Synthesis and anti-influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives.

Article abstract of DOI:10.1248/cpb.51.1386

Polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives on a poly-L-glutamine backbone are described. Aiming for a longer retention time of 4-guanidino-Neu5Ac2en (zanamivir) in bronchi and lungs, we focused on supermolecules bearing 4-gu

Full text of DOI:10.1248/cpb.51.1386

1
386
Chem. Pharm. Bull. 51(12) 1386—1398 (2003)
Vol. 51, No. 12
Synthesis and Anti-influenza Evaluation of Polyvalent Sialidase Inhibitors
Bearing 4-Guanidino-Neu5Ac2en Derivatives
,
a
b
a
a
b
Takeshi MASUDA,* Shuku YOSHIDA, Masami ARAI, Satoru KANEKO, Makoto YAMASHITA, and
Takeshi HONDA
a
a
b
Medicinal Chemistry Research Laboratories, Sankyo Co., Ltd.; and Biological Research Laboratories, Sankyo Co., Ltd.;
1
–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan.
Received July 29, 2003; accepted October 9, 2003; published online October 10, 2003
Polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives on a poly-L-glutamine backbone
are described. Aiming for a longer retention time of 4-guanidino-Neu5Ac2en (zanamivir) in bronchi and lungs,
we focused on supermolecules bearing 4-guanidino-Neu5Ac2en derivatives bound at their C-7 position through
noncleavable alkyl ether linkages. We first found that alkylation of the 7-hydroxyl group of sialic acid derivative 8
proceeded smoothly, and produced 7-O-alkyl-4-guanidino-Neu5Ac2en derivatives 13, which exhibited equipotent
inhibitory activity against not only influenza A virus sialidase but also influenza A virus in the cell culture. Next,
we synthesized poly-L-glutamine bearing 7-O-alkyl-4-guanidino-Neu5Ac2en derivatives linked by amide bonds,
2
6, which showed enhanced antiviral activity against influenza A virus and more potent efficacy in vivo relative to
a monomeric sialidase inhibitor.
Key words anti-influenza drug; sialidase inhibitor; sialic acid; zanamivir; poly-L-glutamine
Influenza virus infection is known to cause substantial fa- sought. To circumvent the short retention time of zanamivir,
tigue and sometimes death worldwide. Treatments were lim- we modifed zanamivir so that it would not be released into
ited before the development of influenza sialidase (neu- the systemic circulation from the respiratory tract. We con-
raminidase) inhibitors, which has lead to a major break- structed a polymer carrying zanamivir equivalents on its
1
—3)
through in the control of influenza.
The influenza siali- sides attached by noncleavable linkages (Fig. 2).
In designing a polymer that carries zanamivir equivalents,
dase is one of two viral coat glycoproteins critical for viral
replication. It is an attractive target for antiviral intervention the important point to consider was how to connect 4-guani-
because its active site is antigenically conserved among all dino-Neu5Ac2en molecules to the polymer backbone with-
4
—6)
clinically relevant strains.
Zanamivir (4-guanidino-Neu5Ac2en)
phosphate
out a loss of inhibitory activity. From X-ray studies of the
and Oseltamivir complex with sialidase and zanamivir reported by Varghese’s
7
—10)
1
1,12)
are two influenza sialidase inhibitors that have
been approved for human use for the treatment of influenza
infection (Fig. 1). They are transition state analogs of sialyl
glycoside linkage hydrolysis. Oseltamivir phosphate, an ethyl
ester prodrug of GS 4071, is administered orally, whereas
Zanamivir is delivered by oral inhalation due to its poor oral
bioavailability. They have been widely used as they are safe,
and effective against both A and B strains, as well as emerg-
Fig. 2
3
)
ing resistant strains.
In developing our sialidase inhibitor, administration by in-
halation was preferred because direct delivery of the medi-
cine to the site of viral replication in the respiratory tract as-
sured the use of a much smaller dose and avoidance of sys-
temic effects. However, zanamivir would require frequent ad-
ministrations because it is rapidly eliminated in the un-
changed form in the urine, and completely eliminated within
1
3)
2
4 h, after a single dose. Therefore, a new class of sialidase
inhibitors to be inhaled at lower and less frequent doses was
Fig. 1
Fig. 3
∗
To whom correspondence should be addressed. e-mail: takemd@shina.sankyo.co.jp
© 2003 Pharmaceutical Society of Japan

December 2003
1387
group, the 7-hydroxyl group of zanamivir was shown to form Chemistry
1
4)
no direct hydrogen bonds with sialidase. Moreover, as it
Synthesis of 7-O-Alkyl Ether Derivatives Related to
was oriented outward of the enzyme active site, it was con- Zanamivir In fact, there were no reports on the direct
1
4)
sidered suitable for further elongatation (Fig. 4). Based on chemical modification of the 7-hydroxyl group of sialic acid
1
6,17)
this knowledge, we anticipated that the 7-hydroxyl group except by acylation.
would help form a suitable link to the polymer. We first tried methylating the 7-hydroxyl group of dihy-
In the course of our previous studies, we have investigated dropyranyl intermediates 2, 4, and 6, which were derived
1
8—20)
the synthesis and biological evaluation of analogues related from intermediate 1.
Despite many attempts, including
to zanamivir modified at position-7 by using chemoenzy- under basic and acidic conditions, we could not obtain 7-
1
5)
matic reactions. Although the replacement of the 7-OH methoxy compounds 3, 5, and 7 (Chart 1). We thought the
moiety with N-amide groups showed a decrease in virus steric hindrance of the 7-hydroxyl group of the glycerol moi-
growth inhibitory activity, replacement with methoxy and ety might have been the cause.
ethoxy groups retained or improved the inhibitory activity
Instead of using dihydropyranyl compounds as substrates,
against both influenza A virus sialidase and influenza A virus we chose tetrahydropyranyl derivative 9, which was reported
1
6)
growth in cell culture (Fig. 3). Therefore, we were interested to have been 7-O-acylated. We found that the alkylation re-
in polymers with 4-guanidino-7-O-alkyl-Neu5Ac2en ana- action proceeded smoothly with sodium hydrogen and an
logues attached as multivalent influenza sialidase inhibitors.
alkyl halide in DMF solution. Instead of sodium hydrogen
Here, we describe the detailed synthesis, characterization, and alkyl halide, activation with potassium hydroxide or
and biological evaluation of 7-O-alkyl ether analogues re- potassium tert-butoxide followed by addition of various di-
lated to zanamivir and polymer-type sialidase inhibitors.
alkylsulfates also afforded 7-O-alkylated sialic acid deriva-
tives 9. Successive methanolysis of 9 gave methyl ester 10.
1
4)
Fig. 4. Perspective View into the Active Site of the N9 Sialidase (Neuraminidase) Complexed with Zanamivir
Left: CPA Model, right: Tube Model.
Chart 1. 7-Modification of Neu5Ac2en Intermediates

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388
Vol. 51, No. 12
Chart 2. 7-O-Alkylation of Sialic Acid Derivative 8
Chart 3. Synthetic Scheme of 7-O-Alkylether Derivatives Related to Zanamivir 13
Chart 4. Synthetic Scheme of 7-O-Hydroxyalkyl Ether Sialic Acid Derivatives 15 and 16
Dialkylsulfate was readily prepared by Ru-oxidation of di- tional methods followed by deprotection which gave hydrox-
alkylsulfite, which was obtained by reacting thionyl chloride yalkyl ether 13j, 13k, and 13l, respectively (Chart 5).
3
0)
and alcohol with triethylamine. 10 was converted to 7-O- Methanolysis of 18 followed by acetonization gave 8,9-O-
alkylated zanamivir derivatives 13 using conventional meth- acetonide-7-O-hydroxyalkyl ether 19. The primary alcohol of
ods via intermediates 4-azide compounds 11, and 4-guani- 19 was tosylated and successive treatment of the resulting
1
8—22)
dino compounds 12 (Chart 3).
compound with sodium azide gave azide compounds 20. De-
To further develop compounds 13, we modified the termi- protection of 20 gave azidealkyl ether derivatives 21 (Chart
nal position of the alkyl groups to allow coupling with the 6). Reaction of 20 with Lindlar catalyst gave amine com-
polymer backbone (Chart 4). Treatment of 8 with potassium pounds 22, and further deprotection of 22 gave aminoalkyl
hydroxide followed by di(benzyloxyethyl)- or di(benzyl- ether derivatives 23. Acetylation of the primary amine of 22
oxypentyl)-sulfate gave 14j and 14k, respectively. An ensu- followed by deprotection gave acetylaminoalkyl ether deriva-
ing reduction with Pd–C gave 7-O-hydroxyethyl ether 15j tives 24 and 25 (Chart 7).
and 7-O-hydroxypentyl ether 15k, respectively. Treatment of
Treatment of 7-O-aminoethyl-4-guanidino-Neu5Ac2en 23j
2
3)
8
with sodium hydride followed by di[10-(tetrahydro-pyran- with excess 1-(acetoxy)benzotriazole and pyridine in water
-yloxy)-decyl]sulfate gave 16. 15j, 15k, and 16 were con- at room temperature for 2 h exclusively afforded acetylamino
2
verted to 4-protected guanidine compounds 18 using conven- derivative 25j without any N-acylguanidine (Chart 8). We

December 2003
1389
Chart 5. Synthetic Scheme of 7-O-Hydroxyalkyl Ether Derivatives 13
Chart 6. Synthetic Scheme of 7-O-Azidealkyl Ether Derivatives 21
Chart 7. Synthetic Scheme of 7-Aminoalkyl Ether 23 and 7-O-Acetylaminoalkyl Ether Derivatives 26
Chart 8. Treatment of 23j with 1-(Acetoxy)benzotriazole and Pyridine in Water

1
390
Vol. 51, No. 12
Chart 9. Synthesis of Polymer Bearng 4-Guanidino-Neu5Ac2en Derivatives as Side Chain 26
Table 1. Sialidase Inhibition and Plaque Reduction Activities of Com-
pounds 13, 21, 23, and 25 (IC50 (ng/ml))
found that selective acetylation of the aminoalkyl ether group
was achieved. This result suggests that the primary amino
group reacted readily perhaps due to its high flexibility. How-
ever, the guanidino group was slow to react probably due to
intra- or intermolecular salt formation with carboxylic acid
and steric hindrance. This selectivity is desirable for the con-
densation of 23 to form the polymer.
Synthesis of a Poly-L-glutamine Polymer Bearing
Zanamivir Molecules As we were able to obtain 7-O-alkyl-
amino-4-guanidino-Neu5Ac2en and subject it to selective
acylation, we next focused on the synthesis of supermole-
cules bearing 4-guanidino-Neu5Ac2en derivatives.
Sialidase inhibition Plaque reduction
assay
assay
R
A/PR/8/34
A/Yamagata/32/89
a)
a)
Zanamivir
OH
5.1—10.2 (1.0)
6.1 (1.2)
1.9—20 (1.0)
We selected poly-L-glutamic acid as a polymer backbone
precursor because poly-L-glutamic acid possesses some ad-
vantages as a drug carrier such as good biodegradability,
high water solubility, the presence of multiple carboxyl
groups that are easily modified chemically, low toxicity, and
1
1
3a
3b
3.0 (0.15)
0.7 (0.14)
26.4 (2.6)
1
1
3c
30.6 (3.0)
14.3 (2.6)
1.5 (0.35)
2.0 (0.23)
3d
2
4)
low immunogenicity.
13e
20.2 (2.0)
49.6 (4.9)
1.8 (0.42)
2.2 (0.51)
Reaction of poly-L-glutamic acid (MW: 50000—100000,
average MW: 71400, average extent of polymerizationϭ470)
with benzotriazole ester and 0.1 or 0.3 eq of the 7-O-
aminoalkyl-4-guanidino-Neu5Ac2en 23j, 23k, or 23l for 1 h
followed by quenching with aqueous ammonia to convert un-
reacted activated esters to carbamoyl groups and purification
1
1
1
1
3f
3g
3h
3i
262 (26)
11.0 (5.8)
3.0 (1.6)
36.6 (3.6)
1
by dialysis afforded poly-L-glutamine conjugate 26. The H-
26.3 (2.6)
10.3 (1.4)
1.9 (1.0)
NMR spectrum of conjugate 26 showed the incorporation of
approximately 10 or 25% ligand 23 onto the polymer back-
bone confirming that a complete reaction had occurred.
Biological Evaluation. Structure–Activity Relation-
ship of 7-O-Alkylated Derivatives Related to Zanamivir
The neuraminidase inhibitory activity and the 50% effective
inhibitory concentration (IC ) against influenza A are shown
13j
1.8 (0.21)
2
2
2
1j
3j
5j
14.8 (2.0)
47.7 (6.5)
9.8 (1.4)
1.5 (0.13)
7.5 (0.63)
2.4 (0.2)
a) Since IC50 values varied depending on the experiment, the relative potencies of
the compounds to zanamivir are shown in parentheses based on the IC50 values. The
IC50 values of zanamivir in enzyme inhibition and plaque reduction were 5.1—
5
0
1
5)
in Table 1. 13a (7-O-methyl ether), 13b (7-O-ethyl ether),
1
3c (7-O-n-propyl ether), 13d (7-O-n-propyl ether), 13e (7- 10.2 ng/ml and 1.9—20 ng/ml, respectively.
O-n-octyl ether), and 13f (7-O-n-dodecyl ether), whose linear
alkyl ether linkages was less than 12 carbons in length exhib-
ited similar inhibitory activity to zanamivir against influenza nificantly affect the binding of compounds 13j, 21j, 23j, and
A virus sialidase. These compounds showed pronounced im- 25j.
provement in inhibitory activity against influenza A virus in-
fection of MDCK cell compared to zanamivir. However, 13g Bearing 4-Guanidino-Neu5Ac2en Derivatives The IC50
7-O-n-tetradecyl ether) which has a linear linkage greater against influenza neuraminidase and influenza A of the syn-
Structure–Activity Relationship of Poly-L-glutamine
(
2
5)
than 12 carbons in length showed a slight decrease in in- thesized polymers 26 are shown in Table 2. The IC values
5
0
hibitory activity against sialidase and virus infection, proba- of each polymer relative to monomeric zanamivir were calcu-
bly due to unfavorable steric and electrostatic interaction lated. The inhibitory activity of all polymers 26 against
within the enzyme binding site. Substituting a cyclic influenza A virus sialidase was less potent than that of
aliphatic moiety, aromatic moiety, and hetero atom for the zanamivir. However, the evaluation of the inhibitory activity
hydroxyl, azide, amino, and acetylamino group did not sig- against influenza A virus infection of MDCK cells demon-

December 2003
1391
Table 2. Sialidase Inhibitory and Plaque Reduction Activities of Com- C-7 position. All polymers displayed less potent influenza A
a)
pounds 26 (IC50 (nM))
sialidase inhibitory activity. However, a much greater effi-
cacy against influenza A in the mice model by intranasal ad-
ministration than zanamivir was observed.
Sialidase inhibitory Plaque reduction
assay
assay
b)
n
p : q
Experimental
A/PR/8/34
A/Yamagata/32/89
General Methods IR spectra were recorded on a Jasco FT-IR 8300 or
900 spectrometer. NMR spectra were recorded on a JEOL EX 270
c)
c)
8
Zanamivir
11—29 (1.0)
9.8 (1.4)
1.3—27 (1.0)
(
(
1
270 MHz) or GSX-400 (400 MHz) spectrometer using tetramethylsilane
TMS) as an internal standard. Mass spectra were recorded on a JEOL HX-
00, SX-102A or AX-505H mass spectrometer. The melting point (mp) was
2
2
2
2
2
2
5j
1
4
1
1
4
9
2.4 (0.2)
5.9 (1.3)
0.066 (0.0077)
0.17 (0.020)
0.072 (0.0085)
0.076 (0.023)
5k
6a
6b
6c
6d
90 (6.0)
114 (5.2)
364 (22.8)
161 (7.3)
62 (2.1)
10 : 1
3 : 1
10 : 1
10 : 1
determined using a Yanagimoto micro-melting point apparatus and was not
corrected. Optical rotations were obtained with a Jasco DIP-370 polarimeter.
Column chromatography was carried out on Silica gel 60 (230—400 mesh,
Art. 9385, Merck). The analytical column for HPLC was an L-Column ODS
(5 mm particle size, 4.6 mm i.d.ϫ150 mm).
5-Acetylamino-3,5-dideoxy-2,7-O-dimethyl-8,9-O-isopropylidene-4-O-
tert-butyldimethylsilyl-D-glycero-b-D-galacto-2-nonulo-pyranosonic Acid
Methyl Ester 9a A solution of 5-acetylamino-3,5-dideoxy-8,9-O-iso-
propylidene-2-O-methyl-4-O-tert-butyldimethylsilyl-D-glycero-b-D-galacto-
2
(
ide (0.67 g, 10.17 mmol). The reaction mixture was stirred at room tempera-
ture for 1 h. Then, dimethyl sulfate (0.96 ml, 10.17 mmol) was added drop-
wise with cooling in an ice bath. The reaction mixture was stirred at room
temperature for 1 h, diluted with aqueous ammonium hydrochloride and ex-
tracted with ethyl acetate. The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and concentrated under reduced pressure.
The crude product was purified by silica gel chromatography (eluent;
CH Cl : MeOHϭ50 : 1) to obtain 9a as an amorphous solid (510 mg,
a) Relative to the monomeric sialidase inhibitor content. b) The ratios were deter-
1
mined by H-NMR. c) Since IC50 values varied depending on the experiment, the rel-
ative potencies of the compounds to zanamivir are shown in parentheses based on the
IC50 values. The IC50 values of zanamivir in enzyme inhibition and plaque reduction
were 11—29 nM and 1.3—27 nM, respectively.
-nonulo-pyranosonic acid methyl ester 8 (1.0 g, 2.03 mmol) in acetonitrile
20 ml) was cooled in an ice bath and slowly mixed with potassium hydrox-
a)
b)
Table 3. Survival Rates of Infected Mice Administered Compound 26c
and Zanamivir
b)
No. of survivors/Total No. of mice
1
0 d after infection
20 d after infection
Zanamivir
1/8
7/7
0/8
7/7
c)
2
6c
2
2
1
1
.01 mmol, 50%). mp 160—161 °C. H-NMR (CDCl ) d: 0.05 (3H, s), 0.06
3
a) Mice were infected with influenza A/PR/8/34 (H1N1) virus. b) Compound 26c
and zanamivir were administered intranasally at doses of 0.3 mmol/kg once beginning
4 h prior to infection. The concentration of 0.3 mmol of 26c was calculated based on
the molar concentration of the monomeric sialidase inhibitor. c) pϭ0.0009 versus
(
1
3H, s), 0.86 (9H, s), 1.33 (3H, s), 1.42 (3H, s), 1.73 (1H, dd, Jϭ13.1,
0.7 Hz), 1.99 (3H, s), 2.28 (1H, dd, Jϭ13.1, 5.1 Hz), 3.22 (3H, s), 3.48—
2
3.56 (1H, m), 3.57 (3H, s), 3.78 (3H, s), 3.80—4.30 (6H, m), 5.16 (1H, d,
Jϭ8.2 Hz). FAB-MS m/z: 506 (MϩH) . HR-FAB-MS m/z Calcd for
C H O NSiNa (MϩNa) : 528.2605. Found: 528.2609. IR (KBr) cm
3
ϩ
zanamivir (Log-rank test).
ϩ
Ϫ1
:
2
3
43
9
23
387, 3291, 3078, 2984, 2954, 2934, 2896, 2857, 1750, 1660, 1554. [a]D
strated that all the polymers were much more active than Ϫ13.4° (cϭ0.10, CH OH).
3
monomeric sialidase inhibitor 25j, 25k and zanamivir, re-
gardless of their content of 4-guanidino-Neu5Ac2en mole-
cules and length of the carbon chain. As a control for the bio-
logical evaluation, unsubsituted poly-L-glutamic acid was
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-
methoxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester
1a 9a (2.31 g, 4.58 mmol) was treated with acetic anhydride (30 ml),
acetic acid (30 ml), sulfuric acid (95%, 3 ml) and the reaction mixture was
stirred at room temperature for 16 h. The solvent was poured onto ice-cold
1
used and showed no inhibitory activity. This enhancement aqueous sodium hydrogen carbonate and methylene chloride. The organic
could be explained by the contribution of multivalence or layer was washed with brine, dried over anhydrous sodium sulfate, and con-
2
6—28)
centrated under reduced pressure. After adding toluene, the mixture was
evaporated, and the residue was dissolved in N,N-dimethylformamide. To
this solution was added sodium azide (2.3 g, 35.4 mmol) and DOWEX 50W
cluster effects.
Efficacy of Intranasally Administered Polymeric Siali-
dase Inhibitor 26c Tested in the Influenza Virus-Infected
ϩ
X8 (H ) (2.3 g). The reaction mixture was stirred at 70 °C for 6 h, and fil-
Mouse Model Furthermore, the efficacy of intranasally ad- tered. The residue was thoroughly washed with ethyl acetate. The filtrate
was combined, washed with aqueous sodium hydrogen carbonate and brine,
dried over anhydrous sodium sulfate, and evaporated under reduced pres-
sure. The crude product was purified by silica gel chromatography (eluent;
ministered polymeric sialidase inhibitor 26c (average MW:
7
8100) was tested in the influenza virus-infected mouse
model in terms of the survival rate of treated and infected
CH Cl : MeOHϭ50 : 1) to obtain 11a as a colorless amorphous solid
2
2
1
mice relative to that of control mice. Compound 26c was ad- (1.28 g, 2.99 mmol, 65%). H-NMR (CDCl ) d: 2.06 (3H, s), 2.06 (3H, s),
3
ministered intranasally once beginning 24 h prior to infec- 2.09 (3H, s), 3.51 (3H, s), 3.65 (1H, dd, Jϭ3.0, 2.0 Hz), 3.79 (3H, s), 4.07
(
1H, ddd, Jϭ8.8, 8.8, 8.8 Hz), 4.22 (1H, dd, Jϭ12.7, 6.8 Hz), 4.40 (1H, dd,
tion. It was found that 26c was much more effective than
zanamivir as shown in Table 3 (7/7 survived in the 26c-
treated infected group while there were no survivors in the
Jϭ8.8, 2.9 Hz), 4.42 (1H, dd, Jϭ8.8, 2.0 Hz), 4.80 (1H, dd, Jϭ12.7, 2.0 Hz),
.32 (1H, ddd, Jϭ6.8, 3.0, 2.0 Hz), 5.94 (1H, d, Jϭ2.9 Hz), 6.47 (1H, d,
5
ϩ
Jϭ8.8 Hz). FAB-MS m/z: 429 (MϩH) . HR-FAB-MS m/z Calcd for
ϩ
Ϫ1
case of zanamivir). This in vivo efficacy is the first such re- C H O N (MϩH) : 429.1621. Found: 429.1628. IR (KBr) cm : 3334,
1
7
25
9
4
sult for polymeric sialidase inhibitors. No in vitro cytotoxic- 3270, 3226, 3073, 2955, 2845, 2099, 1749, 1736, 1684, 1669, 1549, 1439,
2
3
1
374. [a] ϩ117.4° (cϭ0.73, CHCl3).
D
ity or in vivo toxicity in mouse was observed.
(
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)-
In conclusion, we synthesized a series of 7-O-alkyl ether
derivatives related to zanamivir by direct alkylation of the C-
guanidino-6-(2؅,3؅-diacetoxy-1؅-methyloxy)propyl-5,6-dihydro-4H-
pyran-2-carboxylic Acid Methyl Ester 12a A solution of 11a (3.85 g,
7
alcohol of a sialic acid derivative. Alkyl ether moiety less 8.98 mmol) in ethanol (200 ml) was hydrogenated with Lindlar’s catalyst for
than 12 carbons in length showed similar activities against 2 h. The reaction mixture was filtered through Celite and the precipitate was
washed with methanol. The filtrate was combined and evaporated. The
residue was dissolved in tetrahydrofuran (50 ml). This solution was mixed
with N,NЈ-bis-tert-butyloxycarbonyl-1H-pyrazole-1-carboxamidine (9.6 g,
influenza A virus sialidase. These compounds showed im-
proved virus growth inhibitory activity compared to
zanamivir. We also synthesized poly-L-glutamines carrying
3
1.0 mmol) and the reaction mixture was stirred at room temperature for
4
-guanidino-Neu5Ac2en bound via alkyl ether bonds at the 24 h. The reaction mixture was diluted with aqueous ammonium hydrochlo-

1
392
Vol. 51, No. 12
ϩ
ride and extracted with ethyl acetate. The organic layer was washed with (1H, d, Jϭ1.8 Hz). FAB-MS m/z: 361 (MϩH) .
brine, dried over anhydrous sodium sulfate, and concentrated under reduced
5-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-7-O-n-
pressure. The crude product was purified by silica gel chromatography (elu- propyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-pyra-
ent; hexane : ethyl acetateϭ2 : 1) to obtain 12a as a colorless amorphous nosonic Acid n-Propyl Ester 9c A procedure similar to that used for the
1
solid (4.42 g, 6.86 mmol, 76%). H-NMR (CDCl ) d: 1.49 (9H, s), 1.50 (9H, preparation of 10b was employed with di(n-propyl)sulfate and 9c was ob-
3
1
s), 1.97 (3H, s), 2.05 (3H, s), 2.07 (3H, s), 3.52 (3H, s), 3.54—3.56 (1H, m), tained as a colorless amorphous solid (3.37 g, 5.99 mmol, 49%). H-NMR
3
.78 (3H, s), 4.10 (1H, dd, Jϭ10.5, 1.0 Hz), 4.25—4.40 (2H, m), 4.82 (1H,
(CDCl ) d: 0.05 (3H, s), 0.06 (3H, s), 0.86 (9H, s), 0.86—1.00 (6H, m), 1.33
3
dd, Jϭ12.0, 3.2 Hz), 5.12 (1H, ddd, Jϭ11.0, 11.0, 2.5 Hz), 5.29—5.31 (1H, (3H, s), 1.42 (3H, s), 1.60—1.77 (5H, m), 1.98 (3H, s), 2.27 (1H, dd,
m), 5.85 (1H, d, Jϭ2.3 Hz), 6.35 (1H, d, Jϭ8.8 Hz). FAB-MS m/z: 645
Jϭ13.2, 4.6 Hz), 3.23 (3H, s), 3.53—3.68 (3H, m), 3.75—3.98 (2H, m),
4.00—4.30 (6H, m), 5.15 (1H, d, Jϭ7.9 Hz). FAB-MS m/z: 562 (MϩH) .
ϩ
ϩ
(MϩH) .
(
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-(2؅,3؅-dihydroxy-1؅-
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-n-
methoxy)propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13a 12a (53 propoxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid n-Propyl Ester
mg, 0.082 mmol) was treated with methylene chloride (3 ml), and trifluo- 11c Compound 11c was obtained from 10c (1.80 g, 3.21 mmol) using the
roacetic acid (30 ml) and the reaction mixture was stirred at room tempera- same procedure employed for the preparation of 11a (colorless amorphous
1
ture for 4 h. Then, the solvent was removed under reduced pressure. The
solid, 1.10 g, 2.27 mmol, 71%). H-NMR (CDCl
3
) d: 0.89—0.99 (6H, m),
residue was dissolved in methanol (1 ml) and treated with aqueous sodium 1.55—1.80 (4H, m), 2.04 (3H, s), 2.05 (3H, s), 2.09 (3H, s), 3.40—3.50
hydroxide (1 N, 0.3 ml, 0.3 mmol) with cooling in an ice bath. The reaction (1H, m), 3.55—3.65 (1H, m), 3.70—3.77 (1H, m), 3.92—4.02 (1H, m), 4.29
was stirred for 30 min and neutralized with DOWEX 50W X8 (H ). Then, (1H, dd, Jϭ11.9, 7.3 Hz), 4.45—4.56 (2H, m), 4.63—4.71 (1H, m), 5.30—
ϩ
the reaction was filtered and the residue was thoroughly washed with 5.39 (1H, m), 5.68—5.73 (1H, m), 5.98 (1H, d, Jϭ3.3 Hz). FAB-MS m/z:
ϩ
methanol. The filtrate was combined, and the mixture was evaporated under 485 (MϩH) .
reduced pressure. The crude product was purified by reverse phase cosmosil
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)-
chromatography (eluent; water : MeOHϭ1 : 1) to obtain 13a as a colorless guanidino-6-(2؅,3؅-diacetoxy-1؅-propoxy)propyl-5,6-dihydro-4H-pyran-
1
amorphous solid (24 mg, 0.0693 mmol, 84%). H-NMR (D O) d: 2.00 (3H,
2-carboxylic Acid n-Propyl Ester 12c Compound 12c was obtained from
2
s), 3.37 (3H, s), 3.55 (1H, d, Jϭ8.5 Hz), 3.65 (1H, dd, Jϭ5.0, 12.0 Hz), 3.80
1H, dd, Jϭ2.6, 12.0 Hz), 3.90 (1H, m), 4.20 (1H, dd, Jϭ10.0, 10.0 Hz), ration of 12a (colorless amorphous solid, 710 mg, 1.01 mmol, 84%). H-
.40—4.50 (2H, m), 5.85 (1H, d, Jϭ1.8 Hz). FAB-MS m/z: 347 (MϩH) .
-Acetylamino-3,5-dideoxy-7-O-ethyl-8,9-O-isopropylidene-2-O- s), 1.50 (9H, s), 1.55—1.80 (4H, m), 1.95 (3H, s), 2.05 (3H, s), 2.09 (3H, s),
methyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-pyra-
3.49—3.59 (2H, m), 3.70—3.72 (1H, m), 4.03—4.07 (1H, m), 4.29—4.40
nosonic Acid Methyl Ester 10b 8 (6.0 g, 12.2 mmol) in acetonitrile (2H, m), 4.77 (1H, dd, Jϭ12.5, 2.0 Hz), 5.07—5.16 (1H, m), 5.24—5.33
11c (580 mg, 1.20 mmol) using the same procedure employed for the prepa-
1
(
4
ϩ
NMR (CDCl ) d: 0.92 (3H, t, Jϭ7.3 Hz), 0.95 (3H, t, Jϭ7.3 Hz), 1.49 (9H,
3
5
(
(
1
30 ml) was cooled in an ice bath and mixed with potassium hydroxide
4.0 g, 61.0 mmol). The reaction mixture was stirred at room temperature for 8.49—8.54 (1H, m). FAB-MS m/z: 701 (MϩH) .
h. Then, diethyl sulfate (10.3 g, 67.0 mmol) was added dropwise with cool-
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-(2؅,3؅-dihydroxy-1؅-
(1H, m), 5.82 (1H, d, Jϭ2.6 Hz), 6.34—6.40 (1H, m), 7.61—7.65 (1H, m),
ϩ
ing in an ice bath and stirred at room temperature for 1 h. The reaction mix- propoxy)propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13c Com-
ture was diluted with aqueous ammonium hydrochloride and extracted with pound 13c was obtained from 12c (100 mg, 0.143 mmol) using the same
ethyl acetate. The organic layer was washed with brine, dried over anhydrous procedure employed for the preparation of 13a (colorless amorphous solid,
1
sodium sulfate, and concentrated under reduced pressure. The crude ethyl 45 mg, 0.120 mmol, 84%). H-NMR (CDCl
) d: 0.90 (3H, t, Jϭ7.3 Hz),
3
ester was dissolved in methanol, treated with sodium methoxide (4.9 M in
1.52—1.64 (2H, m), 1.99 (3H, s), 3.37—3.43 (1H, m), 3.51—3.69 (4H, m),
methanol, 2.5 ml, 12.0 mmol) at 0 °C, and stirred at room temperature for 3.81—3.87 (1H, m), 4.00—4.06 (1H, m), 4.29—4.34 (1H, m), 4.39—4.44
h. The reaction mixture was diluted with aqueous ammonium hydrochlo- (1H, m), 5.56—5.58 (1H, m). FAB-MS m/z: 375 (MϩH) .
ϩ
1
ride and extracted with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, and concentrated under reduced
5-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-7-O-n-
pentyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-pyra-
pressure. The crude product was purified by silica gel chromatography (elu- nosonic Acid n-Pentyl Ester 9d A procedure similar to that used for the
ent; hexane : ethyl acetateϭ1 : 1) to obtain 10b as an amorphous solid preparation 10b was employed with di(n-pentyl)sulfate and 9d was obtained
1
1
(
0
3.26 g, 6.11 mmol, 50%). H-NMR (CDCl ) d: 0.05 (3H, s), 0.06 (3H, s),
as a colorless amorphous solid (3.70 g, 5.99 mmol, 49%). H-NMR (CDCl
3
)
3
.86 (9H, s), 1.20 (3H, t, Jϭ7.0 Hz), 1.33 (3H, s), 1.42 (3H, s), 1.73 (1H, dd,
d: 0.05 (3H, s), 0.06 (3H, s), 0.86 (9H, s), 0.90—0.94 (6H, m), 1.30—1.38
Jϭ13.1, 10.7 Hz), 1.99 (3H, s), 2.28 (1H, dd, Jϭ13.1, 5.1 Hz), 3.22 (3H, s), (8H, m), 1.32 (3H, s), 1.41 (3H, s), 1.53—1.64 (4H, m), 1.73 (1H, dd,
3
.40—3.60 (3H, m), 3.79 (3H, s), 3.80—4.30 (6H, m), 5.15 (1H, d,
Jϭ13.1, 10.7 Hz), 1.97 (3H, s), 2.26 (1H, dd, Jϭ13.1, 5.1 Hz), 3.22 (3H, s),
3.60—3.71 (3H, m), 3.79—3.85 (1H, m), 3.91 (1H, dd, Jϭ10.5, 1.4 Hz),
ϩ
Jϭ8.3 Hz). FAB-MS m/z: 520 (MϩH) .
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-
(
4.00—4.05 (1H, m), 4.10—4.20 (4H, m), 4.23—4.27 (1H, m), 5.13 (1H, d,
ϩ
ethoxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester Jϭ8.9 Hz). FAB-MS m/z: 618 (MϩH) .
1
1b Compound 11b was obtained from 10b (1.89 g, 3.54 mmol) using the
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-n-
same procedure employed for the preparation of 11a (colorless amorphous pentyloxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester
1
solid, 1.10 g, 2.48 mmol, 70%). H-NMR (CDCl ) d: 1.25 (3H, t, Jϭ7.0 Hz), 11d Compound 10d and successively 11d was obtained from 9d (440 mg,
3
2
4
.09 (3H, s), 2.10 (3H, s), 2.13 (3H, s), 3.50—3.80 (3H, m), 3.80 (3H, s), 0.712 mmol) using the same procedure employed for the preparation of 11a
.05 (1H, m), 4.25 (1H, dd, Jϭ5.0, 12.0 Hz), 4.45—4.55 (2H, m), 4.80 (1H, (colorless amorphous solid, 190 mg, 0.392 mmol, 55%). H-NMR (CDCl )
3
1
dd, Jϭ3.2, 12.0 Hz), 5.35 (1H, m), 5.51 (1H, d, Jϭ8.2 Hz), 6.00 (1H, d, d: 0.90—0.94 (3H, m), 1.30—1.38 (4H, m), 1.56—1.64 (2H, m), 2.06 (3H,
ϩ
Jϭ2.7 Hz). FAB-MS m/z: 443 (MϩH) .
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)-
guanidino-6-(2؅,3؅-diacetoxy-1؅-ethoxy)propyl-5,6-dihydro-4H-pyran-2-
s), 2.07 (3H, s), 2.10 (3H, s), 3.47—3.52 (1H, m), 3.64—3.70 (1H, m), 3.76
(1H, dd, Jϭ4.3, 3.5 Hz), 3.82 (3H, s), 3.96 (1H, ddd, Jϭ8.8, 8.1, 8.1 Hz),
4.23 (1H, dd, Jϭ12.3, 7.1 Hz), 4.49 (1H, dd, Jϭ8.1, 3.0 Hz), 4.55 (1H, dd,
(
carboxylic Acid Methyl Ester 12b Compound 12b was obtained from Jϭ8.8, 3.5 Hz), 4.73 (1H, dd, Jϭ12.3, 3.0 Hz), 5.36 (1H, ddd, Jϭ7.1, 4.3,
1b (580 mg, 1.31 mmol) using the same procedure employed for the prepa- 3.0 Hz), 5.69 (1H, br d, Jϭ8.1 Hz), 6.00 (1H, d, Jϭ3.0 Hz). FAB-MS m/z:
1
1
ϩ
ration of 12a (colorless amorphous solid, 530 mg, 0.80 mmol, 61%). H- 485 (MϩH) .
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)-
3H, s), 2.06 (3H, s), 2.09 (3H, s), 3.50—3.80 (3H, m), 3.80 (3H, s), 4.10 guanidino-6-(2؅,3؅-diacetoxy-1؅-pentyloxy)propyl-5,6-dihydro-4H-
NMR (CDCl ) d: 1.20 (3H, t, Jϭ7.0 Hz), 1.49 (9H, s), 1.50 (9H, s), 1.95
3
(
(
1
1H, dd, Jϭ1.0, 10.5 Hz), 4.25—4.40 (2H, m), 4.80 (1H, dd, Jϭ3.2,
2.0 Hz), 5.10 (1H, ddd, Jϭ2.5, 11.0, 11.0 Hz), 5.30 (1H, m), 5.83 (1H, d,
pyran-2-carboxylic Acid Methyl Ester 12d Compound 12d was obtained
from 11d (109 mg, 0.224 mmol) using the same procedure employed for the
preparation of 12a (colorless amorphous solid, 115 mg, 0.164 mmol, 73%).
H-NMR (CDCl ) d: 0.87—0.91 (3H, m), 1.28—1.35 (4H, m), 1.49 (9H, s),
ϩ
Jϭ2.3 Hz), 6.20 (1H, d, Jϭ8.8 Hz). FAB-MS m/z: 659 (MϩH) .
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-(2؅,3؅-dihydroxy-1؅-
ethoxy)propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13b Com- 1.50 (9H, s), 1.56—1.64 (2H, m), 1.94 (3H, s), 2.06 (3H, s), 2.09 (3H, s),
pound 13b was obtained from 12b (50 mg, 0.076 mmol) using the same pro- 3.52—3.62 (2H, m), 3.70 (1H, dd, Jϭ4.0, 1.6 Hz), 3.78 (3H, s), 3.92—3.98
1
(
3
cedure employed for the preparation of 12a (colorless amorphous solid, (1H, m), 4.07 (1H, dd, Jϭ10.4, 1.6 Hz), 4.26 (1H, dd, Jϭ12.5, 7.6 Hz), 4.83
1
2
(
3
7 mg, 0.075 mmol, 98%). H-NMR (D O) d: 1.05 (3H, t, Jϭ7.0 Hz), 1.97 (1H, dd, Jϭ12.5, 2.4 Hz), 5.07—5.13 (1H, m), 5.28 (1H, ddd, Jϭ7.6, 4.0,
2
3H, s), 3.40 (1H, m), 3.50—3.65 (3H, m), 3.80 (1H, dd, Jϭ5.0, 12.0 Hz), 1.6 Hz), 5.82 (1H, d, Jϭ2.4 Hz), 6.36 (1H, br d, Jϭ8.2 Hz), 8.53 (1H, br d,
.90 (1H, m), 4.15 (1H, dd, Jϭ10.0, 10.0 Hz), 4.40—4.50 (2H, m), 5.55
ϩ
Jϭ8.6 Hz). FAB-MS m/z: 701 (MϩH) .

December 2003
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-(2؅,3؅-dihydroxy-1؅-
1393
(
4.72 (1H, dd, Jϭ12.3, 3.0 Hz), 5.33 (1H, ddd, Jϭ7.1, 4.3, 3.0 Hz), 5.79 (1H,
ϩ
pentyloxy)propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13d Com- br d, Jϭ7.8 Hz), 5.98 (1H, d, Jϭ3.0 Hz). FAB-MS m/z: 583 (MϩH) .
pound 13d was obtained from 12d (42 mg, 0.060 mmol) using the same pro-
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)-
cedure employed for the preparation of 13a (colorless amorphous solid, guanidino-6-(2؅,3؅-diacetoxy-1؅-dodecyloxy)propyl-5,6-dihydro-4H-
1
2
1
3
3
0 mg, 0.0497 mmol, 83%). H-NMR (CD OD) d: 0.87—0.91 (3H, m), pyran-2-carboxylic Acid Methyl Ester 12f Compound 12f was obtained
3
.27—1.35 (4H, m), 1.49—1.57 (2H, m), 1.99 (3H, s), 3.43—3.54 (2H, m),
.55—3.59 (1H, m), 3.61 (1H, dd, Jϭ11.3, 4.6 Hz), 3.85 (1H, dd, Jϭ11.3, preparation of 12a (colorless amorphous solid, 60 mg, 0.0751 mmol, 68%).
.2 Hz), 3.89—3.94 (1H, m), 4.20—4.23 (1H, m), 4.29—4.33 (2H, m), 5.51
1H, d, Jϭ2.4 Hz). FAB-MS m/z: 403 (MϩH) .
from 11f (64 mg, 0.110 mmol) using the same procedure employed for the
1
H-NMR (CDCl ) d: 0.86—0.90 (3H, m), 1.24—1.35 (18H, m), 1.49 (9H,
3
ϩ
(
s), 1.50 (9H, s), 1.51—1.64 (2H, m), 1.94 (3H, s), 2.06 (3H, s), 2.09 (3H, s),
5
-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-7-O-n- 3.50—3.62 (2H, m), 3.69—3.72 (1H, m), 3.78 (3H, s), 4.10—4.15 (1H, m),
octyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-pyra- 4.25 (1H, dd, Jϭ12.4, 7.4 Hz), 4.28—4.37 (1H, m), 4.83 (1H, dd, Jϭ12.4,
nosonic Acid Methyl Ester 10e A procedure similar to that used for the 2.4 Hz), 5.12—5.22 (1H, m), 5.26—5.31 (1H, m), 5.82 (1H, d, Jϭ2.4 Hz),
preparation of compound 10b was employed with di(n-octyl)sulfate and 10e 6.41 (1H, br d, Jϭ9.1 Hz), 8.53 (1H, br d, Jϭ8.7 Hz). FAB-MS m/z: 799
1
ϩ
was obtained as a colorless amorphous solid (3.28 g, 5.43 mmol, 44%). H- (MϩH) .
NMR (CDCl ) d: 0.05 (3H, s), 0.06 (3H, s), 0.86 (9H, s), 0.86—0.90 (3H,
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-[2؅,3؅-dihydroxy-1؅-
3
m), 1.22—1.38 (10H, m), 1.32 (3H, s), 1.41 (3H, s), 1.53—1.64 (2H, m),
dodecyloxy-propyl]-4-guanidino-4H-pyran-2-carboxylic Acid 13f Com-
pound 13f was obtained from 12f (24 mg, 30.2 mmol) using the same proce-
1
3
3
7
.73 (1H, dd, Jϭ13.1, 10.7 Hz), 1.98 (3H, s), 2.26 (1H, dd, Jϭ13.1, 5.1 Hz),
.22 (3H, s), 3.60 (1H, dd, Jϭ5.7, 1.4 Hz), 3.62—3.68 (2H, m), 3.79 (3H, s),
dure employed for the preparation of 13a (colorless amorphous solid, 10 mg,
1
.79—3.86 (1H, m), 3.93 (1H, dd, Jϭ10.6, 1.4 Hz), 4.01 (1H, dd, Jϭ8.6, 16.3 mmol, 54%). H-NMR (CD OD) d: 0.87—0.92 (3H, m), 1.25—1.35
3
.0 Hz), 4.08—4.17 (2H, m), 4.22—4.28 (1H, m), 5.15 (1H, d, Jϭ8.9 Hz). (18H, m), 1.48—1.57 (2H, m), 2.00 (3H, s), 3.43—3.56 (2H, m), 3.57 (1H,
ϩ
FAB-MS m/z: 604 (MϩH) .
dd, Jϭ9.3, 0.8 Hz), 3.61 (1H, dd, Jϭ11.3, 4.7 Hz), 3.83 (1H, dd, Jϭ11.3,
(
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-n- 3.2 Hz), 3.89—3.95 (1H, m), 4.22—4.25 (1H, m), 4.30—4.33 (2H, m), 5.51
ϩ
octyloxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester (1H, d, Jϭ2.5 Hz). FAB-MS m/z: 501 (MϩH) .
1
1e Compound 11e was obtained from 10e (4.1 g, 5.84 mmol) using the
5-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-7-O-n-
tetradecyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-
same procedure employed for the preparation of 10a (colorless amorphous
1
solid, 1.65 g, 3.13 mmol, 53%). H-NMR (CDCl ) d: 0.87—0.91 (3H, m), pyranosonic Acid n-Tetradecyl Ester 9g A procedure similar to that used
3
1
.23—1.35 (10H, m), 1.54—1.64 (2H, m), 2.04 (3H, s), 2.06 (3H, s), 2.09 for the preparation of compound 10a was employed with di(n-tetradecyl)-
(
3H, s), 3.47—3.51 (1H, m), 3.62—3.68 (1H, m), 3.74 (1H, dd, Jϭ4.3, sulfate and 9g was obtained as a colorless amorphous solid (2.0 g,
1
3
.5 Hz), 3.80 (3H, s), 3.95 (1H, ddd, Jϭ8.8, 8.1, 8.1 Hz), 4.22 (1H, dd,
Jϭ12.3, 7.1 Hz), 4.47 (1H, dd, Jϭ8.1, 3.0 Hz), 4.52 (1H, dd, Jϭ8.8, 3.5 Hz), s), 0.86—0.90 (6H, m), 1.22—1.38 (40H, m), 1.32 (3H, s), 1.41 (3H, s),
.72 (1H, dd, Jϭ12.3, 3.0 Hz), 5.33 (1H, ddd, Jϭ7.1, 4.3, 3.0 Hz), 5.79 (1H, 1.53—1.64 (4H, m), 1.73 (1H, dd, Jϭ13.0, 10.9 Hz), 1.98 (3H, s), 2.26 (1H,
3.00 mmol, 25%). H-NMR (CDCl ) d: 0.05 (3H, s), 0.06 (3H, s), 0.86 (9H,
3
4
ϩ
br d, Jϭ7.8 Hz), 5.98 (1H, d, Jϭ3.0 Hz). FAB-MS m/z: 527 (MϩH) .
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)- 3.92 (1H, dd, Jϭ10.6, 1.5 Hz), 4.01 (1H, dd, Jϭ8.4, 7.3 Hz), 4.10—4.21
guanidino-6-(2؅,3؅-diacetoxy-1؅-octyloxy)propyl-5,6-dihydro-4H-pyran- (5H, m), 4.22—4.28 (1H, m), 5.15 (1H, d, Jϭ8.8 Hz). FAB-MS m/z: 870
2
1
dd, Jϭ13.0, 5.0 Hz), 3.22 (3H, s), 3.60—3.71 (2H, m), 3.78—3.85 (1H, m),
(
ϩ
-carboxylic Acid Methyl Ester 12e Compound 12e was obtained from (MϩH) .
1e (1.60 g, 3.04 mmol) using the same procedure employed for the prepara- (4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-n-
1
tion of 12a (colorless amorphous solid, 1.45 g, 1.95 mmol, 64%). H-NMR tetradecyloxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl
(
(
3
CDCl ) d: 0.86—0.90 (3H, m), 1.24—1.35 (10H, m), 1.49 (9H, s), 1.50
Ester 11g Compound 11g was obtained from 9g (1.00 g, 1.14 mmol) using
3
9H, s), 1.51—1.64 (2H, m), 1.94 (3H, s), 2.06 (3H, s), 2.09 (3H, s), 3.50—
the same procedure employed for the preparation of 11d (colorless amor-
1
.62 (2H, m), 3.70 (1H, dd, Jϭ4.0, 1.6 Hz), 3.78 (3H, s), 3.92—3.98 (1H, phous solid, 190 mg, 0.311 mmol, 27%). H-NMR (CDCl ) d: 0.87—0.91
3
m), 4.10 (1H, dd, Jϭ10.4, 1.6 Hz), 4.26 (1H, dd, Jϭ12.5, 7.6 Hz), 4.83 (1H, (3H, m), 1.23—1.35 (22H, m), 1.54—1.64 (2H, m), 2.04 (3H, s), 2.06 (3H,
dd, Jϭ12.5, 2.4 Hz), 5.09—5.15 (1H, m), 5.29 (1H, ddd, Jϭ7.6, 4.0, 1.6 Hz), s), 2.09 (3H, s), 3.47—3.51 (1H, m), 3.62—3.68 (1H, m), 3.74 (1H, dd,
5
.83 (1H, d, Jϭ2.4 Hz), 6.41 (1H, br d, Jϭ9.1 Hz), 8.53 (1H, br d, Jϭ Jϭ4.3, 3.5 Hz), 3.80 (3H, s), 3.95 (1H, ddd, Jϭ8.8, 8.1, 8.1 Hz), 4.22 (1H,
ϩ
8
.7 Hz). FAB-MS m/z: 743 (MϩH) .
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-(2,3-dihydroxy-1-
octyloxy)propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13e Com-
dd, Jϭ12.3, 7.1 Hz), 4.47 (1H, dd, Jϭ8.1, 3.0 Hz), 4.52 (1H, dd, Jϭ8.8,
3.5 Hz), 4.72 (1H, dd, Jϭ12.3, 3.0 Hz), 5.33 (1H, ddd, Jϭ7.1, 4.3, 3.0 Hz),
(
5.79 (1H, br d, Jϭ7.8 Hz), 5.98 (1H, d, Jϭ3.0 Hz). FAB-MS m/z: 611
ϩ
pound 13e was obtained from 12e (1.45 g, 1.95 mmol) using the same proce- (MϩH) .
dure employed for the preparation of 13a (colorless amorphous solid,
(4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)-
1
8
40 mg, 1.46 mmol, 75%). H-NMR (CD OD) d: 0.87—0.91 (3H, m), guanidino-6-(2؅,3؅-diacetoxy-1؅-tetradecyloxy)propyl-5,6-dihydro-4H-
3
1
.25—1.35 (10H, m), 1.49—1.57 (2H, m), 1.99 (3H, s), 3.43—3.56 (2H, pyran-2-carboxylic Acid Methyl Ester 12g Compound 12g was obtained
m), 3.57 (1H, dd, Jϭ8.3, 2.2 Hz), 3.61 (1H, dd, Jϭ11.3, 4.8 Hz), 3.83 (1H,
from 11g (87 mg, 0.142 mmol) using the same procedure employed for the
dd, Jϭ11.3, 3.2 Hz), 3.89—3.95 (1H, m), 4.27—4.32 (2H, m), 4.29—4.37
preparation of 12a (colorless amorphous solid, 62 mg, 0.075 mmol, 53%).
ϩ
1
(
1H, m), 5.53 (1H, d, Jϭ1.8 Hz). FAB-MS m/z: 445 (MϩH) .
H-NMR (CDCl ) d: 0.86—0.90 (3H, m), 1.24—1.35 (22H, m), 1.49 (9H,
3
5
-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-7-O-n- s), 1.50 (9H, s), 1.51—1.64 (2H, m), 1.94 (3H, s), 2.06 (3H, s), 2.09 (3H, s),
dodecyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-pyra-
3.50—3.60 (2H, m), 3.70—3.73 (1H, m), 3.78 (3H, s), 4.10—4.15 (1H, m),
nosonic Acid Methyl Ester 10f A procedure similar to that used for the 4.25 (1H, dd, Jϭ12.4, 7.5 Hz), 4.31—4.40 (1H, m), 4.83 (1H, dd, Jϭ12.4,
preparation of 10a was employed with di(n-dodecyl)sulfate and 10f was ob- 2.4 Hz), 5.09—5.17 (1H, m), 5.26—5.31 (1H, m), 5.82 (1H, d, Jϭ2.4 Hz),
1
tained as a colorless amorphous solid (2.89 g, 4.37 mmol, 36%). H-NMR 6.80 (1H, br d, Jϭ9.4 Hz), 8.55 (1H, br d, Jϭ8.5 Hz). FAB-MS m/z: 827
ϩ
(
1
CDCl ) d: 0.05 (3H, s), 0.06 (3H, s), 0.86 (9H, s), 0.86—0.90 (3H, m),
(MϩH) .
3
.22—1.38 (16H, m), 1.32 (3H, s), 1.41 (3H, s), 1.53—1.64 (2H, m), 1.73
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-(2؅,3؅-dihydroxy-1؅-
tetradecyloxy)propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13g
3H, s), 3.60 (1H, dd, Jϭ5.6 1.4 Hz), 3.62—3.68 (2H, m), 3.79 (3H, s), Compound 13g was obtained from 12g (17 mg, 20.5 mmol) using the same
.79—3.86 (1H, m), 3.93 (1H, dd, Jϭ10.6, 1.4 Hz), 4.01 (1H, dd, Jϭ8.6, procedure employed for the preparation of 12a (colorless amorphous solid,
(1H, dd, Jϭ13.0, 10.9 Hz), 1.98 (3H, s), 2.26 (1H, dd, Jϭ13.0, 5.0 Hz), 3.22
(
3
7
1
.0 Hz), 4.09—4.17 (2H, m), 4.22—4.28 (1H, m), 5.14 (1H, d, Jϭ8.9 Hz). 10 mg, 18.9 mmol, 92%). H-NMR (CD OD) d: 0.88—0.92 (3H, m), 1.25—
3
ϩ
FAB-MS m/z: 660 (MϩH) .
1.35 (22H, m), 1.48—1.57 (2H, m), 1.99 (3H, s), 3.43—3.56 (2H, m), 3.56
(
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-n-do- (1H, dd, Jϭ8.5, 1.9 Hz), 3.61 (1H, dd, Jϭ11.3, 4.7 Hz), 3.81 (1H, dd,
decyloxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester Jϭ11.3, 3.2 Hz), 3.89—3.94 (1H, m), 4.21—4.24 (1H, m), 4.30—4.33 (2H,
ϩ
1
1f Compound 11f was obtained from 10f (0.78 g, 1.18 mmol) using the m), 5.51 (1H, d, Jϭ2.5 Hz). FAB-MS m/z: 529 (MϩH) .
same procedure employed for the preparation of 10b (colorless amorphous
5-Acetylamino-7-O-cyclohexylmethyl-3,5-dideoxy-8,9-O-isopropyl-
idene-2-O-methyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-
1
solid, 240 mg, 0.411 mmol, 35%). H-NMR (CDCl ) d: 0.87—0.91 (3H, m),
3
1
.23—1.35 (18H, m), 1.54—1.64 (2H, m), 2.04 (3H, s), 2.06 (3H, s), 2.09 nonulo-pyranosonic Acid Cyclohexylmethyl Ester 9h A procedure simi-
(
3
3H, s), 3.47—3.51 (1H, m), 3.62—3.68 (1H, m), 3.74 (1H, dd, Jϭ4.3, lar to that used for the preparation of compound 10b was employed with
.5 Hz), 3.80 (3H, s), 3.95 (1H, ddd, Jϭ8.8, 8.1, 8.1 Hz), 4.22 (1H, dd, di(n-tetradecyl)sulfate and 9h was obtained as a colorless amorphous solid
Jϭ12.3, 7.1 Hz), 4.47 (1H, dd, Jϭ8.1, 3.0 Hz), 4.52 (1H, dd, Jϭ8.8, 3.5 Hz), (2.0 g, 2.99 mmol, 25%). H-NMR (CDCl ) d: 0.05 (3H, s), 0.06 (3H, s),
1
3

1
0
394
Vol. 51, No. 12
1
.86 (9H, s), 0.90—1.04 (4H, m), 1.12—1.30 (6H, m), 1.31 (3H, s), 1.41 33 mg, 0.076 mmol, 57%). H-NMR (CD OD) d: 1.99 (3H, s), 2.78—2.92
3
(
3H, s), 1.58—1.80 (13H, m), 1.97 (3H, s), 2.25 (1H, dd, Jϭ13.1, 5.1 Hz), (2H, m), 3.52 (1H, dd, Jϭ11.3, 4.5 Hz), 3.61—3.65 (1H, m), 3.65—3.77
3
3
.22 (3H, s), 3.40—3.51 (2H, m), 3.59—3.62 (1H, m), 3.80—3.86 (1H, m),
(3H, m), 3.87—3.91 (1H, m), 4.29—4.41 (3H, m), 5.66 (1H, d, Jϭ2.2 Hz),
7.13—7.27 (10H, m). FAB-MS m/z: 437 (MϩH) .
5-Acetylamino-7-O-(2؅-benzyloxy)ethyl-3,5-dideoxy-8,9-O-isopropyl-
idene-2-O-methyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-
ϩ
.87—3.91 (1H, m), 3.97—4.05 (3H, m), 4.08—4.15 (2H, m), 4.22—4.26
ϩ
(
1H, m), 5.15 (1H, d, Jϭ9.0 Hz). FAB-MS m/z: 670 (MϩH) .
(
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(1؅-cyclohexylmethoxy-
2
؅,3؅-diacetoxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Cyclo- nonulo-pyranosonic Acid Methyl Ester 14j A procedure similar to that
hexylmethyl Ester 11h Compound 11h was obtained from 9h (1.0 g, used for the preparation of compound 10b was employed with di(2-benzyl-
.49 mmol) using the same procedure employed for the preparation of 11d oxyethyl)sulfate and 14j was obtained as a colorless amorphous solid
1
1
1
(
colorless amorphous solid, 250 mg, 0.422 mmol, 28%). H-NMR (CDCl ) (11.4 g, 18.3 mmol, 30%). H-NMR (400 MHz, CDCl ) d: 0.04 (3H, s), 0.06
3
3
d: 0.90—1.03 (4H, m), 1.10—1.32 (6H, m), 1.53—1.80 (12H, m), 2.04 (3H, (3H, s), 0.86 (9H, s), 1.32 (3H, s), 1.41 (3H, s), 1.59 (1H, dd, Jϭ13.1,
s), 2.05 (3H, s), 2.08 (3H, s), 3.29 (1H, dd, Jϭ8.5, 6.1 Hz), 3.47 (1H, dd,
10.8 Hz), 1.70 (3H, s), 2.30 (1H, dd, Jϭ13.1, 5.0 Hz), 3.18 (3H, s), 3.22—
Jϭ8.5, 6.5 Hz), 3.71 (1H, dd, Jϭ3.8, 3.6 Hz), 3.95 (1H, ddd, Jϭ8.6, 8.0, 3.36 (1H, m), 3.52—3.56 (1H, m), 3.67—3.78 (2H, m), 3.78 (3H, s), 3.79—
7
.8 Hz), 4.00 (2H, d, Jϭ6.6 Hz), 4.28 (1H, dd, Jϭ12.3, 7.4 Hz), 4.48 (1H,
3.82 (1H, m), 4.05—4.29 (4H, m), 4.35 (1H, dd, Jϭ10.3, 2.5 Hz), 4.56 (2H,
dd, Jϭ8.0, 3.0 Hz), 4.52 (1H, dd, Jϭ8.6, 3.6 Hz), 4.65 (1H, dd, Jϭ12.3,
s), 4.70—4.81 (1H, m), 6.43 (1H, br d, Jϭ7.3 Hz), 7.28—7.38 (5H, m). MS
ϩ
3
.0 Hz), 5.34 (1H, ddd, Jϭ7.4, 3.8, 3.0 Hz), 5.77 (1H, br d, Jϭ7.8 Hz), 5.96 (FAB) m/z 625 (MϩH) . HR-MS (FAB) Calcd for C H O NSiNa:
3
1
51 10
ϩ
ϩ
(
1H, d, Jϭ3.0 Hz). FAB-MS m/z: 593 (MϩH) .
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycar-
bonyl)guanidino-6-(1؅-cyclohexylmethoxy-2؅,3؅-diacetoxy)propyl-5,6-di-
648.3180; Found 648.3173 (MϩNa) .
(
5-Acetylamino-3,5-dideoxy-7-O-(2؅-hydroxy)ethyl-8,9-O-isopropyl-
idene-2-O-methyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-
hydro-4H-pyran-2-carboxylic Acid Cyclohexylmethyl Ester 12h Com- nonulo-pyranosonic Acid Methyl Ester 15j Compounds 14j (27.3 g,
pound 12h was obtained from 11h (243 mg, 0.410 mmol) using the same 43.5 mmol) was hydrogenated with palladium carbon (10 g) in methanol
procedure employed for the preparation of 12a (colorless amorphous solid, (500 ml) at room temperature for 3 h and filtrated. The residue was washed
1
2
1
(
32 mg, 0.286 mmol, 70%). H-NMR (CDCl ) d: 0.90—1.03 (4H, m), with methanol thoroughly and evaporated under reduced pressure. The
3
.10—1.32 (6H, m), 1.49 (9H, s), 1.50 (9H, s), 1.56—1.76 (12H, m), 1.94 residue was purufied by silica gel chromatography (eluent; ethyl acetate) to
3H, s), 2.05 (3H, s), 2.08 (3H, s), 3.35—3.42 (2H, m), 3.69 (1H, dd, Jϭ3.6,
1
give compound 15j as an amorphous solid (21.0 g, 39.1 mmol, 90%). H-
1
.3 Hz), 3.95 (1H, dd, Jϭ10.7, 6.7 Hz), 4.03 (1H, dd, Jϭ10.7, 6.7 Hz), 4.07 NMR (CDCl ) d: 0.05 (3H, s), 0.06 (3H, s), 0.85 (9H, s), 1.32 (3H, s), 1.42
3
(
1H, dd, Jϭ10.3, 1.3 Hz), 4.24—4.32 (1H, m), 4.36 (1H, dd, Jϭ12.4, (3H, s), 1.75 (1H, dd, Jϭ13.1, 10.8 Hz), 1.99 (3H, s), 2.27 (1H, dd, Jϭ13.1,
7
.9 Hz), 4.75 (1H, dd, Jϭ12.4, 2.4 Hz), 5.06—5.13 (1H, m), 5.27 (1H, ddd,
4.8 Hz), 3.21 (3H, s), 3.64—3.79 (4H, m), 3.80 (3H, s), 3.80—3.88 (1H, m),
Jϭ7.9, 3.6, 2.4 Hz), 5.79 (1H, d, Jϭ2.3 Hz), 6.80 (1H, br d, Jϭ9.1 Hz), 8.53 3.92—4.02 (2H, m), 4.03—4.18 (3H, m), 4.23—4.31 (1H, m), 5.47 (1H,
ϩ
ϩ
(
1H, br d, Jϭ9.1 Hz). FAB-MS m/z: 809 (MϩH) .
(
br d, Jϭ8.9 Hz). MS (FAB) m/z 536 (MϩH) . HR-MS (FAB) Calcd for
ϩ
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-(1؅-cyclohexylmethoxy-2؅,3؅-di- C H O NSi: 536.2891; Found 536.2880 (MϩH) .
2
4
46 10
hydroxy)propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid
3h Compound 13h was obtained from 12h (113 mg, 0.139 mmol) using acetylamino-4-azide-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl
(4S,5R,6R,1؅S,2؅R)-6-[1؅-(2؆-Acetoxy)ethoxy-2؅,3؅-diacetoxy]propyl-5-
1
the same procedure employed for the preparation of 13a (colorless amor- Ester 17j Compound 17j was obtained from 15j (20.2 g, 37.7 mmol) using
1
phous solid, 56 mg, 0.130 mmol, 94%). H-NMR (CD OD) d: 0.88—1.03 the same procedure employed for the preparation of 11a (colorless amor-
3
1
(
(
(
2H, m), 1.10—1.32 (3H, m), 1.47—1.81 (6H, m), 1.99 (3H, s), 3.24—3.38
2H, m), 3.54 (1H, dd, Jϭ8.3, 1.3 Hz), 3.63 (1H, dd, Jϭ11.3, 4.6 Hz), 3.81
1H, dd, Jϭ11.3, 3.0 Hz), 3.89—4.05 (1H, m), 4.28—4.32 (2H, m), 4.35—
.42 (1H, m), 5.68—5.70 (1H, m). FAB-MS m/z: 429 (MϩH) .
-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-7-O-
phenylethyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-nonulo-
pyranosonic Acid Phenylethyl Ester 9i A procedure similar to that used
phous solid, 15.1 g, 30.2 mmol, 80%). H-NMR (400 MHz, CDCl ) d: 2.05
3
(3H, s), 2.06 (3H, s), 2.09 (3H, s), 2.13 (3H, s), 3.69—3.89 (3H, m), 3.80
(3H, s), 3.92—4.01 (1H, m), 4.20—4.30 (3H, m), 4.58—4.66 (2H, m), 4.75
(1H, dd, Jϭ12.4, 2.9 Hz), 5.34—5.40 (1H, m), 5.97 (1H, d, Jϭ2.7 Hz), 6.14
ϩ
4
ϩ
5
(1H, br d, Jϭ7.9 Hz). MS (FAB) m/z 501 (MϩH) . HR-MS (FAB) Calcd for
ϩ
C H O N : 501.1833; Found 501.1807 (MϩH) .
2
0
29 11
4
(4S,5R,6R,1؅S,2؅R)-6-[1؅-(2؆-Acetoxy)ethoxy-2؅,3؅-diacetoxy]propyl-5-
for the preparation of compound 9a was employed with di(phenylethyl)sul- acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)guanidino-5,6-dihydro-
fate and 9i was obtained as a colorless amorphous solid (343 mg, 0.50 mmol, 4H-pyran-2-carboxylic Acid Methyl Ester 18j Compound 18j was ob-
1
2
(
4%). H-NMR (CDCl ) d: Ϫ0.03 (3H, s), 0.16 (3H, s), 0.88 (9H, s), 1.29
tained from 17j (14.9 g, 29.7 mmol) using the same procedure employed for
3
3H, s), 1.43 (3H, s), 1.47 (1H, dd, Jϭ13.0, 10.8 Hz), 1.70 (3H, s), 2.16 (1H, the preparation of 12a (colorless amorphous solid, 18.7 g, 26.1 mmol, 88%).
dd, Jϭ13.0, 5.0 Hz), 2.82—2.97 (2H, m), 2.98—3.05 (3H, m), 3.07 (3H, s),
.57—3.65 (2H, m), 4.03—4.06 (1H, m), 4.15—4.21 (2H, m), 4.22—4.48
6H, m), 7.21—7.35 (10H, m). FAB-MS m/z: 686 (MϩH) .
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-azide-6-(2؅,3؅-diacetoxy-1؅-
1
H-NMR (400 MHz, CDCl ) d: 1.48 (9H, s), 1.49 (9H, s), 1.95 (3H, s), 2.06
3
3
(
(3H, s), 2.10 (6H, s), 3.72—3.77 (1H, m), 3.78 (3H, s), 3.80—3.87 (2H, m),
4.09 (1H, dd, Jϭ11.0, 2.8 Hz), 4.22—4.28 (3H, m), 4.29—4.40 (1H, m),
4.84 (1H, dd, Jϭ12.6, 2.4 Hz), 5.07—5.17 (1H, m), 5.27—5.33 (1H, m),
ϩ
(
phenethoxy)propyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Phenethyl 5.83 (1H, d, Jϭ2.4 Hz), 6.63 (1H, br d, Jϭ9.0 Hz), 8.55 (1H, br d,
Ester 11i Compound 11i was obtained from 9i (710 mg, 1.03 mmol) using Jϭ8.8 Hz). IR (CDCl ) n
4213, 3425, 2955, 2101, 1740, 1681, 1602,
3
max
Ϫ1
ϩ
the same procedure employed for the preparation of 11a (colorless amor- 1510, 1439 cm . MS (FAB) m/z 717 (MϩH) . HR-MS (FAB) Calcd for
1
ϩ
phous solid, 220 mg, 0.361 mmol, 35%). H-NMR (CDCl ) d: 1.71 (3H, s), C H O N : 717.3215; Found 717.3202 (MϩH) .
3
31 49 15
4
2
3
4
7
.04 (3H, s), 2.07 (3H, s), 2.85—3.09 (4H, m), 3.33—3.39 (1H, m), 3.41—
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-[2؅,3؅-dihydroxy-1؅-
.43 (1H, m), 4.08—4.12 (1H, m), 4.30—4.42 (4H, m), 4.66—4.71 (2H, m), (2؆-hydroxy)ethoxy]propyl-4-guanidino-4H-pyran-2-carboxylic Acid 13j
.75 (1H, dd, Jϭ12.3, 2.6 Hz), 5.33—5.38 (1H, m), 5.82 (1H, d, Jϭ2.7 Hz) Compound 13j was obtained from 18j (50 mg, 0.0846 mmol) using the same
ϩ
.20—7.45 (10H, m). FAB-MS m/z: 609 (MϩH) .
procedure employed for the preparation of 13a (colorless amorphous solid,
1
(
4S,5R,6R,1؅S,2؅R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycar- 15 mg, 0.0397 mmol, 47%). H-NMR (400 MHz, D O) d: 2.04 (3H, s),
bonyl)guanidino-6-(2؅,3؅-diacetoxy-1؅-phenethyl)propyl-5,6-dihydro-4H- 3.55—3.76 (6H, m), 3.90 (1H, dd, Jϭ11.8, 2.9 Hz), 3.97—4.06 (1H, m),
pyran-2-carboxylic Acid Phenethyl Ester 12i Compound 12i was ob- 4.19—4.28 (1H, m), 4.38—4.48 (2H, m), 5.63 (1H, d, Jϭ2.2 Hz). MS (FAB)
2
ϩ
tained from 11i (138 mg, 0.226 mmol) using the same procedure employed m/z 377 (MϩH) . HR-MS (FAB) Calcd for C H O N : 377.1672; Found
1
4
25
8
4
ϩ
for the preparation of 12a (colorless amorphous solid, 140 mg, 0.169 mmol, 377.1683 (MϩH) .
1
7
(
(
5%). H-NMR (CDCl ) d: 1.49 (9H, s), 1.53 (9H, s), 1.89 (3H, s), 2.03
3H, s), 2.06 (3H, s), 2.83—3.00 (4H, m), 3.64—3.72 (2H, m), 3.86—3.93
5-Acetylamino-7-O-(5؅-benzyloxy)pentyl-3,5-dideoxy-8,9-O-isopropyl-
idene-2-O-methyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-
1H, m), 3.98—4.07 (1H, m), 4.11—4.15 (1H, m), 4.22 (1H, dd, Jϭ12.5, nonulo-pyranosonic Acid Methyl Ester 14k A procedure similar to that
.4 Hz), 4.36 (2H, t, Jϭ7.3 Hz), 4.76 (1H, dd, Jϭ12.5, 2.2 Hz), 5.07—5.13 used for the preparation of compound 10b was employed with di(5-benzyl-
3
7
(
1H, m), 5.28 (1H, ddd, Jϭ7.4, 4.3, 2.2 Hz), 5.73 (1H, d, Jϭ2.3 Hz), 7.02 oxypentyl)sulfate and 14k was obtained as a colorless amorphous solid
1
(
1H, br d, Jϭ8.8 Hz), 7.17—7.33 (10H, m), 8.53 (1H, br d, Jϭ9.1 Hz). FAB- (5.70 g, 8.53 mmol, 70%). H-NMR (400 MHz, CDCl ) d: 0.04 (3H, s), 0.06
3
ϩ
MS m/z: 825 (MϩH) .
(3H, s), 0.86 (9H, s), 1.31 (3H, s), 1.33—1.51 (2H, m), 1.41 (3H, s), 1.58—
(
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-[2؅,3؅-dihydroxy-1؅- 1.79 (5H, m), 1.96 (3H, s), 2.27 (1H, dd, Jϭ13.1, 5.0 Hz), 3.22 (3H, s),
phenethoxy-propyl]-4-guanidino-4H-pyran-2-carboxylic Acid 13i 3.42—3.50 (2H, m), 3.55—3.83 (4H, m), 3.77 (3H, s), 3.94—4.06 (2H, m),
Compound 13i was obtained from 12i (110 mg, 0.133 mmol) using the same 4.09—4.28 (3H, m), 4.50 (2H, s), 5.28 (1H, d, Jϭ8.8 Hz), 7.26—7.38 (5H,
ϩ
procedure employed for the preparation of 13a (colorless amorphous solid, m). MS (FAB) m/z 668 (MϩH) . HR-MS (FAB) Calcd for C H O NSi:
34
58 10

December 2003
68.3830; Found 668.3818 (MϩH) .
1395
ϩ
6
dd, Jϭ12.4, 2.9 Hz), 5.28—5.37 (1H, m), 5.81 (1H, br d, Jϭ8.1 Hz), 5.97
ϩ
5
-Acetylamino-3,5-dideoxy-7-O-(5؅-hydroxy)pentyl-8,9-O-isopropyl- (1H, d, Jϭ2.8 Hz). MS (FAB) m/z 613 (MϩH) . HR-MS (FAB) Calcd for
ϩ
idene-2-O-methyl-4-O-tert-butyldimethylsilyl-D-glycero-D-galacto-2-
nonulo-pyranosonic Acid Methyl Ester 15k Compound 15k was ob-
C H O N : 613.3084; Found 613.3094 (MϩH) .
28 45 11 4
(4S,5R,6R,1؅S,2؅R)-6-[1؅-(10؆-Acetoxy)decyloxy-2؅,3؅-diacetoxy]-
tained from 14k (6.52 g, 9.76 mmol) using the same procedure employed for propyl-5-acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)guanidino-5,6-
the preparation of 15j (colorless amorphous solid, 4.72 g, 8.17 mmol, 84%). dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 18l Compound 18l
1
H-NMR (400 MHz, CDCl ) d: 0.04 (3H, s), 0.06 (3H, s), 0.85 (9H, s), 1.31 was obtained from 17l (8.20 g, 13.4 mmol) using the same procedure em-
3
(
3H, s), 1.41 (3H, s), 1.48—1.79 (7H, m), 1.97 (3H, s), 2.26 (1H, dd, ployed for the preparation of 12a (colorless amorphous solid, 7.85 g,
1
Jϭ13.0, 5.0 Hz), 3.22 (3H, s), 3.55—3.87 (6H, m), 3.79 (3H, s), 3.85—4.19 9.47 mmol, 71%). H-NMR (400 MHz, CDCl ) d: 1.25—1.40 (12H, m),
(
(
5
3
4H, m), 4.20—4.29 (1H, m), 5.46 (1H, br d, Jϭ8.7 Hz). MS (FAB) m/z 578
1.49 (9H, s), 1.50 (9H, s), 1.55—1.70 (4H, m), 1.94 (3H, s), 2.04 (3H, s),
ϩ
MϩH) . HR-MS (FAB) Calcd for C H O NSi: 578.3361; Found 2.06 (3H, s), 2.09 (3H, s), 3.50—3.63 (2H, m), 3.67—3.71 (1H, m), 3.78
2
7
52 10
ϩ
78.3360 (MϩH) .
4S,5R,6R,1؅S,2؅R)-6-[1؅-(5؆-Acetoxy)pentyloxy-2؅,3؅-diacetoxy]-
(3H, s), 4.00—4.12 (3H, m), 4.21—4.36 (2H, m), 4.82 (1H, dd, Jϭ12.5,
2.3 Hz), 5.08—5.16 (1H, m), 5.23—5.32 (1H, m), 5.83 (1H, d, Jϭ2.4 Hz),
(
propyl-5-acetylamino-4-azide-5,6-dihydro-4H-pyran-2-carboxylic Acid 6.22 (1H, br d, Jϭ8.7 Hz), 8.52 (1H, br d, Jϭ8.7 Hz). MS (FAB) m/z 829
ϩ
Methyl Ester 17k Compound 17k was obtained from 15k (4.60 g, (MϩH) . HR-MS (FAB) Calcd for C H O N Na: 851.4266; Found
3
9
64 15
4
ϩ
7
.96 mmol) using the same procedure employed for the preparation of 15j 851.4282 (MϩNa) .
1
(
colorless amorphous solid, 2.51 g, 4.62 mmol, 58%). H-NMR (400 MHz,
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-[2؅,3؅-dihydroxy-1؅-
CDCl ) d: 1.35—1.49 (2H, m), 1.56—1.71 (4H, m), 2.04 (3H, s), 2.05 (3H, (10؆-hydroxy)decyloxy]propyl-4-guanidino-4H-pyran-2-carboxylic Acid
3
s), 2.06 (3H, s), 2.09 (3H, s), 3.43—3.52 (1H, m), 3.67—3.77 (2H, m), 3.80
13l Compound 13l was obtained from 18l (100 mg, 0.120 mmol) using the
(
7
(
(
3H, s), 3.80—3.92 (1H, m), 4.05—4.13 (2H, m), 4.21 (1H, dd, Jϭ12.4, same procedure employed for the preparation of 13a (colorless amorphous
1
.2 Hz), 4.50 (1H, dd, Jϭ8.2, 3.0 Hz), 4.58 (1H, dd, Jϭ9.0, 3.2 Hz), 4.72 solid, 33 mg, 0.067 mmol, 56%). H-NMR (400 MHz, D O) d: 1.23—1.40
2
1H, dd, Jϭ12.3, 3.0 Hz), 5.28—5.38 (1H, m), 5.97 (1H, d, Jϭ2.8 Hz), 6.01 (12H, m), 1.44—1.58 (4H, m), 2.00 (3H, s), 3.38—3.48 (1H, m), 3.51—
ϩ
1H, br d, Jϭ8.0 Hz). MS (FAB) m/z 543 (MϩH) . HR-MS (FAB) Calcd for 3.68 (5H, m), 3.85 (1H, dd, Jϭ11.8, 2.3 Hz), 3.90—4.00 (1H, m), 4.09—
ϩ
C H O N K: 581.1861; Found 581.1849 (MϩK) .
4.20 (1H, m), 4.32—4.41 (2H, m), 5.57 (1H, d, Jϭ2.2 Hz). MS (FAB) m/z
489 (MϩH) .
23
34 11
4
ϩ
(
4S,5R,6R,1؅S,2؅R)-6-[1؅-(5؆-Acetoxy)pentyloxy-2؅,3؅-diacetoxy]-
propyl-5-acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)guanidino-5,6-
(4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)-
dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 18k Compound 18k guanidino-6-[(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-yl)-(2ٟ-hydroxy-ethyl-
was obtained from 17k (2.51 g, 4.63 mmol) using the same procedure em- oxy)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 19j
ployed for the preparation of 12a (colorless amorphous solid, 2.50 g, A solution of 18j (18.7 g, 26.1 mmol) in methanol (200 ml) was cooled in an
1
3
(
(
.29 mmol, 71%). H-NMR (400 MHz, CDCl ) d: 1.40—1.55 (2H, m), 1.49
ice bath and mixed with a solution of sodium methoxide in methanol (4.9 N,
3
9H, s), 1.50 (9H, s), 1.60—1.75 (4H, m), 1.94 (3H, s), 2.04 (3H, s), 2.06 2.6 ml, 13.0 mmol). The reaction mixture was stirred for 1 h. Then, the reac-
3H, s), 2.09 (3H, s), 3.50—3.65 (2H, m), 3.67—3.71 (1H, m), 3.78 (3H, s), tion mixture was cooled in an ice bath and neutralized with DOWEX 50W
.02—4.12 (2H, m), 4.21—4.34 (2H, m), 4.83 (1H, dd, Jϭ12.5, 2.3 Hz), X8 (H ). The reaction mixture was filtered and the precipitate was washed
ϩ
4
5
.08—5.16 (1H, m), 5.23—5.32 (1H, m), 5.83 (1H, d, Jϭ2.5 Hz), 6.29 (1H, with methanol. The filtrate was evaporated and the residue was dissolved in
ϩ
br d, Jϭ8.6 Hz), 8.53 (1H, br d, Jϭ8.6 Hz). MS (FAB) m/z 759 (MϩH) .
HR-MS (FAB) Calcd for C H O N : 759.3664; Found 759.3657 (MϩH) .
acetone (200 ml). To the solution, dimethoxypropane (40 ml) and p-toluene
sulfonic acid monohydrate (500 mg, 2.62 mmol) were added and the mixture
was stirred for 2 h. The reaction mixture was combined with sodium hydro-
ϩ
34
55 15
4
(
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-5,6-dihydro-6-[2؅,3؅-dihydroxy-1؅-
(
1
5؆-hydroxy)pentyloxy]propyl-4-guanidino-4H-pyran-2-carboxylic Acid gen carbonate (3 g) with cooling in an ice bath. Then, the reaction mixture
3k Compound 13k was obtained from 18k (70 mg, 0.104 mmol) using was stirred at room temperature for 1 h. The reaction mixture was filtered,
the same procedure employed for the preparation of 13a (colorless amor- the precipitate was washed with ethyl acetate, and the filtrate was evapo-
1
phous solid, 33 mg, 0.0788 mmol, 76%). H-NMR (400 MHz, D O) d: rated. The residue was dissolved in ethyl acetate (200 ml) and mixed with
2
1
3
4
.26—1.38 (2H, m), 1.44—1.58 (4H, m), 2.00 (3H, s), 3.38—3.48 (1H, m),
.51—3.68 (5H, m), 3.85 (1H, dd, Jϭ11.8, 2.3 Hz), 3.90—4.00 (1H, m),
.09—4.20 (1H, m), 4.32—4.41 (2H, m), 5.57 (1H, d, Jϭ2.2 Hz). MS (FAB) washed with aqueous sodium hydrogen carbonate and brine, dried over an-
aqueous hydrogen chloride (1 N, 100 ml). The reaction mixture was stirred
for 5 min, and then extracted with ethyl acetate. The organic layer was
ϩ
m/z 419 (MϩH) .
hydrous sodium sulfate, and concentrated under reduced pressure. The
5
-Acetylamino-3,5-dideoxy-8,9-O-isopropylidene-2-O-methyl-4-O-tert- residue was purified by silica gel chromatography (eluent: ethyl acetate) to
butyldimethylsilyl-7-O-[10؅-(tetrahydro-pyran-2؆-yloxy)decyl]-D-glycero-
give compound 19j as a colorless amorphous solid (12.1 g, 19.1 mmol,
1
D-galacto-2-nonulo-pyranosonic Acid Methyl Ester 16 A solution of 8 73%). H-NMR (400 MHz, CDCl ) d: 1.35 (3H, s), 1.44 (3H, s), 1.48 (9H,
3
(
30.0 g, 61.0 mmol) in N,N-dimethyl formamide (180 ml) was cooled in an s), 1.50 (9H, s), 1.96 (3H, s), 3.59—3.82 (3H, m), 3.79 (3H, s), 3.85—3.89
ice bath and mixed with sodium hydride (5.33 g, 122.0 mmol). The reaction (1H, m), 3.95—4.04 (1H, m), 4.10—4.27 (3H, m), 4.29—4.42 (2H, m),
mixture was stirred at room temperature for 30 min. Then the mixture was 5.10—5.20 (1H, m), 5.83 (1H, d, Jϭ2.3 Hz), 6.65 (1H, br d, Jϭ8.9 Hz), 8.54
ϩ
mixed with 2-(10-bromo-decyloxy)tetrahydropyran (22.9 g, 73.2 mmol) (1H, br d, Jϭ8.6 Hz). MS (FAB) m/z 631 (MϩH) . HR-MS (FAB) Calcd for
ϩ
dropwise and stirred at 50 °C for 3 h. The reaction mixture was diluted with C H O N Na: 653.3110; Found 653.2990 (MϩNa) .
2
8
46 12
4
aqueous ammonium hydrochloride and extracted with ethyl acetate. The or-
(4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycar-
ganic layer was washed with brine and dried over anhydrous sodium sulfate bonyl)guanidino-6-[(2ٟ-azido-ethyloxy)-(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-
and concentrated under reduced pressure. The residue was purified by silica 4؆-yl)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 20j
gel chromatography (eluent; hexane : ethyl acetateϭ2 : 1) to give compound
Compound 19j (12.1 g, 19.2 mmol) was dissolved in anhydrous pyridine
1
1
6 as a colorless amorphous solid (16.6 g, 22.7 mmol, 37%). H-NMR (150 ml), cooled in an ice bath, and mixed with p-toluenesulfonyl chloride
(
(
400 MHz, CDCl ) d: 0.05 (3H, s), 0.06 (3H, s), 0.86 (9H, s), 1.25—1.38
(7.32 g, 38.4 mmol). The reaction mixture was stirred for 2 h at room tem-
perature, then the reaction mixture was diluted with brine and extracted with
ethyl acetate. The organic layer was washed with 1 N aqueous hydrogen chlo-
ride, aqueous sodium hydrogen carbonate, and brine, dried over anhydrous
3
16H, m), 1.31 (3H, s), 1.41 (3H, s), 1.48—1.65 (6H, m), 1.79—1.87 (1H,
m), 1.97 (3H, s), 2.26 (1H, dd, Jϭ13.0, 5.0 Hz), 3.22 (3H, s), 3.34—3.41
(
(
(
1H, m), 3.47—3.53 (1H, m), 3.57—3.95 (7H, m), 3.78 (3H, s), 3.97—4.04
1H, m), 4.08—4.17 (2H, m), 4.21—4.28 (1H, m), 4.57—4.60 (1H, m), 5.14 sodium sulfate, and concentrated under reduced pressure. The residue was
ϩ
1H, br d, Jϭ8.9 Hz). MS (FAB) m/z 754 (MϩNa) . HR-MS (FAB) Calcd dissolved in N,N-dimethylformamide (100 ml) and mixed with sodium azide
ϩ
for C H O NSiNa: 754.4537; Found 754.4520 (MϩNa) .
(2.49 g, 38.4 mmol). The reaction mixture was stirred for 5 h at 50 °C. Then,
37
69 11
(
4S,5R,6R,1؅S,2؅R)-6-[1؅-(10؆-Acetoxy)decyloxy-2؅,3؅-diacetoxy]- the reaction mixture was diluted with brine and extracted with ethyl acetate.
propyl-5-acetylamino-4-azide-5,6-dihydro-4H-pyran-2-carboxylic Acid The organic layer was washed with brine, dried over anhydrous sodium sul-
Methyl Ester 17l Compound 17l was obtained from 16 (16.6 g,
2.7 mmol) using the same procedure employed for the preparation of 11a
fate, and concentrated under reduced pressure. The residue was purified by
2
silica gel chromatography (eluent: hexane : ethyl acetateϭ1 : 2) to give com-
1
1
(
colorless amorphous solid, 8.02 g, 13.1 mmol, 58%). H-NMR (400 MHz, pound 20j as a colorless amorphous solid (10.7 g, 16.3 mmol, 85%). H-
CDCl ) d: 1.24—1.38 (12H, m), 1.53—1.65 (4H, m), 2.04 (3H, s), 2.05 NMR (400 MHz, CDCl ) d: 1.36 (3H, s), 1.45 (3H, s), 1.48 (9H, s), 1.50
3
3
(
3
3H, s), 2.06 (3H, s), 2.09 (3H, s), 3.44—3.52 (1H, m), 3.60—3.68 (1H, m), (9H, s), 1.94 (3H, s), 3.31—3.41 (1H, m), 3.47—3.58 (1H, m), 3.75—3.86
.71—3.75 (1H, m), 3.80 (3H, s), 3.92—4.00 (1H, m), 4.05 (2H, t, (2H, m), 3.78 (3H, s), 3.95—4.05 (1H, m), 4.10—4.32 (5H, m), 5.12—5.22
Jϭ6.8 Hz), 4.21 (1H, dd, Jϭ12.4, 7.2 Hz), 4.43—4.55 (2H, m), 4.75 (1H, (1H, m), 5.82 (1H, d, Jϭ2.4 Hz), 6.47 (1H, br d, Jϭ8.0 Hz), 8.53 (1H, br d,

1
396
Vol. 51, No. 12
ϩ
Jϭ8.6 Hz). MS (FAB) m/z 656 (MϩH) .
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(2؆-azido)ethyloxy-2؅,3؅-dihy-
3.40—3.65 (4H, m), 3.81 (1H, dd, Jϭ13.3, 3.0 Hz), 3.87—3.96 (1H, m),
(
4.20—4.27 (1H, m), 4.30—4.34 (2H, m), 5.51 (1H, d, Jϭ2.4 Hz). MS (FAB)
ϩ
droxy]propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid 21j m/z 514 (MϩH) . HR-MS (FAB) Calcd for C H O N : 514.2989; Found
Compound 21j was obtained from 20j (110 mg, 0.168 mmol) using the same 514.3016 (MϩH) .
procedure employed for the preparation of 13a (colorless amorphous solid, (4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)-
6 mg, 0.114 mmol, 68%). H-NMR (400 MHz, D O) d: 1.99 (3H, s), guanidino-6-[(2ٟ-amino-ethyoxy)-(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-
.33—3.52 (2H, m), 3.55—3.80 (4H, m), 3.86 (1H, dd, Jϭ11.9, 2.6 Hz), yl)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 22j
.92—4.01 (1H, m), 4.17—4.27 (1H, m), 4.33—4.42 (2H, m), 5.57 (1H, d, Compound 20j (640 mg, 0.976 mmol) in ethanol (20 ml) was hydrogenated
Jϭ2.1 Hz). MS (FAB) m/z 401 (MϩH) . HR-MS (FAB) Calcd for with Lindlar’s catalyst for 5 h. The reaction mixture was filtered through
C H O N K: 440.1296; Found 440.1286 (MϩK) .
2
2
40
7
7
ϩ
1
4
3
3
2
ϩ
ϩ
Celite and the precipitate was washed with methanol. The filtrate was com-
bined and evaporated. The residue was purified by silica gel chromatography
14
23
7
7
(
4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)-
guanidino-6-[(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-yl)-(5ٟ-hydroxy-pentyl- (eluent; 2-propanol : ethyl acetate : distilled waterϭ2 : 5 : 1) to give com-
1
oxy)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 19k pound 22j as a colorless amorphous solid (330 mg, 0.523 mmol, 54%). H-
Compound 19k was obtained from 18k (2.45 g, 3.23 mmol) using the same
NMR (400 MHz, CD OD) d: 1.33 (3H, s), 1.41 (3H, s), 1.46 (9H, s), 1.52
3
procedure as that for the preparation of 19j (colorless amorphous solid, (9H, s), 1.95 (3H, s), 2.72—2.80 (2H, m), 3.40—3.50 (1H, m), 3.78 (3H, s),
1
4
(
6 mg, 0.114 mmol, 68%). H-NMR (400 MHz, CDCl ) d: 1.35 (3H, s), 1.43
3.80—3.90 (2H, m), 4.03—4.10 (1H, m), 4.15—4.30 (3H, m), 4.40—4.48
3
3H, s), 1.49 (9H, s), 1.50 (9H, s), 1.50—1.60 (2H, m), 1.65—1.75 (4H, m), (1H, m), 5.00—5.06 (1H, m), 5.88 (1H, d, Jϭ2.4 Hz). MS (FAB) m/z 630
ϩ
1
4
.92 (3H, s), 3.49—3.75 (4H, m), 3.77 (3H, s), 3.75—3.81 (1H, m), 4.00—
.08 (1H, m), 4.10—4.32 (4H, m), 5.12—5.21 (1H, m), 5.85 (1H, d,
(MϩH) .
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(2؆-amino)ethyloxy-2؅,3؅-di-
Jϭ2.3 Hz), 6.63 (1H, br d, Jϭ8.3 Hz), 8.41 (1H, br d, Jϭ8.7 Hz). MS (FAB) hydroxy]propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid
ϩ
m/z 673 (MϩH) . HR-MS (FAB) Calcd for C H O N : 673.3680; Found Trifluoroacetic Acid Salt 23j Compound 23j was obtained from 22j
3
1
53 12
4
ϩ
6
73.3679 (MϩH) .
4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycarbonyl)-
(930 mg, 1.48 mmol) using the same procedure employed for the preparation
of 13a (colorless amorphous solid, 503 mg, 1.02 mmol, 70%). H-NMR
1
(
guanidino-6-[(5
ٟ
-azido-pentyloxy)-(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆- (400 MHz, D O) d: 1.90 (3H, s), 3.02—3.08 (2H, m), 3.43—3.57 (2H, m),
2
yl)methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 20k 3.62 (1H, dd, Jϭ7.3, 2.9 Hz), 3.71 (1H, dd, Jϭ11.7, 3.7 Hz), 3.72—3.80
Compound 20k was obtained from 19k (1.03 g, 1.53 mmol) using the same
(1H, m), 3.83—3.89 (1H, m), 4.12—4.16 (1H, m), 4.21 (1H, dd, Jϭ8.1,
procedure as that for the preparation of 20j (colorless amorphous solid, 2.2 Hz), 4.27 (1H, dd, Jϭ8.8, 2.9 Hz), 5.55 (1H, d, Jϭ2.9 Hz). MS (FAB)
1
ϩ
4
(
(
6 mg, 0.114 mmol, 68%). H-NMR (400 MHz, CDCl ) d: 1.35 (3H, s), 1.43
m/z 376 (MϩH) . HR-MS (FAB) Calcd for C H O N : 376.1832; Found
14 26 7 5
ϩ
3
3H, s), 1.49 (9H, s), 1.50 (9H, s), 1.53—1.75 (6H, m), 1.94 (3H, s), 3.27 376.1842 (MϩH) .
2H, t, Jϭ6.9 Hz), 3.54—3.63 (1H, m), 3.74—3.84 (2H, m), 3.78 (3H, s),
(4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)-
4
.02—4.35 (5H, m), 5.08—5.17 (1H, m), 5.82 (1H, d, Jϭ2.5 Hz), 6.05 (1H, guanidino-6-[(5ٟ-amino-pentyloxy)-(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-
ϩ
br d, Jϭ8.8 Hz), 8.51 (1H, br d, Jϭ8.7 Hz). MS (FAB) m/z 698 (MϩH) .
HR-MS (FAB) Calcd for C H O N : 698.3725; Found 698.3731 (MϩH) .
yl)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 22k
Compound 22k was obtained from 20k (2.01 g, 2.88 mmol) using the same
ϩ
3
1
52 11
7
(
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(5؆-azido)propyloxy-2؅,3؅-di-
procedure employed for the preparation of 22j (colorless amorphous solid,
1
hydroxy]propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid
1k Compound 21k was obtained from 20k (100 mg, 0.143 mmol) using (6H, m), 1.33 (3H, s), 1.40 (3H, s), 1.46 (9H, s), 1.53 (9H, s), 1.94 (3H, s),
1.94 g, 2.88 mmol, quant.). H-NMR (400 MHz, CD OD) d: 1.30—1.65
3
2
the same procedure employed for the preparation of 13a (colorless amor- 2.60—2.67 (1H, m), 3.40—3.50 (1H, m), 3.73—3.83 (2H, m), 3.78 (3H, s),
1
phous solid, 43 mg, 0.0969 mmol, 68%). H-NMR (400 MHz, D O) d: 4.02—4.09 (2H, m), 4.13—4.35 (5H, m), 4.96—5.02 (1H, m), 5.86 (1H, d,
2
1
3
.29—1.43 (2H, m), 1.48—1.65 (4H, m), 2.00 (3H, s), 3.23—3.33 (2H, m),
.36—3.49 (1H, m), 3.52—3.68 (3H, m), 3.79—3.90 (1H, m), 3.89—4.00
Jϭ2.4 Hz), 6.14 (1H, br d, Jϭ8.6 Hz), 8.51 (1H, br d, Jϭ8.7 Hz). MS (FAB)
ϩ
m/z 672 (MϩH) .
(
(
1H, m), 4.10—4.22 (1H, m), 4.30—4.42 (2H, m), 5.55—5.58 (1H, m). MS
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(5؆-amino)pentyloxy-2؅,3؅-di-
ϩ
FAB) m/z 444 (MϩH) . HR-MS (FAB) Calcd for C H O N Na: hydroxy-propyl]-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid
1
7
29
7
7
ϩ
4
66.2026; Found 466.2007 (MϩNa) .
4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؅-tert-butyloxycarbonyl)-
guanidino-6-[(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-yl)-(10ٟ-hydroxy-decy-
Trifluoroacetic Acid Salt 23k Compound 23k was obtained from 22k
(
(1.00 g, 1.49 mmol) using the same procedure employed for the preparation
1
of 13a (colorless amorphous solid, 430 mg, 0.809 mmol, 54%). H-NMR
loxy)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Ester 19l (400 MHz, D O) d: 1.30—1.43 (2H, m), 1.50—1.70 (4H, m), 2.00 (3H, s),
2
Compound 19l was obtained from 18l (7.85 g, 9.47 mmol) using the same 2.92—3.00 (2H, m), 3.38—3.47 (1H, m), 3.55—3.67 (3H, m), 3.80—3.88
procedure employed for the preparation of 19j (colorless amorphous solid, (1H, m), 3.90—3.99 (1H, m), 4.10—4.22 (1H, m), 4.32—4.42 (2H, m), 5.58
1
ϩ
4
(
(
3 mg, 0.0969 mmol, 68%). H-NMR (400 MHz, CDCl ) d: 1.25—1.40 (1H, d, Jϭ2.0 Hz). MS (FAB) m/z 418 (MϩH) . HR-MS (FAB) Calcd for
3
ϩ
12H, m), 1.35 (3H, s), 1.43 (3H, s), 1.49 (9H, s), 1.50 (9H, s), 1.50—1.65 C H O N : 418.2302; Found 418.2307 (MϩH) .
17 32 7 5
4H, m), 1.93 (3H, s), 3.49—3.75 (4H, m), 3.74—3.77 (1H, m), 3.77 (3H,
(4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(10؆-amino)decyloxy-2؅,3؅-di-
hydroxy]propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid
d, Jϭ2.5 Hz), 6.06 (1H, br d, Jϭ9.0 Hz), 8.51 (1H, br d, Jϭ8.7 Hz). MS Trifluoroacetic Acid Salt 23l Compound 22l was obtained from 20l
s), 4.00—4.08 (1H, m), 4.00—4.35 (4H, m), 5.12—5.21 (1H, m), 5.83 (1H,
ϩ
(
7
FAB) m/z 743 (MϩH) . HR-MS (FAB) Calcd for C H O N Na: (100 mg, 0.195 mmol) using the same procedure employed for the prepara-
65.4262; Found 765.4258 (MϩNa) .
3
6
62 12
4
ϩ
tion of 22j. Compound 23l was obtained from 22l (100 mg, 0.195 mmol)
(
4S,5R,6R,1؅S,4؆R)-5-Acetylamino-4-(bis-N,N؆-tert-butyloxycar- using the same procedure employed for the preparation of 23j (colorless
1
bonyl)guanidino-6-[(10ٟ-azido-decyloxy)-(2؆,2؆-dimethyl-[1؆,3؆]diox- amorphous solid, 71 mg, 0.145 mmol, 75%). H-NMR (400 MHz, CD OD)
3
olan-4؆-yl)]methyl-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl Es- d: 1.18—1.40 (12H, m), 1.43—1.61 (4H, m), 1.98 (3H, s), 2.80—2.95 (2H,
ter 20l Compound 20l was obtained from 19l (5.11 g, 6.88 mmol) using m), 3.35—3.46 (1H, m), 3.50—3.68 (3H, m), 3.79—3.89 (1H, m), 3.89—
the same procedure employed for the preparation of 20j (colorless amor- 3.98 (1H, m), 4.09—4.18 (1H, m), 4.30—4.40 (2H, m), 5.55 (1H, m). MS
1
ϩ
phous solid, 3.92 g, 5.10 mmol, 74%). H-NMR (400 MHz, CDCl ) d: (FAB) m/z 488 (MϩH) . HR-MS (FAB) Calcd for C H O N : 488.3084;
3
22 42
7
5
ϩ
1
1
.25—1.40 (12H, m), 1.35 (3H, s), 1.43 (3H, s), 1.49 (9H, s), 1.50 (9H, s), Found 488.3091 (MϩH) .
.55—1.65 (4H, m), 1.93 (3H, s), 3.25 (2H, t, Jϭ6.9 Hz), 3.54—3.63 (1H,
m), 3.68—3.75 (1H, m), 3.75—3.77 (1H, m), 3.78 (3H, s), 4.00—4.11 (2H,
m), 4.14—4.22 (3H, m), 5.08—5.16 (1H, m), 5.82 (1H, d, Jϭ2.5 Hz), 6.00
(4S,5R,6R,1؅S,4؆R)-5-Acetylamino-6-[(2ٟ-acetylamino-ethyloxy)-
(2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-yl)]methyl-4-(bis-N,N؅-tert-butyloxy-
carbonyl)guanidino-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl
(
1H, br d, Jϭ9.0 Hz), 8.51 (1H, br d, Jϭ8.7 Hz). MS (FAB) m/z 768 Ester 24j 22j (76 mg, 0.121 mmol) was treated with acetic anhydride
ϩ
(MϩH) . HR-MS (FAB) Calcd for C H O N Na: 790.4327; Found (1 ml) and pyridine (1 ml). The reaction mixture was stirred at room temper-
3
6
61 11
7
ϩ
7
90.4343 (MϩNa) .
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(10؆-azido)decyloxy-2؅,3؅-di- methylene chloride. The organic layer was washed with aqueous hydrogen
hydroxy]propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic Acid chloride (1 N), aqueous sodium hydrogen carbonate, and brine, dried over an-
1l Compound 21l was obtained from 20l (300 mg, 0.390 mmol) using the hydrous sodium sulfate, and concentrated under reduced pressure. The
ature for 2 h. The reaction mixture was poured into brine and extracted with
(
2
same procedure employed for the preparation of 13a (colorless amorphous residue was purified by silica gel chromatography (eluent; ethyl
1
solid, 101 mg, 0.196 mmol, 50%). H-NMR (400 MHz, CD OD) d: 1.25— acetate : methanolϭ10 : 1) to give compound 24j as a colorless amorphous
3
1
1
.40 (12H, m), 1.47—1.65 (4H, m), 1.99 (3H, s), 3.22—3.30 (2H, m), solid (77 mg, 0.114 mmol, 95%). H-NMR (400 MHz, CDCl ) d: 1.35 (3H,
3

December 2003
1397
s), 1.43 (3H, s), 1.48 (9H, s), 1.49 (9H, s), 1.98 (3H, s), 1.99 (3H, s), 3.25— 3.20—3.40 (2H, m), 3.42—3.70 (4H, m), 3.78—3.88 (1H, m), 3.92—4.00
3
4
.35 (1H, m), 3.41—3.60 (2H, m), 3.79 (3H, s), 3.80—3.90 (1H, m), 3.96— (1H, m), 4.12—4.52 (14H, m), 5.58—5.62 (1H, m).
.40 (7H, m), 5.05—5.16 (1H, m), 5.84 (1H, d, Jϭ2.4 Hz), 6.52 (1H, br d, Polymer Carrying 7-O-Decyl-4-guanidino-Neu5Ac2en 26d Polymer
Jϭ9.2 Hz), 7.44 (1H, m), 8.57 (1H, br d, Jϭ8.8 Hz). MS (FAB) m/z 672 26d was obtained from 23l (25 mg, 0.041 mmol) using the same procedure
ϩ
(
MϩH) .
(
as that for the preparation of 26a (45 mg, 0.26 mmol, 79%). Mol fraction of
1
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(2؆-acetylamino)ethyloxy-
؅,3؅-dihydroxy-propyl]-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic 1.55 (4H, m), 1.85—2.18 (22H, m), 1.98 (3H, s), 2.22—2.48 (22H, m),
sugar unitϭ0.09. H-NMR (400 MHz, D O) d: 1.15—1.35 (12H, m), 1.38—
2
2
Acid 25j. Method A Compound 25j was obtained from 24j (50 mg, 3.20—3.40 (2H, m), 3.42—3.70 (4H, m), 3.78—3.88 (1H, m), 3.92—4.00
.0745 mmol) using the same procedure as that for the preparation of 13a (1H, m), 4.12—4.52 (14H, m), 5.58—5.62 (1H, m).
colorless amorphous, 26 mg, 0.0623 mmol, 84%). Cells and Viruses Madin-Darby canine kidney (MDCK) cells obtained
Method B Compound 23j (10.0 mg, 0.0204 mmol) was dissolved in dis- from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan) were grown in
tilled water (1 ml) and mixed with 1-(acetoxy)benzotriazole (7.2 mg, Earle’s Minimum Essential Medium (MEM) (Invitrogen corp.) supple-
.0408 mmol) and pyridine (0.1 ml). The reaction mixture was stirred for 2 h mented with 10% fetal bovine serum (Hyclone), and the antibiotics peni-
0
(
0
at room temperature, and then concentrated under reduced pressure. The cillin G (50 units/ml) and streptomycin sulfate (50 mg/ml) (Invitrogen corp.).
crude product was purified by reverse phase cosmosil chromatography (elu- Cells were routinely cultured in flasks at 37 °C and 5% CO . The influenza
2
ent; water : MeOHϭ4 : 1) to obtain 25j as a colorless amorphous (7.0 mg, virus strains A/PR/8/34 (H1N1) was provided by Dr. Peter Palese, and A/Ya-
1
0
3
.0168 mmol, 82%). H-NMR (400 MHz, D O) d: 1.95 (3H, s), 1.98 (3H, s), magata/32/89 (H1N1) was obtained from Chiba Serum Institute (Japan).
2
.20—3.40 (2H, m), 3.43—3.52 (1H, m), 3.55—3.70 (3H, m), 3.81 (1H, dd, The A/PR/8/34 (H1N1) virus used for neuraminidase inhibition assays (the
Jϭ11.8, 2.7 Hz), 3.92—3.99 (1H, m), 4.12—4.22 (1H, m), 4.30—4.39 (2H, source of enzyme) was propagated in the allantoic sacs of embryonated eggs
ϩ
m), 5.58 (1H, d, Jϭ2.9 Hz). MS (FAB) m/z 418 (MϩH) . HR-MS (FAB) and purified by sucrose density gradient centrifugation. The A/Yama-
ϩ
Calcd for C H O N : 418.1937; Found 418.1935 (MϩH) .
gata/32/89 (H1N1) virus used for plaque reduction assay was in the allantoic
16
28
8
5
(
4S,5R,6R,1؅R,4؆S)-5-Acetylamino-6-[(5ٟ-acetylamino-pentyloxy)- sacs of embryonated eggs. The viral stocks were stored at Ϫ80 °C. Influenza
2؆,2؆-dimethyl-[1؆,3؆]dioxolan-4؆-yl)]methyl]-4-(bis-N,N؆-tert-butyloxy- virus A/PR/8/34 was adapted to mice by passaging the virus several times in
carbonyl)guanidino-5,6-dihydro-4H-pyran-2-carboxylic Acid Methyl mice (MAIV, Mouse Adapted Influenza Virus).
(
Ester 24k Compound 24k was obtained from 22k (140 mg, 0.201 mmol)
Neuraminidase Inhibition Assay A standard colorimetric assay was
using the same procedure as that for the preparation of 24j (colorless amor- used to measure influenza virus neuraminidase activity. The substrate, 2Ј-(4-
1
phous, 110 mg, 0.154 mmol, 77%). H-NMR (400 MHz, CDCl ) d: 1.35
nitrophenyl)-a-D-N-acetylneuraminic acid, was cleaved by neuraminidase to
3
(
(
(
3H, s), 1.43 (3H, s), 1.49 (9H, s), 1.50 (9H, s), 1.53—1.75 (6H, m), 1.94 yield nitrophenol, which was quantified. The assay mixture contained in-
3H, s), 1.95 (3H, s), 3.21—3.28 (2H, m), 3.40—3.52 (1H, m), 3.74—3.84 hibitors at various concentrations and the substrate in 20 mM 2-(N-mor-
2H, m), 3.78 (3H, s), 4.02—4.35 (5H, m), 5.10—5.19 (1H, m), 5.84 (1H, d, pholino)ethanesulfonic acid (MES) and buffer (50 mM calcium chloride at
Jϭ2.4 Hz), 6.14 (1H, br d, Jϭ8.6 Hz), 8.51 (1H, br d, Jϭ8.7 Hz). MS (FAB) pH 6.0 and 10 mg/ml bovine serum albumin) and was incubated for 5 min at
ϩ
m/z 714 (MϩH) . HR-MS (FAB) Calcd for C H O N : 714.3926; Found 37 °C. The reaction was started by the addition of enzyme. After incubation
3
3
56 12
5
ϩ
7
14.3942 (MϩH) .
4S,5R,6R,1؅R,2؅R)-5-Acetylamino-6-[1؅-(5؆-acetylamino)pentyloxy-
for 20 min, the reaction was terminated by adding 0.2 M glycine/sodium hy-
droxide (pH 10.2). Then, the absorbance at 450 nm was measured using the
(
2
؅,3؅-dihydroxy]propyl-5,6-dihydro-4-guanidino-4H-pyran-2-carboxylic microplate reader. The substrate blanks were subtracted from the sample
Acid 25k Compound 25k was obtained from 24k (110 mg, 0.154 mmol) readings. The IC50 was calculated by plotting the percent inhibition of neu-
using the same procedure as that for the preparation of 13a (colorless amor- raminidase activity versus the inhibitor concentration.
1
phous, 46 mg, 0.100 mmol, 65%). H-NMR (400 MHz, D O) d: 1.20—1.38
Plaque Reduction Assays in MDCK Cells Anti-influenza virus activ-
2
2
9)
(
2H, m), 1.40—1.60 (4H, m), 1.92 (3H, s), 1.99 (3H, s), 3.05—3.17 (2H, ity was measured as described by Hayden et al., with some modifications.
m), 3.35—3.47 (1H, m), 3.52—3.72 (3H, m), 3.75—3.98 (2H, m), 4.09—
Briefly, confluent growing MDCK cells on 35 mm dish were washed with
phosphate buffered saline (PBS) (Invitrogen Corp.), inoculated with virus
4
.18 (1H, m), 4.32—4.40 (2H, m), 5.55—5.58 (1H, m). MS (FAB) m/z 460
ϩ
(MϩH) . HR-MS (FAB) Calcd for C H O N : 460.2407; Found 460.2432
(80 to 150 PFU/well) and incubated in a 5% CO incubator at 37 °C for 1 h.
1
9
34
8
5
2
ϩ
(
MϩH) .
Polymer Carrying 7-O-Ethyl-4-guanidino-Neu5Ac2en 26a To a solu-
tion of poly-L-glutamic acid sodium salt (50 mg, 0.331 mol/unit) in distilled
The virus inoculum was then discarded, and the cell monolayers were over-
laid with MEM supplemented with 0.22% sodium hydrogen carbonate,
10 mM HEPES (pH 7.2) (Invitrogen Corp.), 0.21% bovine serum albumin
water (1 ml), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (70 mg, (Dainippon Pharmaceuticals Co. Ltd.), 1 mg/ml of trypsin, 0.01% DEAE
.364 mmol) and a solution of 1-hydroxybenzotriazole (111 mg, 0.728 Dextran, 0.6% agarose (Sigma Aldrich Corp.) and the test compound. After
0
mmol) were added at room temperature. The reaction mixture was stirred at 30 to 38 h of incubation at 37 °C, the agar overlay was removed and the cell
room temperature for 30 min. Then, the precipitate was separated by filtra- monolayers were stained with 0.1% crystal violet in 19% methanol.
tion, washed with water, and dried under vacuum. The residue was dissolved
in N,N-dimethylformamide (2 ml) and combined with compound 23j plaque number visually or measuring plaque size with PIXEL CATHER &
20 mg, 0.041 mmol) and pyridine (0.1 ml). The reaction mixture was stirred ANALYZER PCA-11 (SYSTEM SCIENCE CO., Japan) at each compound
at room temperature for 2 h. Then, to the reaction, aqueous ammonia (25%, concentration.
ml) was added and the mixture was stirred at room temperature for 2 d. The IC50 was calculated by plotting the percent inhibition of plaque num-
The reaction mixture was concentrated under reduced pressure. To the ber or size versus the inhibitor concentration.
The antiviral efficacy of the test compounds was assessed by counting the
(
3
residue, 2-propanol was added and the precipitate was separated by filtra-
In Vivo Antiviral Experiments Mice (BALB/c CrSlc, female, 5 weeks
tion. The residue was dissolved in water, dialyzed for 2 d in water (MW old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). Compounds
1
0
2000 cut off), and lyophilized to afford 26a as a colorless powder (43 mg, were prepared with saline. Mice were anesthetized with chloroform/diethyl
.268 mmol, 81%). Mol fraction of sugar unitϭ0.076. H-NMR (400 MHz, ether (1 : 1) 24 h before infection and each group of 7 or 8 mice received
1
D O) d: 1.85—2.18 (26.4H, m), 1.98 (3H, s), 2.22—2.48 (26.4H, m), 50 ml of 26c or zanamivir solution intranasally at doses of 0.3 mmol/kg, re-
2
3
.20—3.40 (2H, m), 3.42—3.70 (4H, m), 3.78—3.88 (1H, m), 3.92—4.00
spectively. The control group (nϭ8) was administered saline. Then, 500
plaque-forming units (pfu)/mouse of MAIV in 50 ml of phosphate-buffered
(
1H, m), 4.12—4.52 (16.2H, m), 5.58—5.62 (1H, m).
Polymer Carrying 7-O-Ethyl-4-guanidino-Neu5Ac2en 26b Polymer saline (pH 7.4) containing 0.42% bovine serum albumin was inoculated in-
6b was obtained from 23j (56 mg, 0.116 mmol) using the same procedure tranasally to mice anesthetized with chloroform/diethyl ether (1 : 1) (day 0).
2
as that for the preparation of 26a (52 mg, 0.24 mmol, 73%). Mol fraction of The number of surviving mice was counted everyday for 20 d after infection.
1
sugar unitϭ0.25. H-NMR (400 MHz, D O) d: 1.85—2.18 (8H, m), 1.98 The statistical analyses were performed using the SAS System Release 8.2
2
(
3H, s), 2.22—2.48 (8H, m), 3.20—3.40 (2H, m), 3.42—3.70 (4H, m), for Windows (SAS Institute Inc.,). The Log-Rank test based on the joint
3
.78—3.88 (1H, m), 3.92—4.00 (1H, m), 4.12—4.52 (7H, m), 5.58—5.62
ranking method was carried out to compare the prolonged effects of 26c and
(
1H, m).
zanamivir.
Polymer Carrying 7-O-Pentyl-4-guanidino-Neu5Ac2en 26c Polymer
2
6c was obtained from 23k (22 mg, 0.041 mmol) using the same procedure References and Notes
as that for the preparation of 26a (45 mg, 0.27 mmol, 81%). Mol fraction of
1) Wee C. C., Phillips R. S., Burstin H. R., Cook E. S., Purpolo A. L.,
Brennan T. A., Haas A. S., Amer. J. Med., 110, 181—187 (2001).
2) Bethell R., Smith P., Drugs Future, 23, 1099—1109 (1998).
1
sugar unitϭ0.09. H-NMR (400 MHz, D O) d: 1.15—1.35 (2H, m), 1.38—
2
1
.55 (4H, m), 1.85—2.18 (22H, m), 1.98 (3H, s), 2.22—2.48 (22H, m),

1
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