Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings

Article abstract of DOI:10.1016/S0960-894X(01)00450-4

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5′-position with an N-methyluronamide or with phosphate groups.

Full text of DOI:10.1016/S0960-894X(01)00450-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2295–2300
Synthesis and Purine Receptor Affinity of 6-Oxopurine
Nucleosides and Nucleotides Containing (N)-Methanocarba-
pseudoribose Rings
Gnana Ravi,a Kyeong Lee,a Xiao-duo Ji,a Hak Sung Kim,a Kelly A. Soltysiak,a
Victor E. Marquezb and Kenneth A. Jacobsona,*
aMolecular Recognition Section, LBC, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
bLaboratory of Medicinal Chemistry, National Cancer Institute, Frederick, MD 21702, USA
Received 20 April 2001; accepted 12 June 2001
Abstract—6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclo-
pentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was
coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 50-
position with an N-methyluronamide or with phosphate groups. Published by Elsevier Science Ltd.
We recently examined conformational requirements of
the ribose moiety in adenosine derivatives acting as
agonists or partial agonists at P2 receptors and at sub-
types of adenosine receptors (A1, A2A, and A3 ARs).1ꢀ3
Using a methanocarba modification originally intro-
duced by Marquez and co-workers,4 the ribose-like
moiety of these derivatives was constrained to either a
northern [(N), 20-exo] or southern [(S), 20-endo] con-
formation through fused cyclopropane and cyclo-
pentane rings. Such analogues helped to define the role
of ribofuranosyl puckering in stabilizing the active AR-
bound conformation, and thereby allowed identification
of the (N)-methanocarba conformation as the isomer
which preserved affinity at the A3 and to a lesser extent,
A1 AR. Thus, the (N)-methanocarba analogues, 1 and
2, were highly potent as full agonist (2)3 or partial
agonist (1) at the human A3 AR.2 These analogues
contained substituent groups known to enhance A3
affinity in the ribose series [i.e., N6-(3-iodobenzyl) and
2-chloro]. The affinity of adenosine methanocarba
analogues at the A2A AR was greatly decreased for
the (N)-methanocarba isomer and nearly absent for the
(S)-methanocarba isomer.
a 6-oxo-substitution. Among the nucleoside analogues
synthesized were derivatives of hypoxanthine, guano-
sine, and xanthine. For inosine5,6 and xanthine-7-ribo-
side7 analogues, a 50-methyluronamide modification
(nucleoside numbering) known to enhance the A3 AR
affinity was included.3,5 In comparison, xanthine-7-
riboside derivatives 3 and 4 bound to and activated the
rat A3 AR with partial or full efficacy, respectively,7,8
and were used as reference compounds. In other analo-
gues, phosphate groups were introduced at the 50-posi-
tion with the aim of targeting P2 receptors,9 G proteins
(in the case of a guanosine 50-triphosphate analogue)10
or enzymes involved in purine metabolism (such as IMP
dehydrogenase).11
In this study, we combined the (N)-methanocarba mod-
ification with known purine receptor ligands containing
*Corresponding author. Tel.: +1-301-496-9024; fax: +1-301-480-
8422; e-mail: kajacobs@helix.nih.gov
0960-894X/01/$ - see front matter. Published by Elsevier Science Ltd.
PII: S0960-894X(01)00450-4

2296
G. Ravi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2295–2300
b
Table 1. Affinities of (N)-methanocarba analogues of 6-oxpurine ribosides in radioligand binding assays at rat A1,a rat A2A
receptors,c unless noted, and in other biochemical assay systemsd
, and human A3
Compound
R1
R2
Ki (mM) or % displacement at 100 mM
a
b
c
rA1
rA2A
hA3
5 MRS 1957
6 MRS 1997
7a MRS 2397d
7b MRS 2398d
8 MRS 1941
9 MRS 2351d
10 MRS 1971
11 MRS 1998
12 MRS 2396
H
H
H
CH2OH
CONHCH3
CH2O–phosphate
CH2O–phosphate]2 dimer
CH2OH
CH2O–triphosphate
CH2OH
CONHCH3
<10%
<10%
<10%
<10%
30ꢂ10%
28.8ꢂ5.2
11ꢂ1%
H
21.6ꢂ0.4
20.5ꢂ2.1
<10%
NH2
NH2
—
—
—
<10%
<10%
2.97ꢂ0.15
24.4ꢂ4.8
<10%
<10%
4.35ꢂ0.40
1.86ꢂ0.23
<10%
CH2O–triphosphate
aDisplacement of specific [3H]R-PIA binding to A1 receptors in rat brain membranes, expressed as KiꢂSEMor % ( n=3–5).
bDisplacement of specific [3H]CGS 21680 binding to A2A receptors in rat striatal membranes, expressed as KiꢂSEMor % ( n=3–6).
cDisplacement of specific [125I]AB-MECA binding at human A3 receptors expressed in HEK-293 cells, in membranes, expressed as KiꢂSEMor %
(n=3–5).
dSelected compounds were examined in HL-60 cells for effects on calcium mobilization. Compounds 7a and 7b (100 mM) were inactive in raising
intracellular calcium, while compound 9 (100 mM) caused a rise in intracellular calcium equivalent to 53ꢂ18% of the effect of 1 mMUTP ( n=3).
The structures of (N)-methanocarba purine analogues
(5–12) synthesized for testing in binding and other bio-
chemical assays are shown in Table 1. We introduced
the (N)-methanocarba modification of known potent
adenosine agonists having equivalents of either 50-
hydroxymethyl (5, 8, and 10) or 50-uronamide groups (6
and 11). These 6-oxopurine bases were coupled at either
the 7- (10–12) or 9-position (5–9) to the pseudoribose
moiety, according to well-characterized alkylation pat-
terns of purine bases.7,12 A 50-monophosphate deriva-
tive, 7a, and several 50-triphosphate derivatives were
included (9 and 12).
pyrophosphate failed, and instead a dimer, 7b, was
obtained.
Guanosine derivative 8 was prepared by a Mitsunobu
reaction of 16 and 2-amino-6-chloropurine to furnish
17, which was heated with 2 N NaOH in dioxane at
100 ꢁC to yield the guanosine derivative, 18. Debenzy-
lation at the 50 position using Pd to give compound 19,
followed by TFA deprotection provided the guanosine
derivative, 8. Compound 19 was converted into mono-
phosphate 20 by the phosphoramidite method. This
monophosphate was then converted to phosphor-
imidazolidate by treatment with 1,10-carbonyldiimidazole
followed by 5% Et3N/MeOH, and the phosphor-
imidazolidate reacted with tetrabutylammonium pyr-
ophosphate to provide the triphosphate, 9b.
The synthetic methods used to prepare the new analogues
are outlined in Schemes 1–3.13ꢀ15 6-Monosubstituted
derivatives were prepared from the 6-chloropurine deri-
vative3 as a common intermediate. Refluxing 13 in the
presence of 2-mercaptoethanol and NaOMe in MeOH
gave the protected inosine derivative, 14, which was
converted into triol 5 in acidic conditions or to the 50-
uronamide, 6, via a 50-carboxylic acid, 15. Treatment of
14 with RuCl3, NaIO4, followed by C18 column chro-
matography gave 15, which condensed with MeNH2 in
the presence of BOP-Cl [N,N-bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride] to yield 6. Use of RuCl3 instead of
RuO2 produced a cleaner reaction. The 50-monophos-
phate derivative 7a was prepared by reacting 14 with di-
t-butyl-N,N-diethylphosphoramidite in the presence of
tetrazole and then with mCPBA at ꢀ78 ꢁC, followed by
the treatment with Dowex resin. Attempted synthesis of
the corresponding triphosphate by treating with 1,10-
carbonyldiimidazole followed by tetrabutylammonium
Xanthine derivatives were prepared using a Mitsunobu
coupling of 16 and 1,3-dibutylxanthine to furnish 21,
which was deprotected with BCl3 to yield the xanthine
methanocarba nucleoside, 10. Compound 21 was
debenzylated to furnish 22, which was converted to the
monophosphate 25 and to the triphosphate 12 by the
above phosphorylation procedures. Compound 22
was oxidized using RuO2, NaIO4, K2CO3 to furnish
the acid 23, which was condensed with MeNH2
using BOP-Cl to yield intermediate 24 and finally the
deprotected uronamide, 11.
The nucleoside analogues were evaluated in binding at
three subtypes of ARs. While inosine itself displayed a
measurable affinity at the A3 AR and was even suggested

G. Ravi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2295–2300
2297
Scheme 1. (a) HSCH2CH2OH, NaOMe, MeOH, reflux; (b) RuCl3, NaIO4, CH3CN/CHCl3/H2O=2:2:3; (c) BOP-Cl, Et3N, MeNH2 (2 M/THF),
CH2Cl2/DMF=1:1; (d) CF3CO2H, wet MeOH, 60 ꢁC; (e) (i) 1H-tetrazole, di-t-butyl N,N-diethylphosphoramidite, THF; (ii) mCPBA, CH2Cl2;
(f) DOWEX 50ꢃ8–200, MeOH/H2O=2:1; (g) (i) 1,10-carbonyldiimidazole, DMF; (ii) 5% Et3N/MeOH; (iii) tetrabutylammonium pyrophosphate.
Scheme 2. (a) 2-Amino-6-chloropurine, DIAD, Ph3P, THF; (b) 2 N NaOH, dioxane; (c) Pd black, 10% HCO2H in MeOH; (d) TFA/MeOH/H2O;
(e) (i) tetrazole, di-t-butyl-N,N-diethylphosphoramidite, THF; (ii) mCPBA, CH2Cl2; (f) TFA/MeOH/H2O (g) (i) 1,10-carbonyldiimidazole, DMF;
(ii) 5% Et3N/MeOH; (iii) tetrabutylammonium pyrophosphate.

2298
G. Ravi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2295–2300
Scheme 3. (a) 1,3-Dibutylxanthine, DEAD, Ph3P, THF; (b) BCl3, CH2Cl2; (c) Pd black, 10% HCO2H in MeOH; (d) (i) tetrazole, di-t-butyl-N,N-
diethylphosphoramidite, THF; (ii) mCPBA, CH2Cl2; (e) DOWEX 50ꢃ8–200, MeOH/H2O=1:1; (f) (i) 1,10-carbonyldiimidazole, DMF; (ii) 5%
Et3N/MeOH; (iii) tetrabutylammonium pyrophosphate; (g) RuO2, NaIO4, K2CO3, H2O/CH3CN/CHCl3; (h) CH3NH2ꢄ HCl, Et3N, BOP-Cl;
(i) BCl3/CH2Cl2.
to be an endogenous ligand for this AR,6 the corre-
sponding (N)-methanocarba analogue, 5, bound
weakly. An effort to enhance the affinity in the inosine
series through the introduction of 50-methyluronamide
group slightly increased the affinity of 6 (Ki 29 mM). The
inosine-50-monophosphate analogue, 7a, was inactive at
the adenosine A3 AR. Curiously, the dimer 7b displaced
binding at the A3 AR with a Ki value of 22 mM. Thus,
this was an example of dimerization of an inactive
ligand to increase the affinity, perhaps through cross-
linking two sites on a receptor or by bridging two
proximal receptor molecules. It is unusual for such a
highly anionic molecule to bind to an AR, which may
be the result of an enhancing effect of the (N)-metha-
nocarba ring on A3 affinity.2 It will also be interesting to
evaluate 7b at P2X receptors, at which diinosine poly-
phosphates are potent antagonists.16
The (N)-methanocarba analogue of 1,3-dibutylxanthine-
7-riboside, 10, was equipotent at A1 and A3 ARs and
inactive at the A2A AR. If proven to be an agonist at both
AR subtypes, this profile of selectivity would be desir-
able for a cardioprotective agent.18 Introduction of the
50-N-methyluronamide group, in 11, decreased A1 affi-
nity by 8-fold and increased A1 affinity by 2-fold. A
comparison of compounds 3 and 47 with the corre-
sponding (N)-methanocarba analogues, 10 and 11,
respectively, showed that rat A1 affinities were the same,
and rat A3 affinities of the ribosides were roughly simi-
lar to Ki values at the human A3 AR found in this study.
The xanthine-riboside-50-triphosphate analogue, 12, was
inactive at the A3 AR.
The phosphate-containing analogues were tested for
effects on intracellular calcium concentration in HL-60
leukemia cells, known to express several P2 receptor
subtypes.19 Thus, a P2 receptor agonist would be
expected to cause a rapid rise in intracellular cal-
cium. Compounds 7a and 7b were essentially inactive
at a concentration of 100 mM, either at stimulating
calcium accumulation or inhibiting the rise in calcium
A receptor for guanosine was proposed,17 based on
neuroprotective effects observed for this nucleoside.
However, the guanosine analogues here were examined
only for interaction with ARs. The (N)-methanocarba
analogue, 8, bound weakly, but selectively to the A3 AR
with a Ki value of 21 mM. The corresponding tri-
phosphate, 9, of interest for its potential interaction
with G proteins, was inactive in binding to the A3 AR.
produced by 1 mMUTP (acting at P2Y
receptors).
2
Compound 9 (100 mM) stimulated a rise in intra-
cellular calcium equivalent to 53% of the rise produced

G. Ravi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2295–2300
2299
by 1 mMUTP, suggesting that it may activate a P2Y
receptor.
FAB m/z 333 (MH+). 5 (1R,2R,3S,4R,5S)-4-[6-oxo-9H-purin-
9-yl]-1-(hydroxymethyl)bicyclo-[3.1.0]hexane -2,3-diol): 1H
NMR (CD3OD) d 8.51 (s, 1H), 8.06 (s, 1H), 7.91 (s, 1H), 4.82
(s, 1H), 4.76 (d, J=6.6 Hz, 1H), 4.24 (d, J=11.8 Hz, 1H), 4.88
(d, J=6.8 Hz, 1H), 1.60 (m, 1H), 1.53 (m, 1H), 0.74 (m, 1H).
FAB m/z 279 (MH+). HR-MS (FAB) calcd 279.1093, found
279.1084. Acetonide intermediate of 6: BOP-Cl (26.5 mg,
In conclusion, we have identified non-adenosine analo-
gues containing the (N)-methanocarba modification
which bound weakly, and in some cases selectively, at
the human A3 AR. Since selectivity has been achieved in
these simple derivatives, this could provide the basis for
further development of more potent ligands. The phos-
phate analogues must also be evaluated at a range of
enzymatic and receptor activities. The full evaluation of
the biological activity of the new analogues is in pro-
gress.
0.104 mmol) was added to
a solution of 15 (11.5 mg,
0.035 mmol) and Et3N (0.015 mL, 0.104 mmol) in CH2Cl2/
DMF (2 mL). To the suspension 2.0 M MeNH2/THF
(0.03 mL, 0.06 mmol) was added and the resulting mixture was
stirred for 24 h at rt. Water was added and the solvent was
removed. The residue was purified on silica gel (CHCl3/
1
MeOH=6:1) to give the protected amide (8.1 mg, 68.6%). H
NMR (CD3OD) d 8.11 (s, 1H), 8.01 (s, 1H), 5.74 (d,
J=6.8 Hz, 1H), 5.03 (s, 1H), 2.79 (s, 3H), 2.17 (m, 1H), 1.55
(m, 1H), 1.52 (s, 3H), 1.42 (m, 1H), 1.27 (s, 3H). FAB m/z 346
1
(MH+). 6: H NM R (DO) d 8.18 (s, 1H), 8.10 (s, 1H), 5.08
References and Notes
2
(d, J=6.3 Hz, 1H), 4.96 (s, 1H), 4.17 (d, J=6.6 Hz, 1H), 2.85
(s, 3H), 2.26 (m, 1H), 1.80 (t, J=5.2 Hz, 1H), 1.51 (m, 1H),
1.51 (m, 1H). FAB m/z 306 (MH+). HR-MS (FAB) calcd
1. Nandanan, E.; Jang, S. Y.; Moro, S.; Kim, H.; Siddiqi,
M. A.; Russ, P.; Marquez, V. E.; Busson, R.; Herdewijn, P.;
Harden, T. K.; Boyer, J. L.; Jacobson, K. A. J. Med. Chem.
2000, 43, 829.
2. Jacobson, K. A.; Ji, X.-d.; Li, A. H.; Melman, N.; Siddiqi,
M. A.; Shin, K. J.; Marquez, V. E.; Ravi, R. G. J. Med. Chem.
2000, 43, 2196.
3. Lee, K.; Ravi, R. G.; Ji, X.-d.; Marquez, V. E.; Jacobson,
K. A. Bioorg. Med. Chem. Lett. 2001, 11, 1333.
4. Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.;
Wang, J.; Wagner, R. W.; Matteucci, M. D. J. Med. Chem.
1996, 39, 3739.
5. van Galen, P. J. M.; van Bergen, A. H.; Gallo-Rodriguez,
C.; Melman, N.; Olah, M. E.; IJzerman, A. P.; Stiles, G. L.;
Jacobson, K. A. Mol. Pharmacol. 1994, 45, 1101.
6. Jin, X.; Shepherd, R. K.; Duling, B. R.; Linden, J. J. Clin.
Invest. 1997, 100, 2849.
1
306.1202, found 306.1211. 7a t-butyl intermediate. H NM R
(CD3OD) d 8.45 (s, 1H), 8.20 (s, 1H), 4.92 (s, 1H), 4.44 (dd,
J=5.2, 12 Hz, 1H), 4.04 (d, J=6.3, 1H, 2H), 4.70 (dd, J=5.7,
11.1 Hz, 1H), 1.86 (m, 1H), 1.49 (m, 1H), 0.97 (t, J=6.7 Hz,
1H), 1.34–1.25 (m) 1.09 (m, 1H). FAB m/z 510 (MH+). 7a: 1H
NMR (D2O) d 8.18 (s, 1H), 8.10 (s, 1H), 5.08 (d, J=6.3 Hz,
1H), 4.96 (s, 1H), 4.17 (d, J=6.6 Hz, 1H), 2.85 (s, 3H), 2.26
(m, 1H), 1.80 (t, J=5.2 Hz, 1H), 1.51 (m, 1H), 1.51 (m, 1H).
31P NM R (DO) d 0.51. FAB m/z 357 ((Mꢀ1)ꢀ). HR-MS
2
(FABꢀ) calcd 357.0600, found 357.0607. 7b: 1H NM R (DO) d
8.17 (s, 1H), 8.05 (s, 1H), 4.60 (d, J=10.7 Hz, 1H), 3.93 (d,
2
J=6.6 Hz, 1H), 3.80 (d, J=11.0, 1H), 1.88 (s, 1H), 1.53 (t,
J=4.7 Hz, 1H), 0.97 (t, J=7.1 Hz, 1H). 31P NM R (DO) d
2
ꢀ10.96. FAB m/z 697 ((Mꢀ1)ꢀ). HR-MS (FABꢀ) calcd
697.1173, found 697.1166.
7. Kim, H. O.; Ji, X.-d.; Melman, N.; Olah, M. E.; Stiles,
G. L.; Jacobson, K. A. J. Med. Chem. 1994, 37, 4020.
8. Park, K. S.; Hoffmann, C.; Kim, H. O.; Padgett, W. L.;
Daly, J. W.; Brambilla, R.; Motta, C.; Abbracchio, M. P.;
Jacobson, K. A. Drug Dev. Res. 1998, 44, 97.
9. Williams, M.; Jarvis, M. F. Biochem. Pharmacol. 2000, 59,
1173.
10. Zor, T.; Andorn, R.; Sofer, I.; Chorev, M.; Selinger, Z.
FEBS Lett. 1998, 433, 326.
11. Yalowitz, J. A.; Jayaram, H. N. Anticancer Res. 2000, 20,
2329.
12. Simons, C. Nucleoside Mimetics; Gordon and Breach:
Amsterdam, 2001.
14. Procedures in Scheme 2. To a mixture of 16 (0.2 g,
0.69 mmol), 2-amino-6-chloropurine (0.234 g, 1.38 mmol) and
triphenylphosphine (0.361 g, 1.38 mmol) was added DIAD
(diisopropyl azodicarboxylate, 0.27 mL, 1.38 mmol) dropwise
at 0 ꢁC. The mixture was warmed up to rt and stirred for 6 h.
The solvent was evaporated under vacuum and the residue
was purified by flash chromatography using 6:4 petroleum
ether/ethyl acetate to furnish 17 as a solid (0.2 g, 67%). 1H
NMR (CDCl3) d 8.30 (s, 1H), 7.36 (s, 5H), 5.29 (d,
J=7.15 Hz, 1H), 5.18 (bs, 2H), 4.96 (s, 1H), 4.69–4.53 (ABq,
2H), 4.48 (d, J=7.15 Hz, 1H), 3.92 (d, J=10.16 Hz, 1H), 3.28
(d, J=10.16 Hz, 1H), 1.65–1.60 (m, 1H), 1.55 (s, 3H), 1.28–
1.20 (m, 1H), 1.24 (s, 3H), 0.96–0.88 (m, 1H). 18: To a solution
of 17 (0.1 g, 0.23 mmol) in dioxane (2 mL) was added 2 N
NaOH (8 mL) and heated at 90 ꢁC for 3 h. The mixture was
concentrated to dryness under vacuum and purified by flash
chromatography using 10% MeOH in CHCl3 to furnish 18 as
a solid (0.08 g, 82%). 1H NMR (CDCl3) d 7.96 (s, 1H), 7.37 (s,
5H), 6.29 (bs, 2H), 5.27 (d, J=6.87 Hz, 1H), 4.89 (s, 1H),
4.67–4.50 (m, 3H), 3.92 (d, J=10.16 Hz, 1H), 3.32 (d,
J=10.16 Hz, 1H), 1.70–1.60 (m, 1H), 1.55 (s, 3H), 1.25 (s, 4H),
0.96–0.86 (m, 1H). 19: To a solution of 18 (0.06 g, 0.14 mmol)
in 5% HCOOH/MeOH (5 mL) was added Pd black (0.06 g)
and stirred at rt for 8 h. The reaction mixture was filtered
through Celite and concentrated and purified by flash chro-
matography using 10% MeOH/CHCl3 to furnish 19 as a solid
13. Procedures in Scheme 1. A solution of 13 (40 mg,
0.119 mmol), 2-mercaptoethanol (0.03 mL, 0.475 mmol), 0.5 N
NaOMe/MeOH (0.95 mL, 0.475 mmol) in MeOH (1 mL) was
heated at 90 ꢁC for 3 h and the reaction mixture was neu-
tralized by glacial AcOH. The resulting mixture was con-
centrated and purified on preparative TLC (CHCl3/
MeOH=6:1) to provide the hypoxanthine derivative 14
(37 mg, 98%). 1H NMR (CD3OD) d 8.06 (s, 1H), 7.96 (s, 1H),
5.34 (d, J=7.1 Hz, 1H), 5.04 (s, 1H), 4.69 (d, J=6.9 Hz, 1H),
3.97 (d, J=11.5 Hz, 1H), 3.52 (d, J=11.5 Hz, 1H), 1.72 (m,
1H), 1.51 (s, 3H), 1.24 (s, 3H), 1.16 (m, 1H), 0.97 (m, 1H).
FAB m/z 319 (MH+). 15:
A mixture of 14 (37 mg,
0.116 mmol), RuCl3 (10 mg, 0.048 mmol), NaIO4 (181 mg,
0.847 mmol) in CH3CN/CHCl3/H2O=2:2:3 was vigorously
stirred for 6 h at rt. Solvents were removed and the residue was
subjected to reverse phase C18 column chromatography using
H2O/MeOH (30:1–7:1) as eluents to afford the acid 15
(0.04 g, 86%). 1H NM R (DO) d 789 (s, 1H), 7.83 (s, 1H), 5.28
2
(d, J=6.87 Hz, 1H), 4.77 (s, 1H), 4.59 (d, J=6.87 Hz, 1H),
3.90 (d, J=10.54 Hz, 1H), 3.44 (d, J=10.54 Hz, 1H), 1.64–
1.54 (m, 1H), 1.43 (s, 3H), 1.18 (s, 3H), 1.10–1.00 (m, 1H),
0.94–0.82 (m, 1H). 8: Substrate 19 (0.02 g, 0.06 mmol) was
dissolved in 5% TFA/MeOH (3 mL) and H2O (1 mL) and
stirred at rt for 6 h. The reaction mixture was concentrated to
(20.6 mg, 53.4%). 1H NM R (DO) d 8.15 (s, 1H), 8.11 (s, 1H),
2
5.74 (d, J=7.4 Hz, 1H), 5.03 (s, 1H), 4.87 (d, J=7.1 Hz, 1H),
1.22 (m, 1H), 1.61 (m), 1.55 (s, 3H), 1.34 (m, 1H), 1.28 (s, 3H).

2300
G. Ravi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2295–2300
dryness under vacuum and purified by preparative TLC using
15% MeOH/CHCl3 to furnish 8 (0.012 g, 68%). 1H NM R
(D2O) d 9.20 (s, 1H), 4.69 (d, J=6.32 Hz, 1H), 4.25 (d,
J=11.54 Hz, 1H), 3.92 (d, J=6.32 Hz, 1H), 3.23 (d,
J=11.54 Hz, 1H), 1.66–1.48 (m, 2H), 0.82–0.70 (m, 1H). 20:
To a solution of 19 (0.02 g, 0.06 mmol) in anhydrous THF
(2 mL) was added tetrazole (0.013 g, 0.18 mmol) and di-t-
butyl-N-N-diethylphosphoramidite (0.025 mL, 0.09 mmol) and
stirred at rt for 12 h. The reaction mixture was cooled to
ꢀ78 ꢁC and was added a solution of mCPBA (0.018 g) and
warmed to rt. Methanol (2 mL) was added to the reaction
mixture and concentrated under vacuum and purified by pre-
parative TLC using 10% MeOH/CHCl3 to furnish 0.022 g
product. This compound (0.02 g, 0.038 mmol) was dissolved in
5% TFA/MeOH (3 mL) and H2O (1 mL) and stirred at rt for
6 h. The reaction mixture was concentrated to dryness under
as a solid (0.05 g, 86%). 1H NMR (CDCl3) d 7.88 (s, 1H), 5.46
(d, J=7.14 Hz, 1H), 4.96 (s, 1H), 4.78 (d, J=7.14 Hz 1H),
4.25–3.99 (m, 5H), 3.26 (d, J=11.54 Hz, 1H), 1.77–1.58 m, 5H),
1.53 (s, 3H), 1.44–1.30 (m, 5H), 1.27 (s, 3H), 1.10–0.89 (m, 7H).
25: To a solution of 22 (0.026 g, 0.058 mmol) in anhydrous
THF (2 mL) was added tetrazole (0.012 g, 0.18 mmol) and di-t-
butyl-N,N-diethylphosphoramidite (0.024 mL, 0.09 mmol) and
stirred at rt for 12 h. The reaction mixture was cooled to
ꢀ78 ꢁC and was added a solution of mCPBA (0.017 g) and
warmed to rt. Methanol (2 mL) was added to the reaction
mixture and concentrated under vacuum and purified by pre-
parative TLC using 10% MeOH/CHCl3 to furnish 0.022 g
product 25. 1H NMR (CDCl3) d 8.02 (s, 1H), 5.48 (s, 1H), 5.20
(d, J=6.87 Hz, 1H), 4.54 (d, J=6.87 Hz, 1H), 4.31–3.96 (m,
6H), 1.82–1.23 (m, 34H), 1.10–0.89 (m, 7H). Compound 25
was treated with DOWEX in MeOH/H2O at 90 ꢁC for 2 h and
filtered and concentrated to furnish 0.011 g of mono-
phosphate. To a suspension of this monophosphate (0.011 g,
0.023 mmol) in DMF (1 mL) was added 1,10-carbonyldiimida-
zole (0.019 g, 0.12 mmol) and stirred for 12 h. The reaction
mixture was treated with a solution of 5% Et3N/MeOH and
stirred for 4 h. This mixture was concentrated to dryness under
vacuum, treated with tributylammonium pyrophosphate
(0.063 g, 0.014 mmol) and DMF (1 mL) and stirred for 3 days.
Triethylammonium bicarbonate buffer (3 mL) was added to
the reaction mixture followed by water (2 mL). After lyophili-
zation, the crude material obtained was purified on a Sephadex
vacuum to furnish 0.013 g of 20, which was used directly for
1
the further reaction. H NM R (DO) d 8.15 (s, 1H), 4.71 (s,
2
1H), 4.43 (dd, J=4.94, 11.26 Hz, 1H), 4.02 (d, J=6.59 Hz,
1H), 3.70 (dd, J=4.94, 11.26 Hz, 1H), 1.88–1.78 (m, 1H),
1.54–1.42 (m, 1H), 1.12–0.92 (m, 1H). 31P NM R (DO) d 0.87.
2
9: To a suspension of monophosphate (0.008 g, 0.02 mmol) in
DMF (1 mL) was added 1,10-carbonyldiimidazole (0.016 g,
0.1 mmol) and stirred for 12 h. A reaction mixture was
treated with a solution of 5% Et3N/MeOH and stirred for
4 h. This mixture was concentrated to dryness under vacuum
and were added tributylammonium pyrophosphate (0.055 g,
0.012 mmol) and DMF (1 mL) and stirred for 3 days. Triethyl-
ammonium bicarbonate buffer (3 mL) was added to the reac-
tion mixture followed by water (2 mL) and lyophilized. The
crude material obtained was purified on a Sephadex column
using 0.5 Mammonium bicarbonate and water to furnish
colum using 0.5 Mammonium bicarbonate and water to
1
furnish 3.5 mg of 12. H NM R (DO) d 8.20 (s, 1H), 5.18 (s,
2
1H), 4.80–4.73 (m, 2H), 4.09–3.77 (m, 6H), 1.80–1.22 (m,
10H), 1.04–0.84 (m, 7H). 23: A mixture of 22 (0.03 g,
0.067 mmol), RuO2 (0.18 g, 1.34 mmol), NaIO4 (0.286 g,
1.34 mmol), K2CO3 (0.184 g, 1.34 mmol) in CH3CN/CHCl3/
H2O=2:2:3 (10 mL) was stirred at rt vigorously for 20 h.
Reaction mixture was filtered through Celite. Organic layer
was separated and the aqueous layer was acidified with HCl
and extracted with EtOAc, dried and concentrated under
3.5 mg of 9. 1H NM R (DO) d 8.12 (s, 1H), 4.69 (s, 1H), 4.54–
2
4.44 (m, 1H), 4.07 (d, J=6.32 Hz, 1H), 3.92–3.82 (m, 1H),
1.84–1.78 (m, 1H), 1.46–1.38 (m, 1H), 1.02–0.94 (m, 1H). 31P
NMR (D2O) d ꢀ8.29, ꢀ10.69, ꢀ22.26.
15. Procedures in Scheme 3: To a mixture of 16 (0.1 g,
0.34 mmol), 1,3-dibutylxanthine (0.18 g, 0.68 mmol) and tri-
phenylphosphine (0.178 g, 0.68 mmol) was added DEAD
(diethyl azodicarboxylate, 0.1 mL, 0.68 mmol) dropwise at 0 ꢁC.
The mixture was warmed up to rt and stirred for 6 h. The
solvent was evaporated under vacuum and the residue was
purified by flash chromatography using 4:1 petroleum ether/
1
vacuum to furnish 0.012 g product. H NMR (CDCl3) d 7.64
(s, 1H), 5.69 (d, J=7.14 Hz, 1H), 5.30 (s, 1H), 4.74 (d,
J=7.14 Hz, 1H), 4.09 (t, J=7.42 Hz, 2H), 4.00 (t, J=7.42 Hz,
2H), 1.85–1.59 (m, 6H), 1.55 (s, 3H), 1.44–1.33 (m, 4H), 1.28
(s, 3H), 1.02–0.86 (m, 7H). 11: A mixture of 23 (0.01 g,
ꢄ
0.022 mmol), BOP-Cl (0.011 g, 0.044 mmol), CH3NH2 HCl
1
ethyl acetate to furnish 21 (0.16 g, 88%). H NMR (CDCl3) d
(0.006 g, 0.088 mmol), Et3N (0.012 mL, 0.088 mmol) in THF
(2 mL) was stirred at rt for 8 h. Solvent was removed under
vacuum and the crude material was purified by preparative
TLC using 10% MeOH/CHCl3. The pure material was treated
8.25 (s, 1H), 7.40–7.28 (m, 5H), 5.55 (s, 1H), 5.21 (d,
J=7.14 Hz, 1H), 4.64 (d, J=12.4 Hz, 1H), 4.58 (d, J=12.4 Hz,
1H), 4.49 (d, J=7.14 Hz, 1H), 4.16–3.84 (m, 5H), 3.16 (d,
J=9.89 Hz, 1H), 1.82–1.54 (m, 4H), 1.52 (s, 3H), 1.48–1.28
(m, 6H), 1.23 (s, 3H), 1.02–0.84 (m, 7H). 10: To a solution of
21 (0.025 g, 0.047 mmol) in anhydrous CH2Cl2 (1 mL) was
with 1 MBCl /CH2Cl2 (0.1 mL, 0.1 mmol) at 0 ꢁC for 10 min.
3
Methanol was added to the reaction mixture and the solvent
was evaporated and the crude material was purified by pre-
parative TLC using 15% MeOH/CHCl3 to furnish 0.002 g
product 11. 1H NMR (CDCl3) d 8.29 (s, 1H), 5.14 (s, 1H), 4.87
(s, 1H), 4.72 (s, 1H), 4.18–3.94 (m, 4H), 3.67 (s, 3H), 1.84–1.22
(m, 10H), 1.00–0.82 (m, 7H).
added 1 MBCl /CH2Cl2 (0.23 mL, 0.23 mmol) at 0 ꢁC and the
3
mixture warmed to rt and stirred for 15 min. MeOH (1 mL)
was added to the reaction mixture and concentrated under
vacuum. 10 was purified by preparative TLC using 10%
MeOH/CHCl3 (12 mg, 63%). 1H NMR (CD3OD) d 8.40 (s,
1H), 5.09 (s, 1H), 4.55 (d, J=6.32 Hz, 1H), 4.17 (d,
J=11.54 Hz, 1H), 4.06–3.82 (m, 5H), 3.18 (d, J=11.54 Hz,
1H), 1.70–1.40 (m, 5H), 1.40–1.18 (m, 5H), 0.88 (t, J=7.14 Hz,
6H), 0.72–0.62 (m, 1H). 22: To a solution of 21 (0.07 g,
0.13 mmol) in 5% HCOOH/MeOH (10 mL) was added Pd
black (0.07 g) and stirred at rt for 8 h. The reaction mixture
was filtered through Celite and concentrated and purified by
flash chromatography using 5% MeOH/CHCl3 to furnish 22
16. Dunn, P. M.; Liu, M.; Zhong, Y.; King, B. F.; Burnstock,
G. Br. J. Pharmacol. 2000, 130, 1378.
17. Rathbone, M. P.; Middlemiss, P. J.; Gysbers, J. W.;
Andrew, C.; Herman, M. A.; Reed, J. K.; Ciccarelli, R.; Di
Iorio, P.; Caciagli, F. Prog. Neurobiol. 1999, 59, 663.
18. Jacobson, K. A.; Xie, R.; Young, L.; Chang, L.; Liang,
B. T. J. Biol. Chem. 2000, 275, 30272.
19. Communi, D.; Janssens, R.; Robaye, B.; Zeelis, N.;
Boeynaems, J. M. FEBS Lett. 2000, 475, 39.