Synthesis of pyrrolo[2,3-d]pyrimidine derivatives and their antiproliferative activity against melanoma cell line

Article abstract of DOI:10.1016/j.bmcl.2009.10.051

Synthesis of a new series of diarylureas and amides having pyrrolo[2,3-d]pyrimidine scaffold is described. Their in vitro antiproliferative activities against A375 human melanoma cell line and HS 27 fibroblast cell line were tested and the effect of subst

Full text of DOI:10.1016/j.bmcl.2009.10.051

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6538–6543
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of pyrrolo[2,3-d]pyrimidine derivatives and their antiproliferative
activity against melanoma cell line
Myung-Ho Jung a, Hwan Kim a, Won-Kyoung Choi a, Mohammed I. El-Gamal a,b, Jin-Hun Park a,
Kyung Ho Yoo a, Tae Bo Sim a, So Ha Lee a, Daejin Baek c, Jung-Mi Hah a, Jung-Hyuck Cho a,
Chang-Hyun Oh a,b,
*
a Biomaterials Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
b Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
c Department of Chemistry, Hanseo University, Seosan 356-706, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a new series of diarylureas and amides having pyrrolo[2,3-d]pyrimidine scaffold is described.
Their in vitro antiproliferative activities against A375 human melanoma cell line and HS 27 fibroblast cell
line were tested and the effect of substituents on pyrrolo[2,3-d]pyrimidine was investigated. The newly
synthesized compounds, except N-acetyl derivatives (Id, Ie, and Im), generally showed superior or similar
activity against A375 to Sorafenib. Among all of these derivatives, compounds Iq and Ir having imidazole
and morpholine moieties, respectively, showed the most potent antiproliferative activity against A375.
Ó 2009 Published by Elsevier Ltd.
Received 25 August 2009
Revised 6 October 2009
Accepted 10 October 2009
Available online 15 October 2009
Keywords:
Pyrrolo[2,3-d]pyrimidine
A375
HS 27
Antiproliferative activity
Melanoma
Melanoma is the most aggressive form of skin cancer and is the
fastest growing cancer in the United States.1,2 Early stage mela-
noma can be cured surgically. However, melanoma metastasizing
to major organs (stage IV) is virtually incurable.2 Patients with ad-
vanced melanoma have a median survival time of less than one
year, and the estimated 5-years survival rate is less than 15%.2,3
With the incidence of melanoma rapidly rising in the United States
and other developed countries, there is an urgent need to develop
more effective drugs.4–6
The current treatments involve surgical removal of the tumor,
immunotherapy, radiotherapy, chemotherapy, various combina-
tions, or the use of new treatments in clinical trials. As for immu-
notherapy, interferon alfa-2b (Intron-A)7 is approved by both the
FDA and EMEA for adjuvant treatment of melanoma patients, and
aldesleukin (proleukin)8,9 is also approved for the treatment of
metastatic melanoma in the USA.
now report the synthesis and antiproliferative activity against
A375 human melanoma cell line and HS 27 fibroblast cell line of
these compounds (Fig. 1).
The general method for synthesis of pyrrolo[2,3-d]pyrimidine
ureas and amides is shown in Schemes 2–4. The geminal dicyano
intermediate 2 was prepared from 122 with malononitrile by heat-
ing in DMF in the presence of anhydrous K2CO3. Synthesis of com-
pound 3 can be achieved by refluxing 2 with thiourea in the
presence of potassium tert-butoxide. Cyclization to 4 can be
achieved by neutralization of the thiol potassium salt 3 using 5 N
aqueous HCl followed by heating with 10 N aqueous NaOH. Reduc-
tion of the thiol compound 4 using Raney nickel afforded 7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (5) (Scheme 1).
The N-benzoyl protected compound 6 was prepared by reacting 5
with benzoyl chloride in pyridine. N-Arylation of 6 using 1-iodo-4-
nitrobenzene in the presence of potassium carbonate, copper iodide,
It is recently reported that diarylurea derivatives, such as Sorafe-
nib, are promising, potent, and selective antiproliferative agents for
treatment of melanoma.10–15 There is a considerable number and
variety of diarylureas identified as anticancer agent.16–21
Encouraged by the interesting antiproliferative activity of dia-
rylurea derivatives, a new series of diarylureas and amides con-
taining pyrrolo[2,3-d]pyrimidine moiety was synthesized. We
and L-proline afforded compound 7. The 4-benzoylamino substituted
p-nitrophenyl compound 7 was reduced and deprotected using
tin(II)chloride to provide the diamino compound 8, which was subse-
quently treated with the appropriate isocyanates to provide the cor-
responding urea derivatives (Ia–c). The amide derivatives (Ik–l) were
obtained by condensation of 8 with the corresponding carboxylic acid
derivatives using EDCI/HOBt (Scheme 2).
The N-acetyl compound 9 was obtained by condensation of the
amino compound 5 with acetic acid in the presence of EDCI/HOBt.
* Corresponding author. Tel.: +82 2 958 5160; fax: +82 2 958 5189.
E-mail address: choh@kist.re.kr (C.-H. Oh).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.10.051

M.-H. Jung et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6538–6543
6539
R1
N
O
CF3
H
H3C
O
Cl
N
H
O
N
N
O
N
N
H
N
H
N
N
H
R2
Sorafenib
Pyrrolo[2,3-d ]pyrimidine derivatives
R1 = H, CH3CO, C6H5CO
R2 = Ar, ArNH
Figure 1. Structures of Sorafenib and pyrrolo[2,3-d]pyrimidine derivatives.
CH(OEt)2
c
NH
b
a
H2N
NH2
OEt
OEt
H2N
NC
OEt
CN OEt
Br
N
N
N
N
SK
SH
1
2
3
4
d
NH
H2N
N
N
5
Scheme 1. Reagents and conditions: (a) malononitrile, K2CO3, DMF, 50 °C, 54%; (b) thiourea, potassium tert-butoxide, EtOH, reflux, 61%; (c) 5 N HCl, H2O, 10 N NaOH, 50 °C,
88%; (d) Raney-Ni, H2O, reflux, 87%.
a
b
H
NH
H
H2N
NH
N
N
N
NO2
N
N
O
N
N
N
O
N
N
5
6
7
O
O
d
c
H2N
N
H2N
R2
NH2
N
H
N
H
N
N
N
N
N
N
Ia-c
Ik-l
8
e
N
H2N
N
H
R3
Scheme 2. Reagents and conditions: (a) benzoyl chloride, pyridine, 50 °C, 33%; (b) 1-iodo-4-nitrobenzene, K2CO3, CuI,
80%; (d) aryl isocyanate, THF, Ia: 27%, Ib: 58%, Ic: 35%; (e) benzoic acid derivative, HOBt, EDCI, TEA, DMF, 80 °C, Ik: 86%, Il: 47%.
L
-proline, DMSO, 90 °C, 67%; (c) Sn2ClꢀH2O, EtOH, reflux,
Preparation of the 4-acetylamino substituted p-nitrophenyl com-
pound 10 was accomplished by the same procedure described for
the preparation of 7. Reduction of the nitro compound 10 using
Pd–C/H2 afforded the corresponding amino compound 11, which
was subsequently treated with the appropriate isocyanates to pro-
vide the corresponding urea derivatives (Id–e). The amide deriva-
tive (Im) was obtained by condensation of 11 with 4-chloro-3-
(trifluoromethyl)benzoic acid using EDCI/HOBt (Scheme 3).
The 4-benzoylamino substituted p-nitrophenyl compound 7
was reduced with Pd–C/H2 to the corresponding amino compound
12. Compounds If–j and In–s were also prepared as described for
the preparation of compounds Ia–e and Ik–m, respectively
(Scheme 4).
The antiproliferative activity of the newly synthesized com-
pounds against A375 human melanoma cell line and HS 27 fibro-
blasts was tested.23 The ability of pyrrolo[2,3-d]pyrimidine

6540
M.-H. Jung et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6538–6543
H
H
a
N
b
NH
H3C
N
NH
H2N
H3C
N
NO2
O
N
N
N
N
O
N
N
5
9
10
O
R2
H
H
N
c
d
N
N
H3C
N
H3C
N
NH2
N
H
H
O
N
N
O
O
N
N
N
N
11
Id-e
e
O
H
N
N
R3
H3C
N
H
Im
L-proline, DMSO, 90 °C, 47%; (c) Pd/C, H2, THF,
Scheme 3. Reagents and conditions: (a) acetic acid, HOBt, EDCI, TEA, DMF, 80 °C, 45%; (b) 1-iodo-4-nitrobenzene, K2CO3, CuI,
25%; (d) aryl isocyanate, THF, Id: 70%, Ie: 31%; (e) 4-chloro-3-(trifluoromethyl)benzoic acid, HOBt, EDCI, TEA, DMF, 80 °C, Im: 47%.
H
H
N
N
a
N
N
NO2
NH2
O
N
N
O
N
N
12
7
b
c
O
O
H
H
N
R2
N
N
N
N
R3
N
N
N
H
H
H
O
N
N
O
N
If-j
In-s
Scheme 4. Reagents and conditions: (a) Pd/C, H2, THF, 85%; (b) aryl isocyanate, THF, If: 22%, Ig: 25%, Ih: 54%, Ii: 82%, Ij: 56%; (c) benzoic acid derivative, HOBt, EDCI, TEA, DMF,
80 °C, In: 26%, Io: 33%, Ip: 36%, Iq: 17%, Ir: 17%, Is: 96%.
derivatives to inhibit the growth of A375 and HS 27 cell lines is
summarized in Tables 1 and 2. Sorafenib was selected as the refer-
ence standard, because it has been extensively used in clinical tri-
als for melanoma.4,24
The newly synthesized compounds, except N-acetyl derivatives
(Id, Ie, and Im), generally showed superior or similar activity
against A375 to Sorafenib. Among these compounds, Ia, Ic, If–h,
Ik, and In–s showed similar or slightly improved activity against
A375 compared with Sorafenib.
Regarding the substituents on the 4-position of pyrrolo[2,3-
d]pyrimidine, compounds Ig, Ih, Io, and Ip having benzoylamino
moiety were generally more potent than compounds Ia, Ib, Ik,
and Il having an unsubstituted amino group against A375. This
suggests that the aromatic amide substituent at this position is
more favorable.
The effect of substituents on the phenyl ring of the tail was also
investigated. The introduction of chloro group at para position in
30-trifluoromethylphenyl ring of tail (Ih, Ik, and Io) significantly
enhanced the antiproliferative activity compared with compounds
(Ii, Il, and Ip). This can be attributed to the enhanced affinity pro-
duced by this chloro group to the target protein.
By comparing the activity of derivatives substituted with amide
and urea moieties at pyrrolo[2,3-d]pyrimidine side chain as a lin-
ker, it was found that the derivatives with amide moieties (Ik, In,
Io, Ip, and Is) were generally more potent than that with urea moi-
eties (Ib, Ig, Ih, Ii, and Ij). In addition, most of the newly synthe-
sized amide derivatives (Ik and In–s) demonstrated higher
antiproliferative activity against A375 than that of Sorafenib with
urea moiety. These results were seemed to indicate the effect of
the linker on the activity.
Upon comparing the antiproliferative activity of the newly
synthesized compounds with that of Sorafenib against HS 27
fibroblast cell line, it was found that the new pyrrolo[2,3-
d]pyrimidine derivatives generally showed superior or similar po-
tency against HS 27 to Sorafenib. This assumes little selectivity of
the newly synthesized compounds towards A375. Among all of

M.-H. Jung et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6538–6543
6541
Table 1
Antiproliferative activity of pyrrolo[2,3-d]pyrimidine ureas (Ia–j).
Structure
Compd no.
R1
H
R2
IC50 (lM)
A375P
5.4
HS 27
R1
Cl
Cl
N
N
H
Ia
2.9
NH
Cl
N
N
O
HN
R2
Ib
Ic
H
H
8.1
3.0
>10
CF3
CF3
9.5
CF3
Id
Ie
If
CH3CO
CH3CO
C6H5CO
C6H5CO
C6H5CO
>10
>10
4.1
4.0
4.6
>10
>10
7.8
4.5
6.0
Cl
Cl
Cl
CF3
Cl
Cl
Cl
Ig
Ih
Cl
Cl
CF3
Ii
Ij
C6H5CO
9.5
4.5
CF3
CF3
C6H5CO
9.8
5.6
>10
7.8
CF3
Sorafenib
these derivatives, compound Iq having 4-benzoylamino substi-
tuted 40-amide and imidazole moieties demonstrated superior
activity against A375 and lower potency against HS 27 compared
with Sorafenib.
In conclusion, a new series of pyrrolo[2,3-d]pyrimidine deriva-
tives was synthesized based on our previous literature studies,
by focusing on the structure–activity relationship studies of this
class of compounds. Among all of these derivatives, compounds

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M.-H. Jung et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6538–6543
Table 2
Antiproliferative activity of pyrrolo[2,3-d]pyrimidine amides (Ik–s).
Structure
Compd no.
R1
H
R3
IC50 (lM)
A375P
2.9
HS 27
5.6
R1
Cl
CF3
N
H
N
H
Ik
N
N
N
O
R3
Il
H
9.7
>10
2.0
2.3
4.7
7.1
>10
5.0
5.5
5.9
CF3
Cl
CF3
Im
In
Io
Ip
CH3CO
C6H5CO
C6H5CO
C6H5CO
Cl
Cl
Cl
CF3
CF3
CF3
Iq
C6H5CO
0.8
0.9
2.8
1.6
N
N
CF3
Ir
Is
C6H5CO
N
O
CF3
CF3
C6H5CO
2.0
5.6
6.6
7.8
Sorafenib
Iq25 and Ir25 having 4-benzoylamino substituted 40-amide moie-
ties, and imidazole and morpholine moieties, respectively, showed
the most potent antiproliferative activity against A375 human mel-
anoma cell line.
Further modification of these compounds in order to improve
their potency is currently in progress. Our ultimate goal is to iden-
tify several compounds that are highly potent and highly selective
against melanoma cells.
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supplemented with 10% FBS (Welgene) and 1% penicillin/streptomycin
(Welgene) in a humidified atmosphere with 5% CO2 at 37 °C. A375P cells
were taken from culture substrate with 0.05% trypsin-0.02% EDTA and plated
at a density of 5 ꢁ 103 cells/well in 96 well plates and then incubated at 37 °C
for 24 h in a humidified atmosphere with 5% CO2 prior to treatment of various
concentration (threefold serial dilution, 12 points) of test compounds. The
A357P cell viability was assessed by the conventional 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays
were carried out with CellTiter 96Ò (Promega) according to the manufacturer’s
instructions. The absorbance at 590 nm was recorded using EnVision 2103
(Perkin–Elmer; Boston, MA, US). The IC50 was calculated using GraphPad Prism
4.0 software.
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25. Compound Iq: IR (KBr): 3417, 1504, 1273, 1128, 752 cmꢂ1 1H NMR (300 MHz,
.
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DMSO-d6) d 11.27 (br s, 1H), 10.73 (br s, 1H), 8.68 (s, 1H), 8.50–8.44 (m, 2H),
8.28 (br s, 1H), 8.21 (br s, 1H), 8.10 (d, 2H, J = 7.2 Hz), 8.00–7.89 (m, 5H), 7.75
(br s, 1H), 7.66–7.64 (m, 1H),7.59–7.54 (m, 2H), 6.84 (d, 1H, J = 3.6 Hz). MS (ESI)
m/z 582.1 (M+H)+. Compound Ir: IR (KBr): 3380, 1523, 1314, 1130, 931,
743 cmꢂ1 1H NMR (300 MHz, DMSO-d6) d 11.20 (br s, 1H), 10.57 (br s, 1H),
.
8.67 (s, 1H), 8.30 (s, 1H), 8.16–8.07 (m, 5H), 7.98–7.95 (m, 1H), 7.89–7.88 (m,
1H), 7.67–7.51 (m, 4H), 7.37–7.34 (m, 1H), 6.84 (d, 1H, J = 3.6 Hz), 3.76–3.72
(m, 4H), 2.99–2.98 (m, 4H). MS (ESI) m/z 587.2 (M+H)+.
23. A375P cells were purchased from American Type Culture Collection (ATCC,
Rockville, MD, US) and maintained in DMEM medium (Welgene, Daegu, Korea)