+
+
Cytokinin-Derived cdk Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 411
16: 1.1 mmol of methyl iodide, DMF; column chromatog-
raphy stepwise 0, 1, 2% MeOH in CHCl3; crystallization
CHCl3-Et2O; yield 79%; mp 197-200 °C. Anal. (C11H14N5-
Cl) C, H, N.
18: 1.1 mmol of methyl iodide, DMF; column chromatog-
raphy CHCl3-MeOH (100:4); yield 85%; mp 209-210 °C.
Anal. (C13H18N5Cl) C, H, N.
20: 10 mmol of isopropyl bromide, DMSO; crystallization
from hot MeOH; yield 85%; mp 181-182 °C; 1H NMR (400
MHz, DMSO) 1.48 (6H, d, J ) 6.8, CH3), 4.57 (1H, m, CH),
4.66 (2H, d, J ) 6.1, CH2), 7.19 (1H, tt, J ) 7.2, 1.7, H-p), 7.27
(2H, dd, J ) 7.2, H-m), 7.34 (2H, dd, J ) 7.2, 1.7, H-o), 7.69
(1H, s, H-C8). Anal. (C15H16N5Cl) C, H, N.
R ea ct ion of 2-Ch lor o Der iva t ives of P u r in e w it h
Alk yla m in es: P r ep a r a t ion of 2,6-Bis(a lk yla m in o)-9-
alkylpu r in es, 6-Am in o-2-(alkylam in o)-9-alkylpu r in es, an d
6-Am in o-2-(alkylam in o)pu r in es. 6-(Alkylamino)- or 6-amino-
2-chloro-9-alkylpurine or 6-amino-2-chloropurine (0.5 mmol)
and 3 mL of the appropriate amine were heated for 3 h to 155-
160 °C (sealed ampule). Excess of the amine was evaporated
at a temperature below 60 °C, and the residue was purified
by column chromatography or (and) crystallized.
22: column chromatography stepwise 0, 1, 2, 4% MeOH in
CHCl3; crystallized from CHCl3-Et2O; yield 71%; mp 142-
1
143 °C; H NMR (200 MHz, CDCl3) 1.535 (6H, d, J ) 7.0 Hz,
(CH3)2CH), 3.53-3.60 (2H, m, CH2NHC2), 3.83 (2H, t, J ) 4.2,
CH2O), 3.61 (1H, hept, J ) 7.0, CH(CH3)2), 3.75 (2H, br t,
CH2Ph), 5.20 (1H, br s, OH or NH), 5.26 (1H, br t, NHC2),
6.00 (1H, br s, NH or OH), 7.23-7.36 (5H, m, Ph), 7.48 (1H, s,
HC8). Anal. (C17H22N6O) C, H, N.
23: column chromatography stepwise 0, 1, 2, 4% MeOH in
CHCl3; crystallized from CHCl3-heptane; yield 73%; mp 144-
146 °C; MS (AEI-MS 902) 340 (20, M+ + 1), 295 (60), 282 (15),
253 (10), 106 (11), 91 (100); 1H NMR (200 MHz, CDCl3) 1.213
(3H, d, J ) 6.4, CH3,CHOH), 1.535 (6H, d, J ) 6.8, (CH3)2-
CH), 3.4 (2H, m, CH2NHC2), 4.03 (1H, m, CHO), 4.62 (1H,
hept, J ) 6.8, CH(CH3)2), 4.75 (2H, br d, CH2Ph), 5.20 (1H,
NHC2), 5.95 (1H, NH). Anal. (C18H24N6O) C, H, N.
24: column chromatography stepwise 0, 1, 2, 4% MeOH in
CHCl3; crystallized from CHCl3-Et2O; yield 69%; mp 137-
139 °C; MS(Finnigan MAT 90) 354.2167 (M+, C19H26N6O, calcd
354.2168, 27), 325 (7), 324 (29), 323 (100), 295 (3), 282 (7),
281 (3), 217 (6), 185 (5), 134 (3), 106 (3), 91 (34); 1H NMR (200
MHz, CDCl3) 1.018 (3H, t, J ) 6.8, CH3CH2), 1.522 (6H, d, J
) 6.6, (CH3)2CH), 1.58 (2H, m, CH2CH3), 3.62 (1H, m, CHNH),
3.83 (2H, m, CH2OH), 4.59 (1H, hept, J ) 6.8, CH(CH3)2), 4.75
(2H, br s, CH2Ph), 4.9 (1H, br d, J ) 6.0, NHC2), 5.15 (1H, br
s, OH or NH), 6.07 (1H, br s, NH or OH), 7.22-7.36 (5H, m,
Ph), 7.23 (1H, s, HC8). Anal. (C19H26N6O) C, H, N.
25: for purification see 24; yield 64%; mp 118-120 °C; [R]D
) +35.3 (c ) 0.57, CHCl3). Anal. (C19H26N6O) C, H, N.
26: for purification see 24; yield 62%; mp 102-104 °C; [R]D
) -34.6 (c ) 0.43, CHCl3). Anal. (C19H26N6O) C, H, N.
Alk yla t ion of 2,6-Su b st it u t ed P u r in es a t P osit ion
9 ((n -Bu )4NOH m eth od ). 6-Amino-2-chloropurine or 2-amino-
6-(benzylamino)purine (1 mmol), 40% aqueous tetrabutyl-
ammonium hydroxide (1 mL), methyl iodide (1.2 mmol), and
6 mL of dichloromethane were vigorously shaken for 1 h. After
10 min all the components had dissolved and the product
started to precipitate. It was filtered and washed with
dichloromethane. Crystallization from 1 N HCl afforded the
appropriate hydrochloride, or after alkalization with NH4OH
the free base was prepared.
5: decolorized and crystallized from water; yield 51%; mp
259-260 °C (differs from that given in the literature, 247-
248 °C or over 300 °C8); likewise UV λmax (pH 1) 245 (sh), 289,
(pH 13) 290 disagree with previously described results,8 i.e.,
water (pH 1.1) 249, 298, water (pH 12.4) 263 (sh), 286; MS
and NMR spectra are in accordance with the proposed
structure; MS (AEI-MS 902) 194 (M+, 21),163 (100), 150 (26),
1
134 (42), 121 (11); H NMR (270 MHz, DMSO) 3.30 (2H, t, J
) 6.0, CH2), 3.48 (2H, t, J ) 6.0, CH2), 4.67 (1H, br s, exch
with D2O, OH), 6.02 (1H, t, J ) 5.0, exch with D2O, NH), 6.64
(2H, s, exch with D2O, NH2), 7.67 (1H, s, H-8), 12.14 (1H, br
s, exch with D2O, H-9). Anal. (C7H10N6O‚0.5H2O) C, H, N.
8: crystallized from water; yield 87%; mp 206-208 °C; UV
λ
max (pH 1) 216, 254, 297, (pH 13) 221, 255, 287; MS (AEI-MS
902): 208 (M+, 21), 177 (100), 164 (26), 148 (44), 42 (32); H
NMR (270 MHz, DMSO) 3.33 (5H, m, CH2 + NCH3), 3.51 (2H,
t, J ) 5.9, CH2), 4.734 (1H, br s, exch with D2O, OH), 6.11
(1H, t, J ) 5.6, exch with D2O, NH), 6,69 (2H, s, exch with
D2O, NH2), 7.66 (1H, s, H-8). Anal. (C8H12N6O‚1H2O) C, H,
N.
1
7: hydrochloride; yield 46%; mp over 330 °C; UV λmax (pH
1) 267, (pH 13) 265; TLC 1 M solution of NH3 in CHCl3-MeOH
(95:5), one spot Rf 0.3 identical with the sample prepared from
2,6-dichloro-9-methylpurine according to ref 8. Anal. (C6H6N5-
Cl‚HCl‚0.5H2O) C, H, N.
17: column chromatography stepwise 0, 2, 4% MeOH in
CHCl3 with a trace of concentrated NH4OH; crystallized from
CHCl3-cyclohexane; yield 91%; mp 130-131 °C; UV λmax (pH
1) 251, 294, (pH 13) 260, 288.5; MS (AEI-MS 902) 276 (M+,
39), 261 (13), 245 (27), 233 (22), 189 (26), 178 (18), 177 (100),
148 (32), 69 (12), 41 (43); 1H NMR (400 MHz, CDCl3) 1.71 (3H,
d, J ) 1.2, CH3), 1.73 (3H, dt, J ) 1.2, 1.2, CH3), 2.01 (1H, br
s, OH), 3.58 (2H, m, NCH2CH2O), 3.64 (3H, s, N9-CH3), 3.83
(2H, m, OCH2CH2N), 4.10 (2H, br s, CH2), 5.29 (1H, t, J )
5.4, NHCH2), 5.33 (1H, dqq, J ) 7.2, 1.2, 1.2, -CHd), 5.55 (1H,
br s, N-H), 7.40 (1H, s, -CHd). Anal. (C13H20N6O) C, H, N.
19: column chromatography stepwise 0, 2, 3, 4% MeOH in
CHCl3 with a trace of concentrated NH4OH; crystallized from
benzene; yield 75%; mp 130-31 °C. UV λmax (pH 1) 254, 294.5,
(pH 13) 288; MS (J eol DX 303, direct inlet) 304 (M+, 56), 273
(64), 260 (7), 221 (57), 208 (24), 203 (25), 191 (29), 177 (80),
164 (39), 148 (57), 133 (20), 121 (21), 106 (14), 79 (12), 67 (15),
55 (100); 1H NMR (200 MHz, CDCl3) 0.95-1.30 (5H, m, C6H11),
1.50-1.90 (6H, m, C6H11), 3.32-3.45 (2H, br s, CH2C6H11), 3.55
(2H, m, CH2CH2NH), 3.64 (3H, s, CH3N9), 3.828 (3H, J ) 4.2,
CH2OH), 5.25 (1H, br t, NHC2), 5.65 (1H, br s, NH), 7.403 (1H,
HC8). Anal. (C15H24N6O) C, H, N.
21: column chromatography toluene-MeOH (85:15): after
hydrolysis of tetrahydropyranyl group (MeOH-2 N HCl, 10:
1, 8 h, ambient temperature) crystals from reaction mixture;
yield 65%; mp 180-190 °C; MS (EEI-MS 902) 328 (22, M+),
310 (10, M - H2O), 298 (26, M - CH2O), 284 (18, M - C2H4O),
254 (7, 298 - C2H4O), 240 (5, 284 - C2H4O), 106 (10,
pHCHdNH2+), 91 (100, C7H7), 65 (7, C5H5); 1H NMR (400
MHz, D2O) COSY [3.694 (2H, br s, CH2NHC2), 3.561 (2H, t, J
) 5.4, CH2CH2NHC2)], COSY [3.912 (2H, t, J ) 5.0, CH2N9),
4.257 (2H, t, J ) 5.0, CH2CH2N9)], 4.735 (2H, br s, CH2Ph),
COSY [7.316 (1H, m, Ph), 7.356-7.368 (4H, m, Ph)], 8.008 (1H,
br s, H-C8). Anal. (C14H14N5ClO‚0.25H2O) C, H, N.
14: free base; yield 30%; mp 214-216 °C; UV λmax (pH 1)
252, 288, (pH 13) 260 (sh), 282; MS (AEI-MS 902) 254 (M+,
100), 149 (65), 106 (64), 91 (46), 65 (17), 42 (30); 1H NMR (270
MHz, DMSO) 3.36 (3H, br s, N-CH3), 4.66 (2H, br s, CH2), 5.91
(2H, br s, NH2), 7.17-7.36 (5H, mt, Ar), 7.68 (1H, s, H-8), 8.00
(1H, t, NH). Anal. (C13H14N6‚1H2O) C, H, N.
p 34cd c2/Hist on e H 1 Kin a se Assa y. p34cdc2/cyclin B was
purified from M phase oocytes of the starfish Marthasterias
glacialis by affinity chromatography on p9CKhs1-Sepharose
beads,17 from which it was eluted by free p9CKhs1
. The cdc2
assay mixture contained 10 µL of histone H1 (1 mg/mL), 10
µL of [γ-32P]ATP (15 µmol, 3000 Ci/mmol, 1 mCi/mL), and 10
µL of the inhibitor (0.1-1000 µmol), all in reaction buffer C
(60 mmol of â-glycerolphosphate, 15 mmol of p-nitrophenyl
phosphate, 25 mmol of MOPS, pH 7.2, 5 mmol of EGTA, 15
mmol of MgCl2, 1 mmol of dithiothreitol, 1 mmol of sodium
vanadate, 1 mmol of phenyl phosphate, 10 µg/mL leupeptin,
10 µg/mL aprotinin, 10 µg/mL soybean trypsin inhibitor, 100
µmol of benzamidine). For determination of maximum phos-
phate incorporation, buffer C was used instead of inhibitor.
Nonspecific binding was determined in the absence of histone
H1 in the reaction mixture and substracted from each volume.
The assays were started by addition of radioactive ATP, and
after 10 min incubation at 30 °C, 25 µL aliquots of the
supernatant were spotted onto 2.5 × 3.0 cm pieces of Whatman
P81 phosphocellulose paper. After 20 s, the filters were
washed five times (for at least 5 min each time) in 0.1%
phosphoric acid. The wet filters were transferred into 5 mL
of ACS scintillation cocktail (Amersham), and after mixing,
32P radioactivity was determined using a Packard Tri-Carb