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M. Krecmerová et al. / Bioorg. Med. Chem. 21 (2013) 1199–1208
1205
5.6. (E)-9-(Prop-1-en-1-yl)-9H-purine-2,6-diamine (5)
J1 b,2 = 3.7, Jgem = 14.4, H-10b), 4.50 (m, 2H, P–OCH), 7.29 (br s, 2H,
NH2), 7.92 (s, 1H, H-8), 9.33 (d, 1H, JNH,CH = 10.2, CH@O), 10.36
0
0
13
Isolated from mother liquors after crystallization of 1 using
chromatography on silica gel in system ethyl acetate/acetone/eth-
anol/water (15:3:4:3). Yield 63 mg (0.5%), white solid. 1H NMR
(d, 1H, NH). C NMR (DMSO-d6, ppm) d: 16.95 (C-30), 23.75 and
23.81 (2 ꢁ d, JP,C = 4.6, 2 ꢁ CH3), 23.92 and 23.95 (2 ꢁ d, JP,C = 4.3,
2 ꢁ CH3), 46.77 (C-10), 62.59 (d, JP,C = 165.6, P–C), 70.275 (d,
JP,C = 6.1, P–OCH), 70.32 (d, JP,C = 6.1, P–OCH), 75.44 (d, JP,C = 12.9,
C-20), 116.04 (C-5), 140.58 (C-8), 150.74 (C-4), 156.26 (C-6),
153.14 (C-2), 163.76 (NCH@O). ESIMS, m/z: 850.6 (2 M+Na)+ (45),
437.0 (M+Na)+ (100), 415.1 (13) (MH)+.
0
0
(DMSO-d6, ppm) d: 1.77 (dd, 3H, J3 ,2 = 6.8, J3 ,1 = 1.7, H-30), 5.91
0
0
(br s, 2H, NH2), 6.39 (dd, 1H, J2 ,1 = 14.4, J2 ,3 = 6.8, H-20), 6.79 (br
0
0
0
0
s, 2H, NH2), 6.87 (dd, 1H, J1 ,2 = 14.4, J1 ,3 = 1.7, H-10), 7.95 (s, 1H,
0
0
0
0
13
H-8). C NMR (DMSO-d6, ppm) d: 18.93 (C-30), 112.07 (C-20),
123.74 (C-10), 113.28 (C-5), 138.14 (C-8), 150.21 (C-4), 155.47 (C-
6), 160.65 (C-2). ESIMS, m/z: 189.1 (M)ꢀ (100), HRMS (ESIꢀ): For
C8H9N6 (M)ꢀ calcd: 189.0889; found: 189.0890. Anal. Calcd for
C8H9N6: C, 50.52; H, 5.30; N, 44.18. Found: C, 50.64; H, 5.47; N,
44.06.
5.7.5. Diisopropyl ({2-amino-9-[(2R)-2-
(diisopropoxyphosphorylmethoxy)propyl]-9H-purin-6-
yl}amino)methylphosphate (9)
Yield 1.6 g (6%) of white foam. RF 0.21. 1H NMR (DMSO-d6, ppm)
d: 1.06 (d, 3H, J3 ,2 = 6.1, H-30), 1.175 and 1.185 (2 ꢁ d, 2 ꢁ 3H, CH3),
3.67 and 3.76 (2 ꢁ dd, 2 ꢁ 2H, JP,CH = 9.5, Jgem = 13.6, PCH2), 3.91
0
0
5.7. Introduction of a phosphonomethyl residue I
(m, 1H, H-20), 3.95 (br m, 2H, PCH2–N), 3.98 (dd, 1H, J1 a,2 = 6.7,
0
0
5.7.1. Reaction of unprotected intermediate (1) with
TsOCH2P(O)(OiPr)2. Preparation of diisopropyl ester of (R)-9-[2-
(phosphonomethoxy)propyl]-2,6-diaminopurine (6)
Jgem = 14.2, H-10a), 4.06 (dd, 1H, J1 b,2 = 3.1, Jgem = 14.2, H-10b),
4.52 and 4.60 (2 ꢁ m, 2 ꢁ 2H, P–OCH), 5.90 (br s, 2H, NH2), 6.94
(br s, 1H, NH), 7.67 (s, 1H, H-8). 13C NMR (DMSO-d6, ppm) d:
16.91 (C-30), 23.83, 23.85, 23.96, 23.99 and 24.05 (5 ꢁ d, 8C,
JP,C = 3.9, 8 ꢁ CH3), 35.99 (br d, JP,C = 155.2, P–C–N), 46.57 (C-10),
62.73 (d, JP,C = 165.9, P–C), 70.33 (d, 2C, JP,C = 6.2, P–OCH), 70.37
(d, 2C, JP,C = 6.2, P–OCH), 75.60 (d, JP,C = 13.0, C-20), 113.27 (C-5),
138.33 (C-8), 150.0 (C-4), 154.31 (C-6), 160.16 (C-2). ESIMS, m/z:
587.1 (M+Na)+ (100), 565.1 (39) (MH)+.
0
0
60% Sodium hydride dispersion (5.6 g, 140 mmol) was added to
a stirred mixture of diisopropyl tosyloxymethanephosphonate
(24.5 g, 70 mmol) and intermediate 1 (9.85 g, 47.3 mmol) in DMF
(250 mL) cooled to ꢀ20 °C. Reaction temperature was then gradu-
ally let warm from ꢀ20 °C to room temperature and the mixture
stirred for 24 h. Acetic acid was added for neutralization to pH 7
and the mixture was evaporated. The residue was chromato-
graphed on a column of silica gel (800 mL) in system chloroform/
methanol (9:1). Elution of a complex mixture of by-products
(2.5 g, including 7,8,9) occurred first followed by the desired prod-
5.8. (R)-9-[2-(Phosphonomethoxy)propyl]-N6-
phosphonomethyl-2,6-diaminopurine (10)
uct 6. Yield: 9 g (49%) of 6 as a yellowish foam. [
a
]
D ꢀ46.9 (c 0.326,
Bromotrimethylsilane (6.6 mL, 50 mmol) was added to a solu-
tion of 9 (1.4 g, 2.5 mmol) in acetonitrile (40 mL) and the solution
was set aside in dark for 24 h at room temperature. The mixture
was evaporated, the residue coevaporated with acetonitrile and
in a form of aqueous solution (10 mL) applied onto a column of
Dowex (H+ form, 40 mL). Elution was performed with water
(1000 mL; after 500 mL the first portion of compound 10 was
eluted). The product was evaporated and then lyoplilized from
water to give 540 mg (55%) of 10 as a white solid. The column
was then eluted with 2.5% aqueous ammonia. UV absorbing frac-
tion was evaporated and applied onto a column of Dowex 1 (ace-
tate form, 50 mL). Elution was performed with water 250 mL)
followed by a linear gradient of acetic acid (0–1 M, 1 L) and then
3 M formic acid. UV absorbing fraction (eluted by 3 M–HCOOH)
was evaporated and purified on reverse phase HPLC using isocratic
elution with water. Evaporation of appropriate fractions and
lyophilization from water gave additional portion of 10: 100 mg
(10%). Overall yield of 10 was 640 mg (65%). 1H NMR (D2O, ppm)
CHCl3). NMR data are in agreement with literature.2 ESIMS, m/z:
794.7 (2M+Na)+ (73), 409.1 (M+Na)+ (100), 387.1 (96) (MH)+. Anal.
Calcd for C15H27N4O6P: C, 46.03; H, 7.04; N, 21.75; P, 8.02. Found:
C, 46.27; H, 7.02; N, 21.33; P, 7.81.
5.7.2. Separation and identification of by-products 7, 8, 9
A mixture of by-products from the previous process was evap-
orated and chromatographed on silica gel column in system ethyl
acetate/acetone/ethanol/water (18:3:2:2).
The following fractions were obtained:
5.7.3. Diisopropyl ({(1R)-2-[2-amino-6-(formylamino)-9H-
purin-9-yl]-1-methylethyl}oxy)methylphosphonate (7)
Yield 400 mg (1%) as a colorless oil. RF 0.38. 1H NMR (DMSO-d6,
ppm) d:1.10 and 1.12 (2 ꢁ d, 2 ꢁ 3H, JCH3,CH = 6.2, CH3), 1.16(d, 6H,
CH3), 1.20 (d, 3H, CH3), 3.67 (dd, 1H, JP,CHa = 9.5, Jgem = 13.7, PCHa),
3.78 (dd, 1H, JP,CHb = 9.1, Jgem = 13.7, PCHb), 3.94 (m, 1H, H-20), 4.04
(dd, 1H, J1 a,2 = 7.1, Jgem = 14.3, H-10a), 4.13 (dd, 1H, J1 b,2 = 3.6,
Jgem = 14.3, H-10b), 4.50 (m, 2H, P–OCH), 6.38 (br s, 2H, NH2), 7.93
(s, 1H, H-8), 9.86 (br d, 1H, JNH,CH = 10.0, CH@O), 10.80 (br d, 1H,
d: 1.17 (d, 3H, J3 ,2 = 6.4, H-30), 3.56 (dd, 1H, JP,CHa = 10.1, Jgem = 12.8,
PCHa), 3.78 (dd, 1H, JP,CHb = 9.8, Jgem = 12.8, PCHb), 3.85 (br d, 2H,
0
0
0
0
0
0
JP,CH = 11.7, PCH2–N), 3.96 (m, 1H, H-20), 4.15 (dd, 1H, J1 a,2 = 5.7,
0
0
13
NH). C NMR (DMSO-d6, ppm) d: 16.83 (C-30), 23.74 and 23.82
Jgem = 14.6, H-10a), 4.33 (bdd, 1H, J1 b,2 = 3.0, Jgem = 14.6, H-10b),
0
0
13
(2 ꢁ d, JP,C = 4.5, 2 ꢁ CH3), 23.90 and 23.95 (2 ꢁ d, JP,C = 3.8,
2 ꢁ CH3), 46.80 (C-10), 62.61 (d, JP,C = 165.7, P–C), 70.285 (d,
JP,C = 6.4, P–OCH), 70.335 (d, JP,C = 6.4, P–OCH), 75.33 (d,
JP,C = 12.6, C-20), 114.23 (C-5), 141.77 (C-8), 149.81 (C-4), 154.67
(C-6), 160.09 (C-2), 164.51 (NCH@O). ESIMS, m/z: 850.5
(2M+Na)+ (88), 437.1 (M+Na)+ (100), 415.1 (19) (MH)+.
7.97 (s, 1H, H-8). C NMR (D2O, ppm) d: 15.08 (C-30), 38.70 (br
d, JP,C = 155.5, P–C–N), 49.08 (C-10), 64.39 (d, JP,C = 158.6, P–C),
76.01 (d, JP,C = 13.7, C-20), 114.83 (C-5), 140.39 (C-8), 151.83 (C-
4), 153.19 (C-6), 160.59 (C-2). +ESIMS, m/z: 397.1 (100) (MH)+.
ꢀESIMS, m/z: 395.1 (54) (MꢀH)ꢀ. ꢀESI-HRMS Calcd for
C
10H17O7N6P2 395.0639. Found: 395.0644 (MꢀH)ꢀ.
5.7.4. Diisopropyl ({(1R)-2-[6-amino-2-(formylamino)-9H-
purin-9-yl]-1-methylethyl}oxy)methylphosphonate (8)
5.9. Introduction of a phosphonomethyl residue II. Preparation
of (R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine
(12) from 1 using protection with dimethylaminomethylene
groups
Yield 520 mg (1.3%) as a colorless oil. RF 0.32. 1H NMR (DMSO-
d6, ppm) d: 1.09, 1.12, 1.155, 1.165 and 1.19 (5 ꢁ d, 5 ꢁ 3H,
JCH3,CH = 6.2, CH3), 3.725 (dd, 1H, JP,CHa = 9.5, Jgem = 13.7, PCHa),
3.79 (dd, 1H, JP,CHb = 9.2, Jgem = 13.7, PCHb), 3.96 (m, 1H, H-20),
N,N-Dimethylformamide dimethylacetal (40 mL) was added to
a suspension of 1 (9.9 g; 47.5 mmol) in DMF (100 mL). The mixture
4.07 (dd, 1H, J1 a,2 = 6.6, Jgem = 14.4, H-10a), 4.17 (dd, 1H,
0
0