N6-Substituted adenosines. Cytokinin and antitumor activities

Article abstract of DOI:10.1135/cccc2011114

A series of N⁶-adenosine derivatives were synthesized by alkylation of N⁶-acetyl-2',3',5'-tri-O-acetyladenosine (1) with alkyl halides and alcohols. It was shown that propargyl derivative 2a is a good substrate for copper(I) catalyzed Huisgen [3+2] cycloaddition with azides. This click-reaction can be used for preparation of the libraries of 1,2,3-triazolyl modified adenosines. Biological activities of N⁶-adenosines were studied in two plant and six human cancer cell assays. The remarkable parallel between cytokinin and cytotoxic activities was found. The most cytokinin active compounds 3c-3e at the same time appeared to be the most potent cytotoxic agents.

Full text of DOI:10.1135/cccc2011114

N6-Substituted Adenosines
1361
N6-SUBSTITUTED ADENOSINES. CYTOKININ AND ANTITUMOR
ACTIVITIES
a3
Svetlana V. KOLYACHKINAa1, Vitali I. TARAROVa2, Cyril S. ALEXEEV
,
Dmitry M. KRIVOSHEEVb1, Georgy A. ROMANOVb2, Evgenia V. STEPANOVA ,
Eliso S. SOLOMKO , Andrey N. INSHAKOV and Sergey N. MIKHAILOV
c1
c2
c3
a4,
*
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
1
Vavilov str. 32, 119991 Moscow, Russia; e-mail: kolyachkinasvetlana@gmail.com,
vtarar@yandex.ru, micelle@mail.ru, smikh@eimb.ru
Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow,
2
3
4
b
c
1
2
Russia; e-mail: kdm-86@mail.ru, gar@ippras.ru
N.N. Blokhin Cancer Research Center, Kashirskoy shosse 24, 115478 Moscow, Russia;
1
2
3
e-mail: e_stepanova@nm.ru, room312@eimb.ru, smikh@imb.ac.ru
Received June 3, 2011
Accepted July 21, 2011
Published online November 20, 2011
Dedicated to Professor Antonín Holý on the occasion of his 75th birthday.
A series of N6-adenosine derivatives were synthesized by alkylation of N6-acetyl-2′,3′,5′-tri-
O-acetyladenosine (1) with alkyl halides and alcohols. It was shown that propargyl deriva-
tive 2a is a good substrate for copper(I) catalyzed Huisgen [3+2] cycloaddition with azides.
This click-reaction can be used for preparation of the libraries of 1,2,3-triazolyl modified
adenosines. Biological activities of N6-adenosines were studied in two plant and six human
cancer cell assays. The remarkable parallel between cytokinin and cytotoxic activities was
found. The most cytokinin active compounds 3c–3e at the same time appeared to be the
most potent cytotoxic agents.
Keywords: Nucleosides; Adenosine derivatives; Alkylation; Huisgen [3+2] cycloaddition;
Cytokinins; Anticancer activity; Click chemistry; Antitumor agents.
Cytokinins are a group of plant hormones and related synthetic bio-
regulators that exert multiform effects on plant growth and development.
Endogenous cytokinins stimulate cell division, photomorphogenesis,
chloroplast development, pigment biosynthesis, regulate shoot and root
growth and overall plant architecture, and counteract leaf aging and apical
dominance (for recent reviews see ref.1).
Naturally occurring cytokinins are relatively simple N6-substituted
adenine derivatives. Common natural isoprenoid cytokinins are N6-iso-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011114

1362
Kolyachkina et al.:
pentenyladenine, zeatin and dihydrozeatin. In zeatin, one methyl group of
isopentenyl residue is hydroxylated thus giving rise for two isomeric trans-
and cis-zeatin. Dihydrozeatin has saturated isoprenoid side chain. Aromatic
cytokinins such as N6-benzyladenine (BA) and its m- and o-hydroxylated de-
rivatives, topolins, were found in some plant species. Kinetin (N6-furfuryl-
adenine) can be also regarded as aromatic cytokinin. It was initially isolated
from autoclaved herring sperm DNA and has been proposed to be a product
of decomposition of DNA 2. Later on the problem of its occurrence was thor-
oughly reinvestigated. Kinetin has been found in plant cell extracts and as a
naturally occurring component of DNA 3. Natural cytokinin bases are often
modified (glucosylated, ribosylated, etc.) at different positions of the purine
heterocycle or terminal hydroxyl group of the side chain1a.
The first step of isoprenoid cytokinin biosynthesis is adenosine phosphate-
isopentenyl transferase (EC 2.5.1.27) catalyzed N6-prenylation of adenosine
5′- (mono-, di-, or tri-) phosphates with 3,3-dimethylallyldiphosphate.
Nucleotides are readily dephosphorylated to corresponding nucleoside de-
rivatives. It is believed that two enzymes, namely adenosine nucleosidase
(EC 3.2.2.7) and purine nucleoside phosphorylase (EC 2.4.2.1), are involved
in interconversion of cytokinin nucleoside to cytokinin base1. According to
these transformations N6-substituted adenines and adenosines might have
similar cytokinin activities.
In recent years, cytokinins have been used as constituents of cosmetic
creams to improve skin structure and reduce the signs of aging. Some
cytokinins and their analogs have been shown to have antiproliferative
effect on animal tumor cells, with several of them being used already in
medicinal practice. Several N6-substituted adenosines such as N6-methyl-
adenosine, N6-isopentenyladenosine and some others were isolated from
tRNA 4. N6-Isopentenyladenosine and its analogs have been recently found
to possess profound anticancer activity5.
These important biological properties of cytokinins have stimulated the
search for convenient methods of synthesis of these compounds and their
analogs. For the most synthetic strategies 9-N position of purine moiety
should be protected. Naturally occurring nucleosides are convenient syn-
thetic precursors for cytokinins and their analogs as soon as 9-N position is
already protected. The following general approaches for preparation of
N6-alkylated adenosines could be found in the literature: (i) 1-N-Alkylation
of adenosine with subsequent Dimroth rearrangement in the basic media6,
(ii) one-pot aminations of inosine7, (iii) nucleophilic substitution of
halogen in 6-position with amine8, (iv) reduction of corresponding acyl
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

N6-Substituted Adenosines
1363
9
derivatives with LiAH4 , (v) selective N6-alkylation of N6-acyladenosine de-
rivatives either with alkyl halides under phase-transfer catalysis conditions
or with alcohols utilizing Mitsunobu protocol10.
In spite of a number of available methods for the preparation of N6-sub-
stituted adenosines there is still a room for the development of simple and
reliable methods for the synthesis of this group of important natural com-
pounds. All methods have their limitations. Methods (i) and (ii) do not
require a protection of hydroxyl groups of the sugar moiety. But this syn-
thetic advantage sometimes turns to be a disadvantage for product isola-
tion. It is well-known that free nucleosides have spare solubility in
common solvents, which limits the usage of chromatography for their puri-
fication. The same is true for methods (iii) and (iv). Besides, the preparation
of starting compounds requires intermediate protection of hydroxyl groups.
Base-promoted alkylation of adenosine derivatives has been less thor-
oughly studied. Till now only one example (v) can be found in the litera-
ture utilizing a series of N6-acyl-2′,3′,5′-tri-O-TBDMS adenosine species
which were alkylated with activated alkyl halides under phase-transfer ca-
talysis conditions. The N6/N1-selectivity depends strikingly on the nature of
N6-acyl group, and with the acetyl group only N6-alkylation is observed10a
.
The disadvantage of the protocol is the necessity of TBDMS protection of
ribose hydroxyl groups. This high molecular weight protective group inter-
feres with the atom economy strategy.
In some cases the starting amines and bromides which are needed for the
synthesis of N6-substituted derivatives are not commercially available so it
will be of interest to develop the synthetic scheme utilizing alcohols. In our
research we have been focused on developing of a cheap and simple sub-
strate for regioselective N6-alkylation of adenosine not only with bromides
but also with alcohols under Mitsunobu reaction conditions11.
In our previous publication12, we reported on the preparation of N6-acetyl-
2′,3′,5′-tri-O-acetyladenosine (1) which is a versatile starting compound for
regioselective N6-alkylation under base-promoted and Mitsunobu condi-
tions. In the present work we give additional evidence of synthetic impor-
tance of this substrate. It was used for the synthesis of intermediate
propargyl derivative 2a which was successively used in Huisgen [3+2]
cycloaddition with azides to give a series of yet unknown triazolyl analogs
of aromatic cytokinins. Cytokinin and anticancer activity of N6-substituted
adenosines were studied.
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1364
Chemistry
Kolyachkina et al.:
Adenine molecule has five nitrogen atoms which can be considered as pos-
sible sites for electrophilic attack. Position of its alkylation at different con-
ditions has been unambiguously assigned. Strikingly no products of
alkylation of exocyclic NH2 group were detected at any conditions. In base
free conditions at elevated temperature adenine is alkylated at position 3 13
,
and the resulting product is successively alkylated at position 7 14. When
strong bases are used, the site of alkylation of adenine changes from 3 to
9 15. Adenosine, naturally protected at position 9 with ribose moiety, in
base free conditions is alkylated selectively at position N1 16. To achieve se-
lective N6-alkylation NH2-group of adenosine should be acylated10.
For the purpose of selective preparation of N6-adenosine derivatives we
developed a method of synthesis of N6-acetyl-2′,3′,5′-tri-O-acetyladenosine
(1)12. Tetraacetate 1 can be alkylated at ambient temperature under tra-
ditional base assisted or Mitsunobu conditions (Scheme 1). To promote
reaction of 1 with alkyl halides 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU;
method A) or K2CO3 (method B) were used as bases. DBU is a sterically hin-
dered strong base with pKa 11.6 17 soluble in most organic solvents that al-
lows to conduct alkylation reactions homogeneously. This base is widely
used for the N9-alkylation of adenine instead of the adenine sodium salt14.
The examples of both methods are given for the synthesis of propargyl de-
rivative 2a with high yields 89% (method A) and 85% (method B). Ap-
plying this approach, new adenosine derivative 2b was synthesized with
the yield 70%. Interestingly, the stability of N,O-acetal system was rather
high to allow deacetylation in methanolic ammonia solution.
Tetraacetate 1 is also a useful substrate for Mitsunobu alkylation12. The
Mitsunobu reaction has become a very popular mild chemical trans-
formation, occurring under essentially neutral conditions. In this reaction
alcohol is activated and then coupled to nucleophile11. We found that
tetraacetate 1 can be alkylated under Mitsunobu conditions with retention
of regioselectivity at N6-position. Thus reacting 1 with propargyl alcohol
under activation with Ph3P and diethyl diazadicarboxylate (DEAD) gives
1
only one product (TLC) with H NMR spectra identical to the above pre-
pared compound 2a. Two rounds of the Mitsunobu reaction required to
consume all starting compound 1. Scrupulosity and patience was necessary
to isolate pure 2a from Ph3PO and (NHCOOEt)2 with the yield 74% apply-
ing column chromatography (3 times with different eluents). Though poor
atom economy is the main drawback of Mitsunobu reaction, nevertheless,
when alkyl halides are not available Mitsunobu protocol utilizing parent al-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

N6-Substituted Adenosines
1365
cohols has no alternatives. Its priority is evident in case of cis-2-butene-
1,4-diol which can be used directly for preparation of 2f without protection
of one hydroxyl group. Though the intermediate compound 2f was not
possible to isolate in pure form, ammonolysis of partially purified sub-
stance gave 3f in 52% yield based on starting compound 1.
H
R
Ac
R
Ac
H
N
N
N
7
6
N
N
N
N
5
N
N
N
1
N
8
9
i), ii) or iii)
3
2
iv)
HO
N
N
4
AcO
N
AcO
N
O
O
O
HO OH
3a-3g
AcO OAc
AcO OAc
2a-2g
1
CH2C CH
a R =
b R = CH2OCH2Ph
c R = CH2Ph
d R = CH2CH=CMe2
e R = 2-furfuryl
f
R = (Z)-CH2CH=CHCH2OH
g R = CH2C
C
CH2OH
SCHEME 1
Synthesis of N6-alkyladenosines. Reagents and conditions: i) Method A: RX, DBU, MeCN,
20 °C, 16 h; ii) Method B: RX, K2CO3, DMF, 20 °C, 16 h; iii) Method C: ROH, Ph3P, DEAD,
THF, 20 °C, 20 h; iv) 7 M NH3 in MeOH, 48 h, 20 °C
The propargyl derivative 2a was used for the synthesis of triazolyl
adenosines 5a–5d (Scheme 2). Thus 2a was reacted with a series of azides in
Huisgen [3+2] cycloaddition reaction catalyzed with CuCl in MeCN to give
intermediate 4a–4d. Corresponding azides were prepared in situ starting
with appropriate alkylhalides. The click reaction is carried out at ambient
temperature to afford 4a–4d in good to high yields. It is noteworthy to
Ac
H
Ac
N
N
R'
ii)
N
R'
N
N
N
N
N
N
N
N
N
N
N
N
N
i)
N
N
N
HO
N
AcO
N
AcO
O
O
O
HO OH
5a-5d
5a R' = Me
AcO OAc
AcO OAc
4a-4d
2a
4a R' = Me
5b R' = CH2OCH2Ph
5c R' = CH2Ph
4b R' = CH2OCH2Ph
4c R' = CH2Ph
5d R' = CH2CH2OH
4d R' = CH2CH2OBz
SCHEME 2
Synthesis of N6-triazolylmethyl adenosine analogs. Reagents and conditions: i) alkylazide,
CuCl, MeCN, 20 h, 20 °C; ii) 7 M NH3 in MeOH, 48 h, 20 °C
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

1366
Kolyachkina et al.:
mention that under these conditions the reaction of 2a with NaN3 gave
very polar product(s) of unknown structure. Nucleosides 5a–5d were pre-
pared by standard ammonolysis of 4a–4d.
Biological Results
Cytokinin Activity
The biological activity of synthesized compounds was tested using two
plant assay systems. One system relies on seedlings of transgenic PARR5:GUS
Arabidopsis expressing reporter gene GUS under control of the cytokinin-
dependent promoter of the ARR5 gene. The transcription of transgenic con-
struct was shown to be sensitive and specific toward cytokinins18. Another
assay system is based on Amaranthus seedlings which quickly respond to
cytokinin by accumulation of the red pigment amaranthin19. In both sys-
tems N6-benzyladenine (BA) was used as a reference cytokinin; all com-
pounds were tested at the optimal concentration 5 µM. The results are
presented in Table I and given in percents of activity of BA.
TABLE I
The cytokinin activity of N6-adenosine derivatives 3, 5 and 6 in per cent of BA activity
Compound
GUS test in % to BA
Amaranthin test in % to BA
3a
21.6 0.04
37.2 4.2
91.6 24.3
159.6 20.1
89.2 8.5
59.7 17.5
41.8 1.4
9.2 1.6
22.1 1.8
57.2 5.2
98.9 12.3
94.4 1.8
65.6 3.5
37.9 3.5
50.5 3.5
24.9 6.0
32.8 0.4
17.4 1.1
27.7 5.2
76.8 8.8
26.4 3.5
3b
3c
3d
3e
3f
3g
5a
5b
13.5 3.5
10.5 6.7
9.5 0.04
78.7 19.6
22.8 9.9
5c
5d
6
Without treatment
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N6-Substituted Adenosines
1367
Results obtained with two assay systems were in good accordance. Syn-
thesized compounds 3c, 3d and 3e have shown high activity close to level
of BA (Table I). Compound 3f harboring cis-hydroxylated olefinic side
chain as well as compound 3g with triple bond in the aliphatic chain dis-
played much less activity. Compound 3a with shortened side chain had no
activity at all (control level). The same applies to a group of related com-
pounds 5a–5d harboring complex side chain with the 1,2,3-triazolyl ring.
All these compounds were lacking cytokinin activity. O6-Benzylinosine (6)
with N6 nitrogen replaced with oxygen in the BA and compound 3b with
extended distance between phenyl group and N6-position showed some ac-
tivity.
Thus, assay data fully confirmed the biological activity of ribosides 3c, 3d
and 3f derived from the known cytokinins.
Anticancer Activity
N6-Isopentyladenosine derivatives are known to inhibit DNA synthesis, to
cause cell-cycle arrest and to induce apoptosis. To continue these studies,
concerning the relation between structure and biological activities we have
examined the cytotoxic and apoptosis inducing activities of eight N6-iso-
pentyladenosine derivatives modified in N6-position. The cytotoxic effect
of N6-isopentyladenosine derivatives was assessed by MTT-test in T-cell leu-
kemia, melanoma, lung, ovarian and breast cancer cells (Table II). We have
100
90
80
1
70
2
60
3
4
5
6
50
40
30
20
0
20
40
60
80
100
120
Concentration, µM
FIG. 1
The cytotoxic effect of 3e in Jurkat cells (1), melanoma Mel-Kor cells (2), ovarian cancer cells
SKOV-3 (3), breast cancer cells MCF-7 (4), lung cancer cells A549 (5), endothelial cell
SVEC-4-10 (6)
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

1368
Kolyachkina et al.:
also examined the ability of these compounds to influence the growth of
mouse endothelial cells line SVEC-4-10. Compounds 3c–3e at the concen-
tration 100 µM dramatically reduced the viability of all tested cells. The data
obtained indicate that the most cytotoxic effect was observed in cells of
epithelial origin (lung, ovarian and breast carcinoma) (Fig. 1).
TABLE II
The cytotoxic effect of N6-isopentyladenosine derivatives in cancer cells
IC50, µM
Compound
SVEC-4-10
Jurkat
Mel Kor
SKOV-3
MCF-7
A549
3a
85.8 3.1
49.7 14.3
44.8 8.1
48.0 11.1
45.8 4.0
139.2 59.6
136.8 51.1
140.5 36.7
nd
92.6 27.9
30.7 6.2
39.1 8.1
41.3 26.8
128.3 85.4
>500
73.3 1.19
96.1 39.1
52.5 11.8
57.4 3.13
69.1 9.8
75.8 12.1
22.4 10.9
28.2 5.6
28.4 6.2
77.9 10.5
47.8 11.8
15.1 2.9
13.8 3.2
19.5 2.6
45.1 9.3
18.5 7.7
30.4 15.4
13.6 5.2
3c
3d
3e
10.5 3.7
3f
65.7 22.2
112.8 31.3
3g
87.0 15.1
118.1 35.7
86.2 1.76 383.4 88.0
168.0 22.4 483.2 149.4 255.5 125.2
5a
383.3 72.3
159.4 66.5
nd
5d
213.6 100.7 198.5 48.5
nd 21.0 7.3
236.3 15.1
15.7 4.2
294.0 93.2
7.5 3.8
Gremcitabine
a
nd – Not determined.
TABLE III
The apoptosis induction by N6-adenosine derivatives (100 and 25 µM) in Jurkat cells
% of apoptotic cells
Compound
100 µM
25 µM
3a
3c
3d
3e
3f
24.94
72.4
1
1
21.9 0.4
43.4 4.8
45.9 2.3
40.6 1.2
22.1 0.6
8.5 0.6
8.3 0.9
9.3 1.6
69.9 0.8
61.6 4.7
21.7 2.7
9.8 1.2
8.4 1.6
8.2 1.7
3g
5a
5d
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

N6-Substituted Adenosines
1369
Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) was used as a control anti-
cancer agent. In our experiments IC50 of Gemcitabine was 15.7 4.2 for
MCF-7 cells, 21.0 7.3 for SKOV-3 cells and 7.5 3.8 for A549.
We have also studied the ability of these compounds to induce apoptosis
in Jurkat cells (Table III). Among eight tested compounds, only 3c, 3d and
3e dose-dependently induced apoptosis in cancer cells. Low concentration
(12.5 µM) of these compounds did not induce apoptosis in Jurkat cells.
However, when cells were incubated with high concentrations (above 100 µM)
for 48 h about 70% apoptotic cells were estimated (Fig. 2). The appearance
of apoptotic bodies and chromatin condensation in MCF-7 cells was con-
firmed by immunocytochemistry staining with Hoechst 33258 (not shown).
Our data indicate that the cytotoxic effect was optimal for compounds
3c, 3d and 3e. However, this activity was highest in cancer cells of epi-
thelial origin. The similar cytotoxic effect of 3d was observed in T24 blad-
der cancer cells and other epithelial origin cells8e. Interestingly, compounds
3c, 3d and 3e also displays cytotoxic effect on endothelic SVEC-4-10 cells
thus, giving for the first time a piece of evidence that they may be involved
in tumor angiogenesis.
Recently it was shown that N6-isopentyladenosine derivatives activate
caspase-3 and -7 at the concentration of 100 µM in A549 cells20. Therefore,
we have also studied the ability of these compounds to induce apoptosis.
Flow cytometry analysis showed high level of apoptotic cells. This was con-
firmed by appearance of apoptotic bodies and chromatin condensation on
0.5%
3.1%
2.5%
40.6%
96.1%
0.3%
55.1%
1.8%
2
3
4
5
2
3
4
5
0
10
10
FITC-A
10
10
0
10
10
FITC-A
10
10
–68
FIG. 2
–89
The apoptosis induction in Jurkat cells by treatment with 25 µM of 3e (right) for 48 h versus
control (left) as observed with fluorescently labeled Annexin V-FITC (FITC-A) and propidium
iodide (PI-A)
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

1370
Kolyachkina et al.:
slides stained by Hoechst 33258. We conclude that 3c, 3d and 3e induced
cell death mainly by apoptosis.
However, despite the fact that the cytotoxic activity of 3c, 3d and 3e
was the highest among the tested derivatives, it was weaker than that of
Gemcitabine (nucleoside analog), the widely used anticancer drug for treat-
ment of patients with epithelial cancer21.
The better understanding of mechanisms of N6-isopentyladenosine deriv-
atives action will help to develop a new potential class of anticancer drugs.
In conclusion, we have shown N6-acetyl-2′,3′,5′-tri-O-acetyladenosine (1)
to be a versatile starting compound for the synthesis of N6-substituted
adenosines. This compound can be alkylated either by alkyl halides in base
promoted conditions or by alcohols in Mitsunobu reactions. N6-Propargyl
derivative 2a is a useful starting compound for modification of side chain
in Huisgen [3+2] cycloaddition. Cytokinin and anticancer activity of a se-
ries of compounds has been tested. In all tests the highest activity revealed
N6-adenosine derivatives modified with benzyl (3c), isopentenyl (3d) and
furfuryl (3e) groups. Thus, the remarkable parallel between cytokinin and
cytotoxic activities was found.
EXPERIMENTAL
Biological Materials and Methods
Cytokinin activity assays were performed using model plant systems with Amaranthus
(Amaranthus caudatus L.)19 and transgenic PARR5:GUS Arabidopsis (Arabidopsis thaliana L.)
seedlings18. Both assay systems are specifically sensitive to cytokinins and respond to hor-
mone application in few hours, making possible quantitative determinations. For a positive
control, BA was applied.
Cell lines. T-cell leukemia Jurkat, lung cancer A549, breast cancer MCF-7, ovarian cancer
SKOV-3 cell lines were obtained from ATCC and maintained in RPMI-1640 media (ICN
Pharmaceuticals Inc, USA) supplemented with 10% fetal calf serum (ICN Pharmaceuticals
Inc, USA), 2 mM glutamine (ICN Pharmaceuticals Inc, USA) and 100 U/ml penicillin/strepto-
mycin (ICN Pharmaceuticals Inc, USA). Melanoma cell line Mel Kor was isolated from surgi-
cal species of patients with disseminated melanoma and cultured in RPMI-1640 complete
medium22. Mouse endothelial cell line SVEC-4-10 was kind gift of Dr Grigorian23. The
SVEC-4-10 cells were cultured in DMEM (ICN Pharmaceuticals Inc, USA) supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin/streptomycin. All experiments
were performed with 75–80% confluent culture.
All tested compounds were dissolved in DMSO (Sigma–Aldrich, USA) and used at final
concentration from 1.6 to 100 µM. Clinical grade Gemcitabine (Gemzar, Eli Lilly, Sesto
Fiorentino, FI, Italy) were used as a control.
MTT assay. The cytotoxic potency of compounds was determined in a formazan conver-
sion assay (MTT-test). Cells (5 × 103 in 190 µl of culture medium) were plated into a 96-well
plate (Costar–Corning, USA) and treated with 0.1% DMSO (vehicle control) or with increas-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1361–1378

N6-Substituted Adenosines
1371
ing concentrations of tested compounds for 48 h. After the completion of drug exposure,
50 µg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma–Aldrich, USA)
were added into each well for additional 2 h. Formazan was dissolved in DMSO, and the
absorbance at λ = 540 nm was measured. Cell viability at a given drug concentration was
calculated as the percentage of absorbance in wells with drug-treated cells to that of vehicle
control cells (100%). The IC50 (50% inhibitory concentration) was defined as the concentra-
tion of the compound that inhibited cell viability by 50%.
Flow cytometry analysis. For quantitative estimation of the number of apoptotic cells, the
Annexin V-FITC staining was performed. 6 × 104 cells/ml were incubated with different con-
centrations of tested compounds for 48 h. ApoTarget Annexin V-FITC Apoptosis kit
(Invitrogen, USA) was used for identification of apoptotic cells. The cells were analyzed by
flow cytometry FACSCanto II (Becton Dickinson, USA) using FACSDiva software package.
Statistical analysis. All data on biological activity are averages of three independent experi-
ments made in triplicates. Error bars shown in the figures represent SEM. Statistically com-
parisons were carried out using two-tailed Student’s t test for comparison of means. P values
less than 0.05 were accepted as statistically significant.
Chemical Synthesis
The solvents and materials of reagent grade were used without additional purification.
Column chromatography was performed on silica gel (Kieselgel 60 Merck, 0.063–0.200 mm).
The solvent system used was a gradient of EtOH in CH2Cl2 increasing in polarity from CH2Cl2
to CH2Cl2–EtOH 9:1. TLC was performed on Alugram SIL G/UV254 (Macherey–Nagel) with
UV visualization. Melting points were determined on a Electrothermal apparatus and are un-
corrected. 1H and 13C (with complete proton decoupling) NMR spectra were recorded on
Bruker AMX 400 NMR instrument. Chemical shifts (δ-scale) in ppm were measured relative
to the residual solvent signals as internal standards (CDCl3, 1H: 7.26 ppm, 13C: 77.1 ppm;
DMSO-d6, 1H: 2.50 ppm, 13C: 39.5 ppm). Spin–spin coupling constants (J) are given in Hz.
Double resonance technique was applied for assigning the resonances. UV spectra were re-
corded on a Cary300UV/VIS spectrophotometer (Varian). LC-MS analysis was performed on
Surveyor MSQ instrument (Thermo Finnigan, USA), operating in APCI (atmospheric pressure
chemical ionization) mode with detection of positive and negative ions, and equipped with
Onyx Monollithic C18 25 × 4.6 mm Part No. CHO-7645 column. The eluent was 0.1%
HCOOH in water with a gradient of solution in MeCN. Chromatographic peaks were de-
tected simultaneously with ELSD (evaporative light scattering detector), PAD (photodiode ar-
ray detector), and TIC (total ion current) detector. In all cases, only one peak was revealed
and the chromatographic purity of compounds was more than 99%. N6-Acetyl-2′,3′,5′-tri-
O-acetyladenosine (1), N6-benzyladenosine (3c), N6-isopentenyladenosine (3d), N6-furfuryl-
adenosine (3e) and N6-(4-hydroxy-2-butynyl)adenosine (3g) were prepared according to ref.12
.
N6-Acetyl-N6-(2-propynyl)-2′,3′,5′-tri-O-acetyladenosine (2a)
Method A. To a stirred solution of 1 (500 mg, 1.15 mmol) and DBU (0.34 ml, 2.3 mmol)
in 5 ml of MeCN in one portion BrCH2C≡CH (0.18 ml of 80% solution in toluene, 1.7 mmol)
was added at room temperature. The reaction was monitored by TLC. After 20 h, the reac-
tion mixture was diluted with AcOEt and the solution was washed successively with 20 ml of
0.1 M HCl and brine (3 × 20 ml). The organic layer was dried over Na2SO4 and evaporated.
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Column chromatography of the residue eluting with a gradient of EtOH in CH2Cl2
(1:100→1:25) gave 484 mg (89%, foam) of 2a.
Method B. To a stirred mixture of 1 (500 mg, 1.15 mmol), K2CO3 (0.32 g, 2.3 mmol) in 5 ml
of DMF in one portion BrCH2C≡CH (0.15 ml of 80% solution in toluene, 1.4 mmol) was
added at room temperature. The reaction was monitored by TLC. The mixture was stirred at
ambient temperature for 20 h and then it was diluted with AcOEt. The mixture was washed
with brine (3 × 20 ml). The organic extract was dried over Na2SO4 and evaporated. The resi-
due was applied to column chromatography on silica gel to give 463 mg (85%, foam) of 2a.
Method C. A mixture of 1 (500 mg, 1.15 mmol), Ph3P (453 mg, 1.72 mmol) and propargyl
alcohol (0.1 ml, 1.72 mmol) in 5 ml of THF was stirred at room temperature until
homogeneuos solution was formed. DEAD (0.27 ml, 1.72 mmol) was added in one portion.
The reaction was monitored by TLC. After 20 h, the second addition of reagents (Ph3P,
propargyl alcohol and DEAD) in indicated quantities was made to achieve complete conver-
sion of 1. After 4–5 h, the reaction mixture was evaporated and the residue was applied to
column chromatography. Three column chromatography procedures were performed to iso-
late pure compound: 1) elution with AcOEt, 2) elution with a gradient CHCl3–EtOH
100:1→30:1, and 3) elution with a gradient CH2Cl2–AcOEt 4:1→CH2Cl2–AcOEt 2:1. The
yield of 2a was 405 mg (74%, foam). RF 0.42 (CHCl3–EtOH 25:1). 1H NMR (CDCl3): 2.10 (s,
3 H, AcO), 2.12 (t, 1 H, J = 2.5, HC≡C), 2.13 (s, 3 H, AcO), 2.16 (s, 3 H, AcO), 2.39 (s, 3 H,
AcN), 4.40 (dd, 1 H, J5′b,5′a = –12.9, J5′b,4′ = 5.2, H-5′b), 4.47 (dd, 1 H, J5′a,5′b = –12.9, J5′a,4′
=
3.1, H-5′a), 4.48 (ddd, 1 H, J4′,5′b = 5.2, J4′,5′a = 3.1, J4′,3′ = 4.8, H-4′), 5.10 (d, 2 H, J = 2.5,
CH2N), 5.68 (dd, 1 H, J3′,4′ = 4.8, J3′,2′ = 5.6, H-3′), 5.97 (dd, 1 H, J2′,3′ = 5.6, J2′,1′ = 5.0, H-2′),
6.25 (d, 1 H, J1′,2′ = 5.0, H-1′), 8.22 (s, 1 H, H-2), 8.82 (s, 1 H, H-8). 13C NMR (CDCl3): 20.44
(AcO), 20.54 (AcO), 20.78 (AcO), 24.46 (AcN), 36.46 (N6-CH2), 63.09 (C-5′), 70.54 (C-3′),
71.64 (H-C≡), 73.19 (C-2′), 77.42 (C≡), 80.45 (C-4′), 86.86 (C-1′), 126.97 (C-5), 142.27 (C-8),
152.27 (C-2), 152.60 (C-4), 152.69 (C-6), 169.41 (CO), 169.59 (CO), 170.30 (CO), 170.95
(CO). MS (APCI): m/z [M + H+] calculated for C21H24N5O8: 474.16, found 474.27.
N6-Acetyl-N6-benzyloxymethyl-2′,3′,5′-tri-O-acetyladenosine (2b)
The compound was prepared from 1 (500 mg, 1.15 mmol) by method A. The yield of 2b was
445 mg (70%, foam). RF 0.75 (CH2Cl2–EtOH 25:1). 1H NMR (CDCl3): 2.10 (s, 3 H, AcO), 2.13
(s, 3 H, AcO), 2.16 (s, 3 H, AcO), 2.33 (s, 3 H, AcN), 4.40 (dd, 1 H, J5′b,5′a = –12.5, J5′b,4′ = 4.9,
H-5′b), 4.46 (dd, 1 H, J5′a,5′b = –12.5, J5′a,4′ = 3.3, H-5′a), 4.48 (ddd, 1 H, J4′,5′b = 4.9, J4′,5′a
=
3.3, J4′,3′ = 4.6, H-4′), 4.66 (s, 2 H, OCH2Ph), 5.68 (dd, 1 H, J3′,4′ = 4.6, J3′,2′ = 5.5, H-3′), 5.77
(s, 2 H, OCH2N), 5.98 (dd, 1 H, J2′,3′ = 5.5, J2′,1′ = 5.3, H-2′), 6.25 (d, 1 H, J1′,2′ = 5.3, H-1′),
7.15–7.30 (m, 5 H, H-Ph), 8.20 (s, 1 H, H-2), 8.84 (s, 1 H, H-8). 13C NMR (CDCl3): 20.42
(AcO), 20.51 (AcO), 20.82 (AcO), 24.81 (AcN), 63.119 (C-5′), 70.85 (C-3′), 71.26 (CH2-Ph),
73.19 (C-2′), 78.64 (NCH2O), 80.68 (C-4′), 86.87 (C-1′), 127.55 (C-5), 128.30 (CH-Ph), 128.61
(CH-Ph), 129.81 (C-Ph), 142.37 (C-8), 152.41 (C-2), 153.06 (C-4), 153.26 (C-6), 169.40 (CO),
169.50 (CO), 170.31 (CO), 171.64 (CO). MS (APCI): m/z [M + H+] calculated for C26H30N5O9:
556.20, found 474.32.
N6-(Z)-(4-Hydroxy-2-butenyl)adenosine (3f)
Corresponding tetraacetate 2f was prepared by method C starting with 1 (500 mg, 1.15 mmol).
After column chromatography on silica gel (CH2Cl2–EtOH 100:1→20:1), partially purified
product was dissolved in 7 M ammonia solution in MeOH (4 ml, 28 mmol) and the solution
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N6-Substituted Adenosines
1373
was left for 48 h at ambient temperature to remove acetyl groups. Et2O was added to the re-
sulted slurry and the solid was filtered, washed with Et2O and dried to give 3f (162 mg,
42%). RF 0.43 (CH2Cl2–EtOH 9:1). 1H NMR (DMSO-d6): 3.55 (ddd, 1 H, J5′b,5′a = –12.0, J5′b,4′
=
3.6, J5′b,OH = 7.2, H-5′b), 3.67 (ddd, 1 H, J5′a,5′b = –12.1, J5′a,4′ = 3.8, J5′a,OH = 4.6, H-5′a), 3.96
(ddd, 1 H, J4′,5′b = 3.6, J4′,5′a = 3.8, J4′,3′ = 2.8, H-4′), 4.07–4.23 (m, 5 H, overlapping OCH2C=,
NCH2C=, H-3′), 4.60 (ddd, 1 H, J2′,3′ = 4.9, J2′,1′ = 6.0, J2′,OH = 6.2, H-2′), 4.71 (t, J = 5.3,
ω-OH, exchangeable with D2O), 5.15 (d, 1 H, JOH,3′ = 4.6, 3′-OH, exchangeable with D2O),
5.37 (dd, 1 H, JOH,5′b = 7.2, JOH,5′a = 4.6, 5′-OH, exchangeable with D2O), 5.41 (d, 1 H, JOH,2′
=
6.2, 2′-OH, exchangeable with D2O), 5.88 (d, 1 H, J1′,2′ = 6.0, H-1′), 7.91 (br s, 1 H, NH,
exchangeable with D2O), 8.20 (s, 1 H, H-2), 8.34 (s, 1 H, H-8). 13C NMR (DMSO-d6): 37.85
(CH2N), 57.68 (CH2OH), 62.26 (C-5′), 71.23 (C-3′), 74.27 (C-2′), 86.62 (C-4′), 88.82 (C-1′),
120.19 (C-5), 127.96 (CH=), 132.53 (CH=), 140.73 (C-8), 148.69 (C-4), 153.17 (C-2), 155.04 (C-6).
Preparation of Azides
MeN3 was prepared by reaction of stoichiometric quantities of MeI and NaN3 (5 mmol) in
DMSO (5 ml) at ambient temperature overnight. This 1 M solution was used in the next
step.
PhOCH2OCH2N3, BnN3 and BzCH2N3 were prepared by reaction of PhOCH2OCH2Cl,
BnBr and BzOCH2CH2Br with the 2-fold excess of NaN3 in DMSO at ambient temperature
overnight. The azides were isolated by usual water–AcOEt work-up procedure. Their purity
was confirmed by 1H NMR.
Alkene-Azide Cycloaddition. General Method
A mixture of 2a (500 mg, 1.1 mmol), azide (1.65 mmol) and CuCl (23 mg, 0.22 mmol) in
MeCN (10 ml) was stirred at ambient temperature overnight. The reaction was monitored by
TLC. The mixture was diluted with AcOEt and washed successively with 0.1 M disodium
EDTA aqueous solution and brine. The organic phase was dried over Na2SO4 and evaporated.
The residue was applied to column chromatography on silica gel (CH2Cl2–EtOH 100:1→20:1).
N6-Acetyl-N6-[(1-methyl-1,2,3-triazol-4-yl)methyl]-2′,3′,5′-tri-O-acetyladenosine (4a). Yield 361 mg
(64%, foam). RF 0.33 (CH2Cl2–EtOH 25:1). 1H NMR (CDCl3): 2.10 (s, 3 H, AcO), 2.14 (s, 3 H,
AcO), 2.15 (s, 3 H, AcO), 2.29 (s, 3 H, AcN), 4.03 (s, 3 H, Me), 4.40 (dd, 1 H, J5′b,5′a = –12.5,
J5′b,4′ = 5.0, H-5′b), 4.46 (dd, 1 H, J5′a,5′b = –12.5, J5′a,4′ = 3.1, H-5′a), 4.48 (ddd, 1 H, J4′,5′b
=
5.0, J4′,5′a = 3.1, J4′,3′ = 4.3, H-4′), 5.43 (s, 2 H, CH2), 5.68 (dd, 1 H, J3′,4′ = 4.3, J3′,2′ = 5.3,
H-3′), 5.99 (dd, 1 H, J2′,3′ = 5.3, J2′,1′ = 5.3, H-2′), 6.26 (d, 1 H, J1′,2′ = 5.3, H-1′), 7.70 (s, 1 H,
5-Ht), 8.40 (s, 1 H, H-2), 8.81 (s, 1 H, H-8). 13C NMR (CDCl3): 20.47 (AcO), 20.55 (AcO),
20.86 (AcO), 24.35 (AcN), 36.84 (MeN), 43.16 (N6-CH2), 63.08 (C-5′), 70.67 (C-3′), 73.01
(C-2′), 80.65 (C-4′), 86.87 (C-1′), 125.06 (CHt), 126.93 (C-5), 143.48 (C-8), 144.42 (Ct),
152.46 (C-2), 152.84 (C-4), 153.15 (C-6), 169.40 (CO), 169.57 (CO), 170.36 (CO), 171.83
(CO). MS (APCI): m/z [M + H+] calculated for C22H27N8O8: 531.20, found 531.30.
N6-Acetyl-N6-[(1-benzyloxymethyl-1,2,3-triazol-4-yl)methyl]-2′,3′,5′-tri-O-acetyladenosine (4b).
Yield 624 mg (92%, foam). RF 0.38 (CH2Cl2–EtOH 25:1). 1H NMR (CDCl3): 2.10 (s, 3 H,
AcO), 2.12 (s, 3 H, AcO), 2.15 (s, 3 H, AcO), 2.32 (s, 3 H, AcN), 4.39 (dd, 1 H, J5′b,5′a = –12.4,
J5′b,4′ = 4.8, H-5′b), 4.45 (dd, 1 H, J5′a,5′b = –12.4, J5′a,4′ = 3.3, H-5′a), 4.48 (ddd, 1 H, J4′,5′b
=
4.8, J4′,5′a = 3.3, J4′,3′ = 4.5, H-4′), 4.48 (s, 2 H, CH2), 5.59 (s, 2 H, CH2), 5.63 (s, 2 H, CH2),
5.67 (dd, 1 H, J3′,4′ = 4.5, J3′,2′ = 5.6, H-3′), 5.96 (dd, 1 H, J2′,3′ = 5.6, J2′,1′ = 5.3, H-2′), 6.24 (d,
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1374
Kolyachkina et al.:
1 H, J1′,2′ = 5.3, H-1′), 7.25–7.36 (m, 5 H, Ph), 7.84 (s, 1 H, 5-Ht), 8.20 (s, 1 H, H-2), 8.79 (s,
1 H, H-8). 13C NMR (CDCl3): 20.45 (AcO), 20.54 (AcO), 20.79 (AcO), 24.21 (AcN), 42.95
(N6-CH2), 63.09 (C-5′), 70.65 (C-3′), 71.16 (CH2-Ph), 73.09 (C-2′), 77.64 (NCH2O), 80.58
(C-4′), 86.84 (C-1′), 123.62 (CHt), 127.54 (C-5), 128.28 (CH-Ph), 128.60 (CH-Ph), 129.79
(C-Ph), 142.35 (C-8), 144.93 (Ct), 152.39 (C-2), 152.96 (C-4), 153.16 (C-6), 169.38 (CO),
169.56 (CO), 170.29 (CO), 171.62 (CO). MS (APCI): m/z [M + H+] calculated for C29H33N8O9:
637.24, found 637.40.
N6-Acetyl-N6-[(1-benzyl-1,2,3-triazol-4-yl)methyl]-2′,3′,5′-tri-O-acetyladenosine (4c). Yield 513 mg
(78%, foam). RF 0.42 (CH2Cl2–EtOH 25:1). 1H NMR (CDCl3): 2.11 (s, 3 H, AcO), 2.13 (s, 3 H,
AcO), 2.16 (s, 3 H, AcO), 2.28 (s, 3 H, AcN), 4.40 (dd, 1 H, J5′b,5′a = –12.5, J5′b,4′ = 4.8, H-5′b),
4.45 (dd, 1 H, J5′a,5′b = –12.5, J5′a,4′ = 3.1, H-5′a), 4.47 (ddd, 1 H, J4′,5′b = 4.8, J4′,5′a = 3.1,
J4′,3′ = 4.7, H-4′), 5.43 (s, 2 H, CH2), 5.53 (s, 2 H, CH2), 5.67 (dd, 1 H, J3′,4′ = 4.7, J3′,2′ = 5.4,
H-3′), 5.96 (dd, 1 H, J2′,3′ = 5.4, J2′,1′ = 5.4, H-2′), 6.25 (d, 1 H, J1′,2′ = 5.4, H-1′), 7.17–7.23 (m,
2 H, Ph), 7.31–7.36 (m, 3 H, Ph), 7.62 (s, 1 H, 5-Ht), 8.20 (s, 1 H, H-2), 8.77 (s, 1 H, H-8).
13C NMR (CDCl3): 20.46 (AcO), 20.46 (AcO), 20.73 (AcO), 24.07 (AcN), 42.93 (N6-CH2),
54.12 (NCH2Ph), 63.04 (C-5′), 70.59 (C-3′), 73.01 (C-2′), 80.50 (C-4′), 86.64 (C-1′), 123.74
(CHt), 127.77 (C-5), 127.99 (CH-Ph), 128.59 (CH-Ph), 128.98 (CH-Ph), 134.61 (C-Ph), 142.35
(C-8), 145.50 (Ct), 152.30 (C-2), 152.35 (C-4), 153.09 (C-6), 169.33 (CO), 169.51 (CO),
170.23 (CO), 171.50 (CO). MS (APCI): m/z [M + H+] calculated for C28H31N8O8: 607.23,
found 607.38.
N6-Acetyl-N6-{[1-(2-benzoyloxyethyl)-1,2,3-triazol-4-yl]methyl}-2′,3′,5′-tri-O-acetyladenosine
(4d). Yield 652 mg (93%, foam). RF 0.27 (CH2Cl2–EtOH 25:1). 1H NMR (CDCl3): 2.10 (s, 3 H,
AcO), 2.12 (s, 3 H, AcO), 2.15 (s, 3 H, AcO), 2.28 (s, 3 H, AcN), 4.39 (dd, 1 H, J5′b,5′a = –12.3,
J5′b,4′ = 4.8, H-5′b), 4.44 (dd, 1 H, J5′a,5′b = –12.3, J5′a,4′ = 3.3, H-5′a), 4.46 (ddd, 1 H, J4′,5′b
4.8, J4′,5′a = 3.3, J4′,3′ = 4.5, H-4′), 4.66 (m, 4 H, CH2CH2), 5.55 (s, 2 H, CH2), 5.66 (dd, 1 H,
J3′,4′ = 4.5, J3′,2′ = 5.6, H-3′), 5.95 (dd, 1 H, J2′,3′ = 5.6, J2′,1′ = 5.3, H-2′), 6.23 (d, 1 H, J1′,2′
=
=
5.3, H-1′), 7.42–7.49 (m, 2 H, Bz), 7.50–7.60 (m, 1 H, Bz), 7.82 (s, 1 H, 5-Ht), 7.95–8.01 (m,
2 H, Bz), 8.19 (s, 1 H, H-2), 8.73 (s, 1 H, H-8). 13C NMR (CDCl3): 20.46 (AcO), 20.56 (AcO),
20.79 (AcO), 24.17 (AcN), 43.06 (N6-CH2), 49.20 (CH2), 62.85 (CH2), 63.11 (C-5′), 70.67
(C-3′), 73.09 (C-2′), 80.59 (C-4′), 86.67 (C-1′), 124.18 (CHt), 127.49 (C-5), 128.62 (CH-Ph),
129.37 (C-Ph), 129.81 (CH-Ph), 133.47 (CH-Ph), 142.37 (C-8), 144.91 (Ct), 152.36 (C-2),
152.86 (C-4), 153.14 (C-6), 166.02 (PhCO), 169.39 (CO), 169.58 (CO), 170.31 (CO), 171.62
(CO). MS (APCI): m/z [M + H+] calculated for C30H33N8O10: 665.23, found 665.39.
Preparation of Nucleosides. General Method of Ammonolysis
Corresponding compound was dissolved in 7 M ammonia solution in MeOH (5 mmol per
acetyl group) and left at ambient temperature for 48 h. The resulting slurry was diluted with
Et2O. The insoluble product was filtered, washed with Et2O and dried.
N6-(Benzyloxymethyl)adenosine (3b). Yield 117 mg (56%, solid) from 2a (300 mg, 0.54 mmol).
M.p. 138–140 °C. RF 0.57 (CH2Cl2–EtOH 4:1). 1H NMR (DMSO-d6): 3.57 (ddd, 1 H, J5′b,5′a
–11.9, J5′b,4′ = 4.0, J5′b,OH = 7.2, H-5′b), 3.69 (ddd, 1 H, J5′a,5′b = –11.9, J5′a,4′ = 4.0, J5′a,OH
4.6, H-5′a), 3.97 (ddd, 1 H, J4′,5′b = 4.0, J4′,5′a = 4.0, J4′,3′ = 3.2, H-4′), 4.16 (ddd, 1 H, J3′,4′
3.2, J3′,2′ = 4.8, J3′,OH = 4.9, H-3′), 4.58 (s, 2 H, OCH2Ph), 4.61 (ddd, 1 H, J2′,3′ = 4.9, J2′,1′
=
=
=
=
6.0, J2′,OH = 6.2, H-2′), 5.13 (br s, 2 H, NCH2O), 5.14 (d, 1 H, JOH,3′ = 4.8, 3′-OH, exchange-
able with D2O), 5.26 (dd, 1 H, JOH,5′b = 7.2, JOH,5′a = 4.6, 5′-OH, exchangeable with D2O),
5.42 (d, 1 H, JOH,2′ = 6.2, 2′-OH, exchangeable with D2O), 5.93 (d, 1 H, J1′,2′ = 6.0, H-1′),
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N6-Substituted Adenosines
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7.22–7.36 (m, 5 H, H-Ph), 8.31 (br s, 1 H, H-2), 8.44 (s, 1 H, H-8), 8.70 (br s, 1 H, NH,
exchangeable with D2O). 13C NMR (DMSO-d6): 61.52 (C-5′), 68.88 (PhCH2), 70.50 (C-3′),
70.50 (OCH2N), 73.52 (C-2′), 85.77 (C-4′), 87.81 (C-1′), 119.80 (C-5), 127.17 (Ph), 127.39
(Ph), 128.06 (Ph), 138.54 (Ph), 140.43 (C-8), 149.32 (C-4), 152.10 (C-2), 154.41 (C-6). UV
(H2O), λmax in nm (ε): pH 1, 260 (13 700), 266 (14 900); pH 7, 260 (16 700), 265 (17 500);
pH 13, 260 (14 800), 265 (15 800). MS (APCI): m/z [M + H+] calculated for C18H22N5O5:
388.16, found 388.14; m/z [M – H+ + HCOOH] calculated for C19H22N5O7: 432.15, found
432.06.
N6-[(1-Methyl-1,2,3-triazol-4-yl)methyl]adenosine (5a). Yield 123 mg (60%, solid) from 4a
(300 mg, 0.57 mmol). M.p. 76–78 °C. RF 0.41 (CH2Cl2–EtOH 4:1). 1H NMR (DMSO-d6+D2O):
3.55 (dd, 1 H, J5′b,5′a = –12.2, J5′b,4′ = 3.6, H-5′b), 3.65 (dd, 1 H, J5′a,5′b = –12.2, J5′a,4′ = 2.4,
H-5′a), 3.94 (s, 3 H, Me), 3.97 (ddd, 1 H, J4′,5′b = 3.6, J4′,5′a = 2.4, J4′,3′ = 3.1, H-4′), 4.14 (dd,
1 H, J3′,4′ = 3.1, J3′,2′ = 4.9, H-3′), 4.58 (dd, 1 H, J2′,3′ = 4.9, J2′,1′ = 6.2, H-2′), 4.73 (br s, 2 H,
CH2), 5.87 (d, 1 H, J1′,2′ = 6.2, H-1′), 7.83 (s, 1 H, 5-Ht), 8.19 (br s, 1 H, NH), 8.22 (s, 1 H,
H-2), 8.33 (s, 1 H, H-8). 13C NMR (DMSO-d6): 35.40 (N6-CH2), 36.04 (Me), 61.61 (C-5′),
70.59 (C-3′), 73.48 (C-2′), 85.85 (C-4′), 87.91 (C-1′), 119.80 (C-5), 123.59 (CHt), 139.90 (C-8),
145.52 (Ct), 148.60 (C-4), 152.24 (C-2), 154.30 (C-6). UV (H2O), λmax in nm (ε): pH 1, 260
(23 400), 264 (24 400); pH 7, 260 (24 300), 266 (25 900); pH 13, 260 (14 800), 265 (15 800).
MS (APCI): m/z [M + H+] calculated for C14H19N8O4: 363.15, found 363.07; m/z [M – H+
+
HCOOH] calculated for C15H29N8O6: 407.14, found 407.08.
N6-[(1-Benzyloxymethyl-1,2,3-triazol-4-yl)methyl]adenosine (5b). Yield 163 mg (74%, solid)
from 4b (300 mg, 0.54 mmol). M.p. 87–89 °C. RF 0.38 (CH2Cl2–EtOH 4:1). 1H NMR
(DMSO-d6+D2O): 3.57 (dd, 1 H, J5′b,5′a = –12.1, J5′b,4′ = 3.7, H-5′b), 3.68 (dd, 1 H, J5′a,5′b
=
–12.1, J5′a, 4′ = 3.4, H-5′a), 3.97 (ddd, 1 H, J4′,5′b = 3.7, J4′,5′a = 3.4, J4′,3′ = 2.6, H-4′), 4.16 (dd,
1 H, J3′,4′ = 2.6, J3′,2′ = 5.2, H-3′), 4.52 (s, 2 H, CH2), 4.62 (dd, 1 H, J2′,3′ = 5.2, J2′,1′ = 6.0,
H-2′), 4.78 (br s, 2 H, CH2), 5.74 (s, 2 H, CH2), 5.90 (d, 1 H, J1′,2′ = 6.0, H-1′), 7.20–7.35 (m,
5 H, Ph), 8.08 (s, 1 H, 5-Ht), 8.25 (s, 1 H, H-2), 8.32 (br s, 1 H, NH), 8.38 (s, 1 H, H-8).
13C NMR (DMSO-d6): 35.37 (N6-CH2), 61.61 (C-5′), 70.42 (CH2Ph), 70.58 (C-3′), 73.48 (C-2′),
77.31 (CH2O), 85.84 (C-4′), 87.94 (C-1′), 119.86 (C-5), 123.33 (CHt), 127.64 (CHPh), 127.72
(CHPh), 128.24 (CHPh), 136.78 (CPh), 139.92 (C-8), 145.94 (Ct), 148.52 (C-4), 152.22 (C-2),
154.29 (C-6). UV (H2O), λmax in nm (ε): pH 1, 260 (16 600), 264 (17 600); pH 7, 260
(17 900), 266 (18 500); pH 13, 260 (16 600), 266 (18 000). MS (APCI): m/z [M + H+] calcu-
lated for C21H25N8O5: 469.19, found 469.12; m/z [M – H+ + HCOOH] calculated for
C
22H25N8O7: 513.18, found 513.01.
N6-[(1-Benzyl-1,2,3-triazol-4-yl)methyl]adenosine (5c). Yield 189 mg (84%, solid) from 4c
(300 mg, 0.49 mmol). M.p. 92–94 °C. RF 0.67 (CH2Cl2–EtOH 4:1). 1H NMR (DMSO-d6+D2O):
3.57 (dd, 1 H, J5′b,5′a = –12.3, J5′b,4′ = 3.6, H-5′b), 3.66 (dd, 1 H, J5′a,5′b = –12.3, J5′a, 4′ = 3.1,
H-5′a), 3.97 (ddd, 1 H, J4′,5′b = 3.6, J4′,5′a = 3.1, J4′,3′ = 3.3, H-4′), 4.14 (dd, 1 H, J3′,4′ = 3.3,
J3′,2′ = 4.7, H-3′), 4.58 (dd, 1 H, J2′,3′ = 4.7, J2′,1′ = 6.2, H-2′), 4.75 (br s, 2 H, CH2), 5.49 (s,
2 H, CH2), 5.87 (d, 1 H, J1′,2′ = 6.2, H-1′), 7.20–7.38 (m, 5 H, Ph), 7.94 (s, 1 H, 5-Ht), 8.19
(br s, 1 H, NH), 8.21 (s, 1 H, H-2), 8.32 (s, 1 H, H-8). 13C NMR (DMSO-d6): 35.49 (N6-CH2),
52.61 (CH2Ph), 61.60 (C-5′), 70.58 (C-3′), 73.47 (C-2′), 85.84 (C-4′), 87.92 (C-1′), 119.69
(C-5), 122.88 (CHt), 127.81 (CHPh), 127.96 (CHPh), 128.60 (CHPh), 136.10 (CPh), 139.87
(C-8), 145.70 (Ct), 148.52 (C-4), 152.21 (C-2), 154.28 (C-6). UV (H2O), λmax in nm (ε): pH 1,
260 (10 800), 264 (11 100); pH 7, 260 (11 900), 266 (12 500); pH 13, 260 (10 400), 267
(11 100). MS (APCI): m/z [M + H+] calculated for C20H23N8O4: 439.18, found 439.14; m/z
[M – H+ + HCOOH] calculated for C21H23N8O6: 483.17, found 483.01.
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Kolyachkina et al.:
N6-{[1-(2-Hydroxyethyl)-1,2,3-triazol-4-yl]methyl}adenosine (5d). Yield 189 mg (46%,
solid) from 4d (300 mg, 0.45 mmol). M.p. 97–99 °C. RF 0.12 (CH2Cl2–EtOH 4:1). 1H NMR
(DMSO-d6+D2O): 3.56 (dd, 1 H, J5′b,5′a = –12.2, J5′b,4′ = 3.3, H-5′b), 3.66 (dd, 1 H, J5′a,5′b
=
–12.2, J5′a,4′ = 3.5, H-5′a), 3.73 (t, 3 H, J = 5.3, CH2), 3.97 (ddd, 1 H, J4′,5′b = 3.3, J4′,5′a = 3.5,
J4′,3′ = 3.1, H-4′), 4.14 (dd, 1 H, J3′,4′ = 3.1, J3′,2′ = 5.1, H-3′), 4.32 (t, 3 H, J = 5.3, CH2), 4.58
(dd, 1 H, J2′,3′ = 5.1, J2′,1′ = 6.2, H-2′), 4.76 (br s, 2 H, CH2), 5.87 (d, 1 H, J1′,2′ = 6.2, H-1′),
7.85 (s, 1 H, 5-Ht), 8.19 (br s, 1 H, NH), 8.23 (s, 1 H, H-2), 8.33 (s, 1 H, H-8). 13C NMR
(DMSO-d6): 35.41 (N6-CH2), 51.99 (CH2), 59.83 (CH2), 61.62 (C-5′), 70.59 (C-3′), 73.46
(C-2′), 85.85 (C-4′), 87.92 (C-1′), 119.78 (C-5), 123.12 (CHt), 139.88 (C-8), 145.12 (Ct),
148.48 (C-4), 152.25 (C-2), 154.29 (C-6). UV (H2O), λmax in nm (ε): pH 1, 260 (19 900), 264
(20 800); pH 7, 260 (21 000), 266 (22 800); pH 13, 260 (19 900), 266 (21 900). MS (APCI):
m/z [M + H+] calculated for C15H21N8O5: 393.16, found 392.52; m/z [M – H+ + HCOOH]
calculated for C16H21N8O7: 437.15, found 437.05.
O6-Benzylinosine (6)
To benzylalcohol (5 ml) was carefully added with stirring 60% NaH in paraffin (360 mg,
8.9 mmol) and the mixture was stirred at room temperature for additional 1 h. To the re-
sulted mixture was added 6-chloro-9-(2′,3′,5′-tri-O-acetyribozyl)purine24 (370 mg, 0.89 mmol).
The mixture was stirred at room temperature overnight. After neutralization with AcOH
(0.6 ml) the viscous solution was directly applied for column chromatography. Elution with
a gradient of EtOH in CH2Cl2 (1:100→1:9) gave 260 mg (81%, solid) of 6. M.p. 153–155 °C.
RF 0.63 (CH2Cl2–EtOH 9:1). 1H NMR (DMSO-d6): 3.57 (ddd, 1 H, J5′b,5′a = –12.2, J5′b,4′ = 3.8,
J5′b,OH = 6.0, H-5′b), 3.68 (ddd, 1 H, J5′a,5′b = –12.2, J5′a,4′ = 4.2, J5′a,OH = 5.3, H-5′a), 3.97
(ddd, 1 H, J4′,5′b = 3.8, J4′,5′a = 4.2, J4′,3′ = 3.4, H-4′), 4.17 (ddd, 1 H, J3′,4′ = 3.4, J3′,2′ = 5.1,
J3′,OH = 4.9, H-3′), 4.59 (ddd, 1 H, J2′,3′ = 5.1, J2′,1′ = 5.7, J2′,OH = 6.0, H-2′), 5.14 (dd, 1 H,
JOH,5′b = 6.0, JOH,5′a = 5.3, 5′-OH, exchangeable with D2O), 5.21 (d, 1 H, JOH,3′ = 4.9, 3′-OH,
exchangeable with D2O), 5.49 (d, 1 H, JOH,2′ = 6.0, 2′-OH, exchangeable with D2O), 5.63 (d,
Ja,b = 12.7, CHbHaPh), 5.66 (d, Ja,b = 12.7, CHbHaPh), 5.99 (d, 1 H, J1′,2′ = 5.7, H-1′),
7.32–7.44 (m, 3 H, H-Ph), 7.48–7.53 (m, 2 H, H-Ph), 8.56 (s, 1 H, H-2), 8.61 (s, 1 H, H-8).
13C NMR (DMSO-d6): 61.27 (C-5′), 67.79 (PhCH2), 70.30 (C-3′), 73.76 (C-2′), 85.69 (C-4′),
87.81 (C-1′), 121.13 (C-5), 128.11 (Ph), 128.25 (Ph), 128.43 (Ph), 136.23 (Ph), 142.49 (C-8),
151.52 (C-2), 151.97 (C-4), 159.80 (C-6). UV (H2O), λmax in nm (ε): pH 1, 251 (11 600);
pH 7, 252 (13 300); pH 13, 251 (12 500). MS (APCI): m/z [M + H+] calculated for
C
17H19N4O5: 359.14, found 359.21.
This work was supported by the Molecular and Cellular Biology Program of the Russian Academy of
Sciences, the Russian Foundation for Basic Research and the Russian Federal Program (contract No.
16.512.11.2239).
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