Synthesis and antitumor activities of novel 5-deazaflavin-sialic acid conjugate molecules

Article abstract of DOI:10.1016/S0968-0896(00)00124-3

6-Nitro-5-deazaflavin derivatives bearing O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α- and β-D-galacto-non-2-ulopyranosylonate)alkyl group (sialosylalkyl group) at N(3) or N(10) and 8-amino-5-deazaflavin substituted with the sialosylalkyl group at the amino group were synthesized and their physicochemical properties as well as antitumor effects on KB and L1210 cells have been investigated. The configurations of the glycosides were determined by ¹H NMR and rate of hydrolysis of the glycosidic bond. It has been found that these conjugate molecules show significant antitumor activities. Combination of an 8-amino-5-deazaflavin with the sialosylalkyl group have been found to give rise to significant increase in antitumor activities of the compound. Antitumor effects of 6-nitro-5-deazaflavin-sialic acid conjugate molecules were similar or rather weak in comparison with those of the 6-nitro-5-deazaflavin derivatives without sialosylalkyl group. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00124-3

Bioorganic & Medicinal Chemistry 8 (2000) 2027±2035
Synthesis and Antitumor Activities of Novel 5-Deaza¯avin-Sialic
Acid Conjugate Molecules
Yoshihiro Ikeuchi,a,*,y Motoko Sumiya,a Tetsuji Kawamoto,a,* Naoshige Akimoto,a
Yuji Mikata,b Maki Kishigami,b,{ Shigenobu Yano,b Takuma Sasakic
and Fumio Yonedaa,*,{
aFaculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku Kyoto 606-8501, Japan
bDepartment of Chemistry, Faculty of Science, Nara Women's University, Nara 630-8506, Japan
cDepartment of Experimental Therapeutics, Cancer Research Institute, Kanazawa University, Takaramachi Kanazawa 920-0934, Japan
Received 14 February 2000; accepted 6 May 2000
AbstractÐ6-Nitro-5-deaza¯avin derivatives bearing O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a- and b-
d-galacto-non-2-ulopyranosylonate)alkyl group (sialosylalkyl group) at N(3) or N(10) and 8-amino-5-deaza¯avin substituted with
the sialosylalkyl group at the amino group were synthesized and their physicochemical properties as well as antitumor e€ects on KB
and L1210 cells have been investigated. The con®gurations of the glycosides were determined by 1H NMR and rate of hydrolysis of
the glycosidic bond. It has been found that these conjugate molecules show signi®cant antitumor activities. Combination of an 8-
amino-5-deaza¯avin with the sialosylalkyl group have been found to give rise to signi®cant increase in antitumor activities of the
compound. Antitumor e€ects of 6-nitro-5-deaza¯avin-sialic acid conjugate molecules were similar or rather weak in comparison
with those of the 6-nitro-5-deaza¯avin derivatives without sialosylalkyl group. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Recently, we have developed nitro 5-deaza¯avin
derivatives13 16 as novel class of nitrohetero-aromatic
compounds containing an electrophilic redox coenzyme
ring system. It has been found that a series of nitro 5-
deaza¯avin derivatives shows signi®cant antitumor activ-
ities13 and that 6- and 8-nitro-5-deaza¯avin derivatives
generating stable one-electron reduction product(s) show
marked selective cytotoxicities towards hypoxic cells.14 It
has also been demonstrated that reductively activated 6-
nitro-5-deaza¯avin derivatives bearing pyrrolecarbox-
amide(s) as a DNA minor groove binder induce signi®cant
oxidative DNA damage under anaerobic conditions.15
Furthermore, we have ®rst found that reductively acti-
vated 6- and 8-nitro-5-deaza¯avin derivatives interact
speci®cally with guanine base to give rise to prominent
formation of 8-oxo-7,8-dihydro-20-deoxyguanosine (8-
oxodGuo) under anaerobic conditions.16
Sialic acids1 are commonly present at the non-reducing
terminal positions of carbohydrate chains of glycoproteins
and glycolipids on the cell surface and their crucial roles
played in biological processes involving cell to cell recog-
nition and interaction,2 masking e€ects for cell surface
antigen,3 di€erentiation of cells,4 and neoplastic transfor-
mation5 have been well studied. Combination of biologi-
cally important compounds with a sialic acid derivative
would be anticipated to give rise to selective interaction
towards target biological molecules and target tumor
cells.6 From these points of view, Ogura synthesized var-
ious sialic acid conjugate molecules, sialosyl cholesterol,7
sialyl nucleosides,8 sialyl umbelliferon9 and sialosylphenyl
mitomycin C10 and reported their remarkable physi-
ological activities. Also, taxol-sialyl conjugate11 and sialic
acid containing 2-nitroimidazole nucleoside analogue12 as
a radiosensitizer for hypoxic cells have been prepared
and their biological activities were elucidated.
To develop more potent (bioreductive) antitumor agents
showing a higher selectivity towards tumor cells as well
as hypoxic cells, we have designed 6-nitro-5-deaza¯avin
derivatives 1 and 2 bearing a 2-O-(methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a- and b-d-
galacto-non-2-ulopyranosylonate)ethyl group at N(3)
position and 6-O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-d-glycero-a- and b-d-galacto-non-2-
*Corresponding authors. E-mail: ikeuchi@snowbrand.co.jp
yPresent address: Technology and Research Institute, Snow Brand
Milk Products Co., Ltd, 1-2, Minamidai 1-Chome, Kawagoe, Sai-
tama, 350-1165, Japan. Tel.: +81-492-42-8111; fax: +81-492-42-8696.
{Present address: Fujimoto Pharmaceutical Co., Ltd, Matsubara,
Osaka 580-0011, Japan.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00124-3

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Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
ulopyranosylonate)hexyl group at N(10) position as
novel 5-deaza¯avin-sialic acid conjugate molecules
(Scheme 1). Because among nitro-positional isomer of
nitro 5-deaza¯avin derivatives, 6-nitro-5-deaza¯avin
derivatives showed the most potent antitumor activities13
and the highest selective cytotoxicities towards hypoxic
cells,14 the sialosylalkyl group was introduced into 6-
nitro-5-deaza¯avin derivatives. Also, as a 5-deaza¯avin
derivative modi®ed at benzene ring moiety, 8-amino-5-
deaza¯avin derivatives 3 substituted with 2-[O-(methyl 5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-
and b-d-galacto-non-2-ulopyranosylonate)]ethyl group at
the amino group have been designed (Scheme 1). In the
present paper, we wish to describe synthesis of (nitro) 5-
deaza¯avin-sialic acid conjugate molecules and their phys-
icochemical properties as well as antitumor activities.
Results and Discussion
Synthesis of 5-deaza¯avin-sialic acid conjugate molecules
10-Dodecyl-6-nitro-5-deaza¯avin 6 was prepared by con-
densation of 6-dodecylaminouracil 4 with 2-¯uoro-6-
nitrobenzaldehyde 517 according to Yoneda's method13,18
in 82%. Treatment of 6 with 1-(tert-butyldimethylsil-
oxy)-2-iodoethane19 in the presence of K2CO3 in DMF
at 60 ꢀC a€orded 7 in 82%, which was subjected to
hydrolysis with aqueous 12N hydrochloric acid in etha-
nol to give 8 in 82%. Glycosylation of 8 with methyl (5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-
b-d-galacto-non-2-ulopyranosyl chloride)onate 920 under
the Koenigs-Knorr like reaction condition7,8a,8c,21 gave a
mixture of glycosides 1a and 1b. Chromatographic
separation of the anomeric mixture on thin layer silica
Scheme 1.
Scheme 2. Synthesis of compounds 1a and 1b: (i) DMF, 120 ꢀC; (ii) I(CH2)2OTBDMS, K2CO3, DMF, 60 ꢀC; (iii) 12 N HCl, EtOH, 50 ꢀC;
(iv) AAgOTf, MS 4A, CH2Cl2, rt.

Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
2029
gel plates a€orded the respective single anomers in 16
and 26% (Scheme 2).
and separation of the obtained anomeric mixture of 3a
and 3b in the above similar manner a€orded the
respective single anomers in 22 and 34% (Scheme 4).
Also, 10-(6-hydroxyhexyl)-3-phenyl-5-deaza¯avin 11 was
obtained by condensation of 6-(6-hydroxyhexyl)amino-3-
phenyluracil 10 with 2-¯uoro-6-nitrobenzaldehyde 517 in
72%. Glycosylation of 11 with 920 and separation of the
resulting anomeric mixture of 2a and 2b in the similar
manner described above gave single anomers in 20 and
20% respectively (Scheme 3).
Con®guration of the 5-deaza¯avin-sialic acid conjugate
molecules
It is well understood that in 1H NMR spectra of sialic acid
derivatives, double-doublet peaks for Heq(3) of an a-gly-
coside appear in lower magnetic ®eld than those of the b-
glycoside.7,21,23 Goto also reported that anomeric con®g-
uration of sialic acid derivatives is able to be estimated
from the coupling constants between H(7) and H(8) (J
H(7),H(8)) and the Ád|H(90)-H(9)| values in 1H NMR
spectra of the glycosides.24 To consider anomeric con-
®guration of the sialic acid conjugate molecules 1±3, 1H
NMR spectra of the anomeric pair of compounds were
compared. As Table 1 shows, the chemical shifts for
5-Deaza¯avin derivatives 3 bearing the N-methyl-N-
(sialosylethyl)amino group at C(8) position were prepared
by using 10-dodecyl-8-¯uoro-3-methyl-5-deaza¯avin 13
which was obtained by condensation of 6-dodecylamino-
3-methyluracil 12 with 2,4-di¯uorobenzaldehyde in 86%.
Treatment of 13 with 2-(methylamino)ethanol in DMF
at 100 ꢀC gave 14 in 85%.22 Glycosylation of 14 with 920
Scheme 3. Synthesis of compounds 2a and 2b: (i) DMF, 120 ꢀC; (ii) AgOTf, MS 4A, CH2Cl2, rt.
Scheme 4. Synthesis of compounds 3a and 3b: (i) 2,4-di¯uorobenzaldehyde, DMF, 120 ꢀC; (ii) 2-(methylamino)ethanol, DMF, 100 ꢀC; (iii) AgOTf,
MS 4A, CH2Cl2, rt.

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Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
1
Table 1. Determination of con®guration of 5-deaza¯avin-sialic acid
conjugate molecules 1±3 by means of 1H NMR spectraa and hydro-
lysis reactionb
From above experimental results of H NMR spectra
and hydrolysis rates of the compounds, the anomeric
con®gurations of 1a, 2a and 3a were determined to be a
and 1b, 2b and 3b to be b.
Compound Con®guration
d for Heq(3)
Hydrolysis rate
Conversion
(%)c
(ppm)a
CD spectra of the 5-deaza¯avin-sialic acid conjugate
molecules
1a
1b
2a
2b
3a
3b
a
b
a
b
a
b
2.53, dd, J=12.9, 4.7 Hz
2.22, dd, J=12.7, 5.0 Hz
2.59, dd, J=12.8, 4.6 Hz
2.51, dd, J=12.8, 4.9 Hz
2.56, dd, J=12.8, 4.6 Hz
2.43, dd, J=12.9, 5.0 Hz
99
<3
99
<3
98
Circular dichroism (CD) spectral analysis of sialic acid
derivatives is also considered to be one of the useful
methods for determination of con®guration of the glyco-
sides, if other chromophore in the compound does not
a€ect the Cotton e€ect of carboxyl group at C(1) position
due to n-p* transition.7,21,27 Because (5-deaza)¯avin ring
system is known as a strong chromophore,28 interaction
of 5-deaza¯avin chromophore with sialic acid moiety
would a€ect signi®cantly the CD spectra of the conjugate
molecules. UV±vis and CD spectra of 1±3 were measured
and shown in Figure 2.
<2
a300 MHz, in CDCl3, at 298 K.
3
b[Compound]=5.0Â10 (M), in aqueous 50% acetic acid, at 373 K,
6 h, under nitrogen.
cDetermined by means of an HPTLC method.
Heq(3) of compounds 1a, 2a and 3a have been found to
be larger than those of the other anomers 1b, 2b and 3b.
Although the coupling constants between H(7) and H(8)
(JH(7),H(8)) of compounds 1±3 were not determined due
to complexity of the spectra, signi®cant di€erence of
Ád|H(90)- H(9)| values24,25 has been observed between
the anomeric pair of compounds 1±3.
Signi®cant di€erences of CD spectra between a- and b-
anomer of 1±3 have been found. Negative Cotton e€ect
for a-anomer 1a and 3a around 220±250 nm due to n-p*
transition and positive Cotton e€ect for b-anomer 1b
and 3b were observed (Fig. 2(a), and (c)), however, each
anomer of 2 has been found to show similar pattern
of Cotton e€ects around 200±250 nm (Fig. 2(b)). And
interestingly, marked di€erence of CD spectra for 1±3
were observed in the visible absorption band characteristic
of 5-deaza¯avin chromophore. The result suggests that
there would be signi®cant stereoselective interaction(s)
between sialic acid moiety and 5-deaza¯avin ring system,
which may a€ect recognition abilities for biological target
of the conjugate molecules.
Furthermore, to con®rm the anomeric con®guration of
compounds 1±3, the rates of hydrolysis of each pair of gly-
cosides were compared. Ogura reported that an a-anomer
of sialic acid derivatives is more susceptible to hydrolysis of
glycosidic bond than the corresponding b-anomer.8c Thus
compounds 1±3 were treated with aqueous 50% acetic
acid26 at 100 ꢀC for 6 h in sealed tubes under nitrogen
atmosphere and the reactions were monitored by
measuring the increasing concentration of 8, 11 and 14.
The results of hydrolysis of glycosidic bond of 1a and
1b were exempli®ed in Figure 1. As Table 1 shows,
compounds 1a, 2a, and 3a were hydrolyzed almost com-
pletely after 6 h, on the other hand, 1b, 2b and 3b were
stable under above reaction conditions.
In vitro antitumor activities of the 5-deaza¯avin-sialic
acid conjugate molecules
To evaluate antitumor activities of the 5-deaza¯avin-sialic
acid conjugate molecules and other reference compounds,
compounds 1±3, 8, 11 and 14 were tested for in vitro
antitumor e€ects on human oral epidermoid carcinoma
KB cells and murine leukemia L1210 cells by the MTT
assay developed by Carmichael.29,30 Mitomycin C was
also employed as reference compound. As Table 2 shows,
Table 2. IC50 Values of 5-deaza¯avin-sialic acid conjugate molecules
on KB and L1210 cells growth in vitro
IC50 (mM)a
KB cells
L1210 cells
1a
1b
5.1
2.3
1.7
1.9
2a
2b
14.3
6.4
20.9
6.8
3a
3b
8
11
5.1
3.9
0.74
2.0
13.8
10.6
5.7
9.2
>100
0.6
14
Mitomycin C
>100
0.4
aIC50 (mM) value was given as the concentration at 50% inhibition of
cell growth.
Figure 1. Hydrolysis of glycosidic bond of 1a (circle) and 1b (triangle).

Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
2031
Figure 2. UV±vis and CD spectra of 5-deaza¯avin-sialic acid conjugate molecules 1 (a), 2 (b), and 3 (c).
5-deaza¯avin-sialic acid conjugate molecules 1±3 have
been found to show signi®cant antitumor activities.31 It
has been found that b-anomer 2b shows 2±3 folds more
potent antitumor activities than a-anomer 2a and there
appears to be essentially no signi®cant di€erences of
antitumor e€ects between 1a and 1b, and between 3a
and 3b. Interestingly enough, the conjugate molecules 3
have been found to show much more potent antitumor
activities than 8-amino-5-deaza¯avin derivative 14 without
sialosyl group. The result may suggest that combination of
an 8-amino-5-deaza¯avin with sialosylalkyl group would
give rise to signi®cant e€ects for antitumor activities of

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Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
the compound. Antitumor activities of sialic acid con-
jugates 1 and 2 were similar or rather weak in comparison
with the corresponding 6-nitro-5-deaza¯avin derivatives
8 and 11 without sialosyl group.
3-2[(tert-Butyldimethylsiloxy)ethyl]-10-dodecyl-6-nitro-5-
deaza¯avin (7). A mixture of 10-dodecyl-6-nitro-5-deaza-
¯avin (6, 2.13g, 5.0 mmol), 1-(tert-butyldimethylsiloxy)-2-
iodoethane19 (2.86 g, 10.0 mmol), and well pulverized
K2CO3 (3.46g, 25.0mmol) in 20mL of DMF was warmed
at 60ꢀC for 2 h. The reaction mixture was ®ltered to
remove K2CO3 and the ®ltrate was treated with water,
and then was extracted with ether. The organic layer
was washed successively with water three times and with
brine once. The organic layer was dried over MgSO4
and the solvent was evaporated under reduced pressure.
The residue was subjected to column chromatography
on silica gel (hexane:ethyl acetate=1:1 as eluent) to
a€ord yellow solid. Recrystallization from hexane-ethyl
Conclusion
We have ®rst synthesized 6-nitro- and 8-amino-5-deaza-
¯avin derivatives bearing sialosylalkyl group as novel 5-
deaza¯avin-sialic acid conjugate molecules and evaluated
their antitumor activities. Combination of an 8-amino-
5-deaza¯avin with a sialosylalkyl group has been found
to give rise to signi®cant increase in antitumor activities.
Although antitumor e€ects of 6-nitro-5-deaza¯avin-sialic
acid conjugate molecules 1 and 2 were similar or rather
weak in comparison with 6-nitro-5-deaza¯avin without
sialosyl group, these compounds may be anticipated to
show promoted selective cytotoxicities towards tumor
cells as well as hypoxic cells. The detailed biological
activities of these unique conjugate molecules as well as
biological mechanism for antitumor e€ects of 3 are
under investigation.
acetate to a€ord 2.40 g (82%) of 7 as yellow crystal: mp
1
119±121 ꢀC. IR (KBr) 1709, 1655, 1622, 1563, 1526 cm
.
1H NMR (CDCl3) d 0.05 (6H, s), 0.72±0.96 (12H, m),
1.10±1.40 (16H, m), 1.43±1.61 (2H, m), 1.69±1.95 (2H,
m), 3.89 (2H, t, J=6.4 Hz), 4.25 (2H, t, J=6.4 Hz), 4.77
(2H, br), 7.85±8.09 (3H, m), 9.37 (1H, s). Anal. calcd for
C31H48N4O5Si: C, 63.67; H, 8.27; N, 9.58. Found: C,
63.71; H, 8.38; N, 9.55.
10-Dodecyl-3-(2-hydroxyethyl)-6-nitro-5-deaza¯avin (8).
To a solution of 3-[2(tert-butyldimethylsiloxy)ethyl]-10-
dodecyl-6-nitro-5-deaza¯avin (7, 1.75 g, 3.0 mmol) in
20 mL of ethanol, was added 1 mL of aqueous 12N
hydrochloric acid and the mixture was warmed at 50 ꢀC
for 30 min. The solvent was evaporated under reduced
pressure and the residue was treated with water and
then, was extracted with ethyl acetate. The organic layer
was washed with brine and was dried over MgSO4. The
solvent was evaporated and the obtained yellow solid
was recrystallized from hexane-ethyl acetate to a€ord
Experimental
Melting points were taken using a Mettler thermosystem
FP80HT and are uncorrected. Infrared (IR) spectra were
recorded on a Shimadzu IR-400 spectrophotometer.
Speci®c rotation values were measured on a JASCO
DIP-360 polarimeter. Proton nuclear magnetic resonance
(1H NMR) spectra for compounds 1±3 were recorded
on a Bruker AC-300 (300 MHz) and for other com-
pounds on a JEOL FX200 (200 MHz) spectrometer in
CDCl3 or Me2SO-d6 with tetramethylsilane (TMS) as an
internal standard and chemical shifts are given in ppm.
Low-resolution FAB (fast atom bombardment) mass
spectra (MS (FAB)) were recorded on a JEOL JNS-
DX303. High-resolution FAB (fast atom bombardment)
mass spectra (HRMS (FAB)) were recorded on a JEOL
JMS-HX/HX110A. UV±vis spectra were measured on a
Shimadzu UV-2100. Circular dichroism (CD) spectra
were recorded on a JASCO J-700. Column chromatog-
raphy was carried out on silica gel (Kieselgel 60, 70-230
mesh, Merck) and on thin layer silica gel plates (Silica
Gel 60 F254, HPTLC plate, Merck).
1.16 g (82%) of 8 as yellow ®ne powder: mp 89±91 ꢀC.
1
IR (KBr) 3420, 1707, 1651, 1620, 1561, 1526, 1217cm
.
1H NMR (CDCl3) d 0.88 (3H, t, J=6.4 Hz), 1.10±1.61
(18H, m), 1.72±1.96 (2H, m), 3.87±4.01 (2H, m), 4.28±4.43
(2H, m), 4.78 (2H, br), 7.88±8.04 (2H, m), 8.08 (1H, dd,
J=7.2, 2.0 Hz), 9.43 (1H, s). Anal. calcd for C25H34N4O5:
C, 63.81; H, 7.28; N, 11.91. Found: C, 63.52; H, 7.43; N,
11.75.
6-(6-Hydroxyhexyl)amino-3-phenyluracil (10). A mixture
of 6-chloro-3-phenyluracil (2.22g, 10.0 mmol) and 6-
amino-1-hexanol (2.34 g, 20.0 mmol) in 20mL of 1-buta-
nol was heated to re¯ux for 3 h. The reaction solution was
cooled to room temperature and the deposited precipitate
was collected by ®ltration. The precipitate was washed
with ethanol and was dried under reduced pressure to
10-Dodecyl-6-nitro-5-deaza¯avin (6). A mixture of 6-
dodecylaminouracil18 (4; 2.95g, 10.0 mmol) and 2-¯uoro-
6-nitrobenzaldehyde17 (5; 1.69g, 10.0 mmol) in 30mL of
DMF was heated at 120 ꢀC for 2 h under argon atmos-
phere. The reaction mixture was poured into ice water
and deposited precipitate was collected by ®ltration. The
precipitate was washed with water and was dried under
reduced pressure. Recrystallization from chloroform-
ethanol a€orded 3.50 g (82%) of 6 as yellow powder: mp
216±217 ꢀC. IR (KBr) 1711, 1694, 1676, 1622, 1651,
.
a€ord 2.31 g (68%) of 10 (obtained as 10 2H2O) as white
powder: mp 200±202 ꢀC. IR (KBr) 3335, 1721, 1645,
1586, 1566 cm 1. 1H NMR (Me2SO-d6) d 1.18±1.36 (6H,
m), 1.37±1.61 (2H, m), 2.68 (1H, t, J=7.4 Hz, OH), 2.93±
3.10 (2H, m), 3.28±3.48 (2H, m), 4.59 (1H, s), 6.83 (1H,
br), 7.06±7.20 (2H, m), 7.23±7.49 (3H, m). Anal. calcd for
.
C16H21N3O3 2H2O: C, 56.62; H, 7.42; N, 12.38. Found:
C, 56.63; H, 7.61; N, 12.33.
1524cm 1. H NMR (CDCl3) d 0.88 (3H, t, J=6.6Hz),
1
1.10±1.72 (18H, m), 1.75±1.97 (2H, m), 4.62±4.98 (2H,
m),7.88±8.02 (2H, m), 8.08 (1H, dd, J=7.2, 2.0 Hz), 8.76
(1H, s), 9.42 (1H, s). Anal. calcd for C23H30N4O4: C, 64.77;
H, 7.09; N, 13.14. Found: C, 64.88; H, 7.12; N, 13.01.
10-(6-Hydroxyhexyl)-6-nitro-3-phenyl-5-deaza¯avin (11).
A mixture of 6-(6-hydroxyhexyl)-amino-3-phenyluracil
(10 2H2O, 1.70 g, 5.0 mmol) and 2-¯uoro-6-nitrobenzalde-
hyde (5, 0.85 g, 5.0 mmol)19 in 15 mL of DMF was
.

Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
2033
heated at 120 ꢀC for 3 h. The solvent was evaporated under
reduced pressure and the residue was treated with water
and then, was extracted with chloroform. The organic layer
was washed with brine and was dried over MgSO4. The
solvent was evaporated under reduced pressure and the
residue was subjected to column chromatography on silica
gel (chloroform : methanol=10:1 as eluent). The obtained
yellow solid was recrystallized from ethanol to a€ord 1.56 g
(72%) of 11 as yellow crystal: mp 185±187 ꢀC. IR (KBr)
3472, 1715, 1655, 1626, 1563, 1530, 1219cm 1. 1H NMR
(Me2SO-d6) d 1.30±1.62 (6H, m), 1.66±1.91 (2H, m), 3.35±
3.51 (2H, m), 4.35 (1H, t, J=5.1Hz, OH), 4.62±4.92 (2H,
m), 7.20±7.32 (2H, m), 7.36±7.56 (3H, m), 8.04±8.30 (2H,
m), 8.39 (1H, d, J=8.8 Hz), 9.18 (1H, s). Anal. calcd for
C23H22N4O5: C, 63.59; H, 5.10; N, 12.90. Found: C, 63.54;
H, 5.20; N, 12.62.
by ®ltration and the ®ltrate was condensed under reduced
pressure. The resulting residue was subjected to thin layer
chromatography on silica gel (chloroform:acetone=6:1 as
eluent) to a€ord 80mg (16%) of 1a as yellow amorphous
powder and 120 mg (26%) of 1b as yellow amorphous
powder: a-Anomer (1a): IR (KBr) 1735, 1650, 1620,
20
d
1525 cm 1. ‰ꢀŠ
4.1ꢀ (c=1, CHCl3). 1H NMR (CDCl3)
d 0.88 (3H, t, J=6.6 Hz), 1.02±1.60 (20H, m), 1.84 (3H, s,
NAc), 1.94±2.19 (13H, m, OAc+Hax(30)), 2.53 (1H, dd,
J=12.9, 4.7 Hz, Heq(30)), 3.59±3.69 (2H, m), 3.79 (3H, s,
CO2Me), 3.92±4.20 (2H, m, H(50)+H(60)), 4.25±4.44
(2H, m, H(90)), 4.60±4.94 (4H, m), 4.82±4.91 (1H, m,
H(40)), 5.13 (1H, d, J=9.8 Hz, NH), 5.28±5.50 (2H, m,
H(70)+H(80)), 7.86±8.00 (2H, m), 8.04 (1H, dd, J=7.5,
1.2 Hz), 9.37 (1H, s). MS (FAB) m/z: 943 [(M)+].
HRMS (FAB) for C45H61N5O17 [(M)+] calcd 943.4046,
found 943.4084. b-Anomer (1b): IR (KBr) 1745, 1655,
20
7.3ꢀ (c=1, CHCl3). H NMR
1
10-Dodecyl-8-¯uoro-3-methyl-5-deaza¯avin (13). A mix-
ture of 6-dodecylamino-3-methyluracil18 (12, 3.09 g,
10 mmol) and 2,4-di¯uorobenzaldehyde (1.71 g, 12.0
mmol) in 30 mL of DMF was heated at 120 ꢀC for 2 h.
The reaction mixture was poured into ice-water and
deposited precipitate was collected by ®ltration. The
precipitate was washed with water and was dried under
reduced pressure. Recrystallization from chloroform-
1630, 1535 cm 1. ‰ꢀŠ
d
(CDCl3) d 0.88 (3H, t, J=6.7 Hz), 1.08±1.63 (20H, m), 1.89
(3H, s, NAc), 1.92±2.15 (13H, m, OAc+Hax(30)), 2.22
(1H, dd, J=12.7, 5.0 Hz, Heq(30)), 3.77 (3H, s, CO2Me),
3.81±3.98 (2H, m), 4.02±4.23 (2H, m, H(50)+H(60)), 4.47
(1H, dd, J=10.5, 2.3 Hz, H(90)), 4.50±4.64 (2H, m),
4.67±4.95 (2H, m), 4.80 (1H, dd, J=12.3, 2.5 Hz, H(90)),
5.08-5.21 (1H, m, H(40)), 5.27±5.36 (1H, m, H(80)), 5.42±
5.50 (1H, m, H(70)), 6.05 (1H, d, J=10.2 Hz, NH), 7.88±
8.01 (2H, m), 8.05 (1H, dd, J=7.5, 1.4 Hz), 9.52 (1H, s).
MS (FAB) m/z: 943 [(M)+]. HRMS (FAB) for
C45H61N5O17 [(M)+] calcd 943.4046, found 943.4046.
ethanol a€orded 3.56g (86%) of 13 as yellow crystal: mp
1
221±223 ꢀC. IR (KBr) 1703, 1645, 1620, 1572, 1537cm
.
1H NMR (CDCl3) d 0.88 (3H, t, J=6.4 Hz), 1.14±1.61
(18H, m), 1.71±1.97 (2H, m), 3.48 (3H, s), 4.48±4.87 (2H,
m), 7.18±7.38 (2H, m), 7.95 (1H, dd, J=8.8, 6.2 Hz), 8.86
(1H, s). Anal. calcd for C24H32N3O2F: C, 69.71; H, 7.80;
N, 10.16. Found: C, 69.68; H, 7.65; N, 10.15.
10-[6-O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-ꢀ- and ꢁ-D-glycero-D-galacto-non-2-ulopyrano-
sylonate)hexyl]-6-nitro-3-phenyl-5-deaza¯avin (2). To
a mixture of 10-(6-hydroxyhexyl)-6-nitro-3-phenyl-5-
deaza¯avin (11, 1.09 g, 2.5 mmol), methyl (5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-b-d-galacto-
non-2-ulopyranosyl chloride)onate20 (9, 1.27g, 2.5 mmol),
and well pulverized molecular sieves 4A (6.0 g) in 35 mL
of dry dichloromethane, was added silver tri¯uoro-
methanesulfonate (0.77 g, 3.0 mmol) and the mixture
was stirred in the dark at room temperature under argon
stream for 1 h. The catalyst and molecular sieves were
removed o€ by ®ltration and the ®ltrate was condensed
under reduced pressure. The resulting residue was sub-
jected to thin layer chromatography on silica gel (chloro-
form:acetone=2:1 as eluent ) to a€ord 456 mg (20%) of
2a as yellow amorphous powder and 454 mg (20%) of 2b
10-Dodecyl-8-[N-(2-hydroxyethyl)-N-methylamino]-3-
methyl-5-deaza¯avin (14). A mixture of 10-dodecyl-8-
¯uoro-3-methyl-5-deaza¯avin (13, 2.07 g, 5.0 mmol) and
2-(methylamino)ethanol (1.88 g, 25.0 mmol) in 20 mL of
DMF was heated at 100 ꢀC for 1 h. The reaction mixture
was poured into ice-water and deposited precipitate was
collected by ®ltration. The precipitate was washed with
water and was dried under reduced pressure. Recrys-
tallization from ethanol a€orded 1.99 g (85%) of 14 as
yellow crystal: mp 210 ꢀC. IR (KBr) 3333, 1680, 1632,
1588, 1561, 1524 cm 1. 1H NMR (CDCl3) d 0.88 (3H, t,
J=6.4 Hz), 1.18±1.55 (18H, m), 1.60±1.80 (2H, m), 3.22
(3H, s), 3.39 (3H, s), 3.67±3.78 (2H, m), 3.83±3.92 (1H,
m, OH), 3.94±4.13 (2H, m), 4.48 (2H, br), 6.53 (1H, s),
6.86 (1H, dd, J=9.2, 1.8 Hz), 7.45 (1H, d, J=9.2 Hz),
8.31 (1H, s). Anal. calcd for C27H40N4O3: C, 69.20; H,
8.60; N, 11.96. Found: C, 69.28; H, 8.52; N, 11.98.
as yellow amorphous powder: a-Anomer (2a): IR (KBr)
20
d
1735, 1700, 1660, 1620, 1525 cm 1. ‰ꢀŠ
1.7ꢀ (c=1.0,
1
CHCl3). H NMR (CDCl3) d 1.20±1.31 (4H, m), 1.40±
1.70 (4H, m), 1.87 (3H, s, NAc), 1.97±2.20 (13H, m,
OAc+Hax(30)), 2.59 (1H, dd, J=12.8, 4.6 Hz, Heq(30)),
3.20±3.31 (2H, m), 3.70±3.83 (1H, m, H(50)), 3.81 (3H, s,
CO2Me), 3.98±4.08 (2H, m, H(60)+H(90)), 4.33 (1H, dd,
J=12.3, 2.6 Hz, H(90)), 4.62±4.96 (3H, m, H(40)+CH2),
5.23 (1H, d, J=9.5 Hz, NH), 5.28±5.44 (2H, m,
H(70)+H(80)), 7.28 (1H, d, J=8.7Hz), 7.39±7.58 (5H, m),
7.99 (1H, d, J=4.7 Hz), 8.03±8.12 (1H, m), 9.47 (1H, s).
MS (FAB) m/z: 907 [(M)+]. HRMS (FAB) for
10-Dodecyl-3-[2-O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-D-glycero-ꢀ- and ꢁ-D-galacto-non-2-
ulopyranosylonate)ethyl]-6-nitro-5-deaza¯avin (1). To a
mixture of 10-dodecyl-3-(2-hydroxyethyl)-6-nitro-5-deaza-
¯avin (8, 235 mg, 0.5 mmol), methyl (5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-d-glycero-b-d-galacto-non-2-
ulopyranosyl chloride)onate20 (9, 255 mg, 0.5 mmol), and
well pulverized molecular sieves 4A (1.2 g) in 10 mL of
dry dichloromethane, was added silver tri¯uoromethane-
sulfonate (128 mg, 0.5 mmol) and the mixture was stirred
in the dark at room temperature under argon stream for
1 h. The catalyst and molecular sieves were removed o€
C43H49N5O17 [(M)+] calcd 907.3110, found 907.3132. b-
1
Anomer (2b): IR (KBr) 1735, 1700, 1655, 1620, 1525cm
.
20
d
ꢀ
1.38 (4H, m), 1.52±1.81 (4H, m), 1.96 (3H, s, NAc),
1
‰ꢀŠ
13.4 (c=1, CHCl3). H NMR (CDCl3) d 1.19±

2034
Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
1.97±2.20 (13H, m, OAc+Hax(30)), 2.51 (1H, dd, J=12.8,
4.9 Hz, Heq(30)), 3.30±3.42 (2H, m), 3.55±3.68 (1H, m,
H(50)), 3.80 (3H, s, CO2Me), 3.97±4.20 (2H, m,
H(60)+H(90)), 4.64±5.09 (2H, m), 4.90 (1H, dd, J=12.3,
2.6 Hz, H(90)), 5.20±5.41 (3H, m, H(40)+H(70)+H(80)),
6.38 (1H, d, J=9.1 Hz, NH), 7.28 (1H, d, J=8.1 Hz), 7.38±
7.58 (5H, m), 7.99 (1H, d, J=3.5 Hz), 8.03±8.18 (1H, m),
9.45 (1H, s). MS (FAB) m/z: 907 [(M)+]. HRMS (FAB)
for C43H49N5O17 [(M)+] calcd 907.3110, found 907.3101.
estimated by scanning at 254 nm with CAMAG TLC
Scanner 3 (CAMAG Chemie-Erzeugnisse und Adsorp-
tionstechnik AG).
In vitro antitumor activities of (nitro) 5-deaza¯avin-sialic
acid conjugate molecules
The tetrazolium-based semiautomated colorimetric
assay (MTT assay) developed by Carmichael29 was
modi®ed and used for the in vitro assay. 2000 Cells
(human oral epidermoid carcinoma KB cells or murine
leukemia L1210 cells) in 180 mL of RPML-1640 medium
were seeded in a 96-well ¯at bottom microtest plate and
20 ml of drug solutions with graded concentrations were
simultaneously added in triplicate to each well. The
plate was incubated for 3 days at 37 ꢀC in a humidi®ed
atmosphere of 5% CO2. To each well was added 25 mL
of MTT reagent (2 mg/mL in Dulbecco's phosphate
bu€ered saline without calcium and magnesium). After
another 4 h incubation at 37 ꢀC, the microplate was
centrifuged at 3000 rev/min for 10 min and the medium
was removed by aspiration. To each well was added
0.2 mL of dimethyl sulfoxide and each well was mixed
thoroughly with a mechanical plate mixer to solubilize
the resulting MTT-formazan. Absorbance at 540 nm
(OD540) was measured with a ImmunoReader NJ-2000
(InterMed Japan, Tokyo Japan). By using the OD540 of
each well the 50% inhibitory drug concentration (IC50
value) was determined as was described previously.30
10-Dodecyl-3-methyl-8-[N-methyl-N-[2-O-(methyl 5-acet-
amido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-ꢀ-
and ꢁ-D-galacto-non-2-ulopyranosylonate)ethyl]amino]-
5-deaza¯avin (3). To a mixture of 10-dodecyl-8-[N-(2-
hydroxyethyl)-N-methylamino]-3-methyl-5-deaza¯avin (14,
234mg, 0.5mmol), methyl (5-acetamido-4,7,8,9-tetra- O-
acetyl-3,5-dideoxy-d-glycero-b-d-galacto-non-2-ulopyra-
nosyl chloride)onate20 (9, 255 mg, 0.5 mmol), and well
pulverized molecular sieves, 4A (1.2 g) in 10 mL of dry
dichloromethane, was added silver tri¯uoromethane-
sulfonate (128mg, 0.5 mmol) and the mixture was stirred
in the dark at room temperature under argon stream for
1 h. The catalyst and molecular sieves were removed o€
by ®ltration and the ®ltrate was condensed under reduced
pressure. The resulting residue was subjected to thin layer
chromatography on silica gel (chloroform:acetone=6:1 as
eluent) to a€ord 104 mg (22%) of 3a as yellow amorphous
powder and 160 mg (34%) of 3b as yellow amorphous
powder: a-Anomer (3a): IR (KBr) 1740, 1685, 1625,
20
d
1590, 1525cm 1. ‰ꢀŠ
0.8ꢀ (c=1, CHCl3). H NMR
1
(CDCl3) d 0.88 (3H, t, J=6.6 Hz), 1.07±1.58 (20H, m),
1.88 (3H, s, NAc), 1.94±2.19 (13H, m, OAc+Hax(30)),
2.55 (1H, dd, J=12.8, 4.6 Hz, Heq(30)), 3.21 (3H, s), 3.46
(3H, s), 3.63 (3H, s, CO2Me), 3.68±3.81 (2H, m), 3.86±3.97
(1H, m, H(50)), 3.98±4.12 (2H, m, H(60)+H(90)), 4.19 (1H,
d, J=13.6 Hz, H(90)), 4.42±4.95 (4H, m), 4.78±4.90 (1H,
m, H(40)), 5.23±5.42 (3H, m, H(70)+H(80)+NH), 6.54
(1H, s), 6.95 (1H, dd, J=9.1, 2.0 Hz), 7.67 (1H, d,
J=9.0 Hz), 8.66 (1H, s). MS (FAB) m/z: 941 [(M)+].
HRMS (FAB) for C47H67N5O15 [(M)+] calcd 941.4616,
found 941.4650. b-Anomer (3b): IR (KBr) 1740, 1685,
Acknowledgements
We are grateful to Dr. N. Kawaguchi of Research
Institute of Life Science of Snow Brand Milk Products
Co., Ltd. for measurements of mass spectra of com-
pounds 1±3.
References and Notes
20
1625, 1590, 1525cm 1. ‰ꢀŠ
5.1ꢀ (c=1, CHCl3). H
1
d
NMR (CDCl3) d 0.88 (3H, t, J=6.6 Hz), 1.17±1.58 (20H,
m), 1.97 (3H, s, NAc), 2.00±2.20 (13H, m, OAc+Hax(30)),
2.43 (1H, dd, J=12.9, 5.0 Hz, Heq(30)), 3.26 (3H, s), 3.42
(3H, s), 3.58±3.72 (2H, m), 3.74 (3H, s, CO2Me), 3.83 (1H,
dd, J=10.5, 2.2 Hz, H(50)), 3.88±4.06 (2H, m,
H(60)+H(90)), 4.47±4.68 (4H, m), 5.02 (1H, dd, J=12.3,
2.3 Hz, H(90)), 5.10±5.25 (3H, m, H(40)+H(70)+NH),
5.28±5.32 (1H, m, H(8)), 6.64 (1H, s), 7.08 (1H, dd,
J=9.2, 1.9 Hz), 7.72 (1H, d, J=9.2 Hz), 8.56 (1H, s).
MS (FAB) m/z: 941 [(M)+]. HRMS (FAB) for
C47H67N5O15 [(M)+] calcd 941.4616, found 941.4603.
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3
Solutions (5 mL) of compounds 1±3 (5.0Â10 (M)) in
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concentration of resulting compound 8, 11, or 14 was

Y. Ikeuchi et al. / Bioorg. Med. Chem. 8 (2000) 2027±2035
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0.93 ppm; 3a, 0.07±0.21 ppm; 3b, 0.96±1.14 ppm.
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