Diarylureas and Diarylamides with Pyrrolo[2,3-d]pyrimidine Scaffold as Broad-Spectrum Anticancer Agents

Abstract

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.

Cancer is a generic term for a large group of diseases that can affect any part of the body. Lung, stomach, liver, colon, and breast cancer cause the most cancer deaths each year. About 30% of cancer deaths are due to the five leading behavioral and dietary risks: high body mass index, low fruit and vegetable intake, lack of physical activity, tobacco use, and alcohol intake. More than 70% of all cancer deaths occurred in low- and middle-income countries. Deaths from cancer worldwide are projected to exceed 13 million in 2030.¹⁾ In spite of the extensive efforts and investment in research, the management of human malignancies still constitutes a major challenge for contemporary medicinal chemistry. There has been an urgent need for development of more efficient anticancer agents with minimal side effects.

Diarylureas and diarylamides have been highlighted as potential antiproliferative agents against a variety of cancer cell lines.^2–17) Sorafenib (Nexavar®, Fig. 1) is an example of anticancer diarylureas that has been approved by the U.S. Food and Drug Administration (FDA) for treatment of advanced renal cancer.¹⁸⁾ It has also been approved in Europe for treatment of hepatocellular carcinoma (HCC).¹⁹⁾ Sorafenib is currently subjected to clinical trials for other types of cancer. Imatinib (Gleevec®, Fig. 1) is an example of diarylamides which is used for treatment of chronic myeloid leukemia (CML) with diminished side effects.²⁰⁾

Fig. 1. Structures of Sorafenib, Imatinib, and the Target Compounds

The target pyrrolo[2,3-d]pyrimidine compounds were designed as conformationally restricted analogs of Sorafenib by isosteric replacement of the 4-phenoxypyridine moiety of the lead compound with a pyrrolo[2,3-d]pyrimidine nucleus (Fig. 1). The 50% inhibitory concentration (IC₅₀) values for most of the target compounds over A375P human melanoma cell line only were previously reported.^2,3) Upon further testing over NCI-60 cancer cell line panel of nine different cancer types, the target compounds showed broad-spectrum anticancer activities with high efficacies and potencies. Herein the biological results are reported in details.

Results and Discussion

Chemistry

The target pyrrolo[2,3-d]pyrimidine diarylureas and diarylamides were synthesized by the pathway of reactions illustrated in Charts 2–4.^2,3) But at the beginning, it was essential to synthesize the intermediate compound 7H-pyrrolo[2,3-d]pyrimidin-4-amine (5). It could be successfully achieved by the pathway shown in Chart 1. The geminal dicyano intermediate 2 was prepared from compound 1²¹⁾ with malononitrile by heating in N,N-dimethylformamide (DMF) in the presence of anhydrous K₂CO₃. Synthesis of compound 3 was carried out by refluxing 2 with thiourea in the presence of potassium tert-butoxide. Cyclization to 4 could be achieved by neutralization of the thiol potassium salt 3 using 5 N aqueous HCl followed by heating with 10 N aqueous NaOH. Reduction of the thiol compound 4 using Raney nickel afforded 7H-pyrrolo[2,3-d]pyrimidin-4-amine (5) (Chart 1).

Chart 1

Reagents and conditions: (a) malononitrile, K₂CO₃, DMF, 50°C, 17–18 h, 54%; (b) thiourea, potassium tert-butoxide, EtOH, reflux, 16–17 h; rt, 1–2 h, 61%; (c) 5 N HCl, H₂O, 10 N NaOH, 50°C, 50 min, 88%; (d) Raney-Ni, H₂O, reflux, 4 h, 87%.

Chart 2

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, 40 h, 67% (7a), 40% (7b); (c) SnCl₂·H₂O, EtOH, reflux, overnight, 80% (8a), 50% (8b); (d) aryl isocyanate, THF, rt, 24 h, 27–60%; (e) carboxylic acid derivative, HOBt, EDCI, TEA, DMF, 80°C, 24 h, 10–86%.

Chart 3

Reagents and conditions: (a) acetic acid, HOBt, EDCI, TEA, DMF, 80°C, 24 h, 45%; (b) 1-iodo-4-nitrobenzene, K₂CO₃, CuI, L-proline, DMSO, 90°C, 40 h, 45%; (c) Pd/C, H₂, THF, 50 psi, rt, 2 h, 25%; (d) 4-chloro-3-(trifluoromethyl)phenyl isocyanate, THF, rt, 24 h, 31%; (e) 4-chloro-3-(trifluoromethyl)benzoic acid, HOBt, EDCI, TEA, DMF, 80°C, 24 h, 47%.

Chart 4

Reagents and conditions: (a) Pd/C, H₂, 50 psi, THF, rt, 2 h, 85% (12a), 70% (12b); (b) aryl isocyanate, THF, rt, 24 h, 22–82%; (c) benzoic acid derivative, HOBt, EDCI, TEA, DMF, 80°C, 24 h, 23–36%.

Antiproliferative Activities against 60 Cell Line Panel at the National Cancer Institute (NCI). Single-Dose Testing

Structures of the target compounds were submitted to the (NCI), Bethesda, Maryland, U.S.A.,²²⁾ and the eleven compounds shown in Fig. 2 were selected on the basis of degree of structural variation and computer modeling techniques for evaluation of their antineoplastic activity. The selected compounds were subjected to in vitro anticancer assay against tumor cells in a full panel of 60 cell lines taken from nine different tissues (blood, lung, colon, central nervous system (CNS), skin, ovary, kidney, prostate, and breast). The compounds were tested at a single dose concentration of 10 µM, and the percentages of growth inhibition over the 60 tested cell lines were determined. The mean inhibition percentages for each of the tested compounds over the full panel of cell lines are illustrated in Fig. 2.

Fig. 2. Mean Inhibition Percentages Observed with the Final Compounds in Single-Dose (10 µM) 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 percentages from 100.

Regarding the substituents on the 4-position of pyrrolo[2,3-d]pyrimidine nucleus, compound Vc with benzamido moiety was more active than the corresponding acetamido derivative IIIb. This can be attributed to the steric and/or electronic differences between benzoyl and acetyl groups. This suggests that the aromatic amide substituent at this position is more favorable.

By comparing the activities of derivatives with amide and urea moieties at pyrrolo[2,3-d]pyrimidine side chain as a linker, it was found that compounds IVb and IVd possessing urea moiety were more active than the corresponding amide analogues Va and Vc. This may be attributed to that the longer spacer, urea moiety, may geometrically permit appropriate fitting of the molecule at the receptor site. Or the terminal NH group of the urea moiety may form additional hydrogen bond(s) at the receptor site. Any or both of these effects would enable optimal drug–receptor interaction, and hence higher antiproliferative activity.

Compound IVd with para-disubstituted phenyl ring at N1 position of the pyrrolo[2,3-d]pyrimidine nucleus showed higher mean %inhibition than its meta-disubstituted phenyl positional isomer IVf. This can be rationalized that the direction of terminal moiety can affect the appropriate fitting at the receptor site and hence affinity and activity.

The effect of substituents of the terminal aryl ring on mean %inhibition was also investigated. The introduction of meta-(4′-methylimidazol-1-yl) moiety on the 3′-(trifluoromethyl)phenyl ring (compound IIf) slightly reduced the activity compared with compound IId. On the other hand, para-chloro group improved the activity of compound Vc compared with Vb with 4′-unsubstituted 3′-(trifluoromethyl)phenyl terminal ring. Compound IVa with 2′,3′-dichlorophenyl terminal ring exhibited higher mean %inhibition than the corresponding positional isomer compound IVb with 3′,4′-dichlorophenyl terminal moiety.

Table 1. Structures of the Target Compounds, and Their Yield Percentages and Melting Points

Compound No.	R1	Site of attachment of phenyl ring	R2	Yield%	Melting point (°C)
Ia	H	para		27	232–235 (dec.)
Ib	H	para		35	>300
Ic	H	meta		60	225–226 (dec.)
Id	H	meta		46	>300
IIa	H	para		47	216–218 (dec.)
IIb	H	para		86	215–217 (dec.)
IIc	H	meta		10	204–207 (dec.)
IId	H	meta		69	214–216 (dec)
IIe	H	meta		26	221–223
IIf	H	meta		28	218–220 (dec.)
IIIa	CH3CO	para		31	274–276 (dec.)
IIIb	CH3CO	para		47	241–242 (dec.)
IVa	PhCO	para		22	225–227 (dec.)
IVb	PhCO	para		25	249–250 (dec.)
IVc	PhCO	para		82	234–236 (dec.)
IVd	PhCO	para		54	190–192 (dec.)
IVe	PhCO	para		56	220–221 (dec.)
IVf	PhCO	meta		56	140–141
Va	PhCO	para		23	193–195
Vb	PhCO	para		36	181–183 (dec.)
Vc	PhCO	para		33	193–195 (dec.)

Among all the target compounds, it was found that compounds IVa, IVb, and IVd possessing 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 demonstrated the highest mean %inhibition values; 109.18%, 96.91%, and 107.86%, respectively. So it can be concluded that the presence of those three moieties together is essential for antiproliferative activity this series of pyrrolo[2,3-d]pyrimidine compounds.

Compounds IVa and IVd exerted lethal effect on the NCI-60 cancer cell line panel with mean IC₅₀ values more than 100%. The % inhibitions of these two compounds over each cell line of the NCI-60 panel are illustrated in Fig. 3. At 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.

Fig. 3. %Inhibition Expressed by Compounds IVa and IVd at a Single-Dose Concentration of 10 µM over the NCI-60 Cancer Cell Lines

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 determine 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 benzamido 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.

Table 2. IC₅₀, TGI, and LC₅₀ Values (μM) of the Tested Compounds over the Most Sensitive Cell Line of Each Subpanel

Compd. No.	Results (µM)	Cancer cell line
		CCRF-CEMa)	NCI-H460b)	HCT-15c)	U251d)	LOX IMVIe)	OVCAR-4f)	CAKI-1g)	PC-3h)	HS 578Ti)
IVa	IC50j)	0.53	0.36	0.16	0.57	0.76	0.88	3.84	0.58	0.86
	TGIk)	3.21	1.21	5.42	1.40	2.04	1.91	24.90	1.57	2.60
	LC50l)	>50	NAm)	42.70	3.46	>50	4.14	>50	4.21	>50
IVb	IC50j)	0.99	1.71	1.74	0.94	2.38	0.89	8.79	1.06	1.19
	TGIk)	2.83	5.85	6.97	2.29	16.30	1.80	21.20	2.86	5.77
	LC50l)	38.70	40.10	22.00	7.37	>50	3.67	>50	11.70	>50
IVd	IC50j)	0.81	1.35	0.77	1.20	2.00	1.57	1.69	1.25	1.27
	TGIk)	3.73	3.06	2.19	2.55	6.77	3.48	3.81	3.01	4.61
	LC50l)	>50	6.89	5.30	5.43	>50	7.70	8.60	7.25	>50
IVf	IC50j)	0.66	2.13	2.51	2.09	2.82	2.36	2.04	1.25	2.55
	TGIk)	9.05	9.88	10.20	8.39	15.00	8.18	10.90	5.93	22.60
	LC50l)	>50	>50	32.80	28.60	>50	21.90	>50	29.00	>50
Vb	IC50j)	NAm)	0.008	0.007	0.13	0.23	5.46	0.82	10.10	6.01
	TGIk)	NAm)	12.90	45.70	18.60	2.45	49.20	19.90	41.00	49.70
	LC50l)	NAm)	46.40	>50	>50	19.50	>50	>50	>50	>50
Vc	IC50j)	0.85	0.14	0.007	0.36	0.30	1.44	0.40	1.26	1.48
	TGIk)	18.20	1.74	2.27	2.23	2.75	4.06	5.91	7.41	4.88
	LC50l)	>50	6.08	14.50	5.70	>50	13.80	49.80	>50	>50
Sorafenib	IC50j)	2.00	2.51	2.51	2.00	1.58	3.16	3.16	2.00	2.51
	TGIk)	5.01	5.01	3.16	3.16	2.51	25.12	12.59	5.01	3.98
	LC50l)	100	25.12	7.94	6.31	6.31	100	100	100	100
Imatinib	IC50j)	15.85	15.85	19.95	19.95	19.95	19.95	31.62	19.95	15.85
	TGIk)	63.10	31.62	79.43	50.12	39.81	63.10	79.43	63.10	39.81
	LC50l)	100	79.43	100	100	79.43	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% inhibition; l) LC₅₀ is the concentration causing 50% lethality (50% tumor regression); m) NA means that the datum is not available.

As shown in Table 2, most of the compounds exhibited broad-spectrum activities with high potency (in sub-micromolar and micromolar scale) over all the nine cell lines. Most of the IC₅₀ values were less than 10 µM. Compound IVa, with the highest mean %inhibition value in one-dose testing mode (Fig. 2), showed the highest potency among the six compounds tested in five-dose testing mode. Its IC₅₀ values were in sub-micromolar scale over eight cell lines. It demonstrated higher potency than Sorafenib against eight cell lines. Its TGI results showed higher efficacy than Sorafenib over seven cell lines. And its potency and efficacy against all the nine cell lines were higher than Imatinib. In addition, the diarylamide derivative Vb exhibited one-digit nanomolar IC₅₀ values, 7 and 8 nM, over NCI-H460 non-small cell lung and HCT-15 colon cancer cell lines, respectively. The diarylamide derivative Vc exerted high potency over HCT-15 colon cancer cell line also with IC₅₀ value of 7 nM. Compounds Vb and Vc demonstrated sub-micromolar IC₅₀ values over five and six cell lines, respectively. Both compounds Vb and Vc showed higher potencies and efficacies than Sorafenib and Imatinib against most of the tested cell lines.

Compounds IVa, IVb, IVd, IVf, Vb, and Vc showed high efficacies also over the nine cell lines. The TGI and LC₅₀ values were less than 50 µM 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 4.22 µM. Similarly, compound IVb showed TGI and LC₅₀ values of 1.80 µM and 3.67 µM, 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 mechanism(s) of antiproliferative activity of the most active target compounds, compounds IVa, IVb, and IVd were tested for inhibitory effect over wild type B-RAF, vascular endothelial growth factor receptor 1 (VEGFR1), and platelet-derived growth factor receptor (PDGFR) kinases. The three compounds showed weak potency against these kinases, compared with Sorafenib (Table 3). So it can be concluded that these target compounds inhibit cancer cell growth through other mechanism(s) other than B-RAF, VEGFR1, and PDGFR kinase inhibition.

Table 3. IC₅₀ Values of Compounds IVa, IVb, IVd, and Sorafenib over Potential Kinases

	B-RAF (wild type)	VEGFR1	PDGFR
IVa	>3 µM	>3 µM	>3 µM
IVb	>3 µM	>3 µM	>3 µM
IVd	>3 µM	>3 µM	>3 µM
Sorafenib	25 nM	26 nM	90 nM

Conclusion

A series of diarylureas and diarylamides possessing pyrrolo[2,3-d]pyrimidine scaffold was designed with structural similarity to Sorafenib and synthesized. Eleven final compounds were tested at a single-dose concentration of 10 µM at the NCI over 60 cancer cell line panel of nine different cancer types, and six of them were subsequently tested in five-dose testing mode. Compounds IVa, IVb, IVd, IVf, Vb, and Vc showed broad-spectrum anticancer activities with strong potencies and high efficacies. Among them, compounds IVa, IVb, and IVd possessing 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 exerted the highest mean % inhibition in one-dose testing mode, and high potency and efficacy in five-dose testing experiments. So we can conclude that these moieties together with the pyrrolo[2,3-d]pyrimidine nucleus constitute the pharmacophore of this series of compounds as potential anticancer agents.

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 IC₅₀ values over eight cell lines of eight different cancer types, and high efficacy with TGI and LC₅₀ values less than 4.22 µM over three colon, ovarian, and prostate cancer cell lines. Its potency and efficacy results were promising compared with Sorafenib and Imatinib. So this compound can be considered as promising lead for future development of potential anticancer agents with high potency and efficacy.

Experimental

General

All melting points were obtained on a Walden Precision Apparatus Electrothermal 9300 apparatus and are uncorrected. Mass spectra (MS) were taken in electrospray ionization (ESI) mode on a Waters 3100 Mass Detector (Waters, Milford, MA, U.S.A.). Nuclear magnetic resonance (NMR) spectroscopy was performed using a Bruker ARX-300, 300 MHz (Bruker Bioscience, Billerica, MA, U.S.A.) with TMS as an internal standard. Purities of the target compounds (>95%) were determined by LC-MS analysis using the following system: Waters 2998 photodiode array detector, Waters 3100 mass detector, Waters SFO system fluidics organizer, Waters 2545 binary gradient module, Waters reagent manager, Waters 2767 sample manager, Sunfire™ C18 column (4.6×50 mm, 5 µm particle size); Solvent gradient=95% A at 0 min, 1% A at 5 min; solvent A: 0.035% trifluoroacetic acid (TFA) in water; solvent B: 0.035% TFA in CH₃OH; flow rate=3.0 mL/min; the area under curve (AUC) was calculated using Waters MassLynx 4.1 software. Unless otherwise noted, all solvents and reagents were commercially available and used without further purification.

2-(2,2-Diethoxyethyl)malononitrile (2)

To a suspension of anhydrous potassium carbonate (1.4 g, 10.1 mmol) in dry DMF (3 mL), malononitrile (0.6 g, 9.1 mmol) was slowly added. The resulting mixture was heated at 41°C for 8 h under nitrogen atmosphere. The reaction mixture was cooled to room temperature, then 2-bromo-1,1-diethoxyethane (1, 1.0 g, 5.1 mmol) was added portionwise thereto. The reaction mixture was heated to 50°C for 17–18 h. After completion of the reaction, the reaction mixture was cooled to room temperature, and extracted between toluene (50 mL×3) and water (40 mL). The combined organic layer extracts were dried over anhydrous MgSO₄, the organic solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate–hexane 1 : 6, v/v) to obtain the title product (0.5 g, 54%). IR (KBr): 3350, 2978, 2250, 1748, 1633, 1441, 1375, 1124 cm^－1; ¹H-NMR (CDCl₃, 300 MHz) δ: 4.69 (t, 1H, J=5.3 Hz), 4.03 (t, 1H, J=7.3 Hz), 3.75–3.67 (m, 2H), 3.58–3.53 (m, 2H), 2.32–2.27 (m, 2H), 1.27–1.21 (m, 6H).

4,6-Diamino-5-(2,2-diethoxyethyl)pyrimidine-2-thiol Potassium Salt (3)

A solution of compound 2 (5.0 g, 27.4 mmol) in ethanol (60 mL) was cooled to 0°C, and thiourea (2.4 g, 31.5 mmol) was added portionwise thereto. Then a suspension of potassium tert-butoxide (3.5 g, 31.2 mmol) in ethanol (5.5 mL) was added dropwise. The reaction mixture was heated under reflux for 16–17 h, then cooled to room temperature. Ethanol (30 mL) was added, and the mixture was stirred at room temperature for 1–2 h. The produced solid was filtered out, washed with ethanol (20 mL), and dried to get the pure title compound (5.0 g, 61%). mp: 223–225°C (dec.); IR (KBr): 3434, 1630, 1435, 1314, 1050 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 4.92 (br s, 4H), 4.40 (t, 1H, J=5.3 Hz), 3.65–3.55 (m, 2H), 3.42–3.36 (m, 2H), 2.43–2.41 (m, 2H), 1.11–1.03 (m, 6H).

4-Amino-7H-pyrrolo[2,3-d]pyrimidine-2-thiol (4)

To a solution of compound 3 (7.5 g, 25.3 mmol) in distilled water (40 mL), 5 N HCl aqueous solution (11.8 mL) was added dropwise. After 20 min, distilled water (7 mL) was added thereto. The reaction mixture was heated at 50°C for 50 min, then cooled to room temperature. The reaction mixture was neutralized to pH 7 by dropwise addition of 10 N NaOH aqueous solution. The precipitated solid was filtered out, washed with acetonitrile (50 mL), and vacuum dried to obtain the pure title product (3.7 g, 88%). mp: 218–220°C (dec.); IR (KBr): 3413, 3171, 2884, 1655, 1584, 1133 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.59 (br s, 1H), 11.30 (br s, 2H), 6.88 (d, 1H, J=6.0 Hz), 6.47 (d, 1H, J=5.9 Hz).

7H-Pyrrolo[2,3-d]pyrimidin-4-amine (5)

To a suspension of compound 1 (1.0 g, 6.0 mmol) in water (50 mL) was added Raney nickel (3.0 g). The reaction mixture was heated at reflux for 4 h, and then the hot solution was filtered through celite. The nickel residue was washed with water (100 mL). The combined aqueous filtrate was evaporated to dryness to yield the product (0.7 g, 87.5%). mp: 206–208°C. (dec.). ¹H-NMR (DMSO-d₆, 300 MHz) δ: 12.37 (br s, 1H), 8.22 (br s, 2H), 8.15 (s, 1H), 7.29 (d, 1H, J=1.5 Hz), 6.80 (d, 1H, J=1.1 Hz).

N-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (6)

To a suspension of compound 2 (1.0 g, 7.5 mmol) in pyridine (25 mL) was added benzoyl chloride (1.3 g, 8.9 mmol). The mixture was heated at 50°C overnight. The mixture was cool and evaporated under reduced pressure. The residue was diluted with ethyl acetate (30 mL) and water (50 mL), and then the organic layer was dried over anhydrous Na₂SO₄. The solvent was evaporated under reduced pressure, and purification was achieved by flash column chromatography (silica gel, ethyl acetate–hexane 1 : 2, v/v) to afford the title compound (0.6 g, 33%) as a pale yellow solid. mp: 220–222°C. (dec.); ¹H-NMR (DMSO-d₆, 300 MHz) δ: 12.03 (s, 1H), 11.00 (s, 1H), 8.53 (s, 1H), 8.06 (d, 2H, J=7.3 Hz), 7.65–7.60 (m, 1H), 7.56–7.51 (m, 2H), 7.42–7.41 (m, 1H), 6.59–6.57 (m, 1H).

Synthesis of Compounds 7a, b

A mixture of compound 6 (0.3 g, 1.3 mmol), 1-iodo-4-nitrobenzene or 1-iodo-3-nitrobenzene (0.6 g, 2.4 mmol), anhydrous potassium carbonate (0.5 g, 2.4 mmol), CuI (20.0 mg, 0.1 mmol), and L-proline (30 mg, 0.3 mmol) in dry DMSO (5 mL) was heated at 90°C under nitrogen atmosphere for 40 h. The cooled solution was partitioned between H₂O (20 mL) and ethyl acetate (20 mL). The aqueous layer was extracted with ethyl acetate (3×10 mL). The combined organic layer extracts were washed with brine, and dried over anhydrous Na₂SO₄. The solvent was evaporated under reduced pressure, and purification was achieved by flash chromatography (silica gel, ethyl acetate–hexane 1 : 2, v/v) to afford the target compound 7a or 7b.

N-(7-(4-Nitrophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (7a): Yield: 67%; mp: 223–224°C (dec.); ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.35 (br s, 1H), 8.52–8.43 (m, 3H), 8.38–8.33 (m, 3H), 8.20–8.08 (m, 3H), 7.62–7.60 (m, 2H), 6.89–6.87 (m, 1H).

N-[7-(3-Nitrophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]benzamide (7b): Yield: 40%; mp: 212–214°C (dec.); ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.33 (br s, 1H), 8.98–8.88 (m, 1H), 8.73 (s, 1H), 8.40–8.37 (m, 1H), 8.27–8.24 (m, 1H), 8.20–8.08 (m, 3H), 7.88 (t, 1H, J=8.2 Hz), 7.68–7.64 (m, 1H), 7.59–7.54 (m, 2H), 6.90 (d, 1H, J=3.4 Hz).

Synthesis of Compounds 8a, b

A mixture of compound 7a or 7b (1.0 g, 2.8 mmol) and SnCl₂·H₂O (3.1 g, 13.7 mmol) in ethanol (20 mL) was heated under reflux overnight, and then cooled to room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with ethyl acetate (30 mL) and saturated aqueous NaHCO₃ (100 mL). The organic layer was separated, washed with brine, and dried over anhydrous Na₂SO₄. The solvent was evaporated under reduced pressure, and purification was achieved by flash column chromatography (silica gel, ethyl acetate) to afford the target compounds.

4-Amino-7-(4-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine (8a): Yield: 80%; mp: 231–232°C (dec.); ¹H-NMR (DMSO-d₆, 300 MHz) δ: 8.13–8.06 (m, 1H), 7.32–7.29 (m, 3H), 7.04 (br s, 2H), 6.66–6.63 (m, 3H), 5.21 (br s, 2H).

4-Amino-7-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine (8b): Yield: 50%; mp: 221–224°C (dec.); ¹H-NMR (DMSO-d₆, 300 MHz) δ: 8.09 (s, 1H), 7.40 (d, 1H, J=3.58 Hz), 7.14–7.09 (m, 3H), 7.02–7.01 (m, 1H), 6.82–6.80 (m, 1H), 6.73 (d, 1H, J=3.5 Hz), 6.53–6.51 (m, 1H), 5.33–5.31 (m, 2H).

General Procedure for Synthesis of Compounds Ia–d

To a solution of compound 8a, b (30.0 mg, 0.1 mmol) in anhydrous tetrahydrofuran (THF) (3 mL), a solution of the appropriate aryl isocyanate (0.1 mmol) in anhydrous THF (3 mL) was added dropwise at room temperature under N₂ atmosphere. The reaction mixture was stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure, and purification was carried out by flash column chromatography using the appropriate proportion of ethyl acetate and hexane as mobile phase.

1-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(3,4-dichlorophenyl)urea (Ia): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 9.08 (s, 1H), 9.02 (s, 1H), 8.09 (s, 1H), 7.90 (d, 1H, J=2.4 Hz), 7.72–7.69 (m, 2H), 7.60–7.57 (m, 2H), 7.54 (s, 1H), 7.51–7.49 (m, 2H), 7.13 (br s, 2H), 6.74 (d, 1H, J=3.8 Hz); ESI-MS: 413.0 [M+1]⁺.

1-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(3,5-bis(trifluoromethyl)phenyl)urea (Ib): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 9.46 (s, 1H), 9.20 (s, 1H), 8.15 (s, 2H), 8.10 (s, 1H), 7.73 (d, 2H, J=8.9 Hz), 7.65 (br s, 1H), 7.62 (d, 2H, J=8.9 Hz), 7.51 (d, 1H, J=3.6 Hz), 7.13 (br s, 2H), 6.75 (d, 1H, J=3.6 Hz); ESI-MS: 481.0 [M+1]⁺.

1-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(3,4-dichlorophenyl)urea (Ic): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 9.09 (s, 1H), 9.08 (s, 1H), 8.12 (s, 1H), 7.98 (s, 1H), 7.90 (d, 1H, J=2.4 Hz), 7.52–7.51 (m, 2H), 7.45–7.44 (m, 1H), 7.42–7.41 (m, 1H), 7.39–7.38 (m, 1H), 7.36–7.35 (m, 1H), 7.17 (br s, 2H), 6.78 (d, 1H, J=3.6 Hz). ESI-MS: 413.0 [M+1]⁺.

1-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(3,5-bis(trifluoromethyl)phenyl)urea (Id): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 9.63 (s, 1H), 9.46 (s, 1H), 8.70 (br s, 1H), 8.43 (br s, 2H), 8.15 (br s, 1H), 8.01 (br s, 1H), 7.85 (d, 1H, J=3.6 Hz), 7.78 (br s, 1H), 7.66 (br s, 1H), 7.56–7.48 (m, 2H), 7.42–7.40 (m, 1H) 7.33–7.20 (m, 1H). ESI-MS: 481.0 [M+1]⁺.

General Procedure for Synthesis of Compounds IIa–f

A mixture of compound 8a, b (30 mg, 0.1 mmol), the appropriate carboxylic acid derivative (0.2 mmol), HOBt (36 mg, 0.3 mmol), and EDCI (38 mg, 0.2 mmol) in dry DMF (1.0 mL) was cooled to 0°C under nitrogen atmosphere. Triethylamine (0.03 mL, 0.2 mmol) was added thereto at the same temperature. The mixture was then stirred at 80°C for 24 h. The reaction mixture was cooled and then partitioned between saturated aqueous sodium carbonate and ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×5 mL). The combined organic layer extracts were washed with brine and dried over anhydrous Na₂SO₄. After evaporation of the organic solvent, the residue was purified by column chromatography using the appropriate proportion of ethyl acetate and hexane as mobile phase.

N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamide (IIa): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.63 (s, 1H), 8.32 (br s, 1H), 8.28 (br s, 1H), 8.12 (s, 1H), 8.00–7.97 (m, 1H), 7.94–7.93 (m, 1H), 7.91–7.90 (m, 1H), 7.85–7.83 (m, 1H), 7.83–7.78 (m, 2H), 7.56 (d, 1H, J=3.6 Hz), 7.15 (br s, 2H), 6.77 (d, 1H, J=3.6 Hz); ESI-MS: 397.0 [M+1]⁺.

N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-4-chloro-3-(trifluoromethyl)benzamide (IIb): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.69 (s, 1H), 8.43–8.42 (m, 1H), 8.32–8.29 (m, 1H), 8.11 (s, 1H), 7.97–7.92 (m, 2H), 7.91–7.83 (m, 3H), 7.57 (d, 1H, J=3.6 Hz), 7.18 (br s, 2H), 6.78 (d, 1H, J=3.6 Hz); ESI-MS: 432.0 [M+1]⁺.

N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)pyridazine-4-carboxamide (IIc): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.94 (s, 1H), 9.70–9.67 (m, 1H), 9.52–9.50 (m, 1H), 8.32 (br s, 1H), 8.16–8.13 (m, 1H), 7.79–7.77 (m, 1H), 7.56–7.54 (m, 1H), 7.53–7.52 (m, 1H), 7.35–7.32 (m, 1H), 7.28–7.26 (m, 1H), 7.18 (br s, 2H), 6.81 (d, 1H, J=3.5 Hz); ESI-MS: 332.0 [M+1]⁺.

N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamide (IId): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.69 (s, 1H), 8.46–8.45 (m, 1H), 8.39–8.25 (m, 3H), 8.13 (s, 1H), 7.83–7.69 (m, 3H), 7.54–7.49 (m, 2H), 7.18 (br s, 2H), 6.80 (d, 1H, J=3.5 Hz); ESI-MS: 398.0 [M+1]⁺.

N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-4-chloro-3-(trifluoromethyl)benzamide (IIe): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.74 (s, 1H), 8.42–8.40 (m, 1H), 8.31–8.25 (m, 2H), 8.13 (s, 1H), 7.96–7.93 (m, 1H), 7.79–7.77 (m, 1H), 7.56–7.49 (m, 3H), 7.18 (br s, 2H), 6.80 (d, 1H, J=3.6 Hz); ESI-MS: 432.0 [M+1]⁺.

N-(3-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)benzamide (IIf): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.73 (s, 1H), 8.49–8.47 (m, 1H), 8.43–8.41 (m, 1H), 8.27 (br s, 2H), 8.19 (br s, 1H), 8.13 (s, 1H), 7.78–7.77 (m, 1H), 7.73 (br s, 1H), 7.56–7.53 (m, 2H), 7.35–7.30 (m, 1H), 7.18 (br s, 2H), 6.81 (d, 1H, J=3.6 Hz), 2.19 (s, 3H); ESI-MS: 478.0 [M+1]⁺.

N-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)acetamide (9)

A mixture of compound 5 (0.5 g, 3.7 mmol), acetic acid (0.4 g, 6.6 mmol), HOBt (1.1 g, 8.1 mmol), and EDCI (1.8 g, 9.3 mmol) in dry DMF (15 mL) was cooled to 0°C under nitrogen atmosphere. Triethylamine (1.6 mL, 11.4 mmol) was added thereto at the same temperature. The mixture was then stirred at 80°C for 24 h. The reaction mixture was cooled and then partitioned between water and ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layer extracts were washed with brine and dried over anhydrous Na₂SO₄. After evaporation of the organic solvent, the residue was purified by column chromatography (silica gel, ethyl acetate) to get the pure title compound (0.3 g, 45%) as a pale yellow oil. ¹H-NMR (DMSO-d₆, 300 MHz) δ: 12.00 (br s, 1H), 10.65 (br s, 1H), 8.44 (s, 1H), 7.35 (m, 1H), 6.73–6.68 (d, 1H, J=15.2 Hz), 2.21 (s, 3H).

N-(7-(4-Nitrophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-acetamide (10)

It was prepared by the same method utilized for synthesis of compounds 7a, b. Yield 45%; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.93 (s, 1H), 8.65 (br s, 1H), 8.44–8.30 (m, 2H), 8.28–8.17 (m, 2H), 8.04–8.00 (m, 1H), 7.05–7.00 (m, 1H), 2.07 (s, 3H).

N-(7-(4-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-acetamide (11)

To a solution of compound 10 (0.2 g, 0.7 mmol) in dry THF (10 mL) was added Pd/C (0.1 g) and was hydrogenated at 50 psi for 2 h, and then filtered through celite. The Pd/C residue was washed with further THF (10 mL) and concentration. The crude residue was purified by column chromatography (silica gel, ethyl acetate) to give the title product (45 mg, 25%) as a pale yellow oil. ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.75 (s, 1H), 8.48 (s, 1H), 7.61–7.59 (d, 1H, J=3.6 Hz), 7.34–7.31 (m, 2H), 6.88–6.87 (d, 1H, J=3.6 Hz), 6.72–6.66 (m, 2H), 5.31 (br s, 2H), 2.24 (s, 3H).

Synthesis of Compounds IIIa and b

They were prepared by the same method utilized for synthesis of Ia–d and IIa–f, respectively.

N-(7-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetamide (IIIa): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.81 (s, 1H), 9.24 (s, 1H), 9.08 (s, 1H), 8.54 (s, 1H), 8.12 (br s, 1H), 7.71 (d, 1H, J=3.6 Hz), 7.73–7.70 (m, 3H), 7.65–7.62 (m, 3H), 6.95 (d, 1H, J=3.5 Hz), 2.24 (s, 3H); ESI-MS: 489.0 [M+1]⁺.

N-(4-(4-Acetamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-4-chloro-3-(trifluoromethyl)benzamide (IIIb): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 10.84 (br s, 1H), 10.73 (br s, 1H), 8.57 (br s, 1H), 8.43–8.38 (m, 1H), 8.32–8.29 (m, 1H), 7.97–7.93 (m, 3H), 7.86–7.82 (m, 3H), 6.98–6.97 (d, 1H, J=3.5 Hz), 2.26 (s, 3H); ESI-MS: 474.0 [M+H]⁺.

Synthesis of Compounds 12a, b

They were synthesized in the same way as synthesis of compound 11.

N-(7-(4-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (12a): Yield 85%; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.19 (br s, 1H), 8.62 (s, 1H), 8.09–8.07 (m, 2H), 7.68–7.56 (m, 4H), 7.37–7.34 (m, 2H), 6.71–6.69 (m, 3H), 5.33 (br s, 2H).

N-(7-(3-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (12b): Yield 70%; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.22 (br s, 1H), 8.65 (s, 1H), 8.12–8.09 (m, 2H), 7.70–7.57 (m, 4H), 7.40–7.36 (m, 2H), 6.73–6.70 (m, 3H), 5.35 (br s, 2H).

Synthesis of Compounds IVa–f

This was carried out by the same procedure utilized for synthesis of compounds Ia–d.

N-{{7-{4-[3-(2,3-Dichlorophenyl)ureido]phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVa): IR (KBr): 3310, 1512, 1250, 708 cm^－1; ¹H-NMR(DMSO-d₆, 300 MHz) δ: 11.23 (br s, 1H), 9.72 (br s, 1H), 8.65 (s, 1H), 8.56 (s, 1H), 8.22–8.18 (m, 1H), 8.09 (d, 2H, J=7.7 Hz), 7.85 (d, 1H, J=3.7 Hz), 7.78 (d, 2H, J=8.9 Hz), 7.68–7.63 (m, 4H), 7.59–7.54 (m, 2H), 7.33–7.32 (m, 1H), 6.81 (d, 1H, J=6.6 Hz); ESI-MS: 516.1 [M+1]⁺.

N-{{7-{4-[3-(3,4-Dichlorophenyl)ureido]phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVb): IR (KBr): 3290, 1521, 1230, 689 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.23 (br s, 1H), 9.21–9.18 (m, 2H), 8.65 (s, 1H), 8.10–8.08 (m, 2H), 7.92–7.91 (m, 1H), 7.85 (d, 1H, J=3.6 Hz), 7.70–7.76 (m, 2H), 7.67–7.64 (m, 3H), 7.59–7.54 (m, 3H), 7.39–7.35 (m, 1H), 6.81 (d, 1H, J=3.3 Hz); ESI-MS: 417.1 [M+1]⁺.

N-{{7-{4-[3-(3-(Trifluoromethyl)phenyl)ureido]phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVc): IR (KBr): 3320, 1523, 1339, 1123, 698 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.21 (br s, 1H), 9.48 (br s, 1H), 9.38 (br s, 1H), 8.65 (s, 1H), 8.10–8.05 (m, 3H), 7.84 (d, 1H, J=3.6 Hz), 7.76–7.73 (m, 6H), 7.62–7.50 (m, 3H), 7.33–7.30 (m, 1H), 6.82 (d, 1H, J=3.6 Hz); ESI-MS: 517.1 [M+1]⁺.

N-{{7-{4-[3-(4-Chloro-3-trifluoromethyl-phenyl)-ureido]-phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVd): IR (KBr): 3331, 1522, 1310, 1175, 1032, 711 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.23 (br s, 1H), 9.38 (br s, 1H), 9.22 (br s, 1H), 8.65 (s, 1H), 8.14–8.08 (m, 3H), 7.85–7.83 (m, 1H), 7.77–7.74 (m, 2H), 7.70–7.61 (m, 5H), 7.58–7.47 (m, 2H), 6.81 (d, 1H, J=3.6 Hz); ESI-MS: 551.1 [M+1]⁺.

N-{{7-{4-[3-(3,5-Bis-trifluoromethyl-phenyl)ureido]phenyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl}}benzamide (IVe): IR (KBr): 3311, 1527, 1276, 1124, 879, 681 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.21 (br s, 1H), 9.63 (br s, 1H), 9.37 (br s, 1H), 8.65 (s, 1H), 8.17 (s, 2H), 8.09 (d, 2H, J=7.3 Hz), 7.84 (d, 1H, J=3.7 Hz), 7.78–7.75 (m, 2H), 7.70–7.63 (m, 4H), 7.58–7.56 (m, 2H), 6.82 (d, 1H, J=3.6 Hz); ESI-MS: 585.1 [M+1]⁺.

N-(7-(3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (IVf): ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.25 (br s, 1H), 9.41 (br s, 1H), 9.24 (br s, 1H), 8.67 (s, 1H), 8.17–8.11 (m, 3H), 7.88–7.85 (m, 1H), 7.79–7.76 (m, 2H), 7.73–7.63 (m, 5H), 7.61–7.50 (m, 2H), 6.83 (d, 1H, J=3.6 Hz); ESI-MS: 551.1 [M+1]⁺.

Synthesis of Compounds Va–c

This was carried out by the same procedure utilized for synthesis of compounds IIa–f.

N-[4-(4-Benzoylamino-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl]-3,4-dichlorobenzamide (Va): IR (KBr): 3427, 1620, 1401, 1027, 753 cm^－1; ¹H-NMR(DMSO-d₆, 300 MHz) δ: 11.23 (br s, 1H), 10.59 (br s, 1H), 8.67 (s, 1H), 8.27 (d, 1H, J=1.7 Hz), 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=3.8 Hz); 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, 1523, 1312, 1123, 708 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.24 (br s, 1H), 10.68 (br s, 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=3.7 Hz); 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, 750 cm^－1; ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.27 (br s, 1H), 10.75 (br s, 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=3.6 Hz); ESI-MS: 536.1 [M+1]⁺.

Sixty Cancer Cell Line Screening at the NCI

Screening against a panel of 60 cancer cell lines was carried out at the National Cancer Institute (NCI), Bethesda, Maryland, U.S.A.,²²⁾ applying the standard protocol of the NCI.^23,24)

Kinase Profiling

Reaction Biology Corp. Kinase HotSpot^SM service²⁵⁾ was used for screening of the test compound, and IC₅₀ Profiler Express for IC₅₀ measurement. Assay protocol: In a final reaction volume of 25 µL, kinase (5–10 mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic protein, 10 mM magnesium acetate and [γ³³P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the Mg-ATP mix. After incubation for 40 min at room temperature, the reaction is stopped by the addition of 5 µL of a 3% phosphoric acid solution. Ten microliter of the reaction is then spotted onto a P30 filtermat and washed three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.

Acknowledgment

This work was supported by Korea Institute of Science and Technology (KIST), KIST Project (2E22360). We would like to thank the National Cancer Institute (NCI), Bethesda, Maryland, U.S.A., for performing the anticancer testing of the target compounds over 60 cancer cell lines.

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