Design, synthesis, and biological evaluation of unconventional aminopyrimidine, aminopurine, and amino-1,3,5-triazine methyloxynucleosides

Article abstract of DOI:10.1002/cmdc.201402465

Herein we describe a class of unconventional nucleosides (methyloxynucleosides) that combine unconventional nucleobases such as substituted aminopyrimidines, aminopurines, or aminotriazines with unusual sugars in their structures. The allitollyl or altritollyl derivatives were pursued as ribonucleoside mimics, whereas the tetrahydrofuran analogues were pursued as their dideoxynucleoside analogues. The compounds showed poor, if any, activity against a broad range of RNA and DNA viruses, including human immunodeficiency virus (HIV). This inactivity may be due to lack of an efficient metabolic conversion into their corresponding 5′-triphosphates and poor affinity for their target enzymes (DNA/RNA polymerases). Several compounds showed cytostatic activity against proliferating human CD4⁺ T-lymphocyte CEM cells and against several other tumor cell lines, including murine leukemia L1210 and human prostate PC3, kidney CAKI-1, and cervical carcinoma HeLa cells. A few compounds were inhibitory to Moloney murine sarcoma virus (MSV) in C3H/3T3 cell cultures, with the 2,6-diaminotri-O-benzyl-D-allitolyl- and -D-altritolyl pyrimidine analogues being the most potent among them. This series of unconventional nucleosides may represent a novel family of potential antiproliferative agents.

Full text of DOI:10.1002/cmdc.201402465

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DOI: 10.1002/cmdc.201402465
Design, Synthesis, and Biological Evaluation of
Unconventional Aminopyrimidine, Aminopurine, and
Amino-1,3,5-triazine Methyloxynucleosides
[a]
[a]
[b]
[c]
Gloria Fernꢀndez-Cureses, Sonia de Castro,* Marꢁa-Luisa Jimeno, Jan Balzarini, and
Marꢁa-Josꢂ Camarasa*
[a]
Dedicated to Professor Josꢀ Elguero Bertolini on the occasion of his 80th birthday
Herein we describe a class of unconventional nucleosides
methyloxynucleosides) that combine unconventional nucleo-
for their target enzymes (DNA/RNA polymerases). Several com-
(
pounds showed cytostatic activity against proliferating human
+
bases such as substituted aminopyrimidines, aminopurines, or
aminotriazines with unusual sugars in their structures. The alli-
tollyl or altritollyl derivatives were pursued as ribonucleoside
mimics, whereas the tetrahydrofuran analogues were pursued
as their dideoxynucleoside analogues. The compounds showed
poor, if any, activity against a broad range of RNA and DNA vi-
ruses, including human immunodeficiency virus (HIV). This in-
activity may be due to lack of an efficient metabolic conver-
sion into their corresponding 5’-triphosphates and poor affinity
CD4 T-lymphocyte CEM cells and against several other tumor
cell lines, including murine leukemia L1210 and human pros-
tate PC3, kidney CAKI-1, and cervical carcinoma HeLa cells. A
few compounds were inhibitory to Moloney murine sarcoma
virus (MSV) in C3H/3T3 cell cultures, with the 2,6-diaminotri-O-
benzyl-d-allitolyl- and -d-altritolyl pyrimidine analogues being
the most potent among them. This series of unconventional
nucleosides may represent a novel family of potential antiproli-
ferative agents.
Introduction
Nucleosides, the building blocks of nucleic acids, play crucial
roles in many biological processes such as RNA and DNA syn-
thesis, regulation of gene expression, and in immunomodula-
tion. Naturally occurring nucleosides consist mainly of a nucleo-
base (uracil, thymine, cytosine, adenine, or guanine) and a fura-
tance of the discovery of new, safer, and more selective antitu-
mor and/or antiviral agents.
Several nucleoside analogues show some selectivity against
malignancies and viral infections. Cancer cells that continuous-
ly undergo mitosis (highly proliferative cells) are usually more
sensitive to the cytostatic/cytotoxic activity of nucleoside ana-
[1–3]
nose sugar (b-d-ribofuranose or b-d-2-deoxyribofuranose).
[1]
Synthetic nucleoside analogues, designed to mimic the nat-
urally occurring nucleosides, are important chemotherapeutic
entities with antiviral, antitumor, antibacterial, or antifungal ac-
logues than resting cells.
Nucleosides have been extensively modified, in both the nu-
[11,12]
cleobase and sugar portions.
In general, such modifica-
[
4–9]
tivities.
Nucleosides are key leads in drug development and
tions maintain the relative spatial disposition between the nu-
cleobase and the hydroxymethyl group that is phosphorylated
in the corresponding nucleotide (the biologically active form).
Moreover, several of the modifications are directed to increase
resistance against enzymatic degradation. On one hand, some
nucleoside analogues incorporate unconventional nucleobas-
es; examples include ribavirin, which bears a 1,2,4-triazole car-
have been in clinical use for more than 40 years in the treat-
[3,8–10]
ment of patients with cancer or virus-related diseases.
However, they suffer from several drawbacks and unwanted
side effects such as the development of drug resistance, toxici-
[
6–10]
ty, and poor oral bioavailability.
This highlights the impor-
[13]
boxamide as an adenine or guanine mimetic,
or BVDU,
14]
[
[
a] G. Fernꢁndez-Cureses, S. de Castro, Prof. M.-J. Camarasa
Instituto de Quꢂmica Mꢀdica (IQM-CSIC)
Juan de la Cierva 3, 28006 Madrid (Spain)
E-mail: sonia@iqm.csic.es
which incorporates a 5-bromovinyluracil nucleobase. On the
other hand, some nucleoside analogues incorporate unconven-
tional sugar moieties: for example, methylene cyclopropane as
in synadenol 1 and synguanol 2 (Figure 1), which are potent
mj.camarasa@iqm.csic.es
[15]
[b] M.-L. Jimeno
antivirals against human cytomegalovirus (HCMV), or an oxe-
Centro de Quꢂmica Orgꢁnica, Manuel Lora Tamayo (CENQUIOR-CSIC)
Juan de la Cierva 3, 28006 Madrid (Spain)
tane moiety, as in the antibiotic oxetanocin-A (3) or a cyclobutyl
as in cyclobut-A (4), which are active against HSV, HCMV, and
[
c] J. Balzarini
Rega Institute for Medical Research, KU Leuven
Minderbroedersstraat 10, 3000 Leuven (Belgium)
[16,17]
HIV.
Among the various modifications in the sugar region,
replacement of the furanose ring by an acyclic aliphatic chain
has led to an important group of therapeutic agents: the acy-
clic nucleosides. One of the most prominent representatives of
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402465.
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recognized by HIV-1 re-
verse transcriptase (HIV-
RT) as a purine instead
1
of a pyrimidine deriva-
tive, and is incorporated
into the growing DNA
chain as a purine nucleo-
tide analogue instead of
a
pyrimidine nucleo-
[24]
tide.
Interestingly, and
in contrast to the PMEO-
DAPyms, the pyrimidine
derivatives of PME ana-
Figure 2. General structure I
of target nucleosides.
logues
are
generally
[27]
devoid of antiviral activity.
Inspired by the uniqueness of PMEO-DAPyms and
with an aim to discover new families of nucleoside
analogues (of unknown structures), we designed
a novel class of unconventional nucleosides, of gen-
eral structure I (Figure 2). These compounds combine
in their structure unconventional bases such as dia-
minopyrimidines as in PMEO-DAPym or aminotria-
zines, with unconventional sugars as intact tetrahy-
drofuran rings (but distinct from ribose and deoxyri-
bose). With such nucleosides we aimed to explore
their biological potential. Herein we describe the synthesis,
conformational studies, and cell-based antiviral and cytostatic
evaluations of a series of nucleoside analogues of aminopyri-
midine-, aminopurine-, and amino-1,3,5-triazine-bearing unusu-
al sugar moieties that led to the discovery of a novel family of
potential antiproliferative nucleosides.
Figure 1. Examples of antiviral nucleoside analogues.
this class of nucleosides is acyclovir (ACV, 5), a potent and se-
[
18]
lective antiherpetic drug discovered by the 1988 Nobel Lau-
[
19]
reate, Gertrude B. Elion. ACV and its valyl ester prodrug (vala-
cyclovir) are used in the clinic for the treatment of HSV, VZV,
[
18–22]
and CMV infections.
A special group of acyclic nucleosides are the acyclic nucleo-
[
23]
side phosphonates (ANPs) such as adefovir (PMEA, 6) and te-
nofovir (PMPA, 7), designed as metabolically stable nucleotide
analogues bearing a non-biodegradable phosphonate, via an
ether linkage instead of the hydrolyzable phosphate ester
Results and Discussion
[
10,23]
bond.
They are approved for the treatment of HIV and
Chemistry
[
10,23]
chronic HBV infections.
The ANPs mimic a 5’-monophos-
In an effort to discover new nucleoside analogues as potential
antiviral or anticancer hits, we first pursued the tetrahydrofur-
an nucleosides of 2,6-diaminopyrimidines bearing hydroxy
groups at the 3’ and 4’ positions (compound 13), as ribofuna-
nosyl nucleoside mimics. The procedure shown in Scheme 1
was devised for the synthesis of these key nucleosides. Cou-
phate nucleoside and circumvent the first phosphorylation
step required for the activation of nucleosides, which is often
the bottleneck in the activation sequence to obtain the corre-
sponding triphosphates.
More recently, a novel class of acyclic nucleoside phospho-
nates has been described, designated 6-[2-(phosphonomethox-
y)alkoxy]-2,4-diaminopyrimidines, such as PMEO-DAPym (8) or
[30]
pling of key benzyl intermediate 11
with 2,4-diamino-6-
chloropyrimidine would give nucleoside 12, which, after de-
benzylation, may provide the desired unprotected nucleoside
[
10,23b,24]
[23,24]
PMPO-DAPym (9),
to be active against HIV and HBV.
In these nucleoside phosphonates the base is a pyrimidine
ring (a 2,4-diaminopyrimidine, DAPym) linked to the aliphatic
phosphonate group, via an ether bond, at the C6 position of
the pyrimidine base instead of the N1 or N9 positions of the
13.
[
23b,25]
pyrimidine or purine base.
A unique feature of these ANP
compounds is that they mimic a purine ring that retains the
most important part of the purine base for recognition by the
phosphorylating enzymes (mainly kinases). Therefore, the
PMEO compounds have been considered open-ring mimics of
the PME-2,6-diaminopurine (PME-DAP, 10) and guanine deriva-
[
23b,24–29]
tives, being potent anti-HIV compounds.
Moreover,
PMEO-DAPym-diphosphate (PMEO-DAPym-pp) is specifically
Scheme 1. General synthesis of key nucleosides 12 and 13.
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2 4
Scheme 2. Reagents and conditions: a) H SO , THF, 508C, 48 h; b) NaH 60%,
BnBr, THF, RT, ~16 h; c) NaOAc, AcOH, KBr, AcOOH, 108C, 4 h.
First, it was necessary to prepare the key intermediate 11 as
[31]
shown in Scheme 2. Thus, acid-mediated cyclization of 14,
prepared in five steps from commercially available d-ribose di-
Scheme 4. Reagents and conditions: a) NaH 60%, dry THF, 908C, 16 h; b) H
Pd(C) 10%, MeOH, 308C, 280 kPa, ~16 h.
2
,
[
30]
ethyl dithioacetal, as reported, in the presence of a catalytic
[
31]
amount of sulfuric acid at 508C gave 15a,b in 58% yield as
an intractable mixture of 2,3-cis- and 2,3-trans-tetrahydrofuran
drogenated in the presence of 20% Pd(OH) /C and a catalytic
2
[
31]
isomers. Although van Delft et al. reported that under these
conditions the ratio of 2,3-trans versus 2,3-cis isomer was 20:1,
in our hands all attempts resulted in a 2:1 ratio. Treatment of
amount of hydrochloric acid, the deprotected nucleoside 13a
was obtained in 80% yield as the sole product. Unexpectedly,
no traces of the deprotected 13b isomer were detected.
[
31]
1
5a,b with benzyl bromide in the presence of 60% sodium
Next, in view of the novelty of the structures, the synthesis
of other aminopyrimidine nucleosides was also pursued. Thus,
alkylation of 2-amino-4,6-dichloropyrimidine (18) with 11 a,b
(Scheme 4), using the same reaction conditions as described
above (NaH, THF, 908C), gave a 5:1 mixture of compounds
19a,b, which was separated by preparative centrifugal circular
thin-layer chromatography, to give the individual isomers 19a
and 19b in 71 and 15% respective yields. The structures of
19a and 19b were unequivocally assigned on the basis of
their corresponding spectroscopic data, using a combination
[31]
hydride, followed by Fleming–Tamao oxidation (KBr, AcOOH)
of the corresponding fully protected silane 16a,b, gave 11 a,b
63% overall yield from 15a,b) as an unseparable 6:1 mixture
(
of allitol (11 a) and altritol (11 b) as determined by NMR.
Alkylation of 2,4-diamino-6-chloropyrimidine (17) with the
in situ generated sodium alkoxide of 11 a,b (60% NaH, THF) at
908C (Scheme 3) gave a 4:1 mixture of 12a and 12b in 82%
yield, which could not be separated. Next, removal of the
benzyl groups of 12a,b was attempted. Initial attempts of hy-
1
13
drogenolysis (H , 10% Pd/C) of the mixture 12a,b failed to pro-
of one- and two-dimensional H and C NMR (NOESY, g-HMBC,
g-HSQC). The relative stereochemistry of 19a and 19b was de-
termined by nuclear Overhauser enhancement spectroscopy
2
duce the desired unprotected nucleosides, rendering unreact-
ed starting compound. However, if the mixture 12a,b was hy-
(
NOESY) experiments. A correlation between H2’ and H5’ in
compound 19a confirmed the 2’,3’-trans configuration, where-
as no correlation between these protons were observed in the
isomer 19b, thus indicating a 2’,3’-cis configuration for this
compound. Removal of the benzyl groups of 19a by hydroge-
nolysis in the presence of 10% Pd/C gave the fully deprotected
de-halogenated derivative 20a in 50% yield. However, all at-
tempts to remove the benzyl groups of 19b under various
conditions (H , 10% Pd/C, 5% Pd/C or 20% Pd(OH) , in the
2
2
presence or absence of catalytic amounts of HCl) rendered
complex mixtures without formation of the desired derivative
20b. Compounds 13a and 20b were evaluated for inhibition
of the replication of a wide variety of viruses in cell culture,
but were generally found to show poor, if any, inhibitory activi-
ty.
Next we explored the synthesis of the corresponding amino-
pyrimidine dideoxynucleoside analogues. The synthesis of
compounds 26–28 and 31 (Scheme 5) was carried out by fol-
lowing a procedure similar to that described for the synthesis
[32]
of 13 and 20. Thus, treatment of (2RS,5SR)-21
with two
Scheme 3. Reagents and conditions: a) NaH 60%, dry THF, 908C, 16 h; b) H2,
Pd(OH) /C 20%, MeOH, HCl, 308C, 280 kPa, ~16 h.
2
equivalents of the appropriate aminopyrimidine [2,4-diamino-
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a
a
methoxypyrimidine ring by
benzylate anion to give
a benzyl ether was reported by
[
33]
Delacotte et al.
upon heating
[34]
at 958C. Brown and Sugimoto
reported transetherifications in
the presence of silver oxide as
catalyst.
Hydrogenolysis of benzyl de-
rivative 39 in the presence of
catalytic amounts of Pd(OH) /C
2
and HCl at 308C and 280 kPa re-
sulted in a complex reaction
mixture, and no desired depro-
tected nucleoside 41 was detect-
ed. Instead, analogous treatment
of derivative 40 gave unprotect-
ed 42 (10%), although in low
yield. To avoid the hydrogenoly-
sis step, we replaced the benzyl
group by a tert-butyldiphenylsilyl
(TBDPS) protecting group, re-
movable under acidic conditions,
and used 38 as starting com-
2 2
Scheme 5. Reagents and conditions: a) NaH 60%, DMF or THF, pressure tube, RT, ~16 h; b) H /Pd(OH) , 37% HCl,
308C, 280 kPa, ~16 h.
[35]
6
-chloropyrimidine (17), 2-amino-4,6-dichloropyrimidine (18),
-amino-6-chloropyrimidine (22), or 4-amino-2-chloropyrimi-
pound. Thus, treatment of 38 with pyrimidines 36 or 37,
under the standard conditions, afforded the deprotected nu-
cleosides 41 and 42 in 10 and 12% yield, respectively.
Next, we explored the synthesis of aza analogues of the nu-
cleosides mentioned thus far. Disubstituted triazines are much
less reactive, and substitution of the chlorine atom requires
4
dine (29)] in the presence of 60% sodium hydride in a pressure
tube afforded the corresponding (2’RS,5’SR)-benzyl derivatives
23 (55%), 24 (79%), 25 (58%), and 30 (37%) in moderate to
good yields. Hydrogenolysis of 23–25 and 30 [Pd(OH) /C, HCl
2
[36]
at 308C, 280 kPa] yielded the desired (2RS,5SR)-dideoxy deriva-
tives 26 (51%), 27 (74%), 28 (20%), and 31 (28%), respectively.
All compounds were racemic mixtures (2’RS,5’SR) that could
not be separated.
stronger conditions. Thus, alkylation of 2-chloro-4,6-diamino-
1,3,5-triazine (45) with 21 (Scheme 7) was carried out by treat-
ment with 60% sodium hydride under microwave irradiation
at 1008C for 3 h to give 47 in 62% yield. Hydrogenolysis of 47
(H , 20% Pd(OH) /C, HCl) to remove the benzyl group gave an
A similar alkylation of pyrimidines bearing C=O, or OH sub-
stituents (32–35) under various conditions (large excess of pyr-
imidines in the presence of 60% sodium hydride in acetonitrile
or tetrahydrofuran at 70 or 1008C in a pressure tube or under
microwave irradiation at 1008C) failed to produce the desired
nucleosides; only unreacted starting material was recovered.
Interestingly, when 2-amino-4-chloro-6-methoxypyrimidine (36)
or 4-chloro-6-methoxypyrimidine (37) were alkylated with 21
2
2
unseparable 2:1 mixture of the desired compound 48 and
ameline (49). To avoid the hydrogenolysis step and thus to
prevent the formation of ameline, we used TBDPS-protected
compound 38. Treatment of 38 with 45 gave a complex mix-
ture that could not be identified. Finally, the unprotected de-
rivative 46 was used as starting compound. Thus, 46 was treat-
ed with 2-chloro-4,6-diamino-1,3,5-triazine (45) to give the de-
(60% sodium hydride in a pres-
sure tube at 1008C), the desired
nucleosides 39 and 40 were ob-
tained in 54 and 45% yields, re-
spectively. In both reactions the
dimer compounds 43 and 44 (9
and 8% yield, respectively) were
observed
as
byproducts
(Scheme 6). These unexpected
derivatives may result from
transetherification by the alco-
holate anion of 21. Transetherifi-
cations by alcoholate anions
[
33b,c]
have been described before.
2 2
Scheme 6. Reagents and conditions: a) NaH 60%, DMF or THF pressure tube, 1008C, ~16 h; b) H /Pd(OH) , 37%
A similar transetherification of HCl, 308C, 280 kPa, ~16 h.
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[38]
kawa et al.,
which involves
phosphorylation of unprotected
nucleoside 26 with phosphorous
oxychloride in the presence of
1
,8-bis(dimethylamino)naphtha-
TM
lene (Proton Sponge ) as base,
and trimethyl phosphate as sol-
vent. This was followed by in
situ reaction of the phosphorodi-
chlorate intermediate 57 with
pyrophosphate to yield the
cyclic triphosphate 58, which
was finally hydrolyzed to 59 (8%
yield) upon treatment with a 1m
solution of triethylammonium bi-
carbonate (TEAB).
2 2
Scheme 7. Reagents and conditions: a) NaH 60%, dry DMF, 1008C, microwave, 3 h; b) H /Pd(OH) , 37% HCl, 308C,
280 kPa, ~16 h; c) NaH 60%, dry DMF, pressure tube, 1008C, 48 h.
Conformational studies
With the aim to determine whether the conforma-
tional freedom of the furanose ring is restricted in
these nucleosides, we carried out solution-state con-
formational studies of compounds 20a and 27. For
this type of nucleoside, three conformational features
must be considered: furanose ring pucker, the con-
formation of the exocyclic bonds C4’ꢀC5’ and C1’ꢀ
C7, and the conformation around the glycosidic
bond. Puckering of the furanose ring in solution can
be determined by using the concept of pseudorota-
Scheme 8. Reagents and conditions: a) NaH 60%, DMF, pressure tube, 1008C, ~16 h;
b) H /Pd(OH) , 37% HCl, 308C, 280 kPa, ~16 h.
2
2
[39]
tion, in which the conformation of the sugar ring is
protected derivative 48 in moderate yield (42%) together with
the disubstituted 50 (14%) as minor compound.
fully described by two parameters: a phase angle of pseudoro-
tation (P) and a puckering amplitude (t ). In solution, a two-
m
The results obtained in the synthesis of aminopyrimidine nu-
cleosides were taken into consideration to expand the synthet-
ic application to purine methyloxynucleoside analogues. An ef-
ficient method for the synthesis of 6-oxy-substituted purines
uses 1,4-diazabicyclo[2.2.2]octane (DABCO) as the
state NorthQSouth conformational equilibrium is often accept-
ed as model. Therefore, from the experimental three-bond
3
proton–proton coupling constant values ( JH,H), and by using
[40]
the PSEUROT program, information about the geometry and
[
37]
leaving group to achieve the displacements at C6.
-DABCO purines show a higher rate of displacement
6
[
36]
than chlorine or quinuclidine. Thus, treatment of
[
37]
DABCO purines 51 or 52
(Scheme 8) with the
sodium alkoxide of 21, generated in situ upon treat-
ment with 60% sodium hydride at 1008C overnight,
gave the protected derivatives 53 and 54 in moder-
ate yields (30 and 53%, respectively). Subsequently,
hydrogenolysis (20% Pd(OH) /C and HCl) of 53 and
2
54 afforded the target purine derivatives 55 (41%
yield) and 56 (35% yield). All dideoxynucleoside ana-
logues described so far were racemic mixtures
(2’RS,5’SR).
Finally, we were interested in studying the capacity
of an example of this new kind of methyloxynucleo-
side to interact with DNA polymerases (i.e., the DNA
polymerase activity of HIV RT). Therefore, we pre-
pared compound 59 (the triphosphate derivative of
Scheme 9. Reagents and conditions: a) 1,8-Bis(dimethylamino)naphthalene (Proton Spon-
ge ), PO(OCH ) , POCl , 58C, 16 h; b) nBu N, tributylammonium pyrophosphate, DMF,
3 3 3 3
26; Scheme 9). For the synthesis of the target tri-
TM
phosphate we used methodology reported by Yoshi- 08C, 30 min; c) triethylammonium trimethyl phosphate (0.2m ), RT, 15 min.
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population of both North- and South-type conformers can be
obtained. Five parameters are required to describe such a two-
state conformational equilibrium: P and tm of both North and
South conformers, and the mole fraction (X ) of one of them.
n
For compound 27, eight observables are available, and thus
the system is fully defined. However, for compound 20a, only
three observables are available; therefore, two variables must
be constrained in the calculation. The results obtained for both
compounds are listed in Table 1. The conformational parame-
Table 1. Pseudorotational parameter values for 20a and 27 obtained by
[
40]
PSEUROT software.
Parameter
20a
27
[
a]
[a,b]
Exptl
Calcd
6.68
Exptl
Calcd
J
J
J
J
J
J
J
J
(H2’,H3’a)
(H2’,H3’b)
(H3’a,H4’a)
(H3’a,H4’b)
(H3’b,H4’a)
(H3’b,H4’b)
(H4’a,H5’)
(H4’b,H5’)
6.58
–
5.35
–
5.30
6.20
7.00
6.50
6.50
7.10
5.80
6.00
5.18
6.34
6.89
6.51
6.33
6.89
5.97
6.24
8.1
5.17
–
Figure 3. Molecular modeling conformers of compounds 20a and 27 as de-
termined by PSEUROT software.
[
40]
4.32
4.42
43.0
P
N
[
c]
t
N
40.0
133.5
40.0
39.4
158.5
36.9
0.47
0.16
P
S
[
c]
T
S
H3’a), for compound 20a, only a small correlation was ob-
served with H6’a. These data point to a major anti conforma-
tion for the glycosidic bond in both compounds.
X
RMS
n
0.32
0.13
[
d]
[a] Measured in DMSO at 400 MHz; coupling constant (J) values in Hz.
[b] Values obtained from the spectrum simulation. [c] Constrained values.
[d] Root mean square value in Hz.
Biological evaluations
None of the compounds were significantly effective as inhibi-
tors against a variety of DNA and RNA viruses. Given the struc-
tural similarity of several compounds with antiretroviral 2’,3’-di-
deoxynucleoside analogues (NRTI), the compounds were also
evaluated for their inhibitory activity against HIV-1 and HIV-2 in
ters for compound 20a—P =43.0, P =133.5, X =0.34—indi-
cate that the furanose ring adopts an equilibrium between 4E
N
S
n
and E shifted to the South conformer. For compound 27, the
1
+
conformational parameters (P =8.1, P =158.5) indicate an
CD4 T-lymphocytic CEM cell cultures (Table 2). Unfortunately,
N
S
equilibrium in the usual ranges for dideoxynucleosides (P =0–
they were also devoid of anti-HIV activity at sub-toxic concen-
trations.
N
[
41]
3
6 and PS=144–180).
The conformation around the C5’ꢀC6’ bond can be studied
from the experimental coupling constants J and JH5’,H6’b,
Although none of the compounds showed anti-HIV activity
at sub-toxic concentrations, it could not be excluded that the
antiviral inactivity was due to a lack of metabolic conversion
into the corresponding 5’-triphosphates. Therefore, compound
59, the triphosphate derivative of compound 26, was synthe-
sized and evaluated for its inhibitory activity against HIV-1 RT.
PMEO-DAPym-pp was included as a positive control. As shown
in Table 3, 59 affects HIV-1 RT only with the poly(rU:dA) tem-
plate/primer (as with PMEO-DAPym-pp). Because HIV-1 RT is in-
hibited by compound 59 solely in the presence of this tem-
plate/primer (as was previously demonstrated to be the case
for PMEO-DAPym-pp as well), it is tempting to assume that
this compound—like PMEO-DAPym-pp—acts as an adenine
nucleotide mimic. However, it should be recognized that the
IC50 value for 59 was quite high in the presence of poly(rU:dA)
and dATP; therefore, more research is required to further justi-
fy this assumption.
3
3
H5’,H6’a
by considering an equilibrium between three staggered rotam-
3
ers g+, t, and gꢀ. The experimental values J
=4.2 and
H5’,H6’a
3
3
J
=5.0 Hz for compound 20a and
J
=5.2 and
H5’,H6’a
H5’,H6’b
3
JH5’,H6’b =5.2 Hz for compound 27 suggested no preferred ro-
tamers for this conformational feature in both compounds.
The conformation around the C1’ꢀC2’ bond was studied
3
with the same model. For compound 20a, J
=3.2 and
H1’a,H2’
3
JH1’b,H2’ =6.7 Hz indicated that g+ (47%) and t (43%) rotamers
were preferred. Similar conclusions were obtained for com-
3
pound 27. Thus, the coupling constant values J
=3.7 and
H1’a,H2’
3
JH1’b,H2’ =6.7 Hz indicate that g+ (43%) and t (50%) rotamers
are also preferred (Figure 3).
Finally, the conformation of the “glycosidic bond” was deter-
mined from the cross-peaks observed for the signals corre-
sponding to proton H5 of the base in the NOESY experiments.
Thus, whereas for compound 27, H5 showed cross-peaks with
some protons on the b-face of the furanose (H6’a, H6’b, and
It should be mentioned that compounds 12a,b, 19a, 39, 40,
+
43, 44, and 54 proved cytostatic against the CD4 T-lympho-
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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formation by Moloney murine sarcoma virus (MSV), particularly
Table 2. Anti-HIV-1 and -HIV-2 activity and cytostatic properties of test
compounds in human T-lymphocytes (CEM).
compound 12a,b (EC : 13 mm; Table 5).
50
[
a]
[b]
Compd
EC50 [mm]
CC50 [mm]
HIV-1
>10
>100
>10
>250
>100
>250
>50
HIV-2
>10
>100
>10
>250
>100
>250
>50
Conclusions
1
1
1
1
2
2
2
2
2
2
2
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
2a,b
3a
9a
9b
0a
3
4
5
6
7
8
0
1
9
0
1
2
3
4
7
8
0
3
4
5
6
23ꢁ2.1
>250
35ꢁ8.5
>250
>250
>250
110ꢁ4.9
129ꢁ7.1
>250
>250
>250
>250
>250
52ꢁ2.8
50ꢁ2.8
>50
>50
28ꢁ4.2
21ꢁ3.5
>250
>250
>50
191ꢁ16
13ꢁ0.71
>250
We have described the first series of unconventional aminopyr-
imidine, aminopurine, and amino-1,3,5-triazine methyloxynu-
cleosides of hitherto unknown structure. These unconventional
nucleosides were prepared by alkylation of the corresponding
chloro-substituted nucleobases with conveniently protected
and in situ generated sodium alkoxide of d-allitol, d-altritol, or
>50
>50
>250
>250
>250
>250
>250
>10
>10
>50
>50
>10
>250
>250
>250
>250
>250
>10
>10
>50
>50
>10
2,5-bis-hydroxymethyltetrahydrofuran. The allitollyl or altritollyl
derivatives were pursued as unusual ribonucleoside mimics,
whereas the tetrahydrofuran analogues can be considered as
dideoxynucleoside analogues. The tetrahydrofuran ring in the
polyhydroxylated nucleosides adopts an equilibrium between
E and E shifted to the South conformer, while in the corre-
4
1
sponding dideoxy analogues, the equilibrium is in the usual
3
2
ranges for dideoxynucleosides (between E and E).
>10
>10
None of the compounds showed specific antiviral (including
anti-HIV) activity at sub-toxic concentrations in cell culture.
This might be due to lack of efficient metabolic conversion
into the triphosphate derivatives and/or to a much lower affini-
ty for the target polymerases. On the other hand, several com-
pounds proved cytostatic, in particular against leukemia/lym-
phoma tumor and human prostate carcinoma cell lines. Com-
pounds 12a,b, 31, 40, and 54 inhibited murine fibroblast C3H/
>250
>250
>50
>50
>2
>250
>250
>50
>50
>2
>250
>250
>250
>250
>250
>250
33
(
R)-PMPA
1.1ꢁ0.7
2.7ꢁ1.2
1.3ꢁ1.2
1.9ꢁ0.6
PMEO-DAPym
3T3 cell transformation by Moloney murine sarcoma virus
[
a] 50% Effective concentration; data are the mean ꢁSD of two inde-
pendent experiments. [b] 50% Cytostatic concentration; data are the
(MSV), with 12a,b (EC : 13 mm) being superior to the others.
50
mean ꢁSD of 2–3 independent experiments.
In summary, this series of unconventional methyloxynucleo-
sides may be considered a novel family of candidate antiproli-
ferative agents that warrant further investigation for structural
optimization.
Table 3. Inhibitory activity of compound 59 and DAPym-pp against HIV-
1
RT with various template/primers and radiolabeled substrates.
Experimental Section
[
a]
Template/Primer
Substrate
IC50 [mm]
59
DAPym-pp
Synthesis
poly(rC:dG)
poly(rC:dG)
poly(rA:dT)
poly(rI:dC)
poly(rU:dA)
dGTP
dGTP
dTTP
dCTP
dATP
MgCl
MnCl
MgCl
MgCl
MnCl
2
2
2
2
2
>500
>500
>500
>500
352
>100
14ꢁ2
All experiments that involved water-sensitive compounds were car-
ried out under scrupulously dry conditions. Hygroscopic solids
were vacuum pre-dried in the presence P O for 24 h. THF was dis-
>100
2
5
>100
0.75ꢁ0.66
tilled from Na/benzophenone and stored over molecular sieves
4 ꢄ). Anhydrous DMF was purchased from Sigma–Aldrich and
(
[a] 50% Inhibitory concentration; data are the mean ꢁSD of at least 1–4
stored over molecular sieves (4 ꢄ). Microwave reactions were per-
formed in a Biotage Initiator 2.0 single-mode cavity instrument
from Biotage (Uppsala, Sweden). Experiments were carried out in
sealed microwave process vials using standard absorbance level
independent experiments.
(400 W maximum power). The temperature was measured with an
IR sensor on the outside of the reaction vessel. The reactions were
monitored by analytical TLC on silica gel 60 F254 (Merck) pre-coated
plates (0.2 mm). Products were detected under UV light (l 254 nm)
and/or by heating after treatment with a 5% solution of H SO in
cytic CEM tumor cell cultures (50% cytostatic concentration
ranging between 13 and 50 mm). These particular compounds
were also cytostatic against several other tumor cell lines, in-
cluding murine leukemia L1210, human prostate PC3, kidney
CAKI-1, and cervical carcinoma HeLa cells. There was a trend
toward preferential cytostatic activity against leukemia and
lymphoma tumor cell types versus the solid carcinoma tumor
cell types (Table 4). A few compounds (i.e., 12a,b, 31, 40, and
2
4
EtOH. Preparative TLC was performed on pre-coated silica gel 20ꢅ
2
1
0 cm glass plates (Altech 02013, Silica Gel GF with UV 254,
000 mm layer thickness; Sigma–Aldrich). Separations on silica gel
were performed by preparative centrifugal circular thin-layer chro-
matography (CCTLC) on a Chromatotron Harrison Research 8924
model (Kieselgel 60 PF254 gipshaltig, Merck), with a layer thickness
ꢀ1
54) were able to inhibit murine fibroblast C3H/3T3 cell trans-
of 1 or 2 mm and a flow rate of 4 or 8 mLmin , respectively. Flash
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chromatography was performed by HPFC using
a force flow Isolera One (Biotage) with flash silica
gel SNAP cartridges KP-Sil of 50 g (39ꢅ81 mm) or
Table 4. Inhibitory effects of test compounds on the proliferation of murine leukemia
(L1210) cells, human T-lymphocytes (CEM), human cervical carcinoma (HeLa) cells, human
prostate carcinoma (PC3) cells, and human kidney carcinoma (CAKI-1) cells.
1
00 g (39ꢅ157 mm). Reversed-phase chromatogra-
phy was performed by: a) HPFC in an Isolera One
Biotage) using SNAP 12 g KP-C -HS cartridges, or
[
a]
Compd
IC50 [mm]
L1210
CEM
HeLa
PC3
CAKI-1
(
18
b) solid-phase extraction chromatography (SPE)
under vacuum in the manifold using DSC-Si 1 g
(Supelco) cartridges. Samples were lyophilized
using mixtures of H O/CH CN on a Telstar 8-80 in-
1
1
1
1
2
2
2
2
2
2
2
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
2a,b
3a
9a
9b
0a
3
4
5
6
7
8
0
1
9
0
1
2
3
18ꢁ1
23ꢁ2
22ꢁ1
22ꢁ2
22ꢁ1
175ꢁ21
33ꢁ0
>250
>250
>250
137ꢁ53
76ꢁ17
35ꢁ8
>250
>250
>250
110ꢁ5
129ꢁ7
>250
>250
>250
ꢂ250
>250
52ꢁ3
50ꢁ3
>250
>250
28ꢁ4
21ꢁ4
>250
104ꢁ14
>250
>250
>250
179ꢁ7
208ꢁ60
>250
45ꢁ7
>250
>250
>250
>250
>250
132ꢁ1
182ꢁ16
>250
>250
2
3
202ꢁ68
144ꢁ1
120ꢁ34
127ꢁ7
strument.
130ꢁ12
115ꢁ7
136ꢁ9
The purity of the compounds was determined by
analytical RP-HPLC on: a) a Waters 2690 instrument
equipped with a diode array and a C18 Sunfire
column (4.6 mmꢅ50 mm, 3.5 mm), with A:B mix-
>250
>250
>250
>250
ꢂ250
ꢂ250
>250
>250
ꢂ250
>250
>250
ꢂ250
ꢂ250
156ꢁ81
154ꢁ35
tures used as mobile phase, where A=CH CN
3
ꢂ250
(
0.04% TFA) and B=H O (0.05% TFA); b) an Agilent
2
230ꢁ5
202ꢁ16
107ꢁ21
90ꢁ4
Technologies 1120 Compact LC instrument
equipped with a diode array and an ACE 5 C18 300
column (4.6 mmꢅ150 mm, 3.5 mm), using as
70ꢁ4
83ꢁ1
>250
171ꢁ6
67ꢁ17
69ꢁ6
90ꢁ10
>250
ꢂ250
20ꢁ0
17ꢁ0
>250
>250
240ꢁ14
150ꢁ39
40ꢁ13
33ꢁ7
196ꢁ35
25ꢁ1
ꢂ250
65ꢁ16
175ꢁ68
>250
>250
>250
203ꢁ6
220ꢁ42
>250
>250
mobile phase A/B mixtures in which A=CH CN
3
and B=H O (0.05% TFA). In all cases, the flow rate
2
4
7
8
0
3
4
5
20ꢁ3
ꢀ1
was 1 mLmin , and the samples were analyzed at
235ꢁ20
134ꢁ2
210ꢁ27
l 217 and 254 nm in gradient mode. Gradients
used: a) gradient 1: from A:B mixtures 80:20!
>250
>250
>250
>250
>250
>250
140ꢁ6
29ꢁ6
ꢂ250
>250
0.33ꢁ0.17
0.014ꢁ0.000
ꢂ250
1
7
1
00:0 in 5 min; b) gradient 2: A:B mixtures 40:60!
0:30 in 8 min; c) gradient 3: A:B mixtures 10:90!
00:0 in 10 min; d) gradient 4: A:B mixtures 2:98!
191ꢁ16
13ꢁ1
>250
>250
18ꢁ5
189ꢁ2
84ꢁ35
>250
>250
1.6ꢁ1.0
184ꢁ13
139ꢁ8
183ꢁ10
6
ꢂ250
30:70 in 10 min; e) gradient 5: A:B mixtures
50:50!100:0 in 10 min. HPLC–MS was performed
on an HPLC Waters 2695 instrument connected to
a Waters Micromass ZQ 2000 spectrometer, and
a photodiode array detector. The column used was
a Sunfire C18 (4.6 mmꢅ50 mm, 3.5 mm), and the
[
b]
[
5-FU
0.54ꢁ0.12
2.6ꢁ0.4
0.046ꢁ0.016
c]
5-FUd
–
0.014ꢁ0.002
–
[
a] 50% Inhibitory concentration; data are the mean ꢁSD of at least two to three inde-
pendent experiments. [b] 5-Fluorouracil. [c] 5-Fluorouridine.
ꢀ1
flow rate was 1 mLmin . Solvents used were mix-
tures of A: CH CN (0.08% formic acid) and B: H O
3
2
(
0.1% formic acid). The gradient used was from
0% A to 100% A in 10 min. Detection was at
l 214 and 254 nm. Triphosphate 59 was analyzed by HPLC (Waters
00, UV/Vis detection and ion-exchange column [Waters Spherisorb
SAX, 4.6 mmꢅ250 mm, 10.0 mm]). Mobile phase: 0.5m KH PO solu-
1
Table 5. Inhibitory effect of test compounds against MSV-induced trans-
formation of C3H/3T3 embryo murine fibroblasts in vitro.
6
2
4
[
a]
[b]
ꢀ1
Compd
EC50 [mm]
13ꢁ0
MIC [mm]
tion in isocratic mode for 15 min at a flow rate of 1.5 mLmin ,
with detection at l 220 and 254 nm.
1
1
1
2
2
2
2
2
3
3
3
4
4
5
5
5
5
2a,b
9a
9b
3
4
5
7
8
0
1
9
0
3
3
4
5
6
100 (>20)
>100
>100
Optical rotations were measured in a PerkinElmer 241 polarimeter
at 23ꢁ28C. Electrospray mass spectra were measured on a quadru-
pole mass spectrometer equipped with an electrospray source
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
(Hewlett–Packard, LC–MSHP1100). HRMS (EI+) was carried out on
an Agilent 6520 Accurate-Mass Q-TOF LC–MS spectrometer using
MeOH/H O (0.1% AcOH). NMR spectra were recorded with Varian
2
Inova-300, Varian Inova-400 or Varian System-500 spectrometers
1
63ꢁ13
>20
operating at 300, 400, or 500 MHz for H NMR, and at 75, 100, or at
100 (>20)
>100
100 (>20)
13
1
25 MHz for C NMR, and a Mercury-400 operating at 162 MHz for
60ꢁ8
31
P NMR, with phosphoric acid as internal standard. Chemical shift
>20
values (d) are reported in parts per million. Coupling constants (J)
are reported in Hz, and spin multiplicities are indicated by the fol-
lowing symbols: s (singlet), d (doublet), t (triplet), q (quartet), m
>100
>100
ꢂ100
>100
>100
>100
>100
49ꢁ4
>100
>100
1
13
(
multiplet), bs (broad signal). Complex H and C NMR spectra
1
1
(
R)-PMPA
4.2ꢁ2.7
were assigned by homonuclear H– H COSY and NOESY experi-
ments and by heteronuclear H– C HSQC and HMBC experiments.
1
13
PMEO-DAPym
0.48ꢁ0.12
[
a] 50% Effective concentration. [b] Minimal inhibitory concentration.
2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-dimethylphenylsilyl-d-al-
litol and 2,5-anhydro-3,4-di-O-benzyl-1-deoxy-1-dimethylphenyl-
silyl-d-altritol (15a and 15b): To a solution of 3,4-di-O-benzyl-1-
Data are the mean ꢁSD of at least two to three independent experi-
ments.
[31]
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deoxy-1-dimethylphenylsilyl-d-allitol and 3,4-di-O-benzyl-1-deoxy-1-
(1.97 g, 24.05 mmol) in AcOH (9 mL) the phenylsilane mixture
16a,b (1.02 g, 1.85 mmol) was added. KBr (0.26 g, 2.22 mmol) was
added, and the reaction was cooled to 108C, and AcOOH (18 mL,
32% in AcOH) (18 mL) was slowly added, under exclusion of light.
During the addition oxygen was liberated. The reaction was stirred
at 108C for 4 h, then diluted with EtOAc (20 mL) and poured into
a cooled (08C) solution of saturated Na S O (20 mL). The organic
[31]
dimethylphenylsilyl-d-altritol
(960 mg, 1.99 mmol) in dry THF
(
7 mL), concentrated H SO4 (49.5 mL) was added and stirred at
2
5
08C for 48 h, diluted with EtOAc (15 mL), and washed with satu-
rated NaHCO (3ꢅ10 mL). The layers were separated, and the or-
3
ganic phase dried (Na SO ), filtered and evaporated to dryness. The
2
4
residue was purified by HPFC (Biotage; hexane/EtOAc, 7:3). The
fastest-moving fractions gave 15a and 15b (532 mg, 58%) as an
intractable mixture (ratio 2,3-trans (1a) and 2,3-cis (1b), 2:1), as de-
2
2
3
phase was separated and neutralized with NaHCO , until the com-
3
plete stop of gas liberation. The organic phase was washed with
H O (2ꢅ50 mL), dried (Na SO ), filtered, and concentrated. The resi-
1
termined by H NMR, after purification. Analytical and spectroscop-
2
2
4
[31] 1
ic data (NMR) are similar to those described previously.
H NMR
due was purified by HPFC in the Biotage (hexane/EtOAc, 3:2) to
(
400 MHz, CDCl ): d=0.30, 0.33 (s, 6H, CH , SiCH3trans), 0.31, 0.33 (s,
give the mixture 11 a and 11 b (632 mg, 77%) as an oil. Ratio 2,3-
3
3
1
6
1
1
H, CH , SiCH3cis), 1.00 (dd, 1H, J1a,2 =9.7 Hz, J1a,1b =14.4 Hz, H-
trans (11 a) and 2,3-cis (11 b) (6:1); H NMR (400 MHz, CDCl ): d=
3
3
atrans), 1.12 (dd, 1H, J1b,2 =4.9 Hz, J1a,1b =14.4 Hz, H-1btrans), 1.16 (dd,
H, J1b,2 =5.8 Hz, J1a,1b =14.5 Hz, H-1b ), 1.44 (dd, 1H, J =9.2 Hz,
2.70 (bs, 1H, OH), 3.50 (m, 2H, H-1a, H-6a), 3.70 (dd, 1H, J5,6b
3.2 Hz, J6a,6b =10.4 Hz, H-6b), 3.80 (dd, 1H, J1b,2 =2.7 Hz, J1a,1b
=
=
cis
1a,2
J1a,1b =14.5 Hz, H-1a ), 3.42 (dd, 1H, J =2.9 Hz, J6a,6b =11.9 Hz, H-
12.1 Hz, H-1b), 4.03 (dd, 1H, J2,3 =4.0 Hz, J3,4 =5.2 Hz, H-3), 4.11 (dd,
1H, J3,4 =5.2 Hz, J4,5 =6.1 Hz, H-4), 4.16–4.23 (m, 2H, H-2, H-5), 4.43
(d, 1H, J=11.9 Hz, CH Ph), 4.47 (d, 1H, J=11.8 Hz, CH Ph), 4.53 (d,
cis
5,6a
6
3
atrans), 3.45 (m, 1H, H-3trans), 3.47 (m, 1H, H-3 ), 3.63 (dd, 1H, J
.1 Hz, J6a,6b =11.9 Hz, H-6btrans), 3.72 (dd, 1H, J5,6b =2.9 Hz, J6a,6b
=
=
=
cis
5,6b
2
2
11.9 Hz, H-6a ), 3.75 (t, 1H, J =3.9 Hz, H-4 ), 3.85 (t, 1H, J
1H, J=11.9 Hz, CH Ph), (d, 1H, J=11.8 Hz, CH Ph), 4.61 (s, 2H,
2 2
cis
3,4
cis
3,4
13
5
.5 Hz, H-4trans), 3.95–4.09 (m, 4H, H-2trans,cis, H-5 , H-6b ), 4.18 (m,
CH Ph), 7.22–7.37 ppm (m, 15H, Ar); C NMR (100 MHz, CDCl ): d=
2 3
cis
cis
1
H, H-5trans), 4.41–4.80 (d, 8H, J=11.8 Hz, CH Phtrans,cis), 7.25–
62.2 (C1 ), 63.4 (C1trans), 69.7 (C6trans), 70.3 (C6 ), 72.7, 73.2, 73.6
cis cis
2
7
.53 ppm (m, 30H, Artrans,cis); HPLC (Agilent 1120, gradient 3): t =
(CH Ph ), 72.2, 72.4, 73.6 (CH Phtrans), 78.0, 78.1 (C3trans, C4trans), 78.2,
2 cis 2
R
1
0.84 min (95%); HRMS (ES+) m/z: calcd for C H O Si 462.2224,
78.7 (C3 , C4 ), 81.0 (C2 ), 79.7 (C2 ), 80.9 (C5 ), 83.0 (C5trans),
cis cis trans cis cis
28
34
4
found 462.2226.
127.8–128.7 (CH Artrans,cis), 137.6, 137.9, 138.0 (C Artrans), 137.7, 137.9,
38.1 ppm (C Ar ); HPLC (Agilent 1120, gradient 3): t =9.52 min
1
cis
R
The slowest-moving fractions gave 3,4-di-O-benzyl-1,2-dideoxi-d-
(95%); HRMS (ES+) m/z: calcd For C H O 434.2108, found
27 30 5
[
31]
+
ribo-hex-1-enitol
(260 mg, 40%). MS (ES+) m/z: 351 [M] ;
4
34.2093.
1
H NMR (300 MHz, CDCl ): d=3.10 (bs, 2H, OH), 3.61 (t, 1H, J3,4
=
=
3
J4,5 =6.1 Hz, H-4), 3.71–3.78 (m, 3H, H-5, H-6), 4.08 (dd, 1H, J2,3
General procedure for the synthesis of d-allitolyl-, d-altritolyl-,
and tetrahydrofuran-2’-yl pyrimidine/purine derivatives: To
a cooled (08C) solution of 11a,b, 21, 38, or 46 (0.46 mmol) in dry
THF (10 mL), under argon, 60% NaH (36.8 mg, 0.92 mmol) was
added. The mixture was stirred at room temperature for 1 h; the
corresponding chloropyrimidine/purine (0.92 mmol) was then
added, and the reaction was stirred at 90–1008C, either in a pres-
sure tube or in the microwave, for 3–16 h. After cooling to room
temperature, MeOH was added (to remove excess NaH) and
evaporated to dryness. The residue was dissolved in EtOAc
7
.7 Hz,=6.1 Hz, H-3), 4.52 (d, 2H, J=11.8 Hz, CH Ph), 4.62 (d, 2H,
2
J=11.8 Hz, CH Ph), 5.36–5.46 (m, 2H, H-1), 5.91 (ddd, 1H, J
=
1a,2
2
1
6.7 Hz, J1b,2 =11.0 Hz, J2,3 =7.7 Hz, H-2), 7.25–7.40 ppm (m, 10H,
Ar).
2
,5-Anhydro-3,4,6-tri-O-benzyl-1-deoxy-1-dimethylphenylsilyl-d-
allitol and 2,5-anhydro-3,4,6-tri-O-benzyl-1-deoxy-1-dimethyl-
phenylsilyl-d-altritol (16a and 16b): To a mixture of 15a,b (1.05 g,
2
.26 mmol) and BnBr (0.54 mL, 4.52 mmol) in dry THF (15 mL),
stirred at 08C under argon, 60% NaH (181 mg, 4.52 mmol) was
added. The reaction was stirred at room temperature for ~16 h, di-
luted with Et O (15 mL), and then a saturated solution of NH Cl
(10 mL), washed with 1n HCl (3ꢅ5 mL) and the organic phase
dried (Na SO ), filtered and evaporated to dryness and purified.
2
4
2
4
The purification method, eluents, and yields are indicated in each
case.
(
5 mL) was slowly added. The layers were separated, and the or-
ganic phase was washed with saturated NaCl (2ꢅ10 mL), dried
Na SO ), filtered and evaporated to dryness. The residue was puri-
(
2,6-Diamino-4-(2’,5’-anhydro-3’,4’,6’-tri-O-benzyl-d-allitolyl)pyri-
midine and 2,6-diamino-4-(2’,5’-anhydro-3’,4’,6’-tri-O-benzyl-d-
altritolyl)pyrimidine (12a and 12b): Following the general proce-
dure 11a,b (50 mg, 0.12 mmol) was treated with 2,4-diamino-6-
chloropyrimidine (17) (34 mg, 0.24 mmol), for ~16 h to give, after
purification by CCTLC on the chromatotron (CH Cl /MeOH, 9:1),
2
4
fied by HPFC in the Biotage (hexane/EtOAc, 9:1) to give a mixture
of 16a and 16b; yield 1.02 g (82%) as an oil. Ratio 2,3-trans/2,3-cis,
1
1
4
:1 (by H NMR and HPLC); H NMR (500 MHz, CDCl ): d=0.31, 0.32
3
(
s, 6H, CH , SiCH
), 0.32, 0.33 (s, 6H, CH , SiCH3cis), 1.04 (dd, 1H,
3
3trans 3
J1a,2 =9.0 Hz, J1a,1b =14.5 Hz, H-1atrans), 1.14–1.30 (m, 2H, H-1btrans,cis),
.46 (dd,1H, J1a,2 =8.0 Hz, J1a,1b =14.5 Hz, H-1a ), 3.40–3.51 (m, 4H,
2
2
1
1
a 4:1 mixture of 12a and 12b (54 mg, 82%) as an oil. H NMR
(500 MHz, CDCl ): d=3.52 (dd, 1H, J =4.4 Hz, J6’a,6’b =10.6 Hz, H-
cis
H-3trans, H-6a,btrans, H-6a ), 3.56 (m, 1H, H-6b ), 3.76 (t, 1H, J
=
cis
cis
2,3
3
5’,6’a
J3,4 =4.1 Hz, H-4 ), 3.88 (dd, 1H, J =5.2 Hz, J3,4 =4.5 Hz, H-4trans),
6’atrans), 3.58 (dd, 1H, J5’,6’b =4.0 Hz, J6’a,6’b =10.5 Hz, H-6’btrans), 3.93
(m, 2H, H-3’trans, H-4’trans), 4.06 (m, 1H, H-4’ ), 4.16 (m, 1H, H-3’ ),
cis
2,3
4
.04–4.20 (m, 4H, H-2trans,cis
,
H-5trans,cis), 4.30–4.80 (m, 12H,
cis
cis
13
CH Phtrans,cis), 7.24–7.58 ppm (m, 40H, Artrans,cis); C NMR (125 MHz,
4.26 (m, 2H, H-1’atrans, H-5’trans), 4.33 (m, 2H, H-1’btrans, H-2’trans),
4.48–4.65 (m, 6H, CH Ph ), 5.12 (s, 1H, H-5trans), 5.25 (s, 1H, H-
2
CDCl ): d=ꢀ2.3, ꢀ1.5 (CH , SiCH
), ꢀ2.2, ꢀ1.8 (CH , SiCH3cis),
3
3
3trans
3
2
trans,cis
13
1
7.1 (C1 ), 21.6 (C1 ), 70.5 (C6 ), 70.9 (C6 ), 71.9, 72.1, 73.5
5cis), 7.27–7.37 ppm (m, 15H, Artrans,cis); C NMR (125 MHz, CDCl3):
cis
trans
cis
trans
(
CH Ph ), 72.6, 73.0, 73.5 (CH Ph ), 77.6 (C4 ), 78.8 (C4 , C3 ),
d=64.1 (C1’ ), 65.6 (C1’ ), 70.0 (C6’ ), 70.3 (C6’trans), 71.9, 72.0,
2
trans
2
cis
trans
cis
cis
cis
trans
cis
7
1
1
9.0 (C2trans), 79.4 (C2 ), 80.5 (C5 ), 81.3 (C5trans), 83.6 (C3trans),
27.5–133.8 (CH Artrans,cis), 138.1, 138.3, 138.4, 139.6 (C Artrans), 138.2,
73.5 (CH Ph ), 72.6, 73.4 (CH Ph ), 77.5 (C4’trans), 77.7 (C3’trans),
2 trans 2 cis
cis
cis
78.2 (C4’ ), 78.5 (C3’ ), 78.8 (C5 ), 79.2 (C2’ ), 79.8 (C2’trans), 80.1
cis cis trans cis
38.5, 138.8, 139.5 ppm (C Ar ); HPLC (Waters 2690, gradient 1):
(C5’ ), 81.4 (C5’trans), 127.5–128.5 (CH Artrans,cis), 137.8–138.4 (C
cis
cis
tR =4.58 min (21%), 7.74 (78%); HRMS (ES+) m/z: calcd for
Artrans,cis), 162.6 (C2trans), 162.7 (C2 ), 165.3 (C6trans), 165.4 (C6cis),
cis
C H O Si 552.2688, found 552.2696.
171.0 (C4rans), 171.1 ppm (C4 ); HPLC (Waters 2690, gradient 2):
3
5
40
4
cis
tR =3.51 min (79%) and 3.93 min (19%); HRMS (ES+) m/z: calcd for
2
,5-Anhydro-3,4,6-tri-O-benzyl-d-allitol and 2,5-anhydro-3,4,6-
C H N O 542.2527, found 542.2529.
31
34
4
5
tri-O-benzyl-d-altritol (11 a and 11 b): To a solution of NaOAc
ꢃ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2
-Amino-4-(2’,5’-anhydro-3’,4’,6’-tri-O-benzyl-d-allitolyl)-6-chloro-
CH Oar), 4.25 (dd, 1H, J
=6.5 Hz, JCH2a,CH2b =13.4 Hz, CH Oar),
2a
2’,CH2b
2b
pyrimidine and 2-Amino-4-(2’,5’-anhydro-3’,4’,6’-tri-O-benzyl-d-
altritolyl)-6-chloropyrimidine (19a and 19b): Following the gener-
al procedure 11a,b (200 mg, 0.46 mmol) was treated with 2-amino-
4.48 (m, 2H, CH Ph), 6.07 (s, 1H, H-5), 7.08 (bs, 2H, NH ), 7.27–
2 2
13
7.37 ppm (m, 5H, Ar); C NMR (100 MHz, [D ]DMSO): d=27.3, 27.5
6
(C3’, C4’), 68.5 (CH Oar), 72.2 (CH Ph), 72.5 (CH Obn), 76.5 (C2’),
2
2
2
4
,6-dichloropyrimidine (18) (133 mg, 0.92 mmol) in a pressure tube
for 6 h. Workup and purification by CCTLC in the chromatotron
hexane/Et O, 1:1), gave from the fastest-moving band compound
78.3 (C5’), 94.3 (C5), 127.3, 127.4, 128.3 (CH Ar), 138.5 (C Ar), 160.0
(C2), 162.8 (C6), 170.5 ppm (C4); HPLC (Agilent 1120, gradient 3):
t =8.43 min (98%); HRMS (ES+) m/z: calcd for C H ClN O
(
1
2
R
17
2
3
3
1
9b (40 mg, 15%) as an oil. H NMR (500 MHz, CDCl ): d=3.51 (dd,
349.1211, found 349.1193.
3
1
H, J5’,6’a =3.7 Hz, J6’a,6’b =10.6 Hz, H-6’a), 3.60 (dd, 1H, J5’,6’b =3.6 Hz,
6
-Amino-4-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydrofuran-2’-
J6’a,6’b =10.6 Hz, H-6’b), 4.07 (dd, 1H, J2’,3’ =5.6 Hz, J3’,4’ =4.7 Hz, H-4’),
yl]methoxy}pyrimidine (25): The general procedure was followed
with 21 (85 mg, 0.38 mmol) and 4-amino-6-chloropyrimidine (22)
4
5
.17 (dd, 1H, J3’,4’ =4.7 Hz, J4’,5’ =5.6 Hz, H-3’), 4.26 (dt, 1H, J4’,5’
.6 Hz, J5’,6’a =J5’,6’b =3.6 Hz, H-5’), 4.40 (dt, 1H, J2’,3’ =5.2 Hz, J1’,2’a
=
=
[32]
(59 mg, 0.46 mmol). The crude was purified by CCTLC in the chro-
J1’,2’b =6.7 Hz, H-2’), 4.55 (m, 2H, H-1’), 4.48 (d, 1H, J=11.9 Hz,
CH Ph), 4.50–4.57 (m, 4H, H-1’, CH Ph), 4.57 (d, 1H, J=11.7 Hz,
CH Ph), 4.63 (d, 1H, J=11.9 Hz, CH Ph), 4.74 (d, 1H, J=11.8 Hz,
matotron (EtOAc/MeOH, 9:1) to give 25 (69 mg, 58%) as an oil.
2
2
1
H NMR (400 MHz, CDCl ): d=1.78–2.00 (m, 4H, H-3’, H-4’), 3.49
3
2
2
(dd, 2H, J5’,CH2a =5.3 Hz, JCH2a,CH2b =10.1 Hz, CH Obn), 3.52 (dd, 2H,
2a
CH Ph), 5.13 (bs, 2H, NH ), 6.11 (s, 1H, H-5), 7.27–7.37 ppm (m,
2
2
1
3
J
5’,CH2b =4.9 Hz, JCH2a,CH2b =10.1 Hz, CH2bObn), 4.14–4.31 (m, 3H, H-2’,
1
7
5H, Ar); C NMR (125 MHz, CDCl ): d=65.9 (C1’), 70.1 (C6’), 72.7,
3.4, 73.6 (CH Ph), 77.6 (C3’), 77.7 (C2’), 79.1 (C4’), 80.5 (C5’), 97.4
3
H-5’, CH Oar), 4.38 (dd, 1H, J =3.2 Hz, JCH2a,CH2b =10.2 Hz,
CH Oar), 4.54 (d, 1H, J=12.2 Hz, CH Ph), 4.58 (d, 1H, J=12.2 Hz,
CH Ph),4.83 (bs, 2H, NH ), 5.77 (s, 1H, H-5), 7.27–7.37 (m, 5H, Ar),
2b
2’,CH2a
2
2a
2
(
C5), 127.5–128.5 (CH Ar), 137.9, 138.1, 138.2 (C Ar), 160.8 (C2),
2
2
13
1
1
5
62.2 (C6), 171.1 ppm (C4); HPLC (Agilent 1120, gradient 3): t =
0.63 min (98%); HRMS (ES+) m/z: calcd for C H ClN O
5
61.2051, found 561.2030.
R
8
.23 ppm (s, 1H, H-2); C NMR (100 MHz, CDCl ): d=27.9, 28.0
3
31
32
3
(C3’, C4’), 68.7 (CH Oar), 72.9 (CH Obn), 73.5 (CH Ph), 77.7 (C2’),
2 2 2
7
9.1 (C5’), 87.7 (C5), 127.7, 127.8, 128.4 (CH Ar), 138.4 (C Ar), 158.0
From the slowest-moving band, compound 19a (184 mg, 71%)
(C2), 164.3 (C6), 170.0 ppm (C4); HPLC (Waters 2690, gradient 3):
1
was isolated as an oil. H NMR (400 MHz, CDCl ): d=3.49 (dd, 1H,
tR =3.17 min (99%); HRMS (ES+) m/z: calcd for C H N O
3
3
17 21
3
J5’,6’a =4.2 Hz, J6’a,6’b =10.5 Hz, H-6’a), 3.54 (dd, 1H, J5’,6’b =4.1 Hz,
J6’a,6’b =10.5 Hz, H-6’b), 3.88 (m, 1H, H-3’), 3.95 (m, 1H, H-4’), 4.25
315.1571, found 315.1583.
4
-Amino-2-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydrofuran-2’-
(
m, 1H, H-5’), 4.27 (m, 1H, H-1’a), 4.33 (m, 1H, H-2’), 4.40 (dd, 1H,
yl]methoxy}pyrimidine (30): Following the general procedure
J1 =3.3 Hz, J1’a,1’b =11.2 Hz, H-1’b), 4.48 (d, 1H, J=11.9 Hz,
’b,2’
[32]
2
1
(111 mg, 0.50 mmol) was treated with 4-amino-2-chloropyri-
CH Ph), 4.50 (d, 1H, J=12.0 Hz, CH Ph), 4.54 (d, 1H, J=12.3 Hz,
2
2
midine (29) (78 mg, 0.60 mmol). The crude was purified by re-
versed-phase chromatography using HPFC in the Biotage (H O/
CH Ph), 4.55 (d, 1H, J=12.3 Hz, CH Ph), 4.58 (d, 1H, J=12.0 Hz,
2
2
2
CH Ph), 4.60 (d, 1H, J=11.9 Hz, CH Ph), 5.05 (Bs, 2H, NH ), 5.95 (s,
2
2
2
1
13
CH
3
CN, 1:1) to give 30 (59 mg, 37%) as an oil. H NMR (400 MHz,
1
H, H-5), 7.27–7.37 ppm (m, 15H, Ar); C NMR (100 MHz, CDCl3):
CDCl ): d=1.76–2.02 (m, 4H, H-3’, H-4’), 3.49 (dd, 1H, J
=
5’,CH2a
3
d=66.3 (C1’), 70.3 (C6’), 72.0, 72.2, 73.6 (CH Ph), 77.2 (C4’), 77.5
2
5
.8 Hz, JCH2a,CH2b =10.0 Hz, CH Obn), 3.52 (dd, 1H, J
=5.9 Hz,
2a
5’,CH2b
(
1
(
C3’), 79.2 (C2’), 81.7 (C5’), 97.4 (C5), 127.6–128.6 (CH Ar), 137.7,
37.8, 138.2 (C Ar), 160.8 (C2), 162.1 (C6), 170.9 ppm (C4); HPLC
Agilent 1120, gradient 3): t =10.47 min (98%); HRMS (ES+) m/z:
JCH2a,CH2b =10.0 Hz, CH Obn), 4.16 (m, 1H, H-5’), 4.28 (m, 3H, H-2’,
2b
CH Oar), 4.53 (d, 1H, J=12.1 Hz, CH Ph), 4.58 (d, 1H, J=12.1 Hz,
2
2
R
CH Ph), 5.04 (bs, 2H, NH ), 6.06 (d, 1H, J =5.7 Hz, H-5), 7.27–7.37
2
2
5,6
calcd for C H ClN O 561.2051, found 561.2030.
3
1
32
3
5
13
(m, 5H, Ar), 7.99 ppm (d, 1H, J5,6 =5.7 Hz, H-6); C NMR (100 MHz,
2
2
2
,6-Diamino-4-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydrofuran-
CDCl ): d=28.1, 28.3 (C3’, C4’), 69.1 (CH Oar), 73.1 (CH Obn), 73.5
3
2
2
’-yl]methoxy}pyrimidine (23): Following the general procedure
(CH Ph), 77.5 (C2’), 78.9 (C5’), 99.6 (C5), 127.7, 127.8, 128.5 (CH Ar),
2
[
32]
1
(850 mg, 3.82 mmol) was treated with 2,4-diamino-6-chloro-
138.5 (C Ar), 157.3 (C6), 164.8 (C4), 165.1 ppm (C2); HPLC (Agilent
pyrimidine (17) (828 mg, 5.73 mmol) in a pressure tube for ~16 h.
After workup, the residue was purified by reversed-phase chroma-
tography using HPFC in the Biotage (H O/CH CN, 1:1) to give 23
1120, gradient 3): t =5.74 min (99%); HRMS (ES+) m/z: calcd for
R
C H N O 315.1571, found 315.1583.
17
21
3
3
2
3
1
2-Amino-6-methoxy-4-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahy-
(
693 mg, 55%) as an oil. H NMR (300 MHz, CDCl ): d=1.75–1.99
3
drofuran-2’-yl]methoxy}pyrimidine (39): The general procedure
(
m, 4H, H-3’, H-4’), 3.50 (m, 2H, CH Oar, CH Obn), 4.20 (m, 4H, H-
2a
2a
[32]
was followed with 21
(200 mg, 0.90 mmol) and 2-amino-4-
2
4
’, H-5’, CH Obn, CH Oar), 4.56 (m, 2H, CH Ph), 4.68 (bs, 2H, NH ),
2b 2b 2 2
chloro-6-methoxypyrimidine (36) (176 mg, 1.10 mmol) in dry DMF
(10 mL). The reaction was stirred at a 1008C in a pressure tube for
.86 (bs, 2H, NH ), 5.22 (s, 1H, H-5), 7.27–7.37 ppm (m, 5H, Ar);
2
1
3
C NMR (75 MHz, CDCl ): d=27.9 (C3’, C4’), 68.2 (CH Obn), 73.0
3
2
~
16 h. After the workup the crude was purified by reversed-phase
(
CH Oar), 73.3 (CH Ph), 77.7 (C5), 78.4 (C2’), 78.8 (C5’), 127.5, 127.7,
2
2
chromatography using HPFC in the Biotage (H O/CH CN, 3:2).
2
3
1
28.4 (CH Ar), 138.5 (C Ar), 162.2 (C6), 164.9 (C2), 171.3 ppm (C4);
From the fastest-moving bands 39 (168 mg, 54%) was isolated as
HPLC (Waters 2690, gradient 2): t =3.51 min (79%); HRMS (ES+)
R
1
an oil. H NMR (400 MHz, CDCl ): d=1.80–2.02 (m, 4H, H-3’, H-4’),
3
m/z: calcd for C H N O 330.1698, found 330.1692.
1
7
22
4
3
3
.50 (dd, 1H, J5’,CH2a =5.4 Hz, JCH2a,CH2b =10.1 Hz, CH Obn), 3.53 (dd,
2a
2
-Amino-6-chloro-4-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydro-
1H, J5’,CH2bObn =4.3 Hz, JCH2a,CH2b =10.1 Hz, CH Obn), 3.91 (s, 3H,
2b
furan-2’-yl]methoxy}pyrimidine (24): According to the general
CH ), 4.15–4.35 (m, 4H, CH Oar, H-2’, H-5’), 4.53 (d, 1H, J=12.1 Hz,
3
2
[32]
procedure 21 (259 mg, 1.16 mmol) was treated with 2-amino-
,6-dichloropyrimidine (18) (228 mg, 1.39 mmol) in dry DMF (5 mL)
CH Ph), 4.56 (d, 1H, J=12.1 Hz, CH Ph), 5.50 (s, 1H, H-5), 7.27–
2
2
13
4
7.37 ppm (m, 5H, Ar); C NMR (100 MHz, CDCl ): d=27.8, 27.9 (C3’,
3
in a pressure tube for ~16 h. Workup and purification by reversed-
C4’), 56.2 (CH ), 70.9 (CH Oar), 72.7 (CH Obn), 73.4 (CH Ph), 77.0
3
2
2
2
phase chromatography using HPFC in the Biotage (H O/CH CN,
(C2’), 78.3 (C5), 79.3 (C5’), 127.3, 127.9, 128.4 (CH Ar), 138.3 (C Ar),
157.8 (C2), 169.2 (C6), 171.1 ppm (C4); HPLC (Agilent 1120, gradi-
ent 3): t =6.92 min (97%); HRMS (ES+) m/z: calcd for C H N O
2
3
1
1
:1) gave 24 (321 mg, 79%) as an oil. H NMR (400 MHz,
[
D ]DMSO): d=1.64–1.94 (m, 4H, H-3’, H-4’), 3.39 (dd, 1H, J
6
=
5’,CH2a
R
18 23
3
4
5
.8 Hz, JCH2a,CH2b =10.2 Hz, CH Obn), 3.43 (dd, 1H, J
=4.6 Hz,
345.1704, found 345.1689.
2a
5’,CH2b
JCH2a,CH2b =10.2 Hz, CH Obn), 4.04 (m, 1H, H-5’), 4.15 (m, 2H, H-2’,
2b
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The slowest-moving bands gave 2-amino-4,6-bis{[(2’RS,5’SR)-5’-
6-Methoxy-4-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-2’-
yl]methoxy}pyrimidine (42): The general procedure was followed
with 38 (120 mg, 0.32 mmol) and 4-cloro-6-methoxypyrimidine
(
benzyloxymethyl)tetrahydrofuran-2’-yl]methoxy}pyrimidine (43)
1
[35]
(
42 mg, 9%) as an oil. [a] =0.98 (c=0.25, CHCl3); H NMR
D
(
400 MHz, CDCl ): d=1.80–2.06 (m, 8H, H-3’, H-4’) 3.48 (dd, 2H,
(37) (56 mg, 0.38 mmol). The reaction was heated at 1008C in dry
DMF (5 mL) in a pressure tube for ~16 h. The crude was purified
3
J5’,CH2a =4.8 Hz, JCH2a,CH2b =10.0 Hz, CH Obn), 3.51 (dd, 2H, J
=
2a
5’,CH2b
5
.4 Hz, JCH2a,CH2b =10.0 Hz, CH Obn), 4.14–4.36 (m, 8H, CH Oar, H-2’,
by CCTLC in the chromatotron (EtOAc/MeOH, 10:1) 42 (18 mg,
2b
2
1
H-5’), 4.55 (m, 4H, CH Ph), 5.52 (s, 1H, H-5), 7.27–7.37 ppm (m,
12%) as an oil. H NMR (400 MHz, CDCl ): d=1.79–2.07 (m, 4H, H-
2
3
1
3
1
0H, Ar); C NMR (100 MHz, CDCl ): d=27.9, 28.1 (C3’, C4’), 71.3
3’, H-4’), 2.80 (bs, 1H, OH), 3.50 (dd, 1H, J2’,CH2a =4.4 Hz, JCH2a,CH2b
=
3
(
CH Oar), 72.8 (CH Obn), 73.5 (CH Ph), 77.2 (C2’), 78.9 (C5), 79.5
11.8 Hz, CH OH), 3.74 (dd, 1H, J =3.0 Hz, JCH2a,CH2b =11.8 Hz,
2 2 2
2a
2’,CH2b
(
1
(
C5’), 127.7, 127.8, 128.4 (CH Ar), 138.4 (C Ar), 157.5 (C2’),
69.7 ppm (C4’, C6’); HPLC (Agilent 1120, gradient 3): t =9.06 min
CH OH), 3.93 (s, 3H, CH ), 4.12 (m, 1H, H-2’), 4.33 (m, 2H, CH Oar,
2a 3 2a
H-5’), 4.45 (m, 1H, CH Oar), 6.09 (s, 1H, H-5), 8.40 ppm (s, 1H, H-
2b
13
R
99%); HRMS (ES+) m/z: calcd for C H N O 535.2683, found
2); C NMR (100 MHz, CDCl ): d=26.7, 28.3 (C3’, C4’), 54.3 (OCH ),
3 3
30
37
3
6
5
35.2682.
64.6 (CH OH), 69.0 (CH Oar), 77.9 (C5’), 80.8 (C5), 91.0 (C2’), 157.4
2
2
(
C2), 170.8 (C6), 171.5 ppm (C4); HPLC (Agilent 1120, gradient 3):
tR =4.83 min (97%); HRMS (ES+) m/z: calcd for C H N O
4
40.1101, found 240.1110.
6
2
-Methoxy-4-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydrofuran-
’-yl]mehtoxy}pyrimidine (40): The general procedure was fol-
11
16
2
2
[32]
lowed with 21 (200 mg, 0.90 mmol) and 4-chloro-6-methoxypyri-
midine (37) (159 mg, 1.10 mmol). The reaction was stirred in dry
DMF (10 mL) at a 1008C in a pressure tube for ~16 h. After
workup, the crude was purified by reversed-phase chromatography
using HPFC in the Biotage (H O/CH CN, 1:2). From the fastest-
4,6-Diamino-2-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydrofuran-
2’-yl]methoxy}-1,3,5-triazine (47): The general procedure was fol-
[32]
lowed with 21 (105 mg, 0.47 mmol) in dry DMF (6 mL) and 2-
chloro-4,6-diamino-1,3,5-triazine (45) (96 mg, 0.66 mmol). The reac-
tion was heated at 1008C 3 h in the microwave, additional portions
of 60% NaH (19 mg, 0.47 mmol) and triazine (48 mg, 0.33 mmol)
were added each hour. After cooling to room temperature, MeOH
was added (to remove excess NaH) and evaporated to dryness.
The crude was purified by reversed-phase chromatography using
HPFC in the Biotage (H O/CH CN, 9:1) to give 47 (98 mg, 62%) as
2
3
moving fractions 40 (133 mg, 45%) was isolated as an oil. [a] =
D
1
0
4
.18 (c=0.5, CHCl3); H NMR (400 MHz, CDCl ): d=1.78–2.03 (m,
3
H, H-3’, H-4’), 3.48 (dd, 1H, J5’,CH2a =4.9 Hz, JCH2a,CH2b =10.0 Hz,
CH Obn), 3.52 (dd, 1H, J5’,CH2b =5.3 Hz, JCH2a,CH2b =10.0 Hz,
CH Obn), 3.93 (s, 3H, CH ), 4.18 (m, 1H, H-5’), 4.25–4.44 (m, 3H,
CH Oar, H-2’), 6.07 (s, 1H, H-5), 7.27–7.37 (m, 5H, Ar), 8.41 ppm (s,
1
2
a
2
b
3
2
2
3
1
3
1
H, H-2); C NMR (100 MHz, CDCl ): d=27.9, 28.1 (C3’, C4’), 54.2
an amorphous white solid. [a] =0.18 (c=0.5, MeOH); H NMR
3
D
(
CH ), 69.1 (CH Oar), 72.8 (CH Obn), 73.5 (CH Ph), 77.6 (C2’), 79.1
(400 MHz, [D ]DMSO): d=1.65–1.94 (m, 4H, H-3’, H-4’), 3.40 (dd,
3
2
2
2
6
(
C5’), 90.8 (C5), 127.7, 127.8, 128.4 (CH Ar), 138.4 (C Ar), 157.5 (C2),
1H, J5’,CH2a =5.8 Hz, JCH2a,CH2b =10.2 Hz, CH Obn), 3.44 (dd, 1H,
2a
1
70.9 (C4), 171.3 ppm (C6); HPLC (Agilent 1120, gradient 3): t =
J5’,CH2b =4.6 Hz, JCH2a,CH2b =10.2 Hz, CH Obn), 4.05 (m, 1H, H-5’), 4.16
R
2b
8
.56 min (97%); HRMS (ES+) m/z: calcd for C H N O 330.1592,
(m, 1H, H-2’), 4.20 (dd, 1H, J2’,CH2a =6.1 Hz, JCH2a,CH2b =10.8 Hz,
18
22
2
4
found 330.1580.
CH Oar), 4.31 (dd, 1H, J
=3.7 Hz, JCH2a,CH2b =10.8 Hz, CH Oar),
2’,CH2b 2b
2a
4
2
.48 (m, 2H, CH Ph), 7.23–7.37 (m, 5H, Ar), 7.83 ppm (bs, 4H,
2
The slowest-moving fractions gave 4,6-bis{[(2’RS,5’SR)-5’-(ben-
zyloxymethyl)tetrahydrofuran-2’-yl]methoxy}pyrimidine (44)
13
NH2); C NMR (100 MHz, [D ]DMSO): d=27.2, 27.5 (C3’, C4’), 69.6
6
(CH Oar), 72.2 (CH Ph), 72.5 (CH Obn), 76.3 (C2’), 78.4 (C5’), 127.3,
1
2 2 2
(
38 mg, 8%) as an oil. [a] =0.88 (c=0.25, CHCl3); H NMR
D
1
27.4, 128.2 (CH Ar), 138.5 (C Ar), 163.0 (C4, C6), 166.4 ppm (C2);
(
400 MHz, CDCl ): d=1.77–2.03 (m, 8H, H-3’, H-4’), 3.49 (dd, 2H,
3
HPLC (Agilent 1120, gradient 3): t =5.71 min (93%); HPLC (Agilent
R
J5’,CH2a =4.9 Hz, JCH2a,CH2b =10.0 Hz, CH Obn), 3.51 (dd, 2H, J
=
2a
5’,CH2b
1120, gradient 3): t =5.72 min (99%); HRMS (ES+) m/z: calcd for
R
5
4
.3 Hz, JCH2a,CH2b =10.0 Hz, CH Obn), 4.18 (m, 2H, 2H-5’), 4.29 (m,
2b
C H N O 331.1636, found 331.1644.
16
21
5
3
H, CH Oar, H-2’), 4.38 (m, 2H, CH Oar), 4.53 (d, 1H, J=12.2 Hz,
2
a
2b
CH Ph), 4.56 (d, 1H, J=12.2 Hz, CH Ph), 6.10 (s, 1H, H-5), 7.27–7.37
2-Amino-6-{[(2’RS,5’SR)-5’-(benzyloxymethyl)tetrahydrofuran-2’-
yl]methoxy}-7H-purine (53): The general procedure was followed
2
2
13
(
m, 10H, Ar), 8.37 ppm (s, 1H, H-2); C NMR (100 MHz, CDCl ): d=
3
[32]
[37]
2
7
7.9, 28.1 (C3’, C4’), 69.1 (CH Oar), 72.8 (CH Obn), 73.5 (CH Ph),
7.6 (C2’), 79.1 (C5’), 91.3 (C5), 127.7, 127.8, 128.4 (CH Ar), 138.4 (C
with 21 (50 mg, 0.22 mmol) in dry DMF (5 mL), and 51 (74 mg,
0.26 mmol). The reaction was heated at 1008C in a pressure tube
for ~16 h. After the workup, the crude was purified by HPFC in the
Biotage (CH Cl /MeOH, 9:1) to give 53 (23 mg, 30%) as an amor-
2
2
2
Ar), 157.3 (C2), 170.9 ppm (C4, C6); HPLC (Agilent 1120, gradient 3):
t =10.39 min (97%); HRMS (ES+) m/z: calcd for C H N O
R
30 36
2
6
2
2
1
5
20.2597, found 520.2573.
phous solid. H NMR (400 MHz, [D ]DMSO): d=1.70–2.00 (m, 4H, H-
6
3
3
1
1
’, H-4’), 3.42 (dd, 1H, J5’,CH2a =5.6 Hz, JCH2a,CH2b =10.1 Hz, CH Obn),
2a
2
-Amino-6-methoxy-4-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahy-
.46 (dd, 1H, J5’,CH2b =4.5 Hz, JCH2a,CH2b =10.1 Hz, CH Obn), 4.07 (m,
2b
drofuran-2’-yl]methoxy}pyrimidine (41): The general procedure
H, H-5’), 4.25 (m, 1H, H-2’), 4.32 (dd, 1H, J2’,CH2a =6.4 Hz, JCH2a,CH2b
=
[35]
was followed with 38
chloro-6-methoxypyrimidine (36) (61 mg, 0.38 mmol) in dry DMF
5 mL). The reaction was heated at 1008C in a pressure tube for
16 h. The crude was purified by CCTLC in the chromatotron
(120 mg, 0.32 mmol) and 2-amino-4-
0.9 Hz, CH Oar), 4.41 (dd, 1H, J =3.9 Hz, JCH2a,CH2b =10.9 Hz,
2’,CH2b
2a
CH Oar), 4.48 (m, 2H, CH Ph), 6.25 (s, 2H, NH ), 7.27–7.37 (m, 5H,
Ar), 7.82 (s, 1H, H-8), 12.42 ppm (s, 1H, NH); C NMR (100 MHz,
2b
2
2
(
13
~
[D ]DMSO): d=27.5, 27.9 (C3’, C4’), 67.9 (CH Oar), 72.2 (CH Ph),
1
6
2
2
(
EtOAc/MeOH, 10:1) to give 41 (16 mg, 10%) as an oil. H NMR
7
2.6 (CH Obn), 76.8 (C2’), 78.3 (C5’), 112.4 (C5), 127.3, 127.4, 128.2
2
(
400 MHz, [D ]DMSO): d=1.62–1.94 (m, 4H, H-3’, H-4’), 3.35 (m, 2H,
6
(CH Ar), 138.3 (C Ar), 138.5 (C8), 155.8(C4), 159.6 (C6), 159.7 ppm (C
CH OH), 3.75 (s, 3H, CH ), 3.86 (m, 1H, H-2’), 4.09 (m, 2H, CH Oar,
H-5’), 4.16 (m, 1H, CH Oar), 4.65 (bs, 1H, OH), 5.33 (s, 1H, H-5),
2
3
2a
ꢀ
2); HPLC (Agilent 1120, gradient 3): t =5.62 min (99%); HRMS
R
2b
(
ES+) m/z: calcd for C H N O 355.1647, found: 355.1644.
13
18 21 5 3
6
2
7
.56 ppm (bs, 2H, NH2); C NMR (100 MHz, [D ]DMSO): d=27.2,
7.5 (C3’, C4’), 53.2 (OCH ), 63.9 (CH OH), 68.1 (CH Oar), 76.7 (C5’),
8.1 (C5), 80.3 (C2’), 162.7 (C2), 171.2 (C6), 171.7 ppm (C4); HPLC
6
6-{[(2’RS,5’SR)-5’-(Benzyloxymetyl)tetrahydrofuran-2’-yl]me-
thoxy}-7H-purine (54): Following the general procedure 21
(390 mg, 1.75 mmol) was treated with 52 (650 mg, 2.62 mmol) in
dry DMF (10 mL) at 1008C in a pressure tube for ~16 h. The crude
was purified by HPFC in the Biotage (CH Cl /MeOH, 9:1) to give 54
3
2
2
[32]
[37]
(
Agilent 1120, gradient 3): t =3.77 min (99%); HRMS (ES+) m/z:
R
calcd for C H N O 256.1224, found 255.1219.
11
17
3
4
2
2
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ChemMedChem 0000, 00, 1 – 16
&
11
&
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(
0.60 g, 53%) as an amorphous solid. [a] =0.48 (c=0.9, CHCl );
0.60 mmol) was hydrogenated for ~16 h at 308C and 280 kPa, in
D
3
1
H NMR (500 MHz, [D ]DMSO): d=1.75–2.01 (m, 4H, H-3’, H-4’),
the presence of 20% Pd(OH) /C (10 mg). The reaction was filtered,
6
2
3
1
4
.41 (dd, 1H, J5’,CH2a =5.8 Hz, JCH2a,CH2b =10.3 Hz, CH Obn), 3.46 (dd,
washed with MeOH and evaporated to dryness. The residue was
purified by CCTLC in the chromatotron (CH Cl /MeOH/NH OH,
2a
H, J5’,CH2b =4.5 Hz, JCH2a,CH2b =10.3 Hz, CH Obn), 4.07 (m, 1H, H-5’),
2b
2
1
2
4
.30 (m, 1H, H-2’), 4.43 (dd, 1H, J2’,CH2a =6.6 Hz, JCH2a,CH2b =11.2 Hz,
4:1:0.1) to give 26 (48 mg, 51%) as an oil. H NMR (400 MHz,
[D ]DMSO): d=1.60–1.86 (m, 4H, H-3’, H-4’), 3.33 (m, 2H, CH OH),
CH Oar), 4.46 (m, 2H, CH Ph), 4.58 (dd, 1H, J
=3.8 Hz,
2
a
2
2’,CH2b
6
2
JCH2a,CH2b =11.2 Hz, CH Oar), 7.27–7.37 (m, 5H, Ar), 8.39 (s, 1H, H-2),
3.85 (m, 1H, H-5’), 4.04 (m, 3H, H-2’ and CH Oar), 4.66 (bs, 1H, OH),
2b
2
13
13
8
.47 (s, 1H, H-8), 13.42 ppm (bs, 1H, NH); C NMR (125 MHz,
5.02 (s, 1H, H-5), 5.90 (s, 2H, NH ), 6.03 ppm (s, 2H, NH ); C NMR
2
2
[
D ]DMSO): d=27.4, 27.5 (C3’, C4’), 68.7 (CH Oar), 72.2 (CH Ph),
(100 MHz, [D ]DMSO): d=27.2, 27.4 (C3’, C4’), 63.9 (CH OH), 67.4
6
2
2
6
2
7
2.6 (CH Obn), 76.7 (C2’), 78.3 (C5’), 120.2 (C5), 127.3, 127.4, 128.1
(CH Oar), 76.2 (C5), 76.9 (C2’), 80.2 (C5’), 162.8 (C6), 166.0 (C2),
2
2
(
(
(
CH Ar), 138.6 (C Ar), 141.8 (C8), 150.8 (C2), 153.1 (C4), 159.7 ppm
169.9 ppm (C4); HPLC (Agilent 1120, gradient 4): t =3.16 min
R
C6); HPLC (Agilent 1120, gradient 3): t =6.42 min (99%); HRMS
(97%); HRMS (ES+) m/z: calcd for C H N O 240.1231, found
R
10 16
4
3
ES+) m/z: calcd for C H N O : 340.1531, found 340.1535.
240.1222.
18
20
4
3
General procedure for the catalytic hydrogenation of benzyl-
protected compounds: A solution of the corresponding benzyl de-
rivatives (0.30 mmol) in MeOH (10 mL) and 37% HCl (49 mL,
2-Amino-4-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-2’-yl]-
methoxy}pyrimidine hydrochloride (27): Following the general
procedure compound 24 (46 mg, 0.13 mmol) was hydrogenated.
After the workup the crude was purified by reversed-phase chro-
matography using SPE cartridges (CH Cl /MeOH, 9:1) to give 27
0
.60 mmol) was hydrogenated for ~16 h at 308C and 280 kPa, in
the presence of 20% Pd(OH) /C (10 mg). The reaction was filtered,
2
2
2
1
washed with MeOH, evaporated to dryness, and the residue puri-
fied. The purification method, solvents, and yields are indicated in
each case.
(25 mg, 74%) as an oil. H NMR (400 MHz, [D ]DMSO): d=1.66–1.95
6
(m, 4H, H-3’, H-4’), 3.34 (m, 2H, CH OH), 3.88 (m, 1H, H-5’), 4.17 (m,
2
2H, H-2’), 4.26 (dd, 1H, J2’,CH2a =6.8 Hz, JCH2a,CH2b =11.1 Hz, CH Oar),
2a
4
1
.39 (dd, 1H, J2’,CH2b =3.6 Hz, JCH2a,CH2b =11.1 Hz, CH Oar), 6.43 (d,
H, J5,6 =6.9 Hz, H-5), 8.18 ppm (d, 1H, J5,6 =6.9 Hz, H-6); C NMR
2b
2
,6-Diamino-4-(2’,5’-anhydro-d-allitolyl)pyrimidine dihydrochlor-
ide (13a): A solution of 12a,b (90 mg, 0.17 mmol) in MeOH
10 mL) was hydrogenated for ~16 h at 308C and 280 kPa, in the
13
(
(
100 MHz, [D ]DMSO): d=27.1, 27.3 (C3’, C4’), 63,7 (CH OH), 70.1
CH Oar), 76.0 (C2’), 80.5 (C5’), 98.9 (C5), 147.0 (C6), 156.8 (C4),
6
2
(
2
presence of 20% Pd(OH) /C (10 mg). The reaction was filtered,
washed with MeOH, and evaporated to dryness. The residue was
purified by CCTLC in the chromatotron (CH Cl /MeOH/NH OH,
2
1
71.3 ppm (C2); HPLC (Agilent 1120, gradient 3): t =3.76 min
R
(98%); HRMS (ES+) m/z: calcd for C H N O 225.1115, found
10 15 3 3
2
1
2
4
2
25.1113.
4
:1:0.1) to give 13a (49 mg, 80%) as an oil. H NMR (400 MHz,
[
D ]DMSO): d=3.34 (dd, 1H, J =5.0 Hz, J6’a,6’b =11.6 Hz, H-6’a),
6-Amino-4-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-2’-yl]-
methoxy}pyrimidine hydrochloride (28): Following the general
procedure compound 25 (57 mg, 0.18 mmol) was hydrogenated
for ~16 h. After the workup the crude was purified by CCTLC in
the chromatotron (CH Cl /MeOH, 9:1) to give 28 (10 mg, 20%) as
6
5’,6’a
3
5
3
1
1
7
.42 (dd, 1H, J5’,6’b =4.3 Hz, J6’a,6’b =11.6 Hz, H-6’b), 3.70 (m, 1H, H-
’), 3.74 (m, 1H, H-3’), 3.80 (dd, 1H, J3’,4’ =5.5 Hz, J4’,5’ =4.3 Hz, H-4’),
.89 (m, 1H, H-2’), 4.15 (dd, 1H, J1’a,2’ =6.1 Hz, J1’a,1’b =11.3 Hz, H-
’a), 4.35 (dd, 1H, J
H, H-5); C NMR (100 MHz, [D ]DMSO): d=61.9 (C6’), 68.3 (C1’),
=3.4 Hz, J1’a,1’b =11.3 Hz, H-1’b), 5.35 ppm (s,
1’b,2’
2
2
1
3
1
an oil (hydrochloride); H NMR (400 MHz, [D ]DMSO): d=1.62–1.90
6
6
1.3 (C4’), 71.6 (C3’), 75.8 (C5), 79.8 (C2’), 85.0 (C5’),154.9 (C4), 157.1
(m, 4H, H-3’, H-4’), 3.35 (m, 2H, CH OH), 3.86 (m, 1H, H-5’), 4.09 (m,
2
(
C2), 170.8 ppm (C6); HPLC (Waters 600, gradient 4): t =0.71 min
1H, H-2’), 4.09 and 4.18 (m, 2H, CH Oar), 4.64 (t, 1H, J=5.7 Hz,
R
2
(
2
97%); HRMS (ES+) m/z: calcd for C H N O 272.1126, found
72.1121.
OH), 5.67 (s, 1H, H-5), 6.62 (s, 2H, NH ), 8.06 ppm (s, 1H, H-2);
C NMR (100 MHz, [D ]DMSO): d=27.4, 27.5 (C3’, C4’), 64.1
6
10
16
4
5
2
13
(
1
CH OH), 68.1 (CH Oar), 76.9 (C2’), 80.3 (C5’), 85.8 (C5), 157.9 (C2),
65.5 (C6), 168.9 ppm (C4); HPLC (Waters 600, gradient 2): 0.58 min
2
2
2
-Amino-4-(2’,5’-anhydro-d-allitolyl)pyrimidine (20a): A solution
of 19a (80 mg, 0.14 mmol) in MeOH (10 mL) was hydrogenated for
16 h at 308C and 280 kPa, in the presence of 10% Pd/C (10 mg).
(97%) HRMS (ES+) m/z: calcd for C H N O 225.1119, found
10 15 3 3
~
2
25.1113.
The reaction was filtered, washed with MeOH and evaporated to
dryness. The residue was purified by CCTLC in the chromatotron
4-Amino-2-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-2’-yl]-
methoxy}pyrimidine hydrochloride (31): Compound 30 (34 mg,
0.11 mmol) was hydrogenated according to the general procedure.
The residue was purified by reversed-phase chromatography using
SPE cartridges (CH Cl /MeOH, 9:1) to give 31 (8 mg, 28%) as an oil.
(
CH Cl /MeOH/NH OH, 4:1:0.1) to give 20a (18 mg, 50%) as an oil.
2 2 4
1
H NMR (400 MHz, [D ]DMSO): d=3.33 (dd, 1H, J5’,6’a =5.0 Hz,
6
J6’a,6’b =11.5 Hz, H-6’a), 3.41 (dd, 1H, J5’,6’b =4.2 Hz, J6’a,’6b =11.5 Hz,
H-6’b), 3.68 (dt, 1H, J4’,5’ =4.3 Hz, J5’,6’a =5.0 Hz, J5’,6’b =4.2 Hz, H-5’),
2
2
1
3
.73 (dd, 1H, J2’,3’ =6.6 Hz, J3’,4’ =5.4 Hz, H-3’), 3.78 (dd, 1H, J3’,4’
=
H NMR (500 MHz, [D ]DMSO): d=1.65–1.92 (m, 4H, H-3’, H-4’),
6
5
.4 Hz, J4’,5’ =4.3 Hz, H-4’), 3.89 (td, 1H, J2’,3’ =6.6 Hz, J1’a,2’ =6.7 Hz,
3.33 (m, 2H, CH OH), 3.86 (m, 1H, H-5’), 4.10 (m, 3H, CH Oar, H-2’),
2
2
J1’b,2’ =3.2 Hz, H-2’), 4.13 (dd, 1H, J1’a,2’ =6.7 Hz, J1’a,1’b =11.4 Hz, H-
’a), 4.33 (dd, 1H, J1’b,2’ =3.2 Hz, J1’a,1’b =11.4 Hz, H-1’b), 4.69 (bs, 1H,
4.63 (bs, 1H, OH), 6.06 (d, 1H, J5,6 =5.7 Hz, H-5), 6.84 (bs, 2H, NH ),
2
13
1
7.83 ppm (d, 1H, J5,6 =5.7 Hz, H-6); C NMR (125 MHz, [D ]DMSO):
6
OH), 4.85 (bs, 1H, OH), 4.92 (bs, 1H, OH), 6.00 (d, 1H, J5,6 =5.7 Hz,
H-5), 6.58 (bs, 2H, NH ), 7.94 ppm (d, 1H, J =5.7 Hz, H-6);
d=27.2, 27.5 (C3’, C4’), 63.9 (CH OH), 68.5 (CH Oar), 76.7 (C2’), 80.2
(C5’), 99.4 (C5), 156.1 (C6), 164.7 (C4), 165.4 ppm (C2); HPLC (Agi-
2
2
2
5,6
1
3
C NMR (100 MHz, [D ]DMSO): d=62.1 (C6’), 66.5 (C1’), 71.3 (C4’),
lent 1120, gradient 4): t =4.94 min (99%); HRMS (ES+) m/z: calcd
6
R
7
1
1.8 (C3’), 80.1 (C2’), 84.9 (C5’), 96.4 (C5), 158.4 (C6), 163.2 (C2),
for C H N O 226.1197, found 226.1192.
10
16
3
3
69.4 ppm (C4); HPLC (Agilent 1120, gradient 4): t =2.55 min
R
4
2
,6-Diamino-2-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-
’-yl]methoxy}-1,3,5-triazine dihydrochloride (48): Method A: To
(
98%); HRMS (ES+) m/z: calcd for C H N O 257.1014, found
10 15 3 5
2
57.1012.
a solution of 47 (98 mg, 0.29 mmol) in MeOH (10 mL) and 37% HCl
2
2
,6-Diamino-4-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-
’-yl]methoxy}pyrimidine dihydrochloride (26): A solution of 23
(48 mL, 0.58 mmol) in the presence of Pd(OH) /C 20% (10 mg) was
hydrogenated at 308C and 280 kPa for ~16 h. After filtering, the
2
(
100 mg, 0.30 mmol) in MeOH (10 mL) and 37% HCl (49 mL,
residue was washed with MeOH and evaporated to dryness. The
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residue was triturated with Et O/CH Cl /MeOH, successively
0.92 mmol), previously dried under vacuum for 5 h, in trimethyl
phosphate (2 mL), phosphorous oxychloride (0.14 mL, 1.46 mmol)
was slowly added. The reaction was stirred at 58C for ~16 h. A so-
lution of tributylammonium pyrophosphate (1.6 g, 2.92 mmol) and
tributylamine (0.56 mL, 2.38 mmol) in dry DMF (2.7 mL) was added
and stirring was continued at 08C for 20 min. Then, 1m triethylam-
monium bicarbonate (TEAB) solution (6.7 mL) was added, and the
reaction mixture was stirred at room temperature for 15 min and
evaporated at a reduced pressure, and the residue was lyophilized.
The crude was purified by Sephadex-DEAE A-25 resin ion-exchange
column chromatography (500 mL, linear gradient, 0–0.5m
NH HCO ) to obtain 59 (23 mg, 8%) as an amorphous white solid
2
2
2
washed with several portions of cold Et O, and evaporated to dry-
2
ness to give 21 mg of (2:1) mixture of 48 and ameline (49) as de-
termined by HPLC.
Method B: The general procedure was followed with (2R,5S)-2,5-
bis(hydroxymethyl)tetrahydrofuran (46) (150 mg, 1.13 mmol), in
dry DMF (5 mL) and 2-chloro-4,6-diamino-1,3,5-triazine (45)
(
329 mg, 2.26 mmol). The reaction was heated at 1008C in a pres-
sure tube for 48 h. The crude was purified by preparative TLC
CH Cl /MeOH/AcOH, 9:1:0.1). From the fastest-moving band com-
(
2
2
pound 48 (114 mg, 42%) was isolated as an amorphous white
1
4
3
solid. H NMR (400 MHz, [D ]DMSO): d=1.65–1.92 (m, 4H, H-3’, H-
1
6
after lyophilization. H NMR (400 MHz, D O): d=1.72–1.92 (m, 4H,
2
4
’), 3.35 (m, 2H, CH OH), 3.88 (m, 1H, H-5’), 4.13 (m, 1H, H-2’), 4.23
2
H-3’, H-4’), 3.53–4.14 (m, 6H, CH OP, CH Oar, H-2’, H-5’), 5.56 ppm
2
2
(
dd, 1H, J2’,CH2a =6.3 Hz, JCH2a,CH2b =11.0 Hz, CH Oar), 4.33 (dd, 1H,
31
2a
(
s, 1H, H-5); P NMR (162 MHz, D O): d=1.16, ꢀ8.49, ꢀ21.31 ppm;
2
J
=3.9 Hz, JCH2a,CH2b =11.0 Hz, CH Oar), 8.30 ppm (bs, 4H, NH );
2b 2
2
3
’,CH2b
HPLC (Waters 600, isocratic conditions 0.5m KH PO ): t =6.49 min
1
2
4
R
C NMR (100 MHz, [D ]DMSO): d=27.1, 27.2 (C3’, C4’), 63.9
6
(
98%); HRMS (ES+) m/z: calcd for C H N O P 480.0216, found
10 18 4 12 3
(
CH OH), 70.2 (CH Oar), 75.9 (C2’), 80.4 (C5’), 161.0 (C4, C6),
2
2
4
80.0212.
1
65.1 ppm (C2); HPLC (Agilent 1120, gradient 4): t =4.95 min
R
(
98%); HRMS (ES, +) m/z: calcd for C H N O 241.1179, found
9 15 5 3
2
41.1175.
Biological methods
From the slowest-moving band (2R,5S)-2,5-bis[(4’,6’-diamino-
’,3’,5’-triazin-2’-yl)oxymethyl]tetrahydrofuran (50) (55 mg, 14%)
1
Antiviral activity assays other than HIV: The compounds were evalu-
ated against the following viruses: herpes simplex virus type 1
(HSV-1) strain KOS, thymidine kinase-deficient (TK ) HSV-1 KOS
strain resistant to ACV (ACV ), herpes simplex virus type 2 (HSV-2)
1
was isolated, as an amorphous white solid. H NMR (400 MHz,
D ]DMSO+HCOOH): d=1.69–1.96 (m, 4H, H-3, H-4), 4.12 (m, 6H,
ꢀ
[
6
13
r
H-2, H-5, 2CH Oar), 6.63 ppm (bs, 8H, 4NH ); C NMR (100 MHz,
2
2
ꢀ
[
D ]DMSO+HCOOH): d=27.6 (C3, C4), 68.0 (2CH Oar), 77.0 (C2,
strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK VZV
6
2
C5), 168.3 (C4’, C6’), 170.6 ppm (C2’); HPLC (Agilent 1120, gradi-
ent 4): t =5.46 min (95%); HRMS (ES+) m/z: calcd for C H N O
strain 07-1, human cytomegalovirus (HCMV) strains AD-169 and
Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV)
strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, parain-
fluenza 3, influenza virus A (subtypes H1N1, H3N2), influenza
virus B, Sindbis, reovirus-1, Punta Toro, human immunodeficiency
virus type 1 strain IIIB and human immunodeficiency virus type 2
strain ROD. The antiviral assays (other than anti-HIV) were based
on inhibition of virus-induced cytopathicity or plaque formation in
human embryonic lung (HEL) fibroblasts, African green monkey
cells (Vero), human epithelial cells (HeLa), or Madin–Darby canine
kidney cells (MDCK). Confluent cell cultures in microtiter 96-well
plates were inoculated with 100ꢅCCID50 of virus (with 1ꢅCCID50
being the virus dose to infect 50% of the cell cultures) or with
R
12 18 10
3
3
50.1571, found 350.1563.
2
-Amino-6-{[(2’RS,5’SR)-5’-(hydroxymethyl)tetrahydrofuran-2’-yl]-
methoxy}-7H-purine hydrochloride (55): Compound 53 (52 mg,
.15 mmol) was hydrogenated following the general procedure.
The crude was purified by HPFC in the Biotage (CH Cl /MeOH, 9:1)
0
2
2
1
to give 55 (25 mg, 41%) as an oil. H NMR (500 MHz, [D ]DMSO):
6
d=1.68–1.98 (m, 4H, H-3’, H-4’), 3.35 (m, 2H, CH OH), 3.89 (m, 1H,
2
H-5’), 4.21 (m, 1H, H-2’), 4.30 (dd, 1H, J2’,CH2a =6.6 Hz, JCH2a,CH2b
=
1
1.0 Hz, CH Oar), 4.40 (dd, 1H, J =4.2 Hz, JCH2a,CH2b =11.0 Hz,
2
a
2’,CH2b
CH Oar), 4.70 (bs, 1H, OH), 6.25 (s, 2H, NH ), 7.84 (s, 1H, H-8),
2
b
2
13
1
2.45 ppm (bs, 1H, NH); C NMR (125 MHz, [D ]DMSO): d=27.2,
6
2
0 plaque forming units (PFU) (VZV) in the presence of varying
2
7.6 (C3’, C4’), 63.9 (CH OH), 68.2 (CH Oar), 76.7 (C2’), 80.4 (C5’),
2
2
concentrations of test compounds. Viral cytopathicity or plaque
formation was recorded as soon as it reached completion in the
control virus-infected cell cultures that were not treated with test
compounds. Antiviral activity was expressed as the EC50 or com-
pound concentration required to decrease virus-induced cytopa-
thicity or viral plaque formation by 50%.
113.4 (C5), 137.7 (C8), 155.0 (C4), 159.6 (C6), 172.2 ppm (C2); HPLC
(
Agilent 1120, gradient 4): t =5.61 min (99%); HRMS (ES+) m/z:
R
calcd for C H N O 265.1162, found 265.1175.
11
15
5
3
6
-{[(2’RS,5’SR)-5’-(Hydroxymethyl)tetrahydrofuran-2’-yl]me-
thoxy}-7H-purine hydrochloride (56): Following the general pro-
cedure, 54 (59 mg, 0.17 mmol) was hydrogenated for ~16 h. After
the workup the crude was purified by HPFC in the Biotage (CH Cl /
Anti-HIV-1 and HIV-2 activity assays: Inhibition of HIV-1(III )- and
B
2
2
HIV-2(ROD)-induced cytopathicity in CEM cell cultures was mea-
1
MeOH, 9:1) to give 56 (17 mg, 35%) as an oil. H NMR (500 MHz,
5
sured in microtiter 96-well plates containing ꢃ3ꢅ10 CEM cells
[
D ]DMSO): d=1.73–1.99 (m, 4H, H-3’, H-4’), 3.36 (m, 2H, CH OH),
6 2
per mL infected with 100ꢅCCID50 of HIV per milliliter and contain-
ing appropriate dilutions of the test compounds. After 4–5 days of
3
.90 (m, 1H, H-5’), 4.27 (m, 1H, H-2’), 4.45 (dd, 1H, J2’,CH2a =6.8 Hz,
=3.8 Hz, J
JCH2a,CH2b =11.2 Hz, CH Oar), 4.60 (dd, 1H, J
=
CH2a,CH2b
2a
2’,CH2b
incubation at 378C in a CO -controlled humidified atmosphere,
13
2
11.2 Hz, CH Oar), 8.56 (s, 1H, H-2), 8.70 ppm (s, 1H, H-8); C NMR
2b
CEM giant (syncytium) cell formation was examined microscopical-
ly. The EC50 (50% effective concentration) was defined as the com-
pound concentration required to inhibit HIV-induced giant cell for-
mation by 50%.
(
(
125 MHz, [D ]DMSO): d=27.2, 27.4 (C3’, C4’), 63.8 (CH OH), 69.2
6
2
CH Oar), 76.5 (C2’), 80.4 (C5’), 116.4 (C5), 142.8 (C8), 151.8 (C2),
2
1
54.0 (C4), 158.6 ppm (C6); HPLC (Agilent 1120, gradient 4): t =
R
3
.89 min (91%); HRMS (ES+) m/z: calcd for C H N O 250.1054,
11
14
4
3
found 250.1066.
HIV-1 RT assays in the presence of artificial template/primer: To pre-
pare the template/primers for the RT experiments, 0.15 mm
poly(U), poly(A), poly(C), and poly(I) were mixed with an equal
volume of 0.0375 mm oligo(dA), oligo(dT), oligo(dG), and oligo(dC),
respectively. The final concentrations of the templates in the RT re-
action mixture were 0.015 mm. The reaction mixture (50 mL) con-
2
,6-Diamino-4-{[(2’RS,5’SR)-5’-(triphosphooxymetyl)tetrahydro-
furan-2’-yl]methoxy}pyrimidine triammonium salt (59): To
a cooled (08C) solution of compound 26 (150 mg, 0.54 mmol) and
TM
1
,8-bis(dimethylamino)naphthalene (Proton Sponge
;
196 mg,
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tained 50 mm Tris·HCl (pH 7.8), 5 mm dithiothreitol, 300 mm gluta-
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serum albumin, an appropriate concentration of labeled (tritiated)
substrate dTTP, dCTP, dGTP, or dATP (2 mCi per assay), a fixed con-
centration of the template/primer poly(A):oligo(dT) (0.015 mm),
poly(I):oligo(dC) (0.015 mm), poly(C):oligo(dG) (0.015 mm), and pol-
y(U):oligo(dA) (0.015 mm), 0.06% Triton X-100, 10 mL of inhibitor
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1
(
1 mgmL ) and 1 mL Na P O (0.02m) in trichloroacetic acid (5%
4 2 7
v/v) were added. The solutions were kept on ice for 30 min, after
which the acid-insoluble material was washed and analyzed for ra-
dioactivity. For the experiments in which the 50% inhibitory con-
centration (IC ) of the test compounds was determined, fixed con-
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0
3
3
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3
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Cytostatic activity assays: All assays were performed in 96-well mi-
4
crotiter plates. To each well were added (5–7.5)ꢅ10 tumor cells
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6 h (human kidney carcinoma CAKI-1 and human liver hepatoma
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[
[
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S.d.C. thanks the Spanish Ministry of Science and Innovation for
a postdoctoral Juan de la Cierva contract (JDC-MICINN). The au-
thors also thank the Spanish MINECO (SAF2012-39760-C02), the
Comunidad de Madrid (BIPEDD-2-CM S-2010/BMD-2457), and KU
Leuven (GOA No. 10/014) for financial support, and Ms. Lizette
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Keywords: antiproliferation
·
drug design
·
medicinal
chemistry · nucleosides · nucleotides
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Received: October 28, 2014
Published online on && &&, 0000
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G. Fernꢁndez-Cureses, S. de Castro,*
M.-L. Jimeno, J. Balzarini,
M.-J. Camarasa*
Bucking convention: Herein we de-
scribe unconventional nucleosides that
combine methyloxy-bound pyrimidines
or purines with d-allitolyl, d-altritolyl
groups as unusual ribofuranosyl mimics
or 2,5-bis-hydroxymethyltetrahydrofuran
as their dideoxy analogues. The pyrimi-
dine nucleotides act as adenine nucleo-
tide mimics. This class of unconvention-
al nucleosides represents a new family
of potential antiproliferative agents.
&& – &&
Design, Synthesis, and Biological
Evaluation of Unconventional
Aminopyrimidine, Aminopurine, and
Amino-1,3,5-triazine
Methyloxynucleosides
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