Chemical synthesis of benzamide adenine dinucleotide: Inhibition of inosine monophosphate dehydrogenase (types I and II)

Article abstract of DOI:10.1021/jm9601415

Treatment of 3-(2,3-O-isopropylidene-β-D-ribofuranosyl)benzamide (6) with POCl₃ in (EtO)₃-PO afforded only little phosphorylation product (8, 5%), but the major product was 5'-chlorobenzamide riboside (7, 85%). Reaction of 6 with 2-c

Full text of DOI:10.1021/jm9601415

2422
J . Med. Chem. 1996, 39, 2422-2426
Ch em ica l Syn th esis of Ben za m id e Ad en in e Din u cleotid e: In h ibition of In osin e
Mon op h osp h a te Deh yd r ogen a se (Typ es I a n d II)1
Andrzej Zatorski,† Kyoichi A. Watanabe,‡ Stephen F. Carr,§ Barry M. Goldstein, and Krzysztof W. Pankiewicz*,‡
OncorPharm, Inc., 200 Perry Parkway, Gaithersburg, Maryland 20877, Laboratory of Organic Chemistry,
Sloan-Kettering Institute for Cancer Research, New York, New York 10021, Department of Biochemistry,
Roche Bioscience, Palo Alto, California 94304, Department of Biophysics, University of Rochester Medical Center,
Rochester, New York 14642
Received February 14, 1996X
Treatment of 3-(2,3-O-isopropylidene-â-D-ribofuranosyl)benzamide (6) with POCl3 in (EtO)3-
PO afforded only little phosphorylation product (8, 5%), but the major product was 5′-
chlorobenzamide riboside (7, 85%). Reaction of 6 with 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite followed by 2-cyanoethanol/tetrazole treatment and oxidation with tert-butyl
peroxide gave a 1:1 mixture of the desired 5′-O-bis(2-cyanoethyl) phosphate 9 and the chloro
derivative 7. This mixture was treated with methanolic ammonia and partitioned between
CHCl3 and water. The 2′,3′-O-isopropylidenebenzamide mononucleotide (8) was obtained in
21.2% overall yield from the aqueous layer. Compound 8 was then converted into the
corresponding imidazolide 11b which, upon coupling with 2′,3′-O-acetonide of AMP, afforded
the acetonide of benzamide adenine dinucleotide (15) in 94% yield together with small amounts
of symmetrical pyrophosphates P1,P2-bis(2′,3′-O-isopropylideneadenosin-5′-yl)pyrophosphate (13,
3%) and P1,P2-bis(2′,3′-O-isopropylidene-3-(carbamoylphenyl)-5′-ribosyl)pyrophosphate (14, 2%).
Deprotection of 15 with Dowex 50/H+ in water afforded the desired benzamide adenine
dinucleotide (BAD) in 93% yield. BAD inhibits inosine monophosphate dehydrogenase type I
(IC50 ) 0.78 µM) and type II (IC50 ) 0.88 µM) with same degree of potency.
HO
In tr od u ction
OH
B
O
P
O
IMP-dehydrogenase (IMPDH), the enzyme which
catalyzes the NAD-dependent conversion of inosine 5′-
monophosphate (IMP) to guanosine 5′-monophosphate
(GMP) is positioned at the branch point in the de novo
synthesis of guanine nucleotides.2 Inhibition of IMPDH
has been shown to have antiviral,3,4 immunosuppres-
sive,5,6 antiparasitic,7,8 and anticancer effects.9,10 The
level of IMPDH activity was found to be much greater
in several tumors as compared to the level in normal
tissues.11,12 It has been recently discovered13 that
human IMPDH exists as two isoforms, types I and II.
The two distinct cDNAs have been characterized, and
the two isoforms are found to be regulated differ-
ently.13-16 Type I is constitutively expressed and is the
preponderant isoform in normal cells, while type II is
selectively up-regulated in neoplastic and replicating
cells and emerges as the dominant species. Thus, the
selective inhibition of type II IMPDH is expected to
provide significant therapeutic advantage by eliminating
or reducing potential toxicity caused by inhibition of the
type I isoform.13-16
B
O
N
HO
O
O
P
O
O
O
N
N
HO
OH
N
HO
OH
HO
OH
NH2
CONH2
+
N
1, nicotinamide riboside
NAD
CONH2
N
S
2, tiazofurin
TAD
CONH2
N
3, C-nicotinamide riboside
C-NAD
C-PAD
BAD
CONH2
Tiazofurin, 2-(â-D-ribofuranosyl)thiazole-4-carboxam-
ide (2, Figure 1), is a potent inhibitor of IMPDH. This
oncolytic C-nucleoside requires a unique metabolic
activation. It is phosphorylated by adenosine kinase or
other kinase(s) to the 5′-monophosphate17 and then
converted by NAD-pyrophosphorylase to the NAD ana-
logue, thiazole-4-carboxamide adenine dinucleotide
(TAD).18 TAD has been synthesized18,19 and was found
N
4, C-picolinamide riboside
CONH2
5, benzamide riboside
F igu r e 1.
* Correspondence and reprints (phone 301-527-2063, Fax 301-208-
6997).
† Sloan-Kettering Institute for Cancer Research.
‡ OncorPharm, Inc.
to be much more potent inhibitor of IMPDH than
tiazofurin 5′-monophosphate.
§ Roche Bioscience.
We have reported20-22 the synthesis of the closest
structural analogues of nicotinamide riboside (1), i.e.
University of Rochester Medical Center.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00141-0 CCC: $12.00 © 1996 American Chemical Society

Synthesis of Benzamide Adenine Dinucleotide
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2423
Sch em e 1
5-â-D-ribofuranosylnicotinamide (3) and 6-â-D-ribofura-
nosylpicolinamide (4), the C-nucleoside isosteres of 1.
In contrast to our expectation these compounds showed
only weak inhibitory activity against L1210, P-815, HL-
60, CCRF-CEM, MOLT/4F, and MT-4 cell lines.20-23
However, when our C-nucleosides 3 and 4 were con-
verted into their corresponding NAD analogues, we
CONH2
HO
O
O
O
found24 that C-NAD (IC50 ) 7 µM) and C-PAD (IC50
)
26 µM) were relatively effective competitive inhibitors
of IMPDH. Interestingly, C-NAD caused extremely
potent inhibition of horse liver alcohol dehydrogenase
(ADH, Ki ) 1.1 nM), whereas C-PAD exhibited a much
less potent effect against ADH (Ki ) 20 µM). Crystal-
lographic studies25,26 later showed that the specificity
and affinity of C-NAD for ADH are due to coordination
of the zinc cation at the ADH catalytic site by the
C-NAD pyridine nitrogen. Displacement of the pyridine
nitrogen to the opposite side of the ring, as in C-PAD,
removes the specificity for ADH.25,26
6
CONH2
CONH2
O
P
Cl
RO
O
O
O
R1O
+
O
O
O
O
7
8: R = R1 = H
9: R = R1 = CH2CH2CN
10: R = H; R1 = CH2CH2CN
More recently, the synthesis of 3-â-D-ribofuranosyl-
benzamide (5, benzamide riboside) was published.27 This
compound showed potent toxicity (at nanomolar concen-
tration) toward S49.1 lymphoma cells27 and leukemia
K562 cells.28 It was found that, in K562 cells, nucleoside
5 is anabolized to an NAD analogue, benzamide adenine
dinucleotide (BAD). K562 cells were incubated with
tritium-labeled adenosine and benzamide riboside or
tiazofurin. The cells were centrifuged, washed, homog-
enized, and analyzed on HPLC. BAD was produced in
2-3-fold higher quantities than TAD.29 Such “biologi-
cally prepared” BAD was later used to study inhibition
of lactate, glutamate, and malate dehydrogenases and
was found to be a better general dehydrogenase inhibi-
tor than TAD.30 Finally, BAD was prepared enzymati-
cally (in a minute amount using NAD-pyrophosphory-
lase and ATP) and was shown to inhibit IMPDH (Ki )
0.118 µM).29
We became interested in comparing the inhibitory
effects of our NAD analogues with those of BAD and
TAD as part of our on-going program to design and
synthesize selective inhibitors of IMPDH, particularly
against type II. Since the enzymic preparation of BAD
is inefficient and laborious, we confronted the first
chemical synthesis of BAD. Our initial attempts using
established methods were unsuccessful. Phosphoryla-
tion of the 2′,3′-O isopropylidenebenzamide riboside (6,
Scheme 1) with POCl3 in (MeO)3PO followed by ac-
etonide cleavage was reported to give the desired 5′-
mononucleotide in moderate yield.27 In our hands,
however, such a phosphorylation procedure afforded
only little phosphorylated product (8, 5%). The major
product was 5′-chlorobenzamide riboside (7, 85%), al-
though similar reaction with 2′,3′-O-isopropylidenead-
enosine gave the 2′,3′-O-acetonide of AMP (12, Scheme
2) in good yield. Therefore, for the synthesis of benza-
mide mononucleotide, we used the phosphitylation
procedure. Reaction of 6 with 2-cyanoethyl N,N-diiso-
propylchlorophosporamidite followed by 2-cyanoethanol/
tetrazole treatment and oxidation with tert-butyl per-
oxide gave a 1:1 mixture of the desired 5′-O-bis(2-
cyanoethyl) phosphate 9 and the chloro derivative 7.
This mixture was then treated with methanolic am-
monia to give a 1:1 mixture of 7 and 5′-O-(2-cyanoethyl)
phosphate derivative 10. After concentration in vacuo
the mixture was partitioned between water and CHCl3.
The 5′-chlorobenzamide riboside (7) was recovered from
the organic layer, and derivative 10 was obtained from
the water solution. The second cyanoethyl group was
removed from 10 by prolonged treatment with metha-
nolic ammonia, which required heating at 55 °C for 18
h to furnish the desired 2′,3′-O-isopropylidenebenzamide
mononucleotide (8) in 21.2% overall yield after HPLC
purification.
Our recently improved procedure31 was used for
efficient coupling of 10 with 2′,3′-acetonide of AMP (12,
Scheme 2). We found that preparation of nucleotide
imidazolides prior to the reaction with the correspond-
ing nucleotides gave much better results than commonly
used in situ generation of the nucleotide imidazolides.
Thus, we prepared the imidazolide 11b as the triethy-
lammonium salt and coupled it with nucleotide 12 in
DMF-d7. After 8 days, when the singlet of 11b vanished
(31P NMR), the reaction was quenched by addition of
water and the mixture was lyophilized. The residue was
chromatographed on HPLC column to give the acetonide
of BAD (15) as the major product (94.5%), together with
small amounts of symmetrical pyrophosphates P1,P2-
bis(2′,3′-O-isopropylideneadenosin-5-yl)pyrophosphate (13,
3%) and P1,P2-bis(2′,3′-O-isopropylidene-3-(carbam-
oylphenyl)-5′-ribosyl)pyrophosphate (14, 2%). Treat-
ment of 15 with an excess of Dowex 50/H+ in water
afforded the desired BAD in 93% yield as the free acid
analyzed as a single peak on HPLC. The 1H NMR
spectrum of our synthetic BAD was identical to that of
BAD obtained enzymatically,29 except that our sample
lacked the singlet of unidentified impurity observed at
δ 3.72 in the enzymatically prepared sample. Also, our
sample showed the resonance for the anomeric proton
of benzamide ribose at δ 4.75 (d, J 1′,2′ ) 7.1 Hz), which
was obscured by the water signal in the enzymatic
sample. The assignments of some of the sugar absor-
bances of the enzymatically prepared BAD were not
correct. In particular, the most up-field shifted signal
at δ 4.03 (dd) is not the H3′ of adenosine but rather the
double doublet of the H2′ of the benzamide ribose
(coupling constants J 1′,2′ ) 7.2 Hz and J 2′,3′ ) 5.3 Hz,
see experimental part). The identical coupling con-
stants and a similar, unusual, upfield shift of the H2′
1
were observed in the H NMR spectrum of the benza-
mide riboside.29 In acetonide-protected derivatives 6,
7, 10, 14, and 15, the resonance signal of benzamide

2424 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Zatorski et al.
Sch em e 2
O
O
B
A
R
P
O
HO
P
O
O
O
OH
OH
+
O
O
O
O
12
8: R = OH
O
C
11a: R =
11b: R =
O
N
N
N
N
O
O
O
O
O
P
O
O
P
O
B
A
O
O
O
O
P
O
O
O
P
O
O
O
B
A
HO
OH
HO
OH
O
O
O
O
13
14
O
O
O
P
O
B
O
O
O
P
O
O
A
HO
OH
O
O
15
NH2
N
N
N
HO
OH
A =
N
O
P
O
B
O
O
O
P
O
CONH2
O
A
B =
HO
OH
HO
OH
BAD
ribose H2′ is always present at higher field (0.1-0.3
ppm) than is the H3′ signal. This may be explained by
the strong diamagnetic shielding of the H2′ by the
aromatic benzamide aglycon.
showing equal activity of TAD and BAD.29 These
results demonstrate that neither TAD nor BAD exhibit
selectivity toward IMPDH type II, the target isozyme
predominant in neoplastic cells.32
BAD obtained in the form of a free acid (as a white
powder after lyophilization) was found to be unstable
when kept at room temperature. In two months 50%
of the compound was hydrolyzed into AMP and the 5′-
monophosphate of benzamide riboside. When the same
sample was dissolved in water, no such decomposition
was observed. Furthermore, the sodium or triethylam-
monium salt of BAD were stable as solids.
We examined the inhibitory effect of BAD and TAD
against IMP dehydrogenase type I and type II. The
IC50’s of these compounds for each isoform were mea-
sured in the presence of 100 µM NAD, 50 µM IMP, 100
mM Tris-HCl, 100 mM KCl, 3 mM EDTA, and 25 nM
enzyme at pH 8.0, following the formation of NADH at
340 nm. TAD was equally potent against IMPDH type
I (IC50 ) 0.094 µM) and type II (IC50 ) 0.101 µM). BAD
was more potent than C-NAD and C-PAD but it was 8
times less potent than TAD with equal affinity toward
Exp er im en ta l Section
Gen er a l Meth od s. HPLC was performed on a Dynamax-
60A C18-83-221-C column with flow rate of 5 mL/min or
Dynamax-300A C18-83-243-C column with a flow rate of 20
mL/min of 0.1 M Et3N H2CO3 (TEAB) followed by a linear
gradient of 0.1 M TEAB-aqueous MeCN (70%). Elemental
analyses were performed by M-H-W Laboratories, Phoenix, AZ.
Nuclear magnetic resonance spectra were recorded on a J EOL
Eclipse 270 and Bruker AMX-400 spectrometer with Me4Si or
DDS as the internal standard for 1H and external H3PO4 for
31P. Chemical shifts are reported in ppm (δ), and signals are
described as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), bs (broad singlet), and dd (double doublet). Values
given for coupling constants are first order.
P h osp h or yla tion of 3-(2,3-O-Isop r op ylid en e-â-D-r ibo-
fu r a n osyl)ben za m id e. (A) P r oced u r e w ith P OCl3. To a
cold solution of 627 (58.6 mg, 0.2 mmol) in (EtO)3PO (0.5 mL)
was added a mixture (EtO)3PO (0.5 mL), water (0.6 µL), and
P(O)Cl3 (100 mg, 0.65 mmol, 60 µL). The mixture was kept
at 5-10 °C for 24 h and then added dropwise into a solution
of 2 M TEAB (1 mL) in water (20 mL). Extraction with EtOAc
IMPDH type I (IC50 ) 0.787 µM) and type II (IC50
)
0.884). This is in contrast to previously reported result

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

2426 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Zatorski et al.
(18) Cooney, D. A.; J ayaram, H. N.; Gebeyehu, G.; Betts, C. R.; Kelly,
Ack n ow led gm en t. This work was initiated with
support from the National Institutes of Health [GM
42010 (K.W.P.)] and completed with support from
OncorPharm. This financial support which made this
study possible is gratefully acknowledged.
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