Synthesis of sugar-modified analogs of bredinin (mizoribine), a clinically useful immunosuppressant, by a novel photochemical imidazole ring-cleavage reaction as the key step

Article abstract of DOI:10.1039/b005510g

The imidazole nucleoside bredinin (mizoribine) is a clinically useful immunosuppressant. Derivatization of bredinin by the usual nucleoside chemistry is often troublesome due to the unusual zwitterionic structure of the base moiety. We achieve the synthes

Full text of DOI:10.1039/b005510g

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Synthesis of sugar-modified analogs of bredinin (mizoribine),
a clinically useful immunosuppressant, by a novel photochemical
imidazole ring-cleavage reaction as the key step†1
1
Satoshi Shuto,* Kimiyo Haramuishi, Masayoshi Fukuoka and Akira Matsuda*
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Japan. E-mail: shu@pharm.hokudai.ac.jp
Received (in Cambridge, UK) 10th July 2000, Accepted 1st September 2000
First published as an Advance Article on the web 10th October 2000
The imidazole nucleoside bredinin (mizoribine) is a clinically useful immunosuppressant. Derivatization of
bredinin by the usual nucleoside chemistry is often troublesome due to the unusual zwitterionic structure of the
base moiety. We achieve the synthesis of 5Ј-modified analogs of bredinin via a novel photochemical imidazole ring-
cleavage reaction as the key step. When a solution of 2Ј,3Ј-O-isopropylidenebredinin 5 in 0.1 M AcOH is irradiated
with a high-pressure mercury lamp, an imidazole ring-cleavage reaction occurs to give the 2-aminomalonamide
riboside derivative 16 in 71% yield. Appropriate modifications of the 5Ј-position of 16 and subsequent condensation
with (EtO)3CH to reconstruct the imidazole base moiety follow. Using this imidazole ring-cleavage–reconstruction
strategy, some biologically important 5Ј-modified bredinin analogs, i.e., the 5Ј-phosphate 2, the 5Ј-deoxy derivative 3,
and the 5Ј-O-aminopropylcarbamate 4 are efficiently synthesized.
Introduction
Bredinin (mizoribine, 1) is an imidazole nucleoside anti-
biotic isolated from Eupenicillium brefeldianu2 and now
clinically used as an immunosuppressant,3,4 especially for the
transplantation of viscera3a,b and for autoimmune diseases such
as rheumatism.3c Bredinin as well as its analogs also have anti-
tumor effects in experimental tumor models,5 and recently its
significant antiviral effect was also reported.6 Therefore, chem-
ical modification studies of bredinin may result in the develop-
ment of useful compounds with efficient pharmacological
effects. However, derivatization of bredinin by the conventional
methods used in nucleoside chemistry is often troublesome,5a,7
probably because of the unusual zwitterionic structure of the
base moiety. Thus, despite its biological interest, only a few
studies on the synthesis of derivatives of bredinin have been
reported.5
We planned to synthesize the currently biologically import-
ant 5Ј-modified derivatives of bredinin, the 5Ј-phosphate 2, the
5Ј-deoxy derivative 3, and the 5Ј-O-(3-aminopropyl)carbamate
Fig. 1
4 (Fig. 1). However, our first attempt at synthesizing the target
compounds from 2Ј,3Ј-O-isopropylidenebredinin 5, which was
amino-4-carbamoylimidazol-1-yl-β--ribofuranoside (AICAR,
9) using a novel photoreaction.7 When an acidic aqueous solu-
tion of 9 or its triacetate 10 was irradiated with a UV lamp, an
imidazole ring-cleavage reaction occurred to give the 2-amino-
malonamide derivative 11 or 12, respectively as the major
product. We found that upon heating with (EtO)3CH in DMF,
compounds 11 and 12 were converted into bredinin 1 and its
triacetate 13, respectively, as shown in Scheme 2.7 Accordingly,
2-aminomalonamide ribosides, such as 11 and 12, are con-
sidered synthetic equivalents of bredinin. Although 11 or its tri-
O-acetate 12 might also be useful intermediates for preparing
various bredinin derivatives,5a the yields of 11 and 12 from
AICAR or tri-O-acetyl-AICAR were low (about 30%) and were
obtained on only a 100 mg scale once.5a,7
readily obtained by treating bredinin with TsOH–acetone, was
unsuccessful. When an electron-withdrawing group was intro-
duced at the 5Ј-position, intramolecular attack by the 2-oxygen
of the base moiety quickly occurred. For example, treatment of
5 under the usual mesylation conditions or phosphotriester
method produced none of the desired 5Ј-O-mesylester 6 or
5Ј-phosphate 7. Both reactions instead gave the 5,5Ј-anhydro
derivative 8 as the major product (Scheme 1). We also tried
protecting the phenolic hydroxy group of the base moiety of
bredinin with acyl and silyl groups, but this also proved
unsuccessful.
Previously, we reported the synthesis of bredinin from 5-
† 1H NMR spectral charts of 15, 19, 20, 22 and 4, and H–H COSY
spectral charts of 15, 17, 18, 24 and 4 are available as supplementary
data from BLDSC (SUPPL. NO. 57713, 11 pp.) or the RSC Library.
See Instructions for Authors available via the RSC web page (http://
www.rsc.org/authors).
In this paper, we describe an efficient preparation of 12 and
the corresponding 2Ј,3Ј-O-acetonide 16 (see Scheme 5, below)
from bredinin by a photochemical reaction, and conversion
of 16 into the biologically important 5Ј-modified bredinin
DOI: 10.1039/b005510g
J. Chem. Soc., Perkin Trans. 1, 2000, 3603–3609
This journal is © The Royal Society of Chemistry 2000
3603

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Scheme 3
2-aminomalonamide 14, respectively, are possible. We expected
that the desired 2-aminomalonamide ribosides 11 or 12 might
be obtained efficiently if the photoreaction was carried out
with bredinin 1, or its sugar-protected derivatives, as a sub-
strate, since the acidic hydrolysis process of the amidinium
moiety would not be needed in this case. Thus, we examined the
reaction with bredinin.
We first investigated the photoreaction with bredinin 2Ј,3Ј,5Ј-
tri-O-acetate 13 (Scheme 4), which was readily prepared from
Scheme 1
Scheme 4
bredinin by the usual method with Ac2O–pyridine, in order to
make the detection and purification of the reaction products
easy. When a solution of 13 in 0.1 M HCl was irradiated with a
high-pressure mercury lamp (100 W) under bubbling of argon,
the UV-absorption of the solution rapidly faded, and work-up
gave 12 in 79% yield as a diastereomeric mixture at the
2-position, which was identical with the photoreaction product
of tri-O-acetyl-AICAR 10 previously reported.7
Next, the photoreaction of 2Ј,3Ј-O-isopropylidenebredinin 5
(Scheme 5) was carried out in aq. AcOH to avoid hydrolysis of
the acetonide moiety. Thus, a solution of 5 in 0.1 M AcOH was
irradiated with a high-pressure mercury lamp to give the desired
imidazole ring-cleavage product 16 in 71% yield as a diastereo-
meric mixture at the 2-position, as in the above reaction with
the triacetate 13. In this reaction, the 2,5Ј-cyclized product 18
was also obtained, in 10% yield, as a by-product, the structure
Scheme 2
1
of which was confirmed by H and 13C NMR, HRMS and
derivatives, i.e., the 5Ј-phosphate 2, the 5Ј-deoxy derivative 3,
and the 5Ј-O-(3-aminopropyl)carbamate 4 (Fig. 1), via recon-
struction of the imidazole base moiety.8
elemental analytical data. The configuration at the 2-position
was assigned as S by an NOE experiment (Scheme 5). When the
photoreaction was carried out with a low-pressure instead of
a high-pressure mercury lamp (60 W), the imidazole ring-
cleavage product 16 was formed in 68% yield, with none of the
cyclized product 18 produced. The 2-amino group of the photo-
product 16 was protected with a Boc group by the usual method
for further derivatization.
There is no precedent for such a photochemical ring-cleavage
reaction of imidazole derivatives except for our previous results
with AICAR. Although the mechanism of the photoreaction is
not clear, protonation of the base moiety would significantly
facilitate the reaction (the pKa of bredinin is 6.752a), because
Results and discussion
Photoreaction of bredinin
Although the reaction mechanism of the imidazole ring-
cleavage reaction of AICAR is unclear, it is likely that the
2-aminomalonamide ribosides 11 and 12 are produced via acid-
ic hydrolysis of the amidinium intermediate I (Scheme 3). In
the acidic hydrolysis of I, two pathways, producing the desired
2-aminomalonamide ribosides 11 or 12 and the undesired
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of bredinin’s unusual chemical features; for instance, when
bredinin was treated under Yoshikawa’s phosphorylation con-
ditions,11 which is the most useful method for preparing nucleo-
side 5Ј-phosphates, the desired product 2 was obtained in only
3% yield.12 Attempted phosphorylations at the 5Ј-hydroxy
position of 2Ј,3Ј-O-isopropylidenebredinin 5 by the phos-
photriester method described above, as well as by the
phosphoramidite method, by our hand, were also unsuccessful.
We sought to develop a practical method for preparing 2 via
the above photoreaction of bredinin. Therefore, we investigated
introducing a phosphate unit at the 5Ј-position of N2-protected
photoproduct 17, and found that a phosphoramidite method
with o-xylylene N,N-diethylphosphoramidite (XEPA)13 was
effective in this system (Scheme 7). Treatment of 17 with XEPA
and tetrazole in CH2Cl2, followed by oxidation with aq. I2, gave
the corresponding 5Ј-phosphotriester 19 in 70% yield. The
isopropylidene and Boc groups of 19 were removed simul-
taneously with 90% aq. TFA, and the resulting product, with-
out purification, was heated with (EtO)3CH in DMF at 90 ЊC to
give the bredinin 5Ј-phosphate derivative 20 in 47% yield from
19. Hydrogenation of 20 with Pd-carbon in MeOH furnished
bredinin 5Ј-phosphate 2, which was isolated as a disodium salt
in 89% yield, after successive treatment with Dowex 50 (Hϩ)
and Diaion WK-20 (Naϩ) resins.
Scheme 5
when performed under neutral conditions the photoreaction
proceeded only slowly. Formation of the cyclized product 18 as
a by-product, which is the result of an addition of a 5Ј-hydroxy
group at the 2-position, suggested a possible reaction pathway
giving 16 via hydration at the 2-position followed by deformyl-
ation (Scheme 6). However this mechanism may not be plaus-
Synthesis of 5Ј-deoxybredinin
4-Carbamoylimidazolium-5-olate 25, the aglycone of bredinin,
is known to show potent antitumor effects in vivo stronger than
those of bredinin itself.5c We designed 5Ј-deoxybredinin 3 as a
potential antitumor agent. This compound was expected to
release the aglycone 25 efficiently in tumor cells by the action of
nucleoside phosphorylase (NP) (Scheme 8), since the activity of
this enzyme is significantly increased in tumor cells compared
with normal cells.14,15
The synthesis of 3 is shown in Scheme 7. The N2-(Boc-
amino)malonamide riboside derivative 17 was successively
treated with MsCl–py and NaI–methyl ethyl ketone (MEK) to
give the corresponding 5Ј-deoxy-5Ј-iodo derivative 21 in 90%
yield. The 5Ј-iodide 21 was then subjected to radical reduction
with Bu3SnH–azoisobutyronitrile (AIBN) in benzene to give
the 5Ј-deoxy derivative 22 quantitatively. Removal of the
protecting groups of 22 with 90% TFA and subsequent
reconstruction of the imidazole ring with (EtO)3CH afforded
the target 5Ј-deoxybredinin 3 in 77% yield.
Scheme 6
Synthesis of bredinin 5Ј-O-(3-aminopropyl)carbamate
ible, since, when the N2-formyl derivative 15, prepared from 12
by treatment with formic acid and DCC,9 was subjected to the
above acidic photoreaction conditions with a high-pressure
mercury lamp, deformylation did not occur and none of the
product 12 was obtained (Scheme 4).
This photoreaction of bredinin derivatives is very useful,
since more than 5 g of the photoproduct can be readily
obtained at once, while only 100 mg of the product was
obtained by the reaction with AICAR derivatives. Therefore,
we planned to convert the photoproduct into bredinin deriv-
atives of biological importance.
The 5Ј-O-(3-aminopropyl)carbamate 4 was designed as a
bredinin derivative having an aminoalkyl tether, which can
bond covalently to other molecules. Such a bredinin derivative
would be very useful for biological studies, which include syn-
thesis of haptens for preparing antibodies to bredinin and also
preparation of bredinin-attached resins for affinity chromato-
graphy.
Compound 17 was successively treated with carbonyl-
diimidazole–4-(dimethylamino)pyridine (DMAP) and N-Cbz-
propanediamine–Et3N in CH2Cl2 to give the 5Ј-O-carbamate
derivative 23 in 91% yield. Deprotection and reconstruction of
the base by the above method gave the bredinin 5Ј-O-amino-
propylcarbamate 24, which was hydrogenated with Pd-C in the
presence of HCl in MeOH to afford the target compound 4,
as shown in Scheme 7.
Synthesis of bredinin 5Ј-phosphate
The mechanism of action of bredinin as an immunosuppres-
sant has been studied.3,4,10 In cells, bredinin is metabolized into
the 5Ј-phosphate 2 (Fig. 1) by adenosine kinase,10 which
inhibits cellular inosine monophosphate (IMP) dehydrogenase,
an essential enzyme in the de novo synthesis of guanine ribo-
nucleotides. Bredinin inhibits T lymphocyte proliferation due to
this antimetabolic effect. Accordingly, the 5Ј-phosphate 2, the
active form of bredinin in cells, should be very useful as a
tool in pharmacological studies. However, an efficient method
for preparing 2 has not been developed, probably because
Conclusions
We have found that the imidazole base moiety of bredinin is
cleaved easily by irradiation under aqueous acidic conditions to
give 2-aminomalonamide riboside derivatives and that the base
moiety of bredinin can be reconstructed when the photo-
product is heated with (EtO)3CH. Using this imidazole ring-
J. Chem. Soc., Perkin Trans. 1, 2000, 3603–3609
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Scheme 7
(16.0 g, 84.0 mmol) in acetone (800 mL) was stirred at room
temperature for 2 h, and then the resulting solution was neutral-
ized with NH4OH (28% aq.). The resulting precipitate was
filtered off and washed well with EtOH, and the filtrate was
evaporated. The residue was purified by column chromato-
graphy (SiO2; CHCl3–MeOH, 10:1) to give 5 (9.63 g, 80%) as a
solid; mp 209–212 ЊC (from MeOH) (Found: C, 48.14; H, 5.82;
N, 13.86. C12H17N3O6 requires C, 48.16; H, 5.73; N, 14.04%); 1H
NMR (400 MHz; DMSO-d6 ϩ D2O) δ 8.27 (s, 1 H), 5.73 (d,
1 H, J = 2.6), 5.16 (dd, 1 H, J = 5.9, 2.6), 4.85 (dd, 1 H, J = 5.9,
2.6), 4.15 (m, 1 H), 3.57 (m, 2 H), 1.49 and 1.30 (each s, each
3 H); MS (FAB, positive) m/z 300 (MHϩ).
Scheme 8
5,5Ј-Anhydrobredinin 8
cleavage–reconstruction strategy, several biologically important
5Ј-modified bredinin analogs were efficiently synthesized.
A mixture of 5 (299 mg, 1.0 mmol) and MsCl (88 µL, 1.3 mmol)
in pyridine (8 mL) was stirred at 0 ЊC for 1 h. After addition of
water (1.0 mL), the resulting mixture was evaporated, and the
residue was partitioned between EtOAc and brine. The organic
layer was dried (Na2SO4), evaporated, and purified by column
chromatography (SiO2; CHCl3–MeOH, 100:1, then 20:1) to
give 8 (138 mg, 36%) as a foam; 1H NMR (100 MHz; CDCl3 ϩ
D2O) δ 7.19 (s, 1 H), 5.76 (s, 1H), 5.07 (d, 1 H, J = 5.6), 4.77 (d,
1 H, J = 5.6), 4.69 (d, 1 H, J = 2.2), 4.56 (dd, 1 H, J = 12.9, 2.2),
4.06 (d, 1 H, J = 12.9), 1.54 and 1.53 (each s, each 3 H); MS (CI)
m/z 282 (MHϩ); UV (MeOH) λmax 280 nm.
Experimental
Mps were measured on a Yanagimoto MP-3 micro-melting
1
point apparatus and are uncorrected. H NMR spectra were
recorded at 100, 400, and 500 MHz (1H) and at 100 MHz (13C).
Chemical shifts (δ) and coupling constants (J ) are reported in
ppm downfield from TMS and in Hz, respectively. Assignments
1
of H and 13C NMR described are based on COSY and/or
DEPT spectra. Mass spectra were obtained by fast-atom
bombardment (FAB) or chemical ionization (CI). Thin-layer
chromatography (TLC) was done on Merck 60F254 coated plates.
Silica gel chromatography was done with Merck silica gel 5715
or 9385. Reactions were carried out under an argon atmosphere.
2Ј,3Ј,5Ј-Tri-O-acetylbredinin 13
A mixture of bredinin 1 (5.18 g, 20 mmol) and Ac2O (9.5 mL,
100 mmol) in pyridine (50 mL) was stirred at room temperature
for 3 h. After MeOH was added, the solvent was evaporated,
and the residue was treated with hot CHCl3–MeOH to give
crystalline 13 (5.13 g, 67%); mp 188–190 ЊC (Found: C, 47.05;
2Ј,3Ј-O-Isopropylidenebredinin 5
A suspension of bredinin 1 (10.4 g, 40 mmol) and TsOHؒH2O
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H, 5.09; N, 10.90. C15H19N3O9 requires C, 46.76; H, 4.97; N,
10.90%); H NMR (100 MHz; DMSO-d6) δ 8.35 (s, 1 H), 6.88
(br s, 2 H), 5.80 (m, 2 H), 5.52 (m, 1 H), 4.42–4.11 (m, 3 H),
2.08, 2.07 and 2.03 (each s, each 3 H); MS (CI) m/z 385 (Mϩ);
UV (MeOH) λmax 284 nm.
with a high-pressure mercury lamp (100 W, Pyrex filter) at room
1
temperature under bubbling of argon for 6 h. The resulting
white precipitate was filtered off and washed with water to give
2,5Ј-cyclized product 18 (145 mg, 10%) as crystals, mp 241–
242 ЊC (Found: C, 46.81; H, 5.70; N, 13.80. C12H17N3O6ؒ1/2H2O
1
requires C, 46.75; H, 5.89; N, 13.63%); H NMR (400 MHz;
DMSO-d6/D2O) δ 5.37 (s, 1 H, H-1Ј), 5.04 (s, 1 H, H-2), 4.83
(d, 1 H, H-3Ј, J = 5.9), 4.75 (d, 1 H, H-2Ј, J = 5.9), 4.48 (m, 1 H,
H-4Ј), 4.11 (dd, 1 H, H-5Јa, J = 13.2, 1.0), 3.74 (dd, 1 H, H-5Јb,
J = 13.2, 2.0), 1.40 and 1.28 (each s, each 3 H, CHMe2); NOE
irradiated H-1Ј, observed H-2 (1.1%), H-2Ј (5.8%); irradiated
H-2, observed H-1Ј (3.0%), H-5Јb (6.2%); 13C NMR (100 MHz,
DMSO-d6) δ 169.68 (C), 165.46 (C), 111.89 (C), 88.63 (CH),
87.76 (CH), 85.92 (CH), 84.55 (CH), 80.96 (CH), 72.33 (C),
71.94 (CH2), 25.95 (CH3), 24.43 (CH3); MS (CI) m/z 300
(MHϩ).
2-Amino-N-(2,3,5-tri-O-acetyl-ꢀ-D-ribofuranosyl)malonamide
12
A solution of 13 (4.10 g, 10.6 mmol) in aq. HCl (0.1 M; 500
mL) was irradiated with a high-pressure mercury lamp (100 W,
Pyrex filter) at room temperature under bubbling of argon for
3.5 h. After bring neutralized with aq. NaHCO3 (0.8 M), the
resulting mixture was concentrated to about 50 mL, and then
NaCl was added. The resulting solution was extracted with
CHCl3 (3 times), and the combined organic layer was dried
(Na2SO4) and evaporated. The residue was purified by flash
column chromatography (SiO2; CHCl3–MeOH, 17:1) to give
12 (3.14 g, 79%) as a foam, the 1H NMR spectral data of which
were identical with those reported previously.7a
The filtrate was neutralized with aq. NaHCO3 (0.8 M) and
evaporated. The residue was purified by column chromato-
graphy (SiO2; CHCl3–MeOH, 10:1 then 5:1) to give 16 (1.02 g,
71%) as a foam.
2-Formamido-N-(2,3,5-tri-O-acetyl-ꢀ-D-ribofuranosyl)malon-
amide 15
2-(tert-Butoxycarbonylamino)-N-(2,3-O-isopropylidene-ꢀ-D-
ribofuranosyl)malonamide 17
A mixture of DCC (413 mg, 2.0 mmol) and formic acid (151
µL, 4.0 mmol) in CHCl3 (5 mL) was stirred at 0 ЊC for 10 min.
A solution of 12 (357 mg, 1.0 mmol) in pyridine (3 mL) was
added, and the resulting mixture was stirred at 0 ЊC for 15 min
and then at room temperature for 1 h. The resulting white pre-
cipitate was filtered off, and the filtrate was evaporated. The
residue was partitioned between CHCl3 and brine, and the
aqueous layer was extracted with CHCl3 (5 times). The com-
bined organic layer was dried (Na2SO4), evaporated, and puri-
fied by flash column chromatography (SiO2; CHCl3–MeOH,
30:1, then 25:1) to give 15 (187 mg, 46%) as a foam: 1H NMR
(500 MHz; CDCl3) δ 8.32 and 8.30 (each s, each 0.5 H, CHO),
8.18 and 8.10 (each d, each 0.5 H, 1-NH, J = 8.5 and 8.8,
respectively), 7.65 and 7.61 (each d, each 0.5 H, NHCHO,
J = 6.4 and 6.4, respectively), 7.00 (br s, 1H, CONH2), 6.40 and
6.31 (each br s, each 0.5 H, CONH2), 5.68–5.61 (m, 1 H, H-1Ј),
5.32–5.26 (m, 2 H, H-2Ј and -3Ј), 5.10 and 5.07 (each d, each 0.5
H, H-3, J = 6.4 and 6.4, respectively), 4.30–4.15 (m, 3 H, H-4Ј
A mixture of 16 (867 mg, 3.0 mmol), Boc2O (981 mg, 4.5
mmol), and Et3N (0.63 mL, 4.5 mmol) in 1,4-dioxane (15 mL)
was stirred at room temperature for 2.5 h. The resulting mix-
ture was evaporated, and the residue was partitioned between
EtOAc and brine. The organic layer was dried (Na2SO4), evap-
orated, and purified by column chromatography (SiO2; CHCl3–
MeOH, 20:1) to give 17 (866 mg, 74%) as a foam (Found: C,
48.68; H, 6.85; N, 10.27. C16H27N3O8ؒ1/3H2O requires C, 48.60;
H, 7.05; N, 10.63%); 1H NMR (400 MHz; DMSO-d6) δ 8.61 (br
d, 1 H, 1-NH, J = 8.3), 7.47, 7.45 and 7.40 (each br s, total 2 H,
CONH2), 6.63 (br d, 0.5 H, 2-NH, J = 8.3), 6.61 (br d, 0.5 H,
2-NH, J = 8.8), 5.41 (dd, 0.5 H, H-1Ј, J = 1.5, 8.3), 5.38 (dd, 0.5
H, H-1Ј, J = 1.9, 8.3), 5.19 (t, 0.5 H, 5Ј-OH, J = 4.9), 5.12 (t, 0.5
H, 5Ј-OH, J = 5.4), 4.70 (m, 1 H, H-3Ј), 4.58–4.48 (m, 2 H, H-2
and -2Ј), 4.01 (m, 1 H, H-4Ј), 3.52–3.37 (m, 2 H, H-5Ј), 1.42–
1.26 (m, 15 H, t-Bu and Pri); MS (FAB, positive) m/z 390
(MHϩ).
and H2-5Ј), 2.11, 2.10, 2.09 and 2.08 (each s, total 9 H, Ac); 13
C
2-(tert-Butoxycarbonylamino)-N-[3-oxo-5-O-(2,4,3-benzo-
dioxaphosphepan-3-yl)-2,3-O-isopropylidene-ꢀ-D-ribofuranosyl]-
malonamide 19
NMR (100 MHz; CDCl3) δ 171.01, 170.92, 170.17, 170.01,
169.97, 169.93, 168.40, 167.72, 166.93 and 166.51 (COCH3 and
CONH2), 166.93 and 166.51 (CHO), 82.99 and 82.66 (C-1Ј),
78.80 and 78.75 (C-4Ј), 73.59, 73.23, 70.81 and 70.70 (C-2Ј and
-3Ј), 63.46 (C-5Ј), 56.16 and 55.93 (C-3), 20.76 and 20.52 (acetyl
Me); HRMS (FAB, positive) 404.1292 (MHϩ. C15H22N3O10
requires m/z, 404.1305).
A mixture of 17 (78 mg, 0.20 mmol), tetrazole (32 mg, 0.46
mmol), and 3-diethylamino-2,4,3-benzodioxaphosphepane
XEPA (72 mg, 0.30 mmol) in CH2Cl2 (2 mL) was stirred at
room temperature for 1 h. To the mixture was added a solution
of I2 (120 mg, 0.47 mmol) in aq. THF (95%; 4 mL), and the
whole was stirred at room temperature for 10 min and then
quenched with saturated aq. Na2S2O3. The resulting mixture
was partitioned between CHCl3 and brine. The organic layer
was dried (Na2SO4), evaporated, and purified by column
chromatography (SiO2; CHCl3–EtOAc, 1:1, then CHCl3–
MeOH, 50:1) to give 19 (81 mg, 70%) as a foam; 1H NMR (400
MHz; CDCl3) δ 8.15–8.04 (m, 1 H), 7.40–7.28 (m, 4 H), 7.03 (br
s, 0.5 H), 6.95 (br s, 0.5 H), 6.03–5.90 (m, 2 H), 5.64 (m, 1 H),
5.31–5.16 (m, 4 H), 4.80–4.70 (m, 3 H), 4.36 (m, 1 H), 4.31–4.21
(m, 2 H), 1.52–1.32 (m, 15 H); MS (FAB, positive) m/z 572
(MHϩ).
2-Amino-N-(2,3-O-isopropylidene-ꢀ-D-ribofuranosyl)malon-
amide 16
Reaction using a low-pressure mercury lamp. A solution of 5
(5.96 g, 20.0 mmol) in aq. AcOH (0.1 M; 600 mL) was
irradiated with a low-pressure mercury lamp (60 W, quartz
filter) at room temperature under bubbling of argon for
12 h. After being neutralized with aq. NaHCO3 (0.8 M), the
resulting mixture was evaporated. The residue was purified by
column chromatography (SiO2; CHCl3–MeOH, 10:1, then 5:1)
to give 16 (3.98 g, 68%) as a foam: 1H NMR (400 MHz;
DMSO-d6 ϩ D2O) δ 5.44 (d, 1 H, J = 2.0), 4.71 (d, 1 H, J = 6.4),
4.51 (dd, 1 H, J = 6.4, 2.0), 4.04 (m, 1 H), 3.81 and 3.79 (each s,
each 0.5 H, disappeared after 1 h from D2O-addition), 3.53–
3.43 (m, 2 H), 1.42 and 1.26 (each s, each 3 H); MS (FAB,
positive) m/z 290 (MHϩ). This compound was rather unstable
and therefore was immediately used for the next reaction.
5Ј-O-(3-Oxo-2,4,3-benzodioxaphosphepan-3-yl)bredinin 20
A solution of 19 (1.27 g, 3.4 mmol) in 90% aq. TFA was stirred
at room temperature for 15 min. After water was added, the
solvent was evaporated, and the residue was coevaporated with
water. The residue and Et3N (1.0 mL) were dissolved in MeOH
(15 mL), and then the solvent was evaporated. The residue was
purified by column chromatography (SiO2; CHCl3–MeOH,
10:1, then 5:1) to give a foam (1.01 g). A mixture of the foam
Reaction with high-pressure mercury lamp. A solution of 5
(1.49 g, 5.0 mmol) in aq. AcOH (0.1 M; 500 mL) was irradiated
J. Chem. Soc., Perkin Trans. 1, 2000, 3603–3609
3607

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and (EtO)3CH (734 µL, 4.4 mmol) in DMF (25 mL) was heated
at 90 ЊC for 15 min. The resulting mixture was evaporated, and
the residue was purified by column chromatography (SiO2;
CHCl3–MeOH, 10:1, then 5:1) to give 20 (487 mg, 47%) as a
5Ј-Deoxybredinin 3
Compound 3 (385 mg, 77%) as a solid was obtained from
22 (495 mg, 2.0 mmol) as described above for the synthesis
of 20, after purification by column chromatography (SiO2;
CHCl3–MeOH, 10:1 then 5:1); mp 217–219 ЊC (from MeOH,
decomp.) (Found: C, 44.32; H, 5.52; N, 16.91. C9H13N3O5ؒ1/
7MeOH requires C, 44.62; H, 5.51; N, 16.71%); 1H NMR (400
MHz; DMSO-d6 ϩ D2O) δ 8.29 (s, 1 H), 6.87 (br s, 2 H), 5.48
(d, 1 H, J = 4.8), 4.34 (dd, 1 H, J = 4.8, 5.1), 3.91–3.79 (m, 2 H),
1.24 (d, 3 H, J = 6.2); 13C NMR (100 MHz; D2O) δ 161.71,
155.21, 124.43, 98.52, 86.05, 79.34, 74.43, 72.98, 18.74; MS
(FAB, positive) m/z 244 (MHϩ); UV λmax 278 nm (H2O).
1
solid; H NMR (400 MHz; DMSO-d6 ϩ D2O) δ 8.28 (s, 1 H),
7.46–7.41 (m, 4 H), 5.57 (d, 1 H, J = 4.9), 5.37–5.29 (m, 2 H),
5.11–5.01 (m, 2 H), 4.39 (dd, 1 H, J = 4.9, 4.9), 4.29 (m, 1 H),
4.24–4.19 (m, 2 H), 4.09 (m, 1 H); HRMS (FAB, positive)
442.1036 (MHϩ. C17H21N3O9P requires m/z, 442.1015); UV
(H2O) λmax 280 nm.
Bredinin 5Ј-phosphate sodium salt 2
A mixture of 20 (297 mg, 0.67 mmol) and Pd-C (10%, 100 mg)
in MeOH (10 mL) was stirred at room temperature under
atmospheric pressure of H2 for 30 min. The catalyst was filtered
off, and the filtrate was evaporated. The residue was partitioned
between CHCl3 and water, and the aqueous layer was concen-
trated in vacuo, and then applied to a column of Dowex 50 resin
(Hϩ-form, 1.8 × 8 cm, packed with water). The column was
eluted with water (300 mL), and the appropriate fractions con-
taining 2 were concentrated to about 3 mL in vacuo. The result-
ing solution was applied to a column of Diaion WK-20 resin
(Naϩ-form, 1.8 × 10 cm, packed with water). The column was
eluted with water. The appropriate fractions containing 2 were
evaporated, and the residue was freeze-dried to give pure 2 (230
mg, sodium salt, 89%) as a solid (Found: C, 27.15; H, 4.17; N,
10.04. C9H13N3NaO9Pؒ7/3H2O requires C, 26.81; H, 4.42; N,
10.42%); 1H NMR (400 MHz; D2O) δ 8.38 (s, 1 H), 5.81 (d, 1 H,
J = 4.4), 4.49 (dd, 1 H, J = 4.4, 4.9), 4.41 (dd, 1 H, J = 2.4, 4.9),
4.28 (m, 1 H), 4.14–4.04 (m, 2 H); 31 P NMR (D2O; 125 MHz,
decoupled with 1H) δ 1.85 (s); 13C NMR (125 MHz; D2O)
δ 165.03, 156.36, 127.03, 101.32, 87.43 and 84.41 (d, J = 7.5),
75.61, 70.58 and 64.45; MS (FAB, positive) m/z 362 (MNaϩ);
MS (FAB, negative) m/z 338 [(M Ϫ H)Ϫ]; UV (H2O) λmax 279
nm.
N-{5-O-[3-(Benzyloxycarbonylamino)propylcarbamoyl]-2,3-O-
isopropylidene-ꢀ-D-ribofuranosyl}-3-(tert-butoxycarbonyl-
amino)malonamide 23
A mixture of 17 (97 mg, 0.25 mmol), carbonyldiimidazole (61
mg, 0.50 mmol) and DMAP (67 mg, 0.55 mmol) in CH2Cl2
(5 mL) was stirred at room temperature for 6 h. To the mixture
was added a mixture of 3-(benzyloxycarbonylamino)propyl-
amine hydrochloride16 (257 mg, 1.0 mmol) and Et3N (139 µL,
1.0 mmol) in CH2Cl2 (10 mL), and the whole was stirred at
room temperature for 16 h. After water was added, the resulting
mixture was partitioned, and the organic layer was dried
(Na2SO4), and evaporated. The residue was purified by column
chromatography (SiO2; CHCl3–MeOH, 80:1) to give 23 (138
mg, 91%) as a foam (Found: C, 53.79; H, 6.51; N, 11.00.
1
C28H41N5O11 requires C, 53.92; H, 6.63; N, 11.23%); H NMR
(500 MHz; CDCl3 ϩ D2O) δ 7.37–7.32 (m, 5 H), 5.87 (s, 0.7 H),
5.81 (s, 0.3 H), 5.08 (s, 2 H), 4.72–4.41 (m, 5 H), 3.91 (d, 0.7 H,
J = 12.4), 3.83 (d, 0.3 H, J = 11.9), 3.39–3.18 (m, 4 H), 1.73–
1.31 (m, 17 H); HRMS (FAB, positive) 624.2888 (MHϩ.
C28H42N5O11 requires m/z 624.2881).
5Ј-O-[3-(Benzyloxycarbonylamino)propylcarbamoyl]bredinin 24
2-(tert-Butoxycarbonylamino)-N-(5-deoxy-5-iodo-2,3-O-
isopropylidene-ꢀ-D-ribofuranosyl)malonamide 21
A solution of 23 (249 mg, 0.40 mmol) in 90% TFA (3 mL) was
stirred at room temperature for 10 min, and then the solution
was evaporated. After the residue was coevaporated with water
and then with EtOH (twice), the residue was dissolved in EtOH
(2 mL) and Et3N (1 mL), and the solution was evaporated. The
residue was coevaporated with EtOH (3 times), and then heated
at 40 ЊC in vacuo for 3 h. The resulting syrup was heated at
90 ЊC with (EtO)3CH (67 µL, 0.52 mmol) in DMF (5 mL) for 20
min, and then the solvent was evaporated. The residue was dis-
solved in MeOH (1 mL) and applied to a column of Dowex 50
resin (Hϩ-form, 2 × 15 cm, packed with MeOH). The column
was washed with MeOH (200 mL), and then developed with
50% aq. MeOH. Appropriate fractions were evaporated to give
24 (132 mg, 67%) as a foam (Found: C, 50.26; H, 5.61; N,
13.84. C21H27N5O9ؒ0.5H2O requires C, 50.1; H, 5.81; N,
13.91%); 1H NMR (400 MHz; DMSO-d6 ϩ D2O) δ 8.24 (s, 1 H,
H-2), 5.55 (d, 1 H, J = 5.9), 5.00 (s, 2 H, benzyl CH2), 4.34 (dd,
1 H, H-2Ј, J = 5.9, 4.9), 4.16 (dd, 1 H, H-5Јa, J = 11.7, 2.4),
4.05–3.99 (m, 3 H, H-3Ј, -4Ј, -5Јb), 3.00–2.95 (m, 4 H, NCH2 ×
2), 2.98 (m, 2 H, NCH2CH2CH2); HRMS (FAB, positive)
494.1884 (MHϩ. C21H28N5O9 requires m/z 494.1887); UV
(MeOH) λmax 283 nm.
A mixture of 17 (1.17 g, 3.0 mmol) and MsCl (302 µL, 3.9
mmol) in pyridine (30 mL) was stirred at 0 ЊC for 1 h. After
addition of water, the solvent was evaporated, and the residue
was partitioned between CHCl3 and brine. The organic layer
was dried (Na2SO4) and evaporated. A mixture of the residue
and NaI (1.12 g, 7.5 mmol) in MEK (30 mL) was heated under
reflux for 1 h. The resulting precipitate was filtered off, and the
filtrate was evaporated. The residue was partitioned between
CHCl3 and brine, and the organic layer was dried (Na2SO4) and
evaporated. The residue was purified by column chromato-
graphy (SiO2; CHCl3–EtOAc, 4:1) to give 21 (1.35 g, 90%) as a
solid (Found: C, 38.55; H, 5.28; N, 8.22. C16H26IN3O7 requires
1
C, 38.49; H, 5.25; N, 8.42%); H NMR (400 MHz; CDCl3)
δ 7.81 (d, 0.5 H, J = 8.3), 7.76 (d, 0.5 H, J = 6.8), 6.95 and 6.91
(each br s, each 0.5 H), 6.01 (dd, 1 H, J = 4.9, 4.9), 5.95 (br s,
1 H), 5.59 (m, 1 H), 4.76–4.67 (m, 3 H), 4.23 (m, 1 H), 3.33–3.22
(m, 2 H), 1.53–1.34 (m, 15 H); MS (FAB, positive) m/z 500
(MHϩ).
2-(tert-Butoxycarbonylamino)-N-(5-deoxy-2,3-O-isopropyl-
idene-ꢀ-D-ribofuranosyl)malonamide 22
5Ј-O-(3-Aminopropylcarbamoyl)bredinin hydrochloride 4
A mixture of 21 (998 mg, 2.0 mmol), Bu3SnH (810 µL, 3.0
mmol), and AIBN (100 mg, 0.61 mmol) in benzene (30 mL) was
heated under reflux for 2 h. The solvent was evaporated, and the
residue was purified by column chromatography (SiO2; CHCl3–
A mixture of 24 (49 mg, 0.10 mmol), Pd-C (10%; 50 mg), and
12 M HCl (8.8 µL, 0.105 mmol) in MeOH (5 mL) was stirred at
room temperature under atmospheric pressure of H2 for 30
min. The catalyst was filtered off, and the filtrate was evapor-
ated. The residue was coevaporated with EtOH (3 times), and
then the residue was treated with EtOH to give 4ؒHCl (34 mg,
86%) as a solid; 1H NMR (400 MHz; D2O) δ 8.08 (s, 1 H, H-2),
5.78 (d, 1 H, H-1Ј, J = 3.4), 4.62 (dd, 1 H, H-2Ј, J = 5.4, 3.4),
1
MeOH, 30:1) to give 22 (736 mg, 98%) as a foam; H NMR
(400 MHz; DMSO-d6) δ 8.77–8.68 (m, 1 H), 7.41 (br s, 2 H),
6.63–6.58 (m, 1 H), 5.30–5.21 (m, 1 H), 4.70–4.42 (m, 4 H),
4.02–3.98 (m, 1 H), 1.46–1.16 (m, 15 H), 0.88 (m, 3 H); MS
(FAB, positive) m/z 374 (MHϩ).
3608
J. Chem. Soc., Perkin Trans. 1, 2000, 3603–3609

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4 (a) L. A. Turka, J. Dayton, G. Sinclair, C. B. Thompson and
B. S. Mitchell, J. Clin. Invest., 1991, 87, 940; (b) J. S. Dayton,
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5 (a) S. Shuto, H. Itoh, E. Endo, K. Fukukawa, M. Tsujino and
T. Ueda, Chem. Pharm. Bull., 1987, 35, 3523; (b) N. Yoshida,
M. Nakamura, M. Fukui, S. Morisada, S. Ogino, M. Inaba,
S. Tsukagoshi and Y. Sakurai, Cancer Res., 1983, 43, 5851;
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1982, 42, 1098.
4.50 (dd, 1 H, H-5Јa, J = 12.2, 1.8), 4.41 (dd, 1 H, H-3Ј, J = 5.4,
5.4), 4.31–4.25 (m, 2 H, H-4Ј, -5Јb), 3.23 (t, 1 H, NCH2,
J = 6.8), 3.02 (t, 2 H, NCH2, J = 7.8), 1.86 (m, 2 H, NCH2-
CH2CH2); 13C NMR (125 MHz; DMSO-d6) δ 164.62, 158.30,
155.97, 126.24, 101.46, 87.41, 82.00, 73.64, 70.00, 63.80, 37.56,
37.23, 27.32; HRMS (FAB, positive) 360.1521 (NHϩ. C13H22-
N5O7 requires m/z 360.1519); UV λmax 279 nm (H2O).
6 Y. Kosugi, Y. Saito, S. Mori, J. Watanabe, M. Baba and S. Shigeta,
Antiviral Chem. Chemother., 1994, 5, 366.
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T. Ueda, Chem. Pharm. Bull., 1984, 32, 1644.
Acknowledgements
We are grateful to Drs S. Yokoiyama, K. Arai, and H. Misaki,
Asahi Chemical Industry, for their helpful discussions.
8 A part of this study has been described in a communication: S. Shuto,
K. Haramuishi and A. Matsuda, Tetrahedron Lett., 1996, 37, 187.
9 M. Waki and J. Meienhofer, J. Org. Chem., 1977, 42, 2019.
10 H. Koyama and M. Tsuji, Biochem. Pharmacol., 1983, 32, 200.
11 M. Yoshikawa, T. Kato and T. Takenishi, Bull. Chem. Soc. Jpn.,
1969, 42, 3505.
12 H. Watababe, unpublished results: when bredinin was subjected to
Yoshikawa’s procedure, dehydration of the 4-carbamoyl moiety
occurred, giving the corresponding 4-cyano derivative as the major
product.
13 Y. Watanabe, Y. Komoda, K. Ebisuya and S. Ozaki, Tetrahedron
Lett., 1990, 31, 255.
14 G. Weber, M. E. Burt, R. C. Jackson, N. Prajda, M. S. Lui and
E. Takeda, Cancer Res., 1983, 43, 1019.
Notes and references
1 This report constitutes Part 202 of our series Nucleosides and
Nucleotides. Part 201: T. Umino, N. Minakawa and A. Matsuda,
Tetrahedron Lett., 2000, 41, 6419.
2 (a) K. Mizuno, M. Tsujino, M. Takada, M. Hayashi, K. Atsumi,
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3 (a) H. Amemiya and H. Itoh, in Immunosuppressive Drugs:
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1999, 6, 575.
15 A similar activation of 5Ј-deoxy-5-fluorouridine (doxifluridine), a
clinically useful anticancer drug, by nucleoside phosphorylase in
tumor cells is known: J.-P. Sommadossi, C. Aubert, J.-P. Cano,
J. Gouveia, P. Ribaud and G. Mathe, Cancer Res., 1983, 43, 930.
16 G. J. Atwell and W. A. Denny, Synthesis, 1984, 1032.
J. Chem. Soc., Perkin Trans. 1, 2000, 3603–3609
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