Synthesis and Biological Profiling of Pyrazolo-Fused 7-Deazapurine Nucleosides

Article abstract of DOI:10.1021/acs.joc.0c00928

A series of 8-substituted 1-methyl-1,4-dihydropyrazolo[3′,4′:4,5]pyrrolo[2,3-d]pyrimidine (methylpyrazolo-fused 7-deazapurine) ribonucleosides have been designed and synthesized. Two synthetic approaches to the key heterocyclic aglycon 7, (i) a six-step c

Full text of DOI:10.1021/acs.joc.0c00928

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Article
Synthesis and Biological Profiling of
Pyrazolo-fused 7-Deazapurine Nucleosides
Marianne Fleuti, Katerina Bartova, Lenka Poštová Slav#tínská, Eva Tlouš#ová, Michal
Tichy, So#a Gurská, Petr Pavlis, Petr Džubák, Marián Hajdúch, and Michal Hocek
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00928 • Publication Date (Web): 21 Jul 2020
Downloaded from pubs.acs.org on July 22, 2020
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Synthesis and Biological Profiling of Pyrazolo-fused 7-Deazapurine Nucleosides
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Marianne Fleuti,a,b Kateřina Bártová,a,b Lenka Poštová Slavětínská,b Eva Tloušťová,b Michal Tichý,b
Soňa Gurská,c Petr Pavliš,c Petr Džubák,c Marián Hajdúch,c Michal Hocek*a,b
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a Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8,
CZ-12843 Prague 2, Czech Republic.
b Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2,
CZ-16610 Prague 6, Czech Republic.
c Institute of Molecular and Translational Medicine, Palacky University and University Hospital in
Olomouc, Faculty of Medicine and Dentistry, Hněvotínská 5, CZ-77515 Olomouc, Czech Republic.
* author for correspondence: e-mail: hocek@uochb.cas.cz
Abstract: A series of 8-substituted 1-methyl-1,4-dihydropyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine
(methylpyrazolo-fused 7-deazapurine) ribonucleosides has been designed and synthesized. Two
synthetic approaches to the key heterocyclic aglycon 7, (i) a six-step classical heterocyclization
starting from 5-chloro-1-methyl-4-nitropyrazole and (ii) a three-step cross-coupling and cyclization
approach starting from the zincated 4,6-dichloropyrimidine, gave comparable total yield of 18% vs.
13%. The glycosylation of 7 was attempted by three different methods but only the Vorbrüggen silyl-
base protocol was efficient and stereoselective to give desired β-anomeric nucleoside intermediate
17A. Its nucleophilic substitutions or cross-coupling reactions at position 8 and deprotection of the
sugar moiety gave eight derivatives of pyrazolo-fused deazapurine ribonucleosides, some of which
were weakly fluorescent. Methyl, amino and methylsulfanyl derivatives exerted submicromolar
cytotoxic effects in vitro against a panel of cancer and leukemia cell lines, as well as antiviral effect
against hepatitis C virus in the replicon assay.
TOC graphic:
Me
Me
R
N
N
N
Cl
A
N
N
Cl
Me
N
N
O2N
Cl
N
N
N
O
B
N
HO
N
N
H
M
HO
OH
N
submicromolar cytotoxic
and antiviral activity
Cl
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Introduction
Base-modified nucleosides are a rich source of biologically active compounds, in particular antivirals1
and antineoplastics.2 Among this class of compounds, diverse substituted 7-deazapurine nucleosides3
possess significant cytotoxic,4,5,6 antiviral7 or antiparasitic8 activities. In our group, we have
previously developed the 7-thienyl-7-deazaadenine ribonucleoside AB-61 (1, Figure 1)4 and a
number of other 6-substituted 7-hetaryl-7-deazapurine ribonucleosides (2)5 with nanomolar cytostatic
effects. The mode of action of the parent compound AB-61 involves intracellular phosphorylation
and incorporation to genomic DNA causing double-strand breaks and apoptosis.9 This complex
mechanism of action does not allow to involve any standard methods of rational design of derivatives
to further improve the activities, but based on systematic structure-activity relationship study, we
have noticed that there is a significant space for heteroaromatic modifications in the major-groove
edge of the nucleoside (position 7 of 7-deazapurine). This led us to the design of the second generation
of benzo- and heteroaryl-fused deazapurine nucleosides. While the benzo-,10 naphtho-11, and
benzofuro-fused12 deazapurine nucleosides did not show any significant cytotoxicity, some
heteroaryl-fused derivatives were more active. Two isomeric series of thieno-fused deazapurines 3
and 4 were shown to exert submicromolar cytostatic effects.13 Also, the furo- and methylpyrrolo-
fused14 deazapurine ribonucleosides 5 and 6 showed cytotoxic effects at submicromolar
concentrations. Out of four isomeric series of pyrido-fused deazapurine ribosides,15 only the
4-substituted pyrido[4',3':4,5]pyrimidine nucleosides showed significant antiproliferative acticity. In
each series, some compounds showed good selectivity for cancer and leukemia cell lines and little
toxicity to non-proliferating cells. In furo-, methylpyrrolo-, and pyrido-fused deazapurine
nucleosides, we have proven that the mode of action also involves incorporation to and damage of
DNA followed by apoptosis14 (similarly to AB-61)9. Some of the benzo- and heteroaryl-fused
nucleosides 3-6 also showed moderate to good antiviral activities10-14 against some RNA viruses
(Hepatitis C, Dengue etc.). In this paper, we designed new N-methylpyrrazolo-fused 7-deazapurine
nucleosides, as aza-analogues of previously reported14 cytotoxic N-methylpyrrolo-fused deazapurine
nucleosides 6 (Figure 1).
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R
N
R
S
S
NH2
N
S
N
N
N
N
N
N
N
O
O
O
HO
HO
HO
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HO
OH
HO
OH
Me
HO
OH
3
4
R
R
O
N
1
Y
X
(AB-61)
R
N
N
N
N
N
N
N
N
O
O
N
HO
HO
O
HO
OH
HO
OH
HO
5
6
<M cytotoxic
HO
OH
2
antiviral
nM cytotoxic
R = NH2, OMe,
SMe, Me, Het
X, Y = O, S,
N, NMe
Me
N
R
8
This work:
8b
N
8a
N
3
3a
6
N
4a
N
O
HO
HO
OH
Figure 1. Structures and biological activity of previously synthesized substituted and fused
deazapurine nucleosides and target structures of this study
Results and Discussion
Chemistry
Two approaches were previously used for the synthesis of either benzo- or heteroaryl-fused
deazapurine nucleobases: (a) multi-step heterocyclization approach starting from the chloro-nitro
derivative of the fused (het)aromatic ring (Scheme 1, Approach A)10,16 or (b) cross-coupling followed
by thermal of photochemical cyclization of 5-(het)aryl-6-azidopyrimidines (Approach B).13,14,17 In
this work, our key heterocyclic intermediate was 8-chloro-1-methyl-1,4-dihydropyrazolo[3',4':4,5]-
pyrrolo[2,3-d]pyrimidine (7) and we aimed to try both synthetic approaches and compare them.
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Cl
3
4
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7
8
9
Cl
Me
N
N
Me
N
M
A
B
N
Cl
O2N
N
N
N
Cl
N
Me
N
H
N
I
multistep
heterocycle build-up
N
cross-coupling
cyclization
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Scheme 1. Retrosynthetic strategy for the synthesis of nucleobase
A. Classical Heterocyclization Approach. The classical approach relies on multistep synthesis of
the tricyclic heterocycle starting from the "eastern" portion, the pyrazole ring (Scheme 2). Thus, the
4-nitropyrazole (8) was methylated using iodomethane and K2CO3 in DMF giving 9 in nearly
quantitative yield (96%).18 Then, a selective chlorination19 in position 5 was performed with
hexachloroethane and LiHMDS in DCM to furnish 5-chloro-4-nitro derivative 10 in 88% yield. Then,
the nucleophilic aromatic substitution with an excess of ethyl cyanoacetate (2.0 equiv.) and potassium
tert-butoxide in 1,4-dioxane gave intermediate 11 in 97% yield. Unreacted ethyl cyanoacetate was
removed by vacuum distillation prior to solidification in the freezer and subsequent filtration and
washing with ice-cold DCM. Alternatively, the ethyl cyanoacetate can be removed by
chromatography. A reaction with lower excess ethyl cyanoacetate (1.5 equiv.) resulted in a significant
drop in yield of 11 (76%).
Reduction of nitropyrazolyl-cyanoacetate 11 with zinc dust in acetic acid resulted in
spontaneous cyclization20 during work-up with sat. NaHCO3 of the in-situ formed 4-aminopyrazole
derivative to pyrazolopyrrole derivative 12 in 55% yield. Small traces of zinc in 12 were confirmed
by ED-XRF and did not interfere in the next step. The next step was the formation of the tricyclic
heterocycle 13 through heterocyclization using formamide in presence of sodium methoxide.21
Standard conditions with only formamide and heat (≥ 150 °C),10 as well as other options such as with
formamide and ammonium formate as an additive,16c with formamidine acetate in EtOH at 77 °C,22
or with formamide, DMF and formic acid (4:2:1) at 150 °C were all not successful.23 The pyrazolo-
fused 7-deazahypoxanthine 13 was obtained in 46% yield. Lastly, the chlorination5,24 of 13 with
POCl3 and benzyltriethylammonium chloride (BTEA-Cl) led to the desired nucleobase 7 in 86%
yield. Thus, this classical heterocyclization approach furnished the desired heterocyclic intermediate
7 in six steps from 4-nitro-1H-pyrazole 8 in 18% overall yield.
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Me
N
Me
H
N
N
ii)
.
iii)
i)
.
Cl
N
N
N
.
O2N
O2N
O2N
10
9
8
, 88%
EtO
, 96%
O
O
Me
Me
N
10
11
12
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EtO
N
iv)
.
v)
.
N
N
NC
H2N
N
O2N
H
12
11
, 97%
, 55%
O
Cl
Me
N
Me
N
vi)
.
HN
N
N
N
N
N
N
H
N
H
13
7
, 46%
, 86%
Scheme 2. Reagents and Conditions: (i) MeI, K2CO3, DMF, 20 °C, 18 h; (ii) C2Cl6, LiHMDS (1.0 M
in THF), DCM, 20 °C, 2 h; (iii) ethyl cyanoacetate, KOtBu, 1,4-dioxane, 101 °C, 48 h; (iv) 1) AcOH,
80 °C, 2) Zn, 95 °C, 2 h; (v) 1) NaOMe (25 wt% in MeOH), sonication, 10 min, 2) formamide 90 °C,
48 h, 3) conc. HCl, 0 °C; (vi) N,N-dimethylaniline, BTEA-Cl, POCl3, MeCN, 90 °C, 75 min.
B. Cross-coupling approach. The second approach to the desired nucleobase 7 was based on cross-
coupling of 5-iodo-1-methyl-1H-pyrazole with an organozinc intermediate generated from
4,6-dichloropyrimidine 14 followed by azidation and cyclization (Scheme 3).
Me
N
Cl
Cl
N
N
i)
.
N
N
ii)
N
Cl
Cl
.
15
14
, 42%
Me
Me
N
Cl
Cl
N
N
N
N
N
N
Cl
N
Me
N3
iii)
.
N
N
N
N
N
N
16a
16b
, 70%
N
N
H
7
, 44%
Scheme 3. Reagents and Conditions: (i) 1) (TMP)2Zn·2MgCl2·2LiCl, THF, 0 °C, 1 h, then 20 °C,
1 h; 2) 5-iodo-1-methyl-1H-pyrazole, Pd(PPh3)4, THF, 65 °C, 18 h; (ii) NaN3, LiCl, DMF, 20 °C,
24 h; (iii) 1,4-dibromobenzene, 170 °C, 30 min.
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First, the Turbo-Hauser base (TMP)2Zn·MgCl2·LiCl (TMP = 2,2,6,6-tetramethylpiperidyl)
was prepared in situ with (TMP)MgCl·LiCl and ZnCl2, and it was used for the zincation of 4,6-
dichloropyrimidine 14.25 Then, the in-situ-formed 5-zincated 4,6-dichloropyrimidine underwent
Negishi coupling with 5-iodo-1-methyl-1H-pyrazole. The reaction under standard conditions in
presence of Pd(PPh3)4 in THF gave the desired product 15 in only 5% yield. Extensive optimization
(see Table S1 in SI) resulted in requiring an excess of pyrazole (2.0 equiv.) and high catalyst loading
(25 mol% Pd(PPh3)4) in order to afford 15 in yields ranging from 20 to 42% (depending on the scale).
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Then, the aromatic nucleophilic substitution of 15 with sodium azide in presence of LiCl in
THF gave 70% of 16, which exclusively occurs in the azide form 16a in CDCl3.14 In DMSO, there is
an equilibrium between azide 16a and tetrazole 16b, which is shifted towards the tetrazole 16b (see
Table S2 in SI). Third, thermal cyclization of 16 in 1,4-dibromobenzene at 170 °C for 30 min gave
the desired nucleobase 7 with 30–44% yield. The photocyclization in TFA with UV-light (254 nm,
4 W) for 48 hours resulted in decomposition, while the catalytic cyclization with Rh2(esp)2 in
toluene/TFA (1:1) at 90 °C for 24 hours did not proceed at all (see Table S3 in SI).11,26 The overall
yield of this cross-coupling approach is 13% over three steps.
C. Glycosylation
With the desired nucleobase 7 in hand, we proceeded with its glycosylation. Since the heterocyclic
aglycon 7 was novel, we decided to test and compare the two most commonly used glycosylation
methods, (i) Vorbrüggen glycosylation (a modified Hilbert-Johnson reaction)27 and (ii) nucleobase
anion glycosylation,28 with recently developed (iii) direct glycosylation under modified Mitsunobu
conditions.29
First, we tested the Vorbrüggen procedure, which was previously successfully applied for the
synthesis of other heteroaryl-fused nucleosides.13–15 The nucleobase 7 was silylated with N,O-
bis(trimethylsilyl)acetamide (BSA) before undergoing glycosylation with 1-O-acetyl-2,3,5-tri-O-
benzoyl-β-D-ribofuranose in presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf)
furnishing the nucleoside 17A isolated as a pure β-anomer in good 51% yield (Scheme 4). The
stereochemistry was confirmed by H,H-ROESY using the relations between H-3 of the nucleobase
and H-2’ resp. H-3’ as well as between H-1’ and H-4’ of the sugar moiety.
The anion base glycosylation was performed according to the literature procedure28 starting
from 2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-D-ribofuranose, which was in situ
transformed to a halogenose and reacted with nucleobase 7 in presence of KOH and tris[2-(2-
methoxyethoxy)ethyl]amine (TDA-1). The reaction gave an inseparable mixture of the desired
nucleoside 17B-β with its α-anomer 17B-α in ratio 1:0.7 in total 61% yield (Scheme 4) and only a
very small sample of 17B-β was isolated as pure isomer for characterization.
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8
We also attempted a glycosylation of 7 under modified Mitsunobu conditions.29 No tricyclic
fused-nucleobase was tested in this type of glycosylation so far. We used the optimized protocol
starting from 5-O-trityl-D-ribose30 which was treated with P(nBu3) and 1,1′-
(azodicarbonyl)dipiperidine (ADDP) forming an epoxide. The epoxide was then reacted with pre-
generated sodium salt of nucleobase 7 (generated by treatment with NaH). Unfortunately, only traces
of the desired nucleoside 17C were observed in the complex reaction mixture. All attempts to isolate
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1
pure product 17C failed and only a small sample of crude 17C was partially characterized by H
NMR and HR-MS.
Therefore, we can conclude that from all three tested glycosylation procedures, only the
Vorbrüggen method was efficient and stereoselective to give the desired β-anomer in good yield and
it was easily purified by chromatography. The nucleobase anion glycosylation was not
stereoselective, whereas the Mitsunobu glycosylation gave a complex mixture with only traces of the
desired nucleoside.
Cl
Me
N
N
N
N
N
i) Vorbrüggen glycosylation
O
1. BSA
O
17A
, 51%
BzO
2.
OAc
OBz
BzO
OBz
BzO
Cl
Me
N
BzO
N
N
TMSOTf
ii) Nucleobase-anion
glycosylation
N
N
O
Cl
Me
1. KOH, TDA-1
.
O
N
N
TBDMSO
N
2.
Cl
61%
TBDMSO
N
O
O
N
H
O
O
7
17B-
17B-

 + -anomer
(inseparable mixture)
1. NaH
2. P(nBu)3, ADDP
O
Cl
Me
N
OH
OH
TrO
N
N
HO
N
N
iii) Mitsunobu glycosylation
17C
, traces
O
TrO
OH
HO
Scheme 4. Reagents and Conditions: (i) 1. BSA, MeCN, 60 °C, 1 h; 2. 1-O-acetyl-2,3,5-tri-O-
benzoyl-β-D-ribofuranose, TMSOTf, MeCN, 60 °C, 36 h; (ii) 1. KOH, TDA-1, THF, 23 °C, 30 min,
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0 °C;
2.
2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-D-ribofuranose,
CCl4,
hexamethylphosphorous triamide (HMPT), THF, –30 °C, pre-stirred for 1 h, then 23 °C, 36 h; (iii) 1.
NaH, DMF, 23 °C, 30 min; 2. 5-O-trityl-D-ribofuranose, P(nBu)3, ADDP, pre-stirred for 30 min,
23 °C, 16 h.
4
5
6
7
8
9
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D. Derivatization and Deprotection. The key nucleoside intermediate 17A was used for the
derivatization in position 8 and deprotection to give a series of the desired 8-substituted free
ribonucleosides 19a-h (Scheme 5). The 2-furyl derivative 18a was prepared in 69% yield via Stille
cross-coupling of 17A with 2-(tributylstannyl)furan in presence of PdCl2(PPh3)2 in DMF at 100 °C.
The Suzuki-Miyaura cross-coupling of 17A with 3-furyl- or with 2-benzofurylboronic acid in
presence of K2CO3 and Pd(PPh3)4 in toluene at 100 °C for 48 h gave 18b and 18c in moderate yields
(32% and 38%, respectively). The methyl derivative 18d was prepared by cross-coupling reaction of
17A with AlMe3 and Pd(PPh3)4 in THF at 65 °C for 10 h in good 64% yield. The N,N-dimethylamino
derivative 18e was obtained by a nucleophilic substitution of 17A with dimethylamine in 2-propanol
at 60 °C for 24 h in 47% yield. The protected nucleoside derivatives 18a-e underwent the Zemplén
deprotection of the sugar moiety with NaOMe in methanol at 60 °C for 4–6 h and gave the free
nucleosides 19a-e in very good to excellent yields (72–98%). The preparation of 19f-h included the
nucleophilic substitution at position 8 directly with simultaneous deprotection. The amino derivative
19f was obtained in 80% yield through treatment of 17A with aqueous ammonia/1,4-dioxane (5:2) at
120 °C for 24 h in a screw-cap pressure tube. The reaction of 17A with NaOMe in methanol at 25 °C
after 76 h gave 19g in 80% yield. The methylsulfanyl derivative 19h was obtained from 17A using
NaSMe in 1,4-dioxane at 60 °C for 14 h in 51% yield. The same reaction in methanol gave an
inseparable mixture of 19g and 19h.
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vii), viii), ix)
Cl
Me
N
R
R
O
Me
N
Me
N
N
N
N
N
N
N
N
N
N
N
i), ii), iii),
iv) or v)
N
N
vi)
.
O
O
10
11
12
13
14
15
16
17
18
19
20
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BzO
BzO
HO
BzO
OBz
BzO
18a
OBz
HO
OH
17A
19a
19b
19c
19d
19e
19f
19g
19h
, 87%
, 72%
, 98%
, 73%
, 98%
, 81%
, 69%
, 32%
, 38%
, 64%
, 47%
18b
18c
18d
18e
R =
O
O
O
Me NMe2 NH2 OMe SMe
, 80%
, 51%
a
b
c
d
e
f
g
h
Scheme 5. Reagents and Conditions: (i) 2-(tributylstannyl)furan, PdCl2(PPh3)2, DMF, 100 °C, 10 h;
(ii) furan-3-ylboronic acid, K2CO3, Pd(PPh3)4, toluene, 100 °C, 48 h; (iii) benzofuran-2-ylboronic
acid, K2CO3, Pd(PPh3)4, toluene, 100 °C, 48 h; (iv) AlMe3 (2.0 M in toluene), Pd(PPh3)4, THF, 65 °C,
3 h; (v) Me2NH (2.0 M in THF), propan-2-ol, 60 °C, 24 h ; (vi) NaOMe (25 wt% in MeOH), MeOH,
60 °C, 4–6 h; (vii) aq. NH3/1,4-dioxane (5:2), 120 °C, 24 h; (viii) NaOMe (25 wt% in MeOH),
MeOH, 25 °C, 76 h; (ix) NaSMe, 1,4-dioxane, 60 °C, 14 h.
Spectroscopic Properties of Pyrazolo-fused 7-Deazapurine Nucleosides
Some of the previously synthesized fused 7-deazapurine nucleoside derivatives showed interesting
fluorescent properties useful in applications in nucleic acids probes.11,31 Therefore, we studied the
absorption and emission spectra of the nucleosides 19a-h in methanol (Table 1; for standard deviation
and individual spectra see S5 in SI). We have also determined their quantum yields Φf.32 The
nucleosides 19a-c,e,h exhibited fluorescence with moderate Φf 1.4–7.7%. The quantum yields Φf for
nucleosides with maximal absorbance λabs below 300 nm were not determined (19d, 19f, 19g), as
these values could no longer be compared with quinine sulfate as a standard.
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Table 1. UV and fluorescence maxima of nucleosides 13a-h
4
5
6
7
8
9
Absorption
Emission
Quantum Yield
-1
compd
19a
19b
19c
max. λabs (ε) [nm (M cm-1)]
284 (10700), 323 (12000), 254 (29800)
301 (4600), 250 (24200)
336 (19800), 258 (20900)
281 (6600), 246 (39300)
305 (7400)
max. λem [nm]
Φf
0.077
0.060
0.014
–
506
476
534
434
417
390
398
449
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
28
29
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31
32
33
34
35
36
37
38
39
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47
48
49
50
51
52
53
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55
56
57
58
59
60
19d
19e
0.045
–
19f
281 (7000)
19g
19h
271 (9300)
–
303 (5000)
0.076
UV and fluorescence maxima were measured in MeOH. Fluorescence quantum yields Φf were
determined using quinine sulfate in 0.1 M H2SO4 as a standard (Φf = 0.546 at 25 °C).32
Biological Profiling:
The nucleosides 19a-h were tested for their cytotoxic and antiviral activity. The following cancer cell
lines were used for the study of in vitro cytotoxic activity: A549 (lung cancer), CCRF-CEM and
CEM-DNR (acute T-lymphoblastic leukemia without and with drug resistance), HCT116 and
HCT116p53- (colon carcinoma), K562 and K562-TAX (chronic myelogenous leukemia without or
with multi-drug resistance), U2OS (bone osteosarcoma) in a MTS assay; as well as HeLaS3 ( cervical
cancer), HepG2 (hepatocellular liver carcinoma) and HL60 (acute promyelocytic leukemia) in an
XTT assay.33 For comparison, non-malignant fibroblast cell lines (BJ and MRC-5) were used in the
MTS assay. The required concentrations of 19a-h for the reduction of the respective cell viability by
50% (IC50) were determined by colorimetric assays.34 In the MTS assay,35 after a 3-day treatment of
the cell line with the respective nucleoside, the samples were stained with 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) followed by detection at
492 nm. Initial testing is done with 50 µM concentration. In the XTT assay,36 after incubation for
3 days the samples were treated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide (XTT) prior to visualization. The initial testing concentration was 10 µM.
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Table 2. Cytotoxic activities of nucleosides 13a–h.
4
5
6
XTT assay: IC50
[µM]
MTS assay: IC50 [µM]
7
8
9
CCRF- CEM-
HCT116
K562-
TAX
compd BJ
MRC-5 A549
HCT116
K562
U2OS HeLa HepG2 HL60
CEM DNR
p53-
10
11
12
13
14
15
16
17
18
19
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32
33
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60
19a
19b
19c
19d
19e
19f
> 50
> 50
> 50
27
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
3.9
35
> 50
42
49
> 50
34
> 50 49.8
> 50 > 50
3.6
> 50
33
> 50
> 50
> 50
2.9
> 10 > 10 > 10
> 10 > 10 > 10
> 10 > 10 > 10
> 50 > 50
> 50
-
28
48
48
0.46
2.0
1.8
4.9
1.9
2.2
> 10 3.8
1.4
> 50
47
> 50 > 50
> 50
1.1
> 50
0.83
18
> 50 > 50
> 50
0.58
> 50
0.9
> 50
2.3
> 10 > 10 > 10
> 10 1.15 1.11
> 10 > 10 1.39
> 10 > 10 1.2
3.6
> 50
37
0.54
0.6
2.0
17
1.4
1.2
19g
19h
> 50
3.2
> 50
1.3
19
0.51
0.32
0.43 0.49
2.0
Out of the tested compounds 19a-h, the most cytotoxic were the 8-methyl derivative 19d, 8-amino
derivative 19f and 8-methylsulfanyl derivative 19h that exhibited low micromolar activity in most
leukemia and cancer cell lines and submicromolar activity in CCRF-CEM leukemia cell line.
Compounds 19d and 19f were less cytotoxic in non-cancerous BJ and MRC-5 fibroblasts, while 19h
showed micromolar cytotoxicity in BJ cells. In general, the methylpyrazolo-fused deazapurine
nucleosides 19a-h were found to be less cytotoxic than the previously reported methylpyrrolo-fused
nucleosides14 but still the compounds 19d and 19e show an interesting potential therapeutic window
between the cytotoxicity in the cancer cell lines and non-proliferating cells (Table 2).
The antiviral activity testing of compounds 19a-h was performed against influenza, hepatitis C virus
(HCV), herpes simplex, Coxsackie, human immunodeficiency virus (HIV) and dengue viruses. All
the nucleosides were inactive against most of these viruses. Only the anti-HCV screening37 showed
that the 8-methyl, 8-amino-, 8-methoxy- and 8-methylsulfanyl derivatives 19d,f,g,h exerted
submicromolar activity in 1B and 2A replicon assays (Table 3) with low toxicity against the Huh-7
cells. Unfortunately, the same compounds also exerted cytotoxicity at submicromolar concentrations
against several leukemia and cancer cell lines so the selectivity is not sufficient for further
development of these compounds as antivirals.
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Table 3. Antiviral activities of nucleosides 19a–h.
4
5
HCV replicon 1B
HCV replicon 2A
compd EC50 [µM] CC50 [µM] EC50 [µM] CC50 [µM]
6
7
8
9
19a
19b
19c
19d
19e
19f
13.9
>44.4
34.9
0.6
>44.4
>44.4
>44.4
>44.4
>44.4
35.1
30.4
>44.4
>44.4
0.3
>44.4
>44.4
>44.4
> 44.4
>44.4
> 44.4
> 44.4
>44.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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31
32
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34
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58
59
60
>44.4
> 44.4
0.5
>44.4
0.1
19g
19h
> 44.4
>44.4
0.2
0.3
0.08
Conclusion
We have developed the synthesis of novel pyrazolo-fused 7-deazapurine nucleobase 7 employing two
different synthetic approaches. Approach A involved a multi-step heterocyclization starting from the
chloro-nitro derivative 10 to give the key intermediate 7 in six steps in 18% overall yield. Approach
B started with the Negishi coupling of the zincated 4,6-dichloropyrimidine 14, followed by azidation
and thermal cyclization to give compound 7 with 13% yield over three steps. The heterocyclization
approach is more laborious (6 steps) but only two steps required simple chromatographic separation.
On the other hand, the cross-coupling approach is shorter (3 steps) but needs high catalyst loading to
achieve acceptable conversion, requires tedious chromatography after each step and involves
potentially hazardous thermal cyclization of nitrogen-rich intermediate 16. Three different methods
were tested for glycosylation of the nucleobase 7. Direct glycosylation under modified Mitsunobu
conditions gave only traces of the desired nucleoside. Nucleobase anion glycosylation was not
stereoselective and gave an inseparable mixture of the desired β-nucleoside with α-anomer in total
61% yield. The best result was observed with Vorbrüggen method, which led exclusively to the
desired β-anomeric nucleoside 17A in 51%. The key-intermediate nucleoside 17A was used for final
derivatization and deprotection to give a series of eight 8-substituted methylpyrazolo-fused 7-
deazapurine nucleosides 19a-h. The fused nucleosides exerted fluorescence with low to moderate
quantum yields. Biological testing showed submicromolar cytotoxic activities for the methyl, amino
and methylsulfanyl derivatives (19d, 19f, 19h) against several cancer and leukemia cell lines and
lower toxicity to non-proliferating fibroblasts. The same compounds, as well as the methoxy
derivative 19g, also exerted submicromolar antiviral effect against HCV in the replicon assay.
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7
Cl
Me
N
8
N
8a 8b
N
6
N
3a
3
4a
N
O
5'
O
1'
2'
6
4'
3'
O
8
9
1
O
5
C
2
O
O
2
1
4
3
10
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O
6
4
3
A
6
1
5
4
B
2
5
3
Figure 2: Numbering used in the assignment of NMR signals (example: 17A).
Experimental Section
Unless otherwise stated, all reactions were carried out under argon atmosphere. An oil bath was used
for reactions requiring heating. Thin-layer chromatography (TLC) was performed on Merck silica gel
60 F-254 aluminum sheets. Visualization was obtained by UV light (λmax = 254 nm or 366 nm) and/or
by staining with an anisaldehyde stain (9 mL anisaldehyde in 230 mL ethanol with 8 mL sulfuric acid
and 3 mL glacial acetic acid). High-performance flash chromatography (HPFC) was conducted with
a Combi Flash Rf instrument from Teledyne Isco Inc. using SiO2 (particle size 0.040–0.063 mm, 230–
400 mesh) from Merck in refillable flash columns or HP C18 Redi Sep Rf gold flash columns with
the solvent gradient indicated in the corresponding procedures. High Pressure Liquid
Chromatography – Mass Spectroscopy (HPLC-MS) for reaction control was performed on a 1260
Automated LC/UV Purification System with an InfinityLab Poroshell 120 EC-C18 column
(3.0 × 50 mm, particle size 2.7 µm) coupled with a 6530 Accurate-Mass QTOF LC/MS spectrometer
from Agilent Technologies Inc. The solvent system 0.1% formic acid in water / MeCN at
0.400 mL min–1 flow rate was run (gradient from 95:5 to 0:100 within 8 min, 0:100 for 5 min, 95:5
for 3 min). Melting points (m.p.) were measured by Bohunka Šperlichová at Charles University
Prague on a Büchi Melting Point B-545 apparatus using open glass capillaries and are uncorrected.
Optical rotation of final nucleosides was measured by the analytical laboratory at IOCB Prague using
an AUTOPOL IV automatic polarimeter from Rudolph Research Analytical at 20 °C and 589 nm. The
sample concentration c is given in g mL-1. 1H and 13C{1H} NMR spectra were measured on a Bruker
Avance II 500 MHz spectrometer at 20 °C. Chemical shifts (δ) are reported in ppm using the residual
deuterated solvent signal as an internal reference (CDCl3: δH = 7.26 ppm, δC = 77.16 ppm; DMSO-
d6: δH = 2.50 ppm, δC = 39.52 ppm). Coupling constants (J) are given in Hz and the multiplets are
described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and b (broad). 13C{1H}
NMR experiments are broadband proton-decoupled and were performed using APT pulse sequence.
DFQ-COSY, HSQC and HMBC experiments were used to assign the 1H and 13C NMR signals where
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required. ROESY experiments were used to confirm the relative stereochemistry of nucleosides. To
simplify the assignment, the benzoyl group attached to the 2’-hydroxy group of the ribofuranose ring
was given the letter A, the one attached to 3’-hydroxy group is considered B and the benzoyl group
at the 5’-OH is called C (Figure 2). All spectra can be found in S6 of the supporting information.
Infrared spectra (IR) were recorded on a Bruker ALPHA FT-IR spectrometer with a single-reflexion
Platinum ATR module. The compounds were measured in their initial state of appearance at 25 °C,
and their absorption bands were reported in wavenumbers (ṽ) in the range between 4000–600 cm–1.
Intensities are described as strong (s), medium (m) and weak (w). UV/Vis spectra were measured on
a Varian Cary 100 Bio UV-Visible spectrophotometer in the range 250–800 nm using transparent
1.5 mL quartz cuvettes. Fluorescence spectra were recorded on a Fluoromax 4 spectrofluorimeter
from HORIBA Scientific. The sample concentration was adjusted to have an UV absorbance of 0.05–
0.10. The excitation was performed at the absorption maximum with the highest wavelength λabs with
the slit set at 5 nm. The emission spectra were recorded from λabs + 20 nm to 2 × λabs with a 5 nm slit
opening. The absorption and emission spectra as well as the calculation of the fluorescence quantum
yield Φf can be found in the SI (S5). High-resolution mass spectrometry (HR-MS) was measured by
the MS-Service at IOCB Prague on an LTQ Orbitrap XL instrument from Thermo Fisher Scientific
using electrospray ionization (ESI) or on a Waters GCT Premier instrument using electron ionization
(EI). The purity of the final nucleosides (>95%) was confirmed by analytical HPLC on a Waters 717
Autosampler system with a Waters 2996 Photodiode Array Detector using a Gemini 5 µm C18 110 Å
column (250 × 4.60 mm) from Phenomenex (see Table S4 in the SI). Samples were dissolved in
DMSO (10 µL injection volume). Biological activity screening was performed as described in
previous works.33-37
10
11
12
13
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19
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50
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53
54
55
56
57
58
59
60
8-Chloro-1-methyl-1,4-dihydropyrazolo[3’,4’:4,5]pyrrolo[2,3-d]pyrimidine (7)
Method A: A suspension of 13 (451 mg, 2.38 mmol) in MeCN (4.5 mL) was treated with BTEA-Cl
(1.09 g, 4.80 mmol) and N,N-dimethylaniline (0.33 mL, 2.60 mmol). After 15 min, freshly distilled
POCl3 (1.35 mL, 14.48 mmol) was added dropwise and the brown mixture was stirred at 90 °C for
75 min, until HPLC-MS showed completion of the reaction (tR = 4.20 min, m/z = 208, 210). After
concentration in vacuo, ice was added followed by ammonia (1 M in water) until pH 4–5 was reached,
resulting in precipitation. Filtration and washing with ice-cold water (50 mL) gave crude 7 (425 mg,
86%, NMR pure) as a grey-brown solid.
Method B: A yellow mixture of 16 (72.2 mg, 0.306 mmol) and 1,4-dibromobenzene (365 mg,
1.55 mmol) was heated to 170 °C and stirred for 30 min. After cooling to r.t., the dark mixture was
dissolved in EtOAc. Purification by HPFC (SiO2; cHex/DCM 1:1/EtOAc, gradient 0 → 50% EtOAc)
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gave 7 (28.1 mg, 44%) as a white solid. Rf = 0.29 (SiO2; cHex/EtOAc 1:1); m.p. > 300 °C (dec.);
1H NMR (500 MHz, DMSO-d6): δ = 4.31 (s, 3 H; NMe), 7.72 (s, 1 H; H-3), 8.66 (s, 1 H; H-6),
5
6
13
12.46 ppm (bs, 1 H; NH); C{1H} NMR (125.7 MHz, DMSO-d6): δ = 39.7 (NMe), 103.5 (C-8a),
7
8
9
119.9 (C-3), 126.3 (C-8b), 129.9 (C-3a), 152.0 (C-6), 150.0 and 157.2 ppm (2 C; C-4a, C-8); IR
(ATR): ṽ = 3025 (w), 2851 (w), 1619 (w), 1575 (w), 1531 (m), 1436 (m), 1306 (w), 1234 (m), 1087
(s), 1015 (s), 982 (s), 967 (s), 939 (s), 819 (m), 797 (s), 777 (s), 632 (m), 614 cm–1 (m); HR MS (EI)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
for C8H6N5 Cl+ [M(35Cl)]+: calcd 207.0312, found 207.0313; for C8H6N5 Cl+ [M(37Cl)]+: calcd
35
37
209.0282, found 209.0271.
1-Methyl-4-nitro-1H-pyrazole (9)18
A colorless solution of pre-dried 4-nitro-1H-pyrazole (8, 20.0 g, 177 mmol) in DMF (100 mL) was
stirred at 20 °C for 15 min and treated with K2CO3 (29.4 g, 213 mmol) resulting in a white
suspension. After adding iodomethane (13.2 mL, 212 mmol), the pale-yellow suspension was stirred
at 20 °C for 18 hours. The resulting bright-yellow suspension was treated with water (200 mL) and
extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (600 mL),
dried over Na2SO4 and concentrated in vacuo. Purification by flash chromatography (SiO2;
Hex/EtOAc, gradient 0 → 50% EtOAc) gave 9 (22.1 g, 98%) as a white solid. Rf = 0.49 (SiO2;
1
Hex/EtOAc 1:1); m.p. = 91 °C (Lit.38: 91–92 °C); H NMR (500 MHz, CDCl3): δ = 3.95 (s, 3 H;
NMe), 8.01 (s, 1 H; H-3), 8.12 ppm (s, 1 H; H-5); 13C{1H} NMR (125.7 MHz, CDCl3): δ = 40.2
(NMe), 129.3 (C-5), 135.8 (C-3), 135.7 ppm (C-4); IR (ATR): ṽ = 3112 (m), 1504 (m), 1421 (m),
1398 (s), 1310 (s), 1129 (m), 999 (m), 968 (m), 885 (s), 818 (s), 753 (s), 692 (w), 648 cm–1 (w);
+
HR MS (EI) for C4H5N3O2 [M]+: calcd 127.0382, found 127.0383.
5-Chloro-1-methyl-4-nitro-1H-pyrazole (10)19
LiHMDS (260 mL, 1.0 M in THF, 260 mmol) was added dropwise (over 50 min) to a stirring solution
of 9 (22.04 g, 173.40 mmol) and hexachloroethane (42.6 g, 178.2 mmol) in DCM (340 mL). The
resulting bordeaux-red solution was stirred at 20 °C for 4 hours and then treated with water (400 mL).
After extraction, the organic layer was washed with sat. NaHCO3 (2 × 400 mL) and brine (400 mL),
dried over Na2SO4 and concentrated in vacuo. Purification by flash chromatography (SiO2;
Hex/EtOAc, gradient 0 → 50% EtOAc) gave 10 (24.6 g, 88%) as an off-white solid. Rf = 0.70 (SiO2;
1
Hex/EtOAc 1:1); m.p. = 67 °C (Lit.39: 59–60 °C); H NMR (500 MHz, CDCl3): δ = 3.92 (s, 3 H;
13
NMe), 8.14 ppm (s, 1 H; H-3); C{1H} NMR (125.7 MHz, CDCl3): δ = 37.6 (NMe), 127.6 (C-5),
130.4 (C-4), 136.6 ppm (C-3); IR (ATR): ṽ = 3124 (w), 1493 (s), 1397 (s), 1312 (s), 1180 (m), 1129
(m), 1047 (w), 975 (m), 883 (m), 827 (s), 756 (s), 735 (m), 588 cm–1 (w); HR MS (EI) for
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C4H4N3O2 Cl+ [M(35Cl)]+: calcd 160.9992, found 160.9991; for C4H4N3O2 Cl+ [M(37Cl)]+: calcd
162.9963, found 162.9972.
5
6
7
8
9
Ethyl 2-cyano-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)acetate (11)
10
11
12
13
14
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A white suspension of pre-dried potassium tert-butoxide (14.6 g, 130.4 mmol) in 1,4-dioxane
(120 mL) was treated with ethyl cyanoacetate (13.2 mL, 123.8 mmol) and stirred at 20 °C for 15 min.
Pre-dried 10 (10.0 g, 61.9 mmol) was dissolved in 1,4-dioxane (80 mL) and added via syringe. The
reaction mixture was stirred at 100 °C for 48 hours. This orange suspension was treated with water
(~20 mL) resulting in a red solution; followed by addition of hydrochloric acid (~50 mL, 1 M in water)
until pH = 2 was reached. After extraction with Et2O (3 × 200 mL), the combined organic layers were
washed with brine (400 mL), dried over MgSO4 and concentrated in vacuo giving a brown oil. Excess
of unreacted ethyl cyanoacetate was removed by vacuum distillation at 95 °C / 24 mbar prior to
solidification at –18 °C during 1 week. The solid was then washed with an ice-cold DCM and dried
in vacuo giving 11 (14.3 g, 97%) as colorless crystals. Rf = 0.38 (SiO2; DCM/MeOH 95:5);
1
m.p. = 140 °C; H NMR (500 MHz, CDCl3): δ = 1.36 (t, J2’’,1’’ = 7.2 Hz, 3 H; H-2’’), 4.03 (s, 3 H;
NMe), 4.38 (m, 2 H; H-1’’), 6.18 (s, 1 H; H-1’), 8.16 ppm (s, 1 H; H-3); 13C{1H} NMR (125.7 MHz,
CDCl3): δ = 14.1 (C-2’’), 33.1 (C-1‘), 39.0 (NMe), 65.0 (C-1’’), 111.7 (CN),129.7 (C-5), 133.4 (C-
4), 136.4 (C-3), 161.4 ppm (COO); IR (ATR): ṽ = 3118 (w), 2990 (w), 2945 (w), 2899 (w), 2262 (w),
1743 (s), 1560 (m), 1509 (s), 1497 (s), 1453 (m), 1445 (m), 1398 (m), 1310 (s), 1261 (s), 1221 (m),
1190 (m), 1027 (s), 1000 (w), 984 (m), 894 (w), 856 (m), 825 (s), 795 (w), 759 (m), 705 (w), 644 cm–1
(w); HR MS (ESI) for C9H10N4O4Na+ [M + Na]+: calcd 261.0594, found 261.0594.
Ethyl 5-amino-1-methyl-1,4-dihydropyrrolo[3,2-c]pyrazole-6-carboxylate (12)
A solution of 11 (3.93 g, 16.5 mmol) in glacial acetic acid (37 mL) was heated to 80 °C; and under
an argon stream, treated with zinc dust (5.49 g, 83.9 mmol) in small portions in order to avoid
extensive gas formation. After stirring at 95 °C for 2 hours, HPLC-MS showed completion of the
reaction (tR = 2.53 min, m/z = 209). The greenish suspension was cooled down and the zinc species
was removed by filtration through a celite plug (5 cm) followed by washing with fresh acetic acid
(~50 mL). The filtrate was concentrated in vacuo giving a brown oil. Neutralization with sat. NaHCO3
(~50 mL) resulted in precipitation of the cyclized product. Filtration and washing with cold water
gave 12 (1.91 g, 55%) as a light-grey solid. Rf = 0.33 (SiO2; (DCM with 1% TEA)/MeOH 9:1);
m.p. > 300 °C (dec.); 1H NMR (500 MHz, DMSO-d6): δ = 1.28 (t, J2’,1’ = 7.1 Hz, 3 H; H-2’), 3.96 (s,
3 H; NMe), 4.18 (q, J1’,2’ = 7.1 Hz, 2 H; H-1’), 6.40 (bs, 2 H; NH2), 6.99 (s, 1 H; H-3), 10.01 ppm
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(bs, 1 H; NH); C{1H} NMR (125.7 MHz, DMSO-d6): δ = 14.6 (C-2’), 38.1 (NMe), 58.4 (C-1’),
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118.5 (C-3), 120.3 (C-3a), 134.3 (C-6a), 164.2 ppm (COO); IR (ATR): ṽ = 3337 (broad w), 1652 (w),
1610 (w), 1427 (s), 1379 (m), 1322 m), 1278 (w), 1238 (w), 1121 (w), 1095 (w), 962 (w), 843 (m),
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754 (m), 694 cm–1 (w); HR MS (ESI) for C9H13N4O2 [M]+: calcd 209.1033, found 209.1033.
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1-Methyl-4,7-dihydropyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidin-8(1H)-one (13)
Compound 12 (1.0 g, 4.8 mmol) was treated with sodium methoxide (2.2 mL, 25 wt% in MeOH,
9.62 mmol) and sonicated for 10 min to improve solubility. After adding formamide (3.82 mL,
96.15 mmol), the solution was stirred at 90 °C for 48 hours, until HPLC-MS showed completion of
the reaction (tR = 1.07 min, m/z = 190). Ice was added; followed by addition of conc. HCl (0.85 mL,
35 wt% in water, 9.6 mmol) resulting in precipitation. Filtration and washing with ice-cold water
(100 mL) gave 13 (418 mg, 46%) as a brown solid. Rf = 0.38 (SiO2; DCM/MeOH 9:1); m.p. > 300 °C
(dec.); 1H NMR (500 MHz, DMSO-d6): δ = 4.16 (s, 3 H; NMe), 7.46 (s, 1 H; H-3), 7.99 (s, 1 H; H-6),
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11.75 (bs, 1 H; H-4), 12.19 ppm (bs, 1 H; H-7); C{1H} NMR (125.7 MHz, DMSO-d6): δ = 38.4
(NMe), 93.9 (C-8a), 119.9 (C-3), 125.2 (C-3a), 132.0 (C-8b), 145.8 (C-6), 154.6 and 157.1 ppm (2 C;
C-4a, C-8); IR (ATR): ṽ = 3130 (m), 3066 (m), 3017 (m), 2898 (m), 1669 (s), 1619 (m), 1584 (m),
1537 (m), 1495 (w), 1424 (w), 1362 (m), 1330 (s), 1291 (m), 1236 (w), 1105 (m), 1014 (m), 990 (m),
884 (m), 860 (m), 825 (m), 797 (m), 777 (m), 721 (m), 687 (m), 640 cm–1 (m); HR MS (ESI) for
C8H7N5ONa+ [M + Na]+: calcd 212.0543, found 212.0544.
4,6-Dichloro-5-(1-methyl-1H-pyrazol-5-yl)pyrimidine (15)
ZnCl2 (789 mg, 5.79 mmol) was pre-dried at 250 °C for 20 min and allowed to cool down under
vacuum before being treated with a solution of (TMP)MgCl · LiCl (11.5 mL, 1.0 M solution in
THF/toluene, 11.5 mmol) and stirred at 20 °C for 24 hours. The solution was cooled to 0 °C
(water/ice) and treated with a solution of 4,6-dichloropyrimidine 14 (1.41 g, 9.45 mmol) in THF
(8 mL). After 1 hour, the solution was stirred at 20 °C for an additional hour resulting in the zincated
4,6-dichloropyrimidine. Separately, a solution of 5-iodo-1-methyl-1H-pyrazole (4.00 g, 19.24 mmol)
and Pd(PPh3)4 (2.22 g, 1.92 mmol) in THF (8 mL) was pre-stirred at 20 °C for 20 min before addition
to the zincated pyrimidine. The resulting solution was stirred at 65 °C for 18 hours. After adding
water (2 mL), the mixture was loaded on silica and filtered through a silica plug (1 cm) with DCM
(500 mL). Purification by HPFC (SiO2; cHex/EtOAc, gradient 0 → 50% EtOAc) gave 15 (925.7 mg,
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42%) as a yellow solid. Rf = 0.41 (SiO2; cHex/EtOAc 2:1); m.p. = 86 °C; H NMR (500 MHz,
CDCl3): δ = 3.73 (s, 3 H; NMe), 6.39 (d, J4’,3’ = 2.0 Hz, 1 H; H-4’), 7.62 (d, J3’,4’ = 2.0 Hz, 1 H; H-
3’), 8.86 ppm (s, 1 H; H-2); 13C{1H} NMR (125.7 MHz, CDCl3): δ = 37.2 (NMe), 108.4 (C-4’), 124.9
(C-5), 133.4 (CH-5’), 139.3 (C-3’), 158.5 (C-2), 162.9 ppm (2 C; C-4, C-6); IR (ATR): ṽ = 3127 (w),
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2919 (w), 2850 (w), 1572 (w), 1518 (m), 1505 (m), 1470 (w), 1436 (w), 1404 (m), 1378 (m), 1359
(m), 1291 (w), 1272 (w), 1235 (w), 1200 (w), 1166 (w), 1094 (w), 1063 (w), 1029 (w), 975 (w), 926
(w), 807 (s), 782 (m), 774 (m), 744 (w), 698 (m), 685 (w), 649 (w), 599 cm–1 (w); HR MS (ESI) for
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C8H7N4 Cl2
[M(35Cl) + H]+: calcd 229.00478, found 229.00429; C8H7N4 Cl37Cl+
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[M(35Cl37Cl) + H]+: calcd 231.00183, found 231.00139; for C8H7N4 Cl2 [M(37Cl) + H]+: calcd
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+
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232.9989, found 232.9988.
4-Azido-6-chloro-5-(1-methyl-1H-pyrazol-5-yl)pyrimidine (16a) / 7-chloro-8-(1-methyl-1H-
pyrazol-5-yl)tetrazolo[1,5-c]pyrimidine (16b)
A solution of 15 (271 mg, 1.18 mmol) in THF (6 mL) was treated with sodium azide (77.6 mg,
1.18 mmol) and lithium chloride (52.8 mg, 1.24 mmol) and stirred under light exclusion at 23 °C for
26 hours. After adding water (1 mL), the mixture was concentrated in vacuo. Purification by HPFC
(SiO2; cHex/EtOAc, gradient 0 → 25% EtOAc) gave 16 (196 mg, 70%) as a light-yellow oil.
Rf = 0.40 (SiO2; cHex/EtOAc 2:1); 1H NMR of 16a (500 MHz, CDCl3): δ = 3.73 (s, 3 H; NMe), 6.33
(d, J4’,3’ = 2.0 Hz, 1 H; H-4’), 7.60 (d, J3’,4’ = 2.0 Hz, 1 H; H-3’), 8.76 ppm (s, 1 H; H-2);
13C{1H} NMR of 16a (125.7 MHz, CDCl3): δ = 37.3 (NMe), 108.5 (C-4’), 113.7 (C-5), 132.3 (C-5’),
139.1 (C-3’), 158.4 (C-2), 162.3 and 163.2 ppm (2 C; C-4, C-6); tautomer 16b was not observed in
CDCl3; 1H NMR of 16a (500 MHz, DMSO-d6): δ = 3.67 (s, 3 H; NMe), 6.40 (d, J4’,3’ = 2.0 Hz, 1 H;
H-4’), 7.54 (d, J3’,4’ = 2.0 Hz, 1 H; H-3’), 8.94 ppm (s, 1 H; H-2); 13C{1H} NMR (125.7 MHz,
DMSO-d6): δ = 36.8 (NMe), 108.3 (C-4’), 113.2 C-5), 132.1 (C-5’), 138.3 (C-3’), 158.5 (C-2),
162.6 ppm (2 C; C-4, C-6); 1H NMR of 16b (500 MHz, DMSO-d6): δ = 3.74 (s, 3 H; NMe), 6.63 (d,
J4’,3’ = 2.0 Hz, 1 H; H-4’), 7.68 (d, J3’,4’ = 2.0 Hz, 1 H; H-3’), 10.35 ppm (s, 1 H; H-5); 13C{1H} NMR
of 16b (125.7 MHz, DMSO-d6): δ = 37.6 (NMe), 109.0 (C-4’), 112.6 (c-8), 131.5 (C-5’), 138.7 (C-
3’), 140.4 (C-5), 147.8 and 150.5 ppm (2 C; C-7, C-9); IR (ATR): ṽ = 3374 (broad w), 2931 (w),
2856 (w), 2322 (w), 2143 (s), 2082 (w), 1604 (w), 1581 (w), 1549 (m), 1530 (s), 1464 (w), 1403 (s),
1385 (m), 1369 (m), 1321 (m), 1294 (m), 1276 (m), 1242 (m), 1203 (m), 1180 (m), 1150 (m), 1079
(w), 1057 (w), 976 (m), 936 (w), 897 (m), 848 (w), 786 (m), 752 (m), 701 (m), 649 cm–1 (w); HR MS
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(EI) for C8H6N7 Cl+ [M(35Cl)]+: calcd 235.0373, found 235.0374; for C8H6N7 Cl+ [M(37Cl)]+: calcd
237.0344, found 237.0393.
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8-Chloro-1-methyl-4-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-
d]pyrimidine (17A)
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A suspension of nucleobase 7 (406 mg, 1.96 mmol) in MeCN (18 mL) was treated with BSA
(0.72 mL, 2.95 mmol), sonicated for 30 min to maximize solubility and stirred at 60 °C for 1 hour.
A solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (1.99 g, 3.95 mmol; dried under
vacuum at 60 °C for 6 hours) in MeCN (2 mL) was added, followed by TMSOTf (0.72 mL,
3.95 mmol). After stirring at 60 °C for 36 hours, the resulting mixture was treated with water (20 mL)
and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with sat. NaHCO3
(100 mL) and water (100 mL), dried over MgSO4 and concentrated in vacuo. Purification by HPFC
(SiO2; cHex/EtOAc, gradient 0 → 50% EtOAc) gave 17A (407 mg, 51%) as a pearl-colored foam. A
small amount was recrystallized from DCM/MeOH prior to characterization.
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Rf = 0.64 (SiO2; cHex/EtOAc 1:1); m.p. = 97 °C; H NMR (500 MHz, CDCl3): δ = 4.38 (s, 3 H;
NMe), 4.68 (dd, J5’a,5’b = 12.0 Hz, J5’a,4’ = 3.6 Hz, 1 H; H-5’a), 4.82 (m, 1 H; H-4’), 4.85 (dd, J5’b,5’a
= 12.0 Hz, J5’b,4’ = 2.9 Hz, 1 H; H-5’b), 6.11 (dd, J3’,2’ = 5.9 Hz, J3’,4’ = 4.4 Hz, 1 H; H-3’), 6.33 (t,
J2’,1’ = J2’,3’ = 5.9 Hz, 1 H; H-2’), 6.88 (d, J1’,2’ = 5.9 Hz, 1 H; H-1’), 7.35 (m, 2 H; H-A3, H-A5), 7.42
(m, 2 H; H-B3, H-B5), 7.46 (m, 2 H; H-C3, H-C5), 7.53 (tt, JA4,A3 = JA4,A5 = 7.5 Hz, JA4,A2 = JA4,A6
=
1.3 Hz, 1 H; H-A4), 7.60 (m, 2 H; H-B4, H-C4), 7.67 (s, 1 H; H-3), 7.90 (dd, JA2,A3 = JA6,A5 = 8.4 Hz,
JA2,A4 = JA6,A4 = 1.3 Hz, 2 H; H-A2, H-A6), 8.01 (dd, JB2,B3 = JB6,B5 = 8.4 Hz, JB2,B4 = JB6,B4 = 1.3 Hz,
2 H; H-B2, H-B6), 8.05 (dd, JC2,C3 = JC6,C5 = 8.4 Hz, JC2,C4 = JC6,C4 = 1.3 Hz, 2 H; H-C2, H-C6),
8.69 ppm (s, 1 H; H-6); 13C{1H} NMR (125.7 MHz, CDCl3): δ = 40.2 (NMe), 63.8 (C-5’), 71.3 (C-
3’), 72.6 (C-2’), 80.1 (C-4’), 85.7 (C-1’), 106.0 (C-8a), 120.2 (C-3), 127.5 (C-8b), 128.5, 128.8 and
129.3 (3 C; C-A1, C-B1, C-C1), 128.6 (2 C; C-A3, C-A5), 128.7 and 128.8 (4 C; C-B3, C-B5, C-C3,
C-C5), 129.0 (C-3a), 129.8, 129.9 and 130.0 (6 C; C-A2, C-A6, C-B2, C-B6, C-C2, C-C6), 133.7 and
133.9 (3 C; C-A4, C-B4, C-C4), 149.5 (C-8), 152.5 (C-6), 157.4 (C-4a), 165.2 (C=O A), 165.6 (C=O
B), 166.3 ppm (C=O C); IR (ATR): ṽ = 2927 (very w), 1720 (m), 1601 (w), 1553 (w), 1495 (w), 1450
(w), 1372 (w), 1315 (w), 1262 (s), 1176 (w), 1093 (m), 1067 (m), 1024 (m), 978 (m), 936 (m), 805
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(w), 706 (s), 686 (m), 616 cm–1 (m); HR MS (ESI) for C34H26N5O7 ClNa+ [M(35Cl) + Na]+: calcd
37
674.1413, found 674.1405; for C34H26N5O7 ClNa+ [M(37Cl) + Na]+: calcd 676.1389, found
676.1362.
8-Chloro-1-methyl-4-(2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-β-D-
ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (17B-β) & α-anomer (17B-α)
A solution of the previously synthesized halosugar precursor, 2,3-O-isopropylidene-5-O-tert-
butyldimethylsilyl-β-D-ribofuranose28 (117 mg, 0.37 mmol) in THF (1.8 mL) was cooled to –30 °C
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and treated with CCl4 (47 µL, 0.48 mmol). Then, HMPT (86 µL, 0.48 mmol) was added dropwise.
The resulting green solution was stirred vigorously for 1 hour resulting in the desired halosugar. In a
separate flask, a suspension of nucleobase 7 (50.0 mg, 0.24 mmol) in MeCN (1.5 mL) and powdered
KOH (37.5 mg, 0.67 mmol) was treated with TDA-1 (77 µL, 0.24 mmol) and stirred for 30 min. After
cooling it to 0 °C (water/ice), the halosugar solution was added via syringe. The resulting mixture
was first stirred at 0 °C for 1 hour, then at 23 °C for 36 hours before being treated with water
(0.25 mL). After concentration in vacuo, purification by HPFC (SiO2; cHex/EtOAc, gradient 0 →
20% EtOAc) gave an inseparable mixture of 17B-β/17B-α 1:0.7 (70.4 mg, 59%) as white sticky solid.
Collecting of several early fractions gave a small sample of pure β-anomer 17B-β (3.2 mg, 2%) as a
white amorphous solid, which was used for characterization: Rf = 0.50 (SiO2; cHex/EtOAc 2:1);
1H NMR (500 MHz, CDCl3): δ = 0.00 (s, 3 H; SiMe2), 0.03 (s, 3 H; SiMe2), 0.86 (s, 9 H; SiCMe3),
1.39 (s, 3 H; OCMe2), 1.65 (s, 3 H; OCMe2), 3.81 (dd, J5’a,5’b = 11.3 Hz, J5’a,4’ = 3.9 Hz, 1 H; H-5’a),
3.86 (dd, J5’b,5’a = 11.3 Hz, J5’b,4’ = 3.8 Hz, 1 H; H-5’b), 4.29 (q, J4’,3’ = J4’,5’a = J4’,5’b = 3.7 Hz, 1 H;
H-4’), 4.41 (s, 3 H; NMe), 4.98 (dd, J3’,2’ = 6.5 Hz, J3’,4’ = 3.6 Hz, 1 H; H-3’), 5.21 (dd, J2’,3’ = 6.6 Hz,
J2’,1’ = 3.6 Hz, 1 H; H-2’), 6.49 (d, J1’,2’ = 3.6 Hz, 1 H; H-1’), 7.66 (s, 1 H; H-3), 8.71 ppm (s, 1 H; H-
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6); C{1H} NMR (125.7 MHz, CDCl3): -5.3 (SiMe2), -5.2 (SiMe2), 18.7 (SiCMe3), 25.7 (OCMe2),
26.1 (SiCMe3), 27.5 (OCMe2), 40.2 (NMe), 63.1 (C-5’), 80.6 (C-3’), 83.4 (C-2’), 85.4 (C-4’), 89.7
(C-1’), 105.6 (C-8a), 115.0 (OCMe2), 121.1 (C-3), 127.3 (C-8b), 129.7 (C-3a), 149.4 (C-8), 152.5
(C-6), 156.7 ppm (C-4a); HR MS (ESI) for C22H33N5O4Si35Cl+ [M(35Cl) +H]+: calcd 494.1990, found
494.1992; for C22H32N5O4Si35ClNa+ [M(35Cl) + Na]+: calcd 516.1810, found 516.1812.
8-(Furan-2-yl)-1-methyl-4-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (18a)
A solution of 17A (300 mg, 0.46 mmol) in DMF (4.6 mL) was treated with PdCl2(PPh3)2 (35.7 mg,
0.05 mmol) and 2-(tributylstannyl)furan (0.175 mL, 0.55 mmol). After stirring at 100 °C for
10 hours, DMF was removed in vacuo. EtOAc (2.5 mL) and a solution of KF (32 mg, 0.551 mmol)
in MeOH (2.5 mL) were added. The mixture was concentrated in vacuo and filtered through a silica
plug (5 cm) with cHex (100 mL) followed by EtOAc (500 mL). Purification by HPFC (SiO2;
cHex/EtOAc, gradient 0 → 100% EtOAc) gave 18a (216 mg, 69%) as a pale-yellow foam. Rf = 0.40
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(SiO2; cHex/EtOAc 1:1); m.p. = 71 °C; H NMR (500 MHz, CDCl3): δ = 4.27 (s, 3 H; NMe), 4.72
(m, 1 H; H-5’a), 4.81–4.87 (m, 2 H; H-4’, H-5’b), 6.12 (dd, J3’,2’ = 6.0 Hz, J3’,4’ = 4.3 Hz, 1 H; H-3’),
6.33 (t, J2’,1’ = J2’,3’ = 6.1 Hz, 1 H; H-2’), 6.76 (dd, J4,3 = 3.5 Hz, J4,5 = 1.7 Hz, 1 H; H-4-furyl), 6.99
(d, J1’,2’ = 6.3 Hz, 1 H; H-1’), 7.35, 7.42 and 7.46 (3 × m, 3 × 2 H; H-A3, H-A5, H-B3, H-B5, H-C3,
H-C5), 7.48 (dd, J3,4 = 3.5 Hz, J3,5 = 0.9 Hz, 1 H; H-3-furyl), 7.53, 7.58 and 7.59 (3 × m, 3 H; H-A4,
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H-B4, HC4), 7.70 (s, 1 H; H-3), 7.75 (bd, J5,4 = 1.7 Hz, 1 H; H-5-furyl), 7.91, 8.02 and 8.09 (3 × m,
3 × 2 H; H-A2, H-A6, H-B2, H-B6, H-C2, H-C6), 8.89 ppm (s, 1 H; H-6); 13C{1H} NMR
(125.7 MHz, CDCl3): δ = 41.8 (NMe), 64.0 (C-5’), 71.3 (C-3’), 72.5 (C-2’), 80.1 (C-4’), 85.3 (C-1’),
101.5 (C-8a), 113.9 (C-4-furyl), 119.9 (C-3), 128.5, 128.9 and 129.4 (3 C; C-A1, C-B1, C-C1), 128.6
(C-8b), 128.6, 128.7 and 128.8 (6 C; C-A3, C-A5, C-B3, C-B5, C-C3, C-C5), 129.9 (C-3a), 129.9,
130.0 and 130.1 (6 C; C-A2, C-A6, C-B2, C-B6, C-C2, C-C6), 133.6, 133.8 and 133.9 (3 C; C-A4,
C-B4, C-C4), 145.1 (C-5-furyl), 150.4 (C-2-furyl), 151.2 (C-6), 158.1 (C-4a), 165.2, 165.6 and
166.3 ppm (3 C; C=O), C-8 and C-3-furyl hidden by the noise; IR (ATR): ṽ = 2956 (w), 1720 (s),
1599 (w), 1555 (w), 15332 (w), 1488 (w), 1442 (m), 1414 (w), 1315 (w), 1261 (s), 1176 (w), 1117
(m), 1092 (m), 1067 (m), 1024 (m), 1009 (m), 973 (m), 944 (w), 885 (w), 793 (m), 745 (w), 705 (s),
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685 (w), 616 cm–1 (w); HR MS (ESI) for C38H30N5O8 [M + H]+: calcd 684.2089, found 684.2086;
for C38H29N5O8Na+ [M + Na]+: calcd 706.1908, found 706.1905.
8-(Furan-3-yl)-1-methyl-4-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (18b)
A solution of 17A (300 mg, 0.46 mmol) in toluene (3.1 mL) was treated with furan-3-ylboronic acid
(77.1 mg, 0.69 mmol), K2CO3 (127 mg, 0.92 mmol) and Pd(PPh3)4 (54.88 mg, 0.05 mmol). After
stirring at 100 °C for 48 hours, the suspension was filtered through a celite plug (2 cm). Purification
by HPFC (SiO2; cHex/EtOAc, gradient 0 → 25% EtOAc) gave 18b (101 mg, 32%) as a yellow film.
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Rf = 0.56 (SiO2; cHex/EtOAc 1:1); H NMR (500 MHz, CDCl3): δ = 3.81 (s, 3 H; NMe), 4.70 (m,
1 H; H-5’a), 4.83 (m, 1 H; H-4’), 4.84 (m, 1 H; H-5’b), 6.15 (dd, J3’,2’ = 6.0 Hz, J3’,4’ = 4.3 Hz, 1 H;
H-3’), 6.38 (t, J2’,1’ = J2’,3’ = 6.1 Hz, 1 H; H-2’), 6.85 (dd, J4,5 = 1.8 Hz, J4,2 = 0.8 Hz, 1 H; H-4-furyl),
6.98 (d, J1’,2’ = 6.2 Hz, 1 H; H-1’), 7.34, 7.40 and 7.45 (3 × t, J = 7.7 Hz, 3 × 2 H; H-A3, H-A5, H-
B3, H-B5, H-C3, H-C5), 7.49–7.59 (3 × m, 3 H; H-A4, H-B4, H-C4), 7.61 (t, J5,4 = J5,2 = 1.7 Hz,
1 H; H-5-furyl), 7.66 (s, 1 H; H-3), 7.91 (dd, J2,5 = 1.5 Hz, J2,4 = 0.8 Hz, 1 H; H-2-furyl), 7.92, 8.01
and 8.09 (3 × m, 3 × 2 H; H-A2, H-A6, H-B2, H-B6, H-C2, H-C6), 8.92 ppm (s, 1 H; H-6);
13C{1H} NMR (125.7 MHz, CDCl3): δ = 40.4 (NMe), 63.9 (C-5’), 71.3 (C-3’), 72.4 (C-2’), 79.8 (C-
4’), 85.1 (C-1’), 105.9 (C-8a), 111.2 (C-4-furyl), 120.5 (C-3), 125.0 (C-3-furyl), 128.5, 128.6 and
128.7 (6 C; C-A3, C-A5, C-B3, C-B5, C-C3, C-C5), 128.8, 128.9, 129.1 and 129.4 (5 C; C-A1, C-
B1, C-C1, C-3a, C-8b), 129.8 and 129.9 (6 C; C-A2, C-A6, C-B2, C-B6, C-C2, C-C6), 133.5, 133.8
and 133.8 (3 C; C-A4, C-B4, C-C4), 142.9 (C-2-furyl), 143.9 (C-5-furyl), 150.4 (C-8), 152.8 (C-6),
157.6 (C-4a), 165.2, 165.6 and 166.2 ppm (3 C; C=O); IR (ATR): ṽ = 2924 (w), 2852 (w), 1722 (s),
1602 (w), 1562 (w), 1525 (w), 1492 (w), 1451 (w), 1368 (w), 1315 (w), 1260 (s), 1177 (w), 1119
(m), 1089 (m), 1068 (m), 1024 (m), 1000 (m), 977 (m), 906 (w), 802 (w), 706 (s), 685 (m), 615 (w),
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601 cm–1 (w); HR MS (ESI) for C38H30N5O8 [M + H]+: calcd 684.2089, found 684.2086; for
C38H29N5O8Na+ [M + Na]+: calcd 706.1908, found 706.1903.
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ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (18c)
A solution of 17A (301 mg, 0.46 mmol) in toluene (3.1 mL) was treated with benzofuran-2-ylboronic
acid (112 mg, 0.69 mmol), K2CO3 (128.3 mg, 0.93 mmol) and Pd(PPh3)4 (54.7 mg, 0.05 mmol).
After stirring at 100 °C for 48 hours, the suspension was filtered through a celite plug (2 cm).
Purification by HPFC (SiO2; cHex/EtOAc, gradient 0 → 25% EtOAc) gave 18c (127 mg, 38%) as a
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colorless film. Rf = 0.74 (SiO2; cHex/EtOAc 1:1); H NMR (500 MHz, CDCl3): δ = 4.31 (s, 3 H;
NMe), 4.73 (m, 1 H; H-5’a), 4.84 (m, 1 H; H-4’), 4.85 (m, 1 H; H-5’b), 6.16 (dd, J3’,2’ = 6.0 Hz, J3’,4’
= 4.4 Hz, 1 H; H-3’), 6.40 (t, J2’,1’ = J2’,3’ = 6.1 Hz, 1 H; H-2’), 7.01 (d, J1’,2’ = 6.2 Hz, 1 H; H-1’),
7.32–7.37 (m, 3 H; H-5-benzofuryl, H-C3, H-C5), 7.38–7.49 (m, 5 H; H-6-benzofuryl, H-A3, H-A5,
H-B3, H-B5), 7.52 (m, 1 H; H-C4), 7.55–7.61 (m, 3 H; H-7-benzofuryl, H-A4, H-B4), 7.73 (s, 1 H;
H-3), 7.76 (m, J4,5 = 7.8 Hz, 1 H; H-4-benzofuryl), 7.79 (bs, 1 H; H-3-benzofuryl), 7.93, 8.02 and
8.09 (3 × m, 3 × 2 H; H-A2, H-A6, H-B2, H-B6, H-C2, H-C6), 8.93 ppm (s, 1 H; H-6);
13C{1H} NMR (125.7 MHz, CDCl3): δ = 41.7 (NMe), 64.0 (C-5’), 71.3 (C-3’), 72.4 (C-2’), 80.0 (C-
4’), 85.3 (C-1’), 102.9 (C-8a), 110.0 (C-3-benzofuryl), 111.5 (C-7-benzofuryl), 119.9 (C-3), 122.8
(C-4-benzofuryl), 124.2 (C-5-benzofuryl), 126.6 (C-6-benzofuryl), 128.4, 128.8, 128.9 and 129.4
(6 C; C-3a, C-8b, C-3a-benzofuryl, C-A1, C-B1, C-C1), 128.6, 128.7 and 128.7 (6 C; C-A3, C-A5,
C-B3, C-B5, C-C3, C-C5), 129.8 and 129.9 (6 C; C-A2, C-A6, C-B2, C-B6, C-C2, C-C6), 133.6,
133.7 and 133.8 (3 C; C-A4, C-B4, C-C4), 146.0 (C-8), 152.2 (C-6), 153.4 (C-2-benzofuryl), 155.5
(C-7a-benzofuryl), 158.4 (C-4a), 165.2, 165.6 and 166.3 ppm (3 C; C=O); IR (ATR): ṽ = 2969 (w),
2953 (w), 2882 (w), 2249 (w), 2180 (w), 2153 (w), 2042 (w), 2016 (w), 1987 (w), 1979 (w), 1970
(w), 1922 (w), 1870 (w), 1718 (m), 1583 (m), 1543 (s), 1498 (m), 1451 (w), 1431 (m), 1408 (m),
1364 (m), 1315 (m), 1245 (s), 1119 (m), 1081 (s), 1054 (s), 1046 (s), 1021 (s), 994 (s), 975 (s), 949
(m), 814 (m), 803 (m), 777 (m), 734 (m), 716 (s), 709 (s), 688 (m), 676 (m), 669 (m), 649 (m), 621
+
(m), 610 (s), 598 cm–1 (s); HR MS (ESI) for C42H32N5O8 [M + H]+: calcd 734.2245, found 734.2239;
for C42H31N5O8Na+ [M + H]+: calcd 756.2065, found 756.2060.
1,8-Dimethyl-4-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-
d]pyrimidine (18d)
A solution of 17A (249 mg, 0.38 mmol) in THF (10.1 mL) was treated with Pd(PPh3)4 (23.6 mg,
0.02 mmol) and Me3Al (0.38 mL, 2.0 M in toluene, 0.78 mmol). After stirring at 65 °C for 3 hours,
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the resulting red-brown solution was treated with MeOH (1 mL) before being concentrated in vacuo.
Purification by HPFC (SiO2; cHex/EtOAc, gradient 25 → 75% EtOAc) gave 18d (156 mg, 64%) as
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an off-white foam. Rf = 0.19 (SiO2; cHex/EtOAc 1:2); m.p. = 79 °C; H NMR (500 MHz, CDCl3):
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δ = 2.98 (s, 3 H; CMe), 4.30 (s, 3 H; NMe), 4.68 (m, 1 H; H-5’a), 4.80 (m, 1 H; H-4’), 4.82 (m, 1 H;
H-5’b), 6.12 (dd, J3’,2’ = 5.9 Hz, J3’,4’ = 4.2 Hz, 1 H; H-3’), 6.43 (t, J2’,1’ = J2’,3’ = 6.0 Hz, 1 H; H-2’),
6.93 (d, J1’,2’ = 6.2 Hz, 1 H; H-1’), 7.33 (m, 2 H; H-A3, H-A5), 7.40 (m, 2 H; H-B3, H-B5), 7.45 (m,
2 H; H-C3, H-C5), 7.49–7.61 (m, 3 H; H-A4, H-B4, H-C4), 7.63 (s, 1 H; H-3), 7.90 (m, 2 H; H-A2,
H-A6), 8.00 (m, 2 H; H-B2, H-B6), 8.07 (m, 2 H; H-C2, H-C6), 8.82 ppm (s, 1 H; H-6);
13C{1H} NMR (125.7 MHz, CDCl3): δ = 24.6 (CMe), 40.1 (NMe), 63.9 (C-5’), 71.3 (C-3’), 72.4 (C-
2’), 79.8 (C-4’), 85.1 (C-1’), 106.4 (C-8a), 120.2 (C-3), 128.5 (C-3a), 128.5, 128.6 and 128.7 (6 C;
C-A3, C-A5, C-B3, C-B5, C-C3, C-C5), 128.6, 128.9 and 129.2 (3 C; C-A1, C-B1, C-C1), 129.4 (C-
8b), 129.8 and 129.9 (6 C; C-A2, C-A6, C-B2, C-B6, C-C2, C-C6); 133.5, 133.7 and 133.80 (3 C; C-
A4, C-B4, C-C4), 152.8 (C-6), 156.5 (C-8), 157.0 (C-4a), 165.2 (C=O A), 165.6 (C=O B), 166.2 ppm
(C=O C); IR (ATR): ṽ = 3060 (w), 2923 (w), 2853 (w), 1720 (m), 1601 (w), 1558 (w), 1499 (w),
1451 (m), 1315 (w), 1262 (s), 1177 (m), 1118 (m), 1091 (s), 1067 (m), 1024 (m), 978 (m), 803 (w),
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784 (w), 707 (s), 615 (m), 540 cm–1 (s); HR MS (ESI) for C35H30N5O7 [M + H]+: calcd 632.2140,
found 632.2134; for C35H29N5O7Na+ [M + Na]+: calcd 654.1959, found 654.1953.
8-(N,N-Dimethylamino)-1-methyl-4-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (18e)
A suspension of 17A (136 mg, 0.21 mmol) in 2-propanol (6.71 mL) was treated with dimethylamine
(0.155 mL, 2.0 M in THF, 0.31 mmol) and stirred at 60 °C for 24 hours. Purification by HPFC (SiO2;
cHex/EtOAc, gradient 0 → 100% EtOAc) gave 18e (63.9 mg, 47%) as a colorless film. Rf = 0.54
(SiO2; cHex/EtOAc 1:1); 1H NMR (500 MHz, CDCl3): δ = 3.17 (s, 6 H; NMe2), 4.22 (s, 3 H; NMe),
4.69 (m, 1 H; H-5’a), 4.78 (m, 1 H; H-4’), 4.79 (m, 1 H; H-5’b), 6.11 (dd, J3’,2’ = 6.0 Hz, J3’,4’
=
4.1 Hz, 1 H; H-3’), 6.32 (t, J2’,1’ = J2’,3’ = 6.2 Hz, 1 H; H-2’), 6.93 (d, J1’,2’ = 6.4 Hz, 1 H; H-1’), 7.34
(m, 2 H; H-A3, H-A5), 7.39 (m, 2 H; H-B3, H-B5), 7.46 (m, 2 H; H-C3, H-C5), 7.52, 7.56 and 7.59
(3 × m, 3 H; H-A4, H-B4, H-C4), 7.63 (s, 1 H; H-3), 7.93 (m, 2 H; H-A2, H-A6), 7.99 (m, 2 H; H-
B2, H-B6), 8.11 (m, 2 H; H-C2, H-C6), 8.51 ppm (s, 1 H; H-6); 13C{1H} NMR (125.7 MHz, CDCl3):
δ = 39.7 (NMe), 41.3 (2 C; NMe2), 64.1 (C-5’), 71.3 (C-3’), 72.4 (C-2’), 79.8 (C-4’), 84.9 (C-1’),
94.4 (C-8a), 121.2 (C-3), 126.3 (C-3a), 128.5 and 128.6 (4 C; C-A3, C-A5, C-B3, C-B5), 128.7, 128.9
and 129.5 (3 C; C-A1, C-B1, C-C1), 128.7 (2 C; C-C3, C-C5), 129.9 and 130.0 (6 C; C-A2, C-A6,
C-B2, C-B6, C-C2, C-C6), 130.8 (C-8b), 133.6, 133.7 and 133.8 (3 C; C-A4, C-B4, C-C4), 152.1 (C-
6), 158.2 (C-4a), 159.9 (C-8), 165.2 (C=O A), 165.6 (C=O B), 166.3 ppm (C=O C); IR (ATR):
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ṽ = 2953 (w), 1720 (m), 1584 (w), 1555 (w), 1493 (w), 1450 (w), 1435 (w), 1413 (w), 1367 (w), 1315
(w), 1259 (s), 1177 (w), 1085 (m), 1067 (m), 1024 (m), 993 (w), 973 (w), 948 (w), 835 (w), 803 (w),
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733 (w), 705 (s), 686 (m), 611 cm–1 (w); HR MS (ESI) for C36H33N6O7 [M + H]+: calcd 661.2405,
found 661.2398; for C36H32N6O7Na+ [M + Na]+: calcd 683.2225, found 683.2217.
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8-(Furan-2-yl)-1-methyl-4-(β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine
(19a)
A suspension of 18a (137 mg, 0.20 mmol) in MeOH (10 mL) was treated with NaOMe (14 µL,
25 wt% in MeOH, 0.06 mmol) and stirred at 60 °C for 6 hours. After co-evaporation with MeOH
(3 × 10 mL), purification by HPFC (SiO2; DCM/MeOH, gradient 0 → 10% MeOH) gave 19a
(64.6 mg, 87%) as a light-beige solid. Rf = 0.42 (SiO2; DCM/MeOH 9:1); m.p. = 217 °C; [α]D20 = 0
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(c = 0.167 in DMSO); H NMR (500 MHz, DMSO-d6): δ = 3.59 and 3.62 (2 × dd, Jgem = 11.8 Hz,
J5’,4’ = 4.0 Hz, 2 H; H-5’), 3.94 (td, J4’,5’a = J4’,5’b = 4.0 Hz, J4’,3’ = 2.3 Hz, 1 H; H-4’), 4.13 (dd, J3’,2’
= 5.3 Hz, J3’,4’ = 2.3 Hz, 1 H; H-3’), 4.22 (s, 3 H; NMe), 4.55 (bt, J2’,1’ = J2’,3’ = 6.4 Hz, 1 H; H-2’),
5.08 (bs, 1 H; OH-5’), 5.24 (bs, 1 H; OH-3’), 5.33 (bs, 1 H; OH-2’), 6.41 (d, J1’,2’ = 7.4 Hz, 1 H; H-
1’), 6.87 (dd, J4,3 = 3.4 Hz, J4,5 = 1.7 Hz, 1 H; H-4-furyl), 7.45 (dd, J3,4 = 3.4 Hz, J3,5 = 0.8 Hz, 1 H;
H-3-furyl), 7.92 (s, 1 H; H-3), 8.19 (dd, J5,4 = 1.7 Hz, J5,3 = 0.8 Hz, 1 H; H-5-furyl), 8.83 ppm (s, 1 H;
H-6); 13C{1H} NMR (125.7 MHz, DMSO-d6): δ = 41.3 (NMe), 61.8 (C-5’), 70.6 (C-3’), 71.7 (C-2’),
85.2 (C-4’), 85.4 (C-1’), 100.2 (C-8a), 113.2 (C-4-furyl), 113.6 (C-3-furyl), 120.8 (C-3), 127.7 (C-
8b), 129.6 (C-3a), 145.2 (C-8), 146.0 (C-5-furyl), 151.3 (C-2-furyl), 151.9 (C-6), 157.7 ppm (C-4a);
IR (ATR): ṽ = 3248 (m), 3129 (m), 2923 (m), 2854 (m), 1728 (w), 1667 (w), 1596 (m), 1552 (m),
1531 (m), 1484 (m), 1444 (s), 1415 (s). 1319 (m), 1289 (s), 1258 (m), 1236 (m), 1206 (w), 1180 (w),
1124 (s), 1070 (m), 1021 (s), 968 (s), 936 (m), 883(s), 861 (m), 790 (s), 757 (s), 718 (s), 617 cm–1 (s);
–1
UV/Vis (MeOH): λmax (ε) = 284 (10700), 323 (12000), 254 nm (29800 M cm–1); HR MS (ESI) for
+
for C17H18N5O5 [M + H]+: calcd 372.1303, found 372.1298; for C17H17N5O5Na+ [M + Na]+: calcd
394.1122, found 394.1120.
8-(Furan-3-yl)-1-methyl-4-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (19b)
A suspension of 18b (80.9 mg, 0.12 mmol) in MeOH (6 mL) was treated with NaOMe (8 µL, 25 wt%
in MeOH, 0.03 mmol) and stirred at 60 °C for 6 hours. Co-evaporation with MeOH (3 × 10 mL) and
purification by HPFC (C18; water/MeOH, gradient 0 → 100% MeOH) gave 19b (31.6 mg, 72%) as
an ocher-yellow film. Rf = 0.38 (SiO2; DCM/MeOH 9:1); [α]D20 = –15.0 (c = 0.171 in DMSO);
1H NMR (500 MHz, DMSO-d6): δ = 3.58 (ddd, Jgem = 11.7 Hz, J5’a,OH = 5.1 Hz, J5’a,4’ = 4.0 Hz, 1 H;
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H-5’a), 3.61 (ddd, Jgem = 11.7 Hz, J5’b,OH = 5.3 Hz, J5’b,4’ = 4.1 Hz, 1 H; H-5’b), 3.81 (s, 3 H; NMe),
3.94 (td, J4’,5’a = J4’,5’b = 4.0 Hz, J4’,3’ = 2.3 Hz, 1 H; H-4’), 4.12 (btd, J3’,2’ = J3’,OH = 4.9 Hz, J3’,4’
2.3 Hz, 1 H; H-3’), 4.54 (btd, J2’,1’ = J2’,OH = 7.1 Hz, J2’,3’ = 5.2 Hz, 1 H; H-2’), 5.07 (bt, JOH,5’a
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JOH,5’b = 5.2 Hz, 1 H; OH-5’), 5.26 (bd, JOH,3’ = 4.5 Hz, 1 H; OH-3’), 5.33 (bd, JOH,2’ = 6.7 Hz, 1 H;
OH-2’), 6.39 (d, J1’,2’ = 7.4 Hz, 1 H; H-1’), 7.03 (dd, J4,5 = 1.9 Hz, J4,2 = 0.9 Hz, 1 H; H-4-furyl), 7.88
(s, 1 H; H-3), 7.94 (t, J5,4 = J5,2 = 1.7 Hz, 1 H; H-5-furyl), 8.41 (dd, J2,5 = 1.5 Hz, J2,4 = 0.8 Hz, 1 H;
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H-2-furyl), 8.87 ppm (s, 1 H; H-6); C{1H} NMR (125.7 MHz, DMSO-d6): δ = 40.1 (NMe), 61.8
(C-5’), 70.7 (C-3’), 71.9 (C-2’), 85.2 (C-4’), 85.5 (C-1’), 104.4 (C-8a), 111.4 (C-4-furyl), 121.4 (C-
3), 124.8 (C-3-furyl), 128.1 (C-8b), 128.8 (C-3a), 144.0 (C-2-furyl), 144.3 (C-5-furyl), 150.0 (C-8),
152.4 (C-6), 157.1 ppm (C-4a); IR (ATR): ṽ = 3346 (m), 3249 (m), 2925 (w), 1662 (w), 1602 (w),
1552 (m), 1499 (m), 1447 (m), 1416 (s), 1344 (s), 1317 (s), 1283 (m), 1253 (m), 1208 (m), 1161 (m),
1120 (s), 1083 (s), 1041 (s), 1008 (m), 980 (s), 873 (s), 834 (m), 822 (m), 795 (s), 745 (m), 717 (m),
682 (m), 649 (m), 615 (m), 600 cm–1 (s); UV/Vis (MeOH): λmax (ε) = 301 (4600), 250 nm (24200 M
–
1cm–1); HR MS (ESI) for C17H18N5O5 [M + H]+: calcd 372.1303, found 372.1230; for
+
C17H17N5O5Na+ [M + Na]+: calcd 394.1122, found 394.1119.
8-(Benzofuran-2-yl)-1-methyl-4-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (19c)
A suspension of 18c (121 mg, 0.17 mmol) in MeOH (8.25 mL) was treated with NaOMe (11 µL,
25 wt% in MeOH, 0.05 mmol) and stirred at 60 °C for 6 hours. Co-evaporation with MeOH
(3 × 10 mL) and purification by HPFC (C18; water/MeOH, gradient 0 → 100% MeOH) gave 19c
(69.6 mg, 98%) as a light yellow solid. Rf = 0.47 (SiO2; DCM/MeOH 9:1); m.p. = 257 °C;
[α]D20 = + 19.6 (c = 0.176 in DMSO); 1H NMR (500 MHz, DMSO-d6): δ = 3.58–3.67 (m, 2 H; H-5‘),
3.96 (td, J4‘,5‘a = J4‘,5‘b = 4.0 Hz, J4‘,3‘ = 2.4 Hz, 1 H; H-4‘), 4.14 (dd, J3‘,2‘ = 5.3 Hz, J3‘,4‘ = 2.4 Hz,
1 H; H-3‘), 4.29 (s, 3 H; NMe), 4.57 (dd, J2‘,1‘ = 7.4 Hz, J2‘,3‘ = 5.3 Hz, 1 H; H-2‘), 5.10 (m, 1 H; OH-
5‘), 5.20–5.52 (m, 2 H; OH-2‘, OH-3‘), 6.44 (d, J1‘,2‘ = 7.4 Hz, 1 H; H-1‘), 7.41 (ddd, J5,4 = 7.7 Hz,
J5,6 = 7.2 Hz, J5,7 = 1.0 Hz, 1 H; H-5-benzofuryl), 7.50 (ddd, J6,7 = 8.4 Hz, J6,5 = 7.2 Hz, J6,4 = 1.2 Hz,
1 H; H-6-benzofuryl), 7.87 (dq, J4,5 = 7.7 Hz, J4,6 = 1.1), 7.88 (dq, J7,6 = 8.4 Hz, J7,3 = J7,4 = J7,5
=
0.9 Hz, 1 H; H-7-benzofuryl), 7.90 (d, J3,7 = 1.0 Hz, 1 H; H-3-benzofuryl), 7.97 (s, 1 H; H-3),
8.94 ppm (s, 1 H; H-6); C{1H} NMR (125.7 MHz, DMSO-d6): δ = 41.4 (NMe), 61.8 (C-5‘), 70.6
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(C-3‘), 71.8 (C-2‘), 85.3 (C-4‘), 85.6 (C-1‘), 101.5 (C-8a), 109.4 (C-3-benzofuryl), 111.9 (C-7-
benzofuryl), 121.1 (C-3), 122.7 (C-4-benzofuryl), 124.1 (C-5-benzofuryl), 126.5 (C-6-benzofuryl),
127.7 (C-8b), 128.0 (C-3a-benzofuryl), 129.9 (C-3a), 145.2 (C-8), 152.0 (C-6), 153.4 (C-2-
benzofuryl), 154.9 (C-7a-benzofuryl), 157.9 ppm (C-4a); IR (ATR): ṽ = 3240 (w), 3084 (w), 2920
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(m), 2852 (w), 1739 (w), 1652 (w), 1597 (m), 1559 (m), 1530 (m), 1489 (w), 1447 (s), 1412 (m),
1351 (m), 1331 (m), 1291 (m), 1259 (m), 1215 (w), 1191 (w), 1173 (m), 1135 (m), 1118 (m), 1074
(s), 1038 (s), 1008 (s), 982 (s), 976 (s), 965 (m), 926 (m), 887 (m), 823 (s), 793 (s), 747 (s), 731 (m),
6
–1
685 (m), 663 (m), 612 cm–1 (m); UV/Vis (MeOH): λmax (ε) = 336 (19800), 258 nm (20900 M cm–1);
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HR MS (ESI) for C21H20N5O5 [M + H]+: calcd 422.1459, found 422.1456; for C21H19N5O5Na+
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[M + Na]+: calcd 444.1278, found 444.1276.
1,8-Dimethyl-4-(β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (19d)
A suspension of 18d (135 mg, 0.21 mmol) in MeOH (10 mL) was treated with NaOMe (15 µL,
25 wt% in MeOH, 0.07 mmol) and stirred at 60 °C for 4 hours. Co-evaporation with MeOH
(3 × 10 mL) and purification by HPFC (C18; water/MeOH, gradient 0 → 100% MeOH) gave 19d
(50.4 mg, 73%) as a white solid. Rf = 0.33 (SiO2; DCM/MeOH 9:1); m.p. = 286 °C; [α]D20 = –50.3
(c = 0.173 in DMSO); 1H NMR (500 MHz, DMSO-d6): δ = 2.96 (s, 3 H; CMe), 3.53–3.62 (m, 2 H;
H-5’), 3.91 (td, J4’,5’a = J4’,5’b = 4.1 Hz, J4’,3’ = 2.4 Hz, 1 H; H-4’), 4.10 (m, 1 H; H-3’), 4.29 (s, 3 H;
NMe), 4.51 (td, J2’,1’ = J2’,OH = 6.9 Hz, J2’,3’ = 5.3 Hz, 1 H; H-2’), 5.09 (t, JOH,5’a = JOH,5’b = 5.2 Hz,
1 H; OH-5’), 5.26 (d, JOH,3’ = 4.3 Hz, 1 H; OH-3’), 5.32 (d, JOH,2’ = 6.6 Hz, 1 H; OH-2’), 6.33 (d, J1’,2’
= 7.3 Hz, 1 H; H-1’), 7.79 (s, 1 H; H-3), 8.73 ppm (s, 1 H; H-6); 13C{1H} NMR (125.7 MHz, DMSO-
d6): δ = 24.3 (CMe), 39.7 (NMe), 62.0 (C-5’), 70.8 (C-3’), 71.8 (C-2’), 85.2 (C-4’), 85.6 (C-1’), 105.1
(C-8a), 120.9 (C-3), 128.3 (C-3a), 128.5 (C-8b), 152.6 (C-6), 156.5 (C-4a), 157.0 ppm (C-8); IR
(ATR): ṽ = 3213 (m), 2918 (w), 1606 (w), 1551 (m), 1503 (m), 1477 (m), 1455 (m), 1415 (m), 1374
(m), 1316 (m), 1244 (m), 1218 (m), 1124 (s), 1098 (s), 1077 (s), 1047 (s), 1020 (s), 983 (s), 931 (m),
882 (m), 854 (m), 828 (m), 785 (m), 720 (s), 647 (s), 617 cm–1 (s); UV/Vis (MeOH): λmax (ε) = 281
–1
+
(6600), 246 nm (39300 M cm–1); HR MS (ESI) for C14H18N5O4 [M + H]+: calcd 320.1353, found
320.1351; for C14H17N5O4Na+ [M + Na]+: calcd 342.1173, found 342.1170.
1-Methyl-8-(N,N-dimethylamino)-4-(β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-
d]pyrimidine (19e)
A suspension of 18e (116 mg, 0.18 mmol) in MeOH (8.8 mL) was treated with NaOMe (12 µL,
25 wt% in MeOH, 0.05 mmol) and stirred at 60 °C for 6 hours. Concentration in vacuo, followed by
co-evaporation with MeOH (3 × 10 mL) and purification by HPFC (SiO2; DCM/MeOH, gradient 0
→ 10% MeOH) gave 19e (61.0 mg, 98%) as a light-beige film. Rf = 0.38 (SiO2; DCM/MeOH 9:1);
[α]D20 = + 16.1 (c = 0.261 in DMSO); 1H NMR (500 MHz, DMSO-d6): δ = 3.11 (s, 6 H; NMe2), 3.55
(ddd, Jgem = 11.8 Hz, J5’a,OH = 5.1 Hz, J5’a,4’ = 4.2 Hz, 1 H; H-5’a), 3.58 (ddd, Jgem = 11.8 Hz, J5’b,OH
= 5.5 Hz, J5’b,4’ = 4.2 Hz, 1 H; H-5’b), 3.88 (td, J4’,5’a = J4’,5’b = 4.2 Hz, J4’,3’ = 2.4 Hz, 1 H; H-4’),
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4.09 (btd, J3’,2’ = J3’,OH = 4.9 Hz, J3’,4’ = 2.4 Hz, 1 H; H-3’), 4.18 (s, 3 H; NMe), 4.51 (td, J2’,1’ = J2’,OH
= 7.1 Hz, J2’,3’ = 5.3 Hz, 1 H; H-2’), 5.04 (t, JOH,5’a = JOH,5’b = 5.3 Hz, 1 H; OH-5’), 5.27 (d, JOH,3’
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4.5 Hz, 1 H; OH-3’), 5.27 (d, JOH,2’ = 6.9 Hz, 1 H; OH-2’), 6.27 (d, J1’,2’ = 7.4 Hz, 1 H; H-1’), 7.79
(s, 1 H; H-3), 8.36 ppm (s, 1 H; H-6); 13C{1H} NMR (125.7 MHz, DMSO-d6): δ = 39.8 (NMe), 40.5
(2 C; NMe2), 61.9 (C-5’), 70.6 (C-3’), 71.8 (C-2’), 85.0 (C-4’), 85.5 (C-1’), 92.6 (C-8a), 121.8 (C-3),
126.2 (C-3a), 129.6 (C-8b), 151.8 (C-6), 157.7 (C-4a), 159.6 ppm (C-8); IR (ATR): ṽ = 3261 (broad
w), 2925 (w), 2855 (w), 1732 (w), 1636 (w), 1594 (m), 1559 (m), 1496 (w), 1441 (m), 1412 (m),
1391 (m), 1317 (w), 1263 (m), 1093 (s), 1044 (s), 995 (s), 974 (m), 942 (m), 787 (s), 710 (s), 594 cm–1
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(s); UV/Vis (MeOH): λmax (ε) = 305 nm (7400 M cm–1); HR MS (ESI) for C15H21N6O4 [M + H]+:
calcd 349.1619, found 349.1617; for C15H20N6O4Na+ [M + Na]+: calcd 371.1438, found 371.1436.
8-Amino-1-methyl-4-(β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (19f)
In a screw-cap pressure glass tube, a solution of nucleoside 17A (201 mg, 0.31 mmol) in 1,4-dioxane
(1 mL) was treated with aq. ammonia (2.5 mL, 25 wt% in water, 16.23 mmol). After stirring at 120 °C
for 24 hours (HPLC-MS: tR = 0.82 min, m/z = 321), the solution was cooled down and concentrated
in vacuo. Recrystallisation from DCM/MeOH (9:1, 2.5 mL), followed by filtration and rigorous
washing with ice-cold DCM/MeOH solution (9:1) gave 19f (79.3 mg, 81%) as an ocher-yellow solid.
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Rf = 0.15 (SiO2; DCM/MeOH 9:1); m.p. = 264 °C; [α]D20 = –25.7 (c = 0.198 in DMSO); H NMR
(500 MHz, DMSO-d6): δ = 3.53 and 3.57 (2 dd, Jgem = 11.7 Hz, J5’,4’ = 4.2 Hz, 2 H; H-5’), 3.87 (td,
J4’,5’a = J4’,5’b = 4.2 Hz, J4’,3’ = 2.5 Hz, 1 H; H-4’), 4.08 (dd, J3’,2’ = 5.3 Hz, J3’,4’ = 2.5 Hz, 1 H; H-3’),
4.22 (s, 3 H; NMe); 4.48 (dd, J2’,1’ = 7.2 Hz, J2’,3’ = 5.3 Hz, 1 H; H-2’), 4.78–5.53 (m, 3 H; OH-2’,
OH-3’, OH-5’), 6.20 (d, J1’,2’ = 7.3 Hz, 1 H; H-1’), 6.76 (bs, 2 H; NH2), 7.64 (s, 1 H; H-3), 8.21 ppm
(s, 1 H; H-6); 13C{1H} NMR (125.7 MHz, DMSO-d6): δ = 39.1 (NMe), 61.9 (C-5’), 70.7 (C-3’), 71.8
(C-2’), 84.8 (C-4’), 85.5 (C-1’), 89.7 (C-8a), 120.6 (C-3), 126.2 (C-3a), 129.1 (C-8a), 153.3 (C-6),
156.2 (C-8), 157.3 ppm (C-4a); IR (ATR, neat): ṽ = 3229 (m), 3042 (m), 2935 (m), 2898 (m), 1643
(s), 1612 (m), 1570 (m), 1524 (m), 1505 (m), 1487 (m), 1435 (m), 1359 (m), 1328 (m), 1278 (m),
1239 (m), 1193 (m), 1127 (m), 1101 (s), 1064 (s), 1013 (s), 990 (s), 976 (s), 945 (m), 905 (m), 870
–
(m), 789 (s), 737 (s), 725 (m), 626 (s), 610 cm–1 (s); UV/Vis (MeOH): λmax (ε) = 281 nm (7000 M
+
1cm–1); HR MS (ESI) for C13H17N6O4 [M + H]+: calcd 321.1306, found 321.1303; for
C13H16N6O4Na+ [M + Na]+: calcd 343.1125, found 343.1122.
8-Methoxy-1-methyl-4-(β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine (19g)
A suspension of nucleoside 17A (201 mg, 0.31 mmol) in MeOH (20 mL) was treated with sodium
methoxide (0.15 mL, 25 wt% in MeOH, 0.66 mmol) and stirred at 25 °C for 76 hours, followed by
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concentration in vacuo. Purification by HPFC (C18; water/MeOH, gradient 0 → 100% MeOH) gave
19g (82.1 mg, 80%) as a white solid. Rf = 0.54 (SiO2; DCM/MeOH 9:1); m.p. = 207 °C; [α]D20 = –
37.6 (c = 0.173 in DMSO); 1H NMR (500 MHz, DMSO-d6): δ = 3.53–3.62 (m, 2 H; H-5’), 3.91 (td,
J4’,5’a = J4’,5’b = 4.1 Hz, J4’,3’ = 2.4 Hz, 1 H; H-4’), 4.11 (dd, J3’,2’ = 5.3 Hz, J3’,4’ = 2.4 Hz, 1 H; H-3’),
4.14 (s, 3 H, OMe), 4.16 (s, 3 H; NMe), 4.51 (dd, J2’,1’ = 7.3 Hz, J2’,3’ = 5.3 Hz, 1 H; H-2’), 5.00–5.43
(m, 3 H; OH-2’, OH-3’, OH-5’), 6.28 (d, J1’,2’ = 7.3 Hz, 1 H; H-1’), 7.77 (s, 1 H; H-3), 8.54 ppm (s,
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1 H; H-6); C{1H} NMR (125.7 MHz, DMSO-d6): δ = 38.4 (NMe), 54.5 (OMe), 62.0 (C-5’), 70.8
(C-3’), 72.2 (C-2’), 85.2 (C-4’), 85.9 (C-1’), 91.5 (C-8a), 121.6 (C-3), 126.8 (C-3a), 128.4 (C-8a),
152.9 (C-6), 158.1 (C-4a), 161.3 ppm (C-8); IR (ATR): ṽ = 3197 (w), 2919 (w), 1723 (m), 1679 (w),
1602 (w), 1569 (w), 1537 (w), 1450 (m), 1315 (w), 1266 (s), 1177 (w), 1121 (s), 1096 (m), 1067 (m),
1046 (m), 1025 (s), 987 (m), 973 (m), 940 (w), 803 (w), 786 (w), 707 (s), 686 (m), 620 (m), 600 cm–1
(m); UV/Vis (MeOH): λmax (ε) = 271 nm (9300 M cm–1); HR MS (ESI) for C14H17N5O5Na+
–1
[M + Na]+: calcd 358.1122, found 358.1126.
1-Methyl-8-(methylthio)-4-(β-D-ribofuranosyl)pyrazolo[3',4':4,5]pyrrolo[2,3-d]pyrimidine
(19h)
A solution of 17A (30.5 mg, 0.05 mmol) in 1,4-dioxane (2 mL) was treated with NaSMe (17.3 mg,
0.32 mmol) and stirred at 60 °C for 14 hours. Purification by HPFC (C18; water/MeOH, gradient 0
→ 100% MeOH) gave 19h (8.2 mg, 51%) as a beige oil. Rf = 0.56 (SiO2; DCM/MeOH 9:1);
[α]D20 = –23.3 (c = 0.031 in DMSO); 1H NMR (500 MHz, DMSO-d6): δ = 2.71 (s, 3 H; SMe), 3.53–
3.63 (m, 2 H; H-5’), 3.91 (td, J4’,5’a = J4’,5’b = 4.7 Hz, J4’,3’ = 2.3 Hz, 1 H; H-4’), 4.10 (td, J3’,2’ = J3’,OH
= 4.8 Hz, J3’,4’ = 2.3 Hz, 1 H; H-3’), 4.36 (s, 3 H; NMe), 4.51 (btd, J2’,1’ = J2’,OH = 7.0 Hz, J2’,3’
=
5.3 Hz, 1 H; H-2’), 5.07 (t, JOH,5’a = JOH,5’b = 5.3 Hz, OH-5’), 5.24 (d, JOH,3’ = 4.5 Hz, 1 H; OH-3’),
5.32 (d, JOH,2’ = 6.6 Hz, 1 H; OH-2’), 6.31 (d, J1’,2’ = 7.4 Hz, 1 H; H-1‘), 7.83 (s, 1 H; H-3), 8.73 ppm
(s, 1 H; H-6); 13C{1H} NMR (125.7 MHz, DMSO-d6): δ = 12.3 (SMe), 40.1 (NMe), 61.8 (C-5’), 70.7
(C-3’), 77.9 (C-2’), 85.3 (C-4’), 85.6 (C-1’), 102.6 (C-8a), 121.3 (C-3), 127.8 and 127.9 (2 C; C-3a,
C-8b), 152.1 (C-6), 154.8 (C-4a), 158.5 ppm (C-8); IR (ATR): ṽ = 2964 (w), 1718 (m), 1583 (w),
1555 (w), 1491 (w), 1451 (w), 1413 (w), 1246 (m), 1177 (w), 1084 (m), 1066 (m), 1024 (w), 992 (w),
972 (w), 946 (w), 842 (w), 793 (w), 734 (w), 703 (s), 685 (m), 611 cm–1 (w); UV/Vis (MeOH):
λmax (ε) = 303 nm (5000 M cm–1); HR MS (ESI) for C14H18N5O4S+ [M + H]+: calcd 352.1074, found
–1
352.1071; for C14H17N5O4SNa+ [M + Na]+: calcd 374.0894, found 374.0891.
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Supporting information: FAIR Data is available free of charge via the Internet at http://pubs.acs.org
as Supporting Information and includes the primary NMR FID files for compounds 7, 9–16, 17A,
17B-β, 18a–18e, 19a–19h; optimization of Negishi coupling, azide/tetrazole equilibrium and
cyclization of intermediate 16, HPLC purity of final compounds, UV absorption and fluorescence
emission spectra of the title nucleosides, and copies of NMR spectra.
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