Synthesis and biological properties of C-2, C-8, N-9 substituted 6-(3-chloroanilino)-purine derivatives as cyclin-dependent kinase inhibitors. Part II

Article abstract of DOI:10.1002/1521-4184(200112)334:11<345::AID-ARDP345>3.0.CO;2-1

In this study, C-2, C-8, N-9 substituted 6-(3-chloroanilino)purine derivatives were synthesized and their inhibitory effects on cyclin-dependent kinases (CDK2, 4) as well as their cytotoxicities were evaluated. The effects of substituents at the C-2, C-8, and N-9 positions of the substituted purine were investigated. Among the compounds tested, [6-(3-chloroanilino)-2-(2-hydroxymethyl-4-hydroxypyrrolidyl)- 9-isopropylpurine] (4h) was the most active inhibitor of CDK2 with IC₅₀ of 0.3μM i.e. a two-fold increased inhibitory activity as compared to roscovitine. Results from structure-activity relationship studies should allow the design of more potent and selective CDK2 inhibitors, which may provide an effective therapy for cancer or other CDK-dependent diseases.

Full text of DOI:10.1002/1521-4184(200112)334:11<345::AID-ARDP345>3.0.CO;2-1

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345–350
Chang-Hyun Oha),
6-(3-Chloroanilino)purines 345
Hee-Kwon Kima),
Su-Chul Leea),
Synthesis and biological properties of
C-2, C-8, N-9 substituted 6-(3-chloroanilino)-
purine derivatives as cyclin-dependent
kinase inhibitors. Part II
Changsok Oha),
Boem-Seok Yanga),
Hak June Rheeb),
Jung-Hyuck Choa)
In this study, C-2, C-8, N-9 substituted 6-(3-chloroanilino)purine derivatives were syn-
thesized and their inhibitory effects on cyclin-dependent kinases (CDK2, 4) as well as
their cytotoxicities were evaluated. The effects of substituents at the C-2, C-8, and N-9
positions of the substituted purine were investigated. Among the compounds tested,
[6-(3-chloroanilino)-2-(2-hydroxymethyl-4-hydroxypyrrolidyl)-9-isopropylpurine] (4h)
was the most active inhibitor of CDK2 with IC50 of 0.3µM, i.e. a two-fold increased
inhibitory activity as compared to roscovitine. Results from structure-activity relationship
studies should allow the design of more potent and selective CDK2 inhibitors, which may
provide an effective therapy for cancer or other CDK-dependent diseases.
a)Medicinal Chemistry Research Cen-
ter, Korea Institute of Science and
Technology, Seoul 130-650, Korea
b) Department of Chemistry,
Han Yang University, Ansan 425-791,
Korea
Key Words: CDK2 inhibitor; m-Chloroanilinopurine; Cell cycle regulation; Antiprolifera-
tive effect
Received: April 6, 2001 [FP555]
contact inhibition of cell growth and cellular differentia-
tion[5]. Abnormal up-regulation of cyclin D and cyclin E, thus
Introduction
Cell division is a cellular process that is tightly controlled by
negative and positive regulation. Recently many biochemi-
cal factors which govern the cell cycle process have been
identified[1]. One of the key regulators is a protein kinase
family called cell cycle dependent kinases (CDK)[2]. Among
such protein kinases, CDK1, CDK2, and CDK4 are known
to play a major role during the cell cycle. Each CDK forms
a heteroduplex with one of the cyclins to give an active
protein kinase [3]. That is, CDK4 forms a complex with D
type cyclins, whereas CDK1 complexes with cyclin B, and
CDK2 with cyclin A or cyclin E respectively. The cellular
amount of each cyclin varies in a cell cycle phase depend-
ent manner. In G1 phase, cyclin D1 appears and CDK4/cy-
clin D1 becomes an active kinase in that phase. However,
in G1-S transition, cyclin E exists transiently so that
CDK2/cyclin E is the active CDK in that transition phase.
During S phase progression, CDK2/cyclin Adevelopsmajor
kinase activity by accumulating cyclin A. For initiation and
progression of mitosis cyclin B is required with the rapid
increase of CDK1/cyclin B activity in the G2-M phase.
Whereas cyclins activate CDK activity through their bind-
ing, CDK activity can be inhibited by a specific binding of
CDK inhibitory proteins, called CDKI, such as p16, p21, and
p27 etc.[4]. p16 binds only to CDK4 protein to inhibit the
association between CDK4 and D type cyclin, and its loss
has been ascertained in almost 50% of human cancer cells.
p21 and p27 bind to general CDK/cyclin complexes to
inhibit their kinase activities. p21 is induced by tumor
suppressor protein; p53 and p27 induction is required for
activating CDK2 and CDK4 kinase activity, has been indi-
cated as one of the main cause of several human can-
cers[6]. In transgenic mice, the disruption of the p16 gene[7]
or constant overexpression of cyclin D1 protein[8] gener-
ates tumors. Encouraged by these findings, efforts to de-
velop CDK2 and CDK4 inhibitory compounds as anticancer
drugs have been undertaken in recent years. A number of
chemical agents that act selectively as CDK inhibitors have
been described: Flavopiridol[9], butyrolactone-I[10], and
purine derivatives (olomoucine, roscovitine)[11–13]. The
search for potent inhibitors of CDK’s has led to the discov-
ery of a family of C-2, C-6, N-9-substituted purines with high
CDK selectivity. Recently, olomoucine was found to stimu-
late massive apoptosis in cells which had been arrested in
G2 by the use of DNA-damaging agents[14]
.
OH
OH
OH
O
O
HN
N
COOCH
Cl
3
O
1
N
N
R
N
HO
O
HO
OH
N
H
2
R
N
OH
Olomoucine: R1=H, R2=Me
Roscovitine: R1=Et, R2=i-Pr
Flavopiridol
Butyrolactone-I
These encouraging results led us to investigate other C-2,
C-6, N-9-substituted purines as potential CDK inhibitors.
Here, we report the synthesis of a new potent and selective
CDK2 inhibitor, 6-(3-chloroanilino)-2-(2-hydroxymethyl-4-
hydroxypyrrolidyl)-9-isopropylpurine (4h). On the other
hand, in order to investigate the effect of the C-8 substituent
and an oxime moiety in the C-2 position of the purine, 4j,
7a, and 7b were synthesized and tested for CDK inhibition.
The structure-activity relationships of the synthesized com-
———
Correspondence: Prof. Jung-Hyuck Cho, Medicinal Chemistry
Research Center, Korea Institute of Science and Technology,
Seoul 130-650, Korea.
E-mail: choh@kist.re.kr
Fax: +82 2 958 5189
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/1111/0345 $17.50 +.50/0

346 6-(3-Chloroanilino)purines
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345–350
CI
CI
HN
Cl
HN
N
N
(CH ) CHI
N
N
N
3 2
N
CIC H NH
N
6
4
2
NaH, DMF
n-BuOH
N
N
H
Cl
N
Cl
N
Cl
N
H
1
3
2
CI
HN
N
aminoalcohol
N
N
n-BuOH/DMSO
N
R1
4
1
1
1
1
R :
OH
OH
c =
R :
d =
h=
N NH
R :
a =
e =
N
OH
OH
HN
R :
b =
f =
N
N
N
N
1
1
1
N
1
R :
R :
R :
N
S
g =
R :
OH
HO
OH
N
COOH
1
i=
R :
HO
Scheme 1. General synthesis of 2,6,9-trisubstituted purine derivatives.
Cl
Cl
HN
HN
HN
CI
O
HC
OH
N
NOH
N
N
N
N
N
N
N
N
CICOCOCI
DMSO
H2NOH.HCI
HC
N
N
N
N
N
N
4g
4j
Scheme 2. Synthetic route of oxime substituted purine derivative 4j from 4g.
pounds as well as their cytotoxicities on human cell lines
are reported.
(4-hydroxypiperidine, 2-hydroxymethylpyrrolidine etc.) in
n-butanol/DMSO (4:1) at 150 °C.
The alcohol 4g was converted into the corresponding alde-
hyde by oxidation using oxalyl chloride in DMSO. Com-
pound 4j was prepared by the treatment of the aldehyde
with hydroxyl amine (Scheme 2).
Chemistry
2,6-Dichloropurine (1), an important intermediate for the
preparation of purine derivatives, was synthesized from
adenine [15]
.
On the other hand, in order to investigate the effect of the
C-8 substituent on purine, C-8 substituted derivatives were
synthesized through the reaction of4,5-diaminopyrimidine-
2,6-dione[16,17] with acetic or benzoic acid anhydride as
shown in Scheme 3. Compounds 7a and 7b were prepared
from 6 by the same procedure as described for the prepa-
ration of 4c from 1.
The 2,6-dichloropurine was reacted with 3-chloroaniline in
n-butanol at 90 °C to yield 6-(3-chloroanilino)purine (2). N-9
alkylation of the purine (2) was carried out with alkyl iodide
in the presence of sodium hydride. The final compound (4)
was synthesized by reaction of 3 with a cyclic amino alcohol

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345–350
6-(3-Chloroanilino)purines 347
OH
Cl
N
O
HN
CI
N
N
NH2
NH2
1. R2(CO)2O
2.1N NaOH
POCl3
Cl
N
N
HN
R2
R2
N
N
N
R2
N
H
N
H
HO
N
R1
N
O
N
H
5
6
7
R1:
R2: -CH3
OH
OH
a=
b=
HN
HN
R1:
R2: phenyl
Scheme 3. Synthetic route of C-8 substituted purine derivatives.
solution was added followed by incubation in the dark for
4 h in normal culture. The culture medium was removed
and the formazan crystal formed depending on viable cell
number was dissolved in 100 µl of DMSO:EtOH(1:1) solu-
tion with agitation. The absorbance was measured at O.D.
570 nM.
Biological evaluation
Enzyme activity inhibition assay
The inhibition studies on cell cycle dependent kinase activi-
ties were performed using purified CDK2/cyclin A and
CDK4/cyclin D1 enzymes from infected sf21 insect cells.
For baculoviral overexpressions of proteins, we subcloned
each c-DNA into pBacPak8 baculoviral expression vector
(Clontech, USA). For CDK2 gene, hexa-histidine was
tagged on its N-terminus and for CDK4, glutathione S-
transferase(GST) gene was fused to its N-terminus for their
affinity purifications. CDK2/cyclin A enzyme was purified
using zinc-resin (Novagen, USA) from sf21 insect cell
culture into which CDK2 and cyclin A carrying baculovirus
had been cotransfected. For CDK4/cyclin D1 enzyme,
CDK4 and cyclin D1 carrying virus were cotransfected into
sf21 insect cells and the enzyme was purified using a
glutathione bead column (Pharmacia, Sweden). For both
enzymes, inhibition assays were performed in 20 µl of
50 mM Tris-HCl containing 10 µM ATP, 0.2 µCi of gamma-
P32 ATP, 10 mM MgCl2, 5 mM DTT. 4 µg of Human Rb
c-terminal peptide (a.a.769–928) was used as a substrate
for both enzymes. The reaction was continued for 10 min
in the presence of inhibitors and stopped by adding 80 µl
of 12% phosphoric acid. The stopped mixtures were trans-
ferred to a 96-well PVDF filter (Millipore, USA) prewetted
with 50% EtOH and drained by mild suction. The filter was
washed five times with 10 mM Tris-HCl (pH 8.0) containing
0.1M NaCl. The radioactivity of each well was counted with
a Microbeta counter (Wallac, Finland).
Results and discussion
In this study, C-2,6 and N-9 substituted 6-(3-chloroanil-
ino)purine derivatives were synthesized and their cellular
and enzymatic inhibitory activities are listed in Table 1. The
synthesized compounds were assayed for CDK2 and
CDK4 inhibition respectively.
The effect of substituents at the C-2 position of the purine
was investigated. Among the compounds tested, com-
pound 4h was the most active inhibitor (IC50 = 0.3 µM)
against CDK2. This degree of inhibition by compound 4h is
increased 2-fold when compared to that of roscovitine.
However, the presence of carboxylic acid or oxime on the
C-2 position of purine compound appeared to decrease
inhibition activity against CDK2 (4i and 4j). According to the
above result, it can be assumed that lone pair electrons on
the oxygen atom in the hydroxy group of C-2 position could
be provided for the hydrogen bonding to the residue of
CDK2 enzyme. We may, therefore, conclude that the ter-
minal group at the C-2 position of purine has a great
influence on the inhibition of CDK2. On the other hand, we
introduced methyl and phenyl group at the C-8 position of
purine to investigate the substituent effect. Compounds 7a
and 7b display a moderate low activity compared to com-
pound 4a. It can be suggested that the introduction of the
substitutent on the C-8 position of the purine led to a
lowering potency of inhibition.
Cancer cell growth inhibition assay
Cancer cell growth inhibition activities of synthesized com-
pounds were investigated against three human cancer cell
lines using MTT assays as previously described[15]. Human
stomach (SNU-1), melanoma (SKMEL-2), and leukemia
(K562) cell lines were supplied from the College of Medi-
cine, Seoul National University. Cells were maintained in
RPMI 1640 medium containing 5% fetal bovine serum at
37 °C under 5% CO2. About 2,000 cells were seeded into
96 well plates and left overnight. The following day, various
concentrations of compounds were added to each well in
triplicate and plates were incubated for two days. Cells in
no-drug treated wells grow exponentially in this growth
period. After two days, 50 µl of 0.2% MTT (Sigma, USA)
On the other hand, interesting results were observed in
cellular activities of the synthesized compounds. The de-
crease in the size and polarity of substituent at the C-2
position of the purine leads to a highly potent antiprolifera-
tive activity (4d and 4e). It seems that as the substitutent
has the smaller size and the lower polarity, the more easily
it becomes to permeate the target cell line.
One of the expected problems in studying biological activi-
ties of kinase inhibitory compounds which target the ATP
binding site is their selectivities against other ATP binding
proteins in cells. Many ATP binding proteins including ki-

348 6-(3-Chloroanilino)purines
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345–350
nases play essential roles in cells. Our purine derivatives
also are subject to this selectivity issue. According to our
data (unpublished observations), none of these com-
pounds inhibits either EGFR kinase or p42 MAP kinase
activity (IC50 values over at least 100 µM). However we can
not exclude the possibility of inhibitory actions of these
compounds on other cellular ATP binding proteins. In our
compounds, we could not correlate the activities of CDK2
and CDK4 inhibition very well with those of cancer cell
growth inhibition. The discrepancy between in vitro CDK
inhibitory activity and in vivo cell growth inhibition, espe-
cially apparent in the case of compounds 4e, 4i, and 4j,
might be due to the unidentified cellular side effects of these
compounds besides the inhibitory action of CDK activity.
However, 4d showed a good activity both in CDK enzyme
inhibition and cell growth inhibition. Its cancer cell growth
inhibition activity is as potent as that of doxorubicin. This
suggests that compound 4d could be a starting compound
for further derivatizations to obtain compounds with both a
high potency of CDK inhibition and good selectivity against
other cellular proteins.
Experimental protocols
Melting points (mp) were determined on a Thomas Hoover
apparatus and are uncorrected. 1H-NMR spectra: Varian Gem-
ini 300 spectrometer, tetramethylsilane (TMS) as an internal
standard. The mass spectrometer was a Hewlett Packard
Model HP5989A MS Engine (Palo Alto, CA, USA) mass spec-
trometer interfaced with a HP Model 59987A electrospray sys-
tem.
6-(3-Chloroanilino)-2-chloropurine (2)
3-Chloroaniline (7.2 ml, 56.7 mmol) and 2,6-dichloropurine (1,
4.0 g, 20.0 mmol) were dissolved in n-BuOH (30 ml) and the
solution was heated at 90 °C for 20 h. The resulting solution
was evaporated and diluted with ethyl acetate. The organic
layer was washed with water, 1N-HCl, and brine. Evaporation
of the solvent in vacuo gave a crude solid, which was washed
with cold acetone to give 2 as a white solid. Yield 4.8 g
(81.1%).– 1H-NMR(DMSO-d6): δ (ppm) = 7.16 (d, 1H, J = 7.73
Hz), 7.38 (t, 1H, J = 8.11 Hz), 7.86 (d, 1H, J = 7.43 Hz), 8.07
(s, 1H, C8-H), 8.97 (s, 1H), 10.58 (s, 1H, N9-H), IR (KBr) 3006,
3298 cm –1.– HRMS (EI) calcd for C11H7Cl2N5 280.1476, found
(M+) 280.0435
6-(3-Chloroanilino)-2-chloro-9-isopropylpurine (3)
Finally our synthesized compounds reported here tend to
have more potent inhibitory activities against CDK2 than
CDK4. This tendency was generally observed in other
purine derivative compounds as well [11–13]. At this time it
has not yet become clear whether CDK type specific inhi-
bition has more beneficial anticancer effects than CDK type
independent and potent inhibition. In our limited data, com-
pound 4d has a relatively good inhibition activity on both
CDK2 and CDK4 and this correlates with the best pattern
of cancer cell growth inhibition activity. This fact might
suggest that inhibition of both CDK2 and CDK4 is neces-
sary for effective suppression of cancer cell growth.
To a solution of NaH (0.03 g, 1.46 mmol) in dry DMF (5 ml) at
20 °C was added slowly a solution of 2 (0.28 g, 1.0 mmol) in
DMF under N2 gas. After 1 h isopropyl iodide (0.33 g, 2.0 mmol)
was added dropwise and the reaction mixture was stirred for
24 h. The resulting mixture was diluted with sat. NH4Cl aq.
solution (30 ml) and ethyl acetate (50 ml). The organic layer
was washed with water (50 ml × 3) and brine and dried over
anhydrous Na2SO4. Removal of the solvent gave a crude
residue, which was chromatographed on silica gel using ethyl
acetate as eluent to give 3 as a pale yellow solid. Yield 0.31 g
(94.0%).– 1H-NMR (DMSO-d6): δ (ppm) = 1.63 (d, 6H, J = 9.99
Hz), 4.78 (m, 1H), 7.24–7.35 (m, 4H, phenyl), 8.13 (s, 1H,
C8-H), 8.77 (bs, 1H, C6-NH).– IR (KBr) 1574, 1624, 3418 cm–1.–
HRMS (EI) calcd for C14H13Cl2N5 322.2536, found (M+)
322.0179
Table 1. Inhibitory activity of purine derivatives against CDK2 and
CDK4 enzymatic activity and growth of various human cancer
cells.
6-(3-Chloroanilino)-2-(4-hydroxypiperidyl)-9-isopropyl-
purine (4c)
————————————————————————————–
Compound
Enzyme Inhibition Cell growth inhibition
To a solution of compound 3 (0.2 g, 0.62 mmol) in n-butanol
was added 4-hydroxypiperidine (0.8g, 7.9 mmol) and the mix-
ture was heated in an evacuated sealed tube at 110 °C for 5 h.
After cooling, the solution was diluted with water and ethyl
acetate, and the organic layer was washed with water and brine.
Evaporation of the solvent in vacuo gave a crude solid, which
was recrystallized from ethyl acetate/n-hexane to give 4c as a
white solid. Yield 64.1%, m.p 174–176 °C.– 1H-NMR (CDCl3):
δ (ppm) = 1.56 (d, 6H, J = 8.13 Hz), 1.40 (bs, 4H), 2.04 (bs, 4H),
3.98 (m, 1H), 4.49 (m, 1H), 7.0 (d, 1H, J = 8.86 Hz), 7.24 (t, 1H,
J = 7.43 Hz), 7.36 (d, 1H, J = 8.48 Hz), 7.58(s, 1H).– IR (KBr)
3118, 3334(NH, OH) cm–1.– HRMS(EI) calcd for C19H23ClN6O
406.3833, found (M+) 406.1073
IC50 value (µM)*
IC50 value (µM)*
CDK2 CDK4
SNU-1 SKMEL-2K562
————————————————————————————–
4a
0.4
5.7
4.2
0.9
9.2
0.9
0.9
0.3
9.8
4.2
7.1
>10
>10
4.5
>10
6.9
17.8
16.2
64.6
1.7
31.6 23.4
34.7 29.5
4b
4c
>100
6.0
91.2
7.9
4d
4e
14.5
34.7
40.7
7.4
10.9 12.1
28.1 98.8
75.9 63.1
30.2 22.9
35.5 34.7
16.3 32.4
60.4 39.7
87.4 59.6
9.6 56.7
4f
4g
7.8
6.5
4h
4i
>10
21.8
10.2
78.4
98.5
11.9
2.1
6-(3-Chloroanilino)-2-(2-N-hydroxyliminopyrrolidyl)-
9-isopropylpurine (4j)
4j
>10
>10
>10
7a
>10
>10
0.5
7b
To a solution of oxalyl chloride (39.3 ml, 0.31 mol) in dry
methylene chloride (10 ml) at –78 °C was added slowly a
solution of DMSO (44.4 ml, 0.57 mol) in dry methylene chloride
under N2 gas. After 1 h the reaction mixture was added drop-
wise to a solution of 4g (0.33 g, 2.0 mmol) in dry methylene
chloride at –78 °C and stirred for 1 h. The resulting mixture was
diluted with methylene chloride (50 ml). The organic layer was
washed with water (50 ml × 3), brine, and dried over anhydrous
Roscovitine
Doxorubicin
9.0
–
–
3.0
3.24
————————————————————————————–
*Average value of two or three independent measurements is
shown and the discrepancy between measurements was within
30%.

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345–350
6-(3-Chloroanilino)purines 349
Na2SO4. Removal of the solvent gave a crude residue, which
was recrystallized from ethyl acetate/n-hexane to give 4j as a
white solid. Yield: 16.0% , mp:154–156 °C.– 1H-NMR (CDCl3):
δ (ppm) = 1.57 (d, 1H, J = 8.10 Hz), 2.06 (m, 2H), 2.18 (m, 2H),
2.42 (m, 2H), 3.82 (m, 4H), 4.67 (m, 1H), 5.26 (m, 1H), 6.92 (d,
1H, J = 7.88 Hz), 7.17 (t, 1H, J = 7.43 Hz), 7.27 (s, 1H), 7.61
(s, 1H), 8.21 (m, 3H), 11.88 (s, 1H).– IR (KBr) 1632, 1673(C=N),
3212, 3325(OH) cm–1.– HRMS(EI) calcd for C20H23ClN6O
398.8949, found (M+) 398.2307.
J = 1.18 Hz), 7.60 (s, 1H), 7.89 (s, 1H), 8.10 (s, 1H).– IR (KBr)
3246, 3340 (OH) cm–1.– HRMS(EI) calcd for C20H26ClN7O
415.9672, found (M+) 415.1374
4d: Yield 71.0% , mp: 150–152 °C
1H-NMR (CDCl3): δ (ppm) = 1.54 (d, 6H, J = 6.69 Hz), 2.97 (t,
4H, J = 4.92 Hz), 3.69 (t, 4H, J = 6.60 Hz), 4.68 (m, 1H), 6.97
(d, 1H, J = 9.03 Hz), 7.21 (t, 1H, J = 8.07 Hz), 7.42 (d, 1H,
J = 4.36 Hz), 7.60 (s, 1H), 8.07 (s, 1H), 8.17 (s, 1H).– IR (KBr)
2974, 3256(NH) cm–1.– HRMS (EI) calcd for C18H22ClN7
371.8723, found (M+) 371.1633.
Compounds 4a–4h were prepared by the same procedure as
described for the preparation of 4c.
2,6-Dihydroxy-8-methylpurine (5a)
4e: Yield 62.6% , mp 235–237 °C
4,5-Diamino-2,6-dihydroxypyrimidine (3.0 g, 21.1 mmol) was
refluxed in 100 ml of acetic anhydride. A clear yellow solution
resulted after 20 min and was refluxed for 15 h; at the end of
this time the excess acetic anhydride was distilled under re-
duced pressure and the syrupy residue boiled in 100 ml of 1.5N
NaOH for 30 min. The solution was treated with charcoal and
acidified while hot with glacial acetic acid. Upon cooling, the
solution yielded light yellowish solid. Yield 3.0 g (83.1%), mp
>300 °C.– 1H-NMR (DMSO-d6): δ (ppm) = 2.30 (s, 3H), 10.68
(s, 1H).– HRMS(EI) calcd for C6H6N4O2 166.2417, found (M+)
166.0176
1H-NMR (CDCl3): δ (ppm) = 1.68 (d, 6H, J = 6.81 Hz), 2.71 (m,
4H), 4.20 (m, 4H), 4.67 (m, 1H), 7.08 (d, 1H, J = 6.03 Hz), 7.27
(t, 1H, J = 8.21 Hz), 7.61 (d, 1H, J = 6.18 Hz), 8.21 (s, 1H), 8.52
(s, 1H).– IR (KBr) 3210, 3354 cm–1.– HRMS (EI) calcd for
C18H21ClN6S 388.9487, found (M+) 388.2076.
4f: Yield 42.3% , mp 195–196 °C
1H-NMR (CDCl3): δ (ppm) = 1.57 (d, 6H, J = 8.33 Hz), 3.15 (m,
2H), 3.37 (m, 2H), 3.79 (m, 2H), 4.11 (m, 2H), 4.67 (m, 3H),
5.02 (m, 1H), 7.03 (d, 1H), 7.25 (m, 4H), 7.58 (s, 1H), 8.15 (s,
1H).– IR (KBr) 2936, 3160, 3330 cm–1.– HRMS (EI) calcd for
C20H25ClN6O 400.9478, found (M+) 400.1736
2,6-Dihydroxy-8-phenylpurine (5b)
The title compound 5b was prepared using benzoyl chloride
from 4,5-diamino-2,6-dihydroxypyrimidine in a similar manner
to that described for the preparation of 5a.
Yield 3.0 g (56.1%) mp >300 °C.– 1H-NMR (DMSO-d6): δ (ppm)
= 7.46 (m, 3H), 8.07(d, 2H, J = 8.05 Hz), 10.58 (s, 1H, N9-H).–
HRMS(EI) calcd for C11H8N4O2 228.2784, found (M+) 228.0276
4g: Yield 60.3% , mp 185–186 °C
1H-NMR (CDCl3): δ (ppm) = 1.47 (d, 6H, J = 8.33 Hz), 1.77 (m,
2H), 1.98 (m, 2H), 2.15 (m, 2H), 3.79 (bs, 4H), 4.94 (m, 2H),
4.58 (m, 1H), 6.99 (d, 1H, J = 7.89 Hz), 7.22 (t, 1H, J = 8.09
Hz), 7.44 (d, 1H, J = 7.74 Hz), 7.56 (s, 1H), 8.06 (s, 1H), 8.19
(s, 1H).– IR (KBr) 2867, 2966, 3336 cm–1.– HRMS(EI) calcd for
C19H23ClN6O 386.9247, found (M+) 386.3042.
2,6-Dichloro-8-methylpurine (6a)
2,6-Dihydroxy-8-methylpurine( 5a, 0.30 g, 1.81 mmol) was sus-
pended in a mixture of 10 ml of phosphoryl chloride and 0.7 ml
of diethylaniline and was refluxed for 5 h under N2. After the
mixture was cooled to room temperature, excess phosphoryl
chloride was distilled off under reduced pressure. The residue
was dissolved in 10 g of ice water, and was extracted with ethyl
ether (100 ml × 5). The organic solvent was evaporated in
vacuo to give a crude oil, which was chromatographed on silica
gel using chloroform/methanol (10:1) as an eluent to give 6a as
a white solid. Yield 0.072 g (19.9%), mp 245–249 °C.– 1H-NMR
(DMSO-d6): δ (ppm) = 2.58 (s, 3H, CH3) 10.78 (bs, 1H).
4i: Yield 48.3% , mp 150–152 °C
1H-NMR (CDCl3): δ (ppm) =1.38 (d, 6H, J = 8.33 Hz), 1.48 (bs,
2H), 2.30 (m, 1H), 3.61 (m, 2H), 3.81 (m, 1H), 4.35 (t, 2H,
J = 9.82 Hz), 4.56 (s, 1H), 6.83 (d, 1H, J = 7.68 Hz), 7.05 (t, 1H,
J = 7.99 Hz), 7.22 (d, 1H, J = 7.83 Hz), 7.52 (s, 1H), 7.96 (s,
1H), 8.38 (s, 1H), 10.38 (bs, 1H).– IR (KBr) 1638, 2866, 2972,
3116, 3346 cm–1.– HRMS (EI) calcd for C20H23ClN6O3
430.9275, found (M+) 430.1508.
4h: Yield 47.8% , mp 140–142 °C
1H-NMR (CDCl3): δ (ppm) =1.38 (d, 6H, J = 8.11 Hz), 1.58 (bs,
2H), 2.31 (m, 1H), 3.65 (m, 2H), 3.81 (bs, 1H), 4.36 (t, 2H,
J = 9.82 Hz), 4.56 (s, 1H), 6.89(d, 1H, J = 7.68 Hz), 7.05 (t, 1H,
J = 7.99 Hz), 7.25 (d, 1H, J = 7.80 Hz), 7.48 (s, 1H), 7.96 (s,
1H), 8.38 (s, 1H),– IR (KBr ) 2930, 3336 cm–1.– HRMS (EI) calcd
for C19H23ClN6O2 402.8833, found (M+) 402.1577.
2,6-Dichloro-8-phenylpurine (6b)
The title compound 6b was prepared from 5b (1.0 g, 4.38 mmol)
in a similar manner to that described for the preparation of 6a.
Yield 0.41 g (35.0%), mp 265–269 °C.– 1H-NMR (DMSO-d6): δ
(ppm) = 7.46 (m, 3H), 8.07 (d, 2H, J = 8.05 Hz), 10.88 (s, 1H).
7a: Yield 54.3%, mp 184–187 °C
4a: Yield 74.3%, mp 130–132 °C
1H-NMR (CDCl3): δ (ppm) =1.53 (d, 6H, J = 5.83 Hz), 2.69 (s,
3H), 3.63 (t, 2H, J = 3.27 Hz), 3.89 (t, 2H, J = 4.69 Hz), 4.70
(m, 1H), 7.05 (d, 1H, J = 7.92 Hz), 7.85 (d, 1H, J = 7.86 Hz),
8.07 (t, 1H, J = 1.87 Hz), 8.06 (s, 1H).– HRMS (EI) calcd for
C17H21ClN6O 360.98353, found (M+) 360.2408 C22H23ClN6O
422.9603, found (M+) 422.2046.
1H-NMR (CDCl3): δ (ppm) = 1.55 (d, 6H, J = 4.83 Hz), 3.66 (t,
2H, J = 3.27 Hz), 3.88 (t, 2H, J = 4.69 Hz), 4.70 (m, 1H), 5.77
(t, 1H, J = 5.43 Hz), 5.77 (t, 1H), 7.05 (d, 1H, J = 8.05 Hz), 7.23
(t, 1H, J = 8.55 Hz), 7.45 (d, 1H, J = 7.80 Hz), 7.6(s, 1H), 7.85
(s, 1H), 8.06 (s, 1H) ; IR (KBr) 2938, 3088, 3380 cm–1.–
HRMS (EI) calcd for C16H19ClN6O 346.8724, found (M+)
346.2076.
7b: Yield 74.3% , mp 198–201 °C
4b: Yield 84.3% , mp 116–118 °C
1H-NMR (CDCl3): δ (ppm) = 1.54 (d, 6H, J = 4.79 Hz), 3.43 (t,
2H, J = 3.27 Hz), 3.79 (t, 2H, J = 4.69 Hz), 4.58 (m, 1H), 7.02
(d, 1H, J = 7.44 Hz), 7.33 (q, 1H, J = 3.44 Hz ), 7.45 (m, 3H),
7.85 (d, 1H, J = 7.55 Hz), 7.95 (t, 2H, J = 8.02 Hz), 8.06 (d, 1H,
1H-NMR (CDCl3): δ (ppm) = 1.55 (d, 6H, J = 7.68 Hz), 2.54 (m,
6H), 3.70 (t, 2H), 3.83 (t, 4H, J = 8.38 Hz), 4.63 (m, 1H), 7.01
(d, 1H, J = 7.86 Hz), 7.22 (t, 1H, J = 8.17 Hz), 7.41 (d, 1H,

350 6-(3-Chloroanilino)purines
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 345–350
[10] M. Kitagawa, T. Okabe, H. Oginio, H. Matsumoto, I. Suzuki-
Takahashi, T. Kokubo, H. Higashi, S. Saito, Y. Taya, H.
Yasuda, Y. Ohba, A. Okuyama, Oncogene 1993, 8, 2425–
2432.
J = 8.44 Hz).– HRMS (EI) calcd for C22H23ClN6O 422.9603,
found (M+) 422.2046.
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