Synthesis of Benzamide Adenine Dinucleotide
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2425
(2 × 10 mL) and concentration of the aqueous layer in vacuo
gave the residue that was further purified by HPLC. After
lyophilization the 5′-monophosphate 8 (5 mg, 5%) was obtained
as mono (triethylammonium salt). The organic layer was
concentrated, and the residue was chromatographed on a silica
gel column to give 3-(5-chloro-5-deoxy-2,3-O-isopropylidene-
â-D-ribofuranosyl)benzamide (7) (53 mg, 85%): 1H NMR
(CDCl3) δ 1.35 (s, 3H, iPr), 1.59 (s, 3H, iPr), 3.75 (dd, 1H, H5′,
J 4′,5′ ) 4.2 Hz, J 5′,5′′ ) 12.3 Hz), 3.94 (dd, 1H, H5′′, J 4′,5′′ ) 2.7
Hz), 4.15-4.16 (m, 1H, H4′), 4.63 (pseudo t, 1H, H2′), 4.70 (bs,
1H, CONH2), 4.76 (dd, 1H, H3′, J 2′,3′ ) 6.8 Hz, J 3′,4′ ) 4.1 Hz),
4.85 (d, 1H, H1′, J 1′,2′ ) 5.5 Hz), 6.50 (bs, 1H, CONH2), 7.36
(pseudo t, 1H, H5), 7.47 (d, 1H, H4, J 4,5 ) 7.7 Hz), 7.78 (d,
1H, H6, J 5,6 ) 7.7 Hz), 7.99 (s, 1H, H2); MS (CI) 311 (M+).
Anal. (C15H18NO4Cl) C,H,N.
and then added dropwise into a solution of 2 M TEAB (1 mL)
in water (20 mL). Extraction with EtOAc (2 × 10 mL) and
concentration of the aqueous layer in vacuo gave the residue
that was further purified by HPLC. After lyophilization the
5′-monophosphate 12 (214 mg, 88%) was obtained as a mono-
(triethylammonium salt): 1H NMR (D2O) δ 1.30 (t, 9H, Et3N),
1.47 (s, 3H, iPr), 1.69 (s, 3H, iPr), 3.22 (q, 6H, Et3N), 4.07-
4.09 (m, 2H, H5′,5′′), 4.72-4.73 (m, 1H, H4′′), 5.21 (dd, 1H,
H3′, J 2′,3′ ) 6.0 Hz, J 3′,4′ ) 1.9 Hz), 5.44 (dd, 1H, H2′, J 1′2′
)
3.0 Hz), 6.31 (d, 1H, H1′), 8.32, 8.47 (two 1H singlets, H2, H8).
Anal. (C13H18N5O7P‚Et3N‚3H2O) C, H, N.
3-(2,3-O -I s o p r o p y lid e n e -â-D -r ib o fu r a n o s y l)b e n za -
m id e 5-P h osp h oim id a zolid e (11b) a n d Its Rea ction w ith
2,3-O-Isop r op ylid en ea d en osin e 5′-Mon op h osp h a te. Nu-
cleotide 8 (20 mg, 0.042 mmol as mono(triethylammonium
salt)) was dissolved in DMF-d7 (0.5 mL) followed by addition
of CDI (9 mg, 0.056 mmol), and the reaction was monitored
by 31P NMR. After 45 min the resonance signal of 8 (δ 1.80)
diminished, and the new signal of the anhydride 11a emerged
at δ -6.45, which diminished in time with simultaneous
formation of the resonance of the imidazolide derivative 11b
(δ -8.04). After 2.5 h, the formation of imidazolide 11b was
completed, the mono(triethylammonium salt) of 12 (31 mg,
0.063 mmol) was added, and the mixture was kept at room
temperature for 8 days. 31P NMR analysis showed the
presence of the imidazolide 11b resonance (8.0%), excess of
12 (δ 2.53), and a group of signals at δ 8.52-9.55. Finally,
the reaction mixture was heated at 55 °C until the signal of
11b completly disappeared (15 h). The reaction mixture was
then lyophilized, and the residue was subjected to HPLC to
give nucleotide 12 (tR ) 40.9 min, 9 mg), P1,P2-bis(2′,3′-O-
isopropylideneadenosin-5′-yl)pyrophosphate (13) as the bis-
(triethylammonium salt) [tR ) 49.9 min, 1.5 mg; 1H NMR (D2O)
δ 1.25 (t, 18H, Et3N), 1.43 (s, 6H, iPr), 1.67 (s, 6H, iPr), 3.12
(q, 12H, Et3N), 4.19-4.21 (m, 4H, 5′,5′′), 4.57-4.58 (m, 2H,
(B) P h osp h ityla tion P r oced u r e. To a mixture of 6 (87
mg, 0.3 mmol) and iPr2NEt (75 mg, 0.58 mmol) in dry CH2Cl2
(3 mL) was added 2-cyanoethyl N,N-diisopropylchlorophos-
phoramidite (91.5 mg, 0.38 mmol). After 30 min at room
temperature the starting nucleoside 6 disappeared (TLC,
CHCl3-EtOH, 9:1). The 31P NMR analysis showed the pres-
ence of the unreacted excess of (iPr)2NP(Cl)OCH2CH2CN at δ
184.8 and a 1:1 mixture of the desired product at δ 153.4 and
H-phosphonate [(iPr)2N(H)P(O)OCH2CH2CN] at δ 18.9 (d, J P-H
) 640 Hz, collapsed into a singlet upon decoupling), formed
from the starting phosphitilating reagent as a result of
chlorination of 6. The reaction mixture was diluted with CH2-
Cl2 (3 mL) and washed with an ice-cold 10% solution of
NaHCO3 (5 mL), saturated NaCl (3 × 5 mL), and water (10
mL). The organic layer was dried (Na2SO4) and concentrated
in vacuo, and the residue was dissolved in CH3CN (1 mL). TLC
analysis (CHCl3-EtOH, 9:1) showed the presence of the faster
migrating 5′-chloro derivative 7 and the slower migrating 5′-
O-phosphitilated nucleoside 6. These compounds were not
separated but treated with â-cyanoethanol (60 µL) and a
solution of 1H-tetrazole (42 mg, 0.6 mmol) in CH3CN (1.2 mL).
After 30 min tert-butyl hydroperoxide (100 µL) was added, and
the reaction mixture was stirred for 1 h and concentrated in
vacuo. The residue was dissolved in saturated methanolic
ammonia (4 mL) and kept in a refrigerator overnight. The
reaction mixture was concentrated, and the residue was
dissolved in a mixture of CHCl3 (5 mL) and water (5 mL). The
water layer was separated and washed with CHCl3 (2 × 5 mL).
The organic layers were combined and concentrated, and the
residue was chromatographed on a silica gel column using
CHCl3-EtOH, 19:1, as the eluent to give 7 (37.5 mg, 40%)
identical with that obtained in method A. The water solution
was concentrated in vacuo to give crude 10 (70 mg, 53%). An
analytical sample of 10 was obtained as the triethylammonium
salt after purification by HPLC: 1H NMR (CDCl3) δ 1.23 [t,
9H, (CH3CH2)3N], 1.34 (s, 3H, iPr), 1.63 (s, 3H, iPr), 2.67 (t,
2H, OCH2CH2CN), 2.97 [q, 6H, (CH3CH2)3N], 4.02-4.11 (m,
3H, H5′, OCH2CH2CN), 4.26 (dd, 1H, H5′′, J 4′,5′′ ) 3.2 Hz, J 5′,5′′
) 11.3 Hz), 4.32-433 (m, 1H, H4′), 4.58 (pseudo t, 1H, H2′),
4.88 (dd, 1H H3′, J 2′,3′ ) 6.2 Hz, J 3′,4′ ) 3.0 Hz), 4.98 (d, 1H,
H1′, J 1′′,2′ ) 4.9 Hz), 6.02 (bs, 1H, CONH2), 7.37-7.43 (m, 2H,
H4,5), 7.96 (d, 1H, H6, J 5,6 ) 7.2 Hz), 8.23 (s, 1H, H2), 8.61
(bs, 1H, CONH2); 31P NMR (CDCl3) δ -0.31. Crude 10 was
dissolved in saturated methanolic ammonia (5 mL) and heated
in a steel cylinder at 55 °C for 18 h. The reaction mixture
was concentrated, and the residue was chromatographed by
HPLC to give 10 (56 mg, 40%) as mono(triethylammonium
salt): 1H NMR (D2O) δ 1.27 (t, 9H, Et3N), 1.42 (s, 3H, iPr),
1.67 (s, 3H, iPr), 3.19 (q, 6H, Et3N), 4.08-4.11 (m, 2H, H5′,5′′),
4.42-4.43 (m, 1H, H4′), 4.78 (pseudo t, 1H, H2′), 4.98 (dd, 1H,
H3′, J 2′,3′ ) 6.5 Hz, J 3′,4′ ) 3.5 Hz), 5.02 (d, 1H, H1′, J 1′,2′ ) 5.4
Hz), 7.57 (pseudo t, H5), 7.67 (d, 1H, H4, J 4,5 ) 7.8 Hz), 7.81
(d, 1H, H6, J 5,6 ) 7.8 Hz), 7.86 (s, 1H, H2); 31P NMR (D2O) δ
4.09. Anal. (C15H20NO8P‚Et3N‚2H2O) C, H, N. A small
amount of unreacted 10 (8 mg, 5%) was also eluted from the
column.
H4′), 5.15 (dd, 2H, H3′, J 2′,3′ ) 6.0 Hz, J 3′,4′ ) 5.4 Hz, J 3′,4′
)
2.2 Hz), 5.22 (dd, 2H, H2′, J 1′,2′ ) 3.7 Hz), 6.06 (d, 2H, H1′,
J 1′,2′ ) 3.7 Hz), 8.05, 8.15 (two 2H singlets, H2, H8); 31P NMR
(D2O) δ -9.61], and the desired P1-[3-(2,3-O-isopropylidene-â-
D-ribofuranos-5-yl)carbamoylphenyl]-P2-(2′,3′-O-isopropylide-
neadenosin-5′-yl)pyrophosphate (15) also as the bis(triethy-
lammonium salt) (tR ) 53.5 min, 37.5 mg, 94.5%): 1H NMR
(D2O) δ 1.15 (t, 18H, Et3N), 1.33 [s, 3H, iPr (B)], 1.42 [s, 3H,
iPr (B)], 1.61 [s, 3H, iPr (A)], 1.65 [s, 3H, iPr (A)], 2.90 (q, 12H,
Et3N), 4.10-4.17 [m, 4H, H5′,5′′(A),H5′,5′(B)], 4.24-4.26 [m,
2H, H4′(B)], 4.55-4.58 [m, 2H, H4′(A), H2′(B)], 4.75 [d, 1H,
H1′(B), J 1′,2′ ) 5.6 Hz], 4.85 [dd, 1H, H3′(B), J 2′,3′ ) 6.6 Hz,
J 3′,4′ ) 3.8 Hz], 5.17 [dd, 2H, H3′(A), J 3′,4′ ) 2.1 Hz, J 2′,3′ ) 6.1
Hz], 5.21 [dd, 2H, H2′(A), J 1′,2′ ) 3.6 Hz), 6.08 [d, 2H, H1′(A),
J 1′,2′ ) 3.6 Hz], 7.34 [pseudo t, 1H, H5(B)], 7.42 [d, 1H, H4(B),
J 4,5 ) 7.7 Hz], 7.58 [d, 1H, H6(B), J 5,6 ) 7.7 Hz], 7.62 [s, 1H,
H2(B)], 8.07, 8.29 [two 1H singlets, H2(A), H8(A)]; 31P NMR
(D2O) δ -9.44 (P1), -9.85 (P2, AB system, J P,P ) 21.6 Hz), and
P1,P2-bis[3-(2,3-O-isopropylidene-â-D-ribofuranos-5-yl)carbam-
oylphenyl]pyrophosphate (14) (tR ) 56.6 min, 1 mg): 1H NMR
(D2O) δ 1.27 (t, 18H, Et3N), 1.36 (s, 6H, iPr), 1.63 (s, 6H, iPr),
3.20 (q, 12H, Et3N), 4.16-4.17 (m, 4H, H5′,5′′), 4.35-4.36 (m,
2H, H4′), 4.60 (pseudo t, 1H, H2′), 4.81 (d, 2H, H1′, J 1′,2′ ) 5.6
Hz), 4.92 (dd, 2H, H3′, J 2′,3′ ) 6.6 Hz, J 3′,4′ ) 3.6 Hz), 7.49
(pseudo t, 2H, H5), 7.58 (d, 2H, H4, J 4,5 ) 7.7 Hz), 7.72 (d,
2H, H6, J 5,6 ) 7.7 Hz), 7.76 (s, 2H, H2); 31P NMR (D2O) δ
-9.64.
Compound 15 was de-O-isopropylidenated by treatment
with Dowex 50-X8 (H+) in water, which requires 14 h, and
purified by passage through a Dowex 50-X8 (H+) column to
give BAD (26 mg, 93%): 1H NMR (D2) δ 4.07 [dd, 1H, H2′(B),
J 1′,2′ ) 7.2 Hz, J 2′,3′ ) 5.3 Hz], 4.15-4.26 [m, 6H, H3′(B), H4′-
(B), H5′,5′′(A), H5′,5′′(B)], 4.35 [m, 1H, H4′(A)], 4.44 [pseudo
t, 1H, H3′(A)], 4.59 [pseudo t, 1H, H2′(A)], 4.75 [d, 1H, H1′-
(B), J 1′,2′ ) 7.2 Hz], 6.01 [d, 1H, H1′(A), J 1′,2′ ) 5.3 Hz], 7.41 (t,
1H, H5, J ) 7.7 Hz), 7.57 (d, 1H, H4, J ) 7.7 Hz), 7.66 (d, 1H,
H6), 7.73 [s, 1H H2(B)], 8.31, 8.55 [two 1H singlets, H2, H8-
(A)]; 31P NMR (D2O), AB system, δ 9.36 P1, 9.59 P2, J P,P ) 21.2
Hz. MS was identical with that reported earlier.29
P h osp h or yla tion of 2′,3′-O-Isop r op ylid en ea d en osin e.
To a cold solution of 2′,3′-O-isopropylideneadenosine (153 mg,
0.5 mmol) in (EtO)3PO (1.25 mL) was added a mixture of
(EtO)3PO (1.25 mL), water (1.5 µL), and P(O)Cl3 (250 mg, 1.65
mmol, 150 µL). The mixture was kept at 5-10 °C for 24 h