6-Methyl-7-Aryl-7-Deazapurine Nucleosides as Anti-Trypanosoma cruzi Agents: Structure-Activity Relationship and in vivo Efficacy

Article abstract of DOI:10.1002/cmdc.202100144

Chagas disease is a tropical infectious disease resulting in progressive organ-damage and currently lacks efficient treatment and vaccine options. The causative pathogen, Trypanosoma cruzi, requires uptake and processing of preformed purines from the host because it cannot synthesize these de novo, instigating the evaluation of modified purine nucleosides as potential trypanocides. By modifying the pyrimidine part of a previously identified 7-aryl-7-deazapurine nucleoside, we found that substitution of a 6-methyl for a 6-amino group allows retaining T. cruzi amastigote growth inhibitory activity but confers improved selectivity towards mammalian cells. By keeping the 6-methyl group unaltered, and introducing different 7-aryl groups, we identified several analogues with sub-micromolar antitrypanosomal activity. The 7-(4-chlorophenyl) analogue 14, which was stable in microsomes, was evaluated in an acute mouse model. Oral administration of 25 mg/kg b.i.d. suppressed peak parasitemia and protected mice from infection-related mortality, gave similar reductions as the reference drug of blood parasite loads determined by qPCR, but as benznidazole failed to induce sterile cure in the short time period of drug exposure (5 days).

Full text of DOI:10.1002/cmdc.202100144

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6-Methyl-7-Aryl-7-Deazapurine Nucleosides as Anti-
Trypanosoma cruzi Agents: Structure-Activity Relationship
and in vivo Efficacy
Cai Lin+,[a] Ludmila Ferreira de Almeida Fiuza+,[b] Camila Cardoso Santos,[b]
Daniela Ferreira Nunes,[c] Otacílio Cruz Moreira,[c] Jakob Bouton,[a] Izet Karalic,[a] Louis Maes,[d]
Guy Caljon,[d] Fabian Hulpia,*[a, e] Maria de Nazaré C. Soeiro,*[b] and Serge Van Calenbergh*[a]
Chagas disease is a tropical infectious disease resulting in
progressive organ-damage and currently lacks efficient treat-
ment and vaccine options. The causative pathogen, Trypanoso-
ma cruzi, requires uptake and processing of preformed purines
from the host because it cannot synthesize these de novo,
instigating the evaluation of modified purine nucleosides as
potential trypanocides. By modifying the pyrimidine part of a
previously identified 7-aryl-7-deazapurine nucleoside, we found
that substitution of a 6-methyl for a 6-amino group allows
retaining T. cruzi amastigote growth inhibitory activity but
confers improved selectivity towards mammalian cells. By
keeping the 6-methyl group unaltered, and introducing differ-
ent 7-aryl groups, we identified several analogues with sub-
micromolar antitrypanosomal activity. The 7-(4-chlorophenyl)
analogue 14, which was stable in microsomes, was evaluated in
an acute mouse model. Oral administration of 25 mg/kg b.i.d.
suppressed peak parasitemia and protected mice from infec-
tion-related mortality, gave similar reductions as the reference
drug of blood parasite loads determined by qPCR, but as
benznidazole failed to induce sterile cure in the short time
period of drug exposure (5 days).
Introduction
[a] C. Lin,+ J. Bouton, I. Karalic, Dr. F. Hulpia, Prof. S. Van Calenbergh
Laboratory for Medicinal Chemistry (Campus Heymans)
Ghent University
Chagas disease (CD) is a neglected tropical disease that is
endemic in the Latin American countries.[1–2] CD is caused by
the kinetoplastid parasite Trypanosoma cruzi (T. cruzi) and is
transmitted primarily in endemic regions via triatomine bugs.
While taking a bloodmeal this vector defecates, releasing T.
cruzi trypomastigotes which then establish an infection into the
vertebrate mammalian host.[3] Notwithstanding the vector-
mediated transmission route in endemic areas being most
important, CD may also be contracted by ingesting contami-
nated food or drinks (oral route). Additionally, a diverse range
of non-vector mediated transmission routes, such as congenital
infection, blood transfusion or organ transplantation, plays a
major role in non-endemic countries.[4] This is particularly
worrisome in light of increased migration, often accompanied
by an unawareness of infection status, rendering CD a global
health concern.[5–6]
There are two stages in the progression of CD: the acute
and chronic stage. The initial clinical manifestation in the acute
phase is mild or even oligosymptomatic, but the parasite can
typically be observed through blood examination. If no treat-
ment is provided, the disease enters into a chronic phase and
may remain asymptomatic for years and decades, displaying a
subpatent parasitism due to an effective host’s immune system.
In the later chronic phase, for reasons not yet well known, some
patients (30–40%) develop a progressive cardiomyopathy and/
or digestive disorders besides neurological dysfunction.[7–9] The
continuous damage of the heart muscle ultimately results in
heart failure and sometimes sudden death.[10] Currently, no
vaccine is available, making chemotherapy the only option.
Ottergemsesteenweg 460
9000 Gent (Belgium)
E-mail: fhulpia@its.jnj.com
Serge.VanCalenbergh@UGent.be
[b] L. Ferreira de Almeida Fiuza,+ C. Cardoso Santos,
Prof. M. de Nazaré C. Soeiro
Laboratório de Biologia Celular
Instituto Oswaldo Cruz (FIOCRUZ)
Fundação Oswaldo Cruz, Rio de Janeiro
Avenida Brasil 4365, Manguinhos
21040-360 Rio de Janeiro (Brazil)
E-mail: soeiro@ioc.fiocruz.br
[c] Dr. D. Ferreira Nunes, Dr. O. Cruz Moreira
Plataforma de PCR em Tempo Real RPT09A-Laboratório de Biologia
Molecular e Doenças Endêmicas
Instituto Oswaldo Cruz (FIOCRUZ)
Fundação Oswaldo Cruz, Rio de Janeiro
Avenida Brasil 4365, Manguinhos
21040-360 Rio de Janeiro (Brazil)
[d] Prof. L. Maes, Prof. G. Caljon
Laboratory of Microbiology
Parasitology and Hygiene
University of Antwerp
Universiteitsplein 1
2610 Wilrijk (Belgium)
[e] Dr. F. Hulpia
Janssen Pharmaceutica NV
Turnhoutseweg 30
2340 Beerse (Belgium)
E-mail: fhulpia@its.jnj.com
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100144
This article belongs to the Early-Career Special Collection, “EuroMedChem
Talents”.
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Benznidazole (BZ) and nifurtimox are two nitro-containing
deazaguanosine (5)[28] and 2-amino-6-chloro-7-deazaadenosine
(6) (Figure 1a).[29]
drugs used for the treatment of CD. However, both drugs suffer
from several limitations such as low efficacy mainly at the later
chronic stage, and significant side effects besides the occur-
rence of parasite strains that are naturally resistant to both nitro
derivatives. Drug toxicity is particularly important as it contrib-
utes to poor patient compliance and premature treatment
cessation, thus contributing to the persistence of the
parasitism.[11–13] Therefore, the discovery and development of
new treatment options are of critical importance.[14–15]
In this study, we set out to explore the structure-activity-
relationship (SAR) of analogue 2 with particular attention to
modifications of the pyrimidine part of the 7-deazapurine
system. After having identified a favourable 6-methyl modifica-
tion, we combined this modification with different C7 modifica-
tions (Figure 1b). One promising analogue with favourable
in vitro metabolic stability was selected for efficacy evaluation
in an experimental mouse model of T. cruzi infection.
An attractive drug discovery paradigm is to exploit certain
parasitic auxotrophies. Particularly, T. cruzi cannot synthesize
purine rings from amino acids, resulting in their reliance on Results and Discussion
salvage[16–18] of preformed purine rings from the host for growth
and proliferation, thereby increasing the likeliness for (modified)
Biological evaluation
purine analogues as potential antiparasitic agents.[19–20]
Tubercidin, a natural antibiotic, displays attractive inhibitory
potency against multiple pathogenic organisms, but its applica-
tion is limited because of toxic effects on mammalian cells.[21–22]
Selectivity improvement of 7-deazapurine nucleoside analogues
has been achieved via modifications at C6[23] and/or C7.[22,24]
Introduction of a 4-chlorophenyl substituent (analogue 2,
Figure 1) at C7 of tubercidin was found to confer potent anti-T.
cruzi activity in vitro[22,25] and in vivo,[25] while decreasing
cytotoxicity. Several other nucleoside analogues with modifica-
tions in the pyrimidine part of the purine were reported to
exhibit potent activity against T. cruzi, including puromycin
aminonucleoside (3),[26] 7-iodo-1,7-dideazaadenosine (4),[27] 3-
In vitro evaluation
All prepared compounds were evaluated in vitro for their
antitrypanosomal activity against T. cruzi, employing different
parasite strains belonging to distinct DTUs (Tulahuen expressing
β-galactosidase and Y strains, DTU VI and II, respectively) and
parasite forms relevant for mammalian host (intracellular
amastigotes and bloodstream trypomastigotes) always compar-
ing to the findings obtained with BZ as a reference drug. In
parallel, the in vitro cytotoxicity of the compounds was assessed
in different host cells including the MRC-5 human fibroblast cell
line and primary cardiac cell cultures grown as 2 and 3-D
matrices (Tables 1–3, 6).
Figure 1. (a) Representative antitrypanosomal nucleoside analogues with C7 or pyrimidine ring modifications; (b) SAR exploration of the pyrimidine ring of 2
and exploration of C7-aryl modified 6-methyl-7-deazapurinecleoside.
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Table 1. <Activity of 7-(para-chlorophenyl) nucleoside analogues with modifications in pyrimidine moieties against intracellular T. cruzi amastigotes.
Cpd.
X
R1
R2
R3
T. cruzi[a]
EC50 [μM]
MRC-5[b]
EC50 [μM]
SI[c]
2[22,25]
7
8
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
NH2
NHMe
NMe2
OH
OMe
OEt
SMe
H
Me
Et
Ph
OH
OMe
NH2
Me
H
H
H
H
H
H
H
H
H
H
H
H
NH2
NH2
H
H
H
–
–
–
–
–
–
–
–
–
–
–
–
0.47�0.25
41.6
33.4
>64.0
43.1
26.3
27.0�2.5
>64.0
>64.0
>64.0
61.1
57
>1.54
>1.92
N.D.[d]
1.42
9
10[30]
11
12
13
14
15
16
17
18
19[31]
20
21
22
23
24
25
BZ
50.4
1.92
20.3
11.9
>64.0
>64.0
>64.0
36.3
>64.0
>64.0
22.5
>64.0
>64.0; 20.2
18.2
>3.15
>5.38
>75
1.03
N.D.
N.D.
0.74
N.D.
N.D.
0.76
0.85�0.21
35.3
>64.0
>64.0
30.6
>64.0
9.76�0.45
24.1
–
H
H
F
Cl
NO2
NH2
Me
H
H
H
H
H
H
H
H
7.80
50.8
6.51
>7.57
N.D.
2.34
8.45�0.12
>64.0
9.98
2.28�0.05
>64.0
>64.0
23.4
N.D.
N.D.
[a]Concentration of compound inhibiting growth by 50% (EC50) of intracellular T. cruzi amastigotes (Tulahuen strain-expressing β-galactosidase). EC50 values
are described as mean�SE of 2 or 4 independent experiments. Where SE is not provided, values (EC50 >10 μM) were determined in a single experiment; [b]
Concentration of compound inhibiting growth by 50% (EC50) of MRC-5 fibroblasts; [c] SI=selectivity index, EC50 (MRC-5)/EC50 (T. cruzi); [d] N.D.: not
determined.
First, the activity against intracellular forms (Tulahuen strain)
was investigated. Substitution of the 6-amino group of 2 by
alternative groups (7–12) failed to give analogues with
improved or comparable anti-T. cruzi activity. Likewise, com-
plete removal of the 6-NH2 (13) or its replacement by a phenyl
(16) was not tolerated. Only the 6-methyl analogue 14
displayed sub-micromolar anti-T. cruzi activity, comparable to 2.
This analogue does not exhibit toxicity to the MCR-5 host cells
at the highest concentration tested (>64 μM), marking 14 as an
interesting hit. Remarkably, homologation to a 6-ethyl analogue
(15) resulted in complete loss of antiparasitic activity. Both
deazaguanosine analogues 17 and 18 were devoid of anti-T.
cruzi activity.
Then, we assessed the importance of N1, as we have
previously shown that 1,7-dideazapurine analogues may display
interesting anti-T. cruzi activity.[27] In this series, however, the
combination of a 6-amino group with the 7-para-chlorophenyl
(19) previously proved detrimental for activity.[27] Remarkably,
replacing the 6-amino group of 19 by a methyl group (20)
restored the anti-T. cruzi activity (EC50 ~10 μM). However, this
1,7-dideaza analogue remained 10-fold less active than its 7-
deaza congener 14. Removal of the N1 nitrogen atom allows
modifications at this position which were hardly explored
before. 1-Chloro or nitro analogues showed modest activities
(EC50 <10 μM), while the 1-amino analogue 24 was inactive.
Having identified a 6-methyl group as a potential 6-amino
isostere, we decided to further investigate the SAR of the 7-
substituent of 14 (Table 2).
Removal of the chloro-substituent (26) led to a substantial
activity drop, indicating the importance of para substitution.
Several alternative substituents resulted in less potent ana-
logues (27–35), including 4-F (27), 4-CF3 (28) and 4-Me (32) that
retained low micromolar anti-T. cruzi activity, similar to BZ.
Shifting the halogen to the meta (36, 37) or ortho (38) position
reduced the anti-T. cruzi activity, in line with previous SAR
observations with the 6-amino analogues.[22,25]
Generally, 3,4-disubstituted analogues (39, 40) were more
potent than 2,4-disubstituted (44, 45) or 3,5-disubstituted
analogues (46). Introduction of a fluoro (40) or a methoxy
group (43) in the meta position of 14 gave a slight activity
improvement. Selected bicyclic analogues of 26 (e.g. 49 and
53) also showed reasonable activity. However, the effect of the
aryl substituents on the activity did not show a clear trend. The
fact that the observed SAR is in line with previous findings for
3’-deoxy-7-aryl tubercidin analogues,[25] suggests a similar target
engagement.
Finally, we investigated the effect of 3’-deoxygenation
(Table 3), previously shown to significantly improve antitrypa-
nosomal activity.[25,32–33] However, the 3’-deoxy analogue 54
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Table 2. Activity of 7-aromatic-deazapurine nucleoside analogues against intracellular T. cruzi amastigotes.[a]
Cpd.
R or structure
T. cruzi[a]
EC50 [μM]
MRC-5[b]
EC50 [μM]
SI[c]
14
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
BZ
4-Cl
H
4-F
4-CF3
4-OCF3
4-CN
4-NO2
4-Me
4-Et
4-OMe
4-OH
3-Cl
3-F
2-Cl
3,4-2Cl
3-F-4-Cl
0.85�0.21
40.3
2.89�0.35
2.55�0.11
31.5
33.5
13.4
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
>64.0
29.6;>64.0
>64
>75
>1.5
>22
>25
>2.0
>1.9
>4.7
>22
>1.5
>1.4
>8.5
>4.9
>5.0
>6.9
>40
>104
N.D.[d]
>39
>108
>6.2
>42
>5.8
>25
>5.9
N.D.
2.83�0.03
42.3
44.7
7.47�0.01
12.9�0.78
12.7
9.27�0.06
1.58�0.70
0.61�0.04
>64.0
1.61�0.26
0.59�0.02
10.3�0.09
1.51�0.70
10.9�0.36
2.49�0.11
10.8�2.52
3.06�0.16
60.9
3-CF3-4-Cl
3-Me-4-Cl
3-OMe-4-Cl
2,4-2Cl
2-F-4-Cl
3,5-2Cl
3,5-2F-4-Cl
4-Ph
2-naphthalene
5-(benzo[d][1,3]dioxole)
6-(2,3-dihydrobenzo[b][1,4]dioxine)
6-1H-indole
6-(1-methyl-1H-indole)
1
N.D.
N.D.
13
>64
>64
3.34
2.28�0.05
>64
>64
44.30
N.D.
N.D.
[a] Concentration of compound inhibiting growth by 50% (EC50) of intracellular T. cruzi amastigotes (Tulahuen strain-expressing β-galactosidase). EC50 values
are described as mean�SE of 2 or 4 independent experiments. Where SE is not provided, values (EC50 >10 μM) were determined in a single experiment; [b]
Concentration of compound inhibiting growth by 50% (EC50) of MRC-5 fibroblasts; [c] SI=selectivity index, EC50 (MRC-5)/EC50 (T. cruzi); [d] N.D.: not
determined.
Table 3. Comparison of activity of compound 14 and the corresponding 3’-deoxy- analogue against intracellular T. cruzi amastigotes.[a]
Cpd.
14
Structure
T. cruzi[a]
EC50 [μM]
MRC-5[b]
EC50 [μM]
SI[c]
0.85�0.21
2.58�0.67
>64.0
>64.0
>75
54
>25
[a] Concentration of compound inhibiting growth by 50% (EC50) of intracellular T. cruzi amastigotes (Tulahuen strain-expressing β-galactosidase). EC50 values
are described as mean�SE of 2 or 4 independent experiments; [b] Concentration of compound inhibiting growth by 50% (EC50) of MRC-5 fibroblasts; [c] SI=
selectivity index, EC50 (MRC-5)/EC50 (T. cruzi).
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elicited a 3-fold weaker anti-T. cruzi activity than its ribose
congener 14.
In vitro metabolic stability of 14
In summary, a new series of 6-methyl 7-aryl-7-deazapurine
nucleosides with activity against T. cruzi was discovered. SAR
efforts showed that the 6-methyl group can function as a
suitable bioisostere for the amino functionality, typically present
in purines. Such replacement was previously exploited for
antiviral[23,31,34–35] and cytotoxic nucleoside analogues.[23] Note-
worthy is the complete lack of cytotoxicity against MRC-5
fibroblasts observed for almost all of the 6-methyl-7-(substi-
tuted) phenyl nucleosides, which represents a benefit compared
to our previously reported analogue 7.[22,25] It is noteworthy that
the most promising analogues of this study (14, 40 and 43) all
possess a (modified) 7-(4-chlorophenyl) substituent, as summar-
ized in Figure 2. Given that these three analogues display
comparable in vitro antitrypanosomal activity, the lowest molec-
ular weight compound 14 was selected for further evaluation
(Tables 4-6). Compound 14 was not cardiotoxic (CC50 >400 μM)
and presented a high potency against intracellular forms (Y
strain) present within primary cardiac cell cultures (EC50 =0.77�
0.05 μM) (Table 6). Also, the analogue continued to exert a
promising biological effect when assessed in cardiac spheroids.
The low cardiotoxicity profile of 14 was confirmed in 3D
matrices (CC50 >200 μM) and it displayed similar trypanocidal
effects at a 10 μM concentration as BZ, reducing the T. cruzi (Y
strain) parasite loads in cardiac spheroids by 77.5�6.4 and 87�
4.2% for 14 and BZ respectively, as evidenced by qPCR analysis.
The metabolic stability of 14 in the presence of mouse and
human liver microsomal fractions was evaluated (Table 4). The
percentage of remaining parent compound was monitored at
three time points and compared to the start of the assay,
indicating that 14 remained largely unaffected in the presence
of either NADPH fortification (phase I) or UGT fortification
(phase II). Hence, this analogue was further evaluated in a
mouse model of acute T. cruzi infection.
In vivo evaluation of 14
The in vivo effect of 14 was evaluated in a stringent Y-strain T.
cruzi acute male Swiss mouse model.[36]
Dose titration schedules of the different treatment groups
are summarized Table 5. The nucleoside analogue 14 was
administered at 0.25, 2.5, 25 mg/kg b.i.d. by oral gavage for 5
consecutive days, respectively, while BZ was administered orally
at 10 or 100 mg/kg s.i.d. as suboptimal and optimal doses, for 5
consecutive days as a positive control. Two animal control
groups (untreated and uninfected, as well as untreated and
infected) were also included. To investigate the potential
benefit of a combination treatment regimen, 14 (2.5 mg/kg)
and BZ (10 mg/kg) were co-administrated in a separate group
(G6, Table 5).
Treatment was initiated at the onset of parasitemia (5 days
post-infection (dpi)) and the peak parasitemia readout was
performed at 8 dpi. 14 showed a concentration-dependent
effect (Figure 3). It exerted a peak parasitemia reduction of
47%, 60% and 92% at 8 dpi in treatment regimens of 0.25, 2.5,
25 mg/kg b.i.d., respectively. The efficacy of 14 at 25 mg/kg
b.i.d. (92%) approached that of BZ at 100 mg/kg s.i.d. (>99%),
when measured at 8 dpi and indistinguishable from BZ at 10
dpi and onwards. No toxic effects could be noticed in mice
treated with 14. Unexpectedly, co-administration of 14 (at
2.5 mg/kg) and BZ (at 10 mg/kg) led to a weaker parasitemia
reduction than either compound separately. Treatment with 14
at 25 mg/kg gave 83.3% survival at 34 dpi, whereas lower doses
(0.25, 2.5 mg/kg) did not protect against mortality (Figure 3 (E)
and Table 5). All co-administrated animals succumbed to the
Figure 2. Summary of SAR trends for T. cruzi
Table 4. Evaluation of in vitro metabolic stability of 14 in the presence of male mouse and pooled human S9 microsomal fractions.[a]
Phase I or II
Time [min]
Cpd. 14 remaining [%]
MOUSE
mean
HUMAN
mean
SD
SD
CYP – NADPH
0
100
93
88
86
100
98
–
100
104
95
96
100
97
–
15
30
60
0
15
30
60
2.7
5.0
0.7
–
0.6
1.1
0.8
5.8
10.7
5.4
–
1.8
6.1
3.0
UGT Enzymes
94
97
91
93
[a] Values represent percentages of remaining parent compound. Four time points (0–15–30–60 min) of incubation were monitored. Values are reported as
means and SD of two independent experiments. The reference drug was diclofenac, susceptible to phase I and phase II metabolism (data not shown).
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Figure 3. In vivo efficacy of 14 was evaluated in the mouse model of acute infection with Y-strain T. cruzi. (A–D) Blood parasitemia levels were measured via
direct microscopic counting of the number of parasites in the blood (5 μL) from a tail vein puncture. (E) Cumulative mortality rate of the animals was recorded
until 34 dpi. All treatments were for 5 consecutive days and were initiated at the onset of parasitemia (5 dpi).
Table 5. Administration and efficacy of 14 and BZ in acute CD mice model.[a]
Group
Compound
Dose [mg/kg]
Doses per day
Survival
G1
G2
G3
G4
G5
G6
G7
G8
–
–
14
14
14
14+BZ
BZ
Uninfected
Untreated (vehicle)
0.25
2.5
–
–
2
2
6/6
0/6
1/6
0/6
5/6
0/6
4/5
5/5
25
2
2.5+10 (BZ)
10
100
(2+1)
1
1
BZ
[a] All treatments were executed at the onset of parasitemia (5 dpi) and treated for 5 consecutive days (p.o.).
infection, in line with the parasitemia reduction trend. Unfortu-
nately, none of treated animals had negative parasitemia at 34
dpi, indicating a lack of sterile cure at the used dosing
regimens.
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According to our previous work, the potential reason for
yl)-7-deazaguanosine (17) was prepared from the known 61[23,39]
using the same sequence of Suzuki coupling and O-demeth-
ylation.
in vivo failure may be the lack of activity against the blood-
stream form (BF) trypomastigotes, since 14 was not active
against BF trypomastigotes (Y strain) after 2 and 24 hours of
incubation. Compound 14 displayed EC50 values >81 μM, while
BZ gave 11.5 μM (Table 6). Also, the lack of cure may be due to
the short period of drug exposure in this proof of concept study
(only 5 days of drug therapy). Under this treatment scheme, BZ
also failed to provide sterile cure: all surviving 14- and BZ-
treated animals displayed positive blood parasitism evaluated
by qPCR. Accordingly, the qPCR findings demonstrated a similar
blood parasite load when both groups were analysed, reaching
mean values of 83.9�129.4 and 84.1�90.7 par. eq./mL for 14
and BZ, respectively.
Catalytic dechlorination of 62[38] afforded 63. 62 also served
as a precursor for the introduction of a 6-methyl (65) or 6-ethyl
group[40] (66) (Scheme 2). C7 halogenation was accomplished
with the appropriate halosuccinimides. Subsequent ammonol-
ysis and Suzuki coupling furnished the desired 4-chlorophenyl
analogues 13, 14 and 15.
For the synthesis of the 6-phenyl analogue 16, the phenyl
group was first introduced on 6-chloro-7-deazapurine
(Scheme 3). After iodination in DMF, heterocycle 70 was
glycosylated under Vorbrüggen conditions[37] to give 7-iodo 71
after subsequent deprotection. Final introduction of the para-
chlorophenyl at C7 via Pd-based cross coupling gave the
desired 16.
Chemistry
To probe the importance of the N1 nitrogen, several pyrrolo
[2,3-b]pyridine (1,7-dideazapurine) nucleoside analogues were
prepared (Scheme 4). First, 6-methyl analogue 72[27] was sub-
jected to standard aqueous Suzuki coupling to introduce the 4-
chlorophenyl group. To obtain several 1-substituted nucleoside
analogues, commercially available 5-substituted pyrrolo[2,3-b]
pyridines were employed as the starting material. Bromination
at C3 was effected by NBS/DMF to yield substituted heterocyclic
analogues 73–76.[41–42] Vorbrüggen glycosylation[27] afforded the
desired analogues 77–80 after direct deprotection in saturated
The synthesis of several 6-modified 7-chlorophenyl-7-deaza
nucleoside analogues is depicted in Schemes 1–3. First, 55[37]
was reacted with different nucleophiles to effect 6-substitution
and concomitant deprotection[38] (Scheme 1), providing the 7-
iodo nucleoside intermediates 56–60. Then, aqueous Suzuki
coupling[22,24–25] allowed to construct the target 7-(4-chlorophen-
yl) derivatives 7–8, 10–12. To access inosine analogue 9, 10 was
demethylated with in situ generated TMSI.[37–38] 7-(4-chlorophen-
Table 6. Activity of 14 against T. cruzi (Y strain) bloodstream trypomastigotes and intracellular amastigotes in 2D primary cardiac cells.[a]
Cpd.
Bloodstream trypomastigotes
EC50 [μM]
Intracellular
2D primary cardiac cells
CC50 [μM]
AmastigotesbEC50 [μM]
14
BZ
>81
11.5�1.1*
0.77�0.05
5.76�0.09
481.9
>1000
[a] EC50 values are described as means�SD of 2 independent experiments; [b] The activities of analogues against intracellular amastigotes are estimated as
the reduction of infection index (percentage of infected host cells × number of parasites per host cell); * Hulpia et al., 2018.[25]
Scheme 1. Reagents and conditions: (a) methylamine (40% W/V in water), 82% (56); dimethylamine (40% W/V in water), 72% (57); 0.5 M NaOMe/MeOH or
°
°
NaOEt/EtOH, 50 C, 68% (58); 51% (59); NaSMe, EtOH, r.t., 79% (60); (b) (4-chlorophenyl)boronic acid, Pd(OAc)2, TPPTS, Na2CO3, MeCN/water (1:2 ratio), 100 C,
39% (7), 13% (8), 36% (10), 33% (11), 34% (12), 18% (18); (c) NaI, TMSCl, MeCN, 74% (9), 68% (17).
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Scheme 2. Reagents and conditions: (a) Pd(OH)2/C, H2, K2CO3, MeOH, 84%; (b) NIS, DMF, 38%; (c) (4-chlorophenyl)boronic acid, Pd(OAc)2, TPPTS, Na2CO3,
°
°
MeCN/water (1:2 ratio), 100 C, 32% (13), 63% (14), 49% (15); (d) Me3Al or Et3Al, Pd(PPh3)4, THF, 100 C, 93% (65), 65% (66); (e) (i) NIS for 65, NBS for 66, DMF;
(ii) 7 N NH3/MeOH, 61% (67), 64% (68) for two steps.
group with NBS/DMF afforded 84, which was converted to the
desired 54.
Conclusion
Scheme 3. Reagents and conditions: (a) phenylboronic acid, Pd(PPh3)4,
In a first set of in vitro assays of this study, we have investigated
°
K2CO3, toluene, 100 C, 95%; (b) NIS, DMF, 85%; (c) (i) 1-O-acetyl-2,3,5-tri-O-
°
the influence of modifying the pyrimidine part of a previously
reported 7-deazapurine nucleoside analogue on T. cruzi intra-
cellular amastigotes. We found that a 6-methyl group acts as a
suitable bioisostere and conferred a better selectivity. Next,
keeping the 6-methyl group unaltered, we investigated alter-
native 7-substituents and found two 3,4-substituted phenyl
analogues 40 and 43 that are equipotent to 14. Nucleoside 14
showed a high potency against intracellular forms belonging to
different strains and DTUs (Y and Tulahuen strains) and a low
cardiotoxic profile in primary cardiac cell cultures. Our analyses
employing a variety of in vitro models (fibroblast cell lines, 2D
and 3D cardiac cell cultures and parasite strains belonging to
distinct DTUs) is desirable since the metabolism of mammalian
cells and parasite strains and host-parasite interactions have an
impact on anti-T. cruzi compound activity.[44] In fact, using
different in vitro assays in the drug discovery pipeline may
better predict in vivo outcomes since the parasite invades
different mammalian host cell types, and thus compounds must
be effective in a range of host cell environments. The inclusion
of cardiac organoids for anti-T. cruzi drug evaluation is
recommended as the heart is one of the main target organs for
infection and inflammation in CD and the drug responses may
be influenced by the host cellular metabolism.[44–45]
benzoyl-β-d-ribofuranose, BSA, TMSOTf, MeCN, 80 C; (ii) 7 N NH3/MeOH,
22% for two steps; (d) (4-chlorophenyl)boronic acid, Pd(OAc)2, TPPTS,
°
Na2CO3, MeCN/water (1:2 ratio), 100 C, 23%.
methanolic ammonia. The desired para-chlorophenyl group was
then introduced via Suzuki reaction to yield nucleoside
analogues 21, 23 and 25. For the synthesis of 22, a different Pd-
catalyst system was used to reduce the formation the 7-
dehalogenated product of 78, which was difficult to remove
from the desired 22. To gain access to 1-amino derivative 24,
the 1-nitro group of 79 was reduced by catalytic hydrogenation
(Pt/C) without concomitant dechlorination.
Encouraged by the observed SAR, various aromatic sub-
stituents were introduced at position 7 via Pd-based cross
coupling of the 6-methyl-7-iode precursor 67 (Scheme 5).
To gain access to a 3’-deoxyribofuranose analogue, a
Vorbrüggen reaction was performed involving 4-chloro-5-iodo-
pyrrolo[2,3-d]pyrimidine[43] and 1-O-acetyl-2,5-di-O-benzoyl-3-
deoxy-α/β-D- ribofuranose[25,32–33] to give 81[33] (Scheme 6). A
magnesium-iodine exchange with the Knochel reagent
(iPrMgCl.LiCl)[23,32] and subsequent aqueous workup also re-
moved the 7-iodo group. Palladium catalysed C6 methylation of
the resulting intermediate with Al3Me gave 82. Subsequent
removal of the benzoyl groups, introduction of a 7-bromo
In addition, we also tested the toxicity and anti-T. cruzi
activity of compound 14 using cardiac spheroids that corrobo-
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°
Scheme 4. Reagents and conditions: (a) (4-chlorophenyl)boronic acid, Pd(OAc)2, TPPTS, Na2CO3, MeCN/water (1:2 ratio), 100 C, 61%; (b) NBS, DMF, 95% (73),
°
92% (74), 84% (75), 81% (76); (c) (i) 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose, BSA, TMSOTf, MeCN, 80 C; (ii) 7 N NH3/MeOH, r.t., 24% (77), 32% (78),
°
79% (79), 61% (80) for two steps; (d) (4-chlorophenyl)boronic acid, Pd(OAc)2, TPPTS, Na2CO3, MeCN/water (1:2 ratio), 100 C, 13% (21), 15% (23), 14% (25); (e)
°
Pd (PPh3)4, K2CO3, 1,4-dioxane/water (1:1 ratio), 100 C, 24% (22); (f) Pt/C, H2, MeOH, 68%.
°
Scheme 5. Reagents and conditions: (a) arylboronic acid, Pd(OAc)2, TPPTS, Na2CO3, MeCN/water (1:2 ratio), 100 C, 21%–77%.
°
°
Scheme 6. Reagents and conditions: (a) 1-O-acetyl-2,3,5-tri-O-benzoyl-α/β-d-ribofuranose, BSA, TMSOTf, MeCN, 80 C,74%; (b) (i) iPrMgCl·LiCl, THF, À 10 C; (ii)
°
°
°
0.5 M aq. HCl, 0 C; (ii) Me3Al, Pd(PPh3)4, THF, 100 C, 66% for three steps; (c) 0.1 M NaOMe/MeOH, 83%; (d) NBS, DMF, 50 C, 73%; (e) 4-chlorophenylboronic
°
acid, Pd(OAc)2, TPPTS, Na2CO3, MeCN/water (1:2 ratio), 100 C, 36%.
rated the cardiac 2-D assays, demonstrating lack of cardiotox-
icity and similar antiparasitic effect of 14 as compared to the
reference drug. The use of cardiac spheroids represents a very
promising approach since 3-D cultures more closely reproduce
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Nucleoside analogue 2, 10 were prepared as described
the cellular microenvironments in in vivo systems and has
gained great interest in drug discovery programs due to its
cost-effectiveness.[46] Our in vivo data endorsed the use of 3-D
cardiac cells as a sensitive in vitro model for drug evaluation of
novel trypanosomicidal agents as reported.[45]
previously.[27,47]
In this section, the IUPAC nomenclature and numbering were
applied in the pyrrolo[2,3-d]pyrimidine system, differing with the
purine numbering in the body of the text.
Following confirmation of metabolic stability in vitro, com-
pound 14 was evaluated in
a mouse model of acute
General procedure A for Suzuki coupling reaction (as
described in reference[24–25,48]
)
experimental CD. At the highest dose (25 mg/kg p.o. b.i.d. for
5 days), it elicited comparable peak parasitemia reduction as
the positive control BZ, and treated animals were protected
from infection-caused mortality. Unfortunately, at 34 dpi, none
of the surviving animals showed negative parasitemia evaluated
by light microscopy. qPCR analysis at the endpoint showed that
treatment with BZ at 100 mg/kg and 14 at 25 mg/kg resulted in
similar blood parasite loads, demonstrating comparable effica-
cies under the tested treatment regimens. However, as already
noticed using other nucleoside analogues,[25] a lack of activity
was found when 14 was assayed against bloodstream trypo-
mastigotes which may explain at least in part, the failure of
sterile cure. As a new drug should be active against a
heterogeneity of intracellular multiplicative amastigotes, non-
replicative trypomastigotes and dormant forms,[44] we hoped
that introducing additional modifications may have led to
highly potent antitrypanosomal agents to be further evaluated
in pre-clinical studies.
Iodo-derivative or bromo-derivative (1 eq.), boronic acid (1.5 eq.),
Pd(OAc)2 (0.05 eq.), TPPTS (0.15 eq.) and Na2CO3 (9 eq.) were added
to a 10 mL round-bottom flask, equipped with a stir bar. Then, the
flask was purged with argon thrice after removal of the air in vacuo.
A mixture of MeCN/water (1/2 ratio, 6 mL/mmol SM) was added
into the mixture via syringes. The reaction mixture was stirred at
°
ambient temperature for 5 min, and then heated at 100 C. When
the consumption of SM was observed by HRMS (~1 to 2 h), the
reaction was cooled to ambient temperature. Then, 0.5 M aq. HCl
was added to neutralize the mixture. The solution was evaporated
in vacuo, and the residue resuspended in MeOH and evaporated
three times. Then the residue was adsorbed onto Celite® (from
MeOH) and eluted over a short silica pad (~5 cm) with 20% MeOH/
DCM. The resulting solution was evaporated till dryness and
purified by column chromatography (MeOH/DCM gradient).
4-Methylamino-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (56[23]). 55[23] (600 mg, 0.83 mmol) was dissolved in 1,4-
dioxane (1 mL) and stirred in a 10 mL pressure tube. Methylamine
solution (40% W/V solution in water, 5 mL) was added and then
°
the reaction mixture was stirred at 100 C overnight. After cooling
to room temperature, the reaction mixture was evaporated till
dryness, and purified by flash column chromatography (0!20%
MeOH/DCM) to afford 56 (273 mg, 0.67 mmol) as a yellow foam in
Experimental Section
1
82% yield. H NMR (300 MHz, DMSO-d6) δ: 3.03 (d, J=4.7 Hz, 3 H,
Chemistry
NHCH3), 3.49–3.56 (m, 1 H, H-5’’), 3.58–3.65 (m, 1 H, H-5’), 3.88 (q,
J=3.5 Hz, 1 H, H-4’), 4.06 (q, J=4.2 Hz, 1 H, H-3’), 4.35 (q, J=6.1 Hz,
1 H, H-2’), 5.10–5.16 (m, 2 H, OH-5’ and OH-3’), 5.30 (d, J=6.2 Hz, 1
H, OH-2’), 6.03 (d, J=6.2 Hz, 1 H, H-1’), 6.44 (q, J=4.4 Hz, 1 H, NH),
7.65 (s, 1 H, H-6), 8.19 (s, 1 H, H-2). HRMS (ESI): calculated for
C12H16IN4O4 ([M+H]+): 407.0211, found: 407.0230.
All commercially available reagents and solvents were purchased in
an analytical grade without further purification. All reactions were
monitored under TLC analysis or HRMS or analytical LC–MS.
Precoated F254 aluminium plates from Macherey-Nagel® were used
and visualized by UC for TLC analysis. For analytical LC-MS, the
conditions were that: a C18 column (2.7 mm, 100×4.6 mm) from
Waters Cortecs; a gradient system consisting of 0.2% formic acid in
H2O (v/v)/MeCN; a gradient from 95:5 to 0:100 in 10 min with a
flow rate of 1.44 mL/min. All purification was completed via silica
gel column chromatography or preparative RP-HPLC. The 60 M
silica gel (40–63 μm) from Macherey-Nagel® or an automated flash
unit (Reveleris X2) from Grace/Büchi® with prepacked silica columns
were used for silica gel column chromatography. For preparative
RP-HPLC, the conditions were that: a Luna Omega Polar column
(5 μm, 250×21 mm) from Phenomenex®; a gradient system consist-
ing of 0.2% formic acid in H2O (v/v)/ MeCN; a flow rate of 20 mL/
min. a Waters AutoPurification system was applied in both of
analytical LC–MS and preparative RP-HPLC, equipped with ACQUITY
QDa (mass; 100–1000 amu) and 2998 Photodiode Array (220–
400 nm). A LCT Premier XE time-of-flight (ToF) mass spectrometer
from Waters was applied in exact mass measurements, equipped
with a standard electrospray (ESI) and modular interface from
Lockspray®. A 300 MHz spectrometer from Varian Mercury or a
400 MHz spectrometer from Bruker Avance Neo was used for NMR
4-Dimethylamino-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-
d]-pyrimidine (57[23]). 55[23] (600 mg, 0.83 mmol) was dissolved in
1,4-dioxane (1 mL) and stirred in a 10 mL pressure tube. Dimeth-
ylamine solution (40% W/V solution in water, 5 mL) was added and
°
then the reaction mixture was stirred at 100 C overnight. After
cooling to room temperature, the reaction mixture was evaporated
till dryness, and purified by flash column chromatography (0!20%
MeOH/DCM) to afford 57 (250 mg, 0.60 mmol) as a yellow foam in
1
72% yield. H NMR (300 MHz, DMSO-d6) δ: 3.16 (s, 6 H, 2CH3), 3.52–
3.56 (m, 1 H, H-5’’), 3.60–3.64 (m, 1 H, H-5’), 3.89 (q, J=3.5 Hz, 1 H,
H-4’), 4.06–4.08 (m, 1 H, H-3’), 4.36 (t, J=5.3 Hz, 1 H, H-2’), 5.12 (br.
s., 2 H, OH-5’ and OH-3’), 5.32 (br. s., 1 H, OH-2’), 6.11 (d, J=6.1 Hz,
1 H, H-1’), 7.86 (s, 1 H, H-6), 8.24 (s, 1 H, H-2). ). HRMS (ESI):
calculated for C13H18IN4O4 ([M+H]+): 421.0367, found: 421.0346.
4-Methoxy-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyri-
midine (58[23]). 55[23] (600 mg, 0.83 mmol) was dissolved in 0.5 M
NaOMe in MeOH (10 mL) and the reaction mixture was stirred at
°
50 C for 3 h. Then, the reaction was cooled to room temperature.
1
1
spectra. H-1H 2D NOESY and H-13C gHMBC were used respectively
to ascertain the stereochemistry at C-1’. A Büchi-545 apparatus
(uncorrected) was used for melting points measurements. Purity of
final compounds was measured through integration of UV signal
(total UV chromatogram and wavelength selected at 254 nm) in
analytical LC-MS. Purity of all target compounds was at least 95%.
Then, 0.5 M aq. HCl solution was added to neutralize the reaction
solution. The resulting mixture was evaporated till dryness, and
purified by flash column chromatography (0!10% MeOH/DCM) to
afford 58 (230 mg, 0.56 mmol) as a white solid in 68% yield. 1H
NMR (400 MHz, DMSO-d6) δ: 3.53–3.57 (m, 1 H, H-5’’), 3.62–3.66 (m,
1 H, H-5’), 3.91 (dd, J=3.0, 1.9 Hz, 1 H, H-4’), 4.06–4.09 (m, 4 H, H-3’
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and OCH3), 4.35–4.38 (m, 1 H, H-2’), 5.07–5.09 (m, 1 H, OH-5’), 5.15
(br. s., 1 H, OH-3’), 5.36 (d, J=5.4 Hz, 1 H, OH-2’), 6.13 (dd, J=6.2,
1.8 Hz, 1 H, H-1’), 7.88 (d, J=2.0 Hz, 1 H, H-6), 8.44 (d, J=2.1 Hz, 1
H, H-2). HRMS (ESI): calculated for C12H15IN3O5 ([M+H]+): 408.0051,
found: 408.0063.
4-Ethoxy-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (11). 11 was prepared according to General
procedure A. 59 (110 mg, 0.26 mmol) gave rise to 11 (35 mg,
1
°
0.086 mmol) as a white solid in 33% yield. Melting point: 138 C. H
NMR (300 MHz, DMSO-d6) δ: 1.36 (t, J=7.0 Hz, 3 H, CH3), 3.56 (ddd,
J=11.9, 5.8, 4.2 Hz, 1 H, H-5’’), 3.66 (ddd, J=12.0, 5.2, 4.2 Hz, 1 H, H-
5’), 3.93 (q, J=4.0 Hz, 1 H, H-4’), 4.08–4.20 (m, 1 H, H-3’), 4.41–4.57
(m, 3 H, H-2’ and CH2), 5.08 (t, J=5.6 Hz, 1 H, OH-5’), 5.16 (d, J=
5.0 Hz, 1 H, OH-3’), 5.37 (d, J=6.4 Hz, 1 H, OH-2’), 6.22 (d, J=6.2 Hz,
1 H, H-1’), 7.40–7.52 (m, 2 H, HÀ Ph), 7.67–7.79 (m, 2 H, HÀ Ph), 7.93
(s, 1 H, H-6), 8.45 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 14.2
(CH3), 61.5 (C-5’), 62.2 (CH2), 70.4 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.9
(C-1’), 102.6 (C-4a), 115.1 (C-5), 122.9 (C-6), 128.0 (2 C, C-3Ph and C-
5Ph), 130.2 (2 C, C-2Ph and C-6Ph), 131.1 (C-4Ph), 132.5 (C-1Ph), 150.9 (C-
2), 152.7 (C-7a), 162.2 (C-4). HRMS (ESI): calculated for C19H21ClN3O5
([M+H]+): 406.1144, found: 406.1156.
4-Ethoxy-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimi-
dine (59). 55[23] (300 mg, 0.41 mmol) was dissolved in 0.5 M NaOEt
°
in EtOH (10 mL) and the reaction mixture was stirred at 50 C for
3 h. Then, the reaction was cooled to room temperature. Then,
0.5 M aq. HCl solution was added to neutralize the reaction
solution. The resulting mixture was evaporated till dryness, and
purified by flash column chromatography (0!10% MeOH/DCM) to
1
afford 59 (88 mg, 0.21 mmol) as a white solid in 51% yield. H NMR
(300 MHz, DMSO-d6) δ: 1.40 (t, J=7.2 Hz, 3 H, CH3), 3.54 (ddd, J=
12.0, 5.6, 4.0 Hz, 1 H, H-5’’), 3.63 (ddd, J=12.0, 5.4, 4.0 Hz, 1 H, H-5’),
3.90 (q, J=3.5 Hz, 1 H, H-4’), 4.08 (td, J=4.8, 3.2 Hz, 1 H, H-3’), 4.34–
4.40 (m, 1 H, H-2’), 4.53 (q, J=7.0 Hz, 2 H, CH2), 5.08 (t, J=5.6 Hz, 1
H, OH-5’), 5.15 (d, J=4.7 Hz, 1 H, OH-3’), 5.35 (d, J=6.2 Hz, 1 H, OH-
2’), 6.12 (d, J=6.2 Hz, 1 H, H-1’), 7.86 (s, 1 H, H-6), 8.41 (s, 1 H, H-2).
HRMS (ESI): calculated for C13H17IN3O5 ([M+H]+): 422.0207, found:
422.0199.
4-Oxo-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-
d]-pyrimidine (9). 10 (70 mg, 0.18 mmol) and NaI (135 mg,
0.90 mmol, 5 eq.) were placed in anhydrous MeCN (5 mL). Next,
TMSCl (114 μL, 0.90 mmol, 5 eq.) was slowly added into the reaction
mixture, and the mixture was stirred at ambient temperature for
2 h. The precipitate was filtered, washed with MeCN, and dissolved
in water. Then, aq. sat. NaHCO3 was added to neutralize the
solution. The mixture was evaporated till dryness, and purified by
flash column chromatography (0!20% MeOH/DCM) to afford 9
4-Methylsulfanyl-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-
d]-pyrimidine (60[23]). 55[23] (600 mg, 0.83 mmol) and sodium
thiomethoxide (126 mg, 1.83 mmol, 2.2 eq.) was added in EtOH
(10 mL) and the reaction mixture was stirred at ambient temper-
ature for 4 h. Then, the resulting mixture was evaporated till
dryness, and purified by flash column chromatography (0!10%
MeOH/DCM) to afford 60 (273 mg, 0.66 mmol) as a white solid in
(50 mg, 0.13 mmol) as a white solid in 74% yield. Melting point:
1
°
75 C. H NMR (300 MHz, DMSO-d6) δ: 3.55 (ddd, J=11.8, 5.6, 4.2 Hz,
1 H, H-5’’), 3.65 (ddd, J=12.0, 5.4, 4.0 Hz, 1 H, H-5’), 3.90 (q, J=
3.8 Hz, 1 H, H-4’), 4.07–4.16 (m, 1 H, H-3’), 4.38 (q, J=6.0 Hz, 1 H, H-
2’), 5.04 (t, J=5.6 Hz, 1 H, OH-5’), 5.14 (d, J=5.0 Hz, 1 H, OH-3’), 5.36
(d, J=6.2 Hz, 1 H, OH-2’), 6.10 (d, J=6.2 Hz, 1 H, H-1’), 7.38–7.41 (m,
1 H, HÀ Ph), 7.41–7.45 (m, 1 H, HÀ Ph), 7.78 (s, 1 H, H-6), 7.95–7.98
(m, 2 H, HÀ Ph and H-2), 7.98–8.02 (m, 1 H, HÀ Ph), 12.10 (br. s., 1 H,
NH). 13C NMR (75 MHz, DMSO-d6) δ: 61.4 (C-5’), 70.4 (C-3’), 74.1 (C-
2’), 85.1 (C-4’), 86.8 (C-1’), 105.0 (C-4a), 119.0 (C-5), 119.2 (C-6), 127.9
(2 C, C-3Ph and C-5Ph), 129.6 (2 C, C-2Ph and C-6Ph), 130.7 (C-4Ph),
132.5 (C-1Ph), 144.3 (C-2), 149.0 (C-7a), 158.4 (C-4). HRMS (ESI):
calculated for C17H17ClN3O5 ([M+H]+): 378.0851, found: 378.0840.
1
79% yield. H NMR (300 MHz, DMSO-d6) δ: 2.64 (s, 3 H, CH3), 3.51–
3.58 (m, 1 H, H-5’’), 3.60–3.67 (m, 1 H, H-5’), 3.91 (q, J=3.5 Hz, 1 H,
H-4’), 4.36 (q, J=5.9 Hz, 1 H, H-3’), 5.07 (t, J=5.4 Hz, 1 H, H-2’), 5.16
(d, J=5.0 Hz, 1 H, OH-5’), 5.37 (d, J=6.4 Hz, 1 H, OH-3’), 6.15 (d, J=
6.2 Hz, 1 H, OH-2’), 7.99 (s, 1 H), 8.62 (s, 1 H). HRMS (ESI): calculated
for C12H15IN3O4 S ([M+H]+): 423.9822, found: 423.9809.
4-Methylamino-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (7). 7 was prepared according to General
procedure A. 56 (210 mg, 0.52 mmol) gave rise to 7 (80 mg,
1
°
0.077 mmol) as a white solid in 13% yield. Melting point: 122 C. H
4-Methylsulfanyl-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (12). 12 was prepared according to
General procedure A. 60 (260 mg, 0.61 mmol) gave rise to 12
NMR (300 MHz, DMSO-d6) δ: 2.91 (d, J=4.7 Hz, 3 H, CH3), 3.43–3.57
(m, 1 H, H-5’’), 3.58–3.71 (m, 1 H, H-5’), 3.90 (q, J=3.5 Hz, 1 H, H-4’),
4.03–4.19 (m, 1 H, H-3’), 4.45 (t, J=5.6 Hz, 1 H, H-2’), 5.17 (br. s., 2 H,
OH-5’ and OH-3’), 5.32 (br. s., 1 H, OH-2’), 5.74 (q, J=4.6 Hz, 1 H,
HN), 6.12 (d, J=6.2 Hz, 1 H, H-1’), 7.38–7.49 (m, 2 H, HÀ Ph, 7.49–
7.60 (m, 3 H, HÀ Ph and H-6), 8.24 (s, 1 H, H-2). 13C NMR (75 MHz,
DMSO-d6) δ: 28.0 (CH3), 61.6 (C-5’), 70.6 (C-3’), 73.8 (C-2’), 85.1 (C-4’),
87.0 (C-1’), 100.5 (C-4a), 115.0 (C-5), 121.3 (C-6), 128.9 (2 C, C-3Ph and
C-5Ph), 130.0 (2 C, C-2Ph and C-6Ph), 131.4 (C-4Ph), 133.3 (C-1Ph), 150.2
(C-7a), 151.6 (C-2), 156.8 (C-4). HRMS (ESI): calculated for
C18H20ClN4O4 ([M+H]+): 391.1168, found: 391.1159.
(85 mg, 0.21 mmol) as a white solid in 34% yield. Melting point:
1
°
209 C. H NMR (300 MHz, DMSO-d6) δ: 2.55 (s, 3 H, CH3), 3.55 (ddd,
J=12.0, 5.6, 4.0 Hz, 1 H, H-5’’), 3.64 (ddd, J=12.0, 5.2, 4.2 Hz, 1 H, H-
5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’), 4.05–4.21 (m, 1 H, H-3’), 4.45 (q, J=
5.8 Hz, 1 H, H-2’), 5.04 (t, J=5.6 Hz, 1 H, OH-5’), 5.17 (d, J=5.0 Hz, 1
H, OH-3’), 5.39 (d, J=6.2 Hz, 1 H, OH-2’), 6.25 (d, J=6.2 Hz, 1 H, H-
1’), 7.40–7.57 (m, 4 H, HÀ Ph), 7.85 (s, 1 H, H-6), 8.68 (s, 1 H, H-2). 13C
NMR (75 MHz, DMSO-d6) δ: 11.9 (CH3), 61.4 (C-5’), 70.5 (C-3’), 74.1
(C-2’), 85.2 (C-4’), 86.7 (C-1’), 114.0 (C-4a), 115.4 (C-5), 124.5 (C-6),
127.9 (2 C, CÀ Ph), 131.7 (2 C, CÀ Ph), 132.1 (C-4Ph), 132.4 (C-1Ph),
148.7 (C-7a), 150.4 (C-2), 161.1 (C-4). HRMS (ESI): calculated for
C18H19ClN3O4S ([M+H]+): 408.0779, found: 408.0795.
4-Dimethylamino-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (8). 8 was prepared according to General
procedure A. 57 (250 mg, 0.59 mmol) gave rise to 8 (30 mg,
1
°
0.20 mmol) as a white solid in 39% yield. Melting point: 75 C. H
2-Amino-4-methoxy-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-
7H-pyrrolo[2,3-d]-pyrimidine (18). 18 was prepared according to
NMR (300 MHz, DMSO-d6) δ: 2.75 (s, 6 H, 2CH3), 3.48–3.57 (m, 1 H,
H-5’’), 3.58–3.70 (m, 1 H, H-5’), 3.90 (q, J=3.5 Hz, 1 H, H-4’), 4.06–
4.15 (m, 1 H, H-3’), 4.46 (q, J=6.0“ Hz, 1 H, H-2’), 5.04–5.19 (m, 2 H,
OH-5’ and OH-3’), 5.33 (d, J=6.4 Hz, 1 H, OH-2’), 6.18 (d, J=6.2 Hz,
1 H, H-1’), 7.36–7.56 (m, 4 H, HÀ Ph), 7.71 (s, 1 H, H-6), 8.29 (s, 1 H, H-
2). 13C NMR (75 MHz, DMSO-d6) δ: 40.7 (2 C, CH3), 61.6 (C-5’), 70.5 (C-
3’), 73.8 (C-2’), 85.1 (C-4’), 86.8 (C-1’), 102.0 (C-4a), 115.7 (C-5), 122.1
(C-6), 128.5 (2 C, CÀ Ph), 129.7 (2 C, CÀ Ph), 131.1 (C-4Ph), 134.6 (C-
1Ph), 150.3 (C-2), 152.3 (C-7a), 160.0 (C-4). HRMS (ESI): calculated for
C19H22ClN4O4 ([M+H]+): 405.1324, found: 405.1323.
°
°
General procedure A, except for the use of 90 C instead of 100 C.
61 (300 mg, 0.71 mmol) gave rise to 18 (53 mg, 0.13 mmol) as a
1
°
white solid in 18% yield. Melting point: 202 C. H NMR (300 MHz,
DMSO-d6) δ: 3.44–3.56 (m, 1 H, H-5’’), 3.56–3.69 (m, 1 H, H-5’), 3.84
(q, J=4.0 Hz, 1 H, H-4’), 3.91 (s, 3 H, OCH3), 4.02–4.15 (m, 1 H, H-3’),
4.37 (q, J=6.0 Hz, 1 H, H-2’), 5.00 (t, J=5.6 Hz, 1 H, OH-5’), 5.05 (d,
J=4.4 Hz, 1 H, OH-3’), 5.26 (d, J=6.2 Hz, 1 H, OH-2’), 6.05 (d, J=
6.2 Hz, 1 H, H-1’), 6.32 (br. s., 2 H, NH2), 7.36–7.41 (m, 2 H, HÀ Ph and
H-6), 7.42–7.47 (m, 1 H, HÀ Ph), 7.59–7.63 (m, 1 H, HÀ Ph), 7.64–7.68
(m, 1 H, HÀ Ph). 13C NMR (75 MHz, DMSO-d6) δ: 53.0 (OCH3), 61.6 (C-
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°
5’), 70.5 (C-3’), 73.4 (C-2’), 84.7 (C-4’), 85.8 (C-1’), 95.0 (C-4a), 115.5
(C-5), 118.4 (C-6), 128.0 (2 C, C-3Ph and C-5Ph), 129.5 (2 C, C-2Ph and
C-6Ph), 130.5 (C-4Ph), 133.5 (C-1Ph), 155.7 (C-7a), 159.4 (C-2), 163.2 (C-
4). HRMS (ESI): calculated for C18H20ClN4O5 ([M+H]+): 407.1117,
found: 407.1134.
grey solid in 32% yield. Melting point: 65 C. 1H NMR (300 MHz,
DMSO-d6) δ: 3.58 (ddd, J=11.8, 5.8, 4.4 Hz, 1 H, H-5’’), 3.68 (ddd, J=
12.0, 5.4, 4.2 Hz, 1 H, H-5’), 3.95 (q, J=4.0 Hz, 1 H, H-4’), 4.09–4.22
(m, 1 H, H-3’), 4.51 (q, J=6.0 Hz, 1 H, H-2’), 5.08 (t, J=5.6 Hz, 1 H,
OH-5’), 5.20 (d, J=5.0 Hz, 1 H, OH-3’), 5.42 (d, J=6.2 Hz, 1 H, OH-2’),
6.28 (d, J=5.9 Hz, 1 H, H-1’), 7.41–7.58 (m, 2 H, HÀ Ph), 7.74–7.92 (m,
2 H, HÀ Ph), 8.31 (s, 1 H, H-6), 8.89 (s, 1 H, H-2), 9.38 (s, 1 H, H-4). 13C
NMR (75 MHz, DMSO-d6) δ: 61.5 (C-5’), 70.5 (C-3’), 73.9 (C-2’), 85.3
(C-4’), 86.5 (C-1’), 114.0 (C-5), 116.7 (C-4a), 124.9 (C-6), 128.2 (2 C, C-
2Ph and C-6Ph), 129.0 (2 C, C-3Ph and C-5Ph), 131.2 (C-4Ph), 132.0 (C-
1Ph), 149.1 (C-4), 151.3 (C-2), 151.5 (C-7a). HRMS (ESI): calculated for
C17H17ClN3O4 ([M+H]+): 362.0902, found: 362.0922.
2-Amino-4-oxo-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (17). 18 (53 mg, 0.13 mmol) and NaI
(98 mg, 0.65 mmol, 5 eq.) were placed in anhydrous MeCN (5 mL).
Next, TMSCl (82 μL, 0.65 mmol, 5 eq.) was slowly added into the
reaction mixture, and the mixture was stirred at ambient temper-
ature for 2 h. The precipitate was filtered, washed with MeCN, and
dissolved in water. Then, aq. sat. NaHCO3 was added to neutralize
the solution. The mixture was evaporated till dryness, and purified
by flash column chromatography (0!20% MeOH/DCM) to afford
17 (35 mg, 0.089 mmol) as a white solid in 68% yield. Melting point:
4-Methyl-N7-(2’,3’,5’-tri-O-benzoyl-β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (65). 62[23,49] (9.27 g, 15.5 mmol) and Pd(PPh3)4
(358 mg, 0.31 mmol, 0.02 eq.) were placed into a 250 mL round
bottom flask. The flask was purged with argon thrice after removal
of the air in vacuo. Next, anhydrous THF (62 mL, 4 mL/mmol) was
added via syringe, and AlMe3 solution (2 M in toluene, 9.3 mL,
18.6 mmol, 1.2 eq.) was added dropwise. After the mixture was
stirred for 15 min at ambient temperature, the reaction was
249 C. 1H NMR (300 MHz, DMSO-d6) δ: 3.45–3.55 (m, 1 H, H-5’’),
°
3.56–3.68 (m, 1 H, H-5’), 3.82 (q, J=4.1 Hz, 1 H, H-4’), 4.00–4.12 (m,
1 H, H-3’), 4.31 (q, J=6.2 Hz, 1 H, H-2’), 4.97 (t, J=5.6 Hz, 1 H, OH-
5’), 5.04 (d, J=4.4 Hz, 1 H, OH-3’), 5.26 (d, J=6.2 Hz, 1 H, OH-2’),
5.94 (d, J=6.2 Hz, 1 H, H-1’), 6.32 (br. s, 2 H, NH2), 7.32–7.35 (m, 1 H,
HÀ Ph), 7.35–7.37 (m, 1 H, HÀ Ph), 7.38 (s, 1 H, H-6), 7.96–8.00 (m, 1
H, HÀ Ph), 8.00–8.03 (m, 1 H, HÀ Ph), 10.48 (s, 1 H, NH). 13C NMR
(75 MHz, DMSO-d6) δ: 61.6 (C-5’), 70.4 (C-3’), 73.5 (C-2’), 84.6 (C-4’),
85.8 (C-1’), 97.2 (C-4a), 115.8 (C-6), 118.7 (C-5), 127.8 (2 C, C-3Ph and
C-5Ph), 129.1 (2 C, C-2Ph and C-6Ph), 130.1 (C-4Ph), 133.2 (C-1Ph), 152.6
(C-7a), 152.7 (C-2), 158.8 (C-4). HRMS (ESI): calculated for
C17H18ClN4O5 ([M+H]+): 393.0960, found: 393.0976.
°
refluxed at 100 C until TLC analysis showed the SM was consumed
completely (1 h). After cooling in an ice-water bath, 0.5 M aq. HCl
(10 mL) was added dropwise and slowly. [Caution: methane gas
was generated, with potential excessive foaming]. Next, EA (50 mL)
was added. The layers were separated and the water layer extracted
twice more with EA. Then, the organic layers were combined, dried
over Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (10!50% EA/PET) to give 65 (7.7 g,
13.3 mmol) as a yellow foam in 86% yield. 1H NMR (400 MHz,
CDCl3) δ: 2.71 (s, 3 H, CH3), 4.65–4.71 (m, 1 H, H-5’’), 4.76–4.80 (m, 1
H, H-4’), 4.84–4.89 (m, 1 H, H-5’), 6.16 (dd, J=5.9, 4.4 Hz, 1 H, H-3’),
6.22–6.26 (m, 1 H, H-2’), 6.57 (d, J=3.8 Hz, 1 H, H-1’), 6.73 (d, J=
5.9 Hz, 1 H, H-5), 7.31–7.63 (m, 10 H, H-6 and H-OBz), 7.91–7.94 (m,
2 H, H-OBz), 7.98–8.02 (m, 2 H, H-OBz), 8.11–8.14 (m, 2 H, H-OBz),
8.76 (s, 1 H, H-2). HRMS (ESI): calculated for C33H28N3O7 ([M+H]+):
578.1922, found: 578.1924.
N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidine (63). 62[23,49]
(200 mg, 0.33 mmol) was dissolved in MeOH (5 mL), and solid K2CO3
(91 mg, 0.66 mmol, 2 eq.) was added. The flask was purged with N2,
and a cat. amount of Pd(OH)2/C was added. Then, the N2 gas was
exchanged with H2 (balloon; bubbling). The reaction mixture was
stirred at room temperature until HRMS showed full consumption
of the SM (2 h). Then, the H2 balloon was removed and K2CO3
(136 mg, 0.99 mmol, 3 eq.) was added, and the resulting mixture
stirred at ambient temperature until all benzoyl groups of the SM
were removed (3 h). 0.5 M aq. HCl solution was added to neutralize
the mixture, after which the solution was filtered over Celite®. The
filtrate was evaporated till dryness and purified by column
chromatography (0!10% MeOH/DCM) to afford 63 (70 mg,
0.28 mmol) as a white solid in 84% yield. 1H NMR (300 MHz, DMSO-
d6) δ: 3.55 (ddd, J=12.0, 5.6, 4.0 Hz, 1 H, H-5’’), 3.64 (ddd, J=12.0,
5.4, 4.2 Hz, 1 H, H-5’), 3.93 (q, J=3.7 Hz, 1 H, H-4’), 4.10–4.15 (m, 1
H, H-3’), 4.41–4.47 (m, 1 H, H-2’), 5.06 (t, J=5.4 Hz, 1 H, OH-5’), 5.17
(d, J=5.0 Hz, 1 H, OH-3’), 5.36 (d, J=6.4 Hz, 1 H, OH-2’), 6.22 (d, J=
6.2 Hz, 1 H, H-1’), 6.71 (d, J=3.8 Hz, 1 H, H-5), 7.87 (d, J=3.8 Hz, 1
H, H-6), 8.80 (s, 1 H, H-2), 9.03 (s, 1 H, H-4). HRMS (ESI): calculated
for C11H14N3O4 ([M+H]+): 252.0979, found: 252.0970.
4-Methyl-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimi-
dine (67).[23] 65 (10 g, 17.3 mmol) was dissolved in anhydrous DMF
(120 mL), and NIS (4.67 g, 20.8 mmol, 1.2 eq.) was added. Then, the
reaction mixture was stirred at ambient temperature overnight. EA
(200 mL) and water (200 mL) were added. The layers were
separated and the water layer extracted once more with EA. The
organic layers were combined and washed with brine (200 mL),
dried over Na2SO4, filtered, and evaporated. The residue was
purified by column chromatography (5!30% EA/PET) to give a
yellow oil (10.0 g, 14.2 mmol) in 82% yield. This obtained product
(4 g, 5.68 mmol) was charged in a 250 mL round bottom flask, and
NH3 in MeOH (7 N, 20 mL) was added. The mixture was stirred at
ambient temperature overnight. Then, the reaction solution was
evaporated till dryness, purified by column chromatography (0!
10% MeOH/DCM) to afford 67 (1.95 g, 4.98 mmol) as a slight yellow
solid in 88% yield. 1H NMR (400 MHz, DMSO-d6) δ: 2.89 (s, 3 H, CH3),
3.52–3.58 (m, 1 H, H-5’’), 3.61–3.66 (m, 1 H, H-5’), 3.91 (q, J=3.5 Hz,
1 H, H-4’), 4.09 (q, J=4.1 Hz, 1 H, H-3’), 4.38 (q, J=6.0 Hz, 1 H, H-2’),
5.08 (t, J=5.38 Hz, 1 H, OH-5’), 5.16 (d, J=4.75 Hz, 1 H, OH-3’), 5.36
(d, J=6.38 Hz, 1 H, OH-2’), 6.18 (d, J=6.25 Hz, 1 H, H-1’), 8.06 (s, 1
H, H-6), 8.67 (s, 1 H, H-2). 13C NMR (100 MHz, DMSO-d6) δ: 20.5 (CH3),
53.8 (C-5), 61.4 (C-5’),70.4 (C-3’), 74.1 (C-2’), 85.3 (C-4’), 86.5 (C-1’),
117.8 (C-4a), 131.4 (C-6), 150.0 (C-7a), 150.9 (C-2), 159.5 (C-4). HRMS
(ESI): calculated for C12H15IN3O4 ([M+H]+): 392.0102, found:
392.0105.
5-Iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidine (64).
63 (470 mg, 1.87 mmol) was dissolved in DMF (5 mL), and NIS
(631 mg, 2.81 mmol, 1.5 eq.) was added. The resulting mixture was
°
stirred at 50 C for 2 h. Then, the mixture was evaporated till
dryness and purified by column chromatography (0!10% MeOH/
DCM) to afford 64 (270 mg, 0.71 mmol) as a white solid in 38%
yield. 1H NMR (400 MHz, DMSO-d6) δ: 3.53–3.58 (m, 1 H, H-5’’), 3.62–
3.67 (m, 1 H, H-5’), 3.93 (q, J=3.6 Hz, 1 H, H-4’), 4.11 (q, J=4.2 Hz, 1
H, H-3’), 4.42 (q, J=5.8 Hz, 1 H, H-2’), 5.08 (t, J=5.4 Hz, 1 H, OH-5’),
5.18 (d, J=4.8 Hz, 1 H, OH-3’), 5.40 (d, J=6.4 Hz, 1 H, OH-2’), 6.21
(d, J=6.3 Hz, 1 H, H-1’), 8.12 (s, 1 H, H-6), 8.77 (s, 1 H, H-2), 8.87 (s, 1
H, H-4). HRMS (ESI): calculated for C11H13IN3O4 ([M+H]+): 377.9945,
found: 377.9954.
4-Methyl-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (14). 14 was prepared according to General
5-(4-Chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyr-
imidine (13). 13 was prepared according to General procedure A.
64 (260 mg, 0.69 mmol) gave rise to 13 (80 mg, 0.22 mmol) as a
procedure A. 67 (150 mg, 0.38 mmol) gave rise to 14 (90 mg,
1
°
0.24 mmol) as a white solid in 63% yield. Melting point: 166 C. H
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1
°
NMR (400 MHz, DMSO-d6) δ: 2.46 (s, 3 H, CH3), 3.49–3.59 (m, 1 H, H-
5’’), 3.59–3.73 (m, 1 H, H-5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’), 4.13 (d, J=
3.8 Hz, 1 H, H-3’), 4.46 (dd, J=11.2, 6.0 Hz, 1 H, H-2’), 5.04 (t, J=
5.0 Hz, 1 H, OH-5’), 5.17 (d, J=4.4 Hz, 1 H, OH-3’), 5.37 (d, J=6.4 Hz,
1 H, OH-2’), 6.27 (d, J=6.2 Hz, 1 H, H-1’), 7.45–7.58 (m, 4 H, HÀ Ph),
7.88 (s, 1 H, H-6), 8.70 (s, 1 H, H-2). 13C NMR (100 MHz, DMSO-d6) δ:
22.9 (CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’),
115.7 (C-4a), 115.7 (C-5), 125.0 (C-6), 128.2 (2 C, CÀ Ph), 131.5 (2 C,
CÀ Ph), 132.0 (C-4Ph), 133.2 (C-1Ph), 150.7 (C-7a), 150.9 (C-2), 159.2 (C-
4). HRMS (ESI): calculated for C18H19ClN3O4 ([M+H]+): 376.1059,
found: 376.1068.
0.14 mmol) as a white solid in 49% yield. Melting point: 100 C. H
NMR (400 MHz, DMSO-d6) δ: 1.07 (t, J=7.5 Hz, 3 H, CH3), 2.77 (q, J=
7.5 Hz, 2 H, CH2), 3.48–3.58 (m, 1 H, H-5’’), 3.59–3.70 (m, 1 H, H-5’),
3.93 (q, J=3.8 Hz, 1 H, H-4’), 4.07–4.20 (m, 1 H, H-3’), 4.47 (q, J=
6.1 Hz, 1 H, H-2’), 5.04 (t, J=5.4 Hz, 1 H, OH-5’), 5.17 (d, J=4.8 Hz, 1
H, OH-3’), 5.37 (d, J=6.4 Hz, 1 H, OH-2’), 6.27 (d, J=6.3 Hz, 1 H, H-
1’), 7.41–7.60 (m, 4 H, HÀ Ph), 7.86 (s, 1 H, H-6), 8.75 (s, 1 H, H-2). ).
13C NMR (100 MHz, DMSO-d6) δ: 12.3 (CH3), 28.0 (CH2), 61.5 (C-5’),
70.5 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 115.0 (C-4a), 115.5 (C-
5), 125.2 (C-6), 128.3 (2 C, CÀ Ph), 131.4 (2 C, CÀ Ph), 132.1 (C-4Ph),
133.5 (C-1Ph), 150.8 (C-7a), 151.0 (C-2), 163.8 (C-4). HRMS (ESI):
calculated for C19H21ClN3O4 ([M+H]+): 390.1215, found: 390.1217.
4-Ethyl-N7-(2’,3’,5’-tri-O-benzoyl-β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (66).[50] 62[23,49] (700 mg, 1.17 mmol) and Pd-
(PPh3)4 (135 mg, 0.12 mmol, 0.1 eq.) was placed into a 50 mL round
bottom flask. The flask was purged with argon thrice after removal
of the air in vacuo. Next, anhydrous THF (5 mL, 4 mL/mmol) was
added via syringe, and AlEt3 solution (1 M in hexanes, 1.4 mL,
1.4 mmol, 1.2 eq.) was added dropwise. After the mixture was
stirred for 15 min at ambient temperature, the reaction was
4-Phenyl-7H-pyrrolo[2,3-d]-pyrimidine (69).[50] 4-Chloro-7H-pyrrolo
[2,3-d]pyrimidine (300 mg, 1.95 mmol), phenylboronic acid (480 mg,
3.91 mmol, 2 eq.), K2CO3 (808 mg, 5.85 mmol, 3 eq.) and Pd(PPh3)4
(225 mg, 0.20 mmol, 0.1 eq.) were charged to a 25 mL round-
bottom flask, equipped with a stir bar. Next, the flask was purged
with argon thrice after removal of the air in vacuo. Then, toluene
(8 ml, 4 mL/mmol SM) was added into the mixture via syringe. The
mixture was stirred at ambient temperature for 5 min, and then
°
refluxed at 100 C until TLC analysis showed the SM was consumed
°
completely (1 h). After cooling in an ice-water bath, 0.5 M aq. HCl
(5 mL) was added dropwise and slowly. [Caution: methane gas was
generated, with potential excessive foaming]. Next, EA (10 mL) was
added. The layers were separated and the water layer extracted
twice more with EA. Then, the organic layers were combined, dried
over Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (10!50% EA/PET) to give 66 (450 mg,
0.76 mmol) as a colorless foam in 65% yield. 1H NMR (400 MHz,
CDCl3) δ: 1.38 (t, J=7.6 Hz, 3 H, CH3), 3.02 (q, J=7.6 Hz, 2 H, CH2),
4.68 (dd, J=12.1, 3.9 Hz, 1 H, H-5’’), 4.78 (q, J=3.8 Hz, 1 H, H-4’),
4.87 (dd, J=12.1, 3.2 Hz, 1 H, H-5’), 6.14–6.17 (m, 1 H, H-3’), 6.25 (t,
J=5.8 Hz, 1 H, H-2’), 6.59 (d, J=3.8 Hz, 1 H, H-1’), 6.75 (d, J=5.9 Hz,
1 H, H-5), 7.26–7.42 (m, 5 H, H-6 and H-OBz), 7.46–7.62 (m, 5 H, H-
OBz), 7.93 (d, J=7.4 Hz, 2 H, H-OBz), 8.00 (d, J=7.3 Hz, 2 H, H-OBz),
8.13 (d, J=7.4 Hz, 2 H, H-OBz), 8.79 (s, 1 H, H-2). HRMS (ESI):
calculated for C34H30N3O7 ([M+H]+): 592.2078, found: 592.2067.
heated at 100 C. After the consumption of SM was observed by
analytical LC/MS (1 h), the reaction was cooled to ambient temper-
ature. Then, 0.5 M aq. HCl was added to neutralize the mixture.
Next, the solution was evaporated in vacuo, and the residue was
evaporated till dryness and purified by column chromatography
(10!50% EA/PET) to give 69 (360 mg, 1.84 mmol) as an orange
1
solid in 95% yield. H NMR (300 MHz, DMSO-d6) δ: 6.89 (dd, J=3.5,
1.8 Hz, 1 H, H-5), 7.54–7.61 (m, 3 H, HÀ Ph), 7.66 (dd, J=3.5, 2.5 Hz, 1
H, H-6), 8.16–8.19 (m, 2 H, HÀ Ph), 8.84 (s, 1 H, H-2), 12.26 (br. s., 1 H,
NH). HRMS (ESI): calculated for C12H10N3 ([M+H]+): 196.0869, found:
196.0874.
4-Phenyl-5-iodo-7H-pyrrolo[2,3-d]-pyrimidine (70). 69 (380 mg,
1.95 mmol) was dissolved in anhydrous DMF (5 mL), and NIS
(461 mg, 2.05 mmol, 1.05 eq.) was added. Then, the reaction
mixture was stirred at ambient temperature overnight. Then, water
(cooled in the ice-water bath, 15 mL) was added and the precipitate
generated and was filtered. The solids were washed with ice-cold
water. The solid was collected and dried under high vacuum to
give 70 (530 mg, 1.65 mmol) as a brown solid in 85% yield. 1H NMR
(300 MHz, DMSO-d6) δ: 7.51–7.55 (m, 3 H, HÀ Ph), 7.65–7.69 (m, 2 H,
HÀ Ph), 7.86 (s, 1 H, H-6), 8.82 (s, 1 H, H-2), 12.69 (br. s., 1 H, NH).
HRMS (ESI): calculated for C12H9IN3 ([M+H]+): 321.9836, found:
321.9841.
4-Ethyl-5-bromo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimi-
dine (68). 66 (430 mg, 0.73 mmol) was dissolved in anhydrous DMF
(5 mL), and NBS (168 mg, 0.94 mmol, 1.3 eq.) was added. Then, the
reaction mixture was stirred at ambient temperature overnight. EA
(10 mL) and water (10 mL) were added. The layers were separated
and the water layer extracted once more with EA. The organic
layers were combined and washed with brine (10 mL), dried over
Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (5!30% EA/PET) to give a colorless foam
(356 mg, 0.53 mmol) in 73% yield. This obtained product (350 mg,
0.52 mmol) was charged in a 50 mL round bottom flask, and NH3 in
MeOH (7 N, 5 mL) was added. The mixture was stirred at ambient
temperature overnight. Then, the reaction solution was evaporated
till dryness, purified by column chromatography (0!10% MeOH/
DCM) to afford 68 (165 mg, 0.46 mmol) as a white solid in 88%
4-Phenyl-5-iodo-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimi-
dine (71). 70 (200 mg, 0.62 mmol) was placed in a 25 mL two-neck
round bottom flask, after which the flask was purged with N2 gas.
Anhydrous MeCN (5 mL, 7.5 mL/mmol of SM) was added. BSA (183
μL, 0.75 mmol, 1.2 eq.) was added to the suspension via syringe.
The resulting mixture was stirred until the solid was completely
dissolved. Next, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose
(378 mg, 0.75 mmol, 1.2 eq.) was added, and immediately followed
by TMSOTf (147 μL, 0.81 mmol, 1.3 eq.). The reaction mixture was
1
yield. H NMR (400 MHz, DMSO-d6) δ: 1.31 (t, J=7.6 Hz, 3 H, CH3),
3.23 (qd, J=7.5, 1.1 Hz, 2 H, CH2), 3.55 (ddd, J=11.8, 5.6, 4.0 Hz, 1
H, H-5’’), 3.64 (ddd, J=12.0, 5.2, 4.2 Hz, 1 H, H-5’), 3.92 (q, J=3.8 Hz,
1 H, H-4’), 4.10 (td, J=4.8, 3.3 Hz, 1 H, H-3’), 4.39 (q, J=5.8 Hz, 1 H,
H-2’), 5.08 (t, J=5.4 Hz, 1 H, OH-5’), 5.17 (d, J=4.8 Hz, 1 H, OH-3’),
5.39 (d, J=6.4 Hz, 1 H, OH-2’), 6.22 (d, J=6.1 Hz, 1 H, H-1’), 8.06 (s, 1
H, H-6), 8.75 (s, 1 H, H-2). 13C NMR (100 MHz, DMSO-d6) δ: 13.4 (CH3),
26.7 (CH2), 61.4 (C-5’), 70.5 (C-3’), 74.1 (C-2’), 85.4 (C-4’), 86.4 (C-1’),
87.6 (C-5), 114.6 (C-4a), 126.4 (C-6), 149.8 (C-7a), 151.6 (C-2), 164.0
(C-4). HRMS (ESI): calculated for C13H17BrN3O4 ([M+H]+): 358.0397,
found: 358.0390.
°
stirred at ambient temperature for 15 min, and was heated at 80 C.
When full conversion of 70 was observed via HRMS, the mixture
was cooled to room temperature. Then, EA (10 mL) and aq. sat.
NaHCO3 (10 mL) were added. The layers were separated and the
water layer extracted twice more with EA. Then, the organic layers
were combined, washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by column chromatography
(5!30% EA/PET) to give a yellow foam (140 mg, 0.18 mmol) in
29% yield. This obtained product (330 mg, 0.43 mmol) was charged
in a 50 mL round bottom flask, and NH3 in MeOH (7 N, 5 mL) was
added. The mixture was stirred at ambient temperature overnight.
Then, the reaction solution was evaporated until dryness, purified
by column chromatography (0!10% MeOH/DCM) to afford 71
4-Ethyl-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (15). 15 was prepared according to General
procedure A. 68 (100 mg, 0.28 mmol) gave rise to 15 (53 mg,
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(150 mg, 0.33 mmol) as a white solid in 77% yield. 1H NMR
(400 MHz, DMSO-d6) δ: 3.54–3.60 (m, 1 H, H-5’’), 3.63–3.68 (m, 1 H,
H-5’), 3.95 (q, J=3.5 Hz, 1 H, H-4’), 4.12 (q, J=4.2 Hz, 1 H, H-3’), 4.45
(q, J=6.0 Hz, 1 H, H-2’), 5.11 (t, J=5.3 Hz, 1 H, OH-5’), 5.20 (d, J=
4.6 Hz, 1 H, OH-3’), 5.42 (d, J=6.4 Hz, 1 H, OH-2’), 6.28 (d, J=6.3 Hz,
1 H, H-1’), 7.52–7.56 (m, 3 H, HÀ Ph), 7.66–7.68 (m, 2 H, HÀ Ph), 8.17
(s, 1 H, H-6), 8.89 (s, 1 H, H-2). 13C NMR (100 MHz, DMSO-d6) δ: 54.5
(C-5), 61.4 (C-5’), 70.5 (C-3’), 74.1 (C-2’), 85.4 (C-4’), 86.6 (C-1’), 116.5
(C-4a), 127.6 (2 C, CÀ Ph), 129.6 (CÀ Ph), 130.8 (2 C, CÀ Ph), 133.5 (C-
6), 135.6 (CÀ Ph), 150.9 (C-2), 151.3 (C-7a), 159.7 (C-4). HRMS (ESI):
calculated for C17H17IN3O4 ([M+H]+): 454.0258, found: 454.0255.
Anhydrous MeCN (22 mL, 7.5 mL/mmol of SM) was added. BSA (801
μL, 3.28 mmol, 1.1 eq.) was added into the suspension via syringe.
The resulting mixture was stirred until the solid was completely
dissolved. Next, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose
(1.80 g, 3.57 mmol, 1.2 eq.) was added, and immediately followed
by TMSOTf (646 μL, 3.57 mmol, 1.2 eq.). The reaction mixture was
°
stirred at ambient temperature for 15 min, and was heated at 80 C.
When the full conversion of 73 was observed via HRMS, the mixture
was cooled to ambient temperature. Then, EA (10 mL) and aq. sat.
NaHCO3 (10 mL) were added. The layers were separated and the
water layer extracted twice more with EA. Then, the organic layers
were combined, washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by column chromatography
(5!30% EA/PET) to give a colorless foam (1.17 g, 1.78 mmol). This
obtained product (1.15 g, 1.74 mmol) was charged in a 50 mL
round bottom flask, and NH3 in MeOH (7 N, 10 mL) was added. The
mixture was stirred at ambient temperature overnight. Then, the
reaction solution was evaporated till dryness, purified by column
chromatography (0!10% MeOH/DCM) to afford 77 (285 mg,
0.82 mmol) as a white solid in 24% yield for two steps. 1H NMR
(400 MHz, DMSO-d6) δ: 3.45–3.58 (m, 1 H, H-5’’), 3.59–3.70 (m, 1 H,
H-5’), 3.90 (q, J=3.8 Hz, 1 H, H-4’), 4.04–4.16 (m, 1 H, H-3’), 4.40 (q,
J=6.1 Hz, 1 H, H-2’), 5.04 (t, J=5.4 Hz, 1 H, OH-5’), 5.15 (d, J=
4.9 Hz, 1 H, OH-3’), 5.36 (d, J=6.3 Hz, 1 H, OH-2’), 6.22 (d, J=6.1 Hz,
1 H, H-1’), 7.82 (dd, J=8.8, 2.7 Hz, 1 H, H-4), 8.14 (s, 1 H, H-2), 8.32–
8.41 (m, 1 H, H-6). 13C NMR (100 MHz, DMSO-d6) δ: 61.4 (C-5’), 70.4
(C-3’), 74.0 (C-2’), 85.1 (C-4’), 86.8 (C-1’), 88.2 (C-3), 113.1 (d, J=
22.5 Hz, 1 C, C-4), 119.8 (d, J=7.3 Hz, 1 C, C-3a), 128.0 (C-2), 132.6
(d, J=29.1 Hz, 1 C, C-6), 143.3 (C-7a), 155.8 (d, J=242.0 Hz, 1 C, C-
5). 19F NMR (376 MHz, DMSO-d6)δ: À 136.76. HRMS (ESI): calculated
for C12H13BrFN2O4 ([M+H]+): 347.0037, found: 347.0035.
4-Phenyl-5-(4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (16). 16 was prepared according to General
procedure A. 71 (140 mg, 0.31 mmol) gave rise to a slightly impure
16 (65 mg, 0.15 mmol) as a white solid. 16 was purified by
preparative RP-HPLC gradient: 0.2% formic acid in water:MeCN at a
flow rate of 20 mL/min; The initial gradient composition (95% A/
05% B) was held for 2.0 min, increased to 80% B in 12 min, then
increased to 98% B in 3 min. After RP-HPLC purification, 16 was
obtained as a white solid (31 mg, 0.071 mmol) in 23% yield. Melting
1
°
point: 234 C. H NMR (300 MHz, DMSO-d6) δ: 3.51–3.62 (m, 1 H, H-
5’’), 3.63–3.74 (m, 1 H, H-5’), 3.97 (q, J=3.5 Hz, 1 H, H-4’), 4.09–4.24
(m, 1 H, H-3’), 4.54 (q, J=6.1 Hz, 1 H, H-2’), 5.07 (t, J=5.6 Hz, 1 H,
OH-5’), 5.21 (d, J=5.0 Hz, 1 H, OH-3’), 5.43 (d, J=6.4 Hz, 1 H, OH-2’),
6.38 (d, J=6.2 Hz, 1 H, H-1’), 6.98 (d, J=8.5 Hz, 2 H, HÀ Ph), 7.08–
7.27 (m, 4 H, HÀ Ph), 7.28–7.46 (m, 3 H, HÀ Ph), 8.09 (s, 1 H, H-6), 8.95
(s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 61.5 (C-5’), 70.5 (C-3’),
74.0 (C-2’), 85.3 (C-4’), 86.6 (C-1’), 113.3 (C-4a), 115.5 (C-5), 126.4 (C-
6), 127.5 (4 C, C-3Ph and C-5Ph and C-3’Ph and C-5’Ph), 129.2 (C-4’Ph),
129.5 (2 C, C-2’Ph and C-6’Ph), 130.5 (2 C, C-2Ph and C-6Ph), 131.0 (C-
4Ph), 132.6 (C-1Ph), 137.3 (C-1’Ph), 151.0 (C-2), 152.2 (C-7a), 158.7 (C-4).
HRMS (ESI): calculated for C23H21ClN3O4 ([M+H]+): 438.1215, found:
438.1213.
3-(4-Chloro-phenyl)-5-fluoro-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-
b]pyridine (21). 21 was prepared according to General procedure
A. 77 (100 mg, 0.29 mmol) gave rise to 21 (14 mg, 0.037 mmol) as a
3-(4-Chloro-phenyl)-4-methyl-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-
b]pyridine (20). 20 was prepared according to General procedure
1
°
white solid in 13% yield. Melting point: 131 C. H NMR (400 MHz,
DMSO-d6) δ: 3.44–3.62 (m, 1 H, H-5’’), 3.62–3.75 (m, 1 H, H-5’), 3.92
(q, J=3.9 Hz, 1 H, H-4’), 4.12–4.19 (m, 1 H, H-3’), 4.49 (q, J=6.0 Hz, 1
H, H-2’), 5.05 (t, J=5.6 Hz, 1 H, OH-5’), 5.15 (d, J=4.9 Hz, 1 H, OH-3’),
5.37 (d, J=6.3 Hz, 1 H, OH-2’), 6.27 (d, J=6.0 Hz, 1 H, H-1’), 7.44–
7.57 (m, 2 H, HÀ Ph), 7.70–7.83 (m, 2 H, HÀ Ph), 8.22 (dd, J=9.7,
2.7 Hz, 1 H, H-4), 8.32 (s, 1 H, H-2), 8.35 (dd, J=2.4, 1.2 Hz, 1 H, H-6).
13C NMR (100 MHz, DMSO-d6) δ: 61.5 (C-5’), 70.4 (C-3’), 73.8 (C-2’),
84.9 (C-4’), 86.9 (C-1’), 113.7 (d, J=4.4 Hz, 1 C, C-3), 114.1 (d, J=
21.1 Hz, 1 C, C-4), 118.1 (d, J=6.5 Hz, 1 C, C-3a), 126.5 (C-2), 128.0
(2 C, C-2Ph and C-6Ph), 128.9 (2 C, C-3Ph and C-5Ph), 130.7 (C-4Ph),
131.3 (d, J=29.1 Hz, 1 C, C-6), 132.7 (C-1Ph), 144.9 (C-7a), 155.9 (d,
J=241.2 Hz, 1 C, C-5). 19F NMR (376 MHz, DMSO-d6) δ: À 137.45.
HRMS (ESI): calculated for C18H17BrFN2O4 ([M+H]+): 379.0855, found:
379.0848.
A. 72[27] (85 mg, 0.22 mmol) gave rise to 20 (50 mg, 0.13 mmol) as a
1
°
white solid in 61% yield. Melting point: 96 C. H NMR (400 MHz,
DMSO-d6) δ: 2.29 (s, 3 H, CH3), 3.51–3.56 (m, 1 H, H-5’’), 3.61–3.66
(m, 1 H, H-5’), 3.91 (q, J=3.4 Hz, 1 H, H-4’), 4.10–4.14 (m, 1 H, H-3’),
4.48 (q, J=6.0 Hz, 1 H, H-2’), 5.11 (d, J=4.8 Hz, 1 H, OH-5’), 5.14 (t,
J=5.6 Hz, 1 H, OH-3’), 5.30 (d, J=6.4 Hz, 1 H, OH-2’), 6.29 (d, J=
6.1 Hz, 1 H, H-1’), 6.97 (d, J=4.9 Hz, 1 H, H-5), 7.48 (s, 4 H, HÀ Ph),
7.76 (s, 1 H, H-2), 8.16 (d, J=4.8 Hz, 1 H, H-6). 13C NMR (100 MHz,
DMSO-d6) δ: 19.8 (CH3), 61.7 (C-5’), 70.6 (C-3’), 73.7 (C-2’), 84.9 (C-4’),
87.0 (C-1’), 115.5 (C-3), 118.3 (C-3a), 118.6 (C-5), 124.9 (C-2), 128.0
(2 C, CÀ Ph), 131.5 (C-4Ph), 131.7 (2 C, CÀ Ph), 134.5 (C-1Ph), 140.1 (C-
4), 142.7 (C-6), 147.6 (C-7a). HRMS (ESI): calculated for C19H20ClN2O4
([M+H]+): 375.1106, found: 375.1110.
3-Bromo-5-fluoro-1H-pyrrolo[2,3-b]pyridine (73).[41–42] 5-Fluoro-1H-
pyrrolo[2,3-b]pyridine (500 mg, 3.67 mmol) was dissolved in anhy-
drous DMF (11 mL, 3 mL/mmol of SM), and NBS (686 mg,
3.86 mmol, 1.05 eq.) was added. Then, the reaction mixture was
stirred at ambient temperature overnight. Then, water (cooled in an
ice-water bath, 30 mL) was added and the precipitate generated
and was filtered. The solids were washed with ice-cold water. The
solid was collected and dried under high vacuum to give 73
(753 mg, 3.50 mmol) as a white solid in 95% yield. 1H NMR
(300 MHz, DMSO-d6) δ: 7.71 (dd, J=8.9, 2.6 Hz, 1 H, H-2), 7.82 (d, J=
2.8 Hz, 1 H, H-6), 8.28 (t, J=2.2 Hz, 1 H, H-4), 12.24 (br. s., 1 H, NH).
19F NMR (376 MHz, DMSO-d6) δ: À 138.03. HRMS (ESI): calculated for
C7H5BrFN2 ([M+H]+): 214.9615, found: 214.9623.
3-Bromo-5-chloro-1H-pyrrolo[2,3-b]pyridine (74).[41–42] 5-Chloro-
1H-pyrrolo[2,3-b]pyridine (500 mg, 3.28 mmol) was dissolved in
anhydrous DMF (10 mL, 3 mL/mmol of SM), and NBS (642 mg,
3.61 mmol, 1.1 eq.) was added. Then, the reaction mixture was
stirred at ambient temperature overnight. The water (cooled in an
ice-water bath, 30 mL) was added and the precipitate generated
and was filtered. The solids were washed with ice-cold water. The
solid was collected and dried under high vacuum to give 74
(700 mg, 3.02 mmol) as a white solid in 92% yield. 1H NMR
(300 MHz, DMSO-d6) δ 7.83 (d, J=2.3 Hz, 1 H, H-2), 7.92 (d, J=
2.1 Hz, 1 H, H-6), 8.29 (dd, J=1.9, 1.1 Hz, 1 H, H-4), 12.33 (br. s., 1 H,
NH). HRMS (ESI): calculated for C7H5BrClN2 ([M+H]+): 230.9319,
found: 230.9313.
3-Bromo-5-fluoro-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-b]pyridine
(77). 73 (640 mg, 2.98 mmol) was placed in a 100 mL two-neck
round bottom flask, after which the flask was purged with N2 gas.
3-Bromo-5-chloro-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-b]pyridine
(78). 74 (640 mg, 2.98 mmol) was placed in a 100 mL two-neck
round bottom flask, after which the flask was purged with N2 gas.
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Anhydrous MeCN (22 mL, 7.5 mL/mmol of SM) was added. BSA (801
μL, 3.28 mmol, 1.1 eq.) was added into the suspension via syringe.
The resulting mixture was stirred until the solid was completely
dissolved. Next, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose
(1.80 g, 3.57 mmol, 1.2 eq.) was added, and immediately followed
by TMSOTf (646 μL, 3.57 mmol, 1.2 eq.). The reaction mixture was
2), 8.59 (s, 1 H, H-6), 9.13–9.14 (m, 1 H, H-4), 12.92 (br. s., 1 H, NH).
HRMS (ESI): calculated for C7H5BrN3O2 ([M+H]+): 241.9560, found:
241.9564.
3-Bromo-5-nitro-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-b]pyridine
(79). 75 (620 mg, 2.56 mmol) was placed in a 100 mL two-neck
round bottom flask, after which the flask was purged with N2 gas.
Anhydrous MeCN (19 mL, 7.5 mL/mmol of SM) was added. BSA
(0.7 mL, 2.82 mmol, 1.1 eq.) was added into the suspension via
syringe. The resulting mixture was stirred until the solid was
completely dissolved. Next, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribo-
furanose (1.55 g, 3.07 mmol, 1.2 eq.) was added, and immediately
followed by TMSOTf (0.6 mL, 3.07 mmol, 1.2 eq.). The reaction
mixture was stirred at ambient temperature for 15 min, and was
°
stirred at ambient temperature for 15 min, and was heated at 80 C.
When full conversion of 74 was observed via HRMS, the mixture
was cooled to room temperature. Then, EA (10 mL) and aq. sat.
NaHCO3 (10 mL) were added. The layers were separated and the
water layer extracted twice more with EA. Then, the organic layers
were combined, washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by column chromatography
(5!30% EA/PET) to give a colorless foam (850 mg, 1.26 mmol). This
obtained product (850 mg, 1.26 mmol) was charged in a 50 mL
round bottom flask, and NH3 in MeOH (7 N, 10 mL) was added. The
mixture was stirred at ambient temperature overnight. Then, the
reaction solution was evaporated till dryness, purified by column
chromatography (0!10% MeOH/DCM) to afford 78 (410 mg,
1.13 mmol) as a white solid in 32% yield for two steps. 1H NMR
(400 MHz, DMSO-d6) δ: 3.47–3.58 (m, 1 H, H-5’’), 3.59–3.71 (m, 1 H,
H-5’), 3.91 (q, J=3.5 Hz, 1 H, H-4’), 4.04–4.18 (m, 1 H, H-3’), 4.39 (q,
J=5.9 Hz, 1 H, H-2’), 5.06 (t, J=5.4 Hz, 1 H, OH-5’), 5.17 (d, J=
4.9 Hz, 1 H, OH-3’), 5.39 (d, J=6.3 Hz, 1 H, OH-2’), 6.23 (d, J=6.1 Hz,
1 H, H-1’), 8.01 (d, J=2.3 Hz, 1 H, H-4), 8.15 (s, 1 H, H-2), 8.37 (d, J=
2.3 Hz, 1 H, H-6). 13C NMR (100 MHz, DMSO-d6) δ: 61.4 (C-5’), 70.4 (C-
3’), 74.1 (C-2’), 85.1 (C-4’), 86.6 (C-1’), 88.1 (C-3a), 120.5 (C-3), 124.2
(C-5), 126.4 (C-4), 127.6 (C-2), 142.3 (C-6), 145.0 (C-7a). HRMS (ESI):
calculated for C12H13BrClN2O4 ([M+H]+): 362.9742, found: 362.9738.
°
heated at 80 C. When the full conversion of 75 was observed via
HRMS, the mixture was cooled to ambient temperature. Then, EA
(10 mL) and aq. sat. NaHCO3 (10 mL) were added. The layers were
separated and the water layer extracted twice more with EA. Then,
the organic layers were combined, washed with brine, dried over
Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (5!30% EA/PET) to give a colorless foam
(1.70 g, 2.48 mmol). This obtained product (200 mg, 0.29 mmol)
was charged in a 25 mL round bottom flask, and NH3 in MeOH (7 N,
5 mL) was added. The mixture was stirred at ambient temperature
overnight. Then, the reaction solution was evaporated until dryness,
purified by column chromatography (0!10% MeOH/DCM) to
afford 79 (88 mg, 0.24 mmol) as a light yellow solid in 79% yield for
1
two steps. H NMR (400 MHz, DMSO-d6) δ: 3.58 (ddd, J=12.0, 5.4,
4.0 Hz, 1 H, H-5’’), 3.66 (ddd, J=12.0, 5.2, 4.2 Hz, 1 H, H-5’), 3.95 (q,
J=3.7 Hz, 1 H, H-4’), 4.08–4.19 (m, 1 H, H-3’), 4.40 (q, J=5.8 Hz, 1 H,
H-2’), 5.10 (t, J=5.4 Hz, 1 H, OH-5’), 5.22 (d, J=5.0 Hz, 1 H, OH-3’),
5.46 (d, J=6.3 Hz, 1 H, OH-2’), 6.33 (d, J=6.0 Hz, 1 H, H-1’), 8.36 (s, 1
3-(4-Chloro-phenyl)-5-chloro-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-
b]pyridine (22). 78 (100 mg, 0.28 mmol), phenylboronic acid
(52 mg, 0.33 mmol, 1.2 eq.), K2CO3 (116 mg, 0.84 mmol, 3 eq.) and
Pd(PPh3)4 (16 mg, 0.014 mmol, 0.05 eq.) were charged to a 25 mL
round-bottom flask, equipped with a stir bar. Next, the air was
removed and backfilled with argon. This was repeated thrice. Then,
a mixture solution of 1,4-dioxane and H2O (1:1 ratio, 2 mL) was
added into the reaction mixture via syringes. The mixture was then
stirred at ambient temperature (5 min), and then was refluxed at
H, H-2), 8.65 (d, J=2.4 Hz, 1 H, H-4), 9.21 (d, J=2.4 Hz, 1 H, H-6). 13
C
NMR (100 MHz, DMSO-d6) δ: 61.3 (C-5’), 70.4 (C-3’), 74.4 (C-2’), 85.5
(C-4’), 86.9 (C-1’), 90.9 (C-3a), 118.8 (C-3), 123.6 (C-4), 129.4 (C-2),
139.8 (C-5), 140.1 (C-6), 148.3 (C-7a). HRMS (ESI): calculated for
C12H13BrN3O6 ([M+H]+): 373.9982, found: 373.9974.
3-(4-Chloro-phenyl)-5-nitro-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-b]
pyridine (22). 22 was prepared according to General procedure A.
79 (150 mg, 0.40 mmol) gave rise to 22 (24 mg, 0.059 mmol) as a
°
100 C. The reaction was monitored by HRMS for consumption of
SM (1 h), after which it was allowed to cool to ambient temper-
ature. Then, 0.5 M aq. HCl was added to neutralize the mixture.
Then, the volatiles were removed in vacuo, and the residue purified
by column chromatography (0!10% MeOH/DCM) to give 22
1
°
white solid in 15% yield. Melting point: 112 C. H NMR (400 MHz,
DMSO-d6) δ: 3.52–3.64 (m, 1 H, H-5’’), 3.65–3.77 (m, 1 H, H-5’), 3.97
(q, J=3.8 Hz, 1 H, H-4’), 4.11–4.27 (m, 1 H, H-3’), 4.49 (q, J=6.0 Hz, 1
H, H-2’), 5.09 (t, J=5.5 Hz, 1 H, OH-5’), 5.22 (d, J=5.0 Hz, 1 H, OH-3’),
5.46 (d, J=6.4 Hz, 1 H, OH-2’), 6.39 (d, J=6.0 Hz, 1 H, H-1’), 7.51–
7.65 (m, 2 H, HÀ Ph), 7.75–7.91 (m, 2 H, HÀ Ph), 8.48 (s, 1 H, H-2), 9.01
(d, J=2.4 Hz, 1 H, H-4), 9.22 (d, J=2.4 Hz, 1 H, H-6). 13C NMR
(100 MHz, DMSO-d6) δ: 61.3 (C-5’), 70.4 (C-3’), 74.2 (C-2’), 85.4 (C-4’),
86.9 (C-1’), 116.1 (C-3), 117.1 (C-3a), 124.5 (C-4), 127.6 (C-2), 128.5
(2 C, C-2Ph and C-6Ph), 129.2 (2 C, C-3Ph and C-5Ph), 131.6 (CÀ Ph),
131.7 (CÀ Ph), 139.4 (C-5), 139.7 (C-6), 149.7 (C-7a). HRMS (ESI):
calculated for C18H17ClN3O6 ([M+H]+): 406.0800, found: 406.0795.
(27 mg, 0.068 mmol) as a light yellow solid in 24% yield. Melting
1
°
point: 102 C. H NMR (400 MHz, DMSO-d6) δ: 3.48–3.61 (m, 1 H, H-
5’’), 3.64–3.70 (m, 1 H, H-5’), 3.93 (q, J=3.9 Hz, 1 H, H-4’), 4.09–4.22
(m, 1 H, H-3’), 4.48 (q, J=6.0 Hz, 1 H, H-2’), 5.05 (t, J=5.6 Hz, 1 H,
OH-5’), 5.16 (d, J=4.9 Hz, 1 H, OH-3’), 5.38 (d, J=6.4 Hz, 1 H, OH-2’),
6.28 (d, J=6.0 Hz, 1 H, H-1’), 7.45–7.58 (m, 2 H, HÀ Ph), 7.71–7.82 (m,
2 H, HÀ Ph), 8.31 (s, 1 H, H-2), 8.35 (d, J=2.3 Hz, 1 H, H-6), 8.40 (d,
J=2.3 Hz, 1 H, H-4). 13C NMR (100 MHz, DMSO-d6) δ: 61.5 (C-5’), 70.4
(C-3’), 73.9 (C-2’), 85.0 (C-4’), 86.7 (C-1’), 113.5 (C-3), 118.8 (C-3a),
124.0 (C-5), 126.1 (C-2), 127.3 (C-4), 128.1 (2 C, C-2Ph and C-6Ph),
128.9 (2 C, C-3Ph and C-5Ph), 130.8 (C-4Ph), 132.5 (C-1Ph), 141.3 (C-6),
146.5 (C-7a). HRMS (ESI): calculated for C18H17Cl2N2O4 ([M+H]+):
395.0560, found: 395.0572.
3-(4-Chloro-phenyl)-5-amino-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-
b]pyridine (24). 22 (30 mg, 0.074 mmol) was dissolved in MeOH
(5 mL). The flask was purged with N2, and a catalytic amount of Pt/
C was added. Then, the N2 gas was exchanged with H2 (balloon;
bubbling). The reaction mixture was stirred until the HRMS showed
full consumption of the SM (2 h). The solution was filtered over
Celite® and washed with MeOH. The filtrate was evaporated to
dryness and purified by column chromatography (0!10% MeOH/
3-Bromo-5-nitro-1H-pyrrolo[2,3-b]pyridine (75). 5-Nitro-1H-pyrrolo
[2,3-b]pyridine (500 mg, 3.06 mmol) was dissolved in dry DMF
(15 mL, 5 mL/mmol of SM), and NBS (600 mg, 3.37 mmol, 1.1 eq.)
was added. Then, the reaction mixture was stirred at ambient
temperature overnight. Then, water (cooled in an ice-water bath,
30 mL) was added and the precipitate generated and was filtered.
The solids were washed with ice-cold water. The solid was collected
and dried under high vacuum to give 75 (625 mg, 3.02 mmol) as a
DCM) to afford 24 (19 mg, 0.051 mmol) as a white solid in 68%
1
°
yield. Melting point: 104 C. H NMR (400 MHz, DMSO-d6) δ: 3.47–
3.58 (m, 1 H, H-5’’), 3.62 À 3.67 (m, 1 H, H-5’), 3.89 (q, J=3.6 Hz, 1 H,
H-4’), 4.09–4.16 (m, 1 H, H-3’), 4.51 (q, J=6.2 Hz, 1 H, H-2’), 4.95 (br.
s., 2 H, NH2), 5.08 (d, J=4.8 Hz, 1 H, OH-5’), 5.21 (t, J=5.8 Hz, 1 H,
OH-3’), 5.28 (d, J=6.4 Hz, 1 H, OH-2’), 6.10 (d, J=6.3 Hz, 1 H, H-1’),
1
yellow solid in 84%. H NMR (300 MHz, DMSO-d6) δ: 8.06 (s, 1 H, H-
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7.43–7.53 (m, 3 H, HÀ Ph and H-4), 7.61–7.70 (m, 2 H, HÀ Ph), 7.80 (d,
J=2.4 Hz, 1 H, H-6), 7.97 (s, 1 H, H-2). 13C NMR (100 MHz, DMSO-d6)
δ: 61.8 (C-5’), 70.6 (C-3’), 73.3 (C-2’), 84.8 (C-4’), 87.3 (C-1’), 110.9 (C-
4), 111.9 (C-3), 118.6 (C-3a), 124.5 (C-2), 127.5 (2 C, C-2Ph and C-6Ph),
128.8 (2 C, C-3Ph and C-5Ph), 129.9 (C-4Ph), 132.4 (C-6), 134.1 (C-1Ph),
140.3 (C-5), 141.9 (C-7a). HRMS (ESI): calculated for C18H19ClN3O4([M
+H]+): 376.1059, found: 376.1065.
3’), 5.32 (d, J=6.3 Hz, 1 H, OH-2’), 6.25 (d, J=6.0 Hz, 1 H, H-1’), 7.50
(d, J=8.5 Hz, 2 H, HÀ Ph), 7.75 (d, J=8.5 Hz, 2 H, HÀ Ph), 8.14 (s, 2 H,
H-4 and H-2), 8.17 (s, 1 H, H-6). 13C NMR (100 MHz, DMSO-d6) δ: 18.1
(CH3), 61.7 (C-5’), 70.6 (C-3’), 73.6 (C-2’), 84.9 (C-4’), 87.0 (C-1’), 113.0
(C-3), 118.1 (C-3a), 124.7 (C-2), 125.9 (C-5), 127.9 (3 C, C-2Ph and C-
6Ph and C-4), 128.8 (2 C, C-3Ph and C-5Ph), 130.3 (C-4Ph), 133.4 (C-1Ph),
143.7 (C-6), 146.8 (C-7a). HRMS (ESI): calculated for C19H20ClN2O4 ([M
+H]+): 375.1106, found: 375.1092.
3-Bromo-5-methyl-1H-pyrrolo[2,3-b]pyridine (76). 5-Methyl-1H-
pyrrolo[2,3-b]pyridine (500 mg, 3.06 mmol) was dissolved in dry
DMF (11 mL, 3 mL/mmol of SM), and NBS (741 mg, 4.16 mmol,
1.1 eq.) was added. Next, the reaction mixture was stirred at
ambient temperature overnight. Then, water (cooled in an ice-water
bath, 30 mL) was added and the precipitate generated and was
filtered. The solids were washed with ice-cold water. The solid was
collected and dried under high vacuum to give 76 (560 mg) as a
yellow solid. Since part of 76 was dissolved in the filtrate, the water
was collected and extracted twice with EA (2×20 mL). Then, the
organic layers were combined, washed with brine, dried over
Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (5!30% EA/PET) to give a yellow solid
(90 mg). Total amount of 76 was 650 mg (3.07 mmol) in 81% yield.
1H NMR (300 MHz, DMSO-d6) δ: 2.40 (s, 3 H, CH3), 7.63–7.64 (m, 2 H,
H-6 and H-2), 8.13 (d, J=1.9 Hz, 1 H, H-4), 11.91 (br. s., 1 H, NH).
HRMS (ESI): calculated for C8H8BrN2 ([M+H]+): 210.9865, found:
210.9870.
4-Methyl-5-phenyl-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-d]-pyri-
midine (26).[23] 26 was prepared according to General procedure A.
67 (156 mg, 0.40 mmol) gave rise to 26 (105 mg, 0.31 mmol) as a
white solid in 77% yield. 1H NMR (300 MHz, DMSO-d6) δ: 2.46 (s, 3H,
CH3), 3.48–3.59 (m, 1 H, H-5’’), 3.60–3.72 (m, 1 H, H-5’), 3.93 (q, J=
3.5 Hz, 1 H, H-4’), 4.13 (dd, J=8.2, 4.7 Hz, 1 H, H-3’), 4.47 (q, J=
6.0 Hz, 1 H, H-2’), 5.05 (t, J=5.4 Hz, 1 H, OH-5’), 5.16 (d, J=4.7 Hz, 1
H, OH-3’), 5.37 (d, J=6.4 Hz, 1 H, OH-2’), 6.28 (d, J=5.9 Hz, 1 H, H-
1’), 7.34–7.62 (m, 5 H, HÀ Ph), 7.85 (s, 1 H, H-6), 8.69 (s, 1 H, H-2). 13C
NMR (75 MHz, DMSO-d6) δ: 22.9 (CH3), 61.4 (C-5’), 70.5 (C-3’), 74.0
(C-2’), 85.2 (C-4’), 86.5 (C-1’), 115.8 (C-4a), 117.0 (C-5), 124.7 (C-6),
127.1 (CÀ Ph), 128.2 (2 C, CÀ Ph), 129.8 (2 C, CÀ Ph), 134.2 (CÀ Ph),
150.6 (C-7a), 150.8 (C-2), 159.2 (C-4). HRMS (ESI): calculated for
C18H20N3O4 ([M+H]+): 342.1448, found: 342.1443.
4-Methyl-5-(4-fluoro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (27). 27 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 27 (32 mg,
1
°
3-Bromo-5-methyl-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-b]pyridine
(80). 76 (650 mg, 3.08 mmol) was placed in a 100 mL two-neck
round bottom flask, after which the flask was purged with N2 gas.
Anhydrous MeCN (23 mL, 7.5 mL/mmol of SM) was added. BSA (828
μL, 3.39 mmol, 1.1 eq.) was added into the suspension via syringe.
The resulting mixture was stirred until the solid was completely
dissolved. Next, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose
(1.86 g, 3.70 mmol, 1.2 eq.) was added, and immediately followed
by TMSOTf (670 μL, 3.70 mmol, 1.2 eq.). The reaction mixture was
0.089 mmol) as a white solid in 34% yield. Melting point: 203 C. H
NMR (300 MHz, DMSO-d6) δ: 2.44 (s, 3 H, CH3), 3.55 (ddd, J=12.0,
5.7, 4.0 Hz, 1 H, H-5’’), 3.64 (ddd, J=12.0, 5.3, 4.1 Hz, 1 H, H-5’), 3.93
(q, J=3.8 Hz, 1 H, H-4’), 4.06–4.19 (m, 1 H, H-3’), 4.47 (dd, J=11.4,
6.4 Hz, 1 H, H-2’), 5.04 (t, J=5.4 Hz, 1 H, OH-5’), 5.17 (d, J=5.0 Hz, 1
H, OH-3’), 5.37 (d, J=6.4 Hz, 1 H, OH-2’), 6.27 (d, J=6.2 Hz, 1 H, H-
1’), 7.22–7.37 (m, 2 H, HÀ Ph), 7.47–7.61 (m, 2 H, HÀ Ph), 7.85 (s, 1 H,
H-6), 8.69 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 22.8 (CH3),
61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.5 (C-1’), 115.0 (C-5),
115.2 (2 C, C-3Ph and C-5Ph), 115.9 (C-4a), 124.8 (C-6), 130.6 (C-1Ph),
131.8 (d, J=8.1 Hz, 2 C, C-2Ph and C-6Ph), 150.6 (C-7a), 150.8 (C-2),
159.2 (C-4), 161.6 (d, J=244.2 Hz, 1 C, C-4Ph). 19F NMR (282 MHz,
DMSO-d6) δ: À 115.56–À 115.45 (m, 1 F). HRMS (ESI): calculated for
C18H19FN3O4 ([M+H]+): 360.1354, found: 360.1364.
°
stirred at ambient temperature for 15 min, and was heated at 80 C.
When the full conversion of 76 was observed via HRMS, the mixture
was cooled to ambient temperature. Then, EA (20 mL) and aq. sat.
NaHCO3 (10 mL) were added. The layers were separated and the
water layer extracted twice more with EA. Then, the organic layers
were combined, washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by column chromatography
(5!30% EA/PET) to give a yellow foam (1.70 g, 2.59 mmol). This
obtained product (1.70 g, 2.59 mmol) was charged in a 25 mL
round bottom flask, and NH3 in MeOH (7 N, 10 mL) was added. The
mixture was stirred at ambient temperature overnight. Then, the
reaction solution was evaporated till dryness, purified by column
chromatography (0!10% MeOH/DCM) to afford 80 (650 mg,
1.89 mmol) as a white solid in 61% yield for two steps. 1H NMR
(400 MHz, DMSO-d6) δ: 2.42 (s, 3 H, CH3), 3.52–3.56 (m, 1 H, H-5’’),
3.60–3.65 (m, 1 H, H-5’), 3.89 (q, J=3.8 Hz, 1 H, H-4’), 4.10 (q, J=
4.2 Hz, 1 H, H-3’), 4.42 (q, J=6.1 Hz, 1 H, H-2’), 5.09–5.12 (m, 2 H,
OH-5’ and OH-3’), 5.31 (d, J=6.4 Hz, 1 H, OH-2’), 6.20 (d, J=6.3 Hz,
1 H, H-1’), 7.69 (d, J=1.2 Hz,1 H, H-4), 7.96 (s, 1 H, H-2), 8.19 (d, J=
1.8 Hz, 1 H, H-6). 13C NMR (100 MHz, DMSO-d6) δ: 17.9 (CH3), 61.6 (C-
5’), 70.5 (C-3’), 73.8 (C-2’), 85.0 (C-4’), 86.7 (C-1’), 88.0 (C-3a), 119.5
(C-3), 125.8 (C-2), 126.3 (C-5), 126.8 (C-4), 144.7 (C-6), 145.2 (C-7a).
HRMS (ESI): calculated for C13H16BrN2O4([M+H]+): 343.0288, found:
343.0292.
4-Methyl-5-(4-trifluoromethyl-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (28). 28 was prepared according to
General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 28
(33 mg, 0.081 mmol) as a white solid in 31% yield. Melting point:
1
°
196 C. H NMR (300 MHz, DMSO-d6) δ: 2.48 (s, 3 H, CH3), 3.48–3.59
(m, 1 H, H-5’’), 3.60–3.73 (m, 1 H, H-5’), 3.94 (q, J=3.6 Hz, 1 H, H-4’),
4.14 (dd, J=8.4, 5.0 Hz, 1 H, H-3’), 4.48 (q, J=6.1 Hz, 1 H, H-2’), 5.05
(t, J=5.4 Hz, 1 H, OH-5’), 5.18 (d, J=4.7 Hz, 1 H, OH-3’), 5.40 (d, J=
6.2 Hz, 1 H, OH-2’), 6.29 (d, J=6.2 Hz, 1 H, H-1’), 7.67–7.79 (m, 2 H,
HÀ Ph), 7.79–7.90 (m, 2 H, HÀ Ph), 7.99 (s, 1 H, H-6), 8.73 (s, 1 H, H-2).
13C NMR (75 MHz, DMSO-d6) δ: 23.2 (CH3), 61.4 (C-5’), 70.5 (C-3’),
74.1 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 115.6 (C-5 and C-4a), 124.4 (q, J=
271.8 Hz, 1 H, CF3), 125.1 (q, J=4.6 Hz, 1 H, 2 C, C-3Ph and C-5Ph),
125.7 (C-6), 127.5 (q, J=31.1 Hz, 1 C, C-4Ph), 130.4 (2 C, C-2Ph and C-
6Ph), 138.6 (C-1Ph), 150.8 (C-2), 151.0 (C-7a), 159.3 (C-4). 19F NMR
(282 MHz, DMSO-d6) δ: À 60.83. HRMS (ESI): calculated for
C19H19F3N3O4 ([M+H]+): 410.1322, found: 410.1323.
4-Methyl-5-(4-trifluoromethoxy-phenyl)-N7-(β-d–ribofuranosyl)-
7H-pyrrolo[2,3-d]-pyrimidine (29). 29 was prepared according to
General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 29
3-(4-Chloro-phenyl)-5-methyl-N1-(β-d–ribofuranosyl)-pyrrolo[2,3-
b]pyridine (25). 25 was prepared according to General procedure
(40 mg, 0.094 mmol) as a white solid in 36% yield. Melting point:
A. 79 (150 mg, 0.44 mmol) gave rise to 25 (23 mg, 0.061 mmol) as a
1
°
188 C. H NMR (300 MHz, DMSO-d6) δ: 2.47 (s, 3 H, CH3), 3.49–3.59
1
°
white solid in 14% yield. Melting point: 112 C. H NMR (400 MHz,
(m, 1 H, H-5’’), 3.60–3.74 (m, 1 H, H-5’), 3.93 (q, J=3.4 Hz, 1 H, H-4’),
4.13 (dd, J=8.3, 4.8 Hz, 1 H, H-3’), 4.47 (q, J=6.1 Hz, 1 H, H-2’), 5.04
(t, J=5.4 Hz, 1 H, OH-5’), 5.17 (d, J=4.7 Hz, 1 H, OH-3’), 5.38 (d, J=
DMSO-d6) δ: 2.44 (s, 3 H, CH3), 3.53–3.58 (m, 1 H, H-5’’), 3.64–3.69
(m, 1 H, H-5’), 3.92 (d, J=3.4 Hz, 1 H, H-4’), 4.15 (q, J=4.0 Hz, 1 H,
H-3’), 4.52 (q, J=5.8 Hz, 1 H, H-2’), 5.12–5.17 (m, 2 H, OH-5’ and OH-
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6.4 Hz, 1 H, OH-2’), 6.28 (d, J=6.2 Hz, 1 H, H-1’), 7.46 (d, J=8.5 Hz, 2
H, HÀ Ph), 7.65 (d, J=8.5 Hz, 2 H, HÀ Ph), 7.91 (s, 1 H, H-6), 8.71 (s, 1
H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 22.9 (CH3), 61.5 (C-5’), 70.5
(C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 115.5 (C-5), 115.7 (C-4a),
120.1 (q, J=255.7 Hz, 1 C, OCF3), 120.9 (2 C, C-3Ph and C-5Ph), 125.2
(C-6), 131.6 (2 C, C-2Ph and C-6Ph), 133.7 (C-1Ph), 147.6 (d, J=2.3 Hz,
1 C, C-4Ph), 150.7 (C-2), 150.9 (C-7a), 159.2 (C-4). 19F NMR (282 MHz,
DMSO-d6) δ: À 56.77. HRMS (ESI): calculated for C19H19F3N3O5 ([M+
H]+): 426.1271, found: 426.1244.
5’), 5.16 (d, J=5.0 Hz, 1 H, OH-3’), 5.36 (d, J=6.2 Hz, 1 H, OH-2’),
6.27 (d, J=6.2 Hz, 1 H, H-1’), 7.21–7.35 (m, 2 H, HÀ Ph), 7.36–7.49 (m,
2 H, HÀ Ph), 7.80 (s, 1 H, H-6), 8.68 (s, 1 H, H-2). 13C NMR (75 MHz,
DMSO-d6) δ: 15.6 (CH3), 22.8 (CH3), 27.9 (CH2), 61.5 (C-5’), 70.6 (C-3’),
74.0 (C-2’), 85.2 (C-4’), 86.5 (C-1’), 115.9 (C-4a), 116.9 (C-5), 124.5 (C-
6), 127.6 (2 C, C-3Ph and C-5Ph), 129.7 (2 C, C-2Ph and C-6Ph), 131.5 (C-
1Ph), 142.7 (C-4Ph), 150.6 (C-2), 150.7 (C-7a), 159.2 (C-4). HRMS (ESI):
calculated for C20H24N3O4 ([M+H]+): 370.1761, found: 370.1771.
4-Methyl-5-(4-methoxy-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrro-
lo[2,3-d]-pyrimidine (34). 34 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 34 (68 mg,
4-Methyl-5-(4-cyano-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (30). 30 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 30 (30 mg,
1
°
0.18 mmol) as a white solid in 70% yield. Melting point: 112 C. H
0.082 mmol) as a white solid in 31% yield. Melting point: 119–
NMR (300 MHz, DMSO-d6) δ: 2.45 (s, 3 H, CH3), 3.45–3.59 (m, 1 H, H-
5’’), 3.59–3.72 (m, 1 H, H-5’), 3.81 (s, 3 H, OMe), 3.93 (q, J=3.6 Hz, 1
H, H-4’), 4.08–4.19 (m, 1 H, H-3’), 4.46 (dd, J=11.5, 6.3 Hz, 1 H, H-2’),
5.05 (t, J=5.6 Hz, 1 H, OH-5’), 5.16 (d, J=4.7 Hz, 1 H, OH-3’), 5.36 (d,
J=6.2 Hz, 1 H, OH-2’), 6.26 (d, J=6.2 Hz, 1 H, H-1’), 6.94–7.10 (m, 2
H, HÀ Ph), 7.34–7.49 (m, 2 H, HÀ Ph), 7.76 (s, 1 H, H-6), 8.67 (s, 1 H, H-
2). 13C NMR (75 MHz, DMSO-d6) δ: 22.7 (CH3), 55.1 (OMe), 61.5 (C-5’),
70.6 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.5 (C-1’), 113.7 (2 C, C-3Ph and C-
5Ph), 116.0 (C-4a), 116.6 (C-5), 124.3 (C-6), 126.4 (C-1Ph), 131.0 (2 C, C-
2Ph and C-6Ph), 150.6 (C-2), 150.7 (C-7a), 158.6 (C-4Ph), 159.2 (C-4).
HRMS (ESI): calculated for C19H22N3O5 ([M+H]+): 372.1554, found:
372.1550.
1
°
120 C. H NMR (300 MHz, DMSO-d6) δ: 2.49 (s, 3 H, CH3), 3.47–3.61
(m, 1 H, H-5’’), 3.61–3.73 (m, 1 H, H-5’), 3.94 (q, J=3.7 Hz, 1 H, H-4’),
4.08–4.19 (m, 1 H, H-3’), 4.47 (dd, J=11.5, 6.2 Hz, 1 H, H-2’), 5.05 (t,
J=5.6 Hz, 1 H, OH-5’), 5.18 (d, J=5.0 Hz, 1 H, OH-3’), 5.39 (d, J=
6.4 Hz, 1 H, OH-2’), 6.28 (d, J=6.2 Hz, 1 H, H-1’), 7.74 (d, J=8.2 Hz, 2
H, HÀ Ph), 7.93 (d, J=8.2 Hz, 2 H, HÀ Ph), 8.01 (s, 1 H, H-6), 8.73 (s, 1
H, H-2). 13C NMR (100 MHz, DMSO-d6) δ: 23.3 (CH3), 61.4 (C-5’), 70.5
(C-3’), 74.1 (C-2’), 85.5 (C-4’), 86.7 (C-1’), 109.6 (C-4Ph), 115.4 (C-4a),
115.6 (C-5), 118.9 (CN), 126.0 (C-6), 130.5 (2 C, C-2Ph and C-6Ph),
132.2 (2 C, C-3Ph and C-5Ph), 139.4 (C-1Ph), 150.9 (C-7a), 151.1 (C-2),
159.4 (C-4). HRMS (ESI): calculated for C19H19N4O4 ([M+H]+):
367.1401, found: 367.1394.
4-Methyl-5-(4-hydroxy-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (35). 35 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 35 (70 mg,
4-Methyl-5-(4-nitro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (31). 31 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 31 (31 mg,
1
°
0.20 mmol) as a white solid in 75% yield. Melting point: 75 C. H
1
°
0.080 mmol) as a yellow solid in 31% yield. Melting point: 172 C. H
NMR (300 MHz, DMSO-d6) δ: 2.45 (s, 3 H, CH3), 3.48–3.58 (m, 1 H, H-
5’’), 3.59–3.73 (m, 1 H, H-5’), 3.92 (q, J=3.5 Hz, 1 H, H-4’), 4.04–4.18
(m, 1 H, H-3’), 4.45 (t, J=5.1 Hz, 1 H, H-2’), 5.04 (br. s., 1 H, OH-5’),
5.15 (br. s., 1 H, OH-3’), 5.34 (br. s., 1 H, OH-2’), 6.25 (d, J=6.2 Hz, 1
H, H-1’), 6.77–6.90 (m, 2 H, HÀ Ph), 7.20–7.35 (m, 2 H, HÀ Ph), 7.72 (s,
1 H, H-6), 8.67 (s, 1 H, H-2), 9.52 (s, 1 H, OH). 13C NMR (100 MHz,
DMSO-d6) δ: 22.5 (CH3), 61.5 (C-5’), 70.6 (C-3’), 73.9 (C-2’), 85.2 (C-4’),
86.5 (C-1’), 115.1 (2 C, C-3Ph and C-5Ph), 116.1 (C-4a), 117.1 (C-5),
124.1 (C-6), 124.6 (C-1Ph), 131.0 (2 C, C-2Ph and C-6Ph), 150.5, 150.5,
156.8 (C-4Ph), 159.1 (C-4). HRMS (ESI): calculated for C18H20N3O5 ([M+
H]+): 358.1397, found: 358.1412.
NMR (300 MHz, DMSO-d6) δ: 2.52 (s, 3 H, CH3), 3.50–3.60 (m, 1 H, H-
5’’), 3.61–3.75 (m, 1 H, H-5’), 3.95 (q, J=3.8 Hz, 1 H, H-4’), 4.08–4.22
(m, 1 H, H-3’), 4.48 (dd, J=11.8, 6.2 Hz, 1 H, H-2’), 5.06 (t, J=5.4 Hz,
1 H, OH-5’), 5.19 (d, J=5.0 Hz, 1 H, OH-3’), 5.41 (d, J=6.2 Hz, 1 H,
OH-2’), 6.29 (d, J=5.9 Hz, 1 H, H-1’), 7.75–7.89 (m, 2 H, HÀ Ph), 8.08
(s, 1 H, H-6), 8.25–8.39 (m, 2 H, HÀ Ph), 8.75 (s, 1 H, H-2). 13C NMR
(100 MHz, DMSO-d6) δ: 23.4 (CH3), 61.4 (C-5’), 70.5 (C-3’), 74.1 (C-2’),
85.3 (C-4’), 86.7 (C-1’), 115.2 (C-5), 115.4 (C-4a), 123.5 (2 C, C-3Ph and
C-5Ph), 126.3 (C-6), 130.6 (2 C, C-2Ph and C-6Ph), 141.4 (C-1Ph), 146.3
(C-4Ph), 151.0 (C-2), 151.2 (C-7a), 159.4 (C-4). HRMS (ESI): calculated
for C18H19N4O6 ([M+H]+): 387.1299, found: 387.1315.
4-Methyl-5-(3-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (36). 36 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 36 (23 mg,
4-Methyl-5-(4-methyl-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (32). 32 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 32 (58 mg,
1
°
0.061 mmol) as a white solid in 24% yield. Melting point: 158 C. H
1
°
0.16 mmol) as a white solid in 63% yield. Melting point: 114 C. H
NMR (300 MHz, DMSO-d6) δ: 2.48 (s, 3H, CH3), 3.55 (ddd, J=12.0,
5.6, 4.2 Hz, 1 H, H-5’’), 3.65 (ddd, J=12.0, 5.4, 4.2 Hz, 1 H, H-5’), 3.93
(q, J=3.8 Hz, 1 H, H-4’), 4.13 (dd, J=8.2, 5.0 Hz, 1 H, H-3’), 4.47 (dd,
J=11.5, 6.0 Hz, 1 H, H-2’), 5.04 (t, J=5.6 Hz, 1 H, OH-5’), 5.17 (d, J=
5.0 Hz, 1 H, OH-3’), 5.38 (d, J=6.4 Hz, 1 H, OH-2’), 6.27 (d, J=6.2 Hz,
1 H, H-1’), 7.38–7.54 (m, 3 H, HÀ Ph), 7.56–7.65 (m, 1 H, HÀ Ph), 7.93
(s, 1 H, H-6), 8.71 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 23.0
(CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 115.5
(C-5), 115.6 (C-4a), 125.3 (C-6), 127.0 (CÀ Ph), 128.8 (CÀ Ph), 129.2 (C-
2Ph), 130.0 (C-5Ph), 132.9 (C-3Ph), 136.5 (C-1Ph), 150.7 (C-7a), 150.9 (C-
2), 159.2 (C-4). HRMS (ESI): calculated for C18H19ClN3O4 ([M+H]+):
376.1059, found: 376.1080.
NMR (300 MHz, DMSO-d6) δ: 2.37 (s, 3 H, CH3), 2.45 (s, 3 H, CH3), 3.54
(ddd, J=11.8, 5.7, 3.9 Hz, 1 H, H-5’’), 3.64 (ddd, J=11.8, 5.3, 4.1 Hz,
1 H, H-5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’), 4.13 (td, J=4.9, 3.4 Hz, 1 H,
H-3’), 4.46 (dd, J=11.8, 6.4 Hz, 1 H, H-2’), 5.05 (t, J=5.6 Hz, 1 H, OH-
5’), 5.16 (d, J=5.0 Hz, 1 H, OH-3’), 5.36 (d, J=6.4 Hz, 1 H, OH-2’),
6.27 (d, J=6.2 Hz, 1 H, H-1’), 7.26–7.28 (m, 2 H, HÀ Ph), 7.33–7.45 (m,
2 H, HÀ Ph), 7.79 (s, 1 H, H-6), 8.68 (s, 1 H, H-2). 13C NMR (75 MHz,
DMSO-d6) δ: 20.8 (CH3), 22.8 (CH3), 61.5 (C-5’), 70.6 (C-3’), 74.0 (C-2’),
85.2 (C-4’), 86.5 (C-1’), 115.9 (C-4a), 116.9 (C-5), 124.5 (C-6), 128.8
(2 C, C-3Ph and C-5Ph), 129.7 (2 C, C-2Ph and C-6Ph), 131.3 (C-1Ph),
136.4 (C-4Ph), 150.6 (C-2), 150.7 (C-7a), 159.2 (C-4). HRMS (ESI):
calculated for C19H22N3O4 ([M+H]+): 356.1605, found: 356.1581.
4-Methyl-5-(3-fluoro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (37). 37 was prepared according to General
4-Methyl-5-(4-ethyl-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (33). 33 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 37 (62 mg,
1
°
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 33 (39 mg,
0.17 mmol) as a white solid in 66% yield. Melting point: 201 C. H
1
°
0.11 mmol) as a white solid in 41% yield. Melting point: 105 C. H
NMR (300 MHz, DMSO-d6) δ: 2.49 (s, 3 H, CH3), 3.49–3.60 (m, 1 H, H-
5’’), 3.61–3.73 (m, 1 H, H-5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’), 4.13–4.14
(d, J=3.5 Hz, 1 H, H-3’), 4.47 (q, J=5.8 Hz, 1 H, H-2’), 5.05 (t, J=
4.8 Hz, 1 H, OH-5’), 5.17 (d, J=4.7 Hz, 1 H, OH-4’), 5.38 (d, J=6.2 Hz,
1 H, OH-3’), 6.27 (d, J=6.2 Hz, 1 H, H-1’), 7.13–7.30 (m, 1 H, HÀ Ph),
NMR (300 MHz, DMSO-d6) δ: 1.23 (t, J=7.5 Hz, 3 H, CH3), 2.46 (s, 3 H,
CH3), 2.67 (q, J=7.6 Hz, 2 H, CH2), 3.46–3.59 (m, 1 H, H-5’’), 3.59–
3.70 (m, 1 H, H-5’), 3.93 (q, J=3.5 Hz, 1 H, H-4’), 4.07–4.19 (m, 1 H,
H-3’), 4.46 (dd, J=11.4, 6.4 Hz, 1 H, H-2’), 5.05 (t, J=5.4 Hz, 1 H, OH-
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1
°
7.31–7.43 (m, 2 H, HÀ Ph), 7.51 (td, J=8.0, 6.3 Hz, 1 H, HÀ Ph), 7.92 (s,
1 H, H-6), 8.71 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 23.0
(CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 113.9
(d, J=21.9 Hz, 1 C, CÀ Ph), 115.6 (C-4a), 116.1 (d, J=59.9 Hz, 1 C,
CÀ Ph), 116.2 (C-5), 125.3 (C-6), 126.1 (d, J=2.3 Hz, 1 C, CÀ Ph), 130.2
(d, J=8.1 Hz, 1 C, CÀ Ph), 136.7 (d, J=9.2 Hz, 1 C, CÀ Ph), 150.6 (C-
7a), 150.9 (C-2), 159.2 (C-4), 161.9 (d, J=243.0 Hz, 1 C, CÀ Ph). 19F
NMR (282 MHz, DMSO-d6) δ: À 113.42–À 113.33 (m, 1F). HRMS (ESI):
calculated for C18H19FN3O4 ([M+H]+): 360.1354, found: 360.1354.
242 C. H NMR (300 MHz, DMSO-d6) δ: 2.47 (m, 3 H, CH3), 3.48–3.60
(m, 1 H, H-5’’), 3.60–3.73 (m, 1 H, H-5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’),
4.10–4.20 (m, 1 H, H-3’), 4.47 (q, J=6.0 Hz, 1 H, H-2’), 5.03 (t, J=
5.6 Hz, 1 H, OH-5’), 5.19 (d, J=5.0 Hz, 1 H, OH-3’), 5.39 (d, J=6.2 Hz,
1 H, OH-2’), 6.28 (d, J=5.9 Hz, 1 H, H-1’), 7.76–7.92 (m, 2 H, HÀ Ph),
7.97 (d, J=1.8 Hz, 1 H, HÀ Ph), 8.03 (s, 1 H, H-6), 8.73 (s, 1 H, H-2). 13
C
NMR (75 MHz, DMSO-d6) δ: 23.1 (CH3), 61.4 (C-5’), 70.4 (C-3’), 73.9
(C-2’), 85.2 (C-4’), 86.6 (C-1’), 114.3 (C-5), 115.5 (C-4a), 122.8 (q, J=
274.91 Hz, 1 C, CF3), 125.9 (C-6), 126.5 (q, J=31.1 Hz, 1 C, C-3Ph),
128.5 (q, J=5.8 Hz, 1 C, C-2Ph), 129.4 (d, J=2.3 Hz, 1 C, C-4Ph), 131.5
(C-5Ph), 134.0 (C-1Ph), 135.3 (C-6Ph), 150.8 (C-2), 151.0 (C-7a), 159.2 (C-
4). 19F NMR (282 MHz, DMSO-d6) δ: À 61.154394. HRMS (ESI):
calculated for C19H18ClF3N3O4 ([M+H]+): 444.0932, found: 444.0912.
4-Methyl-5-(2-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (38). 38 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 38 (30 mg,
1
°
0.080 mmol) as a white solid in 31% yield. Melting point: 119 C. H
NMR (300 MHz, DMSO-d6) δ: 2.28 (s, 3 H, CH3), 3.55 (ddd, J=11.8,
5.6, 4.2 Hz, 1 H, H-5’’), 3.65 (ddd, J=11.8, 5.3, 4.0 Hz, 1 H, H-5’), 3.94
(q, J=3.8 Hz, 1 H, H-4’), 4.07–4.18 (m, 1 H, H-3’), 4.47 (dd, J=11.5,
6.1 Hz, 1 H, H-2’), 5.04 (t, J=5.4 Hz, 1 H, OH-5’), 5.18 (d, J=5.0 Hz, 1
H, OH-3’), 5.38 (d, J=6.4 Hz, 1 H, OH-2’), 6.26 (d, J=6.2 Hz, 1 H, H-
1’), 7.39–7.55 (m, 3 H, HÀ Ph), 7.56–7.70 (m, 1 H, HÀ Ph), 7.86 (s, 1 H,
H-6), 8.70 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 21.1 (CH3),
61.5 (C-5’), 70.5 (C-3’), 74.1 (C-2’), 85.2 (C-4’), 86.8 (C-1’), 113.4 (C-5),
116.7 (C-4a), 125.4 (C-6), 127.2 (CÀ Ph), 129.3 (CÀ Ph), 129.8 (CÀ Ph),
132.9 (CÀ Ph), 133.3 (C-1Ph), 134.0 (CÀ Ph), 150.2 (C-7a), 150.9 (C-2),
159.0 (C-4). HRMS (ESI): calculated for C18H19ClN3O4 ([M+H]+):
376.1059, found: 367.1057.
4-Methyl-5-(3-methyl-4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-
7H-pyrrolo[2,3-d]-pyrimidine (42). 42 was prepared according to
General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 42
(56 mg, 0.14 mmol) as a white solid in 55% yield. Melting point:
1
°
208 C. H NMR (300 MHz, DMSO-d6) δ: 2.40 (s, 3 H, CH3), 2.47 (s, 3 H,
CH3), 3.55 (ddd, J=11.8, 5.6, 4.0 Hz, 1 H, H-5’’), 3.64 (ddd, J=12.0,
5.2, 4.0 Hz, 1 H, H-5’), 3.93 (q, J=3.7 Hz, 1 H, H-4’), 4.07–4.19 (m, 1
H, H-3’), 4.46 (dd, J=11.8, 6.2 Hz, 1 H, H-2’), 5.04 (t, J=5.6 Hz, 1 H,
OH-5’), 5.17 (d, J=4.7 Hz, 1 H, OH-3’), 5.37 (d, J=6.2 Hz, 1 H, OH-2’),
6.27 (d, J=5.9 Hz, 1 H, H-1’), 7.35 (dd, J=8.2, 1.8 Hz, 1 H, HÀ Ph),
7.45–7.55 (m, 2 H, HÀ Ph), 7.86 (s, 1 H, H-6), 8.70 (s, 1 H, H-2). 13C
NMR (100 MHz, DMSO-d6) δ: 19.6 (CH3), 23.0 (CH3), 61.5 (C-5’), 70.5
(C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.5 (C-1’), 115.7 (C-5), 115.8 (C-4a),
124.9 (C-6), 128.6 (C-5Ph), 129.0 (C-6Ph), 132.2 (C-3Ph), 132.4 (C-2Ph),
133.2 (C-1Ph), 135.3 (C-4Ph), 150.7 (C-7a), 150.9 (C-2), 159.3 (C-4).
HRMS (ESI): calculated for C19H21ClN3O4 ([M+H]+): 390.1215, found:
390.1198.
4-Methyl-5-(3,4-dichloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyr-
rolo[2,3-d]-pyrimidine (39). 39 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 39 (27 mg,
1
°
0.066 mmol) as a white solid in 25% yield. Melting point: 228 C. H
NMR (300 MHz, DMSO-d6) δ: 2.49 (s, 3H, CH3), 3.55 (ddd, J=12.0,
5.8, 4.1 Hz, 1 H, H-5’’), 3.65 (ddd, J=11.8, 5.3, 4.3 Hz, 1 H, H-5’), 3.93
(q, J=3.8 Hz, 1 H, H-4’), 4.14 (dd, J=8.8, 4.8 Hz, 1 H, H-3’), 4.46 (dd,
J=11.4, 6.0 Hz, 1 H, H-2’), 5.04 (t, J=5.6 Hz, 1 H, OH-5’), 5.18 (d, J=
5.0 Hz, 1 H, OH-3’), 5.38 (d, J=6.2 Hz, 1 H, OH-2’), 6.27 (d, J=5.9 Hz,
1 H, H-1’), 7.53 (dd, J=8.3, 2.2 Hz, 1 H, HÀ Ph), 7.72 (d, J=8.5 Hz, 1
H, HÀ Ph), 7.81 (d, J=2.1 Hz, 1 H, HÀ Ph), 7.97 (s, 1 H, H-6), 8.72 (s, 1
H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 23.1 (CH3), 61.5 (C-5’), 70.5
(C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 114.5 (C-5), 115.6 (C-4a),
125.6 (C-6), 129.9 (C-4Ph), 130.1 (C-6Ph), 130.3 (C-5Ph), 131.0 (C-3Ph),
131.3 (C-2Ph), 135.1 (C-1Ph), 150.7 (C-7a), 151.0 (C-2), 159.3 (C-4).
HRMS (ESI): calculated for C18H18ClN3O4 ([M+H]+): 410.0669, found:
410.0682.
4-Methyl-5-(3-methoxy-4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-
7H-pyrrolo[2,3-d]-pyrimidine (43). 43 was prepared according to
General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 43
(36 mg, 0.089 mmol) as a light yellow solid in 34% yield. Melting
point: 50 C. H NMR (400 MHz, DMSO-d6) δ: 3.52–3.58 (m, 1 H, H-
5’’), 3.62–3.67 (m, 1 H, H-5’), 3.91–3.95 (m, 4 H, H-4’ and CH3), 4.13
(q, J=4.2 Hz, 1 H, H-3’), 4.47 (q, J=6.1 Hz, 1 H, H-2’), 5.05 (t, J=
5.4 Hz, 1 H, OH-5’), 5.18 (d, J=4.9 Hz, 1 H, OH-3’), 5.37 (d, J=6.5 Hz,
1 H, OH-2’), 6.27 (d, J=6.1 Hz, 1 H, H-1’), 7.08 (dd, J=8.0, 1.9 Hz, 1
H, HÀ Ph), 7.25 (d, J=1.9 Hz, 1 H, HÀ Ph), 7.50 (d, J=8.0 Hz, 1 H,
HÀ Ph), 7.90 (s, 1 H, H-6), 8.70 (s, 1 H, H-2). 13C NMR (100 MHz,
DMSO-d6) δ: 23.1 (CH3), 56.2 (OCH3), 61.4 (C-5’), 70.5 (C-3’), 74.0 (C-
2’), 85.2 (C-4’), 86.6 (C-1’), 114.3 (C-2Ph), 115.8 (C-4a), 116.1 (C-5),
120.0 (C-4Ph), 122.7 (C-6Ph), 125.1 (C-6), 129.6 (C-5Ph), 134.6 (C-Ph),
150.6 (C-7a), 150.9 (C-2), 154.1 (C-3Ph), 159.3 (C-4). HRMS (ESI):
calculated for C19H21ClN3O5 ([M+H]+): 406.1164, found: 406.1187.
1
°
4-Methyl-5-(3-fluoro-4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (40). 40 was prepared according to
General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 40
(40 mg, 0.10 mmol) as a white solid in 39% yield. Melting point:
1
°
205 C. H NMR (300 MHz, DMSO-d6) δ: 2.49 (s, 3H, CH3), 3.49–3.60
4-Methyl-5-(2-chloro-4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (44). 44 was prepared according to
General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 44
(35 mg, 0.085 mmol) as a white solid in 33% yield. Melting point:
120–122 C. H NMR (300 MHz, DMSO-d6) δ: 2.30 (s, 3 H, CH3), 3.55
(ddd, J=12.0, 5.5, 4.0 Hz, 1 H, H-5’’), 3.64 (ddd, J=11.8, 5.2, 4.2 Hz,
1 H, H-5’), 3.94 (q, J=3.8 Hz, 1 H, H-4’), 4.07–4.17 (m, 1 H, H-3’), 4.45
(dd, J=11.5, 6.2 Hz, 1 H, H-2’), 5.04 (t, J=5.4 Hz, 1 H, OH-5’), 5.18 (d,
J=5.0 Hz, 1 H, OH-3’), 5.39 (d, J=6.2 Hz, 1 H, OH-2’), 6.25 (d, J=
5.9 Hz, 1 H, H-1’), 7.47–7.59 (m, 2 H, HÀ Ph), 7.80 (dd, J=1.5, 0.9 Hz,
1 H, HÀ Ph), 7.89 (s, 1 H, H-6), 8.71 (s, 1 H, H-2). 13C NMR (75 MHz,
DMSO-d6) δ: 21.2 (CH3), 61.5 (C-5’), 70.5 (C-3’), 74.1 (C-2’), 85.2 (C-4’),
86.8 (C-1’), 112.2 (C-5), 116.6 (C-4a), 125.7 (C-6), 127.4 (C-3Ph), 128.8
(C-5Ph), 132.4 (C-1Ph), 133.5 (CÀ Ph), 134.1 (C-2Ph), 135.0 (CÀ Ph), 150.3
(C-2), 151.0 (C-7a), 159.0 (C-4). HRMS (ESI): calculated for
C18H18Cl2N3O4 ([M+H]+): 410.0669, found: 410.0664.
(m, 1 H, H-5’’), 3.60–3.71 (m, 1 H, H-5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’),
4.13 (dd, J=8.4, 4.8 Hz, 1 H, H-3’), 4.46 (q, J=5.9 Hz, 1 H, H-2’), 5.04
(t, J=5.6 Hz, 1 H, OH-5’), 5.18 (d, J=5.0 Hz, 1 H, OH-3’), 5.38 (d, J=
6.4 Hz, 1 H, OH-2’), 6.27 (d, J=6.2 Hz, 1 H, H-1’), 7.39 (dd, J=8.2,
1.5 Hz, 1 H, HÀ Ph), 7.61 (dd, J=10.5, 2.1 Hz, 1 H, HÀ Ph), 7.64–7.72
(m, 1 H, HÀ Ph), 7.95 (s, 1 H, H-6), 8.71 (s, 1 H, H-2). 13C NMR (75 MHz,
DMSO-d6) δ: 23.1 (CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.2 (C-4’),
86.6 (C-1’), 114.8 (C-5), 115.5 (C-4a), 117.9 (d, J=20.7 Hz, 1 C, CÀ Ph),
118.3 (d, J=17.3 Hz, 1 C, CÀ Ph), 125.6 (C-6), 127.1 (d, J=3.5 Hz, 1 C,
CÀ Ph), 130.4 (CÀ Ph), 135.6 (d, J=8.1 Hz, 1 C, CÀ Ph), 150.7 (C-7a),
151.0 (C-2), 156.9 (d, J=246.5 Hz, 1 C, CÀ Ph), 159.3 (C-4). 19F NMR
(282 MHz, DMSO-d6) δ: À 116.42–À 116.36 (m, 1F). HRMS (ESI):
calculated for C18H18ClFN3O4 ([M+H]+): 394.0964, found: 394.0960.
1
°
4-Methyl-5-(3-trifluoromethyl-4-chloro-phenyl)-N7-(β-d–ribofura-
nosyl)-7H-pyrrolo[2,3-d]-pyrimidine (41). 41 was prepared accord-
ing to General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 41
(51 mg, 0.12 mmol) as a white solid in 45% yield. Melting point:
4-Methyl-5-(2-fluorol-4-chloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (45). 45 was prepared according to
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General procedure A. 67 (100 mg, 0.26 mmol) gave rise to 45
(33 mg, 0.084 mmol) as a white solid in 32% yield. Melting point:
(CÀ Ph), 150.8 (C-2 and C-7a), 159.3 (C-4). HRMS (ESI): calculated for
C24H24N3O4 ([M+H]+): 418.1761, found: 418.1749.
1
°
223 C. H NMR (300 MHz, DMSO-d6) δ: 2.39 (s, 3 H, CH3), 3.48–3.60
4-Methyl-5-(naphthalen-2-yl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo
[2,3-d]-pyrimidine (49). 49 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 49 (25 mg,
(m, 1 H, H-5’’), 3.60–3.76 (m, 1 H, H-5’), 3.94 (q, J=3.7 Hz, 1 H, H-4’),
4.09–4.18 (m, 1 H, H-3’), 4.47 (q, J=6.2 Hz, 1 H, H-2’), 5.04 (t, J=
5.4 Hz, 1 H, OH-5’), 5.18 (d, J=5.0 Hz, 1 H, OH-3’), 5.40 (d, J=6.4 Hz,
1 H, OH-2’), 6.27 (d, J=6.2 Hz, 1 H, H-1’), 7.42 (dd, J=8.2, 2.1 Hz, 1
H, HÀ Ph), 7.55 (t, J=6.2 Hz, 1 H, HÀ Ph), 7.60 (dd, J=9.7, 2.1 Hz, 1 H,
HÀ Ph), 7.94 (s, 1 H, H-6), 8.72 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-
d6) δ: 21.4 (CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.3 (C-4’), 86.7
(C-1’), 108.3 (C-5), 116.4 (d, J=21.9 Hz, 1 C, CÀ Ph), 116.4 (C-4a),
121.2 (d, J=16.1 Hz, 1 C, CÀ Ph), 124.8 (d, J=3.5 Hz, 1 C, CÀ Ph),
126.1 (C-6), 133.3 (d, J=10.4 Hz, 1 C, CÀ Ph), 133.7 (d, J=3.5 Hz, 1 C,
CÀ Ph), 150.5 (C-7a), 151.0 (C-2), 159.2 (C-4), 159.7 (d, J=247.6 Hz,
1 C, CÀ Ph). 19F NMR (282 MHz, DMSO-d6) δ: À 111.36–À 111.30 (m, 1
F). HRMS (ESI): calculated for C18H18ClFN3O4 ([M+H]+): 394.0964,
found: 394.0972.
1
°
0.064 mmol) as a white solid in 25% yield. Melting point: 135 C. H
NMR (300 MHz, DMSO-d6) δ: 2.49 (s, 3 H, CH3), 3.56 (ddd, J=11.8,
5.6, 4.1 Hz, 1 H, H-5’’), 3.66 (ddd, J=11.8, 5.2, 4.2 Hz, 1 H, H-5’), 3.95
(q, J=3.6 Hz, 1 H, H-4’), 4.10–4.22 (m, 1 H, H-3’), 4.51 (dd, J=11.5,
6.0 Hz, 1 H, H-2’), 5.06 (t, J=5.4 Hz, 1 H, OH-5’), 5.18 (d, J=4.7 Hz, 1
H, OH-3’), 5.40 (d, J=6.4 Hz, 1 H, OH-2’), 6.31 (d, J=6.2 Hz, 1 H, H-
1’), 7.48–7.62 (m, 2 H, H-Nap), 7.68 (dd, J=8.5, 1.8 Hz, 1 H, H-Nap),
7.90–8.13 (m, 5 H, H-Nap and H-6), 8.72 (s, 1 H, H-2). 13C NMR
(75 MHz, DMSO-d6) δ: 23.0 (CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’),
85.2 (C-4’), 86.5 (C-1’), 116.0 (C-4a), 116.9 (C-5), 125.0 (C-6), 126.0 (C-
Nap), 126.4 (C-Nap), 127.6 (C-Nap), 127.8 (C-Nap), 128.0 (C-Nap),
128.3 (C-Nap), 131.7 (C-Nap), 132.9 (C-Nap), 150.8 (C-2), 150.9 (C-7a),
159.3 (C-4). HRMS (ESI): calculated for C22H22N3O4([M+H]+):
392.1605, found: 392.1600.
4-Methyl-5-(3,5-dichloro-phenyl)-N7-(β-d–ribofuranosyl)-7H-pyr-
rolo[2,3-d]-pyrimidine (46). 46 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 46 (35 mg,
4-Methyl-5-(benzo[d][1,3]dioxol-5-yl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (50). 50 was prepared according to
General procedure A. 67 (150 mg, 0.38 mmol) gave rise to 50
1
°
0.085 mmol) as a white solid in 33% yield. Melting point: 230 C. H
NMR (300 MHz, DMSO-d6) δ: 2.51 (s, 3 H, CH3), 3.55 (ddd, J=12.0,
5.7, 4.3 Hz, 1 H, H-5’’), 3.65 (ddd, J=12.0, 5.3, 4.1 Hz, 1 H, H-5’), 3.93
(q, J=4.1 Hz, 1 H, H-4’), 4.09–4.18 (m, 1 H, H-3’), 4.46 (dd, J=11.5,
6.0 Hz, 1 H, H-2’), 5.03 (t, J=5.6 Hz, 1 H, OH-5’), 5.18 (d, J=5.0 Hz, 1
H, OH-3’), 5.38 (d, J=6.4 Hz, 1 H, OH-2’), 6.27 (d, J=5.9 Hz, 1 H, H-
1’), 7.56–7.69 (m, 3 H, HÀ Ph), 8.01 (s, 1 H, H-6), 8.72 (s, 1 H, H-2). 13C
NMR (75 MHz, DMSO-d6) δ: 23.1 (CH3), 61.5 (C-5’), 70.4 (C-3’), 74.0
(C-2’), 85.2 (C-4’), 86.6 (C-1’), 114.3 (C-5), 115.5 (C-4a), 126.0 (C-6),
126.6 (CÀ Ph), 128.3 (CÀ Ph), 133.9 (CÀ Ph), 137.9 (C-1Ph), 150.7 (C-2),
151.0 (C-7a), 159.3 (C-4). HRMS (ESI): calculated for C18H18Cl2N3O4
([M+H]+): 410.0669, found: 410.0669.
(95 mg, 0.25 mmol) as a white solid in 65% yield. Melting point:
1
°
232 C. H NMR (400 MHz, DMSO-d6) δ: 2.48 (s, 3 H, CH3), 3.52–3.57
(m, 1 H, H-5’’), 3.61–3.67 (m, 1 H, H-5’), 3.92 (q, J=3.4 Hz, 1 H, H-4’),
4.13 (q, J=4.5 Hz, 1 H, H-3’), 4.45 (q, J=6.0 Hz, 1 H, H-2’), 5.05 (t, J=
5.4 Hz, 1 H, OH-5’), 5.16 (d, J=4.9 Hz, 1 H, OH-3’), 5.36 (d, J=6.4 Hz,
1 H, OH-2’), 6.08 (s, 2 H, CH2), 6.26 (d, J=6.0 Hz, 1 H, H-1’), 6.94 (d,
J=7.9 Hz, 1 H, HÀ Ph), 7.00 (d, J=7.9 Hz, 1 H, HÀ Ph), 7.07 (s, 1 H,
HÀ Ph), 7.78 (s, 1 H, H-6), 8.67 (s, 1 H, H-2). 13C NMR (100 MHz,
DMSO-d6) δ: 22.8 (CH3), 61.5 (C-5’), 70.5 (C-3’), 74.0 (C-2’), 85.1 (C-4’),
86.5 (C-1’), 101.1 (CH2), 108.1 (CÀ Ph), 110.3 (CÀ Ph), 115.9 (C-4a),
116.7 (C-5), 123.3 (CÀ Ph), 124.6 (C-6), 127.9 (CÀ Ph), 146.5 (CÀ Ph),
147.1 (CÀ Ph), 150.5 (C-7a), 150.7 (C-2), 159.2 (C-4). HRMS (ESI):
calculated for C19H20N3O6 ([M+H]+): 386.1347, found: 386.1350.
4-Methyl-5-(4-chloro-3,5-difluoro-4-chloro-phenyl)-N7-(β-d–ribo-
furanosyl)-7H-pyrrolo[2,3-d]-pyrimidine (47). 47 was prepared
according to General procedure A. 67 (100 mg, 0.26 mmol) gave
rise to 47 (37 mg, 0.090 mmol) as a white solid in 35% yield.
4-Methyl-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N7-(β-d–ribofur-
anosyl)-7H-pyrrolo[2,3-d]-pyrimidine (51). 51 was prepared ac-
cording to General procedure A. 67 (120 mg, 0.31 mmol) gave rise
to 51 (55 mg, 0.14 mmol) as a white solid in 44% yield. Melting
1
°
Melting point: 212 C. H NMR (300 MHz, DMSO-d6) δ: 2.54 (s, 3 H,
CH3), 3.55 (ddd, J=12.0, 5.6, 4.2 Hz, 1 H, H-5’’), 3.65 (ddd, J=12.0,
5.4, 4.2 Hz, 1 H, H-5’), 3.93 (q, J=3.8 Hz, 1 H, H-4’), 4.08–4.20 (m, 1
H, H-3’), 4.45 (q, J=5.8 Hz, 1 H, H-2’), 5.04 (t, J=5.6 Hz, 1 H, OH-5’),
5.19 (d, J=5.0 Hz, 1 H, OH-3’), 5.39 (d, J=6.4 Hz, 1 H, OH-2’), 6.27
(d, J=6.2 Hz, 1 H, H-1’), 7.45–7.61 (m, 2 H, HÀ Ph), 8.01 (s, 1 H, H-6),
8.73 (s, 1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 23.3 (CH3), 61.4 (C-
5’), 70.4 (C-3’), 74.1 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 106.8 (CÀ Ph), 113.9
(d, J=45.8 Hz, 1 C, CÀ Ph), 114.0 (C-5), 116.4 (d, J=21.9 Hz, 1 C,
CÀ Ph), 115.4 (C-4a), 126.1 (C-6), 135.6 (t, J=10.2 Hz, 1 C, CÀ Ph),
150.7 (C-7a), 151.1 (C-2), 156.5 (d, J=3.6 Hz, 1 C, C-1Ph), 158.9 (d, J=
4.4 Hz, 1 C, CÀ Ph), 159.5 (C-4). 19F NMR (282 MHz, DMSO-d6) δ:
À 114.34–À 114.31. HRMS (ESI): calculated for C18H17ClF2N3O4 ([M+
H]+): 412.0870, found: 412.0860.
point: 127 C. 1H NMR (400 MHz, DMSO-d6) δ: 2.48 (s, 3 H, CH3),
°
3.52–3.57 (m, 1 H, H-5’’), 3.61–3.66 (m, 1 H, H-5’), 3.92 (q, J=3.5 Hz,
1 H, H-4’), 4.12 (q, J=4.3 Hz, 1 H, H-3’), 4.29 (s, 4 H, 2CH2), 4.45 (q,
J=6.0 Hz, 1 H, H-2’), 5.05 (t, J=5.4 Hz, 1 H, OH-5’), 5.15 (d, J=
4.8 Hz, 1 H, OH-3’), 5.35 (d, J=6.4 Hz, 1 H, OH-2’), 6.25 (d, J=6.1 Hz,
1 H, H-1’), 6.93 (s, 2 H, HÀ Ph), 6.98 (s, 1 H, HÀ Ph), 7.76 (s, 1 H, H-6),
8.67 (s, 1 H, H-2) . 13C NMR (100 MHz, DMSO-d6) δ: 22.8 (CH3), 61.5
(C-5’), 64.1 (2CH2), 70.5 (C-3’), 74.0 (C-2’), 85.1 (C-4’), 86.5 (C-1’),
115.9 (C-4a), 116.5 (C-5), 116.8 (CÀ Ph), 118.3 (CÀ Ph), 122.9 (CÀ Ph),
124.5 (C-6), 127.3 (CÀ Ph), 142.8 (CÀ Ph), 143.0 (CÀ Ph),150.5 (C-7a),
150.7 (C-2), 159.2 (C-4). HRMS (ESI): calculated for C20H22N3O6 ([M+
H]+): 400.1503, found: 400.1509.
4-Methyl-5-([1,1’-biphenyl]-4-yl)-N7-(β-d–ribofuranosyl)-7H-pyrro-
lo[2,3-d]-pyrimidine (48). 48 was prepared according to General
procedure A. 67 (100 mg, 0.26 mmol) gave rise to 48 (60 mg,
4-Methyl-5-(1H-indol-6-yl)-N7-(β-d–ribofuranosyl)-7H-pyrrolo[2,3-
d]-pyrimidine (52). 52 was prepared according to General
procedure A. 67 (120 mg, 0.31 mmol) gave rise to slightly impure
52 (35 mg, 0.092 mmol). 52 was purified by preparative RP-HPLC
gradient: 0.2% formic acid in water:MeCN at a flow rate of 20 mL/
min; The initial gradient composition (95% A/05% B) was held for
2.0 min, increased to 60% B in 13 min, then increased to 100% B in
1
°
0.14 mmol) as a white solid in 55% yield. Melting point: 134 C. H
NMR (300 MHz, DMSO-d6) δ: 2.53 (s, 3 H, CH3), 3.56 (ddd, J=12.0,
5.6, 4.2 Hz, 1 H, H-5’’), 3.66 (ddd, J=12.0, 5.2, 4.2 Hz, 1 H, H-5’), 3.94
(q, J=3.7 Hz, 1 H, H-4’), 4.10–4.22 (m, 1 H, H-3’), 4.49 (dd, J=11.6,
6.2 Hz, 1 H, H-2’), 5.07 (t, J=5.6 Hz, 1 H, OH-5’), 5.17 (d, J=4.7 Hz, 1
H, OH-3’), 5.39 (d, J=6.4 Hz, 1 H, OH-2’), 6.30 (d, J=6.2 Hz, 1 H, H-
1’), 7.33–7.43 (m, 1 H, HÀ Ph), 7.44–7.54 (m, 2 H, HÀ Ph), 7.57–7.67
(m, 2 H, HÀ Ph), 7.70–7.83 (m, 4 H, HÀ Ph), 7.91 (s, 1 H, H-6), 8.71 (s, 1
H, H-2). 13C NMR (100 MHz, DMSO-d6) δ: 23.0 (CH3), 61.5 (C-5’), 70.6
(C-3’), 74.0 (C-2’), 85.2 (C-4’), 86.6 (C-1’), 115.8 (C-4a), 116.6 (C-5),
124.9 (C-6), 126.5 (CÀ Ph), 126.6 (CÀ Ph), 127.5 (CÀ Ph), 127.8 (CÀ Ph),
129.0 (CÀ Ph), 130.3 (CÀ Ph), 133.4 (CÀ Ph), 138.8 (CÀ Ph), 139.7
1 min. After preparative RP-HPLC, 52 was isolated as a white solid
1
°
(25 mg, 0.066 mmol) in 21% yield. Melting point: 153 C. H NMR
(400 MHz, DMSO-d6) δ: 2.45 (s, 3 H, CH3), 3.53–3.57 (m, 1 H, H-5’’),
3.62–3.66 (m, 1 H, H-5’), 3.94 (q, J=3.7 Hz, 1 H, H-4’), 4.13 (dd, J=
4.9, 3.3 Hz, 1 H, H-3’), 4.50 (t, J=5.7 Hz, 1 H, H-2’), 5.22 (br.s. 3 H,
OH-2’, OH-3’and OH-5’) 6.29 (d, J=6.3 Hz, 1 H, H-1’), 6.47 (br.s., 1 H,
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H-indole), 7.12 (d, J=8.1 Hz, 1 H, H-indole), 7.39 (t, J=2.7 Hz, 1 H,
H-indole), 7.47 (s, 1 H, H-indole), 7.61 (d, J=8.1 Hz, 1 H, H-indole),
7.79 (s, 1 H, H-6), 8.68 (s, 1 H, H-2), 11.16 (br. s., 1 H, NH). 13C NMR
(100 MHz, DMSO-d6) δ: 22.8 (CH3), 61.6 (C-5’), 70.6 (C-3’), 73.9 (C-2’),
85.2 (C-4’), 86.5 (C-1’), 101.0 (C-indole), 112.5 (C-indole), 116.2 (C-
4a), 118.3 (C-5), 119.6 (C-indole), 121.5 (C-indole), 124.2 (C-6), 125.8
(C-indole), 126.8 (C-indole), 126.8 (C-indole), 135.8 (C-indole), 150.6
(C-7a and C-2), 159.2 (C-4). HRMS (ESI): calculated for C20H21N4O4
([M+H]+): 381.1557, found: 381.1565.
step. This crude product, Pd(PPh3)4 (116 mg, 0.10 mmol, 0.1 eq.)
were added in a 25 mL two-neck round bottom flask. The flask was
purged with argon three times. Next, anhydrous THF (4 mL, 4 mL/
mmol) was added via syringe, and AlMe3 solution (2 M in toluene,
0.6 mL, 1.2 mmol, 1.2 eq.) was added dropwise. After the mixture
was stirred for 15 min at ambient temperature, the reaction was
°
refluxed at 100 C until the TLC showed the SM was consumed
completely (1 h). After cooling at ice-water bath, 0.5 M aq. HCl
(10 mL) was added dropwise. [Caution: methane gas was gener-
ated, with potential excessive foaming]. Next, EA (50 mL) was
added. The layers were separated and the water layer extracted
twice more with EA. Then, the organic layers were combined, dried
over Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (10!50% EA/PET) to give 82 (400 mg,
4-Methyl-5-(1-methyl-1H-indol-6-yl)-N7-(β-d–ribofuranosyl)-7H-
pyrrolo[2,3-d]-pyrimidine (53). 53 was prepared according to
General procedure A. 67 (150 mg, 0.38 mmol) gave rise to 53
(85 mg, 0.22 mmol) as a white solid in 57% yield. Melting point:
1
°
135 C. H NMR (400 MHz, DMSO-d6) δ: 2.45 (s, 3 H, CH3), 3.53–3.58
1
0.87 mmol) as a yellow foam in 66% yield for two steps. H NMR
(m, 1 H, H-5’’), 3.62–3.67 (m, 1 H, H-5’), 3.82 (s, 3 H, CH3), 3.94 (q, J=
3.2 Hz, 1 H, H-4’), 4.14 (q, J=4.2 Hz, 1 H, H-3’), 4.49 (q, J=5.7 Hz, 1
H, H-2’), 5.06 (t, J=5.4 Hz, 1 H, OH-5’), 5.17 (dd, J=4.8, 1.2 Hz, 1 H,
OH-3’), 5.37 (dd, J=6.4, 1.4 Hz, 1 H, OH-2’), 6.30 (d, J=6.1 Hz, 1 H,
H-1’), 6.47 (dd, J=3.1, 0.6 Hz, 1 H, H-indole), 7.16 (dd, J=8.1, 1.2 Hz,
1 H, H-indole), 7.37 (d, J=3.1 Hz, 1 H, H-indole), 7.53 (s, 1 H, H-
indole), 7.61 (d, J=8.0 Hz, 1 H, H-indole), 7.81 (s, 1 H, H-6), 8.69 (s, 1
H, H-2). 13C NMR (100 MHz, DMSO-d6) δ: 22.9 (CH3), 32.5 (CH3), 61.6
(C-5’), 70.6 (C-3’), 74.0 (C-2’), 85.1 (C-4’), 86.5 (C-1’), 100.3 (C-indole),
110.9 (C-indole), 116.2 (C-4a), 118.3 (C-5), 119.9 (C-indole), 121.5 (C-
indole), 124.3 (C-6), 127.0 (C-indole), 127.1 (C-indole), 130.1 (C-
indole), 136.3 (C-indole), 150.6 (C-7a), 150.7 (C-2), 159.3 (C-4). HRMS
(ESI): calculated for C21H23N4O4 ([M+H]+): 395.1714, found:
395.1711.
(300 MHz, CDCl3) δ: 2.45 (ddd, J=14.1, 5.6, 1.8 Hz, 1 H, CH2), 2.71 (s,
3 H, CH3), 2.80–2.90 (m, 1 H, CH2), 4.53–4.59 (m, 1 H, H-5’’), 4.68–
4.73 (m, 1 H, H-5’), 4.79–4.87 (m, 1 H, H-4’), 5.99 (dt, J=6.2, 1.8 Hz, 1
H, H-2’), 6.49 (d, J=2.1 Hz, 1 H, H-1’), 6.55 (d, J=3.8 Hz, 1 H, H-5),
7.33 (d, J=3.8 Hz, 1 H, H-6), 7.41–7.50 (m, 4 H, H-OBz), 7.52–7.71 (m,
2 H, H-OBz), 8.01–8.10 (m, 4 H, H-OBz), 8.74 (s, 1 H, H-2). HRMS (ESI):
calculated for C26H24N3O5 ([M+H]+): 458.1710, found: 458.1718.
4-Methyl-N7-(3’-deoxy-β-d–ribofuranosyl)-pyrrolo[2,3-d]
pyrimidine (83). 82 (350 mg, 0.77 mmol) was placed in a 25 mL
round bottom flask. Next, 0.5 M NaOMe in MeOH (5 mL) was added,
and the reaction mixture was stirred for 2 h. Then, 0.5 M aq. HCl
was added to neutralize the reaction solution. The resulting mixture
was evaporated till dryness, and purified by flash column
chromatography (0!10% MeOH/DCM) to afford 83 (160 mg,
0.64 mmol) as a white solid in 83% yield. 1H NMR (300 MHz, DMSO-
d6) δ: 1.93 (ddd, J=13.0, 6.3, 3.2 Hz, 1 H, CH2), 2.22 (ddd, J=13.1,
8.7, 6.0 Hz, 1 H, CH2), 2.65 (s, 3 H, CH3), 3.50–3.56 (m, 1 H, H-5’’),
3.62–3.67 (m, 1 H, H-5’), 4.28–4.36 (m, 1 H, H-4’), 4.42–4.45 (m, 1 H,
H-2’), 4.98 (t, J=5.6 Hz, 1 H, OH-5’), 5.60 (d, J=4.4 Hz, 1 H, OH-2’),
6.16 (d, J=2.6 Hz, 1 H, H-1’), 6.72 (d, J=3.8 Hz, 1 H, H-5), 7.78 (d, J=
3.5 Hz, 1 H, H-6), 8.65 (s, 1 H, H-2). HRMS (ESI): calculated for
C12H16N3O3 ([M+H]+): 259.1186, found: 259.1180.
4-Chloro-5-iodo-N7-(2’,5’-di-O-benzoyl-3’-deoxy-β-d–ribofurano-
syl)-pyrrolo[2,3-d]pyrimidine (81).[33] 4-chloro-5-iodo-7H-pyrrolo
[2,3-d]pyrimidine (500 mg, 1.79 mmol) was placed in a 100 mL two-
neck round bottom flask, after which the flask was purged with N2
gas. Anhydrous MeCN (15 mL, 7.5 mL/mmol of SM) was added. BSA
(482 μL, 1.97 mmol, 1.1 eq.) was added into the suspension via
syringe. The resulting mixture was stirred until the solid was
completely dissolved. Next, 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-α/
β-D-ribofuranose (760 mg, 1.97 mmol, 1.1 eq.) was added, and
immediately followed by TMSOTf (357 μL, 1.97 mmol, 1.1 eq.). The
reaction mixture was stirred at ambient temperature for 15 min,
4-Methyl-5-bromo-N7-(3’-deoxy-β-d–ribofuranosyl)-pyrrolo[2,3-d]
pyrimidine (84). 83 (100 mg, 0.40 mmol) was dissolved in DMF
(2 mL), and NBS (107 mg, 0.60 mmol, 1.5 eq.) was added. The
°
and was heated at 80 C. When full conversion of 4-chloro-5-iodo-
°
7H-pyrrolo[2,3-d]pyrimidine was observed via HRMS, the mixture
was cooled to room temperature. Then, EA (15 mL) and aq. sat.
NaHCO3 (15 mL) were added. The layers were separated and the
water layer extracted twice more with EA. Then, the organic layers
were combined, washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by column chromatography
(5!30% EA/PET) to give a colorless foam (800 mg, 1.32 mmol) in
74% yield. 1H NMR (300 MHz, CDCl3) δ: 2.44 (ddd, J=14.1, 5.6,
1.8 Hz, 1 H, CH2), 2.76 (ddd, J=14.1, 10.4, 6.0 Hz, 1 H, CH2), 4.56–
4.61 (m, 1 H, H-5’’), 4.73–4.78 (m, 1 H, H-5’), 4.80–4.88 (m, 1 H, H-4’),
5.90–5.92 (m, 1 H, H-2’), 6.45 (d, J=1.8 Hz, 1 H, H-1’), 7.45–7.51 (m,
4 H, H-OBz), 7.57–7.65 (m, 3 H, H-6 and H-OBz), 8.01–8.09 (m, 4 H,
H-OBz), 8.59 (s, 1 H, H-2). HRMS (ESI): calculated for C20H25ClIN3O5
([M+H]+): 604.0131, found: 604.0126.
resulting mixture was stirred at 50 C for 2 h. Then, the mixture was
evaporated till dryness and purified by column chromatography
(0!10% MeOH/DCM) to afford 84 (96 mg, 0.29 mmol) as a white
1
solid in 73% yield. H NMR (300 MHz, DMSO-d6) δ: 1.89 (ddd, J=
13.2, 6.3, 3.1 Hz, 1 H, CH2), 2.22 (ddd, J=13.2, 8.8, 5.9 Hz, 1 H, CH2),
2.84 (s, 3 H, CH3), 3.53 (ddd, J=12.0, 5.3, 4.1 Hz, 1 H), H-5’’, 3.70
(ddd, J=12.0, 5.4, 3.4 Hz, 1 H, H-5’), 4.29–4.37 (m, 1 H, H-4’), 4.39–
4.44 (m, 1 H, H-4’), 5.05 (t, J=5.4 Hz, 1 H, OH-5’), 5.63 (d, J=4.1 Hz,
1 H, OH-2’), 6.18 (d, J=2.3 Hz, 1 H, H-1’), 8.07 (s, 1 H, H-6), 8.70 (s, 1
H, H-2). HRMS (ESI): calculated for C12H15BrN3O3 ([M+H]+): 328.0291,
found 328.0295.
4-Methyl-5-(4-chloro-phenyl)-N7-(3’-deoxy-β-d–ribofuranosyl)-
pyrrolo[2,3-d]pyrimidine (54). 54 was prepared according to
General procedure A. 84 (90 mg, 0.27 mmol) gave rise to 54 (35 mg,
1
°
4-Methyl-N7-(2’,5’-di-O-benzoyl-3’-deoxy-β-d–ribofuranosyl)-pyr-
rolo[2,3-d]pyrimidine (82). 81 (800 mg, 1.32 mmol) was placed in a
25 mL round bottom flask. Then, the flask was purged with argon.
Next, anhydrous THF (5 mL) was added, and the reaction solution
0.10 mmol) as a white solid in 36% yield. Melting point: 181 C. H
NMR (300 MHz, DMSO-d6) δ: 1.92 (ddd, J=13.0, 6.3, 2.9 Hz, 1 H,
CH2), 2.27 (ddd, J=13.0, 8.8, 6.0 Hz, 1 H, CH2), 2.46 (s, 3 H, CH3), 3.52
(ddd, J=11.9, 5.4, 4.1 Hz, 1 H, H-5’’), 3.67 (ddd, J=11.9, 5.6, 3.4 Hz,
1 H, H-5’), 4.27–4.40 (m, 1 H, H-4’), 4.42–4.55 (m, 1 H, H-2’), 4.98 (t,
J=5.4 Hz, 1 H, OH-5’), 5.63 (d, J=4.4 Hz, 1 H, OH-2’), 6.25 (d, J=
2.3 Hz, 1 H, H-1’), 7.44–7.58 (m, 4 H, HÀ Ph), 7.89 (s, 1 H, H-6), 8.70 (s,
1 H, H-2). 13C NMR (75 MHz, DMSO-d6) δ: 23.0 (CH3), 34.5 (CH2), 62.5
(C-5’), 75.0 (C-2’), 80.2 (C-4’), 90.0 (C-1’), 115.3 (C-4a), 115.5 (C-5),
124.8 (C-6), 128.2 (2 C, CÀ Ph), 131.5 (2 C, CÀ Ph), 131.9 (C-4Ph), 133.3
°
was stirred at À 10 C for 15 min. i-PrMgCl·LiCl (1.3 M in THF,
1.52 mL, 1.98 mmol, 1.1 eq.) was added dropwise slowly. When full
conversion of the SM was observed by TLC (1 h), 0.5 M aq. HCl
solution (5 mL) and EA (10 mL) were added. The layers were
separated, and the water layer was extracted twice more with EA
(2×10 mL). Then, organic layers were combined, dried over Na2SO4,
filtered, and evaporated. The residue was used directly for the next
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(C-1Ph), 150.0 (C-7a), 150.8 (C-2), 159.1 (C-4). HRMS (ESI): calculated
(mice were neither infected nor treated), non-treated (mice were
infected but treated with vehicle À 10% (v/v) Tween 80 in sterile
deionized water), and treatment (mice were infected and treated
with 14 and/or the reference BZ). When a detectable parasitemia
was observed at the onset of parasitemia (i.e., 5 dpi), the
corresponding treatments were initiated through oral administra-
tion. Compound 14 was administrated twice per day in 3 mice
groups, using 10-fold dose titration schedules with 3 concentra-
tions, namely 25, 2.5, 0.25 mg/kg, respectively. The reference drug
BZ was administered orally at 10 (suboptimal) and 100 mg/kg
(optimal dose) once per day, respectively. 14 was co-administered
for C18H19ClN3O3 ([M+H]+): 360.1109, found: 360.1112.
Biology
In vitro evaluation
All in vitro compound evaluations were performed as described
previously, including drug sensitivity assays on intracellular amasti-
gotes of the Tulahuen (β-galactosidase expressing) T. cruzi strain in
MRC-5 fibroblasts, Y-strain T. cruzi in primary mouse cardiac cells
and Y-strain bloodstream trypomastigote forms (BF).[25] Cytotoxicity,
as evaluated on MRC-5 fibroblasts and 2D primary mouse cardiac
cells, was performed as described.[25]
at 2.5 mg/kg (b.i.d) with BZ (once
a day) at 10 mg/kg for
consecutive 5 days, starting at parasitemia onset (5 dpi). All
compounds were formulated freshly before every administration.
5 μL of blood of each individual experimental mouse was obtained
via tail vein puncture and the number of parasites was counted
under a light microscope to determine the blood parasitemia until
15 dpi. The mortality was monitored daily up to 24 days after the
Three-dimensional cardiac cell cultures: cardiac spheroids were
obtained as reported.[45] For cardiotoxicity analyses, non-infected 3-
°
D cultures were incubated for 48 h at 37 C with increasing
consecutive 5 days treatment (34 dpi) and presented as
a
concentrations of BZ and compound 14 (up to 200 μM) and cellular
viability determined using PrestoBlue® (CC) tests.[45] For determi-
nation of anti-T. cruzi activity, 3-D cultures were infected with BF
(strain Y), using 20:1 parasite:host cell ratios. After 48 h, the cultures
were rinsed and either or not incubated with 10 μM compound 14
percentage of cumulative mortality (CM). For qPCR analysis, at 136
dpi, after euthanasia, 500 μL of animal blood was diluted in 1:2
volume of guanidine solution (guanidine-HCl 6 M/EDTA 0.2 M), and
heated for 90 sec. in boiling water. Guanidine-EDTA blood (GEB)
samples were processed using the High Pure PCR Template
Preparation kit, according to manufacturer’s instructions (Roche,
USA). At the final step of the protocol, DNA was eluted in 100 μL of
elution buffer. Quantitative Real-Time PCR Multiplex assays were
performed (40 cycles, threshold set at 0.01) for parasite detection of
the T. cruzi satellite nuclear DNA and the internal amplification
control-IAC (pZErO-2 plasmid containing an insert from the
Arabidopsis thaliana aquaporin gene), as described.[53] The standard
curves for absolute quantification were constructed with 1:10 serial
dilutions of total DNA obtained from a negative GEB sample spiked
with 105 parasite equivalents per milliliter of blood (par. eq./mL).
or BZ (corresponding to the EC90 value of the reference drug upon
[51]
°
intracellular forms) for 96 h at 37 C. To determine the parasite
load, infected spheroids were transferred to a tube for extraction of
total DNA using High Pure PCR Template Preparation Kit, according
to manufacturer’s instructions (Roche, USA). At the final step of the
protocol, DNA was eluted in 100 μL of elution buffer. The absolute
quantification of the parasite‘s DNA was performed in a ABI 7500
Fast Real Time PCR instrument (Applied Biosystems), using the Cruzi
1 (5’-ASTCGGCTGATCGTTTTCGA-3’) and Cruzi 2 (5’-AATTCCTCCAAG-
CAGCGGATA-3’) primers and Cruzi 3 probe (5’-FAM-CACACACTG
GACACCAANFQ-MGB-3’) targeting T. cruzi nuclear satellite DNA, as
reported[52] with minor modifications. qPCR were carried out with
5 μL DNA, using 10 μL FastStart Universal Probe Master Mix [2×]
(Roche), 750 nM primer Cruzi 1, 750 nM primer Cruzi 2 and 50 nM
probe Cruzi 3 (FAM/NFQ-MGB), in a final volume of 20 μL. In
parallel, quantification of cardiac cell DNA was performed using a
TaqMan assay targeting mouse glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH) (Cat. No. Mm99999915–g1, Applied Biosys-
Ethics statement
All animal studies were carried out in strict accordance with the
guidelines established by the FIOCRUZ Committee of Ethics for the
Use of Animals (CEUA L038-2017).
°
tems). Cycling conditions were a first step of 10 minutes at 95 C
°
°
followed by 40 cycles at 95 C for 15 sec. and 58 C for 1 min.
Standard curves were constructed with 1:10 serial dilutions of total
DNA obtained from a pool of 7.5×104 spheroids spiked with 105
epimastigotes. Thus, T. cruzi and cardiac cell quantities were
quantified and expressed as parasite equivalents/cardiac cell.[45]
Supporting Information
Copies of 1H, 13C and 19F NMR spectra of compounds 77, 21, 27,
28, 29, 37, 40, 41, 45, 47 and H-13C gHMBC and 2D NOESY
1
spectra of compounds 67, 14, 16, 20, 77, 78, 79, 25 and 54 can
Microsomal stability assays
be found in the Supporting Information.
The in vitro metabolic stability assays were performed exactly as
described elsewhere.[51]
Abbreviations
In vivo evaluation
BSA
CD
NTD
N,O-bis(trimethylsilyl)acetamide
Chagas disease
neglected tropical disease
For the in vivo evaluation, male Swiss Webster mice were obtained
from the Fundação Oswaldo Cruz (FIOCRUZ) animal facilities (ICTB
/FIOCRUZ, Rio de Janeiro, Brazil). Mice were housed at a maximum
T. cruzi Trypanosoma cruzi
°
of 6 per cage and kept in a standard room at 20 to 24 C under a
TMSOTf trimethylsilyl trifluoromethanesulfonate
TPPTS trisodium 3-bis(3-sulfonatophenyl)
phosphanylbenzenesulfonate
12 h/12 h light/dark cycle. The animals were provided with
sterilized water and chow ad libitum. Mice (18 to 23 g) were
infected by intraperitoneal (i.p.) route with 104 bloodstream
trypomastigotes of the Y strain of T. cruzi. Age-matched non-
infected mice were maintained under identical conditions.[25] These
mice were divided into eight experimental groups: non-infected
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[22] F. Hulpia, G. D. Campagnaro, M. Scortichini, K. Van Hecke, L. Maes, H. P.
Acknowledgements
de Koning, G. Caljon, S. Van Calenbergh, Eur. J. Med. Chem. 2019, 164,
689–705.
C.L. thanks the China Scholarship Council (CSC) for a PhD
scholarship (grant number 201708440366). F.H. thanks the
Flanders Research Foundation (FWO) for a postdoctoral fellowship
(1226921 N). The present work received funding from FWO (S.V.C.,
G.C., L.M.; project number G013118N). S.V.C. thanks the Hercules
Foundation (project AUGE/17/22 “Pharm-NMR”). The present study
was supported by grants from Fundação Carlos Chagas Filho de
Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ),
Conselho Nacional Desenvolvimento científico e Tecnológico
(CNPq), Fundação Oswaldo Cruz, PAEF/CNPq/Fiocruz, CAPES.
M.N.C.S. and O.C.M. are research fellows of CNPq and CNE and
JCNE researcher. The authors thank the excellent technical
assistance of Marcos Meuser Batista, Roberson Donola Girão, Dr
Cristian França da Silva, Dr Dara Denise da Gama Jaen Batista, Dr
Gabriel M de Oliveira, Dr Ana Lia Mazzetti, Natascha Van Pelt, An
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Conflict of Interest
[33] F. Hulpia, G. D. Campagnaro, K. J. Alzahrani, I. A. Alfayez, M. A. Ungogo,
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Keywords: 7-deazapurine nucleosides
relationships · Trypanosoma cruzi · in vivo efficacy
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Manuscript received: February 27, 2021
Accepted manuscript online: April 15, 2021
Version of record online: ■■■, ■■■■
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C. Lin, L. Ferreira de Almeida Fiuza,
C. Cardoso Santos, Dr. D. Ferreir-
a Nunes, Dr. O. Cruz Moreira, J.
Bouton, I. Karalic, Prof. L. Maes,
Prof. G. Caljon, Dr. F. Hulpia*,
Prof. M. de Nazaré C. Soeiro*, Prof. S.
Van Calenbergh*
1 – 24
A SAR investigation on the pyrimi-
dine part of a previously identified 7-
aryl-7-deazapurine nucleoside
provided the most promising 6-
methyl analogue against T. cruzi. A
series of 7-aryl-6-methyl derivatives
was prepared and evaluated, giving
rise to several analogues with sub-mi-
cromolar antitrypanosomal activity.
Compound 14 was found to be meta-
bolically stable and active against T.
cruzi after oral dose of 25 mg/kg b.i.d
in an acute mouse model.
6-Methyl-7-Aryl-7-Deazapurine
Nucleosides as Anti-Trypanosoma
cruzi Agents: Structure-Activity
Relationship and in vivo Efficacy