Synthesis and biological activity of benzo-fused 7-deazaadenosine analogues. 5- and 6-substituted 4-amino- or 4-alkylpyrimido[4,5-b]indole ribonucleosides

Article abstract of DOI:10.1016/j.bmc.2013.06.011

Two series of new 4-aminopyrimido[4,5-b]indole ribonucleosides bearing phenyl or hetaryl group at position 5 or 6 have been prepared by Suzuki or Stille cross-coupling reactions employing X-Phos ligand with (het)arylboronic acids or stannanes. A series of 4-substituted nucleosides has been also prepared by Pd-catalyzed cross-couplings or nucleophilic substitution. Some of these compounds displayed moderate antiviral activities against HCV and dengue viruses.

Full text of DOI:10.1016/j.bmc.2013.06.011

Bioorganic & Medicinal Chemistry 21 (2013) 5362–5372
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of benzo-fused 7-deazaadenosine
analogues. 5- and 6-substituted 4-amino- or 4-alkylpyrimido
[4,5-b]indole ribonucleosides
a
a
a
a
b
b
a,c,
⇑
´
Michal Tichy , Radek Pohl , Eva Tloušt’ová , Jan Weber , Gina Bahador , Yu-Jen Lee , Michal Hocek
a 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
b Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA
c Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of new 4-aminopyrimido[4,5-b]indole ribonucleosides bearing phenyl or hetaryl group at
position 5 or 6 have been prepared by Suzuki or Stille cross-coupling reactions employing X-Phos ligand
with (het)arylboronic acids or stannanes. A series of 4-substituted nucleosides has been also prepared by
Pd-catalyzed cross-couplings or nucleophilic substitution. Some of these compounds displayed moderate
antiviral activities against HCV and dengue viruses.
Received 8 April 2013
Revised 27 May 2013
Accepted 6 June 2013
Available online 17 June 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Nucleosides
Pyrimido[4,5-b]indoles
Suzuki and Stille cross-coupling
Anti-dengue virus activity
1. Introduction
Besides our recent study,7 some other types of pyrimido[4,5-
b]indole heterocycles were reported as tyrosine kinase inhibitors,8
Systematic research in modified purine and deazapurine
nucleosides in our group led to the discovery of two novel classes
of cytostatic 7-deazapurine ribonucleosides. 6-Hetaryl-7-deazapu-
rine ribonucleosides1 bearing either H (1) or F (2) at the position 7
and five-membered hetaryl groups (furyl, thienyl, etc.) at the posi-
tion 6 (Fig. 1) and 7-hetaryl-7-deazaadenosines 32 showed nano-
molar in vitro cytostatic activities against a broad panel of cancer
or leukemia cell lines. Both of these groups of compounds also ex-
erted some non-specific antiviral activities against HCV. On the
other hand, sugar-modified derivatives3 and phosphate prodrugs4
derived from 1 were less active or inactive. Also other groups have
reported biologically active deazapurine nucleosides as inhibitors
of Polio and dengue viruses.5,6 Very recently we have designed
and prepared the first generation of benzo-fused analogues of
7-deazapurines, 4-arylpyrimido[4,5-b]indoleribonucleosides,7 and
found their promising antiviral activity against dengue virus. Here
we report the synthesis and biological activity of other related ben-
zo-fused adenosine analogues, that is, derivatives of 4-amino-
R = aryl or hetaryl
Y
R
N
NH2
R
N
X
R
N
N
N
N
N
N
N
O
HO
HO
HO
O
O
OH OH
1, X = H
OH OH
3
OH OH
4
2
, X = F
nM cytostatic
This work:
alkyl
nM cytostatic
anti-Dengue
R
R
NH2
NH2
N
N
N
N
N
N
N
N
N
5-aryl-,
ribonucleosides.
4-amino-6-aryl-
and
4-alkylpyrimido[4,5-b]indole
HO
HO
HO
O
O
O
OH OH
OH OH
OH OH
⇑
Corresponding author. Tel.: +420 220183324; fax: +420 220183578.
E-mail address: hocek@uochb.cas.cz (M. Hocek).
Figure 1. Structures of recently discovered 7-deazapurine ribonucleoside cytostat-
ics and antivirals 1–4 and the target compounds for this study.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.011

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M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
R2
R2
vascular endothelial growth factor receptor-2 inhibitors, antiangio-
R1
N
R1
N
genic agents9 and microtubule disrupting compounds effective
Cl
N
NH2
against multidrug resistant cells.10
N
N
The pyrimido[4,5-b]indoles were previously prepared by Pd-
catalyzed intramolecular arylation,11 photolysis of tetrazoles12 or
by condensation of 2-amino-indole-3-carboxylates with formam-
ide or orthoformate.13 4-amino-6-methoxypyrimido[4,5-b]indole
20-deoxyribonucleoside was used14 as fluorescent label for single-
nucleotide polymorphism typing, for study of hole transport
through DNA15 and for the construction of DNA logic gates16 and
the corresponding nucleoside triphosphate was successfully incor-
porated to DNA by PCR.17
i)
N
O
BzO
HO
O
OBz OBz
OH OH
10: R1 = Cl; R2 = H
11: R1 = H; R2 = Cl
12: R1 = H; R2 = H;
13: R1 = Cl; R2 = H
: R1 = H; R2 = Cl
: R1 = H; R2 = H
14
15
Scheme 2. Reagents and conditions: (i) aq NH3/dioxane, 100 °C, 24 h.
2. Results and discussion
2.1. Chemistry
Cl
R
NH2
NH2
Our strategy for the synthesis of target 4-amino-6-hetaryl-, 4-
amino-5-hetaryl- and 4-substituted pyrimidoindole ribonucleo-
sides was based on palladium catalyzed cross-coupling reactions18
or nucleophilic substitutions of the corresponding chloro-deriva-
tives. Synthesis of the 4-chloro- and 4,6-dichloropyrimido[4,5-
b]indoles and their ribonucleosides 11 and 12 has been reported7
previously. The remaining key intermediate, 4,5-dichloropyrimi-
do[4,5-b]indole was built up from 2,3-dichloronitrobenzene 5
(Scheme 1) in analogy to previous work.7 The sequence started
by arylation of cyanoacetate followed by reduction of the nitro
group by Zn, cyclization, cyclocondensation with formamide and
reaction with POCl3 to give 4,5-dichloropyrimido[4,5-b]indole (9)
in 39% overall yield. The corresponding ribonucleoside 10 was then
prepared by glycosidation using N,O-bis(trimethylsilyl)acetamide
and TMSOTf.
N
N
i) or ii)
N
N
N
O
N
O
HO
HO
OH OH
OH OH
16a-16f
14
Scheme 3. Reagents and conditions: (i) RM, M = B(OH)2 (1.5 equiv), Pd(OAc)2
(0.05 equiv), X-Phos (0.1 equiv), K2CO3 (3 equiv), DMF, 100 °C, 12 h; (ii) M = SnBu3
(1.2 equiv), Pd(OAc)2 (0.05 equiv), X-Phos (0.1 equiv), DMF, 100 °C, 12 h.
Table 1
Synthesis of 4-amino-6-hetaryl nucleosides
Benzoylated chloropyrimidoindole nucleosides 10, 11 and 12,
were all easily converted to the corresponding 4-amino derivatives
13, 14 and 15 by treatment with aqueous ammonia in dioxane
(3:1) at 100 °C (Scheme 2). Simultaneously, the benzoyl protecting
groups at the ribose were cleaved off. The desired 4-aminopyrim-
ido[4,5-b]indole nucleosides 13, 14 and 15 were isolated just by fil-
tration in good yields (72–96%).
Compd
Reagent
R–
Yield (%)
16a
16b
16c
16d
16e
16f
Phenylboronic acid
(Furan-2-yl)SnBu3
(Furan-3-yl)B(OH)2
(Thiophen-2-yl)SnBu3
(Thiophen-3-yl)B(OH)2
(Benzofuran-2-yl)B(OH)2
Phenyl
2-Furyl
3-Furyl
2-Thienyl
3-Thienyl
2-Benzofuryl
93
68
72
73
77
75
The first class of the target compounds were 4-amino-6-
(het)arylpyrimido[4,5-b]indole nucleosides which were envisaged
to be prepared by the cross-coupling reactions of 6-chloro nucleo-
side 14. Since the chlorine at the position 6 is unreactive, a thor-
ough optimization of catalytic system and reaction conditions
was necessary. Standard procedure using water soluble ligand
TPPTS and Pd(OAc)2 in acetonitrile/water mixture did not work.
Therefore, we tested several Buchwald-type ligands (X-Phos,
S-Phos and RuPhos) and another water soluble ligand Cataxcium
F in mixtures of water and acetonitrile for the Suzuki reaction of
free 4-amino-6-chloronucleoside 14 with phenyl boronic acid. All
these reactions proceeded but only the use of X-Phos led to com-
plete conversion of starting nucleoside to desired product 16a,
which was isolated in excellent yield 93%. Unfortunately, reaction
with 3-furylboronic acid under the same conditions gave just
traces of desired product probably due to decomposition of the
boronic acid in aqueous mixture at high temperature. Therefore,
the acetonitrile/water mixture was replaced by DMF and then a full
conversion of starting nucleoside 14 in reactions with 3-furyl-,
3-thienyl- and 2-benzofurylboronic acid to products 16c, e, f was
observed (Scheme 3, Table 1). For the synthesis of 2-furyl and
2-thienyl derivatives 16b, d, the Stille coupling with the corre-
sponding hetarylstannanes was used to give the desired products
in good yields.
Cl
EtO
O
Cl
Cl
i)
N
O2N
O2N
5
6
ii)
O
O
Cl
HN
N
Cl
EtO
H2N
iii)
N
N
H
8
H
7
Cl
v)
Cl
Cl
N
iv)
5
6
8
Cl
4
N
7
N
N
2
BzO
10
O
N
H
N
9
OBz OBz
Scheme 1. Reagents and conditions: (i) CNCH2COOEt, t-BuOK, THF, reflux, 48 h; (ii)
Zn, AcOH, 55 °C, 150 min; (iii) formamide, 190 °C, 12 h; (iv) POCl3, reflux, 4 h; (v) 1-
O-acetyl-2,3,4-tri-O-benzoyl-b-D-ribofuranose, BSA, TMSOTf, 70 °C, 8 h.

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M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
5364
Cl
R
Cl
R
N
NH2
NH2
R
N
N
N
N
N
N
N
i) or ii)
BzO
N
N
iii)
N
N
i) or ii)
N
O
N
O
N
BzO
HO
O
O
HO
HO
O
OBz OBz
12
OH OH
19a-c
OH OH
13
OH OH
17a,b,e,g
18a-c OBz OBz
Scheme 5. Reagents and conditions: (i) reagent (2 equiv, see Table 3), Pd(PPh3)4
(0.05 equiv), THF, rt; (ii) Me2NH in THF (2 equiv), propan-2-ol, rt, 24 h; (iii) 1 M
MeONa in MeOH (0.3 equiv), MeOH, rt, 24 h.
Scheme 4. Reagents and conditions: (i) RM, M = B(OH)2 (2.0 equiv), Pd(OAc)2
(0.1 equiv), X-Phos (0.2 equiv), K2CO3 (3 equiv), DMF, 120 °C, 12 h; (ii) M = SnBu3
(1.5 equiv), Pd(OAc)2 (0.1 equiv), X-Phos (0.2 equiv), DMF, 120 °C, 12 h.
NH2
H3C
N
NH
I
Table 2
NH2
N
Synthesis of 4-amino-5-substituted nucleosides
H3C
N
N
Entry
Compd
Reagent
R–
Yield (%)
N
O
N
i)
ii)
N
O
N
N
O
1
2
17a
17b
17g
17g
17e
Phenylboronic acid
(Furan-2-yl)SnBu3
Phenyl
2-Furyl
Butyl
Butyl
3-Thienyl
28
33
34
42
24
HO
HO
HO
3
4
(Thiophen-2-yl)SnBu3
(Thiophen-3-yl)B(OH)2
OH OH
OH OH
OH OH
15
20
21
Scheme 6. Reagents and conditions: (i) MeI (2 equiv), DMA, rt, 24 h; (ii) 1 M NaOH,
100 °C, 1.5 h.
The same catalyst and conditions were used also for introduc-
tion of (het)aryl substituents into position 5. The reaction of
5-chloro nucleoside 13 with phenylboronic acid furnished just
28% of desired nucleoside 17a even if 2 equiv of boronic acid were
used and reaction temperature was increased to 120 °C. The Stille
reaction with 2-thiophenyl(tributyl)stannane surprisingly gave
only product of butyl transfer 17g in 42% yield. In reaction with
2-furyl(tributyl)stannane, the 5-butyl nucleoside 17g (34%) was
isolated again, together with the desired 5-furyl nucleoside 17b
(33%). The Suzuki reaction of 13 with 3-thiophenylboronic acid
provided just 5% of desired product 17e. The yield was slightly im-
proved by the addition of the boronic acid in two portions but still
the desired nucleoside 17e was isolated in low yield of 24%
(Scheme 4, Table 2). Apparently, the reactivity of 5-chloro-deriva-
tive 13 in cross-coupling reactions is much lower compared to
6-chloro derivative 14 probably due to steric hindrance of the sub-
stituent at the position 6 with the amino group at position 4.
To examine the influence of amino group at position 4 of pyri-
midoindole core to biological activity of these compounds, a series
of derivatives either replacing the amino group with an alkyl or
substituting the amino function by one or two methyl groups
was designed and prepared. Methyl and cyclopropyl groups were
introduced at position 4 by palladium catalyzed cross-coupling
reaction of protected 4-chloro nucleoside 12 with trimethylalu-
minium or by Negishi reaction with cyclopropylzinc chloride under
standard conditions in the presence of Pd(PPh3)4 in THF at 70 °C.19
In both cases, desired nucleosides 18a and 18b were isolated in
good yields (63% and 85%) (Scheme 5). N,N-Dimethylamino nucle-
oside 18c was synthesized by simple nucleophilic substitution of
12 with dimethylamine at rt in good 74% yield. Deprotection of
nucleosides 18a–c under Zemplen conditions using NaOMe in
MeOH afforded target free nucleosides 19a–c in excelent yields
76–92%.
Table 3
Synthesis of 4-substituted nucleosides
Entry
Reagent
R
Prot.
prod.
Yield
(%)
Unprot.
prod.
Yield
(%)
1a
2a
3b
Me3Al
Cyclopropyl-ZnCl
Me2NH
Me
Cyclopropyl
Me2N
18a
18b
18c
63
63
74
19a
19b
19c
92
89
88
a
Conditions: (i) were used.
Conditions: (ii) were used.
b
Since all the title pyrimidoindole nucleosides exhibited fluores-
cence, we have studied electronic spectra and photophysical prop-
erties in detail. Table 4 summarizes the results showing that the
compounds exerted fluorescence with emission maxima at 341–
485 nm with moderate quantum yields at 0.07–0.36.
2.2. Biological activity profiling
All the title nucleosides 13, 14, 15, 16a–h, 17a–c, 19a–c, 20
were subjected to biological activity screening. The cytotoxic activ-
ity in vitro was studied on the following cell cultures: (i) human
promyelocytic leukemia HL60 cells (ATCC CCL 240); (ii) human
cervix carcinoma HeLa S3 cells (ATCC CCL 2.2); (iii) human T lym-
phoblastoid CCRF-CEM cell line (ATCC CCL 119), and (iv) hepatocel-
lular carcinoma cells HepG2 (ATCC HB 8065). Cell viability was
determined following a 3-day incubation using metabolic 2,3-bis-
(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-
anilide (XTT) based method.21 The title nucleosides did not show
any significant cytotoxicity in these assays. The only three com-
Synthesis of N-methyl nucleoside 21 by nucleophilic substitu-
tion of 12 with methyl amine failed even if 10 equiv of amine
and higher temperature was used. Also the Pd-catalyzed Buch-
wald–Hartwig amination failed. Therefore, the target 4-(N-methyl-
amino)nucleoside 21 was prepared in analogy to literature
procedure20 by methylation of aminonucleoside 15 by MeI in
DMA at position 3 followed by sodium hydroxide induced rear-
rangement of the quaternary salt 20 to nucleoside 21 in 64% yield
(Scheme 6).
pounds with non-negligible activities (in 11–100 lM) are shown
in Table 5. The most active compound was 4-amino-5-chloronucle-
oside 13. All 5- or 6-hetaryl derivatives were inactive except for
benzofuryl derivative 16f. The 4-methylnucleoside 19a was the
only cytotoxic compound in series of 4-substituted derivatives.
All the title nucleosides were also tested against HCV genotype
1A, 1B and 2A replicons. The results are summarized in Table 6.
Several nucleosides showed micromolar activity against HCV, but

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M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
Table 4
Table 6
UV and fluorescence spectral dataa
Anti-HCV activities of nucleosides
Compd
Absorption
Emission
u
Compound
HCV replicon 1A
EC50 CC50
m) m)
HCV replicon 1B
EC50 CC50
m) m)
HCV replicon 2A
EC50 CC50
m) m)
kmax (nm)
e
(L molꢁ1 cmꢁ1
)
kexc (nm)
kmax (nm)
(l
(l
(l
(l
(
l
(l
13
321
292
247
320
273
315
282
242
325
263
280
229
324
257
282
236
269
314
290
248
323
248
320
294
248
289
245
284
255
235
288
255
236
326
298
250
318
290
245
13,769
15,071
71,635
12,786
13,564
10,212
16,538
88,024
2876
33,463
19,049
13,557
1147
18,736
30,297
23,989
36,191
35,291
8993
38,259
10,281
51,832
9163
12,085
86,990
23,021
80,745
16,395
45,473
44,282
26,360
49,620
57,993
10,102
13,086
31,625
16,054
15,592
63,821
300
0.10
447
13
15
0.53
1.16
>44
18.10
17.34
14.47
>44
0.32
11.05
8.75
7.38
4.83
8.81
7.07
0.34
1.71
>44
>44
25.60
28.0
>44
>44
>44
1.28
>44
1.66
>44
30.55
10.03
10.09
6.13
14
15
310
310
0.12
0.19
395
341
16d
16e
16f
17g
19c
19a
24.89
13.77
8.77
>44
>44
30.19
18.00
>44
>44
21.27
0.56
>44
>44
>44
>44
16a
16b
16c
16d
330
320
330
320
0.22
0.24
0.30
0.07
361
442
384
435
>44
>44
Table 7
Anti-dengue activities of nucleosides
Compound
Vero cells, DENV-2 (
lm)
EC50
0.85
18.8
14.8
15.4
39.6
CC50
SI
16e
16f
17a
320
340
320
0.08
0.36
0.20
467
427
452
13
1.14
46.3
62.9
>100
43.5
1.3
2.5
4.3
>6.5
1.1
16e
17g
16f
15
17b
17e
320
320
0.07
0.07
440
356
benzo-fused analogues of adenosine. They were prepared by the
Suzuki or Stille coupling reactions of the corresponding 5- or 6-
chloro derivatives with (het)arylboronic acids or -stannanes. The
cross-coupling reactions using X-phos proceeded very well at posi-
tion 6 but only low conversions were obtained at position 5 due to
steric hindrance. Also a series of 4-alkyl, 4-methylamino and 4-
dimethylamino derivatives was prepared to study the influence
of the replacement of the amino group. The target 5- and 6-(het)ar-
yl derivatives did not show any significant cytotoxicity or antiviral
activity. However, the 4-amino-5-chloro nucleoside 13 showed
significant anti-dengue and anti-HCV activity but accompanied
by cytotoxicity. The 4-methyl nucleoside 19a showed high activity
against HCV with low cytotoxicity. All the title compounds are
fluorescent and thus have some potential for fluorescent labeling
of RNA.
17g
19a
300
310
0.09
0.13
454
386
19b
19c
20
300
300
310
0.13
0.03
0.17
485
449
341
UV spectra were measured in methanol at 25 °C. All 5 ꢂ 10ꢁ5
lM solutions.
a
Table 5
4. Experimental part
Cytotoxic activities of nucleosides
Compound
HL-60
IC50
(
l
m)
NMR spectra were recorded using a 400 MHz (1H at 400 MHz,
13C at 100.6 MHz), 500 MHz (500 MHz for 1H and 125.7 MHz for
13C), or 600 MHz (600 MHz for 1H and 151 MHz for 13C) spectrom-
eter. Melting points were determined using a Kofler block and are
uncorrected. High resolution mass spectra were measured using
electrospray ionization. Reverse phase high performance flash
chromatography (HPFC) purifications were performed with Bio-
tage SP1 apparatus on KP-C18-HS columns. Optical rotations were
HeLa S3
CCRF-CEM
HepG2
13
16f
19a
17
87
54
21
93
11
12
32
82
21
60
15
measured at 25 °C, ½a D
ꢀ
values are given in 10ꢁ1 deg cm2 gꢁ1. UV–
the activity was usually accompanied by some cytotoxicity in MT-4
cells. The 4-amino-5-chloro derivative 13 was the most active but
also the most cytotoxic. However, 4-methyl nucleoside 19a
showed submicromolar activities in the HCV 1A and 1B replicon
vis and fluorescence spectroscopy measurements and quantum
yields determinations were performed in the same way as in a pre-
vious case.22 The purity of final compounds (>95%) was confirmed
by elemental analyses and clean NMR spectra.
assays and cytotoxicity higher than 44 lM.
All the nucleosides were also tested for activity against dengue
virus in Vero cells. Several derivatives showed activity in micromo-
lar concentrations, but only benzofuryl derivative 16f was not
cytotoxic to these cells. 4-Amino-5-chloronucleoside 13 was the
most active compound, but with low selectivity index (Table 7).
4.1. Ethyl 2-(6-chloro-2-nitrophenyl)-2-cyanoacetate (6)
Compound 6 was prepared from 2,3-dichloronitrobenzene (5)
(20 g; 104 mmol) and ethyl cyanoacetate (26.12 ml; 208.4 mmol)
according to literature conditions.7 Compound 6 (21 g; 86%) was
obtained as a dark oil. The crude material was used directly for
the next step. For analysis, the oil was purified by column chroma-
tography (hexane/EtOAc 0–10% EtOAc). 1H NMR is in agreement
with literature.23
3. Conclusions
A new series of 5- and 6-(het)aryl derivatives of 4-aminopyrim-
ido[4,5-b]indole ribonucleosides were designed as substituted

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M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
5366
4.2. Ethyl 2-amino-4-chloro-1H-indole-3-carboxylate (7)
(C-9a); 164.81 (COPh-20); 164.98 (COPh-30); 165.52 (COPh-50). ESI
MS m/z (rel%): 682 (100) [M+H], 684 (66) [M+2+H], 686 (26)
Compound 7 was prepared according to literature conditions7
from crude 6 (27 g; 115 mmol). Compound 7 (21.0 g; 66%) was ob-
tained as brown solid and further purified by column chromatogra-
phy (hexane/chloroform, 0–60% chloroform); mp 147–149 °C. IR
[M+4+H]. HR MS (ESI) for
C36H26Cl2N3O7 [M+H]: calcd
682.11480; found 682.11423; for C36H25Cl2N3O7Na [M+Na]: calcd
704.09660; found 704.09618.
(ATR):
m
= 3 459, 3 347, 1 633, 1 596, 1 492, 1 426, 1 378, 1 330,
4.6. 4-Amino-5-chloro-9-b-
b]indole (13)
D-ribofuranosyl-pyrimido[4,5-
1 318, 1 232, 1 177, 1 143, 731 cmꢁ1. 1H NMR is in agreement with
literature.23 Crude material was used directly for the next step.
Compound 13 was prepared as described for 14 from 4,5-di-
chloro-9-(2,3,5-tri-O-benzoyl-b- -ribofuranosyl)-pyrimido[4,5-
b]indole (10) (0.5 g; 1.4 mmol) to give pure nucleoside 13 (256 mg,
76%) as white crystals; mp 117–122 °C; ½a D ꢁ32.4 (0.26). IR (ATR):
= 3471, 3321, 3188, 2925, 2859, 1627, 1567, 1556, 1449, 1319,
1188, 1120, 1079, 1041, 986, 770. 1H NMR (600.1 MHz, DMSO-
4.3. 5-Chloro-3H-pyrimido[4,5-b]indol-4(9H)-one (8)
D
Compound 8 was prepared according to literature conditions7
from crude 7 (20.5 g; 73.8 mmol). Desired product 8 (15.0 g;
93%) was obtained as brown powder. For analysis, it was purified
by column chromatography (chloroform/MeOH, 3% MeOH); mp
over 300 °C. IR: 3176, 3062, 2959, 2 920, 1669, 1635, 1623, 1587,
1551, 1422, 1354, 1343, 1313, 1084, 992, 766. 1H NMR
(499.8 MHz, DMSO-d6): 7.23 (dd, 1H, J6,5 = 7.8, J6,8 = 1.1, H-6);
7.28 (t, 1H, J7,6 = J7,8 = 7.8, H-7); 7.40 (dd, 1H, J8,7 = 7.8, J8,6 = 1.1,
H-8); 8.13 (d, 1H, J = 3.5, H-2); 12.19 (br s, 1H, NH-3); 12.51 (br
s, 1H, NH-9). 13C NMR (125.7 MHz, DMSO-d6): 99.64 (C-4a);
1110.68 (CH-8); 120.93 (C-4b); 122.57 (CH-6); 125.26 (CH-7);
125.88 (C-5); 137.16 (C-8a); 148.68 (CH-2); 154.51 (C-9a);
157.16 (C-4). ESI MS m/z (rel%): 242 (100) [M+Na], 244 (33)
ꢀ
m
d6): 3.65 (ddd, Jgem = 12.0, J5 b,OH = 6.1, J5 b,4 = 3.7, H-50b); 3.71
0
0
0
(ddd, Jgem = 12.0, J5 a,OH = 4.9, J5 a,4 = 3.1, H-50a); 3.96 (dt,
0
0
0
J4 ,5 = 3.7, 3.1, J4 ,3 = 3.1, H-40); 4.19 (ddd, J3 ,2 = 5.7, J3 ,OH = 4.8,
0
0
0
0
0
0
0
J3 ,4 = 3.1, H-30); 4.75 (ddd, J2 ,1 = 7.3, J2 ,OH = 6.6, J2 ,3 = 5.7, H-20);
0
0
0
0
0
0
0
5.13 (d, 1H, JOH,3 = 4.8, OH-30); 5.19 (d, 1H, JOH,2 = 6.6, OH-20);
0
0
5.33 (dd, 1H, JOH,5 = 6.1, 4.9, OH-50); 6.43 (d, J1 ,2 = 7.3, H-10); 7.26
(br s, 1H, NHaHb); 7.36 (dd, 1H, J6,7 = 7.8, J6,8 = 1.2, H-6); 7.39 (dd,
1H, J7,8 = 8.0, J7,6 = 7.8, H-7); 7.65 (br s, 1H, NHaHb); 7.94 (dd, 1H,
J8,7 = 8.0, J8,6 = 1.2, H-8); 8.32 (s, 1H, H-2). 13C NMR (150.9 MHz,
DMSO-d6): 61.86 (CH2-50); 70.25 (CH-30); 70.55 (CH-20); 85.66
(CH-40); 87.23 (CH-10); 94.50 (C-4a); 111.66 (CH-8); 118.92 (C-
4b); 122.78 (CH-6); 124.47 (C-5); 126.30 (CH-7); 137.98 (C-8a);
155.18 (CH-2); 155.80 (C-9a); 157.77 (C-4). ESI MS m/z (rel%):
351 (100) [M+H], 373 (40) [M+Na]. HR MS (ESI) for C15H16Cl1N4O4
[M+H]: calcd 351.08546; found 351.08552. Anal. Calcd for
0
0
0
[M+2+Na]. HR MS (ESI) for
242.00916; found 242.00915.
C10H6ClN3ONa [M+Na]: calcd
4.4. 4,5-Dichloro-9H-pyrimido[4,5-b]indole (9)
Compound 9 was prepared according to literature conditions7
from crude 8 (15.0 g; 68.3 mmol). Desired product 9 (12.0 g;
74%) was obtained as brown powder. For analysis, it was purified
by column chromatography (chloroform/MeOH, 3% MeOH); mp
252 °C. IR: 3048, 2967, 2804, 1590, 1559, 1541, 1441, 1397,
1306, 1159, 988, 775. 1H NMR (500.0 MHz, DMSO-d6): 7.44 (m,
1H, H-6); 7.59 (m, 2H, H-7, H-8); 8.80 (s, 1H, H-2); 13.17 (br s,
1H, NH). 13C NMR (125.7 MHz, DMSO-d6): 110.45 (C-4a); 111.50
(CH-8); 115.73 (C-4b); 123.91 (CH-6); 127.23 (C-5); 129.47 (CH-
7); 140.42 (C-8a); 151.52 (C-4); 154.16 (CH-2); 156.45 (C-9a). ESI
MS m/z (rel%): 238 (100) [M+H], 240 (66) [M+2+H], 242 (16)
[M+4+H]. HR MS (ESI) for C10H5Cl2N3 [M+H]: calcd 236.9861;
found 236.9860.
C
15H15ClN4O4ꢃ0.55 CH3OH: C, 50.70; H, 4.71; N, 15.21. Found: C,
50.85; H, 4.76; N, 15.09.
4.7. 4-Amino-6-chloro-9-b-
b]indole (14)
D-ribofuranosyl-pyrimido[4,5-
4,6-Dichloro-9-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-pyrimi-
do[4,5-b]indole (11) (2.8 g; 4.1 mmol) was dissolved in dioxane
(10 ml) and 30% aqueous ammonia (30 ml) was added. Reaction
mixture was stirred in screw-cap pressure glass tube at 100 °C for
24 h, then cooled to rt and filtered. After drying under reduced pres-
sure, desired product 14 (1.38 g, 96%) was observed as white crys-
tals; mp 174 °C. ½a D
ꢀ
ꢁ35.8 (0.29). IR (ATR):
m = 3426, 3325, 3159,
2956, 2931, 1721, 1703, 1663, 1644, 1597, 1569, 1451, 1433,
1288, 1269, 1177, 1131, 1096, 1062, 1024, 1008, 905, 848, 829,
794, 708. 1H NMR (500.0 MHz, DMSO-d6): 3.63 (ddd, Jgem = 12.1,
4.5. 4,5-Dichloro-9-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-
pyrimido[4,5-b]indole (10)
J5 b,OH = 6.4, J5 b,4 = 3.7, H-50b); 3.70 (ddd, Jgem = 12.1, J5 a,OH = 4.6,
0
0
0
0
Compound 10 was prepared according to literature conditions7
from crude 9 (1.0 g; 4.2 mmol). Desired product 10 (1.4 g; 52%)
was obtained as white crystals after flash chromatography in hex-
ane/EtOAc 7:1; mp 169–173 °C. IR: 3071, 2925, 1732, 1720, 1560,
1451, 1279, 1261, 1244, 1109, 1088, 1068, 1051, 1034, 1023, 703.
J5 a,4 = 3.3, H-50a); 3.96 (ddd, J4 ,5 = 3.7, 3.3, J4 ,3 = 2.8, H-40); 4.18
0
0
0
0
0
0
(ddd, J3 ,2 = 5.9, J3 ,OH = 4.7, J3 ,4 = 2.8, H-30); 4.77 (ddd, J2 ,1 = 7.5,
0
0
0
0
0
0
0
J2 ,OH = 6.7, J2 ,3 = 5.9, H-20); 5.14 (d, 1H, JOH,3 = 4.7, OH-30); 5.20 (d,
0
0
0
0
1H, JOH,2 = 6.7, OH-20); 5.42 (dd, 1H, JOH,5 = 6.4, 4.6, OH-50); 6.33
0
0
(d, J1 ,2 = 7.5, H-10); 7.39 (dd, 1H, J7,8 = 8.8, J7,5 = 2.1, H-7); 7.46 (br
s, 2H, NH2); 7.88 (d, 1H, J8,7 = 8.8, H-8); 8.30 (s, 1H, H-2); 8.51 (d,
1H, J5,7 = 2.1, H-5). 13C NMR (125.7 MHz, DMSO-d6): 62.01 (CH2-
50); 70.47 (CH-30); 70.82 (CH-20); 85.68 (CH-40); 87.23 (CH-10);
95.29 (C-4a); 113.32 (CH-8); 120.80 (CH-5); 121.76 (C-4b); 124.63
(CH-7); 126.06 (C-6); 134.72 (C-8a); 155.23 (CH-2); 155.88 (C-9a);
157.97 (C-4). ESI MS m/z (rel%): 373 (100) [M+Na], 351 (70)
[M+H]. HR MS (ESI) for C15H16O4N4Cl [M+H]: calcd 351.08546;
found 351.08549. Anal. Calcd for C15H15ClN4O4ꢃ0.2 CH3OH: C,
51.11; H, 4.46; N, 15.69. Found: C, 51.62; H, 4.56; N, 15.89.
0
0
1H NMR (499.8 MHz, DMSO-d6): 4.69 (dd, Jgem = 12.3, J5 b,4 = 4.2, H-
0
0
50b); 4.85 (dd, Jgem = 12.3, J5 a,4 = 3.3, H-50a); 4.90 (ddd, J4 ,3 = 6.7,
0
0
0
0
J4 ,5 = 4.2, 3.3, H-40); 6.37 (t, J3 ,2 = J3 ,4 = 6.7, H-30); 6.54 (dd,
0
0
0
0
0
0
J2 ,3 = 6.7, J2 ,1 = 4.3, H-20); 7.08 (d, J1 ,2 = 4.3, H-10); 7.41 (m, 2H,
H-m-Bz-20); 7.49, 7.50 (2 ꢂ m, 2 ꢂ 2H, H-m-Bz-30,50); 7.53 (m, 2H,
H-6,7); 7.62 (m, 1H, H-p-Bz-20); 7.68 (m, 2H, H-p-Bz-30,50); 7.83
(m, 2H, H-o-Bz-20); 7.92 (m, 2H, H-o-Bz-50); 7.98 (m, 2H, H-o-Bz-
30); 8.10 (m, 1H, H-8); 8.81 (s, 1H, H-2). 13C NMR (150.9 MHz,
DMSO-d6): 63.05 (CH2-50); 70.17 (CH-030); 72.40 (CH-20); 78.80
(CH-40); 86.52 (CH-10); 110.99 (CH-8); 111.70 (C-4a); 116.19 (C-
4b); 125.45 (CH-6); 127.66 (C-5); 128.64, 128.79 (C-i-Bz); 128.90,
128.95, 128.97 (CH-m-Bz); 129.33 (CH-o-Bz-50, C-i-Bz); 129.51
(CH-o-Bz-20); 129.61 (CH-o-Bz-30); 129.84 (CH-7); 133.80, 134.10
(CH-p-Bz); 140.01 (C-8a); 152.14 (C-4); 153.91 (CH-2); 155.62
0
0
0
0
0
0
4.8. 4-Amino-9-b-D-ribofuranosyl-pyrimido[4,5-b]indole (15)
Compound 15 was prepared as describe for 14 from protected
nucleoside 12 (0.8 g; 1.24 mmol) to give pure nucleoside 15

´
5367
M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
(282 mg, 72%) as white crystals; mp 237–240 °C; ½a D
ꢀ
ꢁ47.0 (0.30);
J7,8 = 8.7, J7,5 = 1.9, H-7); 7.86 (m, 2H, H-o-Ph); 7.91 (dd, 1H,
J8,7 = 8.9, J8,5 = 0.4, H-8); 8.29 (s, 1H, H-2); 8.62 (dd, 1H, J5,7 = 1.9,
IR (ATR):
m = 3467, 3444, 3341, 2373, 2169, 2039, 1656, 1596,
1583, 1567, 1449, 1316, 1198, 1089, 1075, 1024, 740. 1H NMR
J5,8 = 0.4, H-5). C NMR (125.7 MHz, DMSO-d6): 61.15 (CH2-50);
13
0
(499.8 MHz, DMSO-d6): 3.63 (ddd, 1H, Jgem = 12.0, J5 b,OH = 6.9,
70.61 (CH-30); 70.89 (CH-20); 85.69 (CH-40); 87.32 (CH-10); 96.13
(C-4a); 112.13 (CH-8); 119.36 (CH-5); 121.08 (C-4b); 123.81 (CH-
7); 127.00 (CH-p-Ph); 127.25 (CH-o-Ph); 128.96 (CH-m-Ph);
133.82 (C-6); 135.83 (C-8a); 140.78 (C-i-Ph); 154.74 (CH-2);
155.77 (C-9a); 158.03 (C-4). ESI MS m/z (rel%): 393 (100) [M+H].
HR MS (ESI) for C21H21N4O4 [M+H]: calcd 393.15573; found
393.15563. Anal. Calcd for C21H20N4O4: C, 61.05; H, 5.44; N,
13.56. Found: C, 60.92; H, 5.35; N, 13.44.
J5 b,4 = 3.7, H-50b); 3.71 (ddd, 1H, Jgem = 12.0, J5 a,OH = 4.5,
0
0
0
J5 a,4 = 3.2, H-50a); 3.96 (ddd, 1H, J4 ,5 = 3.7, 3.2, J4 ,3 = 3.0, H-40);
0
0
0
0
0
0
4.19 (ddd, 1H, J3 ,2 = 5.8, J3 ,OH = 4.7, J3 ,4 = 3.0, H-30); 4.83 (ddd,
0
0
0
0
0
1H, J2 ,1 = 7.3, J2 ,OH = 6.7, J2 ,3 = 5.8, H-20); 5.15 (d, 1H, JOH,3 = 4.7,
0
0
0
0
0
0
OH-30); 5.20 (d, 1H, JOH,2 = 6.7, OH-20); 5.48 (dd, 1H, JOH,5 = 6.9,
0
0
4.5, OH-50); 6.33 (d, 1H, J1 ,2 = 7.3, H-10); 7.29 (ddd, 1H, J6,5 = 7.8,
J6,7 = 7.3, J6,8 = 1.0, H-6); 7.32 (br s, 2H, NH2); 7.39 (ddd, 1H,
J7,8 = 8.3, J7,6 = 7.3, J7,5 = 1.2, H-7); 7.82 (dd, 1H, J8,7 = 8.3, J8,6 = 1.0,
J8,5 = 0.7, H-8); 8.34 (dd, 1H, J5,6 = 7.8, J5,7 = 1.2, J5,8 = 0.7, H-5);
8.35 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6): 62.13 (CH2-
50); 70.56 (CH-30); 70.76 (CH-20); 85.60 (CH-40); 87.28 (CH-10);
95.95 (C-4a); 111.67 (CH-8); 120.29 (C-4b); 121.22 (CH-6);
121.47 (CH-5); 124.90 (CH-7); 136.29 (C-8a); 154.53 (CH-2);
155.25 (C-9a); 157.93 (C-4). ESI MS m/z (rel%): 317 (11) [M+H]
339 (100) [M+Na]. HR MS (ESI) for C15H17N4O4 [M+H]: calcd
317.12443; found 317.12448; for C15H16N4O4Na [M+Na]: calcd
339.10638; found 339.10638. Anal. Calcd for C15H16N4O4ꢃ1 H2O:
C, 53.89; H, 5.43; N, 16.76. Found: C, 54.05; H, 5.28; N, 16.62.
0
0
4.12. 4-Amino-6-furan-2-yl-9-b-D-ribofuranosyl-pyrimido[4,5-
b]indole (16b)
Free aminonucleoside 14 (150 mg, 0.43 mmol) and 2-(tributyl-
stannyl)furane (230 mg, 0.64 mmol) were used. Desired product
16b (111 mg, 68%) was obtained as white powder; mp 219–
222 °C; ½a D
ꢀ
ꢁ34.8 (0.28). IR (ATR):
m = 3343, 3215, 3128, 1633,
1597, 1570, 1463, 1311, 1084, 1043, 1012, 800, 736. 1H NMR
(500.0 MHz, DMSO-d6): 3.64 (dd, Jgem = 11.9, J5 b,4 = 3.7, H-50b);
0
0
3.72 (dd, Jgem = 11.9, J5 a,4 = 3.2, H-50a); 3.97 (ddd, J4 ,5 = 3.7, 3.2,
0
0
0
0
J4 ,3 = 2.9, H-40); 4.20 (dd, J3 ,2 = 5.8, J3 ,4 = 2.9, H-30); 4.82 (dd,
0
0
0
0
0
0
4.9. General procedure for the Suzuki coupling
J2 ,1 = 7.2, J2 ,3 = 5.8, H-20); 5.45 (br s, 3H, OH-20,30,50); 6.33 (d,
0
0
0
0
J1 ,2 = 7.2, H-10); 6.62 (dd, 1H, J4,3 = 3.3, J4,5 = 1.8, H-4-furyl); 7.04
(dd, 1H, J3,4 = 3.3, J3,5 = 0.8, H-3-furyl); 7.46 (br s, 2H, NH2); 7.737
(dd, 1H, J7,8 = 8.6, J7,5 = 1.7, H-7); 7.743 (dd, 1H, J5,4 = 1.8, J5,3 = 0.8,
H-5-furyl); 7.87 (d, 1H, J8,7 = 8.6, H-8); 8.29 (s, 1H, H-2); 8.64 (d,
1H, J5,7 = 1.7, H-5). 13C NMR (125.7 MHz, DMSO-d6): 62.11 (CH2-
50); 70.58 (CH-30); 70.94 (CH-20); 85.67 (CH-40); 87.38 (CH-10);
95.93 (C-4a); 104.96 (CH-3-furyl); 112.01 (CH-8); 112.19 (CH-4-
furyl); 116.58 (CH-5); 120.58 (CH-7); 120.80 (C-4b); 124.36 (C-
6); 135.67 (C-8a); 142.33 (CH-5-furyl); 154.14 (C-2-furyl);
154.83 (CH-2); 155.79 (C-9a); 157.98 (C-4). ESI MS m/z (rel%):
383 (100) [M+H]. HR MS (ESI) for C19H19N4O5 [M+H]: calcd
383.13500; found 383.13516. Anal. Calcd for C19H18N4O5ꢃ1 CH3OH:
C, 57.97; H, 5.35; N, 13.52. Found: C, 58.06; H, 5.32; N, 13.60.
0
0
Free aminonucleoside 13 or 14 (0.5 mmol), boronic acid
(1.5 equiv), K2CO3 (3 equiv), Pd(OAc)2 (0.05 equiv) and X-Phos
(0.1 equiv) were dissolved in DMF and heated to 100 °C for 12 h.
The volatiles were removed in vacuo and the residue was purified
by RP-HPFC on C-18 (0?100% MeOH in water). Products were ob-
tained as white powders or crystals after recrystallization from
MeOH/H2O mixtures.
4.10. General procedure for the Stille coupling
Free aminonucleoside 13 or 14 (0.5 mmol), tributylstannane
(1.5 equiv), Pd(OAc)2 (0.05 equiv) and X-Phos (0.1 eqiuv) were dis-
solved in DMF and heated to 100 °C for 12 h. The volatiles were re-
moved in vacuo and the residue was first put on silica column
containing 15% of KF. Column was washed with 3 L of hexane, than
with gradient of MeOH in DCM (2–40% MeOH). Fractions contain-
ing crude product were evaporated and purified by RP-HPFC on C-
18 (0?100% MeOH in water). Products were obtained as white
powders or crystals after recrystallization from MeOH/H2O
mixtures.
4.13. 4-Amino-6-furan-3-yl-9-b-D-ribofuranosyl-pyrimido[4,5-
b]indole (16c)
Free aminonucleoside 14 (100 mg, 0.29 mmol) and furan-3-yl
boronic acid (48 mg, 0.43 mmol) were used. Desired product 16c
(111 mg, 72%) was obtained as white powder; mp 121–122 °C;
½
a D
ꢀ
ꢁ41.6 (0.33). IR (ATR):
m = 3330, 3164, 1652, 1593, 1571,
1464, 1307, 1077, 1046, 1029, 794, 596. 1H NMR (500.0 MHz,
4.11. 4-Amino-6-phenyl-9-b-
b]indole (16a)
D-ribofuranosyl-pyrimido[4,5-
DMSO-d6): 3.64 (bddd, Jgem = 11.8, J5 b,OH = 6.1, J5 b,4 = 3.3, H-50b);
0
0
0
3.72 (bdt, Jgem = 11.8, J5 a,OH = J5 a,4 = 3.3, H-50a); 3.97 (td,
0
0
0
J4 ,5 = 3.3, J4 ,3 = 2.7, H-40); 4.20 (ddd, J3 ,2 = 5.7, J3 ,OH = 4.7,
0
0
0
0
0
0
0
Nucleoside 14 (50 mg, 0.14 mmol), phenylboronic acid (25 mg,
0.21 mmol), K2CO3 (58 mg, 0.42 mmol), Pd(OAc)2 (1.6 mg,
0.007 mmol) and X-Phos (6.7 mg, 0.014 mmol) were dissolved in
acetonitrile/water mixture (3:2, 5 ml) and heated to 100 °C for
16 h. The volatiles were removed in vacuo and the residue was
purified by RP-HPFC on C-18 (0?100% MeOH in water). Product
16a (52 mg, 93%) was obtained as white powder after recrystalliza-
J3 ,4 = 2.7, H-30); 4.82 (ddd, J2 ,1 = 7.3, J2 ,OH = 6.7, J2 ,3 = 5.7, H-20);
0
0
0
0
0
0
0
5.13 (d, 1H, JOH,3 = 4.7, OH-30); 5.19 (d, 1H, JOH,2 = 6.7, OH-20);
0
0
5.47 (bdd, 1H, JOH,5 = 6.1, 3.3, OH-50); 6.33 (d, J1 ,2 = 7.3, H-10);
7.19 (dd, 1H, J4,5 = 1.8, J4,2 = 0.8, H-4-furyl); 7.43 (br s, 2H, NH2);
7.67 (dd, 1H, J7,8 = 8.6, J7,5 = 1.7, H-7); 7.76 (dd, 1H, J5,4 = 1.8,
J5,2 = 1.5, H-5-furyl); 7.83 (dd, 1H, J8,7 = 8.6, J8,5 = 0.4, H-8); 8.25
(dd, 1H, J2,5 = 1.5, J2,4 = 0.8, H-2-furyl); 8.28 (s, 1H, H-2); 8.51 (dd,
1H, J5,7 = 1.7, J5,8 = 0.4, H-5). 13C NMR (125.7 MHz, DMSO-d6):
62.10 (CH2-50); 70.56 (CH-30); 70.89 (CH-20); 85.64 (CH-40); 87.33
(CH-10); 95.93 (C-4a); 109.30 (CH-4-furyl); 112.01 (CH-8); 117.99
(CH-5); 120.83 (C-4b); 122.64 (CH-7); 125.52 (C-6); 126.55 (C-3-
furyl); 135.35 (C-8a); 138.94 (CH-2-furyl); 144.16 (CH-5-furyl);
154.68 (CH-2); 155.59 (C-9a); 157.90 (C-4). ESI MS m/z (rel%):
383 (30) [M+H]; 405 (100) [M+Na]. HR MS (ESI) for C19H19N4O5
[M+H]: calcd 383.13500; found 383.13495; for C19H18N4O5Na
[M+Na]: calcd 405.11694; found 405.11683. Anal. Calcd for
0
0
0
tion from MeOH/H2O mixture; mp 216–218 °C; ½a D
IR (ATR): = 3437, 3329, 3156, 1651, 1629, 1594, 1570, 1468,
1405, 1316, 114, 1045, 1022, 988, 717. 1H NMR (499.8 MHz,
ꢀ ꢁ49.8 (0.20).
m
DMSO-d6): 3.65 (ddd, Jgem = 12.0, J5 b,OH = 6.8, J5 b,4 = 3.7, H-50b);
0
0
0
3.73 (ddd, Jgem = 12.0, J5 a,OH = 4.5, J5 a,4 = 3.2, H-50a); 3.98 (ddd,
0
0
0
J4 ,5 = 3.7, 3.2, J4 ,3 = 2.9, H-40); 4.20 (ddd, J3 ,2 = 5.7, J3 ,OH = 4.7,
0
0
0
0
0
0
0
J3 ,4 = 2.9, H-30); 4.84 (ddd, J2 ,1 = 7.3, J2 ,OH = 6.8, J2 ,3 = 5.7, H-20);
0
0
0
0
0
0
0
5.15 (d, 1H, JOH,3 = 4.7, OH-30); 5.22 (d, 1H, JOH,2 = 6.8, OH-20);
0
0
5.50 (dd, 1H, JOH,5 = 6.8, 4.5, OH-50); 6.36 (d, J1 ,2 = 7.3, H-10); 7.35
(m, 1H, H-p-Ph); 7.48 (m, 4H, NH2, H-m-Ph); 7.71 (dd, 1H,
0
0
0

´
M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
5368
C19H18N4O5ꢃ1 H2O: C, 57.00; H, 5.03; N, 13.99. Found: C, 56.85; H,
½
a D
ꢀ
ꢁ53.3 (0.30). IR (ATR):
m = 3342, 3202, 2940, 2372, 1632, 1594,
4.89; N, 13.96.
1569, 1452, 1084, 1042, 799, 750. 1H NMR (500.0 MHz, DMSO-d6):
3.66 (bddd, Jgem = 12.0, J5 b,OH = 6.1, J5 b,4 = 3.6, H-50b); 3.74 (bdt,
0
0
0
Jgem = 12.0, J5 a,OH = J5 a,4 = 3.6, H-50a); 4.00 (td, J4 ,5 = 3.6,
0
0
0
0
0
4.14. 4-Amino-6-thiophen-2-yl-9-b-
pyrimido[4,5-b]indole (16d)
D-ribofuranosyl-
J4 ,3 = 2.8, H-40); 4.22 (bdd, J3 ,2 = 5.6, J3 ,4 = 2.8, H-30); 4.86 (bdd,
0
0
0
0
0
0
J2 ,1 = 7.2, J2 ,3 = 5.6, H-20); 5.16 (br s, 1H, OH-30); 5.24 (br s, 1H,
0
0
0
0
OH-20); 5.47 (bdd, 1H, JOH,5 = 6.1, 3.6, OH-50); 6.37 (d, J1 ,2 = 7.2,
H-10); 7.27 (ddd, 1H, J5,4 = 7.5, J5,6 = 7.2, J5,7 = 1.0, H-5-benzofuryl);
7.31 (ddd, 1H, J6,7 = 8.1, J6,5 = 7.2, J6,4 = 1.5, H-6-benzofuryl); 7.54
(d, 1H, J3,7 = 1.0, H-3-benzofuryl); 7.55 (br s, 2H, NH2); 7.64 (dtd,
1H, J7,6 = 8.1, J7,3 = J7,5 = 1.0, J7,4 = 0.7, H-7-benzofuryl); 7.68 (ddd,
1H, J4,5 = 7.5, J4,6 = 1.5, J4,7 = 0.7, H-4-benzofuryl); 7.96 (dd, 1H,
J7,8 = 8.6, J7,5 = 1.5, H-7); 7.98 (dd, 1H, J8,7 = 8.6, J8,5 = 0.6, H-8);
8.33 (s, 1H, H-2); 8.89 (dd, 1H, J5,7 = 1.5, J5,8 = 0.6, H-5). 13C NMR
(125.7 MHz, DMSO-d6): 62.09 (CH2-50); 70.57 (CH-30); 70.91 (CH-
20); 85.73 (CH-40); 87.36 (CH-10); 95.90 (C-4a); 101.03 (CH-3-ben-
zofuryl); 111.14 (CH-7-benzofuryl); 112.17 (CH-8); 117.97 (CH-5);
120.94 (C-4b); 120.97 (CH-3-benzofuryl); 121.60 (CH-7); 123.32
(CH-5-benzofuryl); 123.44 (C-6); 124.42 (CH-6-benzofuryl);
129.37 (C-3a-benzofuryl); 136.60 (C-8a); 154.33 (C-7a-benzofu-
ryl); 155.02 (CH-2); 155.97 (C-9a); 156.49 (C-2-benzofuryl);
158.03 (C-4). ESI MS m/z (rel%): 433 (92) [M+H]; 455 (100)
[M+Na]. HR MS (ESI) for C23H21N4O5 [M+H]: calcd 433.15065;
found 433.15073; calcd 455.13259; for C23H20N4O5Na [M+H]:
found 455.13266. Anal. Calcd for C23H20N4O5 1.3 H2O: C, 60.60;
H, 5.00; N, 12.29. Found: C, 60.85; H, 4.89; N, 12.04.
0
0
0
Free aminonucleoside 14 (100 mg, 0.29 mmol) and 2-(tributyl-
stannyl)thiophene (160 mg, 0.43 mmol) were used. Desired
product 16d (83 mg, 73%) was obtained as white powder; mp
228–230 °C; ½aꢀD-50.0 (0.18). IR (ATR):
m = 3396, 3301, 1688,
1674, 1659, 1545, 1511, 1502, 1401, 1381, 1243, 1160, 965, 762.
1H NMR (499.8 MHz, DMSO-d6): 3.64 (br d, Jgem = 12.4, H-50b);
3.72 (bdd, Jgem = 12.4, J5 a,4 = 3.1, H-50a); 3.97 (q, J4 ,5 = J4 ,3 = 3.1,
0
0
0
0
0
0
H-40); 4.20 (dd, J3 ,2 = 5.7, J3 ,4 = 3.1, H-30); 4.82 (dd, J2 ,1 = 7.3,
0
0
0
0
0
0
J2 ,3 = 5.7, H-20); 5.20 (br s, 2H, OH-20,30); 5.47 (br s, 1H, OH-50);
0
0
6.33 (d, J1 ,2 = 7.3, H-10); 7.17 (dd, 1H, J4,5 = 5.1, J4,3 = 3.6, H-4-thie-
nyl); 7.51 (br s, 2H, NH2); 7.51 (dd, 1H, J5,4 = 5.1, J5,3 = 1.2, H-5-thi-
enyl); 7.63 (dd, 1H, J7,8 = 8.6, J7,5 = 1.8, H-7); 7.69 (dd, 1H, J3,4 = 3.6,
J3,5 = 1.2, H-3-thienyl); 7.89 (dd, 1H, J8,7 = 8.6, J8,5 = 0.4, H-8); 8.29
(s, 1H, H-2); 8.63 (dd, 1H, J5,7 = 1.8, J5,8 = 0.4, H-5). 13C NMR
(125.7 MHz, DMSO-d6): 62.11 (CH2-50); 70.57 (CH-30); 70.89 (CH-
20); 85.68 (CH-40); 87.28 (CH-10); 95.91 (C-4a); 112.26 (CH-8);
117.93 (CH-5); 121.05 (C-4b); 122.92 (CH-7); 123.54 (CH-3-thie-
nyl); 124.98 (CH-5-thienyl); 127.67 (C-6); 128.50 (CH-4-thienyl);
135.73 (C-8a); 144.41 (C-2-thienyl); 154.90 (CH-2); 155.85 (C-
9a); 158.01 (C-4). ESI MS m/z (rel%): 399 (65) [M+H]; 421 (100)
[M+Na]. HR MS (ESI) for C19H19N4O4S [M+H]: calcd 399.11215;
found 399.11219. Anal. Calcd for C19H18N4O4S ꢃ1.1 H2O: C, 54.56;
H, 4.87; N, 13.40; S, 7.67. Found: C, 54.31; H, 4.78; N, 13.28; S, 7.58.
0
0
4.17. 4-Amino-5-phenyl-9-b-
b]indole (17a)
D-ribofuranosyl-pyrimido[4,5-
4.15. 4-Amino-6-thiophen-3-yl-9-b-
pyrimido[4,5-b]indole (16e)
D
-ribofuranosyl-
Nucleoside 13 (200 mg, 0.57 mmol), phenylboronic acid
(139 mg, 1.14 mmol)), K2CO3 (236 mg, 1.14 mmol), Pd(OAc)2
(12.0 mg, 0.05 mmol) and X-Phos (54.0 mg, 0.11 mmol) was dis-
solved in DMF (5 ml) and heated to 120 °C for 16 h. The volatiles
were removed in vacuo and the residue was purified by RP-HPFC
on C-18 (0?100% MeOH in water). Product 17a (62 mg, 28%)
was obtained as white powder after recrystallization from
Nucleoside 16e was prepared according to general procedure.
Free aminonucleoside 14 (50 mg, 0.14 mmol) and thiophene-3-yl
boronic acid (27.0 mg, 0.21 mmol) were used. Desired product
16e (43 mg, 77%) was obtained as white powder; mp 167–
169 °C; ½a D
ꢀ
ꢁ38.9 (0.30). IR (ATR):
m
= 3373, 3364, 1640, 1630,
MeOH/H2O mixture; mp 185–157 °C; ½a D
= 3469, 3378, 2934, 1578, 1561, 1456, 1321, 1092, 1029, 794,
765, 705. 1H NMR (499.8 MHz, DMSO-d6): 3.66 (dd, Jgem = 12.0,
ꢀ ꢁ28.2 (0.26). IR (ATR):
1469, 1080, 782. 1H NMR (600.1 MHz, DMSO-d6): 3.65 (ddd,
m
Jgem = 12.0, J5 b,OH = 6.7, J5 b,4 = 3.7, H-50b); 3.72 (ddd, Jgem = 12.0,
0
0
0
J5 a,OH = 4.5, J5 a,4 = 3.4, H-50a); 3.99 (ddd, J4 ,5 = 3.7, 3.4, J4 ,3 = 2.8,
J5 b,4 = 3.8, H-50b); 3.73 (dd, Jgem = 12.0, J5 a,4 = 3.1, H-50a); 3.98
0
0
0
0
0
0
0
0
0
0
0
H-40); 4.21 (bdd, J3 ,2 = 5.5, J3 ,4 = 2.8, H-30); 4.83 (bdd, J2 ,1 = 7.3,
(dt, J4 ,5 = 3.8, 3.1, J4 ,3 = 3.1, H-40); 4.22 (dd, 1H, J3 ,2 = 5.8,
0
0
0
0
0
0
0
0
0
0
0
0
J2 ,3 = 5.5, H-20); 5.14 (br s, 1H, OH-30); 5.20 (br s, 1H, OH-20);
J3 ,4 = 3.1, H-30); 4.86 (dd, J2 ,1 = 7.3, J2 ,3 = 5.8, H-20); 5.28, 5.43
0
0
0
0
0
0
0
0
5.47 (dd, 1H, JOH,5 = 6.7, 4.5, OH-50); 6.34 (d, J1 ,2 = 7.3, H-10); 7.46
(br s, 2H, NH2); 7.66 (dd, 1H, J5,4 = 5.0, J5,2 = 3.0, H-5-thienyl);
7.79 (dd, 1H, J7,8 = 8.6, J7,5 = 1.7, H-7); 7.82 (dd, 1H, J4,5 = 5.0,
J4,2 = 1.4, H-4-thienyl); 7.85 (dd, 1H, J8,7 = 8.6, J8,5 = 0.5, H-8); 7.97
(dd, 1H, J2,5 = 3.0, J2,4 = 1.4, H-2-thienyl); 8.29 (s, 1H, H-2); 8.63
(dd, 1H, J5,7 = 1.7, J5,8 = 0.5, H-5). 13C NMR (150.9 MHz, DMSO-d6):
62.09 (CH2-50); 70.54 (CH-30); 70.87 (CH-20); 85.62 (CH-40); 87.31
(CH-10); 96.03 (C-4a); 111.99 (CH-8); 118.55 (CH-5); 119.96 (CH-
2-thienyl); 120.90 (C-4b); 123.22 (CH-7); 126.74 (CH-5-thienyl);
126.93 (CH-4-thienyl); 128.93 (C-6); 135.46 (C-8a); 142.14 (C-3-
thienyl); 154.69 (CH-2); 155.70 (C-9a); 157.93 (C-4). ESI MS m/z
(rel%): 399 (100) [M+H]; 421 (95) [M+Na]. HR MS (ESI) for
(2 ꢂ br s, 3H, OH-20,30,50); 6.46 (d, J1 ,2 = 7.3, H-10); 7.13 (dd, 1H,
J6,7 = 7.4, J6,8 = 1.0, H-6); 7.45 (dd, 1H, J7,8 = 8.3, J7,6 = 7.4, H-7);
7.51 (br m, 2H, H-o-Ph); 7.56 (m, 3H, H-m,p-Ph); 7.92 (dd, 1H,
J8,7 = 8.3, J8,6 = 1.0, H-8); 8.22 (s, 1H, H-2). 13C NMR (125.7 MHz,
DMSO-d6): 62.07 (CH2-50); 70.45 (CH-30); 70.62 (CH-20); 85.62
(CH-40); 87.31 (CH-10); 95.58 (C-4a); 111.20 (CH-8); 119.21 (C-
4b); 123.39 (CH-6); 124.86 (CH-7); 128.61 (CH-p-Ph); 128.67
(CH-m-Ph); 130.04 (CH-o-Ph); 135.39 (C-5); 136.72 (C-8a);
141.73 (C-i-Ph); 154.37 (CH-2); 155.86 (C-9a); 157.37 (C-4). ESI
MS m/z (rel%): 393 (100) [M+H]; 415 (89) [M+Na]. HR MS (ESI)
for C21H21N4O4 [M+H]: calcd 393.15573; found 393.15573. Anal.
Calcd for C21H20N4O4 1 H2O: C, 61.46; H, 5.40; N, 13.65. Found:
C, 61.32; H, 5.52; N, 13.48.
0
0
0
0
0
C
19H19N4O4S [M+H]: calcd 399.11215; found 399.11211. Anal.
Calcd for C19H18N4O4S ꢃ1.15 H2O: C, 54.45; H, 4.88; N, 13.37.
Found: C, 54.39; H, 4.92; N, 13.47.
4.18. 4-Amino-5-furan-2-yl-9-b-D-ribofuranosyl-pyrimido[4,5-
b]indole (17b)
4.16. 4-Amino-6-benzofuran-2-yl-9-b-
pyrimido[4,5-b]indole (16f)
D-ribofuranosyl-
Compound 17b was prepared according to general procedure
for Stille coupling. Free aminonucleoside 13 (150 mg, 0.43 mmol),
Pd(OAc)2 (12 mg, 0.05 mmol), X-Phos (50 mg, 0.1 mmol) and 2-
(tributylstannyl)furane (230 mg, 0.64 mmol) were used. RP-HPFC
purification furnished nucleoside 17b (54 mg, 33%) as white solid;
Nucleoside 16f was prepared according to general procedure.
Free aminonucleoside 14 (150 mg, 0.43 mmol) and benzofuran-2-
yl boronic acid (140.0 mg, 0.86 mmol) were used. Desired product
16f (138 mg, 75%) was obtained as white crystals; mp 265–268 °C;
mp 112–113 °C; ½a D
ꢀ
ꢁ37.8 (0.21). IR (ATR):
m = 3329, 3285, 2373,

´
5369
M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
2351, 2170, 1557, 1451, 1073, 1040, 788, 739. 1H NMR (499.8 MHz,
1023, 991, 863. 1H NMR (500.0 MHz, DMSO-d6): 0.88 (t, 3H,
Jvic = 7.4, CH3CH2CH2CH2); 1.33 (m, 2H, CH3CH2CH2CH2); 1.63 (m,
2H, CH3CH2CH2CH2); 3.22 (dd, 2H, Jvic = 8.4, 7.1, CH3CH2CH2CH2);
0
0
0
DMSO-d6): 3.66 (ddd, 1H, Jgem = 12.0, J5 b,OH = 6.4, J5 b,4 = 3.7, H-
50b); 3.73 (ddd, 1H, Jgem = 12.0, J5 a,OH = 4.7, J5 a,4 = 3.2, H-50a);
0
0
0
3.98 (ddd, 1H, J4 ,5 = 3.7, 3.2, J4 ,3 = 3.0, H-40); 4.21 (ddd, 1H,
3.63 (ddd, Jgem = 11.9, J5 b,OH = 6.5, J5 b,4 = 4.0, H-50b); 3.71 (ddd,
0
0
0
0
0
0
0
J3 ,2 = 5.8, J3 ,OH = 4.8, J3 ,4 = 3.0, H-30); 4.82 (ddd, 1H, J2 ,1 = 7.3,
Jgem = 11.9, J5 a,OH = 4.8, J5 a,4 = 3.2, H-50a); 3.95 (dt, J4 ,5 = 4.0, 3.3,
0
0
0
0
0
0
0
0
0
0
0
0
J2 ,OH = 6.6, J2 ,3 = 5.9, H-20); 5.15 (d, 1H, JOH,3 = 4.8, OH-30); 5.22
J4 ,3 = 3.3, H-40); 4.20 (br m, 1H, H-30); 4.83 (bddd, J2 ,1 = 7.1,
0
0
0
0
0
0
0
0
(d, 1H, JOH,2 = 6.6, OH-20); 5.41 (dd, 1H, JOH,5 = 6.4, 4.7, OH-50);
J2 ,OH = 6.3, J2 ,3 = 4.3, H-20); 5.12 (br s, 1H, OH-30); 5.17 (d, 1H,
0
0
0
0
0
6.44 (d, 1H, J1 ,2 = 7.3, H-10); 6.74 (dd, 1H, J3,4 = 3.2, J3,5 = 0.8, H-3-
furyl); 6.77 (dd, 1H, J4,3 = 3.2, J4,5 = 1.9, H-4-furyl); 7.31 (dd, 1H,
J6,7 = 7.4, J6,8 = 1.0, H-6); 7.46 (dd, 1H, J7,8 = 8.3, J7,6 = 7.4, H-7);
7.94 (dd, 1H, J5,4 = 1.9, J5,3 = 0.8, H-5-furyl); 8.01 (dd, 1H, J8,7 = 8.3,
J8,6 = 1.0, H-8); 8.27 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
62.00 (CH2-50); 70.41 (CH-30); 70.65 (CH-20); 85.66 (CH-40); 87.22
(CH-10); 95.56 (C-4a); 110.77 (CH-3-furyl); 111.92 (CH-4-furyl);
112.93 (CH-8); 119.61 (C-4b); 124.05 (CH-6); 125.22 (C-5);
124.78 (CH-7); 136.86 (C-8a); 144.01 (CH-5-furyl); 152.53 (C-2-
furyl); 154.68 (CH-2); 155.93 (C-9a); 157.79 (C-4). ESI MS m/z
(rel%): 383 (43) [M+H]; 405 (100) [M+Na]. HR MS (ESI) for
JOH,2 = 6.3, OH-20); 5.39 (dd, 1H, JOH,5 = 6.5, 4.8, OH-50); 6.39 (d,
0
0
0
0
J1 ,2 = 7.1, H-10); 6.76 (br s, 2H, NH2); 7.10 (dd, 1H, J6,7 = 7.4,
J6,8 = 0.8, H-6); 7.31 (dd, 1H, J7,8 = 8.2, J7,6 = 7.4, H-7); 7.68 (dd,
1H, J8,7 = 8.2, J8,6 = 0.8, H-8); 8.26 (s, 1H, H-2). 13C NMR
0
0
(125.7 MHz,
DMSO-d6):
13.99
(CH3CH2CH2CH2);
21.82
(CH3CH2CH2CH2); 33.27 (CH3CH2CH2CH2); 35.64 (CH3CH2CH2CH2);
62.04 (CH2-50); 70.37 (CH-30); 70.44 (CH-20); 85.46 (CH-40); 87.34
(CH-10); 96.82 (C-4a); 109.68 (CH-8); 119.33 (C-4b); 123.00 (CH-
6); 125.27 (CH-7); 135.86 (C-5); 137.13 (C-8a); 153.68 (CH-2);
155.63 (C-9a); 158.24 (C-4). ESI MS m/z (rel%): 373 (100) [M+H].
HR MS (ESI) for C19H25N4O4 [M+H]: calcd 373.18703; found
373.18707. Anal. Calcd for C19H24N4O4 0.85 CH3OH: C, 59.66; H,
6.91; N, 14.02. Found: C, 59.86; H, 6.52; N, 13.65.
C19H19N4O5 [M+H]: calcd 383.13500; found 383.13505. Anal. Calcd
for C19H18N4O5: C, 59.68; H, 4.74; N, 14.65. Found: C, 59.96; H,
4.86; N, 14.89.
4.21. 4-Methyl-9-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-
4.19. 4-Amino-5-thiophen-3-yl-9-b-
pyrimido[4,5-b]indole (17e)
D-ribofuranosyl-
pyrimido[4,5-b]indole (18a)
(Me)3Al (310 ll, 2 M in toluene) was added to solution of nucle-
Free nucleoside 13 (200 mg, 0.57 mmol), K2CO3 (236 mg,
1.71 mmol), Pd(OAc)2 (12.8 mg, 0.053 mmol), X-Phos (54.0 mg,
0.110 mmol) and one third of all amount of 3-furanboronic acid
(146 mg, 1.14 mmol) were dissolved in anhydrous DMF (20 ml)
and heated to 120 °C for 3 h. Second third of boronic acid was
added and reaction was stirred at 120 °C for 3 h. Then, last third
of boronic acid was added and reaction was heated for another
3 h at 120 °C. Solvent was evaporated under reduced pressure
and crude product was purified by RP-HPFC (MeOH/H2O,
0?100% MeOH). Nucleoside 17e was obtained (54 mg, 24%) as
oside 12 (200 mg, 0.31 mmol) and Pd(PPh3)4 (17.9 mg,
0.015 mmol) in THF (8 ml) and the reaction mixture was stirred
at 70 °C for 12 h. Volatiles were removed under reduced pressure
and crude product was purified by HPFC (10–50% EtOAc in hexane)
to give 18a (120 mg, 63%) as white solid; mp 155–158 °C; IR (ATR):
m
= 1733, 1497, 1457, 1420, 1278, 1263, 1136, 1113, 1091, 1072,
727, 707. 1H NMR (499.8 MHz, DMSO-d6): 2.94 (s, 3H, CH3); 4.68
(dd, 1H, Jgem = 12.5, J5 b,4 = 4.1, H-50b); 4.82 (dd, 1H, Jgem = 12.5,
0
0
J5 a,4 = 3.2, H-50a); 4.89 (ddd, 1H, J4 ,3 = 6.6, J4 ,5 = 4.1, 3.2, H-40);
0
0
0
0
0
0
6.36 (t, 1H, J3 ,2 = J3 ,4 = 6.6, H-30); 6.65 (dd, 1H, J2 ,3 = 6.6,
0
0
0
0
0
0
J2 ,1 = 4.8, H-20); 6.99 (d, 1H, J1 ,2 = 4.8, H-10); 7.40 (m, 2H, H-m-
Bz-20); 7.44 (m, 1H, H-6); 7.45 (m, 1H, H-7); 7.49, 7.51 (2 ꢂ m,
2 ꢂ 2H, H-m-Bz-30,50); 7.61 (m, 1H, H-p-Bz-20); 7.67, 7.68 (2 ꢂ m,
2 ꢂ 1H, H-p-Bz-30,50); 7.82 (m, 2H, H-o-Bz-20); 7.95 (m, 2H, H-o-
Bz-50); 7.99 (m, 2H, H-o-Bz-30); 8.01 (m, 1H, H-8); 8.20 (m, 1H,
H-5); 8.78 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6): 23.00
(CH3); 63.24 (CH2-50); 70.39 (CH-30); 72.08 (CH-20); 78.61 (CH-
40); 85.96 (CH-10); 111.65 (CH-8); 112.77 (C-4a); 119.84 (C-4b);
122.69 (CH-6); 123.40 (CH-5); 127.71 (CH-7); 128.63, 128.87 (C-
i-Bz); 128.97, 129.01, 129.03 (CH-m-Bz); 129.45 (CH-o-Bz-50, C-i-
Bz); 129.53 (CH-o-Bz-20); 129.67 (CH-o-Bz-30); 133.84, 134.17
(CH-p-Bz); 137.98 (C-8a); 154.01 (CH-2); 154.45 (C-9a); 160.64
(C-4); 164.84 (COPh-20); 165.10 (COPh-30); 165.66 (COPh-50). ESI
MS m/z (rel%): 628 (39) [M+H] 650 (100) [M+Na]. HR MS (ESI)
for C37H30N3O7 [M+H]: calcd 628.20783; found 628.20790; for
0
0
0
0
white crystals; mp 141 °C; ½a D
ꢀ
ꢁ39.4 (0.32). IR (ATR):
m = 3464,
3313, 3200, 2919, 2870, 2372, 2346, 1629, 1577, 1559, 1454,
1320, 1077, 1042, 738. 1H NMR (500.0 MHz, DMSO-d6): 3.65 (dd,
1H, Jgem = 12.0, J5 b,4 = 3.5, H-50b); 3.73 (dd, 1H, Jgem = 12.0,
0
0
J5 a,4 = 3.2, H-50a); 3.98 (dt, 1H, J4 ,5 = 3.5, 3.2, J4 ,3 = 3.2, H-40);
0
0
0
0
0
0
4.21 (dd, 1H, J3 ,2 = 5.8, J3 ,4 = 3.2, H-30); 4.84 (dd, 1H, J2 ,1 = 7.3,
0
0
0
0
0
0
J2 ,3 = 5.8, H-20); 5.20-5.50 (br m, 3H, OH-20,30,50); 6.44 (d, 1H,
0
0
J1 ,2 = 7.3, H-10); 7.14 (dd, 1H, J6,7 = 7.4, J6,8 = 1.0, H-6); 7.36 (dd,
1H, J4,5 = 4.8, J4,2 = 1.3, H-4-thienyl); 7.42 (dd, 1H, J7,8 = 8.3,
J7,6 = 7.4, H-7); 7.74 (dd, 1H, J2,5 = 2.9, J2,4 = 1.3, H-2-thienyl); 7.80
(dd, 1H, J5,4 = 4.8, J5,2 = 2.9, H-5-thienyl); 7.91 (dd, 1H, J8,7 = 8.3,
J8,6 = 1.0, H-8); 8.23 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6):
62.04 (CH2-50); 70.43 (CH-30); 70.60 (CH-20); 85.60 (CH-40); 87.27
(CH-10); 95.71 (C-4a); 111.33 (CH-8); 119.75 (C-4b); 123.65 (CH-
6); 124.71 (CH-7); 125.65 (CH-2-thienyl); 127.10 (CH-5-thienyl);
130.34 (C-5); 130.58 (CH-4-thienyl); 136.75 (C-8a); 142.07 (C-3-
thienyl); 154.38 (CH-2); 155.76 (C-9a); 157.56 (C-4). ESI MS m/z
(rel%): 399 (100) [M+H]. HR MS (ESI) for C19H19N4O4S [M+H]: calcd
399.11215; found 399.11218. Anal. Calcd for C19H18N4O4S 1.0 H2O:
C, 54.80; H, 4.84; N, 13.45; S, 7.70. Found: C, 54.68; H, 4.86; N,
13.55; S, 7.60.
0
0
C37H29N3O7Na [M+Na]: calcd 650.18977; found 650.18975.
4.22. 4-Cyclopropyl-9-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-
pyrimido[4,5-b]indole (18b)
Nucleoside 18b was prepared in analogy to literature condi-
tions.24 THF (2 ml) was added to dried zinc chloride (84.5 mg,
0.62 mmol) under argon atmosphere. Mixture was cooled to
ꢁ10 °C and cyclopropylmagnesium chloride (1.24 ml, 0.5 M in
THF) was added dropwise. After 40 min of stirring the solution of
nucleoside 12 (200 mg, 0.31 mmol) and Pd(PPh3)4 (35.8 mg,
0.03 mmol) in THF (5 ml) was added. The mixture was stirred at
40 °C for 2 h. The reaction mixture was dilluted with water
(20 ml) and extracted with ethyl-acetate (3 ꢂ 50 ml). The collected
organic phase was dried over Na2SO4 and concentrated under re-
duced pressure. Flash chromatography (20–40% EtOAc in hexane)
4.20. 4-Amino-5-butyl-9-b-
b]indole (17g)
D-ribofuranosyl-pyrimido[4,5-
Compound 17g was prepared according to general conditions
for Stille coupling. Free aminonucleoside 13 (150 mg, 0.43 mmol)
and 2-(tributylstannyl)thiophene (230 mg, 0.64 mmol) were used.
Unexpected product 17g (66 mg, 42%) was obtained as white pow-
der; mp 125–127 °C; ½a D
ꢀ
ꢁ31.2 (0.24). IR (ATR):
m = 3542, 3384,
3363, 2965, 2373, 2169, 1637, 1591, 1559, 1456, 1327, 1056,

´
M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
5370
furnished 18b (128 mg, 63%); mp 202–204 °C; ½a D
ꢀ
ꢁ51.8 (0.24); 1H
4.25. 4-Methyl-9-b-D-ribofuranosyl-pyrimido[4,5-b]indole (19a)
NMR (600.1 MHz, DMSO-d6): 1.24–1.33 (m, 4H, CH2-cyclopropyl);
2.91 (ttd, 1H, Jvic = 7.7, 4.7, 5J = 0.5, CH-cyclopropyl); 4.68 (dd, 1H,
Deprotection of 18a (80 mg, 0.12 mmol) according to the gen-
eral procedure afforded compound 19a (36 mg, 92%) as white so-
Jgem = 12.4, J5 b,4 = 4.1, H-50b); 4.82 (dd, 1H, Jgem = 12.4, J5 a,4 = 3.2,
0
0
0
0
H-50a); 4.89 (ddd, 1H, J4 ,3 = 6.6, J4 ,5 = 4.1, 3.2, H-40); 6.36 (t, 1H,
lid: mp 212–215 °C; ½a D
ꢀ
ꢁ25.6 (0.21); IR (ATR):
m = 3341, 3253,
0
0
0
0
J3 ,2 = J3 ,4 = 6.6, H-30); 6.66 (dd, 1H, J2 ,3 = 6.6, J2 ,1 = 4.7, H-20);
1598, 1457, 1433, 1111, 1056, 1039, 1012, 994, 749, 739. 1H
0
0
0
0
0
0
0
0
6.99 (d, 1H, J1 ,2 = 4.7, H-10); 7.41 (m, 2H, H-m-Bz-20); 7.42 (m,
1H, H-6); 7.45 (m, 1H, H-7); 7.49, 7.50 (2 ꢂ m, 2 ꢂ 2H, H-m-Bz-
30,50); 7.61 (m, 1H, H-p-Bz-20); 7.67, 7.68 (2 ꢂ m, 2 ꢂ 1H, H-p-Bz-
30,50); 7.83 (m, 2H, H-o-Bz-20); 7.95 (m, 2H, H-o-Bz-50); 7.98 (m,
2H, H-o-Bz-30); 8.02 (m, 1H, H-8); 8.41 (m, 1H, H-5); 8.72 (d, 1H,
5J = 0.5, H-2). 13C NMR (150.9 MHz, DMSO-d6): 11.22 (CH2-cyclo-
propyl); 15.04 (CH-cyclopropyl); 63.24 (CH2-50); 70.38 (CH-30);
72.05 (CH-20); 78.55 (CH-40); 85.94 (CH-10); 111.64 (CH-8);
112.20 (C-4a); 119.75 (C-4b); 122.59 (CH-6); 123.01 (CH-5);
127.46 (CH-7); 128.62, 128.84 (C-i-Bz); 128.91, 128.95, 128.97
(CH-m-Bz); 129.40 (CH-o-Bz-50); 129.42 (C-i-Bz); 129.49 (CH-o-
Bz-20); 129.62 (CH-o-Bz-30); 133.77, 134.09 (CH-p-Bz); 137.92 (C-
8a); 154.21 (CH-2); 154.23 (C-9a); 164.80 (COPh-20); 165.03
(COPh-30); 165.31 (C-4); 165.60 (COPh-50). ESI MS m/z (rel%): 654
(62) [M+H] 676 (100) [M+Na]. HR MS (ESI) for C39H32N3O7
[M+H]: calcd 654.22348; found 654.22349.
NMR (600.1 MHz, DMSO-d6): 2.95 (s, 3H, CH3); 3.66 (bdt, 1H,
0
0
Jgem = 12.0, J5 b,4 = J5 b,OH = 4.0, H-50b); 3.73 (bdt, 1H, Jgem = 12.0,
0
0
0
J5 a,4 = J5 a,OH = 3.3, H-50a); 3.98 (ddd, 1H, J4 ,5 = 4.0, 3.3, J4 ,3 = 3.1,
0
0
0
0
0
0
0
H-40); 4.23 (dd, 1H, J3 ,2 = 5.9, J3 ,4 = 3.1, H-30); 4.83 (dd, 1H,
0
0
0
0
J2 ,1 = 7.2, J2 ,3 = 5.9, H-20); 5.22 (br m, 1H, OH-50); 5.27, 5.31
0
0
0
0
(2 ꢂ br s, 2 ꢂ 1H, OH-20,30); 6.47 (dd, 1H, J1 ,2 = 7.2, J1 ,3 = 0.4, H-
10); 7.43 (ddd, 1H, J6,5 = 8.0, J6,7 = 7.3, J6,8 = 1.0, H-6); 7.57 (ddd,
1H, J7,8 = 8.3, J7,6 = 7.3, J7,5 = 1.2, H-7); 8.04 (ddd, 1H, J8,7 = 8.3,
J8,6 = 1.0, J8,5 = 0.7, H-8); 8.21 (ddd, 1H, J5,6 = 8.0, J5,7 = 1.2,
J5,8 = 0.7, H-5); 8.84 (d, 1H, 5J = 0.3, H-2). 13C NMR (150.9 MHz,
DMSO-d6): 22.93 (CH3); 61.88 (CH2-50); 70.30 (CH-30); 70.57 (CH-
20); 85.56 (CH-40); 87.08 (CH-10); 112.32 (C-4a); 112.95 (CH-8);
119.93 (C-4b); 122.09 (CH-6); 123.07 (CH-5); 127.43 (CH-7);
137.77 (C-8a); 153.82 (CH-2); 154.83 (C-9a); 160.12 (C-4). ESI
MS m/z (rel%): 316 (15) [M+H] 338 (100) [M+Na]. HR MS (ESI)
for C16H18N3O4 [M+H]: calcd 316.12918; found 316.12924; for
C16H17N3O4Na [M+Na]: calcd 338.11113; found 338.11105. Anal.
Calcd for C16H17N3O4 0.6 H2O: C, 58.92; H, 5.62; N, 12.88. Found:
C, 58.67; H, 5.28; N, 12.58.
0
0
0
0
4.23. 4-(N,N-Dimethylamino)-9-(2,3,5-tri-O-benzoyl-b-
ribofuranosyl)-pyrimido[4,5-b]indole (18c)
D-
4.26. 4-Cyclopropyl-9-b-D-ribofuranosyl-pyrimido[4,5-b]indole
(19b)
Dimethylamine (230 ll, 2 M in THF) was added to solution of
nucleoside 12 (200 mg, 0.31 mmol) in propan-2-ol (10 ml) and
the reaction mixture was stirred at rt for 24 h. Volatiles were re-
moved under reduced pressure and crude product was purified
by HPFC (15% EtOAc in hexane) to give 18c (150 mg, 74%) as
Deprotection of 18b (80 mg, 0.12 mmol) according to the gen-
eral procedure afforded compound 19b (37 mg, 89%) as white so-
lid: mp 230–231 °C, ½a D
ꢀ
ꢁ31.3 (0.26); IR (ATR):
m
= 3389, 3228,
.
white solid; mp 64–67 °C; IR (ATR):
m = 1724, 1576, 1556,
2185, 2000, 1597, 1085, 1053, 1020, 752 cmꢁ1
1H NMR
1512, 1269, 1251, 1099, 1070, 710. 1H NMR (499.8 MHz,
(499.8 MHz, DMSO-d6): 1.22-1.36 (m, 4H, CH2-cyclopropyl); 2.92
(tt, 1H, Jvic = 8.0, 4.7, CH-cyclopropyl); 3.66, 3.73 (2 ꢂ br d,
DMSO-d6): 3.24 (s, 6H, (CH3)2N); 4.67 (dd, 1H, Jgem = 12.3,
J5 b,4 = 4.2, H-50b); 4.81 (dd, 1H, Jgem = 12.3, J5 a,4 = 3.3, H-50a);
0
0
0
0
2 ꢂ 2H, Jgem = 11.7, H-50); 3.98 (td, 1H, J4 ,5 = 3.8, J4 ,3 = 3.0, H-40);
0
0
0
0
4.86 (ddd, 1H, J4 ,3 = 6.6, J4 ,5 = 4.2, 3.3, H-40); 6.35 (t, 1H,
0
0
0
0
4.23 (dd, 1H, J3 ,2 = 5.9, J3 ,4 = 3.0, H-30); 4.83 (dd, 1H, J2 ,1 = 7.3,
0
0
0
0
0
0
J3 ,2 = J3 ,4 = 6.6, H-30); 6.64 (dd, 1H, J2 ,3 = 6.6, J2 ,1 = 4.8, H-20);
0
0
0
0
0
0
0
0
J2 ,3 = 5.9, H-20); 5.22 (br s, 3H, OH-20,30,50); 6.46 (d, 1H, J1 ,2 = 7.3,
H-10); 7.42 (ddd, 1H, J6,5 = 8.0, J6,7 = 7.3, J6,8 = 1.0, H-6); 7.56 (ddd,
1H, J7,8 = 8.3, J7,6 = 7.3, J7,5 = 1.2, H-7); 8.03 (ddd, 1H, J8,7 = 8.3,
J8,6 = 1.0, J8,5 = 0.7, H-8); 8.42 (ddd, 1H, J5,6 = 8.0, J5,7 = 1.2,
J5,8 = 0.7, H-5); 8.77 (d, 1H, 5J = 0.2, H-2). 13C NMR (12.57 MHz,
DMSO-d6): 10.98, 11.03 (CH2-cyclopropyl); 14.97 (CH-cyclopro-
pyl); 61.89 (CH2-50); 70.30 (CH-30); 70.51 (CH-20); 85.55 (CH-40);
87.07 (CH-10); 111.86 (C-4a); 112.95 (CH-8); 119.86 (C-4b);
122.05 (CH-6); 122.77 (CH-5); 127.25 (CH-7); 137.76 (C-8a);
154.04 (CH-2); 154.63 (C-9a); 164.87 (C-4). ESI MS m/z (rel%):
342 (15) [M+H] 364 (100) [M+Na]. HR MS (ESI) for C18H20N3O4
[M+H]: calcd 342.14483; found 342.14486; for C18H19N3O4Na
[M+Na]: calcd 364.12678; found 364.12668. Anal. Calcd for
0
0
0
0
6.97 (d, 1H, J1 ,2 = 4.8, H-10); 7.29 (ddd, 1H, J7,8 = 8.3, J7,6 = 7.3,
J7,5 = 1.3, H-7); 7.34 (ddd, 1H, J6,5 = 8.0, J6,7 = 7.3, J6,8 = 1.1, H-6);
7.42 (m, 2H, H-m-Bz-20); 7.48, 7.51 (2 ꢂ m, 2 ꢂ 2H, H-m-Bz-
30,50); 7.61 (m, 1H, H-p-Bz-20); 7.67, 7.69 (2 ꢂ m, 2 ꢂ 1H, H-p-
Bz-30,50); 7.84 (m, 2H, H-o-Bz-20); 7.93 (ddd, 1H, J8,7 = 8.3,
J8,6 = 1.1, J8,5 = 0.7, H-8); 7.95 (ddd, 1H, J5,6 = 8.0, J5,7 = 1.3,
J5,8 = 0.7, H-8); 7.97 (m, 2H, H-o-Bz-30); 7.98 (m, 2H, H-o-Bz-
50); 8.40 (s, 1H, H-2). 13C NMR (125.7 MHz, DMSO-d6): 40.10
((CH3)2N); 63.36 (CH2-50); 70.41 (CH-30); 72.11 (CH-20); 78.44
(CH-40); 85.92 (CH-10); 98.31 (C-4a); 111.10 (CH-8); 120.13 (C-
4b); 121.99 (CH-6); 123.19 (CH-5); 125.23 (CH-7); 128.66,
128.85 (C-i-Bz); 128.92, 128.97, 128.98 (CH-m-Bz); 129.44 (CH-
o-Bz-50, C-i-Bz); 129.50 (CH-o-Bz-20); 129.60 (CH-o-Bz-30);
133.79, 134.08, 134.10 (CH-p-Bz); 136.48 (C-8a); 153.51 (CH-
2); 156.13 (C-9a); 160.12 (C-4); 164.81 (COPh-20); 165.03
(COPh-30); 165.63 (COPh-50). ESI MS m/z (rel%): 657 (79)
[M+H] 679 (100) [M+Na]. HR MS (ESI) for C38H33N4O7 [M+H]:
calcd 657.23438; found 657.23432; for C38H32N4O7Na [M+Na]:
calcd 679.21632; found 679.21619.
0
0
C
18H19N3O4 ꢃ1.8 H2O: C, 57.84; H, 6.09; N, 11.24. Found: C, 57.94;
H, 6.12; N, 11.20.
4.27. 4-(N,N-Dimethylamino)-9-b-
pyrimido[4,5-b]indole (19c)
D-ribofuranosyl-
Deprotection of 18c (80 mg, 0.12 mmol) according to the gen-
eral procedure afforded compound 19c (37 mg, 88%) as white so-
4.24. General procedure for deprotection of 4-substituted
nucleosides
lid: mp 98–101 °C; ½a D
ꢀ
ꢁ28.5 (c 0.29, DMSO). IR (ATR):
m = 3279,
3243, 1580, 1556, 1113, 1070, 1043, 749. 1H NMR (499.8 MHz,
DMSO-d6): 3.24 (s, 6H, (CH3)2N); 3.64 (bdd, 1H, Jgem = 11.6,
J5 b,4 = 4.0, H-50b); 3.72 (bdd, 1H, Jgem = 11.6, J5 a,4 = 3.2, H-50a);
Protected nucleoside 18a–18c (0.2 mmol) was disolved in
methanol (10 ml) and 1 M solution of MeONa in MeOH (0.3 equiv)
was added. Reaction mixture was stirred at rt overnight. Solvent
was evaporated under reduced pressure and crude products were
purified using RP-HPFC (0?100% of MeOH in H2O).
0
0
0
0
3.96 (ddd, 1H, J4 ,5 = 4.0, 3.2, J4 ,3 = 3.1, H-40); 4.21 (dd, 1H,
0
0
0
0
J3 ,2 = 5.9, J3 ,4 = 3.1, H-30); 4.84 (dd, 1H, J2 ,1 = 7.3, J2 ,3 = 5.9, H-
0
0
0
0
0
0
0
0
20); 5.21 (br s, 2H, OH-20,30); 5.35 (br s, 1H, OH-50); 6.42 (d, 1H,
J1 ,2 = 7.3, H-10); 7.34 (ddd, 1H, J6,5 = 8.0, J6,7 = 7.3, J6,8 = 1.1, H-6);
0
0

´
5371
M. Tichy et al. / Bioorg. Med. Chem. 21 (2013) 5362–5372
7.43 (ddd, 1H, J7,8 = 8.1, J7,6 = 7.3, J7,5 = 1.3, H-7); 7.91 (ddd, 1H,
J8,7 = 8.1, J8,6 = 1.1, J8,5 = 0.5, H-8); 7.95 (ddd, 1H, J5,6 = 8.0,
J5,7 = 1.3, J5,8 = 0.5, H-5); 8.43 (s, 1H, H-2). 13C NMR (125.7 MHz,
DMSO-d6): 40.12 ((CH3)2N); 62.04 (CH2-50); 70.41 (CH-30); 70.66
(CH-20); 85.53 (CH-40); 87.28 (CH-10); 98.19 (C-4a); 112.20 (CH-
8); 120.03 (C-4b); 121.45 (CH-6); 122.94 (CH-5); 125.12 (CH-7);
136.49 (C-8a); 153.22 (CH-2); 156.33 (C-9a); 160.25 (C-4). ESI
MS m/z (rel%): 345 (100) [M+H] 367 (75) [M+Na]. HR MS (ESI)
for C17H21N4O4 [M+H]: calcd 345.15573; found 345.15577. Anal.
Calcd for C17H20N4O4: C, 56.35; H, 6.12; N, 15.46. Found: C,
56.22; H, 6.01; N, 15.33.
cells (European Collection of Cell Cultures). Briefly, two-fold serial
dilutions of compounds were added in triplicate in a 96-well plate
with 20,000 Vero cells plated day ago in DMEM with
supplemented with 2% fetal bovine serum, 100 U of penicillin/
mL, 100 g of streptomycin/mL (all reagents Biotech). After 1 h
L-glutamine
l
incubation dengue type 2 virus (DENV-2, obtained from Dr. Jochen
Bodem, University of Wurzburg) was added at multiplicity of infec-
tion 0.3 IU/cell. After three days incubation at 37 °C in 5% CO2 incu-
bator, cells were fixed with 4% paraformaldehyde and
permeabilized in 0.2% Triton X-100 for 4 min. Cells were washed,
incubated with DENV-2 specific antibody (harvested from HB-46
cells, obtained from ATCC) overnight at 4 °C, followed by 1.5 h
incubation with Cy3-labeled donkey anti-mouse IgG (Jackson
ImmunoResearch Europe) and documented using fluorescence
microscope with camera (Carl Zeiss). Images were processed in Im-
ageJ program (NIH) and EC50s were calculated using nonlinear
regression analysis with GraphPad Prism version 5.04 for Windows
(GraphPad Software).
Cytotoxicity of the compounds was evaluated by incubating se-
rial dilutions of each compound with Vero cell monolayers. After
48 h incubation XTT solution was added (Sigma) for 4 h and forma-
tion of orange formazan solution was spectrophotometrically
quantified using Victor X3 plate reader (Perkin Elmer). The concen-
tration of compound with 50% cytotoxic effect (CC50) was calcu-
lated as above mentioned EC50s.
4.28. 4-(N-Methylamino)-9-b-D-ribofuranosyl-pyrimido[4,5-
b]indole (21)
Nucleoside 21 was prepared in analogy to literature procedure.20
Aminonucleoside 15 (100 mg, 0.32 mmol) was dissolved in DMA
(5 ml) and MeI (0.2 ml, 0.62 mmol) was added. The reaction mixture
was stirred overnight at rt, poured into diethylether and precipitated
hydroiodide 20 was filtered out and dried under vacuum. IR (ATR):
m
= 3397, 3302, 2985, 1667, 1502, 1462, 1411, 1243, 1119, 1034,
966, 786, 760. 1H NMR (500.0 MHz, DMSO-d6): 3.70 (ddd, 1H,
Jgem = 11.8, J5 b,OH = 5.1, J5 b,4 = 3.9, H-50b); 3.73 (ddd, 1H, Jgem = 11.8,
0
0
0
J5 a,OH = 5.1, J5 a,4 = 3.2, H-50a); 3.89 (s, 3H, CH3); 4.00 (ddd, 1H,
0
0
0
J4 ,5 = 3.9, 3.2, J4 ,3 = 2.8, H-40); 4.21 (ddd, 1H, J3 ,2 = 5.7, J3 ,OH = 4.9,
0
0
0
0
0
0
0
J3 ,4 = 2.8, H-30); 4.69 (ddd, 1H, J2 ,1 = 7.5, J2 ,OH = 6.6, J2 ,3 = 5.7, H-
0
0
0
0
0
0
0
20); 5.19 (t, 1H, JOH,5 = 5.1, OH-50); 5.28 (d, 1H, JOH,3 = 4.9, OH-30);
4.30. UV–vis and fluorescence spectroscopy
0
0
5.29 (d, 1H, JOH,2 = 6.6, OH-20); 6.44 (d, 1H, J1 ,2 = 7.5, H-10); 7.51
(ddd, 1H, J6,5 = 7.7, J6,7 = 7.3, J6,8 = 1.0, H-6); 7.57 (ddd, 1H, J7,8 = 8.3,
J7,6 = 7.3, J7,5 = 1.2, H-7); 8.22 (d, 1H, J8,7 = 8.3, H-8); 8.62 (d, 1H,
J5,6 = 7.7, H-5); 8.88 (s, 1H, H-2); 8.94 (br s, 2H, NH2). 13C NMR
(125.7 MHz, DMSO-d6): 38.21 (CH3); 61.62 (CH2-50); 70.08 (CH-30);
71.03 (CH-20); 86.07 (CH-40); 87.08 (CH-10); 95.58 (C-4a); 114.21
(CH-8); 119.26 (C-4b); 121.81 (CH-5); 123.21 (CH-6); 126.88 (CH-
7); 136.37 (C-8a); 149.87 (CH-2); 151.11 (C-4); 152.32 (C-9a). ESI
MS m/z (rel%): 331 (100) [M+H]. HR MS (ESI) for C16H19O4N4
[M+H]: calcd 331.14008; found 331.14006.
0
0
0
The UV–vis spectra were measured on Varian CARY 100 Bio
Spectrophotometer
(e is the molar extinction coefficient in
L molꢁ1 cmꢁ1
)
in MeOH. The fluorescence measurements (in
MeOH) were performed on a spectrofluorometer Aminco Bowman
series 2 with 220–850 nm range, xenon source, excitation and
emission wavelength scans, spectral bandwidth 1–16 nm, PMT
detector, scan rate 3–6000 nm/min, Saya–Namioka grating mono-
chromator. We used the comparative method of Williams et al.
for recording fluorescence quantum yield of a sample USA (a
10 mM solution of quinine sulfate in 0.1 M H2SO4 (in H2O) was
chosen as a standard: UST = 0.54). Thus, the fluorescence quantum
yield of a sample USA is calculated by the formula of Eq. 1, where
the subscripts ST and SA denote standard and sample respectively,
Crude hydroiodide salt 20 was dissolved in 1 M NaOH (5 ml) and
heated to 100 °C for 1.5 h. Reaction mixture was cooled to rt, filtered
and the filtrate was neutralized with 2 M HCl. The product crystal-
lized from solution after standing at 4 °C for 48 h. Filtration fur-
nished desired compound 21 (66 mg, 64%); mp 242–244 °C; ½a D
ꢀ
U is the fluorescent quantum yield, the terms F(SA) and F(ST) are the
ꢁ61.8 (0.14); IR (ATR):
m
= 3395, 3119, 2261, 1626, 1608, 1576,
integrated fluorescence intensities of the sample and the standard,
respectively; A(SA) and A(ST) are the optical densities of the sample
and the standard solution at the wavelength of excitation, respec-
tively; and n(SA) and n(ST) are the values of the refractive index for
the solvents used for the sample, respectively.
1027, 1011, 999, 740 cmꢁ1
.
1H NMR (600.1 MHz, DMSO-d6): 3.10
0
(d, 3H, Jvic = 4.6, CH3N); 3.63 (bddd, 1H, Jgem = 11.8, J5 b,OH = 6.6,
J5 b,4 = 3.8, H-50b); 3.71 (bddd, 1H, Jgem = 11.8, J5 a,OH = 4.8,
0
0
0
J5 a,4 = 3.8, H-50a); 3.96 (td, 1H, J4 ,5 = 3.8, J4 ,3 = 2.9, H-40); 4.19 (dd,
0
0
0
0
0
0
1H, J3 ,2 = 5.7, J3 ,4 = 2.9, H-30); 4.83 (bdd, 1H, J2 ,1 = 7.3, J2 ,3 = 5.7,
H-20); 5.15, 5.20 (2 ꢂ br s, 2 ꢂ 1H, OH-20,30); 5.47 (bdd, 1H,
0
0
0
0
0
0
0
0
Â
Ã
Â
Ã
Â
Ã
2
UFðSAÞ
¼
U
FðSTÞ ꢂ FðSAÞ=FðSTÞ ꢂ AðSTÞ=AðSAÞ ꢂ n =n
ð1Þ
ðSAÞ
ðSTÞ
JOH,5 b = 6.6, 4.8, OH-50); 6.35 (d, 1H, J1 ,2 = 7.3, H-10); 7.29 (br q,
1H, Jvic = 4.6, NH); 7.32 (ddd, 1H, J6,5 = 7.9, J6,7 = 7.2, J6,8 = 1.0, H-6);
7.40 (ddd, 1H, J7,8 = 8.3, J7,6 = 7.2, J7,5 = 1.2, H-7); 7.84 (ddd, 1H,
J8,7 = 8.3, J8,6 = 1.0, J8,5 = 0.6, H-8); 8.35 (ddd, 1H, J5,6 = 7.9, J5,7 = 1.2,
J5,8 = 0.6, H-5); 8.39 (d, 1H, 5J = 0.4, H-2). 13C NMR (150.9 MHz,
DMSO-d6): 28.05 (CH3N); 62.14 (CH2-50); 70.56 (CH-30); 70.83
(CH-20); 85.60 (CH-40); 87.31 (CH-10); 96.38 (C-4a); 111.79 (CH-8);
119.96 (C-4b); 121.18 (CH-5,6); 124.84 (CH-7); 136.09 (C-8a);
154.51 (C-9a); 154.53 (CH-2); 157.14 (C-4). ESI MS m/z (rel%): 331
(100) [M+H]. HR MS (ESI) for C16H19O4N4 [M+H]: calcd 331.14008;
found 331.14005. Anal. Calcd for C16H18N4O4 1 H2O: C, 55.17; H,
5.79; N, 16.08. Found: C, 55.23; H, 5.71; N, 16.02.
0
0
0
Acknowledgments
This work was supported by the Academy of Sciences of the
Czech Republic (RVO: 61388963), Czech Science Foundation
(P207/11/0344) and by Gilead Sciences, Inc.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.011.
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