Synthesis, cytostatic, antimicrobial, and anti-HCV activity of 6-substituted 7-(het)aryl-7-deazapurine ribonucleosides

Article abstract of DOI:10.1021/jm4018948

A series of 80 7-(het)aryl- and 7-ethynyl-7-deazapurine ribonucleosides bearing a methoxy, methylsulfanyl, methylamino, dimethylamino, methyl, or oxo group at position 6, or 2,6-disubstituted derivatives bearing a methyl or amino group at position 2, were

Full text of DOI:10.1021/jm4018948

Article
pubs.acs.org/jmc
Synthesis, Cytostatic, Antimicrobial, and Anti-HCV Activity of
6‑Substituted 7‑(Het)aryl-7-deazapurine Ribonucleosides
Petr Naus,† Olga Caletkova,† Petr Konecny,‡ Petr Dzubak,‡ Katerina Bogdanova,‡ Milan Kolar,‡
̌
́
̌
́
̌
́
̌
́
́
̌
Jana Vrbkova,‡ Lenka Slavetínska,† Eva Tloust’ova,† Pavla Perlíkova,† Marian Hajduch,‡
́
̌
́
̌
́
́
́
́
and Michal Hocek*,†,§
†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences & IOCB Research
Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic
‡Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in
̌ ́
Olomouc, Hnevotínska 5, CZ-775 15 Olomouc, Czech Republic
§Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
S
* Supporting Information
ABSTRACT: A series of 80 7-(het)aryl- and 7-ethynyl-7-deazapurine ribonucleo-
sides bearing a methoxy, methylsulfanyl, methylamino, dimethylamino, methyl, or
oxo group at position 6, or 2,6-disubstituted derivatives bearing a methyl or amino
group at position 2, were prepared, and the biological activity of the compounds
was studied and compared with that of the parent 7-(het)aryl-7-deazaadenosine
series. Several of the compounds, in particular 6-substituted 7-deazapurine
derivatives bearing a furyl or ethynyl group at position 7, were significantly
cytotoxic at low nanomolar concentrations whereas most were much less potent or
inactive. Promising activity was observed with some compounds against
Mycobacterium bovis and also against hepatitis C virus in a replicon assay.
(het)aryl, ethynyl, and halogen groups, as well as 7-
unsubstituted derivatives, were studied.
INTRODUCTION
■
Purine nucleosides and their analogues display a wide range of
antiviral and antitumor activities,1 but there is still a room for
development of new nucleoside-based anticancer drugs2
targeting malignant versus nonmalignant cells as well as drugs
for the treatment of resistant tumors. We have previously
observed significant cytotoxic activity among certain 6-(het)-
arylpurine3 and 6-(het)aryl-7-deazapurine4 nucleosides. More
recently we discovered 7-hetaryl-7-deazaadenine ribonucleo-
sides 15 (Chart 1) with nanomolar cytostatic activities against
leukemia and cancer cell lines and found6 that some sugar-
modified compounds derived from 1 are still cytotoxic but less
so than the parent compounds. The mechanism of action of 1
has not yet been fully explored. We previously reported5 that
these nucleosides interfere with RNA synthesis, although their
triphosphates are only weak inhibitors of RNA polymerases. To
analyze the SAR of these compounds and their mode of action,
we have now prepared and tested a series of base-modified
derivatives of 1 (Chart 1). The first group (compounds 2−7)
was characterized by replacement of the amino group (H-bond
donor) at position 6 by diverse H-bond acceptors (methoxy,
methylsulfanyl, dimethylamino, oxo), methylamino (which can
serve as either a donor or acceptor), and the isosteric but
nonpolar methyl group. The second group of derivatives
(compounds 8−11) were 2,6-disubstituted 7-deazaguanine, 7-
deazadiaminopurine, and 2-methyl-7-deazaadenine nucleosides.
In each class, a series of 7-substituted derivatives bearing
As a group, these derivatives may be viewed as analogues of
the antibiotic tubercidin.7 Many other tubercidin analogues
have been studied,8 including 7-halogen, 7-carboxamido, and 7-
alkynyl derivatives,9 some of which showed cytotoxic,
antiparasitic, and antiviral activities. Several related 6-
substituted analogues of tubercidin10 were likewise cytostatic,
whereas 6-substituted analogues of toyocamycin possessed anti-
HCV11 activity. 6-Methyl-7-deazapurine ribonucleoside 6h12
was also recently reported to have an antiviral effect.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic strategy for preparation of 6-
monosubstituted 7-(hetaryl)-7-deazapurine nucleosides 2−7
was based on aqueous-phase Suzuki cross-coupling reactions of
the corresponding 6-substituted 7-iodo-7-deazapurine ribonu-
cleosides 13−16. These key intermediates were obtained in
good yields from 4-chloro-5-iodo-7-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine 1213 through nu-
cleophilic substitution at position 6 with NaOMe, NaSMe,
CH3NH2, or (CH3)2NH (Scheme 1). Concomitant nucleo-
philic debenzoylation of the sugar proceeded in all cases under
the reaction conditions, affording directly free nucleosides.
Received: December 10, 2013
© XXXX American Chemical Society
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Chart 1. Previously Reported Cytostatic Nucleosides 1 and
the Design of Derivatives in This Study
Scheme 2. Synthesis of 7-Halogenated 6-Methyl-7-
deazapurine Ribonucleosides
a
a
Conditions: (i) 1. iPrMgCl·LiCl, THF, −10 °C. 2. aq workup
(quant); (ii) AlMe3, Pd(PPh3)4, THF, 100 °C (92%); (iii) MeONa,
MeOH, rt (97%); (iv) for 19: NBS, DMF, rt (66%); for 20: NIS,
DMF, rt (58%).
was converted to 6-methyl derivative 18 by palladium-catalyzed
reaction with trimethylaluminum in 92% yield, and the follow-
up Zemplen deprotection furnished free 6-methyl-7-deazapur-
́
ine ribonucleoside 6h12 in 97% yield. Compound 6h was then
brominated by N-bromosuccinimide (NBS) in DMF to give 7-
bromo derivative 19 in 66% yield. Analogous 7-iodo derivative
20 was prepared by treatment with N-iodosuccinimide (NIS),
but the reaction was more sluggish, giving 20 in 58% yield with
18% recovery of unreacted 6h.
a
Scheme 1. Nucleophilic Substitution at Position 6
The halogenated nucleoside intermediates 13−16 and 19
served as starting points for the synthesis of the target 7-
(het)aryl 7-deazapurine ribonucleosides via the Suzuki−
Miyaura reactions with (het)aryl boronic acids (Scheme 3).
Scheme 3. Suzuki Cross-Coupling Reactions of 13−16 and
19
a
Conditions: (i) for 13: MeONa/MeOH; for 14: MeSNa/EtOH; for
15: MeNH2/EtOH; for 16: aq Me2NH/dioxane.
The synthesis of 7-halogenated 6-methyl-7-deazapurine
ribonucleosides was more complicated due to the need for
cross-coupling methylation at position 6, which on 6-chloro-7-
iodo-7-deazapurine nucleoside 12 would not proceed chemo-
selectively. Attempted “classical” SNAr methylation of 12 using
either Wittig reagent14 or enolates of 1,3-dicarbonyl com-
pounds (acetylacetone,15 ethyl acetoacetate16) failed to give
desired product. Attempted methylation via iron-catalyzed17
reaction of 12 with methylmagnesium chloride also failed
resulting in deiodination, presumably by iodine−magnesium
exchange. Thus we decided to first selectively hydrodeiodinate
12 followed by C-6 methylation and reintroduction of the
halogen at position 7 (Scheme 2). Deiodination of 12 was
achieved by iodine−magnesium exchange reaction using
Knochel’s Turbo-Grignard reagent18 (iPrMgCl·LiCl) and
subsequent hydrolysis of magnesium intermediate to give 6-
chloro-7-deazapurine riboside 1719 in almost quantitative yield
without need for purification. This process is a useful alternative
route to 6-chloro-7-deazapurine ribonucleosides, because the
glycosylation of the 7-unsubstituted 6-chloro-7-deazapurine
base is troublesome,19,20 whereas that of 7-halogenated 6-
The Pd-catalyzed reactions were conducted under aqueous
conditions21 in the presence of TPPTS and sodium carbonate.
The reactions in a water/acetonitrile 2:1 mixture at 100 °C
proceeded smoothly within 3 h to give the desired library of 30
(5 × 6) 7-(het)aryl derivatives 2a−f, 3a−f, 4a−f, 5a−f, and
6a−f in moderate to good yields. In most cases, except for
methylamino derivatives 4a−f, the reaction mixtures also
chloro-7-deazapurines is convenient under Vorbruggen con-
̈
ditions using commercialy available 1-O-acetyl-2,3,5-tri-O-
benzoyl-β-D-ribofuranose.13 The 6-chloro-7-deazapurine 17
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a
contained minor byproducts of concomitant reductive deiodi-
nation, 2h, 3h, 5h, or 6h, which were easily removed by
chromatography. For methylsulfanyl derivatives 3a, 3c, and 3f,
the cross-coupling reactions did not reach full conversions of
starting iodide 14, and separation from the products required
crystallization. This could be due to sulfur poisoning of the
palladium catalytic species, resulting in a decreased reaction rate
and hence premature decomposition of labile hetarylboronic
acids22 (furan23 and thiophene24 boronic acids are prone to fast
protodeboronation).
Ethynyl derivatives 2g−6g were prepared from the
corresponding iododeazapurines 13−16 and 20 by Sonogashira
reaction with (trimethylsilyl)acetylene followed by cleavage of
the trimethylsilyl group by treatment with K2CO3 in methanol
(Scheme 4). It is noteworthy that methyl derivative 6g had to
be synthesized from a more reactive iododeazapurine 20
because the bromo derivative 19 failed to react under these
conditions.
Scheme 6. Preparation of 7-(Het)aryl-7-deazainosines 7a−f
a
Conditions: (a) NaI, TMSCl, MeCN, rt.
such as 13 to yield 7-iodo-7-deazainosine. Instead, cleavage of
methyl ethers 2a−f with in situ generated iodotrimethylsilane25
(from TMSCl and NaI) in acetonitrile was found to give 7-
(het)aryl-7-deazainosines 7a−f in good yields. Only with 2-
furyl derivative 7a, shorter reaction time and modified workup
had to be used due to decomposition during standard
conditions.
Synthetic pathway toward 2-methyl-7-deazaadenosines began
with the preparation of 2-methyl-7-deazahypoxanthine 21 as
reported26 (Scheme 7). Deoxychlorination of 21 via a slight
a
Scheme 4. Preparation of Ethynyl Derivatives 2g−6g
a
Scheme 7. Preparation of 2-Methyl-7-deazaadenosine 9h
a
Conditions: (i) 1. trimethylsilylacetylene, PdCl2(PPh3)2, CuI, NEt3,
DMF, 100 °C. 2. K2CO3, MeOH, rt. The yields are given as overall
isolated yields over the two steps.
For comparison of biological activity, 7-unsubstituted 7-
deazapurine ribonucleosides 2h−4h10 were prepared from 6-
chlorodeazapurine nucleoside 17 by substitution/deprotection
(Scheme 5). Dimethylamino compound 5h10 was isolated as a
byproduct from Suzuki reactions in the synthesis of compounds
5a−e (Scheme 3).
Scheme 5. Preparation of 7-Unsubstituted 7-Deazapurine
Ribonucleosides 2h−4h
a
a
Conditions: (i) POCl3, N,N-dimethylaniline, BTEACl, MeCN, 100
°C (82%); (ii) NIS, CH2Cl2, rt (87%); (iii) 1. BSA, MeCN. 2.
TMSOTf, 50 °C (35%); (iv) aq NH3 (27% w/w), dioxane, 120 °C
(92%); (v) H2, Pd/C, NEt3, DMF, rt (97%).
modification of literature conditions27 produced 6-chloro-2-
methyl-7-deazapurine 2228 (82%) whose further iodination
using NIS provided 23 in 87% yield. Nucleobase 23 was
converted to nucleoside 25 via a silyl-Hilbert−Johnson reaction
with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose 24 under
a
Conditions: (i) for 2h: MeONa/MeOH; for 3h: MeSNa/EtOH; for
Vorbruggen conditions. Thus, silylation of 23 with BSA and
̈
4h: MeNH2/EtOH.
treatment with 24 in the presence of TMSOTf catalyst afforded
crystalline nucleoside 25 in 35% yield. The formation of 25 was
slow due to extensive decomposition of 24 at the usual reaction
temperature of 80 °C. Therefore, the reaction was run at only
50 °C with 2 equiv of 24 being added to the reaction in three
portions. Essentially this is a protocol developed by Seela for
analogous ribosylation of 7-halogenated 2-pivaloylamino-6-
chloro-7-deazapurine29 (we also used these conditions to
obtain iodides 27 and 28, vide infra). Amination of 25 with
benzoyl deprotection by treatment with ammonia gave 7-iodo-
2-methyl-7-deazaadenosine 26 in 92% yield. Reduction of iodo
7-(Het)aryl-7-deazainosines (7a−f) synthesized directly from
7-iodo-7-deazainosine13 using Pd-catalyzed Suzuki or Stille
cross-couplings were obtained in very low yields. Therefore,
7a−f were prepared by O-demethylation of 6-methoxy
derivatives 2a−f (Scheme 6). Application of the Seela’s basic
hydrolysis13 (aq KOH, dioxane, reflux) to 7-(het)aryl-6-
methoxy-7-deazapurine ribosides 2a−f surprisingly failed to
give even trace of desired products 7a−f, although this protocol
perfectly works for demethylation of 7-halogenated congeners
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a
derivative 26 by catalytic hydrogenation on Pd/C furnished 2-
methyl-7-deazaadenosine 9h.30
Scheme 10. Synthesis of 7-Deazaguanosines 11a−f
7-(Het)aryl-substituted 2-amino-7-deazaadenosines 8a−f, 2-
methyl-7-deazaadenosines 9a−f, and 2-amino-6-methoxy-7-
deazapurine ribonucleosides 10a−f were synthesized once
again by aqueous Suzuki cross-couplings from iodo derivatives
26−2829 with the coresponding boronic acids (Scheme 8). The
Scheme 8. Suzuki Cross-Coupling Reactions of 26−28
a
Conditions: (i) NaI, TMSCl, MeCN, rt.
reactions from 7-iodo-7-deazaguanosine was found to be of
little use because of low conversions.
Biological Activity Profiling. Cytotoxic/Cytostatic Activ-
ity. In vitro cytotoxic/cytostatic activity of final nucleosides 2−
26 was initially evaluated against six cell lines derived from
human solid tumors including lung (A549 cells) and colon
(HCT116 and HCT116p53−/−) carcinomas, as well as
leukemia cell lines (CCRF-CEM, CEM-DNR, K562, and
K562-TAX) and, for comparison, nonmalignant BJ and
MRC-5 fibroblasts. Concentrations inhibiting the cell growth
by 50% (IC50) were determined using a quantitative metabolic
staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT)31 following a 3-day treatment.
In addition, the antiproliferative effect was tested against a
human T-lymphoblastic leukemia line CCRF-CEM, promyelo-
cytic leukemia HL-60, and cervical carcinoma HeLa S3 growing
in liquid suspension. Cell viability was determined following a
3-day incubation using 2,3-bis(2-methoxy-4-nitro-5-sulfophen-
yl)-2H-tetrazolium-5-carboxanilide (XTT) assay.32
reactions leading to 2,6-diamino compounds 8a−f and 2-
methyl derivatives 9a−f proceeded cleanly without significant
side reactions, but those leading to 2-amino-6-methoxy 7-
deazapurines 10a−f were accompanied by reductive dehaloge-
nation of iodide 28 (<14% as determined by NMR), and this
byproduct was removed during reverse phase chromatography.
7-Ethynyl nucleosides 9g and 10g were synthesized by the
Sonogashira reaction of 7-iodo-7-deazapurine nucleosides 26 or
28 followed by desilylation (Scheme 9).
Table 1 summarizes the cytotoxic activities of 2−26 in
comparison with the previously published5 7-hetaryl-7-
deazaadenosines 1a and 1c. In the 6-substituted series, the 7-
(2-furyl) derivatives 2a, 3a, 4a, and 6a were the most potent,
showing low nanomolar cytostatic effects similar to those of 1a
and 1c, whereas the IC50 values of the bulkier 6-dimethylamino
derivative 5a were in micromolar range. These derivatives,
however, were also the most cytotoxic to fibroblasts, suggesting
a nonspecific cytotoxic effect. On the other hand, analogous 7-
(3-furyl)-, 7-(2-thienyl)-, and 7-(3-thienyl) derivatives 2−6b,
2−6c, and 2−6d were less active or inactive. The 6-methoxy
derivatives 2b−d still showed significant activities but with low
toxicity to fibroblasts showing promissing in vitro therapeutic
index. The more bulky 7-phenyl 2−6e and 7-benzofuryl 2−6f
derivatives were generally inactive. On the other hand, 6-
substituted 7-deazapurine nucleosides bearing a small ethynyl
group at position 7 (2−6g) displayed considerable activity
against several cell lines; in particular, the 6-methylsulfanyl
derivative had a low nanomolar IC50 value against most tumor
cells but had little effect on fibroblasts. The 7-unsubstituted
derivatives 2−5h (analogous to tubercidin) did not show a
significant cytotoxic effect with the exception of 6-methyl
derivative 6h12, which was the most cytotoxic compound of the
entire set to both cancer cells and fibroblasts. The 7-
deazahypoxanthine derivatives 7, as well as all the 2-substituted
derivatives 8−11, were devoid of any significant cytotoxic
effect. The unusual specific effect of 2-furyl and ethynyl
substitution on cytostatic activity was the major difference from
the rest of the 7-deazaadenosine 1a−g, where all compounds
bearing five-membered heterocyclic substituents exerted similar
low nanomolar effects. On the other hand, the SAR analysis of
a
Scheme 9. Sonogashira Reaction of 26 and 28
a
Conditions: (i) 1. trimethylsilylacetylene, PdCl2(PPh3)2, CuI, NEt3,
DMF, 100 °C. 2. K2CO3, MeOH, rt. The yields are given as overall
isolated yields over the two steps.
Similarly to the synthesis of inosines 7a−f, 7-(het)aryl 7-
deazaguanosines 11a−f were obtained from the corresponding
6-methoxy compounds 10a−f by demethylation using TMSCl/
NaI reagent (Scheme 10). The products 11a−f were isolated in
only moderate unoptimized yields, as they were somewhat
unstable during prolonged treatment under reaction and
workup conditions. Again similarly to inosines 7a−f, attempted
direct synthesis of 7-deazaguanosines 11a−f by cross-coupling
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a
Table 2. Anti-HCV Activities of Selected Compounds
replicon 1B
replicon 2A
replicon 1B
replicon 2A
EC50 (μM)
CC50 (μM)
EC50 (μM)
CC50 (μM)
EC50 (μM)
CC50 (μM)
EC50 (μM)
CC50 (μM)
2a
2b
2c
2d
2e
2f
0.02
0.33
>44
>44
>44
>44
>44
>44
>44
38.58
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
6.62
0.03
8.28
>44
>44
>44
10.23
>44
0.05
>44
>44
>44
>44
29.64
>44
>44
>44
0.14
>44
13.57
>44
>44
1.35
>44
>44
>44
>44
>44
0.10
0.97
>44
0.15
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
43.60
6b
6c
4.43
6.67
>44
>44
>44
>44
>44
>44
0.23
20.85
35.61
>44
>44
>44
>44
>44
>44
>44
>44
39.25
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
0.12
13.81
28.17
>44
>44
>44
n.t.
>44
1.08
6d
6e
4.47
>44
0.55
34.83
6.76
>44
20.98
2.64
6f
40.20
21.60
0.02
8.75
20.74
22.34
>44
6g
0.23
2g
3a
3b
3c
3d
3e
3f
0.18
6h
20
19
7b
7d
7f
0.02
0.07
2.16
4.72
12.01
4.54
5.42
10.93
23.27
8.03
14.35
7.21
17.45
b
8a
12.45
21.29
10.03
5.58
n.t.
3g
3h
14
4a
4b
4f
0.21
8b
8c
n.t.
n.t.
29.89
19.21
0.06
n.t.
n.t.
8f
11.49
>44
18.25
n.t.
>44
>44
39.10
n.t.
9d
9f
28.73
6.14
35.93
2.05
9g
8.00
4g
4h
5a
5b
5c
5d
5e
5f
0.35
26
10a
10b
10c
10d
10f
11b
11c
2.45
>44
n.t.
>44
n.t.
0.82
2.44
1.49
19.74
6.07
n.t.
n.t.
7.88
n.t.
n.t.
16.76
38.41
26.88
19.20
0.07
14.45
8.98
n.t.
n.t.
20.66
40.71
n.t.
>44
>44
n.t.
25.66
18.80
a
5g
5h
16
6a
Compounds not listed in this table were inactive (EC50 and CC50 >44
μM). n.t. = not tested.
b
0.27
0.69
0.07
the entire panel of compounds shows that the H-bond-
donating NH2 group at position 6 can be replaced by H-bond
acceptor groups or even by an isosteric but nonpolar methyl
group, with some analogues retaining high cytotoxic activity.
Multidrug resistance is a major therapeutic problem in cancer
chemotherapy. It is associated with overexpression of drug
transporters, typically multidrug resistance protein 1 (mrp-1),
and multidrug resistance gene 1 (mdr-1), which transport drugs
extracellularly in an ATP-dependent manner.33 To assess
efficacy of our compounds in multidrug resistant cancers, we
have tested their cytotoxic potency in drug-resistant sublines
derived from chemosensitive CCRF-CEM T-lymphoblastic and
K562 myeloid leukemia cells. CEM daunorubicine-resistant
(CEM-DNR-bulk) and K562 paclitaxel-resistant (K562-TAX)
lines were originally prepared by increasing the concentration
of cytotoxic compounds and then characterization. While
CEM-DNR-bulk cells stabily overexpress mrp-1, K562-TAX
cells express only mdr-1 gene.34 Unfortunately, the vast
majority of cytotoxic compounds with submicromolar activity
showed decreased potency in drug-resistant sublines over-
expressing multidrug transporters. Similarly to the multidrug
resistance phenomenon, functional inactivation of p53 tumor
suppressor is associated with poor prognosis and therapeutic
response in many cancers.35 Therefore, we have evaluated in
vitro therapeutic potency of nucleoside derivatives using
isogenic colorectal cancer cells bearing wild type (HCT116)
or deleted p53 gene (HCT116p53−/−). Interestingly, the
compounds were equally toxic for tumor cells bearing wild-type
or null alleles of p53 gene, indicating potential therapeutic
activity in p53 mutant tumors.
Antimicrobial Activity. Antibacterial activity was tested
against Enterococcus faecalis CCM 4224, Staphylococcus aureus
CCM 3953, Escherichia coli CCM 3954, Pseudomonas aeruginosa
CCM 3955, methicillin-resistant staphylococci (Staphylococcus
aureus MRSA 4591 and Staphylococcus haemolyticus A/16568),
multiresistant strains of Escherichia coli C/16702, and
Pseudomonas aeruginosa A/16575. None of the tested
compounds exerted any activity in these assays. None of the
compounds showed antifungal activity against Candida albicans,
Candida parapsilosis, Candida crusei, and Candida tropicalis. On
the othe hand, significant antimycobacterial effects of several
compounds against Mycobacterium bovis were found (Table 1,
last column). The most active (but also most cytotoxic) was 6-
methyl-7-deazapurine ribonucleoside 6h. Bulky and nonpolar 7-
benzofuryl-6-methyldeazapurine nucleoside 6f and 2,6-diami-
no-7-thienyldeazapurine 8c showed low micromolar antimyco-
bacterial activity with no cytotoxicity, which makes them
promising candidates for further development as antitubercu-
lotics.
Antiviral Activity. Compounds were also tested in Huh-7
cells harboring subgenomic reporter replicons derived from
HCV subtypes 1B and 2A. Partial inhibition of the replicon
reporter was observed with most of the compounds (Table 2).
Activity correlated with cytotoxicity in most cases, suggesting
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that the activity detected by the replicon assay was a reflection
of interference with host target(s). Several 2,6-disubstituted 7-
deazapurine nucleosides (e.g., 8f, 26, 10a, 10c, and 10f)
showed low micromolar anti-HCV effects without significant
cytotoxicity.
Cell Cycle Analysis. Cell cycle analysis of selected
compounds with low-micromolar cytotoxicity in cancer cells
(2a, 2b, 2c, 2d, 3a, 4a, 5a, 5d, 5g, 13, and 16) was performed
on CCRF-CEM lymphoblasts at the concentration correspond-
ing to the IC50 after 24 h of incubation and compared to that of
the previously published compound 1c (Table 3). Some of the
(compared to thienyl), resulting in lower expected log P values.
However, this difference suggests that the mechanism of action
of compounds 1a−d may not necessarily be the same as that of
2−6. All the 7-deazahypoxanthine derivatives, as well as all the
2-substituted derivatives, were inactive. The compounds
inhibited RNA synthesis in treated cells and induced apoptosis
at their cytotoxic concentrations. Nonetheless, molecular
target(s) identification requires additional investigation.
None of the title compounds showed antibacterial or
antifungal effects, but some compounds (i.e., 6f and 8c)
showed promising activity against Mycobacterium bovis. Most of
the compounds also exerted a significant (and mostly
nonspecific) anti-HCV effect in a replicon assay accompanied
by cytotoxicity, but some derivatives showed a low micromolar
anti-HCV effect without in vitro toxicity.
Table 3. Summary of Cell Cycle, Apoptosis (sub-G1),
Mitosis (pH3+), RNA (BrU+), and DNA (BrdU+) Synthesis
a
Analysis
% of total cell populations
EXPERIMENTAL SECTION
■
sub-G1
G1
S
G2/M pH3Ser10+ BrdU+ BrU+
General. NMR spectra were recorded on a 400 MHz (1H at 400
MHz, 13C at 100.6 MHz), a 500 MHz (1H at 500 MHz, 13C at 125.7
MHz), or a 600 MHz (1H at 600 MHz, 13C at 150.9 MHz)
spectrometer. Melting points were determined on a Kofler block and
control
1c
9.6
18.6
6.8
41
44.8
39.2
44.3
44.2
48.7
46.5
45.3
45.8
53.6
47.4
46.8
49.2
46.7
14.2
10.1
12.9
12.3
9.8
1.4
1.4
1.4
1.6
1.1
2.9
0.7
1.4
1.5
0.7
1.3
0.9
1.3
50.1
52.4
45.3
44.2
38.6
44.4
29.5
47.2
50.6
36.1
43.4
38.9
48.7
43.3
15.2
13.8
0.7
0.1
0.5
5.0
1.0
0.4
0.7
8.6
0.2
4.8
50.6
42.8
43.5
41.5
40.8
43.5
42.2
31
2a
20
2b
2c
8.5
are uncorrected. Optical rotations were measured at 25 °C, and [α]D
values are given in 10−1 deg cm2 g−1. High resolution mass spectra
were measured using electrospray ionization. Reverse-phase high
performance flash chromatography (HPFC) was performed on KP-
C18-HS columns with Biotage SP1 system, and the sample was loaded
as a solution in pure water or in water/DMSO (5:1) mixture. FT IR
spectra were measured on a Bruker Alpha spectrometer using the ATR
technique. The purity of all tested compounds was confirmed by
HPLC analysis and was >95%.
7.5
2d
3a
75
12.7
11.3
12.1
15.4
11.2
14.7
12.9
13.5
7.9
4a
10.6
10.2
6.8
5a
5d
5g
41.4
38.5
37.9
39.8
8.9
13
6.0
5-Iodo-4-methylsulfanyl-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (14). A mixture of 4-chloro-5-iodo-7-(2,3,5-tri-O-benzoyl-
β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine 1213 (5.37 g, 7.42
mmol) and sodium thiomethoxide (1.1 g, 15.7 mmol) in EtOH (150
mL) was stirred at rt for 4 h. The volatiles were removed in vacuo, and
the residue was coevaporated several times with water and crystallized
from water to furnish 14 (2.94 g, 94%) as a white solid. Mp 217−219
°C. [α]20D −69.8 (c 0.242, DMSO). 1H NMR (500 MHz, DMSO-d6):
2.63 (s, 3H, CH3S); 3.55 (ddd, 1H, Jgem = 12.0 Hz, J5′a,OH = 5.5 Hz,
16
9.4
a
In CCRF-CEM cells treated with selected nucleosides at IC50 for 24
h.
compounds induced major apoptosis (2d) as early as after 24 h.
However, the major mechanism of action was inhibition of
RNA synthesis (BrU incorporation), and all tested compounds
(1c−16) were more active than parent compound 1c. DNA
synthesis was barely inhibited by compounds 3a, 5d, and 13,
and this most probably occurred via action at the level of
cellular signaling and metabolic pathways. Cell cycle alterations
(G1, S, G2/M) were not observed in treated cells under
specified experimental conditions.
J
J
5′a,4′ = 4.0 Hz, H-5′a); 3.63 (ddd, 1H, Jgem = 12.0 Hz, J5′b,OH = 5.2 Hz,
5′b,4′ = 4.0 Hz, H-5′b); 3.91 (bq, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.6 Hz, H-
4′); 4.09 (m, 1H, H-3′); 4.36 (m, 1H, H-2′); 5.09 (bt, 1H, JOH,5′a
=
J
OH,5′b′ = 5.4 Hz, OH-5′); 5.18 (bd, 1H, JOH,3′ = 3.9 Hz, OH-3′); 5.40
(bs, 1H, OH-2′); 6.15 (d, 1H, J1′,2′ = 6.2 Hz, H-1′); 7.99 (s, 1H, H-6);
8.62 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6): 12.13 (CH3S);
53.17 (C-5); 61.56 (CH2-5′); 70.62 (CH-3′); 74.38 (CH-2′); 85.54
(CH-4′); 86.77 (CH-1′); 117.20 (C-4a); 130.87 (CH-6); 148.74 (C-
7a); 150.86 (CH-2); 161.82 (C-4). IR (ATR): ν 3386, 3132, 1556,
1449, 1220, 1113, 1066, 954, 492 cm−1. MS (ESI) m/z 424 (M + H),
446 (M + Na). HRMS (ESI) for C12H15IN3O4S [M + H] calcd:
423.98279; found: 423.98216. Anal. (C12H14IN3O4S·3/4H2O): C, H,
N.
CONCLUSIONS
■
Synthesis of a large series (80 derivatives) of diverse 6-
substituted 7-(het)aryl- or 7-ethynyl-7-deazapurine ribonucleo-
sides was developed, and a systematic biological activity
screening was performed in comparison with the parent 7-
(het)aryl-7-deazaadenosine series published previously.5 Several
title nucleosides, in particular 6-methoxy-, 6-methylsulfanyl-, 6-
methylamino-, and 6-methyl-7-(2-furyl)-deazapurine nucleo-
sides 2a, 3a, 4a, and 6a, as well as some 7-ethynyl derivatives
3g, 5g, and 6g, have been found to possess cytostatic effects at
low nanomolar concentrations with the potency comparable to
1a and 1c. On the other hand, most other derivatives were
much less active compared to analogously substituted 7-
deazaadenosines 1. A striking difference was found in activities
of certain 7-(2-furyl)- and 7-(2-thienyl)-7-deazapurine deriva-
tives (compare highly active derivatives 3a−6a with inactive
derivatives 3c−6c, whereas the parent compounds 1a and 1c
showed comparably high cytostatic/cytotoxic effects). This can
be attributed to the higher polarity of the furyl substituent
5-Iodo-4-methylamino-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (15). A mixture of 4-chloro-5-iodo-7-(2,3,5-tri-O-benzoyl-
β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine 1213 (2.4 g, 3.31
mmol) in methylamine (33 wt % in absolute EtOH, 25 mL) was
stirred in a pressure tube at 100 °C for 5 h. After cooling, the mixture
was evaporated to dryness and purified by column chromatography
(SiO2, 3% MeOH in chloroform) to afford 15 (1.24 g, 92%) as a white
solid. Crystallization from MeOH afforded colorless needles. Mp 218−
223 °C. [α]20D −61.3 (c 0.419, DMSO). 1H NMR (500 MHz, DMSO-
d6): 3.02 (d, 3H, JCH3,NH = 4.7 Hz, CH3); 3.52 (ddd, 1H, Jgem = 12.0
Hz, J5′a,OH = 6.1 Hz, J5′a,4′ = 3.8 Hz, H-5′a); 3.61 (ddd, 1H, Jgem = 12.0
Hz, J5′b,OH = 5.1 Hz, J5′b,4′ = 3.8 Hz, H-5′b); 3.88 (bq, 1H, J4′,5′a = J4′,5′b
= J4′,3′ = 3.5 Hz, H-4′); 4.06 (td, 1H, J3′,2′ = J3′,OH = 4.9 Hz, J3′,4′ = 3.1
Hz, H-3′); 4.35 (td, 1H, J2′,1′ = J2′,OH = 6.4 Hz, J2′,3′ = 5.1 Hz, H-2′);
5.12 (d, 1H, JOH,3′ = 4.7 Hz, OH-3′); 5.16 (dd, 1H, JOH,5′a = 6.1 Hz,
JOH,5′b′ = 5.1 Hz, OH-5′); 5.31 (d, 1H, JOH,2′ = 6.5 Hz, OH-2′); 6.03
H
dx.doi.org/10.1021/jm4018948 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(d, 1H, J1′,2′ = 6.3 Hz, H-1′); 6.44 (q, 1H, JNH,CH3 = 4.7 Hz, NH); 7.66
(s, 1H, H-6); 8.19 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
27.90 (CH3NH); 51.25 (C-5); 61.74 (CH2-5′); 70.70 (CH-3′); 74.10
(CH-2′); 85.37 (CH-4′); 86.99 (CH-1′); 103.73 (C-4a); 127.17 (CH-
6); 149.70 (C-7a); 152.07 (CH-2); 156.62 (C-4). IR (ATR): ν 3387,
3323, 1603, 1550, 1305, 1090, 866, 596 cm−1. MS (ESI) m/z 407 (M
+ H), 429 (M + Na). HRMS (ESI) for C12H16IN4O4 [M + H] calcd:
407.02162; found: 407.02111. Anal. (C12H15IN4O4): C, H, N.
= 3.2 Hz, H-3′); 4.38 (td, 1H, J2′,1′ = J2′,OH = 6.3 Hz, J2′,3′ = 5.1 Hz, H-
2′); 5.10 (t, 1H, JOH,5′a = JOH,5′b = 5.5 Hz, OH-5′); 5.18 (d, 1H, JOH,3′
=
4.8 Hz, OH-3′); 5.38 (d, 1H, JOH,2′ = 6.4 Hz, OH-2′); 6.18 (d, 1H, J1′,2′
= 6.2 Hz, H-1′); 8.06 (s, 1H, H-6); 8.66 (s, 1H, H-2). 13C NMR
(125.7 MHz, DMSO-d6): 20.74 (CH3); 54.05 (C-5); 61.61 (CH2-5′);
70.67 (CH-3′); 74.30 (CH-2′); 85.52 (CH-4′); 86.66 (CH-1′); 118.04
(C-4a); 131.65 (CH-6); 150.27 (C-7a); 151.17 (CH-2); 159.76 (C-4).
IR (ATR): ν 1584, 1562, 1347, 1335, 1207, 1122, 1078, 1059, 1031,
1000, 893 cm−1. MS (ESI) m/z 392 (M + H), 414 (M + Na). HRMS
(ESI) for C12H15N3O4I [M + H] calcd: 392.01018; found: 392.01014.
Anal. (C12H14N3O4I): C, H, N.
4-Dimethylamino-5-iodo-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (16). A mixture of 4-chloro-5-iodo-7-(2,3,5-tri-O-
benzoyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine 1213 (5.79
g, 8 mmol), aq dimethylamine (40% w/w, 10 mL) in dioxane (10
mL) was stirred in a steel bomb at 120 °C for 8 h. After cooling, the
mixture was evaporated to dryness and the residue was coevaporated
several times with water. Crystallization from water afforded 16 (2.84
g, 84%) as white needles. Mp 195−197 °C. [α]D −39.0 (c 0.290,
5-(Furan-2-yl)-4-methoxy-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (2a). An argon-purged mixture of 1313 (794 mg, 1.95
mmol), furan-2-boronic acid (328 mg, 2.93 mmol), Na2CO3 (620 mg,
5.85 mmol), Pd(OAc)2 (22 mg, 98 μmol), and TPPTS (136 mg, 0.24
mmol) in water/MeCN (2:1, 10 mL) was stirred at 100 °C for 3 h.
After cooling, the mixture was neutralized using aq HCl (1 M) and
concentrated to dryness in vacuo and the residue purified by reverse
phase HPFC (C-18, 0→100% MeOH in water). Repurification by
column chromatography (SiO2, 2.5% MeOH in chloroform) furnished
2a (507 mg, 75%) as a beige solid, which was crystallized from water/
1
DMSO). H NMR (500 MHz, DMSO-d6): 3.16 (s, 6H, (CH3)2N);
3.54 (ddd, 1H, Jgem = 11.9 Hz, J5′a,OH = 5.8 Hz, J5′a,4′ = 3.8 Hz, H-5′a);
3.62 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH = 5.2 Hz, J5′b,4′ = 3.9 Hz, H-5′b);
3.89 (bq, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.5 Hz, H-4′); 4.07 (td, 1H, J3′,2′
=
J3′,OH = 5.0 Hz, J3′,4′ = 3.2 Hz, H-3′); 4.36 (td, 1H, J2′,1′ = J2′,OH = 6.3
Hz, J2′,3′ = 5.1 Hz, H-2′); 5.13 (t, 1H, JOH,5′a = JOH,5′b = 5.5 Hz, OH-
5′); 5.13 (d, 1H, JOH,3′ = 4.7 Hz, OH-3′); 5.33 (d, 1H, JOH,2′ = 6.4 Hz,
OH-2′); 6.11 (d, 1H, J1′,2′ = 6.3 Hz, H-1′); 7.86 (s, 1H, H-6); 8.24(s,
1H, H-2). 13C NMR (125.7 MHz, DMSO-d6): 43.28 ((CH3)2N);
53.85 (C-5); 61.66 (CH2-5′); 70.65 (CH-3′); 74.12 (CH-2′); 85.36
(CH-4′); 86.77 (CH-1′); 106.62 (C-4a); 129.62 (CH-6); 150.46
(CH-2); 152.13 (C-7a); 160.31 (C-4). IR (ATR): ν 1580, 1536, 1422,
1222, 1127, 1060, 1013, 947, 760, 600 cm−1. MS (ESI) m/z 421 (M +
H), 443 (M + Na). HRMS (ESI) for C13H18N4O4I [M + H] calcd:
421.03672; found: 421.03663. Anal. (C13H17N4O4I): C, H, N.
1
MeOH. Mp 155−157 °C. [α]D −78.2 (c 0.317, DMSO). H NMR
(500 MHz, DMSO-d6): 3.58 (dd, 1H, Jgem = 11.9 Hz, J5′a,4′ = 3.6 Hz,
H-5′a); 3.65 (dd, 1H, Jgem = 11.9 Hz, J5′b,4′ = 3.7 Hz, H-5′b); 3.94 (bq,
1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.5 Hz, H-4′); 4.11 (s, 3H, CH3O); 4.13 (dd,
1H, J3′,2′ = 5.0 Hz, J3′,4′ = 3.2 Hz, H-3′); 4.44 (bt, 1H, J2′,1′ = J2′,3′ = 5.6
Hz, H-2′); 5.14 (bs, 1H, OH-5′); 5.18 (bs, 1H, OH-3′); 5.38 (bs, 1H,
OH-2′); 6.22 (d, 1H, J1′,2′ = 6.2 Hz, H-1′); 6.57 (dd, 1H, J4,3 = 3.3 Hz,
J4,5 = 1.9 Hz, H-4-furyl); 6.93 (dd, 1H, J3,4 = 3.3 Hz, J3,5 = 0.9 Hz, H-3-
furyl); 7.67 (dd, 1H, J5,4 = 1.9 Hz, J5,3 = 0.9 Hz, H-5-furyl); 7.98 (s,
1H, H-6); 8.47 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
54.05 (CH3O); 61.65 (CH2-5′); 70.79 (CH-3′); 74.41 (CH-2′); 85.51
(CH-4′); 87.08 (CH-1′); 101.50 (C-4a); 107.17 (C-5); 107.42 (CH-
3-furyl); 111.92 (CH-4-furyl); 121.09 (CH-6); 141.81 (CH-5-furyl);
148.27 (CH-2-furyl); 151.52 (CH-2); 152.54 (C-7a); 162.76 (C-4). IR
(ATR): ν 1590, 1563, 1120, 1080, 1063, 1012, 795, 744 cm−1. MS
(ESI) m/z 348 (M + H), 370 (M + Na). HRMS (ESI) for
C16H18N3O6 [M + H] calcd: 348.11901; found: 348.11899. Anal.
(C16H17N3O6·1/4H2O): C, H, N.
5-Bromo-4-methyl-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (19). To a stirred solution of 6h12 (1061 mg, 4 mmol) in
DMF (10 mL) was added dropwise N-bromosuccinimide (748 mg, 4.2
mmol) in DMF (4 mL) within 3 min. The mixture was stirred at rt for
40 min followed by addition of solid Na2SO3 (50 mg) and dilution
with water (66 mL). This solution was separated (in portions
according to column capacity) using reverse-phase HPFC (C-18, 0→
100% MeOH in water) to obtain a crude product, which after
crystallization from iPrOH afforded 19 (913 mg, 66%) as white solid.
Mp 188−189 °C. [α]D −61.1 (c 0.229, DMSO). 1H NMR (500 MHz,
5-(Furan-2-yl)-4-methylsulfanyl-7-(β-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (3a). An argon-purged mixture of 14 (313
mg, 0.74 mmol), furan-2-boronic acid (124 mg, 1.11 mmol), Na2CO3
(235 mg, 2.22 mmol), Pd(OAc)2 (8 mg, 36 μmol) and TPPTS (53
mg, 0.093 mmol) in water/MeCN (2:1, 4 mL) was stirred at 100 °C
for 1 h. After cooling the mixture was neutralized using aq HCl (1 M)
and concentrated by evaporation. The residue was coevaporated with
silica and purified by column chromatography (SiO2, 1→3% MeOH in
CHCl3). Repurification by reverse-phase HPFC (C-18, 0→100%
MeOH in water) and crystallization from water/MeOH afforded 3a
(123 mg, 46%) as a yellowish solid. Mp 150−152 °C. [α]20D −70.5 (c
0.237, DMSO). 1H NMR (500 MHz, DMSO-d6): 2.59 (s, 3H, CH3S);
3.56 (ddd, 1H, Jgem = 12.0 Hz, J5′a,OH = 5.6 Hz, J5′a,4′ = 3.8 Hz, H-5′a);
3.65 (ddd, 1H, Jgem = 12.0 Hz, J5′b,OH = 5.3 Hz, J5′b,4′ = 3.9 Hz, H-5′b);
DMSO-d6): 2.85 (s, 3H, CH3); 3.55 (ddd, 1H, Jgem = 11.9 Hz, J5′a,OH
5.5 Hz, J5′a,4′ = 4.0 Hz, H-5′a); 3.64 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH
=
=
5.2 Hz, J5′b,4′ = 4.1 Hz, H-5′b); 3.91 (bq, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.6
Hz, H-4′); 4.10 (td, 1H, J3′,2′ = J3′,OH = 4.9 Hz, J3′,4′ = 3.3 Hz, H-3′);
4.38 (td, 1H, J2′,1′ = J2′,OH = 6.2 Hz, J2′,3′ = 5.1 Hz, H-2′); 5.10 (t, 1H,
JOH,5′a = JOH,5′b = 5.4 Hz, OH-5′); 5.19 (d, 1H, JOH,3′ = 4.8 Hz, OH-3′);
5.40 (d, 1H, JOH,2′ = 6.3 Hz, OH-2′); 6.21 (d, 1H, J1′,2′ = 6.2 Hz, H-1′);
8.06 (s, 1H, H-6); 8.70 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-
d6): 21.14 (CH3); 61.58 (CH2-5′); 70.64 (CH-3′); 74.33 (CH-2′);
85.54 (CH-4′); 86.66 (CH-1′); 88.18 (C-5); 115.68 (C-4a); 126.38
(CH-6); 149.75 (C-7a); 151.65 (CH-2); 159.54 (C-4). IR (ATR): ν
1593, 1563, 1345, 1211, 1119, 1092, 1058, 1032, 968, 921, 787 cm−1.
MS (ESI) m/z 344 (M + H), 366 (M + Na). HRMS (ESI) for
C12H15N3O4Br [M + H] calcd: 344.02405; found: 344.02407. Anal.
(C12H14N3O4Br): C, H, N.
3.93 (bq, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.6 Hz, H-4′); 4.12 (td, 1H, J3′,2′
=
J3′,OH = 5.0 Hz, J3′,4′ = 3.3 Hz, H-3′); 4.42 (td, 1H, J2′1′ = J2′,OH = 6.2
Hz, J2′,3′ = 5.1 Hz, H-2′); 5.10 (t, 1H, JOH,5′a = JOH,5′b = 5.4 Hz, OH-
5′); 5.19 (d, 1H, JOH,3′ = 4.8 Hz, OH-3′); 5.42 (d, 1H, JOH,2′ = 6.3 Hz,
OH-2′); 6.23 (d, 1H, J1′,2′ = 6.1 Hz, H-1′); 6.60 (dd, 1H, J4,3 = 3.3 Hz,
J4,5 = 1.9 Hz, H-4-furyl); 6.71 (dd, 1H, J3,4 = 3.3 Hz, J3,5 = 0.9 Hz, H-3-
furyl); 7.78 (dd, 1H, J5,4 = 1.9 Hz, J5,3 = 0.9 Hz, H-5-furyl); 8.01 (s,
1H, H-6); 8.68 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
12.21 (CH3S); 61.58 (CH2-5′); 70.68 (CH-3′); 74.42 (CH-2′); 85.54
(CH-4′); 86.92 (CH-1′); 106.61 (C-5); 109.18 (CH-3-furyl); 111.68
(CH-4-furyl); 113.63 (C-4a); 125.13 (CH-6); 142.90 (CH-5-furyl);
146.82 (C-2-furyl); 148.92 (C-7a); 150.91 (CH-2); 161.69 (C-4). IR
(ATR): ν 3166, 2937, 2903, 1547, 1446, 1062, 1029, 976, 594 cm−1.
MS (ESI) m/z 364 (M + H), 386 (M + Na). HRMS (ESI) for
C16H18N3O5S [M + H] calcd: 364.09672; found: 364.09609.
5-Iodo-4-methyl-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (20). To a stirred solution of 6h (796 mg, 3 mmol) in
DMF (7 mL) was added dropwise N-iodosuccinimide (709 mg, 3.15
mmol) in DMF (3 mL) within 3 min. The mixture was stirred at rt for
16 h followed by addition of solid Na2SO3 (50 mg) and dilution with
water (50 mL). This solution was directly separated (in portions
according to column capacity) using reverse phase HPFC (C-18, 0→
100% MeOH in water) to afford 20 (685 mg, 58%) as a white solid
after crystallization from water. HPFC also recovered starting 6h (145
1
mg, 18%). Mp 187−188 °C. [α]D −58.3 (c 0.365, DMSO). H NMR
(500 MHz, DMSO-d6): 2.88 (s, 3H, CH3); 3.55 (ddd, 1H, Jgem = 11.9
Hz, J5′a,OH = 5.6 Hz, J5′a,4′ = 3.9 Hz, H-5′a); 3.64 (ddd, 1H, Jgem = 11.9
Hz, J5′b,OH = 5.3 Hz, J5′b,4′ = 4.0 Hz, H-5′b); 3.91 (btd, 1H, J4′,5′a = J4′,5′b
= 3.9 Hz, J4′,3′ = 3.2 Hz, H-4′); 4.09 (td, 1H, J3′,2′ = J3′,OH = 5.0 Hz, J3′,4′
5-(Furan-2-yl)-4-methylamino-7-(β-D-ribofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidine (4a). An argon-purged mixture of 15 (300 mg, 0.74
I
dx.doi.org/10.1021/jm4018948 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mmol), furan-2-boronic acid (124 mg, 1.11 mmol), Na2CO3 (235 mg,
2.22 mmol), Pd(OAc)2 (8 mg, 36 μmol), and TPPTS (53 mg, 0.093
mmol) in water/MeCN (2:1, 4 mL) was stirred at 100 °C for 1 h.
After cooling, the mixture was neutralized using aq HCl (1 M) and
concentrated to dryness. The residue was coevaporated with silica and
purified by column chromatography (SiO2, 1→3% MeOH in CHCl3)
to afford product 4a (220 mg, 86%) as a white solid, which was
2′); 6.26 (d, 1H, J1′,2′ = 6.1 Hz, H-1′); 6.62 (dd, 1H, J4,3 = 3.3 Hz, J4,5 =
1.9 Hz, H-4-furyl); 6.66 (dd, 1H, J3,4 = 3.3 Hz, J3,5 = 0.9 Hz, H-3-
furyl); 7.80 (dd, 1H, J5,4 = 1.9 Hz, J5,3 = 0.9 Hz, H-5-furyl); 8.07 (s,
1H, H-6); 8.71 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
23.03 (CH3); 61.63 (CH2-5′); 70.71 (CH-3′); 74.31 (CH-2′); 85.50
(CH-4′); 86.79 (CH-1′); 106.79 (C-5); 108.50 (CH-3-furyl); 111.75
(CH-4-furyl); 115.36 (C-4a); 125.82 (CH-6); 142.93 (CH-5-furyl);
147.62 (C-2-furyl); 150.82 (C-7a); 151.38 (CH-2); 159.71 (C-4). IR
(ATR): ν 1571, 1438, 1348, 1201, 1120, 1080, 1031, 968, 892, 792,
736, 637 cm−1. MS (ESI) m/z 332 (M + H), 354 (M + Na). HRMS
(ESI) for C16H18N3O5 [M + H] calcd: 332.12410; found: 332.12406.
Anal. (C16H17N3O5·3/4H2O): C, H, N.
crystallized from water/MeOH. Mp 114−117 °C. [α]20 −70.6 (c
D
0.299, DMSO). 1H NMR (500 MHz, DMSO-d6): 3.04 (d, 3H, JCH3,NH
= 4.8 Hz, CH3); 3.54 (dm, 1H, Jgem = 12.0 Hz, H-5′a); 3.64 (dm, 1H,
Jgem = 11.9 Hz, H-5′b); 3.91 (q, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.6 Hz, H-
4′); 4.10 (td, 1H, J3′,2′ = J3′,OH = 4.9 Hz, J3′,4′ = 3.3 Hz, H-3′); 4.41 (td,
1H, J2′1′ = J2′,OH = 6.3 Hz, J2′,3′ = 5.2 Hz, H-2′); 5.18 (d, 1H, JOH,3′
=
5-Ethynyl-4-methoxy-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (2g). An argon-purged mixture of 13 (407 mg, 1 mmol),
PdCl2(PPh3)2 (35 mg, 0.05 mmol), CuI (19 mg, 0.1 mmol),
trimethylsilylacetylene (1.4 mL, 10 mmol), and triethylamine (0.5
mL) was stirred in DMF (2 mL) at rt for 16 h. The volatiles were
removed in vacuo, and the residue was coevaporated several times with
EtOH/toluene and loaded on silica by coevaporation. Column
chromatography (SiO2, 0→1.5% MeOH in CHCl3) afforded
trimethylsilylethynyl derivative contaminated by triethylammonium
iodide. This material was directly deprotected by treatment with
K2CO3 (207 mg, 1.5 mmol) in MeOH (5 mL) at rt for 5 h, followed
by coevaporation with silica and final column chromatography (SiO2,
2.5% MeOH in CHCl3) afforded 2g (268 mg, 88% in two steps),
which was crystallized from MeOH. Mp 205−207 °C. [α]D −78.4 (c
0.333, DMSO). 1H NMR (500 MHz, DMSO-d6): 3.56 (ddd, 1H, Jgem
= 11.9 Hz, J5′a,OH = 5.7 Hz, J5′a,4′ = 3.7 Hz, H-5′a); 3.65 (ddd, 1H, Jgem
= 11.9 Hz, J5′b,OH = 5.3 Hz, J5′b,4′ = 3.9 Hz, H-5′b); 3.92 (td, 1H, J4′,5′a
= J4′,5′b = 3.8 Hz, J4′,3′ = 3.5 Hz, H-4′); 4.06 (s, 3H, CH3O); 4.10 (td,
1H, J3′,2′ = J3′,OH = 5.0 Hz, J3′,4′ = 3.4 Hz, H-3′); 4.11 (d, 1H, JCH,6 = 0.4
Hz, C≡CH); 4.38 (td, 1H, J2′,1′ = J2′,OH = 6.2 Hz, J2′,3′ = 5.0 Hz, H-2′);
5.12 (t, 1H, JOH,5′a = JOH,5′b = 5.5 Hz, OH-5′); 5.18 (d, 1H, JOH,3′ = 4.9
4.8 Hz, OH-3′); 5.25 (m, 1H, OH-5′); 5.37 (d, 1H, JOH,2′ = 6.4 Hz,
OH-2′); 6.09 (d, 1H, J1′,2′ = 6.2 Hz, H-1′); 6.61 (dd, 1H, J4,3 = 3.3 Hz,
J4,5 = 1.9 Hz, H-4-furyl); 6.66 (dd, 1H, J3,4 = 3.3 Hz, J3,5 = 0.8 Hz, H-3-
furyl); 6.85 (q, 1H, JNH,CH3 = 4.8 Hz, NH); 7.76 (dd, 1H, J5,4 = 1.9 Hz,
J5,3 = 0.8 Hz, H-5-furyl); 7.82 (s, 1H, H-6); 8.22 (s, 1H, H-2). 13C
NMR (125.7 MHz, DMSO-d6): 28.24 (CH3NH); 61.92 (CH2-5′);
70.81 (CH-3′); 74.12 (CH-2′); 85.45 (CH-4′); 87.31 (CH-1′); 100.03
(C-4a); 105.59 (CH-3-furyl); 106.35 (C-5); 112.20 (CH-4-furyl);
120.52 (CH-6); 142.39 (CH-5-furyl); 148.79 (C-2-furyl); 150.34 (C-
7a); 152.27 (CH-2); 156.84 (C-4). IR (ATR): ν 3649, 2934, 1624,
1319, 1021, 585, 564 cm−1. MS (ESI) m/z 347 (M + H), 369 (M +
Na). HRMS (ESI) for C16H19N4O5 [M + H] calcd: 347.13554; found:
347.13496.
4-Dimethylamino-5-(furan-2-yl)-7-(β-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (5a). An argon-purged mixture of iodide 16
(420 mg, 1 mmol), furan-2-boronic acid (168 mg, 1.5 mmol), Na2CO3
(318 mg, 3 mmol), Pd(OAc)2 (11 mg, 49 μmol), and TPPTS (71 mg,
0.125 mmol) in water/MeCN (2:1, 5 mL) was stirred at 100 °C for 3
h. After cooling, the mixture was neutralized using aq HCl (1 M),
concentrated to dryness in vacuo and the residue was purified by
reverse phase HPFC (C-18, 0→100% MeOH in water). Repurification
by column chromatography (SiO2, 2.5% MeOH in chloroform)
furnished 5a (176 mg, 49%) as a beige foam. Reverse phase HPFC
also provided product of reduction 5h (73 mg, 25%). Mp 97−103 °C.
[α]D −42.8 (c 0.358, DMSO). 1H NMR (500 MHz, DMSO-d6): 2.84
(s, 6H, (CH3)2N); 3.54 (ddd, 1H, Jgem = 11.9 Hz, J5′a,OH = 6.0 Hz, J5′a,4′
= 3.8 Hz, H-5′a); 3.62 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH = 5.1 Hz, J5′b,4′
= 3.8 Hz, H-5′b); 3.90 (btd, 1H, J4′,5′a = J4′,5′b = 3.8 Hz, J4′,3′ = 3.2 Hz,
Hz, OH-3′); 5.41 (d, 1H, JOH,2′ = 6.3 Hz, OH-2′); 6.14 (d, 1H, J1′,2′
=
6.0 Hz, H-1′); 8.05 (s, 1H, H-6); 8.47 (s, 1H, H-2). 13C NMR (125.7
MHz, DMSO-d6): 54.04 (CH3O); 61.55 (CH2-5′); 70.63 (CH-3′);
74.45 (CH-2′); 77.17 (CCH); 81.80 (CCH); 85.56 (CH-4′);
87.31 (CH-1′); 94.82 (C-5); 105.16 (C-4a); 130.01 (CH-6); 151.43
(C-7a); 151.93 (CH-2); 162.95 (C-4). IR (ATR): ν 1596, 1574, 1061,
1005, 653, 604, 535 cm−1. MS (ESI) m/z 306 (M + H), 328 (M +
Na). HRMS (ESI) for C14H15N3O5Na [M + Na] calcd: 328.0904;
found: 328.0892. Anal. (C14H15N3O5·1/4H2O): C, H, N.
H-4′); 4.10 (m, 1H, H-3′); 4.42 (td, 1H, J2′,1′ = J2′,OH = 6.4 Hz, J2′,3′
=
5.1 Hz, H-2′); 5.14 (d, 1H, JOH,3′ = 4.8 Hz, OH-3′); 5.15 (dd, 1H,
JOH,5′a = 5.9 Hz, JOH,5′b = 5.1 Hz, OH-5′); 5.35 (d, 1H, JOH,2′ = 6.5 Hz,
OH-2′); 6.17 (d, 1H, J1′,2′ = 6.4 Hz, H-1′); 6.51 (dd, 1H, J3,4 = 3.2 Hz,
J3,5 = 0.9 Hz, H-3-furyl); 6.58 (dd, 1H, J4,3 = 3.2 Hz, J4,5 = 1.9 Hz, H-4-
furyl); 7.75 (dd, 1H, J5,4 = 1.9 Hz, J5,3 = 0.9 Hz, H-5-furyl); 7.75 (s,
1H, H-6); 8.25 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
39.44 ((CH3)2N); 61.73 (CH2-5′); 70.74 (CH-3′); 74.10 (CH-2′);
85.35 (CH-4′); 86.98 (CH-1′); 102.05 (C-4a); 106.87 (C-5); 107.61
(CH-3-furyl); 111.74 (CH-4-furyl); 122.92 (CH-6); 142.56 (CH-5-
furyl); 149.33 (C-2-furyl); 150.71 (CH-2); 152.01 (C-7a); 159.64 (C-
4). IR (ATR): ν 1574, 1515, 1450, 1420, 1410, 1203, 1120, 1077, 1058
cm−1. MS (ESI) m/z 361 (M + H), 383 (M + Na). HRMS (ESI) for
C17H21N4O5 [M + H] calcd: 361.15065; found: 361.15057.
5-(Furan-2-yl)-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-
4(3H)-one (7a). To a stirred slurry of 2a (139 mg, 0.40 mmol) and
NaI (300 mg, 2 mmol) in dry MeCN (5 mL) was slowly added
TMSCl (253 μL, 2 mmol), and the mixture was stirred at rt for 4 h.
The precipitate was filtered off, washed carefully with MeCN, and
dissolved in water, and pH of the solution was quickly adjusted to 7
using solid K2CO3. The mixture was evaporated to dryness, and
crystallization from water gave 7a (50 mg, 38%) as a beige solid.
Reverse phase HPFC (C-18, 0→100% MeOH in water) of the mother
liquors provided additional 7a (31 mg, 23%). Total yield of 7a was
1
61%. Mp 184−186 °C. [α]D −83.8 (c 0.216, DMSO). H NMR (500
MHz, DMSO-d6): 3.56 (ddd, 1H, Jgem = 11.8 Hz, J5′a,OH = 5.4 Hz, J5′a,4′
= 3.7 Hz, H-5′a); 3.63 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH = 5.3 Hz, J5′b,4′
= 3.7 Hz, H-5′b); 3.90 (q, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.5 Hz, H-4′);
4.09 (td, 1H, J3′,2′ = J3′,OH = 4.9 Hz, J3′,4′ = 3.3 Hz, H-3′); 4.35 (td, 1H,
5-(Furan-2-yl)-4-methyl-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (6a). An argon-purged mixture of 19 (258 mg, 0.75
mmol), furan-2-boronic acid (126 mg, 1.125 mmol), Na2CO3 (239
mg, 2.25 mmol), Pd(OAc)2 (8 mg, 35.6 μmol), and TPPTS (53 mg,
93 μmol) in water/MeCN (2:1, 5 mL) was stirred at 100 °C for 1 h.
After cooling, the mixture was neutralized using aq HCl (1 M),
concentrated to dryness in vacuo and the residue was purified by
reverse phase HPFC (C-18, 0→100% MeOH in water) to furnish 6a
J
2′,1′ = J2′,OH = 6.3 Hz, J2′,3′ = 5.1 Hz, H-2′); 5.08 (t, 1H, JOH,5′a = JOH,5′b
= 5.3 Hz, OH-5′); 5.15 (d, 1H, JOH,3′ = 4.8 Hz, OH-3′); 5.36 (d, 1H,
JOH,2′ = 6.4 Hz, OH-2′); 6.08 (d, 1H, J1′,2′ = 6.2 Hz, H-1′); 6.51 (dd,
1H, J4,3 = 3.3 Hz, J4,5 = 1.8 Hz, H-4-furyl); 7.37 (dd, 1H, J3,4 = 3.3 Hz,
J3,5 = 0.9 Hz, H-3-furyl); 7.60 (dd, 1H, J5,4 = 1.9 Hz, J5,3 = 0.9 Hz, H-5-
furyl); 7.68 (s, 1H, H-6); 7.96 (d, 1H, J2,NH = 3.8 Hz, H-2); 12.10 (d,
1H, JNH,2 = 3.7 Hz, NH). 13C NMR (125.7 MHz, DMSO-d6): 61.61
(CH2-5′); 70.75 (CH-3′); 74.60 (CH-2′); 85.41 (CH-4′); 86.93 (CH-
1′); 104.08 (C-4a); 108.41 (CH-3-furyl); 111.14 (C-5); 111.75 (CH-
4-furyl); 116.71 (CH-6); 141.42 (CH-5-furyl); 144.94 (CH-2); 148.68
(C-2-furyl); 148.82 (C-7a); 158.39 (C-4). IR (ATR): ν 1696, 1599,
1045, 788, 723 cm−1. MS (ESI) m/z 334 (M + H), 356 (M + Na).
1
(207 mg, 83%) as an orange foam. [α]D −75.3 (c 0.219, DMSO). H
NMR (500 MHz, DMSO-d6): 2.65 (s, 3H, CH3); 3.57 (bdt, 1H, Jgem
=
11.9 Hz, J5′a,OH = J5′a,4′ = 4.5 Hz, H-5′a); 3.66 (bdt, 1H, Jgem = 11.9 Hz,
J5′b,OH = J5′b,4′ = 4.4 Hz, H-5′b); 3.94 (bq, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.6
Hz, H-4′); 4.13 (m, 1H, H-3′); 4.45 (btd, 1H, J2′,1′ = J2′,OH = 6.2 Hz,
J2′,3′ = 5.3 Hz, H-2′); 5.10 (bt, 1H, JOH,5′a = JOH,5′b = 5.4 Hz, OH-5′);
5.20 (d, 1H, JOH,3′ = 4.8 Hz, OH-3′); 5.41 (d, 1H, JOH,2′ = 6.4 Hz, OH-
J
dx.doi.org/10.1021/jm4018948 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HRMS (ESI) for C15H15N3O6Na [M + Na] calcd: 356.0853; found:
356.0843. Anal. (C15H15N3O6·H2O): C, H, N.
Na). HRMS (ESI) for C12H15IN4O4Na [M + Na] calcd: 429.0030;
found: 429.0029. Anal. (C12H15N4O4I·H2O): C, H, N.
2,4-Diamino-5-(furan-2-yl)-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (8a). An argon-purged mixture of 2,4-diamino-5-iodo-7-
(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine 2729 (305 mg, 0.75
mmol), furan-2-boronic acid (126 mg, 1.125 mmol), Na2CO3 (239
mg, 2.25 mmol), Pd(OAc)2 (8 mg, 0.036 mmol), and TPPTS (53 mg,
0.093 mmol) in water/MeCN (2:1, 5 mL) was stirred at 100 °C for 3
h. After cooling, the mixture was neutralized using aq HCl (1 M) and
concentrated to dryness in vacuo, and the residue was purified by
reverse phase HPFC (C-18, 0→100% MeOH in water) to afford 8a
(209 mg, 80%), which was crystallized from water as white needles.
Mp 234−236 °C. [α]D −71.9 (c 0.313, DMSO). 1H NMR (500 MHz,
DMSO-d6): 3.52 (ddd, 1H, Jgem = 11.9 Hz, J5′a,OH = 6.0 Hz, J5′a,4′ = 4.1
Hz, H-5′a); 3.61 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH = 5.2 Hz, J5′b,4′ = 4.1
Hz, H-5′b); 3.84 (td, 1H, J4′,5′a = J4′,5′b = 4.1 Hz, J4′,3′ = 3.3 Hz, H-4′);
4.06 (td, 1H, J3′,2′ = J3′,OH = 4.9 Hz, J3′,4′ = 3.3 Hz, H-3′); 4.34 (td, 1H,
J2′,1′ = J2′,OH = 6.4, J2′,3′ = 5.2 Hz, H-2′); 5.08 (d, 1H, JOH,3′ = 4.6 Hz,
OH-3′); 5.18 (dd, 1H, JOH,5′a = 6.0 Hz, JOH,5′b = 5.2 Hz, OH-5′); 5.28
(d, 1H, JOH,2′ = 6.3 Hz, OH-2′); 5.77 (bs, 2H, NH2-2); 5.96 (d, 1H,
4-Chloro-5-iodo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (23). A
mixture of compound 2226 (4.51 g, 26.9 mmol) and N-
iodosuccinimide (6.7 g, 29.8 mmol) in dichloromethane (80 mL)
was stirred at rt for 4 h. The volatiles were removed under reduced
pressure, and the residue was crystallized from MeCN, affording 23
1
(6.89 g, 87%) as pinkish-orange needles. Mp 229−231 °C. H NMR
(500 MHz, DMSO-d6): 2.60 (s, 3H, CH3); 7.80 (d, 1H, J6,NH = 2.5 Hz,
H-6); 12.67 (bs, 1H, NH). 13C NMR (125.7 MHz, DMSO-d6): 25.07
(CH3); 51.58 (C-5); 113.55 (C-4a); 133.02 (CH-6); 150.62 (C-4);
152.49 (C-7a); 160.04 (C-2). IR (ATR): ν 1607, 1550, 1463, 1402,
1245, 1196, 964, 897, 808, 779, 632, 609, 573 cm−1. MS (ESI) m/z
294 (M + H), 316 (M + Na). HRMS (ESI) for C7H6N3ClI [M + H]
calcd: 293.92894; found: 293.92895.
4-Chloro-5-iodo-2-methyl-7-(2,3,5-tri-O-benzoyl-β-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidine (25). To a slurry of iodide 23 (3.39
g, 11.55 mmol) in dry MeCN (60 mL) was added N,O-
bis(trimethylsilyl)acetamide [BSA] (3.44 mL, 13.87 mmol), and the
mixture was stirred at rt for 20 min (clear solution was formed). Then
TMSOTf (4.2 mL, 23.24 mmol) was added, 1-O-acetyl-2,3,5-tri-O-
benzoyl-β-D-ribofuranose 24 (11.67 g, 23.13 mmol) was introduced in
three portions (3 × 3.89 g, once per 8 h), and the mixture was stirred
at 50 °C for 24 h. After cooling, the mixture was treated with saturated
aq NaHCO3 (150 mL) and extracted with CHCl3 (300 mL, then 2 ×
50 mL). Combined organic phases were dried over MgSO4,
concentrated in vacuo, and coevaporated with silica. Column
chromatography (SiO2, dichloromethane→2.5% EtOAc in dichloro-
methane) afforded fractions containing product contaminated to
various extents with non-nucleoside sugar byproducts. Crystallization
of these fractions from hexane/EtOAc afforded 25 (3.03 g, 35%) as a
white crystalline solid. Mp 180−182 °C. 1H NMR (500 MHz, DMSO-
d6): 2.58 (s, 3H, CH3); 4.68 (dd, 1H, Jgem = 12.2 Hz, J5′a,4′ = 5.0 Hz, H-
5′a); 4.78 (dd, 1H, Jgem = 12.2 Hz, J5′b,4′ = 3.9 Hz, H-5′b); 4.87 (ddd,
1H, J4′,3′ = 5.7 Hz, J4′,5′a = 5.0 Hz, J4′,5′b = 3.9 Hz, H-4′); 6.23 (t, 1H,
J
1′,2′ = 6.4 Hz, H-1′); 6.47 (bs, 2H, NH2-4); 6.55−6.59 (m, 2H, H-3,4-
furyl); 7.40 (s, 1H, H-6); 7.73 (dd, 1H, J5,4 = 1.7 Hz, J5,3 = 1.0 Hz, H-
5-furyl). 13C NMR (125.7 MHz, DMSO-d6): 61.95 (CH2-5′); 70.77
(CH-3′); 73.50 (CH-2′); 84.91 (CH-4′); 86.35 (CH-1′); 93.16 (C-
4a); 104.60 (CH-3-furyl); 106.73 (C-5); 112.00 (CH-4-furyl); 116.99
(CH-6); 141.56 (CH-5-furyl); 149.70 (C-2-furyl); 154.12 (C-7a);
157.84 (C-4); 160.13 (C-2). IR (ATR): ν 1609, 1579, 1478, 1444,
1126, 1010, 875, 792, 738 cm−1. MS (ESI) m/z 348 (M + H), 370 (M
+ Na). HRMS (ESI) for C15H18N5O5 [M + H] calcd: 348.13025;
found: 348.13034. Anal. (C15H17N5O5): C, H, N.
4-Amino-5-(furan-2-yl)-2-methyl-7-(β-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (9a). An argon-purged mixture of 26 (305
mg, 0.75 mmol), furan-2-boronic acid (126 mg, 1.125 mmol), Na2CO3
(239 mg, 2.25 mmol), Pd(OAc)2 (8 mg, 0.036 mmol), and TPPTS
(53 mg, 0.093 mmol) in water/MeCN (2:1, 5 mL) was stirred at 100
°C for 3 h. After cooling, the mixture was neutralized using aq HCl (1
M) and concentrated to dryness in vacuo, and the residue was purified
by reverse phase HPFC (C-18, 0→100% MeOH in water) to afford
product 9a (203 mg, 78%) as a beige solid, which was crystallized from
water/MeOH to give beige needles. Mp 205−207 °C. [α]D −74.7 (c
J
3′,4′ = J3′,2′ = 5.9 Hz, H-3′); 6.30 (dd, 1H, J2′,3′ = 6.1 Hz, J2′,1′ = 4.5 Hz,
H-2′); 6.63 (d, 1H, J1′,2′ = 4.5 Hz, H-1′); 7.40−7.52 (m, 6H, H-m-Bz);
7.60−7.70 (m, 3H, H-p-Bz); 7.84−7.97 (m, 6H, H-o-Bz); 8.17 (s, 1H,
H-6). 13C NMR (125.7 MHz, DMSO-d6): 25.07 (CH3); 54.44 (C-5);
63.52 (CH2-5′); 70.93 (CH-3′); 73.82 (CH-2′); 79.34 (CH-4′); 86.89
(CH-1′); 114.83 (C-4a); 128.50 and 128.78 (C-i-Bz); 128.94, 128.96,
and 128.98 (CH-m-Bz); 129.31 (C-i-Bz); 129.35 and 129.57 (CH-o-
Bz); 133.68 (CH-6); 133.75, 134.08, and 134.19 (CH-p-Bz); 151.39
(C-7a); 151.48 (C-4); 160.86 (C-2); 164.66, 164.87, and 165.58
(CO). IR (ATR): ν 1729, 1720, 1593, 1415, 1267, 1248, 1212, 1103,
1071, 708 cm−1. MS (ESI) m/z 738 (M + H), 760 (M + Na). HRMS
(ESI) for C33H25ClIN3O7Na [M + Na] calcd: 760.0318; found:
760.0314.
1
0.269, DMSO). H NMR (500 MHz, DMSO-d6): 2.39 (s, 3H, CH3);
3.54 (ddd, 1H, Jgem = 12.0 Hz, J5′a,OH = 6.8 Hz, J5′a,4′ = 3.7 Hz, H-5′a);
3.64 (ddd, 1H, Jgem = 12.0 Hz, J5′b,OH = 4.8 Hz, J5′b,4′ = 3.9 Hz, H-5′b);
3.91 (td, 1H, J4′,5′a = J4′,5′b = 3.8 Hz, J4′,3′ = 2.9 Hz, H-4′); 4.09 (btd,
1H, J3′,2′ = J3′,OH = 4.9 Hz, J3′,4′ = 2.9 Hz, H-3′); 4.47 (td, 1H, J2′,1′
=
J2′,OH = 6.4 Hz, J2′,3′ = 5.1 Hz, H-2′); 5.15 (bd, 1H, JOH,3′ = 4.6 Hz,
OH-3′); 5.31 (bd, 1H, JOH,2′ = 6.5 Hz, OH-2′); 5.34 (dd, 1H, JOH,5′a
=
6.8 Hz, JOH,5′b = 4.8 Hz, OH-5′); 6.03 (d, 1H, J1′,2′ = 6.7 Hz, H-1′);
6.60 (dd, 1H, J4,3 = 3.3 Hz, J4,5 = 1.9 Hz, H-4-furyl); 6.65 (dd, 1H, J3,4
= 3.3 Hz, J3,5 = 0.9 Hz, H-3-furyl); 6.86 (s, 2H, NH2); 7.74 (s, 1H, H-
6); 7.77 (dd, 1H, J5,4 = 1.9 Hz, J5,3 = 0.9 Hz, H-5-furyl). 13C NMR
(125.7 MHz, DMSO-d6): 25.32 (CH3); 62.04 (CH2-5′); 70.96 (CH-
3′); 73.61 (CH-2′); 85.55 (CH-4′); 87.32 (CH-1′); 97.50 (C-4a);
105.19 (CH-3-furyl); 106.21 (C-5); 112.09 (CH-4-furyl); 120.39 (C-
6); 142.04 (CH-5-furyl); 148.99 (C-2-furyl); 151.93 (C-7a); 157.33
(C-4); 160.82 (C-2). IR (ATR): ν 1641, 1565, 1499, 1417, 1316,
1123, 1099, 1046, 1014, 983, 873, 792, 740 cm−1. MS (ESI) m/z 347
(M + H), 369 (M + Na). HRMS (ESI) for C16H19N4O5 [M + H]
calcd: 347.1350; found: 347.1340. Anal. (C16H18N4O5·1/2H2O): C, H,
N.
4-Amino-5-iodo-2-methyl-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (26). A suspension of nucleoside 25 (2.99 g, 4.05 mmol)
in dioxane (20 mL) and aq ammonia (27% w/w, 15 mL) was stirred in
a steel bomb at 120 °C for 24 h. After cooling, the volatiles were
evaporated in vacuo, the residue was purified by reverse phase HPFC
(C-18, 0→100% MeOH in water), and final recrystallization from
MeOH/water (1:1) afforded 26 (1.51 g, 92%) as long colorless
1
needles. Mp 236−239 °C. [α]D −57.0 (c 0.316, DMSO). H NMR
(500 MHz, DMSO-d6): 2.37 (s, 3H, CH3); 3.52 (ddd, 1H, Jgem = 12.0
Hz, J5′a,OH = 6.6 Hz, J5′a,4′ = 3.7 Hz, H-5′a); 3.60 (ddd, 1H, Jgem = 12.0
Hz, J5′b,OH = 4.7 Hz, J5′b,4′ = 3.8 Hz, H-5′b); 3.88 (td, 1H, J4′,5′a = J4′,5′b
= 3.7 Hz, J4′,3′ = 2.7 Hz, H-4′); 4.05 (td, 1H, J3′,2′ = J3′,OH = 4.8 Hz, J3′,4′
= 2.7 Hz, H-3′); 4.40 (td, 1H, J2′,1′ = J2′,OH = 6.6 Hz, J2′,3′ = 5.1 Hz, H-
2′); 5.12 (d, 1H, JOH,3′ = 4.5 Hz, OH-3′); 5.28 (d, 1H, JOH,2′ = 6.5 Hz,
OH-2′); 5.29 (dd, 1H, JOH,5′a = 6.6 Hz, JOH,5′b = 4.7 Hz, OH-5′); 5.97
(d, 1H, J1′,2′ = 6.8 Hz, H-1′); 6.60 (bs, 2H, NH2); 7.56 (s, 1H, H-6).
13C NMR (125.7 MHz, DMSO-d6): 25.35 (CH3); 51.79 (C-5); 61.93
(CH2-5′); 70.90 (CH-3′); 73.71 (CH-2′); 85.53 (CH-4′); 87.10 (CH-
1′); 101.42 (C-4a); 126.94 (CH-6), 151.19 (C-7a); 157.22 (C-4);
160.74 (C-2). IR (ATR): ν 1627, 1588, 1560, 1460, 1416, 1289, 1119,
1086, 1030, 786, 657 cm−1. MS (ESI) m/z 407 (M + H), 429 (M +
2-Amino-5-(furan-2-yl)-4-methoxy-7-(β-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (10a). An argon-purged mixture of 2-amino-
5-iodo-4-methoxy-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
2829 (822 mg, 2 mmol), furan-2-boronic acid (336 mg, 3 mmol),
Na2CO3 (636 mg, 6 mmol), Pd(OAc)2 (22 mg, 98 μmol), and TPPTS
(142 mg, 0.25 mmol) in water/MeCN (2:1, 10 mL) was stirred at 100
°C for 3 h. After cooling, the mixture was neutralized using aq HCl (1
M) and evaporated to dryness in vacuo, and the residue was purified
by reverse phase HPFC (C-18, 0→100% MeOH in water).
Repurification by column chromatography (SiO2, 2.5% MeOH in
K
dx.doi.org/10.1021/jm4018948 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
chloroform) furnished 10a (426 mg, 59%) as a beige solid, which was
crystallized from MeOH. Mp 204−206 °C. [α]D −63.7 (c 0.331,
DMSO). 1H NMR (500 MHz, DMSO-d6): 3.53 (ddd, 1H, Jgem = 11.9
Hz, J5′a,OH = 5.5 Hz, J5′a,4′ = 3.9 Hz, H-5′a); 3.59 (ddd, 1H, Jgem = 11.9
Hz, J5′b,OH = 5.3 Hz, J5′b,4′ = 3.9 Hz, H-5′b); 3.85 (btd, 1H, J4′,5′a = J4′,5′b
= 3.9 Hz, J4′,3′ = 3.1 Hz, H-4′); 3.99 (s, 3H, CH3O); 4.06 (btd, 1H,
ACKNOWLEDGMENTS
■
This work was supported by the Czech Science Foundation
(P207/11/0344), Technology Agency of the Czech Republic
(TE01020028), and by Gilead Sciences, Inc. The infrastructural
part of this project (Institute of Molecular and Translational
Medicine) was supported from the Operational Programme
Research and Development for Innovations (project CZ.1.05/
2.1.00/01.0030) and RVO:61388963. The authors thank Drs.
Joy Feng and Gina Bahador (Gilead Sciences, Inc.) for anti-
HCV testing.
J
3′,2′ = J3′,OH = 4.8 Hz, J3′,4′ = 3.0 Hz, H-3′); 4.34 (td, 1H, J2′,1′ = J2′,OH =
6.4 Hz, J2′,3′ = 5.1 Hz, H-2′); 5.04 (t, 1H, JOH,5′a = JOH,5′b = 5.4 Hz,
OH-5′); 5.07 (d, 1H, JOH,3′ = 4.4 Hz, OH-3′); 5.27 (d, 1H, JOH,2′ = 6.3
Hz, OH-2′); 6.03 (d, 1H, J1′,2′ = 6.5 Hz, H-1′); 6.36 (s, 2H, NH2);
6.51 (dd, 1H, J4,3 = 3.3 Hz, J4,5 = 1.8 Hz, H-4-furyl); 6.82 (dd, 1H, J3,4
= 3.3 Hz, J3,5 = 0.9 Hz, H-3-furyl); 7.43 (s, 1H, H-6); 7.59 (dd, 1H, J5,4
= 1.8 Hz, J5,3 = 0.9 Hz, H-5-furyl). 13C NMR (125.7 MHz, DMSO-d6):
53.28 (CH3O); 61.79 (CH2-5′); 70.83 (CH-3′); 73.73 (CH-2′); 84.95
(CH-4′); 85.89 (CH-1′); 93.78 (C-4a); 106.50 (CH-3-furyl); 107.54
(C-5); 111.71 (CH-4-furyl); 116.53 (C-6); 141.13 (CH-5-furyl);
149.21 (C-2-furyl); 155.61 (C-7a); 159.86 (C-2); 163.29 (C-4). IR
(ATR): ν 1626, 1597, 1575, 1486, 1442, 1411, 1308, 1213, 1043, 1004,
790, 739 cm−1. MS (ESI) m/z 363 (M + H), 385 (M + Na). HRMS
(ESI) for C16H18N4O6Na [M + Na] calcd: 385.11186; found:
385.11168. Anal. (C16H18N4O6·1/4H2O): C, H, N.
2-Amino-5-(furan-2-yl)-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidin-4(3H)-one (11a). To a stirred slurry of 10a (174 mg, 0.48
mmol) and NaI (360 mg, 2.4 mmol) in dry MeCN (5 mL) was slowly
added TMSCl (304 μL, 2.4 mmol), and the mixture was stirred at rt
for 4 h. The precipitate was filtered off, washed carefully with MeCN,
and dried. The solid was then dissolved in water/MeOH, the pH of
the solution was quickly adjusted to 7 using solid K2CO3, and the
mixture was evaporated to dryness. The residue was desalted by
reverse phase HPFC (C-18, 0→100% MeOH in water) and repurified
by column chromatography (SiO2, 6% MeOH in CHCl3) to obtain
11a (35 mg, 21%) as a yellowish foam. [α]D −66.5 (c 0.200, DMSO).
1H NMR (500 MHz, DMSO-d6): 3.52 (ddd, 1H, Jgem = 11.9 Hz, J5′a,OH
ABBREVIATIONS USED
■
BrdU, 5-bromo-2′-deoxyuridine; BrU, 5-bromouridine; HPFC,
high performance flash chromatography; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TCA,
trichloroacetic acid; TDA-1, tris(2-(2-methoxyethoxy)ethyl)-
amine; TMS, trimethylsilyl; TTPTS, triphenylphosphine-
3,3′,3″-tris(sulfonate); XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfo-
phenyl)-2H-tetrazolium-5-carboxanilide
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= 5.4 Hz, J5′a,4′ = 4.0 Hz, H-5′a); 3.58 (ddd, 1H, Jgem = 11.9 Hz, J5′b,OH
= 5.3 Hz, J5′b,4′ = 3.9 Hz, H-5′b); 3.82 (td, 1H, J4′,5′a = J4′,5′b = 3.9 Hz,
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999, 786 cm−1. MS (ESI) m/z 349 (M + H), 371 (M + Na). HRMS
(ESI) for C15H16N4O6Na [M + Na] calcd: 371.09621; found:
371.09615.
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