methoxypurine (11). 6-Methoxypurine (1.0 g, 7.0 mmol), anhydrous
potassium carbonate (1.6 g, 12.0 mmol), 1,4-dichlorobutyne (1.1 g, 9.0 mmol) was stirred at r.t. for 4
h. The crude product was isolated as described in the general procedure-A. Further purification on a
column of silica gel using ethyl acetate/hexane 1:1 v/v as the eluent furnished a brownish white solid
1
(1.1g), 70% yield, m.p. 141–143 °C. H-NMR: δ 8.57 (1H, s, H-2), 8.45 (1H, s, H-8), 5.25 (2H, t,
13
J = 2.0 Hz, N-CH2), 4.48 (2H, t, J = 2.0 Hz, CH2Cl), 4.10 (3H, s, OCH3). C-NMR: δ 160.31 (C-6),
151.79 (C-2), 151.50 (C-4), 143.07 (C-8), 120.45 (C-5), 80.30 (C-2', C-3'), 53.95 (OCH3), 32.92
(C-1'), 30.52 (C-4'). Anal. Calcd. for C10H9N4OCl: C 50.75, H 3.83, N 23.67; Found: C 50.60, H 3.90,
N 23.55.
N9-[4'-chloro-2'-butynyl-1'-yl]-2-chloro-6-methoxypurine (12). Brownish white solid, 61% yield, m.p.
1
143–145 °C. H-NMR: δ 8.48 (1H, s, H-8), 5.23 (2H, t, J = 2.0 Hz, N-CH2), 4.45 (2H, t, J = 2.0 Hz,
CH2Cl), 4.11 (3H, s, OCH3). 13C-NMR: δ 160.85 (C-6), 152.64 (C-2), 151.61 (C-4), 143.82 (C-8),
119.75 (C-5), 80.66 (C-3'), 79.88 (C-2'), 54.98 (OCH3), 33.18 (C-1'), 30.50 (C-4'). Anal. Calcd. for
C10H8N4OCl2: C 44.30, H 2.97, N 20.67; Found: C 44.15, H 3.15, N 20.55.
3.4.General procedure B for N9-[(E)-2',3'-dibromo)-4'-chloro-2'-butenyl-1'-yl]-6-chloropurine (13)
A suspension of 9a (0.242 g, 1.0 mmol), pyridiniumtribromide(0.4 g, 1.3 mmol) in anhydrous
dichloromethane (100 mL) was cooled to –10 °C while stirring. Anhydrous methanol (50 mL) was
added drop wise during 15–20 minutes. The reaction mixture was allowed to warm-up and stirred for

Molecules 2011, 16
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20 h in a fume hood. The clarified reaction mixture was evaporated on a rotary evaporator at 30–35 °C
without any quenching with sodium thiosulfate. The product was purified on a column of silica gel
using ethyl acetate - light petroleum ether 1:1, 2:1 v/v as the eluents. It was further crystallized from
ethyl acetate-light petroleum ether as a white crystalline solid 0.22 g, 54% yield, m.p. 88–90 °C.
1H-NMR: δ 8.80 (1H, s, H-2), 8.70 (1H, s, H-8), 5.40 (2H, s, N-CH2), 4.71(2H, s, CH2Cl). 13C-NMR: δ
151.70 (C-2), 151.30 (C-6), 149.0 (C-4), 146.7 (C-8), 130.70 (C-5), 121.30 (C-3'), 119.40 (C-2'), 50.40
(C-1'), 49.70 (C-4'). Anal. Calcd. for C9H6N4Br2Cl2: C 26.96, H 1.51, N 13.98; Found: C 26.80, H
1.65, N 14.15. The pyridinium salts were retained on the column.
N9-[(E)-2',3'-Dibromo-4'-chloro-2'-butenyl-1'-yl]-6-methoxypurine (14). Compound 11 was used as
the starting material and the above bromination procedure was followed. A crystalline white solid
1
resulted, 65% yield, m.p. 127–129 °C. H-NMR: δ 8.56 (1H, s, H-2), 8.41 (1H, s, H-8), 5.45 (2H, s,
N-CH2), 4.72 (2H, s, CH2Cl), 4.11 (3H, s,OCH3). 13C-NMR: δ 160.29 (C-6), 152.01 (C-4), 151.94
(C-2), 143.93 (C-8), 121.59 (C-5), 121.33 (C-3'), 119.46 (C-2'), 53.97 (OCH3), 50.42 (C-1'), 49.75
(C-4'). LC-MS (m/z): 397[M+1]+, 100%. Anal. Calcd. for C10H9N4OBr2Cl: C 30.29, H 2.29, N 14.13;
Found: C 30.10, H 2.34, N 14.05.
Synthesis of N9-(tetrahydropyran-2-yl)-6-chloropurine (25). A suspension of 6-chloropurine (5.0 g,
32 mmol), 3,4-dihydro-2H-pyran (5.4 g, 64 mmol) and pyridinium-p-toluenesulfonate (PPTS) (3.3 g,
13 mmol) in anhydrous dichloromethane (250 mL) was stirred at room temperature in an atmosphere
of argon for 64 h. The original suspension transformed in to a clear liquid and the starting material
disappeared. The reaction mixture was evaporated and the residue was chromatographed on a column
of silica gel. The THP ether was eluted with 100% ethyl acetate and that was isolated as a semi-solid.
It solidified as a pale yellow soft solid upon cooling in a freezer (7.0 g), 91% yield. Homogeneous on
TLC on silica gel plate, Rf 0.58, mobile phase 100% ethyl acetate. The starting material 6-chloropurine
1
under the same conditions was slow moving, Rf 0.12. H-NMR: δ 8.92 (1H, s, H-2), 8.81 (1H, H-8),
THP protons: 5.81–5.77 (1H, m), 4.01–3.99 (1H, m), 3.77–3.68 (1H, m), 2.37–2.28 (1H, m),
2.04–1.96 (2H, m), 2.04 (1H, m), 2.03 (2H, m).
3.5. General Procedure C for Synthesis of N9-(tetrahydropyran-2-yl)-6-(4-methoxyphenyl)purine (26)
Suzuki-Miyaura cross coupling reaction: A stirred suspension of 6-chloro-9-(tetrahydropyran-2-yl)
purine 25, (3.5 g, 15 mmol), 4-methoxyphenyl boronic acid (3.35 g, 22 mmol), anhydrous potassium
carbonate (3.1 g, 22 mmol),tetrakis(triphenylphosphine)palladium (0), Pd (PPh3)4, (0.85 g, 0.74 mmol)
in anhydrous toluene (150 mL) was gradually heated to 100 °C during 1 hr and then maintained at that
temperature for 18 h. The TLC, silica gel plate, indicated the disappearance of the starting material Rf
0.45 and the formation of the new product Rf0.54, mobile phase ethyl acetate-hexane 7:3 v/v. The
reaction mixture was filtered while warm to remove the potassium and boron salts. The filtrate was
concentrated and the resulting residue was crystallized from ethyl acetate-methanol 1:1 v/v as a
1
brownish white solid (4.0g), 88% yield, m.p.146–148 °C. H-NMR: δ 8.93 (1H, s, H-2), 8.87–8.82
(2H, m, Ar-H), 8.70 (1H, s, H-8), 7.18–7.13 (2H, m, Ar-H), 4.06–4.03(1H, m, THP), 3.87

Molecules 2011, 16
5855
(3H,s,OCH3), 3.76–3.71(1H, m, THP), 2.37–2.31 (1H, m, THP), 2.04–1.99(2H, m, THP), 1.81–1.73
(1H, m, THP), 1.67–1.79 (2H, m,THP).
Synthesis of 6-(4-Methoxyphenyl)purine (28). To a suspension of compound 26 (3.2 g,10 mmol) in
methanol (100 mL) was added acetyl chloride (0.2 mL, 2.8 mmol) and the contents stirred overnight at
room temperature for 24 h. The reaction mixture was evaporated, the residue was treated with water
(100 mL) and the pH was adjusted to 7.5–8.0 with a saturated solution of sodium bicarbonate in water.
The resulting solid was filtered, washed with DI water (100 mL) followed by cold ethanol (25 mL),
hexane (25 mL) and dried under vacuum, brownish white solid (1.9 g), 81% yield. No further
purification was necessary as the product was found to be homogeneous on silica gel TLC, Rf 0.3,
1
mobile phase ethyl acetate-hexane 7:3 v/v. H-NMR: δ 8.97 (1H, s, H-2), 8.82–8.79 (2H, m, Ar-H),
8.75 (1H, s, H-8), 8.0 (1H, broad, NH), 7.2–7.17 (3H, m, Ar-H), 3.88 (3H, s, OCH3).
N9-[(Z)-4'-Chloro-2'-butenyl-1'-yl]-6-(4-methoxyphenyl)purine (15a). A suspension of 6-(4-methoxy-
phenyl)purine (0.68 g, 3.0 mmol), anhydrous potassium carbonate (1.0 g, 7.2 mmol), cis-1,4-dichloro-
2-butene (0.42 g, 3.4 mmol) was stirred at room temperature under argon atmosphere for 4h. The
reaction mixture was worked-up as described in the general procedure-A. The resulting residue was
chromatographed on a column of silica gel. Fractions 3–4 (100 mL) each from ethyl acetate-hexane
(1:1) v/v furnished a brownish white solid 15a (0.41 g), 43% yield, m.p. 129–131 °C. 1H-NMR: δ 8.92
(1H, s, H-2), 8.87–8.82 (2H, m, Ar-H), 8.61 (1H, s, H-8), 7.16–7.13 (2H, s, Ar-H), 5.92–5.89 (2H, m,
HC=CH), 5.09–5.07 (2H, d, J = 9.0 Hz, N-CH2), 4.54–4.52 (2H, m, CH2Cl), 3.86(3H, s, OCH3).
13C-NMR: δ 161.61 (C-13), 152.42 (C-4), 151.86 (C-6), 145.44 (C-8), 131.07 (C-11, C-15), 129.74
(C-3'), 129.62 (C-10), 128.08 (C-2'), 127.82 (C-5), 114.05 (C-12, C-14), 55.32 (OCH3), 39.68 (C-1'),
39.22(C-4'). LC-MS (m/z): 315 [M+1]+, 100%. Anal. Calcd. for C16H15N4OCl: C 61.05, H 4.80, N
17.80; Found: C 61.10, H 4.92, N 17.90.
N9,N9''-bis[(Z)-2'-Butenyl-1'4'-diyl]-6-(4-methoxyphenyl)purine (15b). The fractions 5–8 from the
above column chromatography yielded a brownish white solid (0.1 g), 11% yield, m.p. 163–165 °C.
1H-NMR: δ 8.93 (2H, s, H-2, H-2''), 8.89–8.84 (4H, m, Ar-H), 8.72 (2H, s, H-8,H-8''), 7.17–7.14 (4H,
m, Ar-H), 5.99–5.96 (2H, m, HC=CH), 5.29–5.27 (4H, d, J = 10.0 Hz, 2 × N-CH2), 3.87 (6H, s, C-13-
OCH3, C-13''-OCH3). 13C-NMR: δ 161.63 (C-13, C-13''), 152.45 (C-4, C-4''), 151.98 (C-6, C-6''),
151.73 (C-2, C-2''), 145.66 (C-8, C-8''), 131.10 (C-11, C-11'', C-15, C-15''), 129.70 (C-10, C-10''),
128.03 (C-2', C-3'), 127.85 (C-5, C-5''), 114.09 (C-12, C-12'', C-14, C-14''), 55.34 (C-13-OCH3, C-13''-
OCH3), 40.20 (C-1', C-4'). LC-MS (m/z): 505 [M+1]+, 100%. Anal. Calcd. forC28H24N8O2: C 66.65, H
4.80, N 22.21; Found: C 66.52, H 4.92, N 22.33.
3.6. Synthesis of N9-(tetrahydropyran-2-yl)-6-(4-fluorophenyl)purine (27)
Suzuki-Miyaura cross coupling reaction: 9-(Tetrahydropyran-2-yl)-6-chloropurine (4.8 g, 20 mmol),
4-fluorophenyl boronic acid (4.2 g, 30 mmol), Pd(PPh3)4, (1.0 g, 0.9 mmol) in anhydrous
dimethoxy-ethane (200 mL) was added a 2.7 molar saturated Na2CO3 solution in water (11.2 mL,
30 mmol). The reaction mixture was gradually heated to reflux during 2 h in an oil bath and then

Molecules 2011, 16
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maintained at reflux for 7 h. Reaction was worked-up and purified as above to yield a brownish
1
white solid (5.0 g), 83% yield. m.p. 146–148 °C. H-NMR: δ 9.0 (1H, s, H-2), 8.93–8.89 (2H, m,
Ar-H), 8.88 (1H, s, H-8), 7.46–7.42 (2H, m, Ar-H), THP protons: 5.85–5.82 (1H, m), 4.06–4.03
(1H, m), 3.77–3.72 (1H, m), 2.38–2.35 (1H, m), 2.06–1.99 (2H, m), 1.84–1.72 (1H, m), 1.63–1.61
(2H, m).
6-(4-Fluorophenyl)purine (29). To a stirred suspension of 9-(tetrahydropyran-2-yl)-6-(4-fluorophenyl)
purine (27, 3.75 g, 13 mmol) in methanol (200 mL) at room temperature was added acetyl chloride
(0.4 mL, 5.6 mmol) and the reaction was worked-up as described in 26. A brownish white solid
resulted (2.6 g), 96% yield. The product was found homogeneous on TLC on a silica gel plate, Rf 0.3,
mobile phase ethyl acetate-hexane (7:3) v/v. 1H-NMR: δ 8.94 (3H, s, H-2 and 2H Ar-H), 8.92 (1H, br,
NH), 8.65 (1H, s, H-8), 7.45–7.40 (2H, m, Ar-H).
N9-[(Z)-4'-Chloro-2'-butenyl-1'-yl]-6-(4-fluorophenyl)purine (16a).
A
suspension of 6-(4-
fluorophenyl) purine 29 (0.65 g, 3.0 mmol), anhydrous potassium carbonate (1.0 g, 7.2 mmol), cis-1,4-
dichloro-2-butene (0.413 g, 3.3 mmol) in DMF (50 mL) was stirred under argon at room temperature
for 3.5 h. The reaction was worked-up as described in general procedureA.The crude reaction mixture
contained a major product 16a and a minor dimeric product 16b. The above crude product upon
crystallization from ethyl acetate-methanol furnished the dimeric product 16b as feathery brownish
white needeles. The mother liquor was concentrated and chromatographed on a column of silica gel
using ethyl acetate-light petroleum ether (1:1, 2:1v/v)as the eluents that furnished a brownish white
1
solid of 16a (0.4 g), 43% yield, m.p. 94–96 °C. H-NMR: δ 8.99 (1H, s, H-2), 8.93–8.90 (2H, m,
Ar-H), 8.67 (1H, 2, H-8), 7.45–7.41 (2H, m, Ar-H), 5.93–5.91 (2H, m, HC=CH), 5.10–5.09 (2H, d,
1
J = 5.5 Hz, N-CH2), 4.54–4.52 (2H, m, CH2Cl). 13C-NMR: δ 164.80 (d, JCF = 249 Hz, C-13), 152.13
(C-4), 151.75 (C-2), 151.39 (C-6), 146.09 (C-8), 131.85 (d, 4JCF = 3.8 Hz, C-10), 131.75 (d, 3JCF = 8.8
2
Hz, C-11, C-15), 129.99 (C-5), 129.82 (C-3'), 127.97 (C-2'), 115.73 (d, JCF = 21.4 Hz, C-12, C-14),
39.74 (C-1'), 39.21 (C-4'). LC-MS (m/z): 303 [M+1]+, 10%, 267 [M+1]+-HCl, 100%. Anal. Calcd. for
C15H12N4FCl: C 59.51, H 4.0, N 18.51; Found: C 59.40, H 4.10, N 18.45.
N9,N9''-bis[(Z)-2'-Butenyl-1',4'-diyl]-6-(4-fluorophenyl)purine (16b). 0.27 g, 18% yield, m.p.
1
198–200 °C. H-NMR: δ 8.99 (2H, s, H-2, H-2''), 8.94–8.92 (4H, m, Ar-H), 8.77 (2H, s, H-8,H-8''),
7.46–7.42 (4H, m, Ar-H), 6.01–5.99 (2H, m, HC=CH), 5.31–3.30 (4H, d, J = 5.5 Hz, 2 × N-CH2).
1
13C-NMR: δ 164.83 (d, JCF = 249 Hz, C-13, C-13''), 152.27 (C-4, C-4''), 151.76 (C-2, C-2''), 151.44
4
3
(C-6, C-6''), 146.34 (C-8, C-8''), 131.90 (d, JCF = 3.8 Hz, C-10, C-10''), 131.79 (d, JCF = 18.9 Hz,
2
C-11, C-11'', C-15, C-15''), 130.09 (C-5, C-5''), 128.04 (C-2', C-3'), 115.80 (d, JCF = 21.4 Hz, C-12,
C-12'', C-14,C-14''), 40.29 (C-1', C-4'). LC-MS (m/z): 481 [M+1]+, 100%. Anal. Calcd. forC26H18N8F2:
C 64.99, H 3.78, N 23.32; Found: C 65.10, H 3.82, N 23.45.
N9-[(E)-4'-Chloro-2'-butenyl-1'-yl]-6-(4-methoxyphenyl)purine (17). Cream white needles, 63% yield, m.p.
112–114 °C. 1H-NMR: δ 8.92 (1H, s, H-2), 8.87–8.82 (2H, m, Ar-H), 8.61 (1H, s, H-8), 7.16–7.13 (2H, s,
Ar-H), 6.19–6.09 (1H, m, HC=CH), 5.83–5.73 (1H, m, HC=CH), 4.99–4.98 (2H, d, J = 9.0 Hz, N-CH2),
13
4.23–4.20 (2H, m, CH2Cl), 3.86 (3H, s, OCH3). C-NMR: δ 161.62 (C-13), 152.47 (C-4), 151.91 (C-6),

Molecules 2011, 16
5857
151.82 (C-2), 145.66 (C-8), 131.09 (C-11, C-15), 129.59 (C-10), 129.50 (C-3'), 129.02 (C-2'), 127.83 (C-
5), 114.06 (C-12,14), 55.33 (C-13-OCH3), 44.08 (C-1'), 43.78 (C-4'). LC-MS (m/z): 277 [M+1]+, 100%.
Anal. Calcd. for C16H15N4OCl: C 61.05, H 4.80, N 17.80; Found: C 61.15, H 4.95, N 17.95.
N9-[(E)-4'-Chloro-2'-butenyl-1'-yl]-6-(4-fluorophenyl)purine (18a). A suspension of 6-(4-fluoro-
phenyl) purine (0.5 g, 2.3 mmol), anhydrous potassium carbonate (0.65 g, 4.7 mmol), trans-1,4-
dichloro-2-butene (0.33 g, 2.6 mmol) in DMF (40 mL) was stirred at room temperature under argon
for 22 h. The reaction has been worked-up as described in the general procedureA.The resulting
product was chromatographed on a column of silica gel using ethyl acetate - light petroleumether (1:1)
v/v as an eluent. Fractions of 100 mL were collected. The fractions 3-4 yielded 18a as a cream white
solid, homogeneous on silicagel TLC, mobile phase ethyl acetate-hexane 7:3 v/v, Rf 0.69 (0.4g), 57%
1
yield, m.p. 73–75 °C. H-NMR: δ 8.98 (1H, s, H-2), 8.94–8.91 (2H, m, Ar-H), 8.68 (1H, s, C-8),
7.45–7.42 (2H, m, Ar-H), 6.16–6.12 (1H, m, HC=CH), 5.82–5.79 (1H, m, HC=CH), 5.01–5.0 (2H, d,
1
J = 5.0 Hz, N-CH2), 4.22–4.20 (2H, m, CH2Cl). 13C-NMR: δ 164.80 (d, JCF = 250 Hz, C-13), 152.16
4
(C-4), 151.80 (C-2), 151.43 (C-6),146.27 (C-8), 131.85 (d, JCF = 2.52 Hz, C-10), 131.75 (d,
3JCF = 8.8 Hz, C-11, C-15),129.96 (C-5), 129.59 (C-3'), 128.87 (C-2'),115.71 (d, 2JCF = 21.4 Hz, C-12,
C-14), 44.04 (C-1'), 43.85(C-4'). LC-MS (m/z): 303 [M+1]+, 100%. Anal. Calcd. for C15H12N4FCl: C
59.51, H 4.0, N 18.51; Found: C 59.25, H 4.16, N 18.65.
N9,N9''-bis[(E)-2'-Butenyl-1',4'-diyl]-6-(4-fluorophenyl)purine (18b). From the above chromatography,
fractions 6–10 yielded this dimer 18b, Rf 0.1, brownish white solid, (0.15 g), 13% yield, m.p.
1
217–219 °C. H-NMR: δ 8.95 (2H, s, H-2, H-2''), 8.93–8.90 (4H, m, Ar-H), 8.65 (2H, s, H-8, H-8''),
7.46–7.42 (4H, m, Ar-H), 5.98 (2H, m, HC=CH), 4.97–4.96 (4H, m, 2 × N-CH2). 13C-NMR: δ 164.86
1
(d, JCF = 252 Hz, C-13, C-13''), 152.61 (C-4, C-4''), 151.75 (C-6, C-6''), 151.61 (C-2, C-2''), 144.04
3
4
(C-8, C-8''), 131.44 (d, JCF = 8.8 Hz, C-11, C-11'', C-15, C-15''), 131.32 (d, JCF = 2.52 Hz, C-10,
2
C-10''), 130.06 (C-5, C5''), 127.90 (C-2', C-3'), 115.06 (d, JCF = 21.4 Hz, C-12, C-12'' C-14, C-14''),
43.88 (C-1', C-4'). LC-MS (m/z): 481 [M+1]+, 100%. Anal. Calcd. for C26H18N8F2: C 64.99, H 3.78, N
23.32; Found: C 64.82, H 3.85, N 23.45.
N9-[4'-Chloro-2'-butynyl-1'-yl]-6-(4-methoxyphenyl)purine (19). White solid, 80% yield, m.p.
1
119–121 °C. H-NMR: δ 8.93 (1H, s, H-2), 8.87–8.82 (2H, m, Ar-H), 8.70 (1H, s, H-8), 7.18–7.13
(2H, m, Ar-H), 5.31 (2H, t, J = 3.5 Hz, N-CH2), 4.50 (2H, m, CH2Cl), 3.87 (3H, s, OCH3). 13C-NMR:
δ 161.70 (C-13), 152.67 (C-4), 152.0 (C-2), 151.58 (C-6), 145.19 (C-8), 131.13 (C-11, C-15), 129.49
(C-10), 127.67 (C-5), 114.11 (C-12, C-14), 80.34 (C-3'), 80.28 (C-2'), 55.34 (OCH3), 32.77 (C-1'),
30.55 (C-4'). LC-MS (m/z): 313 [M+1]+, 100%. Anal. Calcd. for C16H13N4OCl: C 61.44, H 4.19, N
17.91; Found: C 61.35, H 4.30, N 17.85.
N9-[4'-Chloro-2'-butynyl-1'-yl]-6-(4-fluorophenyl)purine (20a). Abrownish white solid, 64% yield,
1
m.p. 128–130 °C. H-NMR: δ 9.02 (1H, s, H-2), 8.92–8.89 (2H, m, Ar-H), 8.75 (1H, s, H-8),
7.46–7.42 (2H, m, Ar-H), 5.33 (2H, t, J = 2.0 Hz, N-CH2), 4.50 (2H, t, J = 2.0 Hz, CH2Cl). 13C-NMR:
δ 164.86 (d, 1JCF = 249 Hz, C-13), 151.99 (C-2), 151.83 (C-4), 151.65 (C-6), 145.80 (C-8), 131.79 (d,
4
2
3JCF = 8.8 Hz, C-11, C15), 131.68 (d, JCF = 2.52 Hz, C-10), 129.87 (C-5), 115.75 (d, JCF = 21.4 Hz,

Molecules 2011, 16
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C-12, C-14), 80.43 (C-3'), 80.14 (C-2'), 32.86 (C-1'), 30.52 (C-4'). LC-MS (m/z): 301 [M+1]+, 100%.
Anal. Calcd. for C15H10N4FCl: C 59.91, H 3.35, N 18.63; Found: C 60.0, H 3.40, N 18.55.
N9,N9''-[2'-butynyl-1',4'-diyl]-6-(4-fluorophenyl)purine (20b). A light brown solid, 11% yield, m.p.
1
225–227 °C. H-NMR: δ 8.99 (2H, s, H-2, H-2''), 8.93–8.90 (4H, m, Ar-H), 8.76 (2H, s, H-8, H-8''),
7.47–7.43 (4H, m, Ar-H), 6.01–5.30 (4H, s, 2 × N-CH2). LC-MS (m/z): 479 [M+1]+, 100%.Anal.
Calcd. for C26H16N8F2: C 65.27, H 3.37, N 23.42; Found: C 65.15, H 3.45, N 23.55.
4. Conclusions
On the purine base selection, a strong electron withdrawing 2,6-dichloro system on the purine base
is helpful for the cytotoxicity, ex: compounds 5a, 5b, 8b, 10a, 10b. This also suggests that 2,6-
dichloropurine base has significant potential for further work on the synthesis of anti-cancer
compounds. The simultaneous electron withdrawing chlorine at the 2-position and electron donating
methoxy group at 6-position on the purine ring did not contribute to the cytotoxic activity of the
examples studied, compounds 7, 12. A 6-methoxy group on the purine base did not contribute to the
cytotoxic activity in compounds 6 or 11, however, a 6-methoxypurine bearing a chloromethyl vinylic
dibromide moiety (compound 14) exhibited excellent cytotoxic activity. This is the first report of
activity in such a molecule and hence has a potential for further exploration in this direction. The 6-(4-
methoxyphenyl)purine moiety elicited very potent activity (compound 19). A riboside of this base also
exhibited excellent cytostatic activity [10]. The consistency of this purine base in eliciting tumor
inhibiting properties for wide range of cell lines is a very important finding from our work and from
that reported in[10]. It is important to note that in our case the linker is an acyclic unsaturated butyne
with a chloromethyl group and in the other case [10] it is a natural ribose sugar. Hence this purine base
may be considered as potential candidate for future anti-cancer drug development work. The 6-(4-
fluorophenyl)purine unit did not elicit any significant cytotoxicity in the compounds studied (16a, 20a,
20b). This suggests electron withdrawing 4-fluoro group on the phenyl ring was not helpful, a striking
difference with the beneficial 4-methoxy group.
In the linker selection, the methylchloromethylbutyne linker emerged as a viable non-sugar
unsaturated linker (compounds 10a, 19). Even the dimeric compound with the butyne linker, 10b
exhibited cytotoxicity indicating this linker with the triple bond is important. Similarly the
methylchloromethyl-cis-butyne linker compound 5a, its dimer 5b demonstrated excellent cytotoxic
activity. The methylchloromethyl-trans-butene linker compound 17 and the dimer 8belicited good
cytotoxic activity, but in other cases 8a did not contribute any activity. The mechanism of action
although not determined yet for our molecules may be different from purine nucleosides with natural
sugars. Further useful structural modifications of the active purines 5a, 5b, 14 and 19 is in progress.
Acknowledgments
We thank the Antitumor Evaluation and Biological Testing branch of the National Cancer Institute
(NCI, Bethesda, MD, USA) for the biological evaluation of the compounds described in this paper. We
also thank Mr. Quiang Zhang of Xavier RCMI Core for providing mass spectral data of the
compounds. The work described herein was supported by a grant 5SC1GM84808 from NIH/NIGMS.

Molecules 2011, 16
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Sample Availability: Samples of the compounds described in the experimental are available from the
authors.
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