Diarylureas and diarylamides with pyrrolo[2,3-d]pyrimidine scaffold as broad-spectrum anticancer agents

Article abstract of DOI:10.1248/cpb.c13-00249

A series of diarylureas and diarylamides possessing pyrrolo[2,3-d] pyrimidine scaffold was designed and synthesized. The in vitro antiproliferative activities of a selected group of the target compounds against NCI-60 cell line panel were tested and compared with Sorafenib and Imatinib as reference compounds. Most of the compounds showed strong and broad-spectrum antiproliferative activities. Compounds IVa, IVb, and IVd with benzamido moiety at position 4 of the pyrrolo[2,3-d]pyrimidine nucleus, para-disubstituted phenyl ring at N1-position of pyrrolo[2,3-d]pyrimidine scaffold, and urea linker showed strong and broad-spectrum anticancer results with high potencies and efficacies. In addition, the amide derivatives Vb and Vc demonstrated one-digit nanomolar IC₅₀ values over two and one cell line(s), respectively. Amid all the target compounds, compound IVa demonstrated the best results in both one-dose and five-dose testing modes. It showed 109.18% mean % inhibition over the NCI-60 cancer cell line panel at 10 μM concentration, submicromolar 50% inhibitory concentration (IC₅₀) values over eight cell lines of eight different cancer types, and high efficacy with total growth inhibition (TGI) and 50% lethal concentration (LC₅₀) values less than 4.22 μM over three colon, ovarian, and prostate cancer cell lines. It showed superior potency and efficacy to Sorafenib and Imatinib over most of the tested cell lines.

Full text of DOI:10.1248/cpb.c13-00249

January 2014
Regular Article
Chem. Pharm. Bull. 62(1) 25–34 (2014)
25
Diarylureas and Diarylamides with Pyrrolo[2,3-d]pyrimidine Scaffold as
Broad-Spectrum Anticancer Agents
,a,b
Mohammed Ibrahim El-Gamala,b,c and Chang-Hyun Oh*
a Center for Biomaterials, Korea Institute of Science and Technology; P.O. Box 131, Cheongryang, Seoul 130–650,
b
Republic of Korea: Department of Biomolecular Science, University of Science and Technology; 113 Gwahangno,
c
Yuseong-gu, Daejeon 305–333, Republic of Korea: and Department of Medicinal Chemistry, Faculty of Pharmacy,
University of Mansoura; Mansoura 35516, Egypt.
Received April 1, 2013; accepted October 8, 2013
A series of diarylureas and diarylamides possessing pyrrolo[2,3-d]pyrimidine scaffold was designed
and synthesized. The in vitro antiproliferative activities of a selected group of the target compounds against
NCI-60 cell line panel were tested and compared with Sorafenib and Imatinib as reference compounds. Most
of the compounds showed strong and broad-spectrum antiproliferative activities. Compounds IVa, IVb, and
IVd with benzamido moiety at position 4 of the pyrrolo[2,3-d]pyrimidine nucleus, para-disubstituted phenyl
ring at N1-position of pyrrolo[2,3-d]pyrimidine scaffold, and urea linker showed strong and broad-spectrum
anticancer results with high potencies and efficacies. In addition, the amide derivatives Vb and Vc demon-
strated one-digit nanomolar IC50 values over two and one cell line(s), respectively. Amid all the target com-
pounds, compound IVa demonstrated the best results in both one-dose and five-dose testing modes. It showed
109.18% mean % inhibition over the NCI-60 cancer cell line panel at 10µM concentration, submicromolar
50% inhibitory concentration (IC50) values over eight cell lines of eight different cancer types, and high ef-
ficacy with total growth inhibition (TGI) and 50% lethal concentration (LC50) values less than 4.22µM over
three colon, ovarian, and prostate cancer cell lines. It showed superior potency and efficacy to Sorafenib and
Imatinib over most of the tested cell lines.
Key words diarylurea; diarylamide; pyrrolo[2,3-d]pyrimidine; anticancer; cytotoxicity
Cancer is a generic term for a large group of diseases that is currently subjected to clinical trials for other types of can-
can affect any part of the body. Lung, stomach, liver, colon, cer. Imatinib (Gleevec®, Fig. 1) is an example of diarylamides
and breast cancer cause the most cancer deaths each year. which is used for treatment of chronic myeloid leukemia
About 30% of cancer deaths are due to the five leading behav- (CML) with diminished side effects.20)
ioral and dietary risks: high body mass index, low fruit and
The target pyrrolo[2,3-d]pyrimidine compounds were
vegetable intake, lack of physical activity, tobacco use, and designed as conformationally restricted analogs of Sorafenib
alcohol intake. More than 70% of all cancer deaths occurred by isosteric replacement of the 4-phenoxypyridine moiety of
in low- and middle-income countries. Deaths from cancer the lead compound with a pyrrolo[2,3-d]pyrimidine nucleus
worldwide are projected to exceed 13 million in 2030.1) In (Fig. 1). The 50% inhibitory concentration (IC50) values for
spite of the extensive efforts and investment in research, the most of the target compounds over A375P human melanoma
management of human malignancies still constitutes a major cell line only were previously reported.2,3) Upon further test-
challenge for contemporary medicinal chemistry. There has ing over NCI-60 cancer cell line panel of nine different cancer
been an urgent need for development of more efficient anti- types, the target compounds showed broad-spectrum anti-
cancer agents with minimal side effects.
cancer activities with high efficacies and potencies. Herein the
Diarylureas and diarylamides have been highlighted as biological results are reported in details.
potential antiproliferative agents against a variety of cancer
cell lines.2–17) Sorafenib (Nexavar®, Fig. 1) is an example of
Results and Discussion
anticancer diarylureas that has been approved by the U.S.
Chemistry The target pyrrolo[2,3-d]pyrimidine dia-
Food and Drug Administration (FDA) for treatment of ad- rylureas and diarylamides were synthesized by the pathway
vanced renal cancer.18) It has also been approved in Europe of reactions illustrated in Charts 2–4.2,3) But at the beginning,
for treatment of hepatocellular carcinoma (HCC).19) Sorafenib it was essential to synthesize the intermediate compound 7H-
Fig. 1. Structures of Sorafenib, Imatinib, and the Target Compounds
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: choh@kist.re.kr

26
Vol. 62, No. 1
Reagents and conditions: (a) malononitrile, K2CO3, DMF, 50°C, 17–18h, 54%; (b) thiourea, potassium tert-butoxide, EtOH, reflux, 16–17h; rt, 1–2h, 61%; (c) 5N HCl,
H2O, 10N NaOH, 50°C, 50min, 88%; (d) Raney-Ni, H2O, reflux, 4h, 87%.
Chart 1
Reagents and conditions: (a) benzoyl chloride, pyridine, 50°C, overnight, 33%; (b) 1-iodo-4-nitrobenzene or 1-iodo-3-nitrobenzene, K2CO3, CuI, L-proline, DMSO, 90°C,
40h, 67% (7a), 40% (7b); (c) SnCl2·H2O, EtOH, reflux, overnight, 80% (8a), 50% (8b); (d) aryl isocyanate, THF, rt, 24h, 27–60%; (e) carboxylic acid derivative, HOBt,
EDCI, TEA, DMF, 80°C, 24h, 10–86%.
Chart 2
pyrrolo[2,3-d]pyrimidin-4-amine (5). It could be successfully acetate (TEA) (Chart 2). In both pathways for synthesis of
achieved by the pathway shown in Chart 1. The geminal di- compounds Ia–d and IIa–f, the aniline amino groups reacted
cyano intermediate 2 was prepared from compound 121) with while the amino group at position 4 of the pyrrolo[2,3-d]-
malononitrile by heating in N,N-dimethylformamide (DMF) in pyrimidine nucleus remained unaffected.
the presence of anhydrous K2CO3. Synthesis of compound 3
N-Acetylation of compound 5 was carried out by condensa-
was carried out by refluxing 2 with thiourea in the presence of tion with acetic acid in the presence of HOBt/EDCI/TEA to
potassium tert-butoxide. Cyclization to 4 could be achieved by produce 4-acetamidopyrrolo[2,3-d]pyrimidine (9). Preparation
neutralization of the thiol potassium salt 3 using 5N aqueous of the 4-acetylamino substituted p-nitrophenyl compound 10
HCl followed by heating with 10N aqueous NaOH. Reduction was accomplished by the same procedure described for the
of the thiol compound 4 using Raney nickel afforded 7H- preparation of 7a, b. Reduction of the nitro compound 10
pyrrolo[2,3-d]pyrimidin-4-amine (5) (Chart 1).
using Pd/C in hydrogen atmosphere afforded the correspond-
The N-benzoyl protected compound 6 was prepared by ing amino compound 11, which was subsequently treated
heating 5 with benzoyl chloride in pyridine. N-Arylation of with 4-chloro-3-(trifluoromethyl)phenyl isocyanate to provide
6 using 1-iodo-3(4)-nitrobenzene in the presence of potas- the corresponding urea derivative IIIa. The amide derivative
sium carbonate, copper iodide, and L-proline afforded the N- IIIb was obtained by condensation of 11 with 4-chloro-3-
(nitrophenyl) compounds 7a, b. The 4-benzoylamino substitut- (trifluoromethyl)benzoic acid using HOBt/EDCI/TEA (Chart
ed nitrophenyl compounds 7a, b were reduced and deprotected 3).
using tin(II) chloride to provide the diamino compounds 8a, b,
The 4-benzamido substituted nitrophenyl compounds 7a, b
which were subsequently treated with the appropriate iso- were reduced with Pd/C in hydrogen atmosphere to the cor-
cyanates to provide the corresponding urea derivatives Ia–d. responding amino compound 12a, b. Compounds IVa–f and
The amide derivatives IIa–f were obtained by condensation of Va–c were also prepared as described for the preparation of
8a, b with the corresponding carboxylic acid derivatives using compounds Ia–d and IIa–f, respectively (Chart 4). Table 1
1-hydroxybenzotriazole (HOBt)/1-ethyl-(3-(3-dimethylamino)- illustrates structures of the final compounds, their yield per-
propyl)-carbodiimide hydrochloride (EDCI)/triethylamine centages, and their melting points.

January 2014
27
Reagents and conditions: (a) acetic acid, HOBt, EDCI, TEA, DMF, 80°C, 24h, 45%; (b) 1-iodo-4-nitrobenzene, K2CO3, CuI, L-proline, DMSO, 90°C, 40h, 45%; (c)
Pd/C, H2, THF, 50 psi, rt, 2h, 25%; (d) 4-chloro-3-(trifluoromethyl)phenyl isocyanate, THF, rt, 24h, 31%; (e) 4-chloro-3-(trifluoromethyl)benzoic acid, HOBt, EDCI, TEA,
DMF, 80°C, 24h, 47%.
Chart 3
Reagents and conditions: (a) Pd/C, H2, 50 psi, THF, rt, 2h, 85% (12a), 70% (12b); (b) aryl isocyanate, THF, rt, 24h, 22–82%; (c) benzoic acid derivative, HOBt, EDCI,
TEA, DMF, 80°C, 24h, 23–36%.
Chart 4
Antiproliferative Activities against 60 Cell Line Panel that the aromatic amide substituent at this position is more
at the National Cancer Institute (NCI). Single-Dose Test- favorable.
ing Structures of the target compounds were submitted to
By comparing the activities of derivatives with amide and
the (NCI), Bethesda, Maryland, U.S.A.,22) and the eleven com- urea moieties at pyrrolo[2,3-d]pyrimidine side chain as a
pounds shown in Fig. 2 were selected on the basis of degree linker, it was found that compounds IVb and IVd possessing
of structural variation and computer modeling techniques for urea moiety were more active than the corresponding amide
evaluation of their antineoplastic activity. The selected com- analogues Va and Vc. This may be attributed to that the lon-
pounds were subjected to in vitro anticancer assay against ger spacer, urea moiety, may geometrically permit appropriate
tumor cells in a full panel of 60 cell lines taken from nine fitting of the molecule at the receptor site. Or the terminal
different tissues (blood, lung, colon, central nervous system NH group of the urea moiety may form additional hydrogen
(CNS), skin, ovary, kidney, prostate, and breast). The com- bond(s) at the receptor site. Any or both of these effects would
pounds were tested at a single dose concentration of 10µM, enable optimal drug–receptor interaction, and hence higher
and the percentages of growth inhibition over the 60 tested antiproliferative activity.
cell lines were determined. The mean inhibition percentages
for each of the tested compounds over the full panel of cell N1 position of the pyrrolo[2,3-d]pyrimidine nucleus showed
lines are illustrated in Fig. 2.
higher mean %inhibition than its meta-disubstituted phenyl
Compound IVd with para-disubstituted phenyl ring at
Regarding the substituents on the 4-position of pyrrolo[2,3- positional isomer IVf. This can be rationalized that the direc-
d]pyrimidine nucleus, compound Vc with benzamido moiety tion of terminal moiety can affect the appropriate fitting at the
was more active than the corresponding acetamido derivative receptor site and hence affinity and activity.
IIIb. This can be attributed to the steric and/or electronic dif-
The effect of substituents of the terminal aryl ring on mean
ferences between benzoyl and acetyl groups. This suggests %inhibition was also investigated. The introduction of meta-

28
Vol. 62, No. 1
Table 1. Structures of the Target Compounds, and Their Yield Percentages and Melting Points
Site of attachment of
Compound No.
R1
R2
Yield%
27
Melting point (°C)
232–235 (dec.)
phenyl ring
Ia
H
H
H
H
H
para
Ib
Ic
para
meta
meta
para
35
60
46
47
>300
225–226 (dec.)
>300
Id
IIa
216–218 (dec.)
IIb
IIc
IId
H
H
H
para
meta
meta
86
10
69
215–217 (dec.)
204–207 (dec.)
214–216 (dec)
IIe
IIf
H
H
meta
meta
para
26
28
31
221–223
218–220 (dec.)
274–276 (dec.)
IIIa
CH3CO
IIIb
IVa
IVb
IVc
CH3CO
PhCO
PhCO
PhCO
para
para
para
para
47
22
25
82
241–242 (dec.)
225–227 (dec.)
249–250 (dec.)
234–236 (dec.)
IVd
PhCO
para
54
190–192 (dec.)

January 2014
29
Table 1. Continued.
Site of attachment of
phenyl ring
Compound No.
R1
R2
Yield%
56
Melting point (°C)
220–221 (dec.)
IVe
PhCO
para
IVf
Va
Vb
Vc
PhCO
PhCO
PhCO
PhCO
meta
para
para
para
56
23
36
33
140–141
193–195
181–183 (dec.)
193–195 (dec.)
10 µM concentration, both compounds showed lethal effects
(>100% inhibition) over 32 and 25 cell lines, respectively.
Both compounds demonstrated broad-spectrum cytotoxicities
over all the nine tested cancer types.
Five-Dose Testing Compounds IVa, IVb, IVd, IVf, Vb,
and Vc with promising results in single-dose testing were
further tested in a five-dose testing mode, in order to deter-
mine their IC50, total growth inhibition (TGI), and 50% lethal
concentration (LC50) values over the 60 cancer cell lines. The
results of these six compounds over the most sensitive cell
line of the nine tested cancer subpanels are shown in Table
2. The results of Sorafenib and Imatinib were obtained from
NCI datawarehouse index23) and are inserted in Table 2. It is
noteworthy that all the selected six compounds possess ben-
zamido moiety at position 4 of the pyrrolo[2,3-d]pyrimidine
nucleus and para-disubstituted phenyl ring at N1-position of
pyrrolo[2,3-d]pyrimidine scaffold.
Fig. 2. Mean Inhibition Percentages Observed with the Final Com-
pounds in Single-Dose (10µM) 60-Cancer Cell Line Screening
Mean %inhibition represents the mean inhibition percentages over the 60 cell
lines. The inhibition percentages were calculated by subtracting the growth per-
centages from 100.
As shown in Table 2, most of the compounds exhibited
(4′-methylimidazol-1-yl) moiety on the 3′-(trifluoromethyl)- broad-spectrum activities with high potency (in sub-micromo-
phenyl ring (compound IIf) slightly reduced the activity lar and micromolar scale) over all the nine cell lines. Most of
compared with compound IId. On the other hand, para-chloro the IC50 values were less than 10µM. Compound IVa, with the
group improved the activity of compound Vc compared with highest mean %inhibition value in one-dose testing mode (Fig.
Vb with 4′-unsubstituted 3′-(trifluoromethyl)phenyl terminal 2), showed the highest potency among the six compounds
ring. Compound IVa with 2′,3′-dichlorophenyl terminal ring tested in five-dose testing mode. Its IC50 values were in sub-
exhibited higher mean %inhibition than the corresponding micromolar scale over eight cell lines. It demonstrated higher
positional isomer compound IVb with 3′,4′-dichlorophenyl potency than Sorafenib against eight cell lines. Its TGI results
terminal moiety.
showed higher efficacy than Sorafenib over seven cell lines.
Among all the target compounds, it was found that com- And its potency and efficacy against all the nine cell lines
pounds IVa, IVb, and IVd possessing benzamido moiety were higher than Imatinib. In addition, the diarylamide de-
at position 4 of the pyrrolo[2,3-d]pyrimidine nucleus, para- rivative Vb exhibited one-digit nanomolar IC50 values, 7 and
disubstituted phenyl ring at N1-position of pyrrolo[2,3-d]- 8nM, over NCI-H460 non-small cell lung and HCT-15 colon
pyrimidine scaffold, and urea linker demonstrated the highest cancer cell lines, respectively. The diarylamide derivative Vc
mean %inhibition values; 109.18%, 96.91%, and 107.86%, exerted high potency over HCT-15 colon cancer cell line also
respectively. So it can be concluded that the presence of those with IC50 value of 7nM. Compounds Vb and Vc demonstrated
three moieties together is essential for antiproliferative activity sub-micromolar IC50 values over five and six cell lines, respec-
this series of pyrrolo[2,3-d]pyrimidine compounds.
tively. Both compounds Vb and Vc showed higher potencies
Compounds IVa and IVd exerted lethal effect on the and efficacies than Sorafenib and Imatinib against most of the
NCI-60 cancer cell line panel with mean IC50 values more tested cell lines.
than 100%. The % inhibitions of these two compounds over
Compounds IVa, IVb, IVd, IVf, Vb, and Vc showed high
each cell line of the NCI-60 panel are illustrated in Fig. 3. At efficacies also over the nine cell lines. The TGI and LC50

30
Vol. 62, No. 1
Fig. 3. %Inhibition Expressed by Compounds IVa and IVd at a Single-Dose Concentration of 10µM over the NCI-60 Cancer Cell Lines
Table 2. IC50, TGI, and LC50 Values (μM) of the Tested Compounds over the Most Sensitive Cell Line of Each Subpanel
Cancer cell line
Compd. No. Results (µM)
CCRF-CEMa) NCI-H460b) HCT-15c)
U251d)
LOX IMVIe) OVCAR-4f ) CAKI-1g)
PC-3h)
HS 578Ti)
j)
IVa
IC50
TGIk)
0.53
3.21
0.36
1.21
NAm)
1.71
5.85
40.10
1.35
3.06
6.89
2.13
9.88
0.16
5.42
0.57
1.40
3.46
0.94
2.29
7.37
1.20
2.55
5.43
2.09
8.39
28.60
0.13
18.60
>50
0.36
2.23
5.70
2.00
3.16
6.31
19.95
50.12
100
0.76
2.04
0.88
1.91
4.14
0.89
1.80
3.67
1.57
3.48
7.70
2.36
8.18
21.90
5.46
49.20
3.84
0.58
1.57
0.86
2.60
>50
1.19
5.77
>50
1.27
4.61
>50
2.55
22.60
>50
6.01
49.70
>50
1.48
4.88
>50
2.51
3.98
100
24.90
l)
LC50
>50
42.70
1.74
>50
>50
4.21
j)
IVb
IC50
0.99
2.38
8.79
1.06
TGIk)
2.83
6.97
16.30
21.20
2.86
l)
LC50
38.70
0.81
22.00
0.77
>50
>50
11.70
1.25
j)
IVd
IC50
2.00
1.69
TGIk)
3.73
2.19
6.77
3.81
3.01
l)
LC50
>50
5.30
>50
8.60
7.25
j)
IVf
IC50
0.66
2.51
2.82
2.04
1.25
TGIk)
9.05
10.20
32.80
0.007
45.70
15.00
10.90
5.93
l)
LC50
>50
>50
>50
>50
29.00
10.10
41.00
j)
Vb
IC50
NAm)
NAm)
NAm)
0.85
0.008
12.90
46.40
0.14
0.23
0.82
TGIk)
2.45
19.90
l)
LC50
>50
19.50
0.30
>50
>50
>50
j)
Vc
IC50
0.007
1.44
0.40
1.26
TGIk)
18.20
1.74
2.27
2.75
4.06
5.91
7.41
l)
LC50
>50
6.08
14.50
2.51
>50
13.80
3.16
49.80
3.16
>50
j)
Sorafenib
Imatinib
IC50
2.00
2.51
1.58
2.00
TGIk)
5.01
5.01
3.16
2.51
25.12
12.59
5.01
l)
LC50
100
25.12
15.85
31.62
79.43
7.94
6.31
100
100
100
j)
IC50
15.85
63.10
19.95
79.43
19.95
39.81
79.43
19.95
63.10
31.62
79.43
19.95
63.10
15.85
39.81
100
TGIk)
l)
LC50
100
100
100
100
100
a) Leukemia cell line; b) non-small cell lung cancer cell line; c) colon cancer cell line; d) CNS cancer cell line; e) melanoma cell line; f ) ovarian cancer cell line; g) renal
cancer cell line; h) prostate cancer cell line; i) breast cancer cell line; j) IC50 is the concentration producing 50% inhibition; k) TGI is the concentration producing 100% inhibi-
tion; l) LC50 is the concentration causing 50% lethality (50% tumor regression); m) NA means that the datum is not available.
values were less than 50µM over most of the cell lines. Of
special interest, compound IVa with the best results was
able to induce TGI and LC50 at U251 CNS cancer cell line,
OVCAR-4 ovarian cancer cell line, and PC-3 prostate cancer
cell line at concentrations below 4.22µM. Similarly, compound
IVb showed TGI and LC50 values of 1.80µM and 3.67µM,
respectively, over OVCAR-4 ovarian cancer cell line. The six
tested compounds showed higher potencies and efficacies than
Sorafenib and Imatinib against most of the tested cell lines.
Kinase Screening In an attempt to study the possible
Table 3. IC50 Values of Compounds IVa, IVb, IVd, and Sorafenib over
Potential Kinases
B-RAF
(wild type)
VEGFR1
PDGFR
IVa
>3µM
>3µM
>3µM
25nM
>3µM
>3µM
>3µM
26nM
>3µM
>3µM
>3µM
90nM
IVb
IVd
Sorafenib
mechanism(s) of antiproliferative activity of the most active growth factor receptor (PDGFR) kinases. The three com-
target compounds, compounds IVa, IVb, and IVd were tested pounds showed weak potency against these kinases, compared
for inhibitory effect over wild type B-RAF, vascular endothe- with Sorafenib (Table 3). So it can be concluded that these
lial growth factor receptor 1 (VEGFR1), and platelet-derived target compounds inhibit cancer cell growth through other

January 2014
31
mechanism(s) other than B-RAF, VEGFR1, and PDGFR ki- 1.0g, 5.1mmol) was added portionwise thereto. The reaction
nase inhibition.
mixture was heated to 50°C for 17–18h. After completion of
the reaction, the reaction mixture was cooled to room tem-
perature, and extracted between toluene (50mL×3) and water
Conclusion
A
series of diarylureas and diarylamides possessing (40mL). The combined organic layer extracts were dried over
pyrrolo[2,3-d]pyrimidine scaffold was designed with struc- anhydrous MgSO4, the organic solvent was evaporated under
tural similarity to Sorafenib and synthesized. Eleven final reduced pressure, and the residue was purified by column
compounds were tested at a single-dose concentration of 10µM chromatography (silica gel, ethyl acetate–hexane 1:6, v/v) to
at the NCI over 60 cancer cell line panel of nine different can- obtain the title product (0.5g, 54%). IR (KBr): 3350, 2978,
1
cer types, and six of them were subsequently tested in five- 2250, 1748, 1633, 1441, 1375, 1124cm－1; H-NMR (CDCl3,
dose testing mode. Compounds IVa, IVb, IVd, IVf, Vb, and 300MHz) δ: 4.69 (t, 1H, J=5.3Hz), 4.03 (t, 1H, J=7.3Hz),
Vc showed broad-spectrum anticancer activities with strong 3.75–3.67 (m, 2H), 3.58–3.53 (m, 2H), 2.32–2.27 (m, 2H),
potencies and high efficacies. Among them, compounds IVa, 1.27–1.21 (m, 6H).
IVb, and IVd possessing benzamido moiety at position 4 of
4,6-Diamino-5-(2,2-diethoxyethyl)pyrimidine-2-thiol
the pyrrolo[2,3-d]pyrimidine nucleus, para-disubstituted phe- Potassium Salt (3) A solution of compound 2 (5.0g,
nyl ring at N1-position of pyrrolo[2,3-d]pyrimidine scaffold, 27.4mmol) in ethanol (60mL) was cooled to 0°C, and thiourea
and urea linker exerted the highest mean % inhibition in one- (2.4g, 31.5mmol) was added portionwise thereto. Then a sus-
dose testing mode, and high potency and efficacy in five-dose pension of potassium tert-butoxide (3.5g, 31.2mmol) in etha-
testing experiments. So we can conclude that these moieties nol (5.5mL) was added dropwise. The reaction mixture was
together with the pyrrolo[2,3-d]pyrimidine nucleus constitute heated under reflux for 16–17h, then cooled to room tempera-
the pharmacophore of this series of compounds as potential ture. Ethanol (30mL) was added, and the mixture was stirred
anticancer agents.
at room temperature for 1–2h. The produced solid was filtered
Compound IVa demonstrated the best results in both one- out, washed with ethanol (20mL), and dried to get the pure
dose and five-dose testing modes. It showed 109.18% mean title compound (5.0g, 61%). mp: 223–225°C (dec.); IR (KBr):
% inhibition over the NCI-60 cancer cell line panel at 10µM 3434, 1630, 1435, 1314, 1050cm－1; 1H-NMR (DMSO-d6,
concentration, submicromolar IC50 values over eight cell lines 300MHz) δ: 4.92 (brs, 4H), 4.40 (t, 1H, J=5.3Hz), 3.65–3.55
of eight different cancer types, and high efficacy with TGI (m, 2H), 3.42–3.36 (m, 2H), 2.43–2.41 (m, 2H), 1.11–1.03 (m,
and LC50 values less than 4.22µM over three colon, ovarian, 6H).
and prostate cancer cell lines. Its potency and efficacy results
4-Amino-7H-pyrrolo[2,3-d]pyrimidine-2-thiol (4) To a
were promising compared with Sorafenib and Imatinib. So solution of compound 3 (7.5g, 25.3mmol) in distilled water
this compound can be considered as promising lead for future (40mL), 5N HCl aqueous solution (11.8mL) was added drop-
development of potential anticancer agents with high potency wise. After 20min, distilled water (7mL) was added thereto.
and efficacy.
The reaction mixture was heated at 50°C for 50min, then
cooled to room temperature. The reaction mixture was neu-
tralized to pH 7 by dropwise addition of 10N NaOH aqueous
Experimental
General All melting points were obtained on a Walden solution. The precipitated solid was filtered out, washed with
Precision Apparatus Electrothermal 9300 apparatus and are acetonitrile (50mL), and vacuum dried to obtain the pure
uncorrected. Mass spectra (MS) were taken in electrospray title product (3.7g, 88%). mp: 218–220°C (dec.); IR (KBr):
1
ionization (ESI) mode on a Waters 3100 Mass Detector 3413, 3171, 2884, 1655, 1584, 1133cm－1; H-NMR (DMSO-
(Waters, Milford, MA, U.S.A.). Nuclear magnetic resonance d6, 300MHz) δ: 11.59 (brs, 1H), 11.30 (brs, 2H), 6.88 (d, 1H,
(NMR) spectroscopy was performed using a Bruker ARX-300, J=6.0Hz), 6.47 (d, 1H, J=5.9 Hz).
300MHz (Bruker Bioscience, Billerica, MA, U.S.A.) with
7H-Pyrrolo[2,3-d]pyrimidin-4-amine (5) To a suspen-
TMS as an internal standard. Purities of the target compounds sion of compound 1 (1.0g, 6.0mmol) in water (50mL) was
(>95%) were determined by LC-MS analysis using the fol- added Raney nickel (3.0g). The reaction mixture was heated
lowing system: Waters 2998 photodiode array detector, Waters at reflux for 4h, and then the hot solution was filtered through
3100 mass detector, Waters SFO system fluidics organizer, celite. The nickel residue was washed with water (100mL).
Waters 2545 binary gradient module, Waters reagent man- The combined aqueous filtrate was evaporated to dryness
ager, Waters 2767 sample manager, Sunfire™ C18 column to yield the product (0.7g, 87.5%). mp: 206–208°C. (dec.).
(4.6×50mm, 5µm particle size); Solvent gradient=95% A at 1H-NMR (DMSO-d6, 300MHz) δ: 12.37 (brs, 1H), 8.22
0min, 1% A at 5min; solvent A: 0.035% trifluoroacetic acid (brs, 2H), 8.15 (s, 1H), 7.29 (d, 1H, J=1.5Hz), 6.80 (d, 1H,
(TFA) in water; solvent B: 0.035% TFA in CH3OH; flow rate= J=1.1 Hz).
3.0mL/min; the area under curve (AUC) was calculated using
N-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (6) To
Waters MassLynx 4.1 software. Unless otherwise noted, all a suspension of compound 2 (1.0g, 7.5mmol) in pyridine
solvents and reagents were commercially available and used (25mL) was added benzoyl chloride (1.3g, 8.9mmol). The
without further purification.
mixture was heated at 50°C overnight. The mixture was cool
2-(2,2-Diethoxyethyl)malononitrile (2) To
a
suspen- and evaporated under reduced pressure. The residue was dilut-
sion of anhydrous potassium carbonate (1.4g, 10.1mmol) in ed with ethyl acetate (30mL) and water (50mL), and then the
dry DMF (3mL), malononitrile (0.6g, 9.1mmol) was slowly organic layer was dried over anhydrous Na2SO4. The solvent
added. The resulting mixture was heated at 41°C for 8h was evaporated under reduced pressure, and purification was
under nitrogen atmosphere. The reaction mixture was cooled achieved by flash column chromatography (silica gel, ethyl
to room temperature, then 2-bromo-1,1-diethoxyethane (1, acetate–hexane 1:2, v/v) to afford the title compound (0.6g,

32
Vol. 62, No. 1
1
33%) as a pale yellow solid. mp: 220–222°C. (dec.); H-NMR J=2.4Hz), 7.72–7.69 (m, 2H), 7.60–7.57 (m, 2H), 7.54 (s, 1H),
(DMSO-d6, 300MHz) δ: 12.03 (s, 1H), 11.00 (s, 1H), 8.53 (s, 7.51–7.49 (m, 2H), 7.13 (brs, 2H), 6.74 (d, 1H, J=3.8Hz); ESI-
1H), 8.06 (d, 2H, J=7.3Hz), 7.65–7.60 (m, 1H), 7.56–7.51 (m, MS: 413.0 [M+1]+.
2H), 7.42–7.41 (m, 1H), 6.59–6.57 (m, 1H).
1-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-
Synthesis of Compounds 7a, b A mixture of compound (3,5-bis(trifluoromethyl)phenyl)urea (Ib): 1H-NMR (DMSO-
6 (0.3g, 1.3mmol), 1-iodo-4-nitrobenzene or 1-iodo-3-ni- d6, 300MHz) δ: 9.46 (s, 1H), 9.20 (s, 1H), 8.15 (s, 2H), 8.10
trobenzene (0.6g, 2.4mmol), anhydrous potassium carbon- (s, 1H), 7.73 (d, 2H, J=8.9Hz), 7.65 (brs, 1H), 7.62 (d, 2H,
ate (0.5g, 2.4mmol), CuI (20.0mg, 0.1mmol), and L-proline J=8.9Hz), 7.51 (d, 1H, J=3.6Hz), 7.13 (brs, 2H), 6.75 (d, 1H,
(30mg, 0.3mmol) in dry DMSO (5mL) was heated at 90°C J=3.6Hz); ESI-MS: 481.0 [M+1]+.
under nitrogen atmosphere for 40h. The cooled solution was
1-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-
1
partitioned between H2O (20mL) and ethyl acetate (20mL). (3,4-dichlorophenyl)urea (Ic): H-NMR (DMSO-d6, 300MHz)
The aqueous layer was extracted with ethyl acetate (3×10mL). δ: 9.09 (s, 1H), 9.08 (s, 1H), 8.12 (s, 1H), 7.98 (s, 1H), 7.90 (d,
The combined organic layer extracts were washed with brine, 1H, J=2.4Hz), 7.52–7.51 (m, 2H), 7.45–7.44 (m, 1H), 7.42–7.41
and dried over anhydrous Na2SO4. The solvent was evaporated (m, 1H), 7.39–7.38 (m, 1H), 7.36–7.35 (m, 1H), 7.17 (brs, 2H),
under reduced pressure, and purification was achieved by flash 6.78 (d, 1H, J=3.6Hz). ESI-MS: 413.0 [M+1]+.
chromatography (silica gel, ethyl acetate–hexane 1:2, v/v) to
afford the target compound 7a or 7b.
1-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-
(3,5-bis(trifluoromethyl)phenyl)urea (Id): 1H-NMR (DMSO-
N-(7-(4-Nitrophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- d6, 300MHz) δ: 9.63 (s, 1H), 9.46 (s, 1H), 8.70 (brs, 1H),
1
benzamide (7a): Yield: 67%; mp: 223–224°C (dec.); H-NMR 8.43 (brs, 2H), 8.15 (brs, 1H), 8.01 (brs, 1H), 7.85 (d, 1H,
(DMSO-d6, 300MHz) δ: 11.35 (brs, 1H), 8.52–8.43 (m, 3H), J=3.6Hz), 7.78 (brs, 1H), 7.66 (brs, 1H), 7.56–7.48 (m, 2H),
8.38–8.33 (m, 3H), 8.20–8.08 (m, 3H), 7.62–7.60 (m, 2H), 7.42–7.40 (m, 1H) 7.33–7.20 (m, 1H). ESI-MS: 481.0 [M+1]+.
6.89–6.87 (m, 1H).
General Procedure for Synthesis of Compounds IIa–f
N-[7-(3-Nitrophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]- A mixture of compound 8a, b (30mg, 0.1mmol), the appro-
1
benzamide (7b): Yield: 40%; mp: 212–214°C (dec.); H-NMR priate carboxylic acid derivative (0.2mmol), HOBt (36mg,
(DMSO-d6, 300MHz) δ: 11.33 (brs, 1H), 8.98–8.88 (m, 1H), 0.3mmol), and EDCI (38mg, 0.2mmol) in dry DMF (1.0mL)
8.73 (s, 1H), 8.40–8.37 (m, 1H), 8.27–8.24 (m, 1H), 8.20–8.08 was cooled to 0°C under nitrogen atmosphere. Triethylamine
(m, 3H), 7.88 (t, 1H, J=8.2Hz), 7.68–7.64 (m, 1H), 7.59–7.54 (0.03mL, 0.2mmol) was added thereto at the same tempera-
(m, 2H), 6.90 (d, 1H, J=3.4 Hz).
ture. The mixture was then stirred at 80°C for 24h. The reac-
Synthesis of Compounds 8a, b A mixture of compound tion mixture was cooled and then partitioned between saturat-
7a or 7b (1.0g, 2.8mmol) and SnCl2·H2O (3.1g, 13.7mmol) in ed aqueous sodium carbonate and ethyl acetate. The organic
ethanol (20mL) was heated under reflux overnight, and then layer was separated, and the aqueous layer was extracted with
cooled to room temperature. The reaction mixture was con- ethyl acetate (3×5mL). The combined organic layer extracts
centrated under reduced pressure, and the residue was diluted were washed with brine and dried over anhydrous Na2SO4.
with ethyl acetate (30mL) and saturated aqueous NaHCO3 After evaporation of the organic solvent, the residue was puri-
(100mL). The organic layer was separated, washed with brine, fied by column chromatography using the appropriate propor-
and dried over anhydrous Na2SO4. The solvent was evaporated tion of ethyl acetate and hexane as mobile phase.
under reduced pressure, and purification was achieved by flash
column chromatography (silica gel, ethyl acetate) to afford the (trifluoromethyl)benzamide (IIa): 1H-NMR (DMSO-d6,
target compounds. 300MHz) δ: 10.63 (s, 1H), 8.32 (brs, 1H), 8.28 (brs, 1H),
N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-
4-Amino-7-(4-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine 8.12 (s, 1H), 8.00–7.97 (m, 1H), 7.94–7.93 (m, 1H), 7.91–7.90
1
(8a): Yield: 80%; mp: 231–232°C (dec.); H-NMR (DMSO-d6, (m, 1H), 7.85–7.83 (m, 1H), 7.83–7.78 (m, 2H), 7.56 (d, 1H,
300MHz) δ: 8.13–8.06 (m, 1H), 7.32–7.29 (m, 3H), 7.04 (brs, J=3.6Hz), 7.15 (brs, 2H), 6.77 (d, 1H, J=3.6Hz); ESI-MS:
2H), 6.66–6.63 (m, 3H), 5.21 (brs, 2H).
4-Amino-7-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine
(8b): Yield: 50%; mp: 221–224°C (dec.); H-NMR (DMSO-d6, chloro-3-(trifluoromethyl)benzamide (IIb): H-NMR (DMSO-
300MHz) δ: 8.09 (s, 1H), 7.40 (d, 1H, J=3.58Hz), 7.14–7.09 d6, 300MHz) δ: 10.69 (s, 1H), 8.43–8.42 (m, 1H), 8.32–8.29
(m, 3H), 7.02–7.01 (m, 1H), 6.82–6.80 (m, 1H), 6.73 (d, 1H, (m, 1H), 8.11 (s, 1H), 7.97–7.92 (m, 2H), 7.91–7.83 (m, 3H),
397.0 [M+1]+.
N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-4-
1
1
J=3.5Hz), 6.53–6.51 (m, 1H), 5.33–5.31 (m, 2H).
7.57 (d, 1H, J=3.6Hz), 7.18 (brs, 2H), 6.78 (d, 1H, J=3.6Hz);
General Procedure for Synthesis of Compounds Ia–d ESI-MS: 432.0 [M+1]+.
To a solution of compound 8a, b (30.0mg, 0.1mmol) in an-
N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-
hydrous tetrahydrofuran (THF) (3mL), a solution of the ap- pyridazine-4-carboxamide (IIc): 1H-NMR (DMSO-d6,
propriate aryl isocyanate (0.1mmol) in anhydrous THF (3mL) 300MHz) δ: 10.94 (s, 1H), 9.70–9.67 (m, 1H), 9.52–9.50 (m,
was added dropwise at room temperature under N2 atmo- 1H), 8.32 (brs, 1H), 8.16–8.13 (m, 1H), 7.79–7.77 (m, 1H),
sphere. The reaction mixture was stirred at room temperature 7.56–7.54 (m, 1H), 7.53–7.52 (m, 1H), 7.35–7.32 (m, 1H),
for 24h. The solvent was evaporated under reduced pressure, 7.28–7.26 (m, 1H), 7.18 (brs, 2H), 6.81 (d, 1H, J=3.5Hz); ESI-
and purification was carried out by flash column chromatog- MS: 332.0 [M+1]+.
raphy using the appropriate proportion of ethyl acetate and
hexane as mobile phase.
N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-
(trifluoromethyl)benzamide (IId): 1H-NMR (DMSO-d6,
1-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3- 300MHz) δ: 10.69 (s, 1H), 8.46–8.45 (m, 1H), 8.39–8.25 (m,
1
(3,4-dichlorophenyl)urea (Ia): H-NMR (DMSO-d6, 300MHz) 3H), 8.13 (s, 1H), 7.83–7.69 (m, 3H), 7.54–7.49 (m, 2H), 7.18
δ: 9.08 (s, 1H), 9.02 (s, 1H), 8.09 (s, 1H), 7.90 (d, 1H, (brs, 2H), 6.80 (d, 1H, J=3.5Hz); ESI-MS: 398.0 [M+1]+.

January 2014
33
N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-4-
chloro-3-(trifluoromethyl)benzamide (IIe): H-NMR (DMSO- in the same way as synthesis of compound 11.
Synthesis of Compounds 12a, b They were synthesized
1
d6, 300MHz) δ: 10.74 (s, 1H), 8.42–8.40 (m, 1H), 8.31–8.25
N-(7-(4-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
1
(m, 2H), 8.13 (s, 1H), 7.96–7.93 (m, 1H), 7.79–7.77 (m, 1H), benzamide (12a): Yield 85%; H-NMR (DMSO-d6, 300MHz)
7.56–7.49 (m, 3H), 7.18 (brs, 2H), 6.80 (d, 1H, J=3.6Hz); ESI- δ: 11.19 (brs, 1H), 8.62 (s, 1H), 8.09–8.07 (m, 2H), 7.68–7.56
MS: 432.0 [M+1]+.
(m, 4H), 7.37–7.34 (m, 2H), 6.71–6.69 (m, 3H), 5.33 (brs, 2H).
N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-
N-(7-(3-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
1
(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)benzamide benzamide (12b): Yield 70%; H-NMR (DMSO-d6, 300MHz)
(IIf): 1H-NMR (DMSO-d6, 300MHz) δ: 10.73 (s, 1H), δ: 11.22 (brs, 1H), 8.65 (s, 1H), 8.12–8.09 (m, 2H), 7.70–7.57
8.49–8.47 (m, 1H), 8.43–8.41 (m, 1H), 8.27 (brs, 2H), 8.19 (m, 4H), 7.40–7.36 (m, 2H), 6.73–6.70 (m, 3H), 5.35 (brs, 2H).
(brs, 1H), 8.13 (s, 1H), 7.78–7.77 (m, 1H), 7.73 (brs, 1H),
Synthesis of Compounds IVa–f This was carried out by
7.56–7.53 (m, 2H), 7.35–7.30 (m, 1H), 7.18 (brs, 2H), 6.81 (d, the same procedure utilized for synthesis of compounds Ia–d.
1H, J=3.6Hz), 2.19 (s, 3H); ESI-MS: 478.0 [M+1]+.
N-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)acetamide
N-{{7-{4-[3-(2,3-Dichlorophenyl)ureido]phenyl}-7H-
pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVa): IR (KBr):
(9)
A
mixture of compound 5 (0.5g, 3.7mmol), acetic acid (0.4g, 3310, 1512, 1250, 708cm－1; 1H-NMR(DMSO-d6, 300MHz)
6.6mmol), HOBt (1.1g, 8.1mmol), and EDCI (1.8g, 9.3mmol) δ: 11.23 (brs, 1H), 9.72 (brs, 1H), 8.65 (s, 1H), 8.56 (s,
in dry DMF (15mL) was cooled to 0°C under nitrogen atmo- 1H), 8.22–8.18 (m, 1H), 8.09 (d, 2H, J=7.7Hz), 7.85 (d, 1H,
sphere. Triethylamine (1.6mL, 11.4mmol) was added thereto J=3.7Hz), 7.78 (d, 2H, J=8.9Hz), 7.68–7.63 (m, 4H), 7.59–7.54
at the same temperature. The mixture was then stirred at 80°C (m, 2H), 7.33–7.32 (m, 1H), 6.81 (d, 1H, J=6.6Hz); ESI-MS:
for 24h. The reaction mixture was cooled and then partitioned 516.1 [M+1]+.
between water and ethyl acetate. The organic layer was sepa-
N-{{7-{4-[3-(3,4-Dichlorophenyl)ureido]phenyl}-7H-
rated, and the aqueous layer was extracted with ethyl acetate pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVb): IR (KBr):
1
(3×50mL). The combined organic layer extracts were washed 3290, 1521, 1230, 689cm－1; H-NMR (DMSO-d6, 300MHz) δ:
with brine and dried over anhydrous Na2SO4. After evapora- 11.23 (brs, 1H), 9.21–9.18 (m, 2H), 8.65 (s, 1H), 8.10–8.08 (m,
tion of the organic solvent, the residue was purified by column 2H), 7.92–7.91 (m, 1H), 7.85 (d, 1H, J=3.6Hz), 7.70–7.76 (m,
chromatography (silica gel, ethyl acetate) to get the pure title 2H), 7.67–7.64 (m, 3H), 7.59–7.54 (m, 3H), 7.39–7.35 (m, 1H),
1
compound (0.3g, 45%) as a pale yellow oil. H-NMR (DMSO- 6.81 (d, 1H, J=3.3Hz); ESI-MS: 417.1 [M+1]+.
d6, 300MHz) δ: 12.00 (brs, 1H), 10.65 (brs, 1H), 8.44 (s, 1H),
7.35 (m, 1H), 6.73–6.68 (d, 1H, J=15.2Hz), 2.21 (s, 3H).
N- {{ 7 - { 4 - [ 3 - ( 3 - ( T r i fl u o r o m e t h y l ) p h e n y l ) u r e i d o ] p h e n y l } - 7 H-
pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVc): IR (KBr):
N-(7-(4-Nitrophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 3320, 1523, 1339, 1123, 698cm－1; 1H-NMR (DMSO-d6,
acetamide (10) It was prepared by the same method uti- 300MHz) δ: 11.21 (brs, 1H), 9.48 (brs, 1H), 9.38 (brs, 1H),
1
lized for synthesis of compounds 7a, b. Yield 45%; H-NMR 8.65 (s, 1H), 8.10–8.05 (m, 3H), 7.84 (d, 1H, J=3.6Hz),
(DMSO-d6, 300MHz) δ: 10.93 (s, 1H), 8.65 (brs, 1H), 7.76–7.73 (m, 6H), 7.62–7.50 (m, 3H), 7.33–7.30 (m, 1H), 6.82
8.44–8.30 (m, 2H), 8.28–8.17 (m, 2H), 8.04–8.00 (m, 1H), (d, 1H, J=3.6Hz); ESI-MS: 517.1 [M+1]+.
7.05–7.00 (m, 1H), 2.07 (s, 3H).
N-{{7-{4-[3-(4-Chloro-3-trifluoromethyl-phenyl)-ureido]-
N-(7-(4-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVd):
1
acetamide (11) To a solution of compound 10 (0.2g, IR (KBr): 3331, 1522, 1310, 1175, 1032, 711cm－1; H-NMR
0.7mmol) in dry THF (10mL) was added Pd/C (0.1g) and (DMSO-d6, 300MHz) δ: 11.23 (brs, 1H), 9.38 (brs, 1H), 9.22
was hydrogenated at 50 psi for 2h, and then filtered through (brs, 1H), 8.65 (s, 1H), 8.14–8.08 (m, 3H), 7.85–7.83 (m, 1H),
celite. The Pd/C residue was washed with further THF (10mL) 7.77–7.74 (m, 2H), 7.70–7.61 (m, 5H), 7.58–7.47 (m, 2H), 6.81
and concentration. The crude residue was purified by column (d, 1H, J=3.6Hz); ESI-MS: 551.1 [M+1]+.
chromatography (silica gel, ethyl acetate) to give the title
N-{{7-{4-[3-(3,5-Bis-trifluoromethyl-phenyl)ureido]-
1
product (45mg, 25%) as a pale yellow oil. H-NMR (DMSO- phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVe):
1
d6, 300MHz) δ: 10.75 (s, 1H), 8.48 (s, 1H), 7.61–7.59 (d, 1H, IR (KBr): 3311, 1527, 1276, 1124, 879, 681cm－1; H-NMR
J=3.6Hz), 7.34–7.31 (m, 2H), 6.88–6.87 (d, 1H, J=3.6Hz), (DMSO-d6, 300MHz) δ: 11.21 (brs, 1H), 9.63 (brs, 1H), 9.37
6.72–6.66 (m, 2H), 5.31 (brs, 2H), 2.24 (s, 3H).
(brs, 1H), 8.65 (s, 1H), 8.17 (s, 2H), 8.09 (d, 2H, J=7.3Hz),
Synthesis of Compounds IIIa and b They were prepared 7.84 (d, 1H, J=3.7Hz), 7.78–7.75 (m, 2H), 7.70–7.63 (m, 4H),
by the same method utilized for synthesis of Ia–d and IIa–f, 7.58–7.56 (m, 2H), 6.82 (d, 1H, J=3.6Hz); ESI-MS: 585.1 [M+
respectively.
1]+.
N-(7-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-
N-(7-(3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-
phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetamide
(IIIa): phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (IVf):
1H-NMR (DMSO-d6, 300MHz) δ: 10.81 (s, 1H), 9.24 (s, 1H-NMR (DMSO-d6, 300MHz) δ: 11.25 (brs, 1H), 9.41 (brs,
1H), 9.08 (s, 1H), 8.54 (s, 1H), 8.12 (brs, 1H), 7.71 (d, 1H, 1H), 9.24 (brs, 1H), 8.67 (s, 1H), 8.17–8.11 (m, 3H), 7.88–7.85
J=3.6Hz), 7.73–7.70 (m, 3H), 7.65–7.62 (m, 3H), 6.95 (d, 1H, (m, 1H), 7.79–7.76 (m, 2H), 7.73–7.63 (m, 5H), 7.61–7.50 (m,
J=3.5Hz), 2.24 (s, 3H); ESI-MS: 489.0 [M+1]+.
2H), 6.83 (d, 1H, J=3.6Hz); ESI-MS: 551.1 [M+1]+.
Synthesis of Compounds Va–c This was carried out by
N-(4-(4-Acetamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
phenyl)-4-chloro-3-(trifluoromethyl)benzamide (IIIb): 1H- the same procedure utilized for synthesis of compounds IIa–f.
NMR (DMSO-d6, 300MHz) δ: 10.84 (brs, 1H), 10.73 (brs,
N-[4-(4-Benzoylamino-pyrrolo[2,3-d]pyrimidin-7-yl)-
1H), 8.57 (brs, 1H), 8.43–8.38 (m, 1H), 8.32–8.29 (m, 1H), phenyl]-3,4-dichlorobenzamide (Va): IR (KBr): 3427, 1620,
1
7.97–7.93 (m, 3H), 7.86–7.82 (m, 3H), 6.98–6.97 (d, 1H, J= 1401, 1027, 753cm－1; H-NMR(DMSO-d6, 300MHz) δ: 11.23
3.5Hz), 2.26 (s, 3H); ESI-MS: 474.0 [M+H]+.
(brs, 1H), 10.59 (brs, 1H), 8.67 (s, 1H), 8.27 (d, 1H, J=1.7Hz),

34
Vol. 62, No. 1
K. H., Sim T. B., Lee S. H., Baek D., Hah J.-M., Cho J.-H., Oh
C.-H., Bioorg. Med. Chem. Lett., 19, 6538–6543 (2009).
4) Nam B. S., Kim H., Oh C.-H., Lee S. H., Cho S. J., Sim T. B., Hah
J.-M., Kim D. J., Choi J. H., Yoo K. H., Bioorg. Med. Chem. Lett.,
19, 3517–3520 (2009).
5) Song D.-Q., Du N.-N., Wang Y.-M., He W.-Y., Jiang E.-Z., Cheng
S.-X., Wang Y.-X., Li Y.-H., Wang Y.-P., Li X., Jiang J.-D., Bioorg.
Med. Chem., 17, 3873–3878 (2009).
6) Choi W.-K., Oh C.-H., Bull. Korean Chem. Soc., 30, 2027–2031
(2009).
7) Kim H. J., Jung M.-H., Kim H., El-Gamal M. I., Sim T. B., Lee
S. H., Hong J. H., Hah J.-M., Cho J.-H., Choi J. H., Yoo K. H., Oh
C.-H., Bioorg. Med. Chem. Lett., 20, 413–417 (2010).
8) Yu H., Jung Y., Kim H., Lee J., Oh C.-H., Yoo K. H., Sim T., Hah
J.-M., Bioorg. Med. Chem. Lett., 20, 3805–3808 (2010).
9) Lee J., Kim H., Yu H., Chung J. Y., Oh C.-H., Yoo K. H., Sim T.,
Hah J.-M., Bioorg. Med. Chem. Lett., 20, 1573–1577 (2010).
10) Yao P., Zhai X., Liu D., Qi B. H., Tan H. L., Jin Y. C., Gong P.,
Arch. Pharm. Chem. Life Sci., 343, 17–23 (2010).
8.11–8.08 (m, 2H), 8.00–7.95 (m, 3H), 7.89–7.85 (m, 4H),
7.59–7.54 (m, 3H), 6.84 (d, 1H, J=3.8Hz); ESI-MS: 502.0 [M+
1]+.
N-[4-(4-Benzoylamino-pyrrolo[2,3-d]pyrimidin-7-yl)-
phenyl]-(3-trifluoromethyl)benzamide (Vb): IR (KBr): 3223,
1
1523, 1312, 1123, 708cm－1; H-NMR (DMSO-d6, 300MHz) δ:
11.24 (brs, 1H), 10.68 (brs, 1H), 8.67 (s, 1H), 8.34–8.30 (m,
2H), 8.11–8.08 (m, 2H), 8.02–7.97 (m, 2H), 7.89–7.76 (m, 4H),
7.69–7.64 (m, 1H), 7.59–7.54 (m, 3H), 6.84 (d, 1H, J=3.7Hz);
ESI-MS: 502.1 [M+1]+.
N-[4-(4-Benzoylamino-pyrrolo[2,3-d]pyrimidin-7-yl)-
phenyl]-4-chloro-3-(trifluoromethyl)benzamide (Vc): IR (KBr):
3300, 1523, 1312, 1138, 750cm－1; 1H-NMR (DMSO-d6,
300MHz) δ: 11.27 (brs, 1H), 10.75 (brs, 1H), 8.68 (s, 1H),
8.43–8.41 (m, 1H), 8.11–8.08 (m, 3H), 7.98–7.85 (m, 4H),
7.68–7.64 (m, 2H), 7.59–7.54 (m, 3H), 6.84 (d, 1H, J=3.6Hz);
ESI-MS: 536.1 [M+1]+.
Sixty Cancer Cell Line Screening at the NCI Screen-
ing against a panel of 60 cancer cell lines was carried out
11) El-Gamal M. I., Jung M.-H., Lee W. S., Sim T., Yoo K. H., Oh
C.-H., Eur. J. Med. Chem., 46, 3218–3226 (2011).
at the National Cancer Institute (NCI), Bethesda, Maryland, 12) Choi W.-K., El-Gamal M. I., Choi H. S., Baek D., Oh C.-H., Eur. J.
U.S.A.,22) applying the standard protocol of the NCI.23,24)
Med. Chem., 46, 5754–5762 (2011).
13) Kim H. J., Cho H. J., Kim H., El-Gamal M. I., Oh C.-H., Lee S.
H., Sim T., Hah J.-M., Yoo K. H., Bioorg. Med. Chem. Lett., 22,
3269–3273 (2012).
14) El-Gamal M. I., Oh C.-H., Bull. Korean Chem. Soc., 33, 1571–1576
Kinase Profiling Reaction Biology Corp. Kinase Hot-
SpotSM service25) was used for screening of the test compound,
and IC50 Profiler Express for IC50 measurement. Assay protocol:
In a final reaction volume of 25µL, kinase (5–10mU) is in-
(2012).
cubated with 25mM Tris pH 7.5, 0.02mM EGTA, 0.66mg/mL
15) Jung M.-H., El-Gamal M. I., Abdel-Maksoud M. S., Sim T., Yoo K.
myelin basic protein, 10mM magnesium acetate and [γ33P-
H., Oh C.-H., Bioorg. Med. Chem. Lett., 22, 4362–4367 (2012).
ATP] (specific activity approx. 500cpm/pmol, concentration
as required). The reaction is initiated by the addition of the
Mg-ATP mix. After incubation for 40min at room tempera-
ture, the reaction is stopped by the addition of 5µL of a 3%
phosphoric acid solution. Ten microliter of the reaction is then
spotted onto a P30 filtermat and washed three times for 5min
in 75mM phosphoric acid and once in methanol prior to dry-
ing and scintillation counting.
16) Choi W.-K., El-Gamal M. I., Choi H. S., Hong J. H., Baek D., Choi
K., Oh C.-H., Bull. Korean Chem. Soc., 33, 2991–2998 (2012).
17) Cho H. J., El-Gamal M. I., Oh C.-H., Kim G., Hong J. H., Choi H.
S., Yoo K. H., Bull. Korean Chem. Soc., 33, 3635–3639 (2012).
18) Wilhelm S., Carter C., Lynch M., Lowinger T., Dumas J., Smith R.
A., Schwartz B., Simantov R., Kelley S., Nat. Rev. Drug Discov., 5,
835–844 (2006).
19) DGNews website: ‹http://www.pslgroup.com/news/content.nsf/med
icalnews/852571020057CCF685257384005A45B1?OpenDocument&
id=&count=10›.
Acknowledgments This work was supported by Korea
Institute of Science and Technology (KIST), KIST Project
(2E22360). We would like to thank the National Cancer Insti-
tute (NCI), Bethesda, Maryland, U.S.A., for performing the
anticancer testing of the target compounds over 60 cancer cell
lines.
20) Capdeville R., Buchdunger E., Zimmermann J., Matter A., Nat. Rev.
Drug Discov., 1, 493–502 (2002).
21) Bio M. M., Xu F., Waters M., Williams J. M., Savary K. A.,
Cowden C. J., Yang C., Buck E., Song Z. J., Tschaen D. M., Volate
R. P., Reamer R. A., Grabowski E. J. J., J. Org. Chem., 69, 6257–
6266 (2004).
22) NCI website: ‹www.dtp.nci.nih.gov›.
23) DTP Data Search: ‹http://dtp.nci.nih.gov/dtpstandard/dwindex/index.
jsp›.
24) NCI-60 DTP Human Tumor Cell Line Screen Process: ‹http://www.
dtp.nci.nih.gov/branches/btb/ivclsp.html›.
References
1) WHO website: ‹http://www.who.int/mediacentre/factsheets/fs297/en/›.
2) Jung M.-H., Oh C.-H., Bull. Korean Chem. Soc., 29, 2231–2236
(2008).
25) Reaction Biology Corp. website: ‹http://www.reactionbiology.com›.
3) Jung M.-H., Kim H., Choi W.-K., El-Gamal M. I., Park J.-H., Yoo