Synthesis and anti-HIV activity of conformationally restricted bicyclic hexahydroisobenzofuran nucleoside analogs

Article abstract of DOI:10.1039/b818707j

A chiral synthesis of a series of hexahydroisobenzofuran (HIBF) nucleosides has been accomplished via glycosylation of a stereo-defined (syn-isomer) sugar motif 16 with the appropriate silylated bases. All nucleoside analogs were obtained in 52-71% yield as a mixture of α- and β-anomeric products increasing the breadth of the novel nucleosides available for screening. The structure of the novel bicyclic HIBF nucleosides was established by a single crystal X-ray structure of the β-HIBF thymine analog 22b. Furthermore, the sugar conformation for these nucleosides was established as N-type. Among the novel HIBF nucleosides synthesized, twenty-five compounds were tested as inhibitor of HIV-1 in human peripheral blood mononuclear (PBM) cells and seven were found to be active (EC₅₀ = 12.3-36.2 μM). Six of these compounds were purine analogs with β-HIBF inosine analog 22o being the most potent (EC₅₀ = 12.3 μM) among all compounds tested. The striking resemblance between didanosine (ddI) and 22o may explain the potent anti-HIV activity.

Full text of DOI:10.1039/b818707j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and anti-HIV activity of conformationally restricted bicyclic
hexahydroisobenzofuran nucleoside analogs†
Alba D´ıaz-Rodr´ıguez,a,b Yogesh S. Sanghvi,c Susana Ferna´ndez,a Raymond F. Schinazi,d
Emmanuel A. Theodorakis,*b Miguel Ferreroa and Vicente Gotor*a
Received 22nd October 2008, Accepted 5th January 2009
First published as an Advance Article on the web 11th February 2009
DOI: 10.1039/b818707j
A chiral synthesis of a series of hexahydroisobenzofuran (HIBF) nucleosides has been accomplished via
glycosylation of a stereo-defined (syn-isomer) sugar motif 16 with the appropriate silylated bases. All
nucleoside analogs were obtained in 52–71% yield as a mixture of a- and b-anomeric products
increasing the breadth of the novel nucleosides available for screening. The structure of the novel
bicyclic HIBF nucleosides was established by a single crystal X-ray structure of the b-HIBF thymine
analog 22b. Furthermore, the sugar conformation for these nucleosides was established as N-type.
Among the novel HIBF nucleosides synthesized, twenty-five compounds were tested as inhibitor of
HIV-1 in human peripheral blood mononuclear (PBM) cells and seven were found to be active (EC50
=
12.3–36.2 mM). Six of these compounds were purine analogs with b-HIBF inosine analog 22o being the
most potent (EC50 = 12.3 mM) among all compounds tested. The striking resemblance between
didanosine (ddI) and 22o may explain the potent anti-HIV activity.
Introduction
Despite prodigious efforts in combating the HIV infection, it
remains the leading cause of death worldwide.1 Among the various
therapeutic treatments available for HIV-1, nucleoside reverse
transcriptase inhibitors (NRTIs) has proven to be an important
class of drugs with a variety of nucleoside analogs at the forefront.
For example, zidovudine (AZT), zalcitabine (ddC), didanosine
(ddI), stavudine (d4T, 1 in Fig. 1), lamivudine (3TC), abacavir
(ABC), viread (TDF) and emtricitabine (FTC) have been approved
by the FDA as anti-HIV drugs.2 However, the therapeutic potential
of these compounds is often compromised by drug resistance
and toxicity.3 In order to overcome these limitations and design
Fig. 1 Designed features of modified nucleosides.
improved inhibitors of HIV-1, several investigators4 have modified
the carbohydrate moiety of the nucleosides resulting in enhanced
understanding of the structure–activity relationship (SAR).
The anti-HIV studies with various carbohydrate-modified
nucleosides have established three salient SAR features that
contribute to the observed activity. First, the placement of a
2¢,3¢-double bond in the structures of d4T (1), abacavir, reverset
(D-d4FC, 2) and elvucitabine (L-d4TC) is essential to their anti-
HIV-1 activity.5 For instance, molecular modeling studies with
d4T-triphosphate indicate that the double bond is positioned in
close proximity to the aromatic side chain of Tyr 115 in the active
site of HIV-RT, thus contributing to desirable p–p interactions.6
Second, the acid labile nature of the 2¢,3¢-dideoxypurine analogs
can be overcome by introducing an electronegative fluorine atom
or other hydrophobic groups at the 2¢ or 3¢-positions (structure 3)
of the carbohydrate moiety.7 Such groups improve the stability of
these compounds at a lower pH and increase their lipophilicity for
uptake, both essential features for oral delivery of drugs. Third,
nucleoside analogs with a variety of conformational restrictions
imposed on the sugar ring (4, 5 and 6 in Fig. 1) have resulted in
modulation of the enzyme–substrate recognition.8 In particular,
planar pseudo-sugar ring systems are usually preferred. This con-
cept has led to the synthesis of several bicyclic nucleoside analogs
with some exhibiting N-type (2¢-exo/3¢-endo) sugar conformation
and antiviral activity.9
aDepartamento de Qu´ımica Orga´nica e Inorga´nica and Instituto Universi-
tario de Biotecnolog´ıa de Asturias, Universidad de Oviedo, 33006-Oviedo
(Asturias), Spain. E-mail: vgs@uniovi.es; Fax: (+34) 98-510-3448
bDepartment of Chemistry and Biochemistry, University of California,
San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA. E-mail:
etheodor@ucsd.edu
cRasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA
dEmory University School of Medicine/Veterans Affair Medical Center,
Atlanta, Georgia 30033, USA
† Electronic supplementary information (ESI) available: 1H, 13C NMR
spectral data. The level of purity is indicated by the inclusion of copies of
1H, 13C and 19F NMR spectra. In addition, some 2D NMR experiments
are shown, which were used to assign the peaks. CCDC reference number
666543. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/b818707j
Based on our ongoing interest10 in the design of conforma-
tionally restricted nucleoside analogs for various drug-discovery
approaches, we decided to explore the possibility of introducing
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these three attributes to one molecule and ultimately discover new
compounds with antiviral activity. More specifically, we envisioned
that a hexahydroisobenzofuran (HIBF) nucleoside, represented
by structure 7 (Fig. 2), could contain all three characteristics
discussed above. Earlier, it was reported that 3-hydroxymethyl-
1,3-dihydrobenzo[c]furan nucleosides 5 were found to be inactive
as inhibitors of HIV.11 This lack of activity was attributed to the
steric effect of the rigid benzene ring, which reduces the ability
of the molecule to access the enzyme-binding site. Replacement
of the benzene ring with an HIBF system may offer multiple
advantages, such as: (i) possible interactions with Tyr 115 in the
active site of HIV-RT due to the 7¢–8¢ double bond; (ii) stabilization
of the glyocosidic linkage at a lower pH due to the C–C bond
at the 2¢-position; (iii) adoption of the preferred N-type sugar
conformation for improved recognition by cellular enzymes, due
to the rigid bicyclic motif of HIBF; and (iv) improved lipophilicity
for enhanced cellular uptake. With this in mind, we embarked on
the synthesis of HIBF type nucleosides.
Results and discussion
Synthesis of HIBF nucleosides
The bicyclic core of the designed nucleosides was synthesized
as shown in Scheme 1. Commercially available D-mannitol was
converted to butenolide 10 via a sequence of steps that includes: (a)
acetonide protection of the 1,2 and 5,6 diol; (b) oxidative cleavage
of the 3,4 diol; (c) Wittig olefination of the resulting aldehyde and
(d) acid-catalyzed acetonide deprotection and lactonization of the
resulting hydroxy ester.13,14 Initial attempts to silylate butenolide
10 under basic conditions (imidazole, DMAP) led to complete
racemization of the starting material, due to the relative acidity
of the 4¢ hydrogen. However, silylation under acidic conditions
(NH4NO3, TBDPSCl, DMF)15 produced the desired compound 11
as a single isomer in excellent yield. The Diels–Alder cycloaddition
between 11 and butadiene proceeded exclusively from the opposite
face of the bulky TBDPS group and afforded 12 as a single
enantiomer in 85% yield.16 DIBAL-H reduction of lactone 12
proceeded exclusively from the less sterically hindered a-face
forming b-lactol 13 in 92% yield.
Fig. 2 Concept for the design and synthesis of HIBF nucleosides.
Although many variants of the bicyclic nucleosides have been
studied, HIBF nucleosides have not received much attention.
As illustrated in Fig. 2, our synthetic approach is based on
glycosidation of an appropriately protected HIBF motif 8 with
a variety of nucleobases. This strategy is advantageous for a
number of reasons. The chirally defined starting material 8 could
be synthesized in few steps from inexpensive D-mannitol. The
chemically inert HIBF moiety is not expected to interfere in
the glycosylation step. The glycosylation reaction would most
likely result in the formation of both a- and b-anomers of-
fering both varieties of nucleosides in one step. Appropriately
protected 8 could be synthesized in bulk and a large number of
nucleoside analogs could be synthesized in parallel mode in a
relatively short time. As such, the overall approach is convergent
and amenable to scale-up, should any of these nucleosides be
required in larger amounts for further studies. Moreover, the
HIBF nucleoside family of compounds offers a unique SAR
link between the 2¢,3¢-dideoxynucleosides and the structural
requirements for appending a second ring without abolishing
the antiviral activity. Herein we report in detail the synthesis
of several HIBF nucleosides and their antiviral and cytotoxicity
profiles.
Scheme 1 Synthesis of bicyclic scaffold 16. Reagents and conditions:
(a) 0.01 equiv SnCl2, 2,2-dimethoxypropane–DME (2 : 3), reflux, 4 h,
78%; (b) 2.0 equiv NaIO4, sat. aq. NaHCO3–CH2Cl2 (1 : 20), 0 ◦C, 2 h,
73%; (c) 1.5 equiv Ph3P CHCO2Me, MeOH, 0 ◦C, 16 h, 70%; (d) conc.
=
H2SO4–MeOH (1 : 100), 25 ◦C, 1 h, 90% (e) 1.2 equiv TBDPSCl, 5.0 equiv
NH4NO3, DMF, 25 ◦C, 12 h, 92%; (f) 0.33 equiv AlCl3, CH2Cl2, 60 ◦C, 6 d,
85%; (g) 1.1 equiv DIBAL-H (1 M), CH2Cl2, -78 ◦C 1 h, 92%; (h) 3.0 equiv
Ac2O, pyridine, 25 ◦C, 12 h, 90%; (i) 1.2 equiv TBAF (1 M), THF, 25 ◦C,
30 min, 87%; (j) 3.0 equiv Ac2O, pyridine, 25 ◦C, 12 h, 89%; (k) 1.2 equiv
TBAF (1 M), THF, 25 ◦C, 25 min, 96%; (l) 4.0 equiv Ac2O, pyridine,
25 ◦C, 12 h, 92%; (m) 1.3 equiv Bu4NOH (33% in H2O), THF, 25 ◦C,
6 h; (n) 4.0 equiv of Ac2O, pyridine, 25 ◦C, 12 h, 70% (over two steps).
It should be noted that while this work was in progress, a com-
munication appeared in the literature12 describing the synthesis of
the uridine analog 7 (B = Uracil). The reported synthesis departs
from uridine and employs a ring-closure metathesis reaction to
form the cyclohexene ring. As such, this route is not convergent
and suffers from a multi-step process that results in the formation
of cis- and trans-isomeric products. Moreover, the biological
activities of 7 have not been reported.
Initially we targeted lactol 13 as the glycosyl donor for coupling
with appropriately protected nucleobases. These efforts proved to
be unsuccessful, presumably due to the steric hindrance of the
bulky TBDPS group. To overcome this problem we decided to
change the protecting group at the 5¢ center to an acetate func-
tionality. To this end, compound 13 was first acetylated at the C1¢
center and then underwent a TBAF-induced desilylation to form
alcohol 15 in 78% combined yield. Acetylation of 15 then formed
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bicyclic scaffold 16 in 89% yield. Alternatively, desilylation of 13
formed diol 17 that, without further purification, was converted
to compound 16 (2 steps, 88% combined yield). It is worth noting
that, upon standing, diol 17 can undergo conversion to lactol
18, in which the C2¢ stereocenter was isomerized to form a more
stable 6,6 trans bicyclic motif. In fact, treatment of 17 under basic
conditions (Bu4NOH, H2O) followed by acetylation of the reaction
mixture led to isolation of diacetate 19 in 70% combined yield.
The glycosidation of diacetate 16 proceeded under Hilbert–
Johnson conditions17 (silylated base, DBU, TMSOTf) to produce
a mixture of a- and b-nucleosides in 52–71% overall yield
(Scheme 2). The ratio of a/b-anomers was determined by LC-
ESI-MS studies used in combination with 1H NMR on the crude
reaction mixture. In addition, spectroscopic studies including
NOESY measurements confirmed that the b-anomer was the
major compound. Unambiguous confirmation of the structure of
the synthesized compounds was obtained by a single crystal X-ray
analysis of 22b, the fully deprotected product of 20b (see Fig. 3).
Scheme 3 Deprotection of HIBF nucleosides. Reagents and conditions:
(a) 2 M NH3–MeOH, rt for obtaining a–d, f, h; (b) NH3 sat–MeOH, 110 ◦C
for obtaining i, j, m; (c) 2 M NH3–MeOH, 90–100 ◦C for obtaining k, l.
Yields are reported in the Experimental section.
group was easily removed in all cases at room temperature. Under
these conditions all pyrimidine nucleosides (20a–e, 21a–b and 21d–
e) formed the corresponding 5¢-deprotected HIBF nucleosides in
excellent yields. These conditions were also successfully used for
the selective deprotection of 6-Cl-purine 20f leading to compound
22f. However, treatment of 20f with saturated NH3–MeOH at
110 ◦C formed the 6-aminopurine analog 22i, while under less
drastic conditions produced the 6-methoxypurine 22k.
Treatment of 2,6-dichloropurine 21g under ammonia led to a
mixture of monoamino (23j) and diaminopurine derivative (23m)
in quantitative combined yield. The mixture of compounds 23j and
23m was purified by silica gel column chromatography (gradient
50–100% EtOAc–hexane). This transformation is reproducible
with both anomeric nucleosides (see Scheme 4).
Scheme 2 Glycosylation of 16 with pyrimidines and purines. Reagents
and conditions: (a) 0.5 equiv 16, 1.0 equiv base, 3.5 equiv BSA, MeCN,
75 ◦C, 1 h; (b) 1.6 equiv DBU, 25 ◦C; (c) 3.0 equiv TMSOTf, 0–25 ◦C,
2–3 h, 52–71% (over three steps).
Scheme 4 Synthesis of a-diamino purine bicyclic nucleoside 23. Reagents
and conditions: NH3 sat–MeOH, 110 ◦C, 36 h, 40% of 23j and 60% of 23m.
Alternatively, the diaminopurine 22m could be obtained by
reaction of 20g with NaN3 and Me3P (Scheme 5).19 Under these
conditions the double bond remained unchanged.
The inosine compound 22o was obtained via enzymatic deami-
nation of adenosine analogue 22i (Scheme 6).20 This reaction was
slower than the reported deamination of natural adenosine and
only afforded a 50% yield of the desired product after 72 h. We
attribute this sluggish reactivity to the lipophilic nature of the
HIBF nucleoside 22i. Nonetheless, it is remarkable to observe
that the presence of the cyclohexene ring is not incompatible to the
recognition by the enzyme. The recognition of 22i as a substrate for
adenosine deaminase further confirms that the 5¢-hydroxyl group
was accessible to the active site in the enzyme for its transformation
to 22o.21
Fig. 3 Single X-ray structure of compound 22b.
The coupled nucleosides were deprotected under NH3–MeOH
conditions as shown in Scheme 3.18 We observed that the 5¢-acetate
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Scheme 5 Purine transformations in b-nucleoside 20g. Reagents and
conditions: (a) 4 equiv NaN3, DMF, 70 ◦C, 4 h, 80%; (b) 2.2 equiv Me3P,
THF–H2O (1 : 1), 24 h, 77%.
Fig. 4 Percentage versus time curves for d4A and HIBF-ddA (22i) in
pH = 2 buffer at 37 ◦C.
Scheme 6 Synthesis of inosine 22o through enzymatic deamination.
Reagents and conditions: 1% ADA, phosphate buffer pH = 7, 3% DMSO,
35 ◦C, 72 h, 50%.
Biological evaluation
Antiviral assays. A set of twenty-five compounds were tested
against HIV-1 (strain LAI) and compared to those of 3¢-azido-
3¢-deoxythymidine (AZT, zidovudine) in an assay with human
peripheral blood mononuclear (PBM) cells. Some of the 5¢-O-
acetyl HIBF nucleosides were also included in the testing as
potential prodrugs because acyl groups are known to hydrolyze in
the presence of cellular esterases generating the free nucleosides.
Furthermore, some nucleosides were tested as mixtures of a/b-
anomers because these were obtained as an inseparable mixture.
A summary of the data, expressed as the effective concentration
required to inhibit viral replication by 50% (EC50) and 90% (EC90),
is presented in Table 2. HIBF nucleoside analogs 20g/21g, 22j,
22l, 22o, 23d, 23f, and 23j demonstrate modest activity when
compared with the AZT as a control. The potencies (EC50) of the
compounds were in the order of 22o > 22j > 23d > 22l > 20g/21g >
23f > 23j. In general, the purine nucleosides were found to be
more potent than the corresponding pyrimidine nucleosides, with
the exception of b-5-(2-bromovinyl)uridine 23d. Among various
HIBF purine nucleoside derivatives tested for anti-HIV-1, two a-
anomers (6-Cl-purine 23f, 2-Cl-adenosine 23j) and a mixture of
a/b-anomers (2,6-di-Cl-purines 20g/21g) also exhibited moderate
activities. The HIBF nucleoside 22o containing the inosine base
is the most potent nucleoside analog in this series of compounds
(EC50 = 12.3 mM). Interestingly, the 2-Cl-adenosine derivative
22j showed closely related antiviral activity as inosine analog 22o
(EC50 16.5 mM vs. 12.3 mM). The similar anti-HIV-1 activities of
these two nucleosides could be attributed to possible deamination
of the 22j furnishing a 2-chloro-inosine analog with a close
structural resemblance to 22o. This observation is in agreement
with the transformation of the adenosine analog 22i to 22o in the
presence of adenosine deaminase (Scheme 6). In fact, the masked
activity of the inosine analog is also evident in 2-Cl-6-OMe-purine
22l and 6-Cl-purine 23f. Both nucleosides could potentially be
hydrolyzed under physiological conditions exposing the anti-HIV
motif of the 2-chloro-inosine analog and its activity. It is note-
worthy that the natural configuration of b-2-Cl-adenine derivative
22j exhibited 2-fold greater potency than its corresponding a-
anomer 23j (EC50 16.5 mM vs. 36.2 mM). Therefore, the observed
Single crystal X-ray data for compound 22b is given in Table 1.
HIBF nucleoside analog 22b crystallized in the form of colorless
needles.
Fig. 3 is a PyMOL 0.9922 drawing, which corresponds to single-
crystal X-ray diffraction studies of compound 22b.23 Note that
the sugar conformation is North type with the 2¢-exo and 3¢-endo
configurations.
Evaluation of HIBF nucleosides
Acid stability studies. The acid stability of the glycosidic
linkage in 22i (HIBF-ddA) was studied by a direct comparison
with 2¢,3¢-didehydro-2¢,3¢-dideoxynucleoside (d4A, Fig. 4). The
cleavage of the◦glycosidic bond was studied at pH 2 buffered
solution and 37 C.24 The degradation of the nucleoside to the free
adenine was followed by high performance liquid chromatography
(HPLC), where samples were collected at different time points.
The data indicated that under experimental conditions d4A was
degraded at a significantly faster rate compared to the HIBF
nucleoside 22i (t1 d4A is about 20 min while t1 of 22i is about
2
2
6 h). The data confirmed that placement of a C–C bond at the
2¢-position enhances significantly the stability of HIBF nucleosides
towards lower pH.
Table 1 Crystal data for HIBF nucleoside analog 22b
Formula
C14H18N2O4
Molecular weight
Crystal system
Space group
278.30
Tetragonal
P42/n
22.892 (12)
22.892 (12)
5.224 (4)
8
˚
a, A
˚
b, A
˚
c, A
Z
Final R
Reflections
0.074
7109
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Table 2 Effect of analogs against HIV-1 (strain LA1) in human peripheral blood mononuclear (PBM) cells
Anti-HIV-1 activity in PBM cellsa Cytotoxicity (IC50, mM)b
EC50, mM EC90, mM
Analog
Base
PBM cells
CEM cells
VERO cells
AZT
20a+21a
20b+21b
20d+21d
20e+21e
20g+21g
22a
b-T
0.0015
60.7
82.02
0.006
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
43.1
> 100
> 100
> 100
> 100
> 100
> 100
27.2
> 100
> 100
41.4
> 100
31.9
54.2
> 100
30.6
14.3
> 100
> 100
> 100
> 100
5.9
50.6
> 100
> 100
> 100
> 100
18.9
> 100
> 100
> 100
> 100
> 100
> 100
34.7
76.2
> 100
50.3
> 100
19.6
98.3
> 100
61.9
> 100
> 100
> 100
> 100
> 100
a/b-U
a/b-T
a/b-BVU
a/b-N-Ac-C
a/b-2,6-di-Cl-purine
b-U
> 100
> 100
24.6
90.5
> 100
> 100
77.8
70.3
> 100
16.5
20.2
> 100
12.3
> 100
62.4
> 100
> 100
> 100
> 100
> 100
> 100
39.1
95.6
> 100
44.3
> 100
74.2
46.9
> 100
67.1
> 100
67.0
22b
b-T
> 100
> 100
> 100
95.6
> 100
> 100
> 100
> 100
20.1
22c
b-5-F-U
22d
b-BVU
22f
b-6-Cl-purine
b-A
22i
22j
b-2-Cl-A
b-2-Cl-6-OMe-purine
b-2,6-di-NH2-purine
b-I
22l
22m
22o
23b
a-T
a-BVU
a-6-Cl-purine
a-A
> 100
34.2
23d
19.0
28.5
90.6
36.2
23f
21.9
23i
≥ 100
> 100
> 100
> 100
> 100
> 100
> 100
23j
a-2-Cl-A
a-2,6-di-NH2-purine
a/b-T
23m
24b
> 100
≥ 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
24h
24i
24j
a/b-C
a/b-A
a/b-2-Cl-A
76.2
a HIV drug susceptibility assay was done as previously described in ref. 25. b Cytotoxicity assays in PBM, CEM and Vero cells were done as previously
described in ref. 26.
activity of the mixture a/b-2,6-di-Cl-purine 20g/21g could be
due to the presence of b-anomer in the mixture tested. Clearly,
there is a correlation between the six active purine nucleoside
analogs with the inosine analog being most active. The structural
resemblance of the ddI and inosine analog 22o is remarkable
and warrants further studies with the HIBF series of purine
nucleosides.
Conclusions
In summary, the present study provides for the first time a
direct access to a variety of bicyclic nucleoside analogs starting
with D-mannitol. The designed HIBF nucleosides contain the
three essential SAR elements, which are: (i) the presence of
a double bond in the carbohydrate portion of the structure;
(ii) the stabilization of the glycosidic linkage at lower pH and
(iii) the adoption of a preferred N-conformation. The NMR and
X-ray studies confirmed that the sugar adopts an N-conformation
that may be responsible for the observed anti-HIV-1 activity with
some of the compounds. The anti-HIV-1 activity exhibited by
purine analogs 22o, 22j and 22l is of particular interest because
of the close resemblance with the US FDA approved anti-HIV-1
drug ddI. Despite the modified bicyclic carbohydrate motif, the
adenosine analog 22i was deaminated with ADA indicating its
ability to mimic the natural adenosine. Therefore, it is reasonable
to postulate that the observed anti-HIV-1 activity with purine
analogs is perhaps dependent on the recognition by cellular
ADA. Interestingly, the antiviral activity and cytotoxicity was
modulated by the nature of the substituent on the purine ring.
The intracellular metabolism of these nucleosides deserves further
attention with particular emphasis on specificity for the ADA.
The anti-HIV-1 activity of ddI is due to its transformation
to dideoxyadenosine triphosphate, which inhibits the activity of
HIV-1 RT by competing with natural substrate, 2¢-deoxyadenosine
triphosphate as well as by its incorporation into viral DNA causing
termination of the viral chain elongation. Based on the observed
anti-HIV-1 activity with various purine analogs, we propose that
Cytotoxicity assays. The compounds were evaluated for
their potential toxic effects on uninfected phytohemagglutinin-
stimulated human PBM cells, and also in CEM and Vero (African
green monkey kidney) cells. Primary human PBM cells were
selected, because these lymphocytes represent slow-growing cells,
and were also used in the antiviral assay as host cells. The
lymphocytic CEM cells were selected, as they are a continuous
nontransformed cell line that replicates quickly and is commonly
used for anti-HIV-1 assays. Vero cells were used, as they not only
grow rapidly but are also anchored on the surface. The toxicities
in these cells may translate into the observed anti-HIV-1 activity.
It is noteworthy that the observed cytotoxicity in PBM and CEM
cells with inosine analog 22o could be eliminated by introduction
of suitable functionality on the purine ring. For example, b-2-Cl-
6-OMe-purine 22l is not cytotoxic to the PBM and CEM cells.
No significant toxicity was detected for most of the compounds
devoid of the anti-HIV activity.
In summary, results of both antiviral and cytotoxicity studies
indicate that these compounds could offer valuable leads into the
development of novel NRTIs with improved therapy for HIV-1
treatment.
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the HIBF nucleosides are mimics of ddI and exert their activity
via incorporation into viral DNA. Because these compounds are
stable at lower pH, we believe that they could offer superior
bioavailability and longer half-life in vivo after oral administration.
The transformation of some of the lead compounds (e.g. 22o) into
their triphosphate analogs is currently under progress for further
biological studies. The synthetic methodology described in this
study offers a facile and efficient entry into a variety of bicyclic
nucleoside analogs with a wider range of therapeutic applications,
such as synthesis of novel nucleosides as antiviral compounds and
building blocks for the construction of antisense oligonucleotides.
Synthesis of lactol 13. A solution of 12 ◦(12.8 g, 31.5 mmol)
in dry CH2Cl2 (150 mL) was cooled to -78 C under argon and
treated with DIBAL-H (35 mL, 35 mmol, 1 M in CH2Cl2) for 1 h.
The reaction was quenched with MeOH (10 mL) and the solution
was brought to r.t. over a 20 min period. Saturated Rochelle salt
was added (stirred for 15 min) and the crude product was extracted
with EtOAc (4 ¥ 100 mL). Lactol 13 was isolated in 92% yield as
yellow oil. Rf (33% hexane–EtOAc): 0.29; [a]2D0 = -16.9 (c 1.9,
CH2Cl2); MS (ESI+, m/z): 409 [(M + H)+, 100%]; Exact mass
found for C25H32O3Si: 408.2120.
Synthesis of acetate 14. Lactol 13 (6.65 mmol) was dissolved
in pyridine (40 mL) and Ac2O (1.8 mL, 19.95 mmol) was added
dropwise. The reaction was stirred overnight and then quenched
with saturated NaHCO3. The solution was extracted with CH2Cl2
(3 ¥ 60 mL). The combined organic extracts were dried (Na2SO4).
The residue was purified by flash chromatography (10% hexane–
Experimental section
General techniques
All reagents were bought from Aldrich and Acros at highest
commercial quality and used without further purification except
where noted. Air- and moisture-sensitive liquids and solutions
were transferred via syringe or stainless steel cannula. Organic
solutions were concentrated by rotary evaporation below 45 ◦C at
approximately 20 mmHg. All non-aqueous reactions were carried
out under anhydrous conditions using flame-dried glassware
within an argon atmosphere in dry, freshly distilled solvents,
unless otherwise noted. THF and CH2Cl2 were purified by passage
through a bed of activated alumina. Pyridine (Py) was distilled
from CaH2 prior to use. Yields refer to chromatographically
and spectroscopically (1H NMR) homogeneous materials, unless
otherwise stated. Reactions were monitored by TLC carried out
on 0.25 mm E. Merck silica gel plates (60F-254) using UV
light as visualizing agent and 7% ethanolic phosphomolybdic
acid, or p-anisaldehyde solution and heat as developing agents.
E. Merck silica gel (60, particle size 0.040–0.063 mm) was used
for flash chromatography. LC-ESI-MS analyses were carried
out in a chromatograph with UV detector at 280 nm using
a VYDAC C-18 column, Cat. Number: 218TP54, (4.6 mm ¥
250 nm); flow 1 mL min-1; r.t.; gradient MeOH–H2O as eluent.
NMR spectra were recorded on Varian Mercury 300, 400 and/or
Unity 500 MHz or Bruker AV-400 or 600 MHz instruments
and calibrated using residual undeuterated solvent as an internal
reference. The following abbreviations were used to explain the
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad. IR spectra were recorded on a Nicolet 320
Avatar FT-IR spectrometer and values are reported in cm-1 units.
Optical rotations were recorded on a Jasco P-1010 polarimeter
and values are reported as follows: [a]Tl (c: g/100 mL, solvent).
High resolution mass spectra (HRMS) were recorded on a VG
7070 HS mass spectrometer under electron spray ionization (ESI)
conditions. X-Ray data were recorded on a Bruker SMART APEX
3 kW Sealed Tube X-ray diffraction system.
EtOAc) to obtain 14 (90%). Rf (25% hexane–EtOAc): 0.65; [a]D20
22.8 (c 0.6, CHCl3); IR (film): n 3069, 3025, 2925, 2849, 1744,
1421, 1226, 1099 cm-1; MS (ESI+, m/z): 468 [(M + NH4)+, 100%],
473 [(M + Na)+, 45], and 489 [(M + K)+, 15].
=
Synthesis of acetate 15. Compound 14 (3.34 mmol) was
dissolved in 60 mL of dry THF and then treated with TBAF
(4.3 mL, 4.3 mmol, 1 M in THF). The reaction was kept
under argon at r.t. for 30 min. The solution was extracted with
EtOAc (3 ¥ 30 mL). The combined organic extracts were dried
using Na2SO4 and then filtered. The residue was purified by
flash chromatography (30% hexane–EtOAc) to give 15 (89%). Rf
(EtOAc): 0.74; [a]2D0 = 47.4 (c 0.9, CHCl3); IR (film): n 3019,
2922, 2845, 1728, 1427, 1369, 1233, 932 cm-1; MS (ESI+, m/z):
230 [(M + NH4)+, 50%], 235 [(M + Na)+, 75], and 251 [(M + K)+,
20].
Synthesis of diacetate 16. A solution of 17 (31 mmol) in
pyridine (100 mL) was treated with Ac2O (12 mL, 124 mmol).
The reaction was stirred overnight and quenched with saturated
NaHCO3. The solution was extracted with CH2Cl2 (4 ¥ 200 mL).
The combined organic extracts were dried using Na2SO4 and then
filtered. The residue was purified by flash chromatography (15%
hexane–EtOAc) to afford 16 (93%). Rf (50% EtOAc–hexane): 0.53;
[a]2D0 = -21.8 (c 1.9, CHCl3); IR (film): n 3030, 2918, 2838, 1737,
1641, 1440, 1239, 933 cm-1; MS (ESI+, m/z): 272 [(M + NH4)+,
20%] and 277 [(M + Na)+, 100].
Synthesis of diol 17. Lactol 13 (31.5 mmol) was dissolved in
THF (100 mL) under argon and then treated with TBAF (37.8 mL,
37.8 mmol, 1 M in THF) at r.t. for 25 min. The solution was
quenched with brine and extracted with EtOAc (3 ¥ 100mL). The
residue was chromatographed using a gradient of 30–70% EtOAc–
hexane to give 17 (96%). Rf (EtOAc): 0.44; [a]2D0 = -16.9 (c 1.9,
CH2Cl2); MS (ESI+, m/z): 193 [(M + Na)+, 55%].
The bicyclic motif of the synthetic hexahydroisobenzofuran
nucleosides is abbreviated below as HIBF. The assignment of the
NMR data is based on the nucleoside numbering (1¢–5¢) and the
second ring as 6¢–9¢ positions (Fig. 2).
Synthesis of diacetate 19. A solution of 13 (31.5 mmol) in
THF (100 mL) was treated with TBAOH (47.3 mmol, 33% in
H2O) at r.t. for 6 h. Then, solvents were removed in vacuo. The
orange residue was treated in pyridine (100 mL) with Ac2O (12 mL,
124 mmol). The reaction was stirred overnight and then quenched
with saturated NaHCO3. The solution was extracted with CH2Cl2
(4 ¥ 200 mL). The combined organic extracts were dried using
Na2SO4, and then filtered. The residue was purified by flash
Synthesis of lactone 12. It was obtained following a previously
described procedure.14 Rf (33% hexane–EtOAc): 0.41; [a]2D0 = 19.4
(c 3.40, CH2Cl2); IR (film): n 3070, 3043, 2932, 2857, 1775, 1112,
704 cm-1; MS (ESI+, m/z): 407 [(M + H)+, 100%]; Exact mass
found for C25H30O3Si: 406.1964.
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chromatography (20% hexane–EtOAc) to give 19 (70%). Rf (50%
EtOAc–hexane): 0.55; IR (film): n 3026, 2931, 2839, 1734, 1633,
1233, 936 cm-1; MS (ESI+, m/z): 272 [(M + NH4)+, 80%] and 277
[(M + Na)+, 100].
1239, 1190, 1076, cm-1; MS (ESI+, m/z): 405 [(M35Cl35Cl + Na)+,
100%], 407 [(M35Cl37Cl + Na)+, 80], and 409 [(M37Cl37Cl + Na)+,
10%]; LC-ESI-MS (UV 280 nm): ratio b/a 2.4 : 1 tR(min) = 23.62,
24.27; Exact mass found for C16H16Cl2N4O3: 382.0600.
21g. Rf (50% hexane–EtOAc): 0.52; [a]2D0 = -40.8 (c 1.3,
CHCl3); MS (ESI+, m/z): 405 [(M35Cl35Cl + Na)+, 100%], 407
[(M35Cl37Cl + Na)+, 80], and 409 [(M37Cl37Cl + Na)+, 10]; LC-ESI-
MS (UV 280 nm): coupling ratio b/a 2.4 : 1 tR(min) = 23.62,
24.27; Exact mass found for C16H16Cl2N4O3: 382.0651.
General procedure for condensation of diacetate 16 with
heterocyclic bases
To a stirred solution of the different bases (1.81 mmol) in dry
MeCN (5.0 mL), BSA was added (1.5 mL, 6.338 mmol). The
reaction mixturewas heated at70◦C for 1.5h. To the resulting clear
mixture was added at r.t. a solution of diacetate 16 (0.9 mmol) in
dry MeCN (3 mL), followed by DBU (0.434 mL, 2.899 mmol). This
solution was cooled to 0 ◦C and then TMSOTf was added dropwise
(1.05 mL, 5.798 mmol). The resulting mixtures were stirred for 2–
3 h. Each mixture was poured into an ice-cool saturated NaHCO3
solution and extracted with CH2Cl2 (3 ¥ 25 mL). Residues were
purified by silica gel column chromatography to give 20 and 21
using a gradient of 33–100% EtOAc–hexane.
General protocols for deprotection of HIBF nucleoside derivatives
20–21
Method A (deprotection of 5¢-OAc group). Nucleoside derivatives
20a–f+21a–f (0.1 mmol) were dissolved in a 2 M NH3–MeOH
solution (1.5 mL) and stirred at r.t. overnight. Residues were
purified by silica gel column chromatography using a gradient
of 33–100% EtOAc–hexane. Method B. Nucleoside derivatives
20f+21f, 20g and 21g (0.1 mmol) were treated with a NH3–
MeOH saturated solution (1.5 mL) and stirred at 110 ◦C in a
Parr reactor for 36 h. Residues were purified by silica gel column
chromatography (gradient 50–100% EtOAc–hexane) obtaining
22i, 23i (95–97%), 22j–22m, 23j–23m (quantitative combined
yield). Method C. Nucleoside derivatives 20f and 20g (0.11 mmol)
were treated with a 2 M NH3–MeOH solution (1.8 mL) and stirred
at 90–100 ◦C in a Parr reactor for 30 h. Residues were purified
by silica gel column chromatography using a gradient 50–100%
EtOAc–hexane.
HIBF-a/b-uracil (20a+21a). Rf (EtOAc): 0.55; [a]2D0 = -23.4
(c 0.7, CHCl3); IR (KBr): n 3200, 3032, 2922, 2844, 1743, 1686,
1461, 1271, 1237, 1042, 705 cm-1; MS (ESI+, m/z): 307 [(M + H)+,
70%], 324 [(M + NH4)+, 80], and 329 [(M + Na)+, 100]; LC-ESI-
MS (UV 280 nm): coupling ratio b/a 1.1 : 1 tR(min) = 17.46,
17.91; Exact mass found for C15H18N2O5: 306.1206.
HIBF-a/b-thymine ((20b+21b). Rf (50% hexane–EtOAc):
0.22; [a]2D0 = -41.26 (c 1.0, CHCl3); IR (KBr): n 3197, 3032, 2926,
1744, 1684, 1662, 1473, 1271, 1237, 1044 cm-1; MS (ESI+, m/z):
321 [(M + H)+, 100%], 338 [(M + NH4)+, 90], and 343 [(M + Na)+,
95]; LC-ESI-MS (UV 280 nm): coupling ratio b/a 1.2 : 1 tR(min) =
19.11, 19.29; Exact mass found for C16H20N2O5: 320.1371.
HIBF-b-uracil (22a). From 20a using Method A. Rf (EtOAc):
0.40; [a]2D0 = +0.7 (c 0.3, CHCl3); MS (ESI+, m/z): 287 [(M + Na)+,
100%]; Exact mass found for C13H15N2O4: 264.1104.
HIBF-b-5-fluorouracil (20c). Rf (50% hexane–EtOAc): 0.29;
[a]2D0 = +10.0 (c 1.3, CHCl3); IR (KBr): n 3189, 3075, 3044, 2950,
2857, 1718, 1661, 1469, 1262, 1230, 1093, 1064 cm-1; 19F NMR
(CDCl3, 282 MHz): d 167.1; MS (ESI+, m/z): 325 [(M + H)+,
30%], 342 [(M + NH4)+, 60], and 347 [(M + Na)+, 100]; LC-
ESI-MS (UV 280 nm): tR(min) = 19.31; Exact mass found for
C15H17FN2O5: 324.1122.
HIBF-b-thymine (22b). From 20b using Method A. Rf (50%
hexane–EtOAc): 0.16; [a]2D0 = -13.6 (c 0.3, CHCl3); MS (ESI+,
m/z): 301 [(M + Na)+, 100%]; Exact mass found for C14H18N2O4:
278.1257.
HIBF-b-5-fluorouracil (22c). From 20c using Method A. Rf
(50% hexane–EtOAc): 0.28; [a]2D0 = +28.6 (c 0.8, CHCl3); IR
(KBr): n 3508, 3448, 3156, 3040, 2925, 2851, 1682, 1652, 1266,
1093, 1066, 1026 cm-1; 19F NMR (CDCl3, 282 MHz): d 167.0;
MS (ESI+, m/z): 305 [(M + Na)+, 100%]; Exact mass found for
C13H15FN2O4: 282.1014.
HIBF-b-5-bromovinyluracil (20d). Rf (50% hexane–EtOAc):
0.41; [a]2D0 = -22.3 (c 0.6, CHCl3); IR (KBr): n 3424, 2926, 2874,
1793, 1747, 1700, 1472, 1368, 1280, 1166, 1109, 1041 cm-1; MS
(ESI+, m/z): 433 [(M79Br + Na)+, 100%], 435 [(M81Br + Na)+, 97];
Exact mass found for C17H19BrN2O5: 410.0872.
HIBF-b-5-bromovinyluracil (22d). From 20d using Method A.
Rf (50% hexane–EtOAc): 0.32; [a]2D0 = -24.8 (c 0.6, CHCl3); MS
(ESI+, m/z): 369 [(M79Br + H)+, 100%] and 371 [(M81Br + H)+,
96]; Exact mass found for C15H17BrN2O4: 368.0395.
HIBF-a/b-cytosine (20e+21e). Rf (EtOAc): 0.24; [a]2D0 = -25.8
(c 0.4, MeOH); MS (ESI+, m/z): 370 [(M + Na)+, 100%]; LC-ESI-
MS (UV 280 nm): coupling ratio b/a 1.2 : 1 tR(min) = 19.09,
19.63; Exact mass found for C17H21N3O5: 347.1480.
HIBF-b-6-chloropurine (22f). From 20f using Method A. Rf
(EtOAc): 0.50; [a]2D0 = -37.2 (c 1.3, CHCl3); MS (ESI+, m/z): 329
[(M35Cl + Na)+, 100%] and 331 [(M37Cl + Na)+, 20]; Exact mass
found for C14H15ClN4O2: 306.0682.
HIBF-a/b-6-chloropurine (20f+21f). Rf (50% hexane–
EtOAc): 0.37; [a]2D0 = -25.8 (c 1.0, CHCl3); IR (KBr): n 3389,
2942, 2890, 1708, 1633, 1489, 1245, 1193, 1094, 920 cm-1; MS
(ESI+, m/z): 371 [(M35Cl + Na)+, 100], 373 [(M37Cl + Na)+, 30];
LC-ESI-MS (UV 280 nm): coupling ratio b/a 1.5 : 1 tR(min) =
21.19, 21.43; Exact mass found for C16H17ClN4O3: 348.0982.
HIBF-b-6-aminopurine (22i). From 20f using Method B. Rf
(5% CH2Cl2–MeOH): 0.20; [a]2D0 = -19.6 (c 0.8, DMSO); MS
(ESI+, m/z): 288 [(M + H)+, 100%] and 310 [(M + Na)+, 60]; Exact
mass found for C14H17N5O2: 287.0713.
HIBF-a/b-2,6-dichloropurine (20g+21g). The two anomers
were separated. 20g: Rf (50% hexane–EtOAc): 0.52; [a]2D0 = -7.3
(c 0.3, CHCl3); IR (KBr): n 3370, 2940, 2879, 1728, 1627, 1480,
HIBF-b-6-amino-2-chloropurine (22j). From 20g using
Method B. Rf (10% EtOAc–MeOH): 0.44; [a]D20
=
-8.5
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(c 0.3, DMSO); MS (ESI+, m/z): 322 [(M35Cl + H)+, 100%],
324 [(M37Cl + H)+, 20], 344 [(M35Cl + Na)+, 80], and 346
[(M37Cl + Na)+, 10].
residue obtained was purified by silica gel flash chromatography
(gradient 30–50% EtOAc–hexane) to give 20n in 76% yield. Rf
(50% hexane–EtOAc): 0.47; MS (ESI+, m/z): 419 [(M + Na)+,
100%].
HIBF-b-6-methoxypurine (22k). From 20f using Method C.
Rf (EtOAc): 0.39; MS (ESI+, m/z): 325 [(M + Na)+, 100%]; Exact
mass found for C15H18N4O3: 302.0774.
Synthesis of HIBF-b-hypoxanthine 22o. Nucleoside 22i
(20 mg, 0.070 mmol) was dissolved in a phosphate buffer
pH = 7/3% DMSO (400 mL) and treated with 1 mg of ADA
dissolved in that buffer (100 mL). The residue was purified by
flash chromatography (gradient 5–20% MeOH–EtOAc) to give
22o (50%). Rf (10% EtOAc–MeOH): 0.10; MS (ESI+, m/z): 289
[(M + H)+, 100%] and 311 [(M + Na)+, 60].
HIBF-b-2-chloro-6-methoxypurine (22l). From 20g using
Method C. Rf (EtOAc): 0.44; MS (ESI+, m/z): 359 [(M35Cl + Na)+,
100%] and 361 [(M37Cl + Na)+, 20]; Exact mass found for
C15H17ClN4O3: 336.1102.
HIBF-b-2,6-diaminopurine (22m). From 20g using Method B.
Rf (10% EtOAc–MeOH): 0.17; [a]2D0 = -19.1 (c 2.5, DMSO); MS
(ESI+, m/z): 303 [(M + H)+, 100%].
Antiviral assays
The procedures for the antiviral assays in human peripheral blood
mononuclear (PBM) cells have been reported previously.25 Briefly,
uninfected phytohemagglutinin-stimulated human PBM cells were
infected with HIV-1 (strain LA1) (about 63 000 disintegrations
of RT activity per minute per 107 cells per 10 ml of medium).
The HIBF nuclesoside analogs were then added to duplicate
or triplicate cultures. Uninfected and untreated PBM cells were
grown in parallel at equivalent cell concentrations as controls.
The cultures were maintained in a humidified 5% CO2–95%
air incubator at 37 ◦C for 6 days after infection, at which
point all cultures were sampled for supernatant RT activity. The
supernatant was clarified, and the viral particles were then pelleted
at 40 000 rpm for 30 min by using a rotor and suspended in
virus-disrupting buffer. The RT assay was performed in 96-well
microdilution plates by using (rA)n·(dT)12–18 as the template primer.
The RT results were expressed in disintegrations per minute per
millilitre of originally clarified supernatant.
HIBF-a-thymine (23b). From 21b using Method A. Rf (50%
hexane–EtOAc): 0.10; [a]2D0 = -34.5 (c 0.3, CHCl3); MS (ESI+,
m/z): 301 [(M + Na)+, 100%]; Exact mass found for C14H18N2O4:
278.1260.
HIBF-a-5-bromovinyluracil (23d). From 21d using Method A.
Rf (50% hexane–EtOAc): 0.14; MS (ESI+, m/z): 369 [(M79Br + H)+,
100] and 371 [(M81Br + H)+, 96]; Exact mass found for
C15H17BrN2O4: 368.0874.
HIBF-a-6-chloropurine (23f). From 21f using Method A. Rf
(EtOAc): 0.40; [a]2D0 = -9.7 (c 0.8, CHCl3); MS (ESI+, m/z): 329
[(M35Cl + Na)+, 100%] and 331 [(M37Cl + Na)+, 30]; Exact mass
found for C14H15ClN4O2: 306.0548.
HIBF-a-6-aminopurine (23i). From 21f using Method B. Rf
(5% CH2Cl2–MeOH): 0.09; [a]2D0 = -9.6 (c 2.5, MeOH); MS (ESI+,
m/z): 288 [(M + H)+, 100%] and 310 [(M + Na)+, 60]; Exact mass
found for C14H17N5O2: 287.0772.
Cytotoxicity assays
HIBF-a-6-amino-2-chloropurine (23j). From 21g using
Method B. Rf (6.6% EtOAc–MeOH): 0.37; [a]2D0 = -25.9 (c 1.0,
DMSO); MS (ESI+, m/z): 322 [(M35Cl + H)+, 100%] and 324
[(M37Cl + H)+, 20].
The HIBF nucleoside analogs were evaluated for their poten-
tial toxic effects on uninfected phytohemagglutinin-stimulated
human PBM cells and also in CEM and Vero (African green
monkey kidney) cells as described previously.26 PBM cells were
obtained from the whole blood of healthy HIV-1 and hepatitis B
virus-seronegative volunteers and collected by single-step Ficoll-
Hypaque discontinuous gradient centrifugation. CEM (CEM-
CCRF) cells were a T-lymphoblastoid cell line that was obtained
from the American Type Culture Collection, Rockville, Md. The
CEM cells were maintained in RPMI 1640 medium supplemented
with 20% heat-inactivated fetal bovine serum, penicillin (100
U ml-1), and streptomycin (100 mg ml-1). The PBM and CEM
cells were cultured with and without drug for 6 days, at which
time portions were counted for cell proliferation and viability by
the trypan blue exclusion method. Only the effects on cell growth
are reported, since these correlated well with cell viability. The
toxicity of the compounds in Vero cells was assessed after 3 days
of treatment with a hemacytometer.
HIBF-a-2,6-diaminopurine (23m). From 21g using Method B.
Rf (10% EtOAc–MeOH): 0.12; [a]2D0 = -59.8 (c 0.8, MeOH); MS
(ESI+, m/z): 303 [(M + H)+, 100%].
HIBF-a/b-uracil (24a). From 20a+21a using Method A. Rf
(EtOAc): 0.40b, 0.36a; MS (ESI+, m/z): 287 [(M + Na)+, 100%].
HIBF-a/b-cytosine (24h). From 20h+21h using Method A. Rf
(EtOAc–MeOH 10%): 0.20; [a]2D0 = -28.6 (c 0.7, MeOH); MS
(ESI+, m/z): 264 [(M + H)+, 70%] and 286 [(M + Na)+, 100]; Exact
mass found for C13H17N3O3: 263.1318.
Synthesis of HIBF-b-2,6-diaminopurine (20m). A solution of
20n (30 mg, 0.076 mmol) in a mixture THF–H2O was treated
with Me3P (15.4 mL, 0.174 mmol). Immediately, N2 was given off
and the solution became yellowish. The reaction was stirred for
26 h and then solvents were removed. The residue was purified by
silica gel chromatography (2–10% MeOH–EtOAc) to give 20m in
73% yield. Rf (20% EtOAc–MeOH): 0.49; MS (ESI+, m/z): 367
[(M + Na)+, 100%].
Acknowledgements
Financial support of this work by the Spanish Ministerio de
Educacio´n y Ciencia (MEC) (Project MEC-07-CTQ-61126) is
gratefully acknowledged. A. D.-R. thanks MEC for a predoctoral
fellowship. We also thank Dr A. G. DiPasquale and Professor
Synthesis of HIBF-b-2,6-diazidopurine (20n). A solution of
20g (0.262 mmol, 100 mg) in DMF (1.8 mL) was treated with NaN3
(1.048 mmol, 68 mg) at 70 ◦C for 4 h. Solvent was removed and the
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A. L. Rheingold (UCSD X-Ray Facility) for the reported crys-
tallographic studies and Dr Y. Su (UCSD Mass Spectrometry
Facility) for mass analysis and the NSF (CHE-0116662, CHE-
9709183 and CHE-0741968) for NMR and MS instrumentation
grants. R. F. S. is supported in part by NIH grants 5P30-AI-50409
(CFAR), 5R37-AI-041980 and by the Department of Veterans
Affairs. We are grateful to Kim L. Rapp and Jason Grier for their
excellent technical assistance.
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