Pyrimidine-Based Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1829
(s, 1 H). MS m/z: 295 (M+ + 1). HRMS for C12H9ClFN5O
(M+): calcd, 294.0552; found, 294.0543.
7.6 Hz, 3 H), 2.21 (s, 3 H), 2.56 (q, J ) 7.6 Hz, 2 H), 7.08 (d,
J ) 8.3 Hz, 1 H), 7.25 (s, 1 H), 7.42 (m, 1 H), 7.80 (s, 1 H),
8.62 (bs, 1 H), 10.29 (bs, 1 H), 13.05 (bs, 1 H). MS m/z: 270
(M+ + 1). HRMS for C14H15N5O (M+): calcd, 270.1349; found,
270.1336.
6-[(3,4-Dich lor oben zyl)am in o]-1,5-dih ydr o-4H-pyr azolo-
[3,4-d ]p yr im id in -4-on e, 14. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3,4-dichlorobenzyl-
amine, compound 14 was obtained according to the procedure
6-[(3-Iodo-4-m eth ylph en yl)am in o]-1,5-dih ydr o-4H-pyr a-
zolo[3,4-d ]p yr im id in -4-on e, 23. Starting from 6-chloro-1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3-iodo-4-meth-
ylaniline, compound 23 was obtained according to the proce-
1
described for 12. H NMR (DMSO): δ 4.49 (d, J ) 5.9 Hz, 2
H), 6.99 (bs, 1 H), 7.31 (dd, J ) 8.4 Hz, J ) 1.7 Hz, 1 H), 7.56-
7.59 (m, 2 H), 7.74 (bs, 1 H), 10.39 (bs, 1 H), 12.88 (bs, 1 H).
MS m/z: 311 (M+ + 1). Anal. (C12H9Cl2N5O) C, H. N: calcd,
22.58; found, 22.14.
1
dure described for 12. H NMR (DMSO): δ 2.32 (s, 3 H), 7.26
(d, J ) 8.5 Hz, 1 H), 7.44 (dd, J ) 8.3 Hz, J ) 2.0 Hz, 1 H),
7.87 (s, 1 H), 8.16 (d, J ) 1.8 Hz, 1 H), 8.82 (s, 1 H), 10.41 (s,
1 H). MS m/z: 368 (M+ + 1). Anal. (C12H10IN5O) H, N. C: calcd,
39.26; found, 38.80. HRMS (M+): calcd, 368.0003; found,
368.0002.
6-[(4-Br om ob en zyl)a m in o]-1,5-d ih yd r o-4H -p yr a zolo-
[3,4-d ]p yr im id in -4-on e, 15. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 4-bromobenzyl-
amine, compound 15 was obtained according to the procedure
1
described for 12. H NMR (DMSO): δ 4.46 (d, J ) 5.9 Hz, 2
6-[(3-Br om o-4-m et h ylp h en yl)a m in o]-1,5-d ih yd r o-4H -
p yr a zolo[3,4-d ]p yr im id in -4-on e, 24. Starting from 6-chloro-
1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3-bromo-
4-methylaniline, compound 24 was obtained according to the
procedure described for 12. 1H NMR (DMSO): δ 2.29 (s, 3 H),
7.29 (d, J ) 8.0 Hz, 1 H), 7.36 (dd, J ) 8.3 Hz, J ) 2.2 Hz, 1
H), 7.82 (s, 1 H), 8.01 (d, J ) 2.3 Hz, 1 H), 8.84 (s, 1 H), 10.42
(s, 1 H), 13.17 (s, 1 H). MS m/z: 321 (M+ + 1). HRMS for
H), 6.93 (bs, 1 H), 7.27 (d, J ) 8.5 Hz, 1 H), 7.51 (d, J ) 8.5
Hz, 1 H), 7.72 (s, 1 H), 10.50 (bs, 1 H), 12.86 (s, 1 H). MS m/z:
321 (M+ + 1). HRMS for C12H10BrN5O (M+): calcd, 320.0142;
found, 320.0140.
6-[(2-Ch lor ob en zyl)a m in o]-1,5-d ih yd r o-4H -p yr a zolo-
[3,4-d ]p yr im id in -4-on e, 16. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 2-chlorobenzyl-
amine, compound 16 was obtained according to the procedure
C
12H10BrN5O (M+): calcd, 320.0412; found, 320.0136.
1
described for 12. H NMR (DMSO): δ 4.56 (d, J ) 6.0 Hz, 2
6-[(3-Ch lor o-4-m et h ylp h en yl)a m in o]-1,5-d ih yd r o-4H -
H), 6.94 (bs, 1 H), 7.27-7.38 (m, 3 H), 7.45 (dd, J ) 7.3 Hz, J
) 1.8 Hz, 1 H), 7.73 (bs, 1 H), 12.89 (bs, 1 H). MS m/z: 276
(M+ + 1). Anal. (C12H10ClN5O) C, H, N.
p yr a zolo[3,4-d ]p yr im id in -4-on e, 25. Starting from 6-chloro-
1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3-chloro-
4-methylaniline, compound 25 was obtained according to the
procedure described for 12. 1H NMR (DMSO): δ 2.27 (s, 3 H),
7.28-7.31 (m, 2 H), 7.90 (s, 1 H), 8.95 (s, 1 H), 10.44 (s, 1 H).
MS m/z: 276 (M+ + 1). HRMS for C12H10ClN5O (M+): calcd,
276.0647; found, 276.0634.
6-[(4-Meth oxyben zyl)a m in o]-1,5-d ih yd r o-4H-p yr a zolo-
[3,4-d ]p yr im id in -4-on e, 17. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 4-methoxybenzyl-
amine, compound 17 was obtained according to the procedure
1
described for 12. H NMR (DMSO): δ 3.71 (s, 3 H), 4.40 (d, J
6-[(3,4-Dim et h ylp h en yl)a m in o]-1,5-d ih yd r o-4H -p yr a -
zolo[3,4-d ]p yr im id in -4-on e, 26. Starting from 6-chloro-1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3,4-dimethyl-
aniline, compound 26 was obtained according to the procedure
) 5.7 Hz, 2 H), 6.80 (bs, 1 H), 6.89 (d, J ) 8.7 Hz, 1 H), 7.25
(d, J ) 8.7 Hz, 1 H), 7.73 (bs, 1 H), 12.88 (bs, 1 H). MS m/z:
272 (M+ + 1). Anal. (C13H13N5O2) C, H, N.
1
6-[(2,4-Dim eth ylben zyl)am in o]-1,5-dih ydr o-4H-pyr azolo-
[3,4-d ]p yr im id in -4-on e, 18. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 2,4-dimethylben-
zylamine, compound 18 was obtained according to the procedure
described for 12. H NMR (DMSO): δ 2.17 (s, 3 H), 2.20 (s, 3
H), 7.08 (d, J ) 8.2 Hz, 1 H), 7.28 (s, 1 H), 7.38 (m, 1 H), 7.80
(s, 1 H), 8.62 (bs, 1 H), 10.28 (bs, 1 H), 13.05 (s, 1 H). MS m/z:
256 (M+ + 1). Anal. (C13H13N5O) C, H, N.
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described for 12. H NMR (DMSO): δ 2.23 (s, 3H), 2.25 (s, 3
Clon in g, Exp r ession , a n d P u r ifica tion of DNA P oly-
m er a se III fr om Sta ph ylococcu s a u r eu s. DNA polymerase
III (pol III) was cloned by PCR from S. aureus genomic DNA.
The pol III gene was cloned into pET15. The pol III/pET15
was transformed into BL21 cells. The cells were grown at 29
°C in M9ZB media containing ampicillin and chloramphenicol
to an OD of 1.0. The cells were induced by adding 1 mM IPTG
and grown for 3 h. The cells were centrifuged at 2500g for 10
min. The cell pellet was resuspended in 50 mM Tris, pH 7.4,
0.1% Triton X100, 1 mM EDTA, 20% glycerol, and complete
protease inhibitor cocktail (Boehringer Mannheim) and were
lysed by freeze-thawing three times in a dry ice and methanol
bath. The lysate was centrifuged at 10000g for 1 h.
The supernatant was loaded onto a HiTrapQ column that
was equilibrated in 50 mM Tris, pH 7.4, 20% glycerol, and 1
mM EDTA. Following loading, the column was washed with
20 column volumes of buffer. The pol III was eluted using a
linear NaCl gradient from 0 to 0.5 M. The pol III activity eluted
between 0.3 and 0.4 M NaCl. The peak of activity was loaded
onto a HiTrap Blue column equilibrated in 50 mM Tris, pH
7.4, 20% glycerol, 1 mM EDTA, and 1 mg/mL BSA. The column
was washed with 10 column volumes of equilibration buffer,
followed by a wash with 20 column volumes of equilibration
buffer containing 0.5 M NaCl. The pol III was eluted by
washing the column with equilibration buffer containing 3 M
NaCl.
H), 4.40 (d, J ) 5.5 Hz, 2 H), 6.71 (bs, 1 H), 6.96 (d, J ) 7.8
Hz, 1 H), 7.00 (s, 1 H), 7.13 (d, J ) 7.8 Hz, 1 H), 7.73 (s, 1 H),
10.24 (bs, 1 H), 12.87 (bs, 1 H). MS m/z: 270 (M+ + 1). Anal.
(C14H15N5O) C, H, N.
6-[(4-Isopr opylben zyl)am in o]-1,5-dih ydr o-4H-pyr azolo-
[3,4-d ]p yr im id in -4-on e, 19. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 4-isopropylbenzyl-
amine, compound 19 was obtained according to the procedure
1
described for 12. H NMR (DMSO): δ 1.16 (s, 3 H), 1.17 (s, 3
H), 2.81-2.88 (m, 1 H), 4.43 (d, J ) 5.7 Hz, 2 H), 6.83 (bs, 1
H), 7.18-7.25 (m, 4 H), 7.72 (bs, 1 H), 10.35 (bs, 1 H), 12.87
(bs, 1 H). MS m/z: 284 (M+ + 1). Anal. (C15H17N5O) C, H, N.
6-{[(3-(Tr iflu or om et h yl)b en zyl]a m in o}-1,5-d ih yd r o-
4H-pyr azolo[3,4-d]pyr im idin -4-on e, 20. Starting from 6-chlo-
ro-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3-tri-
fluoromethylbenzylamine, compound 20 was obtained accord-
ing to the procedure described for 12. 1H NMR (DMSO): δ
4.58 (d, J ) 6.0 Hz, 2 H), 7.01 (bs, 1 H), 7.54-7.64 (m, 3 H),
7.67 (s, 1 H), 7.74 (bs, 1 H), 12.87 (bs, 1 H). MS m/z: 310 (M+
+ 1). Anal. (C13H10F3N5O) C, H, N.
6-[(3-Meth oxyben zyl)a m in o]-1,5-d ih yd r o-4H-p yr a zolo-
[3,4-d ]p yr im id in -4-on e, 21. Starting from 6-chloro-1,5-dihy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3-methoxybenzyl-
amine, compound 21 was obtained according to the procedure
1
described for 12. H NMR (DMSO): δ 3.72 (s, 3 H), 4.46 (d, J
) 5.9 Hz, 2 H), 6.81 (dd, J ) 8.3 Hz, J ) 1.9 Hz, 1 H), 6.88-
6.90 (m, 3 H), 7.24 (t, J ) 8.2 Hz, 1 H), 7.73 (bs, 1 H), 12.87
(bs, 1 H). MS m/z: 272 (M+ + 1). Anal. (C13H13N5O2) C, H, N.
6-[(3-E t h yl-4-m et h ylp h en yl)a m in o]-1,5-d ih yd r o-4H -
p yr a zolo[3,4-d ]p yr im id in -4-on e, 22. Starting from 6-chloro-
1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one and 3-ethyl-4-
methylaniline, compound 22 was obtained according to the
DNA P olym er a se III En zym e In h ibition Assa y. The
reaction mix consisted of 30 mM Tris-HCl, pH 7.5, 20%
glycerol, 4 mM DTT, 10 mM MgOAc, 0.003 mM dATP, 0.003
mM dGTP, 0.001 mM dCTP, 0.001 mM [3H]dTTP, and 0.35
mg/mL “activated” calf thymus DNA (Worthington Enzymes)
in a final volume of 0.1 mL. The assay was initiated by the
addition of enzyme and incubated for 30 min at 30 °C. The
assay was stopped by the addition of cold 10% TCA/0.1%
1
procedure described for 12. H NMR (DMSO): δ 1.15 (t, J )