Y.J. Esvan et al. / European Journal of Medicinal Chemistry 118 (2016) 170e177
175
pressure. Residue was purified by flash chromatography (EtOAc/
cyclohexane 7:3 to 10:0) yielding the isoquinoline 9 (276 mg,
1.42 mmol, 76%) as a purple powder. Rf ¼ 0.3 (EtOAc/cyclohexane
calcd for C11H8N5O2 (M þ H)þ 242.0673, found 242.0682. HPLC
(method I): purity ꢁ 98%, tR ¼ 15.8 min.
7:3). Mp 138e140 ꢂC; IR (ATR): 3500-2965, 1626, 1436, 1177 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6): 4.73 (2H, d, J ¼ 5.2 Hz), 5.68 (1H, t,
J ¼ 5.2 Hz, OH), 7.80 (1H, d, J ¼ 6.0 Hz), 8.12 (1H, s), 8.25 (1H, s), 8.51
(1H, d, J ¼ 6.0 Hz), 9.37 (1H, s); 13C NMR (100 MHz, DMSO-d6): 60.5
(CH2), 119.2, 125.9, 126.1, 143.3, 152.2 (CHarom), 126.7, 134.2, 134.8,
139.3 (Carom); HRMS (ESIþ) calcd for C10H9ClNO (M þ H)þ 194.0373,
found 194.0366.
4.1.13. Pyrido[3,4-g]quinazoline-2,10-diamine (13)
To a suspension of compound 12 (50 mg, 0.21 mmol) in 40 mL of
1:1 mixture of anhydrous dichloromethane and anhydrous meth-
anol was added palladium on charcoal (10% wt, 9 mg, 0.008 mmol).
The mixture was stirred under 1 atm. H2 for 3 h, and filtered
through Celite. The Celite pad was washed with a mixture of
CH2Cl2/MeOH 1:1. Combined filtrates were concentrated under
reduced pressure to give the title compound without further pu-
rification (43 mg, 0.21 mmol, quantitative yield) as a red powder.
Rf ¼ 0.2 (EtOAc). Mp > 250 ꢂC (decomposition); IR (ATR): 3400-
3000, 1646, 1614 cmꢀ1; 1H NMR (500 MHz, DMSO-d6): 6.24 (2H, br
s, NH2), 7.02 (2H, br s, NH2), 7.85 (1H, s), 7.97 (1H, d, J ¼ 6.2 Hz), 8.20
(1H, d, J ¼ 6.2 Hz), 9.23 (1H, s), 9.36 (1H, s); 13C NMR (100 MHz,
DMSO-d6): 113.6, 115.4, 139.4, 154.8, 164.8 (CHarom), 118.9, 121.0,
124.4, 135.1, 136.0, 158.7 (Carom); HRMS (ESIþ) calcd for C11H10N5
4.1.10. 6-Chloroisoquinoline-7-carbaldehyde (10)
To a solution of compound 9 (3.73 g, 19.3 mmol, 1 equiv) in
CHCl3 (175 mL) was added MnO2 (5.02 g, 57.8 mmol, 3 equiv). The
mixture was stirred at reflux for 36 h, filtered through a pad of
Celite and washed with EtOAc. The filtrate was concentrated in
vacuo. The crude product was triturated with cyclohexane and
diisopropyl ether yielding compound 10 (3.28 g, 17.1 mmol, 88%) as
a purple powder. Rf ¼ 0.2 (CH2Cl2/EtOAc 9:1). Mp 155e157 ꢂC; IR
(M
þ
H)þ 212.0936, found 212.0941. HPLC (method II):
(ATR): 1692, 1617, 1410 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 7.90
;
purity ꢁ 95%, tR ¼ 8.1 min.
(1H, d, J ¼ 6.0 Hz), 8.30 (1H, s), 8.68 (1H, d, J ¼ 6.0 Hz), 8.78 (1H, s),
9.57 (1H, s), 10.44 (1H, s, CHO); 13C NMR (100 MHz, DMSO-d6):
119.5, 127.9, 132.5, 146.2, 154.4 (CHarom), 126.0, 131.0, 135.1, 138.0
(Carom), 189.6 (CO); HRMS (ESIþ) calcd for C10H7ClNO (M þ H)þ
192.0216, found 192.0225.
4.1.14. Pyrido[3,4-g]quinazolin-2-amine (14)
To an ice/water bath cooled solution of compound 13 (57 mg,
0.27 mmol, 1 equiv) in a 5 M aqueous solution of HCl (2 mL), was
added dropwise a solution of NaNO2 (24 mg, 0.35 mmol, 1.3 equiv)
in water (2 mL) and the mixture was stirred at 0 ꢂC for 1 h. Then, an
aqueous solution of H3PO2 (50% wt, 0.5 mL) was added and the
mixture was stirred at room temperature for 24 h. The mixture was
then diluted with water and basified by addition of a saturated
aqueous NaHCO3 solution. The aqueous layer was extracted with
EtOAc and the organic layers were dried over MgSO4 and concen-
trated. Residue was purified by flash chromatography on Et3N
neutralized silica gel using CH2Cl2/MeOH/Et3N 96:3:1 as eluant.
The residue was dissolved in EtOAc and further washed with a
diluted NaHCO3 aqueous solution. The organic phase was dried
over MgSO4 and evaporated yielding the title compound 14 (15 mg,
0.08 mmol, 29%) as a yellow powder. Rf ¼ 0.3 (CH2Cl2/MeOH/Et3N
96:3:1). Mp > 220 ꢂC (decomposition); IR (ATR): 3350-3100, 1653,
1624, 1579, 1558 cmꢀ1; 1H NMR (400 MHz, DMSO-d6): 7.32 (2H, br
s, NH2), 7.78 (1H, d, J ¼ 6.0 Hz), 7.84 (1H, s), 8.36 (1H, d, J ¼ 6.0 Hz),
8.73 (1H, s), 9.40 (1H, s), 9.49 (1H, s); 13C NMR (100 MHz, DMSO-
d6): 118.0, 119.3, 130.5, 142.7, 154.8, 165.4 (CHarom), 119.7, 124.0,
137.5, 149.4, 160.3 (Carom); HRMS (ESIþ) calcd for C11H9N4 (M þ H)þ
197.0827, found 197.0835. HPLC (method III): purity ꢁ 99%,
tR ¼ 4.8 min.
4.1.11. 6-Chloro-5-nitroisoquinoline-7-carbaldehyde (11)
To a solution of compound 10 (1.35 g, 7.05 mmol, 1 equiv) in
concentrated sulfuric acid (15 mL) was added dropwise a 65% nitric
acid solution (0.6 mL, 8.45 mmol, 1.2 equiv). After stirring overnight
at room temperature the reaction mixture was poured into ice/
water, and filtered through a pad of Celite. Portions of solid NaHCO3
were added to the filtrate (until pH > 7) before extraction with
EtOAc. The organic layers were washed with brine, dried over
MgSO4 and concentrated under reduced pressure. The crude ma-
terial was triturated with diethyl ether yielding compound 11
(1.45 g, 6.13 mmol, 87%) as a pale brown powder. Rf ¼ 0.4 (CH2Cl2/
EtOAc 9:1). Mp 186e187 ꢂC; IR (ATR): 1692, 1613, 1523, 1268 cmꢀ1
;
1H NMR (500 MHz, DMSO-d6): 7.73 (1H, dt, J1 ¼ 6.0 Hz; J2 ¼ 0.8 Hz),
8.86 (1H, d, J ¼ 6.0 Hz), 9.07 (1H, d, J ¼ 0.8 Hz), 9.78 (1H, d,
J ¼ 0.8 Hz),10.39 (1H, s, CHO); 13C NMR (100 MHz, DMSO-d6): 113.3,
134.5, 148.6, 154.9 (CHarom), 125.7, 127.2, 129.0, 130.7, 145.2 (Carom),
187.8 (CO); HRMS (ESIþ) calcd for C10H6ClN2O3 (M þ H)þ 237.0067,
found 237.0078.
4.1.12. 10-nitropyrido[3,4-g]quinazolin-2-amine (12)
A 10 mL CEM Discover microwave tube was charged with
compound 11 (100 mg, 0.42 mmol, 1 equiv) and guanidine car-
bonate (100 mg, 0.52 mmol, 1.3 equiv) in 4 mL of N,N-dime-
4.2. Crystallography
thylformamide (99.8%; for peptide synthesis) resulting in
suspension. The reaction flask was purged with argon for 30 min,
sealed and irradiated for 45 (Discover Mode, Control
a
Recombinant CLK1 was expressed and purified as previously
described [33]. Apo crystals were obtained using sitting vapour
diffusion method at 4 ꢂC and the condition containing 30% 1,2-
propanediol, 10% glycerol and 50 mM Na/K phosphate. Soaking
was performed overnight using the reservoir solution supple-
mented with 5e10 mM compounds and 25% glycerol. Diffraction
data were collected at Diamond Light Source, beamline I03 using X-
ray at 0.97625 Å wavelength, and were processed and scaled with
Mosflm [34] and Scala [35], respectively. Structures were solved by
molecular replacement method using Phaser [36] and the co-
ordinates of published CLK1 structure [33]. Model rebuilding was
performed in COOT [37], and the structures were refined using
REFMAC [38]. Geometric correctness was verified using MOLPRO-
BITY [39]. The data collection and refinement statistics are sum-
marized in the table below.
s
Type ¼ Standard, P ¼ 300 W, T ¼ 166 ꢂC). After cooling, the reaction
was diluted with EtOAc and filtered through a pad of Celite. The
organic layer was washed with water, dried over MgSO4 and
concentrated under reduced pressure. Residue was purified by flash
chromatography using CH2Cl2/MeOH (99:1 to 97:3) as eluant,
yielding the pure compound 12 (50 mg, 0.21 mmol, 49%) as an
orange powder. Rf ¼ 0.3 (EtOAc). Mp > 245 ꢂC (decomposition); IR
(ATR): 3300-3000, 1700, 1623, 1608, 1576, 1507 cmꢀ1 1H NMR
;
(500 MHz, DMSO-d6): 7.52 (1H, d, J ¼ 6.0 Hz, H9), 8.00 (1H, br s, NH),
8.09 (1H, br s, NH), 8.56 (1H, d, J ¼ 6 Hz, H8), 8.99 (1H, s, H5), 9.53
(1H, s, H6), 9.58 (1H, s, H4); 13C NMR (100 MHz, DMSO-d6): 112.2
(C9), 134.2 (C5), 146.1 (C8), 155.0 (C6), 165.8 (C4), 119.5 (C4a), 121.8
(C5b), 128.5 (C9b), 136.2 (C10), 141.7 (C10a), 161.2 (C2); HRMS (ESIþ)