MURAMYLDIPEPTIDE DERIVATIVES

DERIVES DE MURAMYLDIPEPTIDE

English Abstract

ABSTRACT OF THE DISCLOSURE
Muramyldipeptide derivatives represented by
the formula:
<IMG>
wherein Ala represents alanine; Acyl represents an acyl
group; R1 and R2 each represents a hydrogen atom or, when
taken together, may form an alkylidene group; R3 represents
a hydrogen atom, an acyl group or an alkyl group; and R4
represents a hydrogen atom or an alkyl group. These
compounds have excellent immunoadjuvant activity, prophyl-
actic and therapeutic effects against microbial infections,
and antitumor activity.

Claims

The embodiments of the invention in which an ex-
clusive property or privilege is claimed are defined as
follows:
1. A process for the preparation of a muramyldipeptide
derivative represented by the formula (I):
<IMG>
wherein Ala represents alanine; Acyl represents an acyl group
having 2 to 6 carbon atoms; R1 and R2 each represents a hydro-
gen atom or, when taken together, may form an alkylidene group
having 1 to 6 carbon atoms; R3 represents a hydrogen atom, an
acyl group having 2 to 40 carbon atoms or an alkyl group
having 1 to 40 carbon atoms; and R4 represents a hydrogen atom
or an alkyl group having 1 to 6 carbon atoms, comprising a
process selected from the group consisting of:
(a) a process to produce compounds of the formula (I)
wherein R1 and R2 when taken together form an alkylidene
group having 1 to 6 carbon atoms, R3 is an acyl group having
2 to 40 carbon atoms and R4 is an alkyl group having 1 to 6
carbon atoms, comprising condensing a compound of the formula
IV
33

<IMG> IV
wherein R31 represents an acyl group having 2 to 40 carbon
atoms, R5 represents a hydrogen atom or an alkyl group having
1 to 6 carbon atoms, and R6 represents an alkyl group having
1 to 6 carbon atoms,
with an alanyl-isoglutamine alkyl ester,
(b) a process to produce compounds of the formula (I)
wherein R1 and R2 when taken together form an alkylidene
group having 1 to 6 carbon atoms, R3 is a hydrogen atom,
and R4 is an alkyl group having 1 to 6 carbon atoms, com-
prising process (a) above further including reacting the
resultant product of process (a) with. a metal alcoholate;
(c) a process to produce compounds of the formula (I)
wherein R1 and R2 when taken together form an alkylidene
group having 1 to 6 carbon atoms, R3 is an alkyl group
having 1 to 40 carbon atoms, and R4 is an alkyl group having
1 to 6 carbon atoms, comprising process (b) above further
including reacting the resultant product of process (b) with
an alkyl halide;
34

(d) a process to produce compounds of the formula (I)
wherein R1 and R2 each is a hydrogen atom, R3 is an acyl group
having 2 to 40 carbon atoms and R4 is an alkyl group having
1 to 6 carbon atoms, comprising process (a) above further
including the step of cleavage of the alkylidene group;
(e) a process to produce compounds of the formula (I)
wherein R1 and R2 when taken together from an alkylidene
group having 1 to 6 carbon atoms, R3 is an acyl group having
2 to 40 carbon atoms, and R4 is a hydrogen atom, comprising
process (a) above further including the step of hydrolysis
of the ester group;
(f) a process to produce compounds of the formula (I)
wherein R1 and R2 each is a hydrogen atom, R3 is an acyl
group having 2 to 40 carbon atoms, and R4 is a hydrogen
atom, comprising process (a) above further including the
steps of cleavage of the alkylidene group and hydrolysis
of the ester group;
(g) a process to produce compounds of the formula (I)
wherein R1 and R2 each is a hydrogen atom, R3 is a hydrogen
atom, and R4 is an alkyl group having 1 to 6 carbon atoms
comprising process (b) above further including the step
of cleavage of the alkylidene group;
(h) a process to produce compounds of the formula [I)
wherein R1 and R2 when taken together form an alkylidene
group having 1 to 6 carbon atoms, R3 is a hydrogen atom,
and R4 is a hydrogen atom, comprising process (b) above

further including the step of hydrolysis of the ester group;
(i) a process to produce compounds of the formula (I)
wherein R1, R2, R3 and R4 each is a hydrogen atom comprising
process (b) above further including the steps of cleavage
of the alkylidene group and hydrolysis of the ester group;
(j) a process to produce compounds of the formula (I)
wherein R1 and R2 each is a hydrogen atom, R3 is an alkyl group
having 1 to 40 carbon atoms, and R4 is an alkyl group having
1 to 6 carbon atoms, comprising process (c) above further
including the step of cleavage of the alkylidene group;
(k) a process to produce compounds of the formula (I)
wherein R1 and R2 when taken together form an alkylidene
group having 1 to 6 carbon atoms, R3 is an alkyl group having
1 to 40 carbon atoms, and R4 is a hydrogen atom, comprising
process (c) above further including the step of hydrolysis
of the ester group, and
(l) a process to produce compounds of the formula (I)
wherein R1, R2 and R4 each is a hydrogen atom and R3 is an
alkyl group having 1 to 40 carbon atoms, comprising process
(c) above further including the steps of cleavage of the
alkylidene group and hydrolysis of the ester group.
36

2. A muramyldipeptide derivative represented by the
formula:
<IMG> I
wherein Ala represents alanine; Acyl represents an acyl group
having 2 to 6 carbon atoms; R1 and R2 each represents a
hydrogen atom or, when taken together, may form an alkylidene
group having 1 to 6 carbon atoms; R3 represents a hydrogen
atom, an acyl group having 2 to 40 carbon atoms or an alkyl
group having 1 to 40 carbon atoms; and R3 represents a hydro-
gen atom or an alkyl group having 1 to 6 carbon atoms,
when produced by the process of claim 1 or an obvious chemi-
cal equivalent.
3. Process (a) of claim 1.
4. The compound of formula (I) as claimed in claim 2
wherein R1 and R2 when taken together form an alkylidene
group having 1 to 6 carbon atoms, R3 is an acyl group having
2 to 40 carbon atoms and R4 is an alkyl group having 1 to 6
carbon atoms when produced by the process of claim 3 or an
obvious chemical equivalent.
37

5. Process (b) of claim 1.
6. Process (c) of claim 1.
7. Process (d) of claim 1.
8. The compound of the formula (I) as claimed in claim
2 wherein R1 and R2 each is a hydrogen atom, R3 is an acyl
group having 2 to 40 carbon atoms and R4 is an alkyl group
having 1 to 6 carbon atoms when produced by the process of
claim 7.
9. Process (e) of claim 1.
10. Process (f) of claim 1.
11. Process (g) of claim 1.
12. Process (h) of claim 1.
13. Process (i) of claim 1.
14. Process (j) of claim 1.
15. Process (k) of claim 1.
16. Process (l) of claim 1,
17. A process to produce 2-acetamido-2-deoxy-l-S-
decanoyl-3-0-(D-2-propanoyl-L-alanyl-D-isoglutamine methyl
ester)-l-thio-.beta.-D-glucopyranose comprising condensing
2-acetamido-2-deoxy-l-S-decanoyl-4,6-0-isopropylidene-3-0-
(D-2-propanoyl)-l-thio-.beta.-D-glucopyranose with alanyl-iso-
glutamine methyl ester and subjecting the resultant product
to cleavage of its alkylidene group.
38

18. 2-Acetamido-2-deoxy-l-S-decanoyl-3-0-(D-2-propanoyl-
L-alanyl-D-isoglutamine methyl ester)-l-thio-.beta.-D-glucopyranose,
when prepared by the process of claim 17 on an obvious chemical
equivalent.
19. A process to produce 2-acetamido-l-S-acetyl-2-deoxy-
3-0-(D-2-propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-
thio-.beta.-D-glucopyranose, comprising condensing 2-acetamido-2-
deoxy-l-S-acetyl-4,6-0-isopropylidene-3-O-(D-2 propanoyl)-l-
thio-.beta.-D-glucopyranose with alanyl-isoglutamine methyl ester
and subjecting the resultant product to cleavage of its al-
kylidene group.
20. 2-Acetamido-l-S-acetyl-2-deoxy-3-O-(D-2-propanoyl-
L-alanyl-D-isoglutamine methyl ester)-l-thio-.beta.-D-glucopyranose,
when prepared by the process of claim 19 or an obvious chemical
equivalent.
21. A process to prepare 2-acetamido-2-deoxy-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-.beta.-D-
glucopyranose, comprising condensing 2-acetamido-2-deoxy-l-S-
acetyl-4,6-0-isopropylidene-3-O-(D-2-propanoyl)-l-thio-.beta.-D-
glucopyranose with alanyl-isoglutamine methyl ester and then
subjecting the resultant product to reaction with a metal al-
coholate and to cleavage of its alkylidene group.
22. 2-Acetamido-2-deoxy-3-O-(D-2-propanoyl-L-alanyl-D-
isoglutamine methyl ester)-l-thio-.beta.-D-glucopyranose, when
prepared by the process of claim 21 or an obvious chemical
equivalent.
39

23. A process to prepare 2-acetamido-2-deoxy-3-O-
(D-2-propanoyl-L-alanyl-D-isoglutamine)-1-thiO-.beta.-D-glucopyra-
nose comprising condensing 2-acetamido-2-deoxy-1-S-acetyl-4,
6-O-isopropylidene-3-O-(D-2-propanoyl)-1-thio-.beta.-D-glucopyranose
with alanyl-isoglutamine methyl ester, and then subjecting the
resultant product to reaction with a metal alcoholate, to
cleavage of its alkylidene group and to hydrolysis of its ester
group.
24. 2-Acetamido-2-deoxy-3-O-(D-2-propanoyl-L-alanyl-D-
isoglutamine)-l-thio-.beta.-D-glucopyranose, when prepared by the
process of claim 23 or an obvious chemical equivalent.
25. A process to prepare 2-acetamido-2-deoxy-l-S-
hexadecanoyl-3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine methyl
ester)-l-thio-.beta.-D-glucopyranose, comprising condensing
2-acetamido-2-deoxy-l-S-hexadecanoyl-4,6-O-isopropylidene-3-
O-(D-2-propanoyl)-l-thio-.beta.-D-glucopyranose with
alanyl-isoglutamine methyl ester and subjecting the resultant
product to cleavage of its alkylidene group.
26. 2-Acetamido-2-deoxy-l-S-hexadecanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-.beta.-D-
glucopyranose, when prepared by the process of claim 25 or
an obvious chemical equivalent.
27. A process to prepare 2-acetamido-2-deoxy-l-S-hexa-
decanoyl-3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine)-l-thio-
.beta.-D glucopyranose, comprising the process of claim 25 further
including subecting the resultant product to hydrolysis of
its ester group.

28. 2-Acetamido-2-deoxy-1-S-hexadecanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine)-1-thio-.beta.-D-glucopyranose,
when prepared by the process of claim 27 or an obvious chemi-
cal equivalent.
29. A process to prepare 2-acetamido-2-deoxy-1-S-
eicosanoyl-3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine)-1-
thio-.beta.-D-glucopyranose, comprising condensing 2-acetamino-2-
deoxy-1-S-eicosanoyl-4,6-O-isopropylidene-3-O-(D-2-propanoyl)
-1-thio-.beta.-D-glucopyranose with alanyl-isoglutamine alkyl
ester and subjecting the resultant product to cleavage of its
alkylidene group and then to hydrolysis of its ester group.
30. 2-Acetamido-2-deoxy-1-S-eicosanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine)-1-thio-.beta.-D-glucopyranose,
when prepared by the process of claims 29 or an obvious
chemical equivalent.
31. The process claimed in claim 1 wherein R3 represents
an acyl group having 2 to 6 carbon atoms or an alkyl group
having 1 to 40 carbon atoms.
32. The process claimed in claim 3, 5 or 6 wherein R3
represents an acyl group having 2 to 5 carbon atoms or an
alkyl group having 1 to 40 carbon atoms.
33. The process claimed in claim 1 wherein R3 represents
an acyl group having 2 to 6 carbon atoms or an alkyl group
having 1 to 6 carbon atoms.
41

Description

MURAMYLDIPEPTIDE DERIVATIVES
FIELD OF THE INVENTION
This invention relates to novel muramyldipeptide
derivatives having excellent immunoadjuvant activity and
prophylactic and therapeutic effects against microbial
infections and antitumor activity and, more speclfically,
this invention relates to a muramyldipeptide derivative
represented by the formula ~
R10--CH2
~ O~SR3 (I)
R2~
NH--A c~ 1
CONH
1 2
C~3C~ICO-Ala-NH-C~CH2CH~COOR4
. I
wherein Ala represents alanine; Acyl represents an acyl
group having 2 to 40 carbon atoms; ~1 and R2 each represents
a hydrogen atom, or Rl and R2, when taken together, may
: form an alkylidene group having 1 to 6 carbon atoms; R3
represents a hy~rogen atom, an acyl group having 2 to 40
carbon atoms or an alkyl group having 1 to 40 carbon atoms;
and R4 represents a hydrogen atom or an alkyl gxoup having
: 1 to 20 carbon atoms.
^' ~

3~
BACKGROUND OF THE INVENTION
Hitherto, certain types of muramyldipeptide
derivatives have been known to have useful immunotherapeutic
activities. For example, U.S. Paten-t 4,101,536 discloses
muramyldipeptide derivatives and the salts thereof having
potent immunoadjuvant activi-ty and antltumor activity and
being applicable as an agent for the immunotherapy of
cancer for human and animals~ These known compaunds,
however, have a free hydroxy group at the l-position of
muramic acid of M-acetylmuramyldipeptide and do not possess
an S-substituted moiety at the.l-position.
. SIJMMARY O`F THE INVENTION
As a result of extensive studies on compounds
useful as adjuvant substances, we have found that the
compounds of the above formula (I) which are obtained by
introducing a sul~ur atom to ~he l-position of muramic
acid o N-acylmuramyldipeptides, the minimum unit consti-
tuting bact~rial cell wall of human type Mycobacterium
tubercul_sis, BCG and other mycobacteria as well as cellular
parastic bacteria, have excellent immunoadjuvant activity,
and thus completed the present invention.
The compounds according to the present invention
are of low toxicity and possess excellent immunoadjuvant
activity and, therefore, can be useful for various diseases
attributed to reduction in i~munity function~ for example,
., ,

O~
as prophylactic and therapeutic ayents against microbial
infectlons, antitumor agents and the like.
DETAILED DESCRIPTION OF THE INVENTION
The muramyldipeptide derivatives of the present
s invention are represented by the formula (I)
R10--CH
~ 9R3
R20 \~ ~
~ ~H-Acyl
fONH2
CH3CHCO-Ala-NEI-CHCH2CH2COOR~
wherein Ala represents alanine; Acyl represents an acyl
group having 2 to 40 carbon atoms; Rl and R2 each represents
a hydrogen atom, or Rl and R2, when taken together, may
: 10 form an alkylidene group having 1 to 6 carbon atoms; R3
represents a hydrogen atom, an acyl group having 2 to 40
carbon atoms or an alkyl group having 1 to 40 carbon atoms;
and R4 represents a hydrogen atom or an alkyl group having
1 to 20 carbon atoms.
lS A preferred class of compounds according to the
present invention is that having the formula (I) above
wherein Rl and R2 each represents a hydrogen atomi R3
represents an acyl group having 2 to 40 carbon atoms; and

0~
R4 represents an alkyl group having 1 to 20 carbon atoms.
Particularly preferred compounds of the present
invention are 2-acetamido-2-deoxy-1-S-decanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-~-
D-glucopyranose, 2-acetamido-1-S-acetyl-2-deoxy-3-O-(D-
2-propanyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-
~-D-glucopyranose, 2-acetamido-2-deoxy-3-O-(D-2-propanoyl~
L-alanyl-D-isoglutamine methyl ester)-l-thio-~-D-glucopyranose,
2-acetamido-2-deoxy-3-O-(D-2-propanoyl-L-alanyl-D-iso-
glutamine)-l-thio-~-D-glucopyranose, 2-acetamido-2-deoxy-
l-S-hexadecanoyl-3-O-(D-2-prop~noyl-L-alanyl-D-isoglutamine
methyl ester)-l-thio-~-D-glucopyranose, 2-acetamido-2-
deoxy-l-S-hexadecanoyl-3-O-(D-2-propanoyl-1-alanyl-D-iso-
; glutamine)-l-thio-~-D-glucopyranose, and 2-acetamido-2-
deoxy-1-S-eicosanoyl-3 O-(D-2-propanoyl-L-alanyl-D-iso-
glutamine)-l-thio-~-D-glucopyranose.
The effects of the compounds of -the present
invention were confirmed by the following method.
(1) Adjuvant Activity
The adjuvant activity was confirmed by the enhancing
activity on the induction of delayed-type hypersensitivity
to N-acetyltyrosine-3-azobenzene-4'-arsonic acid (herein-
after referred to as ABA-N-Ac-Tyr) in guinea pig.
That is, a water-in-oil emulsion prepared by
25 mixing 50 ~g/animal of ABA-N-Ac-Tyr and 100 ~g/animal of
- 4 -

~ S)~3
each of the compounds of the present invention with
Freund's incomplete adjuvant ~hereinafter referred to as
FIA) was administered to the soles of guinea pigs.
Two weeks later, 100 ~g of 3-azobenzene-4'-arsonic acid
bonded to bovine serum albumin (hereinafter referred to
as ABA-BSA3 was intradermally injected and the diameter of
skin reaction (i.e., erythema and induration) was measured
24 hours and 48 hours after the injection. The diameter
of the skin reaction is considered a measure of cellular
immunity. The results obtained are shown in Table 1 below.
Table
Adjuvant Activity on Induction of
- Delayed-Type Hypersensitivity
Skin Reaction
Compound No.* Dose 24 Hours 48 Hours
_ _
(~g~ (mm+SE) (mm+SE)
2 10021.0+1.0 21.0+0.7
3 10022.1+0.9 20.9+0.7
4 10023.1+1.2 21.0-~1.3
8 10014.5 ~a ~ 710.3+1.0
9 10013.5+0.8 12.4+0.7
10~17.4+1.7 13.6+1.8
11 10015.6+1.9 11.6+1.4
14 10020.4+0.7 21.0+1.4
- 25 15 10016.9+0.8 14.0+1.0
16 10011. 3+n ~ 85.6+1.4
17 100~1.8+0.8 23.1+1.1
Control
~ABA-Tyr+FIA) 4.5~0.3 2.9+0.7

o~
(2) Prophylactic ~ffect Against Microbial Infection
Animals: Groups of 20 STD-ddy male mice, 5 weeks
of age, were used.
Bac-teria: E. coli E77156 which has been maintained
in a freezed-culture was used.
A~ents: Compounds of the present invention were
dissolved or suspended in Dulbecco phosphate
buffered saline (PBS: pH 7.4) at a concen-
tration of 500 ~g/ml just before use.
Treatment and Infection:
The animals weEe treated subcutaneously
with 0.2 ml of each of the above solution
or suspension (equivalent to 100 ~g/mouse),
follow~d by the subcutaneous challenge with
E. coli 24 hours later.
The effect was judged from the percent survival
o~ the mice seven days after the infection. As is apparent
Erom the results shown in Table 2 belowl the compounds of
the present invention exhibit excellent prophylactic effect
'20 against microbial infection.
-- 6 --
. . ..
, ~ ~

c)~
Table 2
Prophylactic Effect in Mice Infected with E. coli E 77156
~ Survival 7 days
after Infection
Inoculum Size (cells/mouse)
of E. coli E 77155
. _
Compound Number of
5No. Mice Used 4.~ x lo66.17 x 106 6 88 x lo6
4 20 55
14 20 80
17 20 60
18 20 90
10 19 20 70
21 20 50
22 20 55
23 20 60
: 24 20 65
15 25 20 55
PBS con~rol 20 5 5 5
(3) Antitumor Activity Against Meth-A System
A mixture of tumor cells (~eth-A, 1.5 x 10 6 ) and
100 ~g of Compound 20 of -the present invention suspended in
PBS was inoculated intradermally in BALB/c mice.
Suppress.ion of Meth-A growth in BALB/c on the 27th day after
;
:`
- 7 -
,~ ,

o~
inoculation was found to be 5/9. The value is expressed
in te.rms of the number of tumor-free mice/the number of
mice tested.
The test compounds used i'n the above experiments
(1), (2) and (3) are as follows.
Compound 2: 2-Acetamido-l-S-acetyl-2-deoxy-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl
ester)-l-thio-~-D-glucopyranose
Compound 3: 2-Acetamido-2-deoxy-3-O-(D-2-propanoyl-L-
alanyl-D-isoglutamine methyl ester)-l-thio-
~-D-glucopyranos~
Compound 4- 2-~cetamido-2-deoxy-3-O-(D-2-propanoyl-L-
aranyl-D-isoglutamine)-l-thio-~-D-glucopyranose
Compound 8: l-S-n-Butyl 2-acetamido-2-deoxy-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-
l-thio-~-D-glucopyranoside
Compound 9: l-S-Hexadecanyl 2-acetamido-2-deoxy-3-O-(D-
2-propanoyl-L alanyl-D-isoglutamine methyl
ester)-l-thio-~-D-glucopyranoside
Compound 10: l-S-n-Butyl 2-acetamido-2-deoxy-3-O-(D-2-
. propanoyl-L-alanyl-D~isoglutamine)-l-thio-
~-D-glucopyranoside
Compound 11: l-S-Hexadecanyl 2-acetamido-2-deoxy-3-O-(D-
2-propanoyL-L-alanyl-D-isoglutamine)-l-thi
~-D-glucopyranoside
- ~ _
:, :
i

o~
Compound 14: 2-Acetamido~2-deoxy-1-S-hexadecanoyl-3-O-(D-
2-propanoyl-L-alanyl-D-isoglutamine methyl
ester)-l-thio-~-D-glucopyranose
Compound 15: 2-Acetamido-2-deoxy-4,6-O-isopropylidene-3-
O-(D-2-propanoyl-L-alanyl-D-isoglutamine)-
l-thio-~-D-glucopyranose
Compound 16: 2-Acetamido-2-deoxy-1-S-hexadecanoyl-4,6-O-
isopropylidene-3-O-(D-2-propanoyl-L-alanyl-
D-isoglutamine)-l-thio-~-D-glucopyranose
Compound 17: 2-Acetamido-2-deoxy-1-S-hexadecanoyl-3-O-
(D-2-propanoyl-L.alanyl-D-isoglutamine)-l-
thio-~-D-glucopyranose
Compound 18: 2-Acetamido-2-deoxy-1-S-decanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methy].
ester)-l-thio-~-D-glucopyrano5e
Compound 19: 2-Acetamido-2-deoxy-1-S-octadecanoyl-3-O-
(D-2-propanoyl-L-alanyl-D-isoglutamine me~hyl
ester)-l-thio-~-D-glucopyranose
Compound 20: 2-Acetamido-2-deoxy-1-S-triacontanoyl-3-O-
(D-2-propanoyl-L-alanyl-D isoglutamine methyl
ester)-l-thio-~-D-glucopyranose
Compound 21: 2-Acetamido-2-deoxy-1-S-n-butanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-
l-thio-~-D-glucopyranose
.

~L2~40C~
Compound 220 2 Acetamido-2-deoxy-1-S-dodecanoyl-3-0-
(D-2-propanoyl-L-alanyl-D-isoglutamine)-l-
thio-~-D-glucopyranose.
Compound 23: 2-Acetamido-2-deoxy-1-S-tetradecanoyl-3-0-
(D-2-propanoyl-L-alanyl-D-isoglutamine) 1-
thio-~-D-glucopyranose
Compound 24: 2-Acetamido-2-deoxy-1-S-tetradecanoyl-3-0-
(D-2-propanoyl-L-alanyl-D-isoglutamine methyl
ester)-l-thio-~-D-glucopyranose.
Compound 25: 2-Acetamido-2-deoxy-1-S-eicosanoyl-3-0-(D-
2-propanoyl-L-alanyl-D-isoglutamine)-l-thio-
~-D-glucopyranose.
The compounds of the present invention can be
prepared according to the following reaction scheme.
- .
R5 X ~ C~2- rO ) R6 ~ ~
NHAcyl NHAcyl
CH3cHcooR7 ~ CH3cHcooR7
(II) (III)
- 10 -

12~
R5~<OC'~ ~ RSX~CH ~
NHAcyl NHAcyl
CH3CHCOOR7 CH3CHCOOX7
~IV) (V)
R OCH2
R~<o/~o ~ ~ R6>o~¢
NHAcy 1
I NHAcyl
CH3cHcooR7
CH 3CHCOOH
(VI) (VII)
> ( I )
I
I'

;3~0~
In the above reaction scheme, R5 represents a hydrogen
atom or an alkyl group having 1 to 6 carbon atom, and R6
and R7 each represents an alkyl group having 1 to 6
carbon atom.
That is, the compound (II) is oxidized in the
presence of a solvent, such as dichloromethane, using a
chromic anhydride-pyridine complex at a temperature of
about 0C to 60C to obtain the compound (III).
The compound (III) is reacted with a metal alcoholate in
an alcohol solvent to obtain the compound (IV). The
compound (IV) is dissolved in a solvent, such as a mixed
. solvent of dichloromethane and carbon tetrachloride, and
then reacted with hexamethylphosphorus triamide [P(NMe2)3]
at a low temperature, preferably -40C or lower, to obtain
the compound (V). The compound (V) is dissolved in a
solvent or a mixture of solvents, for example, a mixture
of dichloromethane and acetone, and reacted wi-th an alkali
thio-organic acid, for example, potassium thioacetate
(C~3COSK), to obtain the compound (VI)~ The compound (VI)
is reacted with an alkali hydroxide in the presence of
a solvent, such as dioxane, to hydrolyze the S-acetyl
group and alkoxycarbonyl group and then the hydrolysate
is reacted with an organic acid anhydride such as acetic
anhydride, and triethylamine to acylate the SH group
thereby obt.aining the compound (VII).
'~
- 12 -

L~Lo~i
The compound (VII) -thus obtained is then condensed
with an alanyl-isoglutamine alk~l ester to obtain the
desired compound of the formula (I) wherein Rl and R2 are
bonded together to form an alkylidène group; R3 is an acyl
group; and R4 is an alkyl group.
The above condensation reaction can be carried
out by methods commonly employed for peptide syntheses,
i.e., carbodiimide method, eintops method, active ester
method, acid anhydride method and the like. For example,
the compound (VII) is dissolved in a solvent such as
dioxane, and dicyclohexylcarbodiimide and N-hydroxysuccinic
acid imide or l-hydroxybenzotriazole are added to the
solution at about 0C to 60C to form an active ester of
the compound (~II). To the resulting reaction mixture is
added an L-alanyl-D-isoglutamine alkyl ester at about 0C
to 60C to obtain the above-described compound (I).
The S-acyl group of the compound (I) thus obtained
can be converted into a free SH group by reacting the
compound (I) with a metal alcoholate in an alcoholic
solution at about 0C to room tempera~ure, followed by
neutralization with a cation exchange resin, etc.
The thus obtained SH-compound may be converted into an S-
acylated compound by reacting with an acyl halide in a
solvent, such as dichloromethane, in the presence of an
acid acceptor such as pyridine, at about -10C to 50C.
- 13

$ L~ O~
Further, the S~alkylated compound can be obtained by
reacting the above S-acylated compound with a sodium alcoholate
in an alcohol and, without conducting neutralization, the
resulting product is then xeacted with an alkyl halide at
0 to 40C. The alkylidene group can be released by
reacting with a 50 - 90% aqueous solution of acetic acid
at room temperature to 100C. The conversion of the
alkoxycarbonyl group to a carboxyl group can be accomplished
by reacting with an aqueous alkali hydroxide solution in
an alcoholic solution at 0 to room temperature and then
neutralizing with a cation exchange resin, etc.
The present in~ention will now be described in
greater detail with reference to examples but the scope
of the invention is not limited thereto. Unless otherwise
indicated, a mixture of solvents is by volume.
Example
(1) 164.7 mg of chromic anhydride and 0.25 ml of
pyridine were added to 5 ml of dichloromethane and the
mixture was stirred for 15 minutes. To the resulting
mixture were added 180 mg of benzyl 2-acetamido-2-deoxy~
4,6-O-isopropylidine-3-~-[D-l-(methoxycarbonyl)ethyl] a-
D-glucopyranoside (compound II) dissolved in 3 ml of
dichloromethane and then 0.16 ml of acetic anhydride.
-The reaction mixture was stirred at 45C for
3 hours and passed through a column of silica gel (Wako~C-
r~
- 14 -
:"

~,z~
200, 20 g), and the column was eluted with ethyl acetate
to obtain 170 mg (yield: 92%) of 2-acetamido-1-o-benzoyl-
2-deoxy-4,6 O-isopropylidene 3-o-[D-l-(methoxycarbonyl)-
ethyl]-~-D-glucopyranose (compound III) having a melting
point of 134-137C.
[~]25 +144 (c=1.5, methanol)
Elemental Analysis for C22H29NOg:
Calcd. (%): C, 58.53; H, 6.47; N, 3.10
Found (%): C, 58.39; H, 6.58; N, 3.02
(2) To a solution of 4.0 g of the compound (III) in
60 ml of absolute methanol, about 30 mg of metai sodium
was added under cooling. After allowing to stand at room
temperature for 10 minutes, the mixture was treated with
Amberlite IRC-50. The solution was concentrated and the
resulting syrup was purified by column chromatography
using silica gel (50 g) (developing agent (a): chloroform,
developiny agent (b): chloroform-methanol = 50:1).
From the eEfluent (b), there was obtained 2.8 g ~yield: 93%)
of 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-~D-l-(methoxy-
carbonyl)ethyl]-D-glucopyranose (compound IV) having a
melting point of 180 - 184C.
[a]D5 -t44.3O (c=0.47, chloroform, equilibrium)
Elemental Analysis for C15H25NO8:
Calcd. (%): C, 51.86; H, 7.25; N, 4.03
; 25 Found (%): C, 51.81; H, 7.29; N, 3.96
-rf2 ,9 D ~ ~R ~ -
- 15 -

9~
(3) 2.5 g of the compound (IV) was dissolved in
a mixture of 50 ml of anhydrous dichloromethane and 2.17 g
of anhydrous carbon tetrachloride and the mixture was
cooled to -50C~ To the resulting mixture was added a
solution of 1.6 g of hexamethylphosphorus triamide in
25 ml of anhydrous dichloromethane over 15 minukes while
stirring at -50C, and the reaction was conducted for 1
hour. The reaction mixture was concentrated under reduced
pressure (bath temperature: 40C). At this stage, the
thin layer chromatography revealed the presence of a
mix-ture of ~- and ~-chlorides and oxazoline. By repeating
the above procedure of addition of anhydrous dichloromethane
followed by concentration three to four times, all the
chlorides were led to the a-chloro derivative. The a-
chloride was dissolved in a mixed solution of 15 ml ofanhydrous dichloromethane and 15 ml of anhydrous acetone,
a~d 2.4 g of potassium thioacetate was added thereto,
followed by stirring the mixture at room temperatLlre
overnight. After it was confirmed that all the chloride
disappeared by thin la~er chromatography, the reaction
; mLxture was conc~ntrated under reduced pressure, and the
residue was extracted with chloroform, washed with water,
and dried over sodium sulfate. The solvent was removed,
and the residue was crystallized from diethyl ether to
25 obtain 1.94 g (yield: 66.4%~ of 2-acetamido-1-S-acetyl-
- 16 ~
:,., "

~2~
2-deoxy-4,6-O-isopropylidene-3-O-[D-l-(methoxycarbonyl)-
ethyl]-l-thio-~-D-glucopyranose (compound VIj as a needle-
like crystal. The filtxate was purified by silica gel (30 g)
column chromatography (developing agent (a): chloroform;
(b): chloroform-methanol = 150:1). From the effluent (b)
was obtained 500 mg (yield: 17.1%) of the compound ~VI).
Total yield: 2.44 g (83.5~); melting point: 171 - 173C.
[~]D +8.1 (c=0.4, chloroform)
Elemental Analys~s for C17H27NO8S:
Calcd. (~): C, 50.36; H, 6.71; N, 3.45
Found (%): C, 50.28.; H, 6.59; N, 3.45
(4) 200 mg.of the compound (VI) was dissolved in
10 ml of 1,4-dioxane, and 12 ml of 0.2M potassium hydroxide
was added thereto. After allowing the mixture to stand
at room temperakure for 10 minutes, the mixture was treated
with Amberlite IRC-50 to remove the base. The resin was
separated by filtration and washed with methanol.
The filtrate and the washing were combined and concentrated
under reduced pressure. The resulting syrup was dissolved
2~ in methanol, and 1 ml of triethylamine and 0.8 ml of
anhydrous acetic acid were added to the solution under
cooling, followed by reacting at room temperature for 30
minutes. The reaction mixture was concentrated under
reduced pressure, and the resulting syrup was purified
by silica gel (20 g) column chromatography ~developing
.
- 17
..

~.2'~3 ?*~)~
agent (a): chloroform; (b): chloroform-methanol=50:1).
From the effluent (b), there was obtained 150 mg (yield: 78%)
of 2-acetamido-1-S acetyl-3-O-(D-l-carboxyethyl)-2
deoxy-4,6-O-isopropylidene-l-tllio-a-D-glucopyranose
(compound VII) as needle-like crystals. Recrystallization
from diethyl ether gave a product having a melti,ng point
of 193 - 200C (decomposition).
~a]D +10.5 (c=0.3, chloroform~
Elemental Analysis for C16H25NO8S:
Calcd. (~): C, 49.09; H, 6.44; N, 3.58
Found (%): C, 49.13; H, 6.51; N, 3.48
(5) 160 m~ of the compound (VII) was dissolved in
5 ml of anhydrous 1,4-dioxane, and 210 mg of dicyclohexyl-
carbodiimide and 100 mg of N-hydroxysuccinimide were added
to the solution, and the mixture was stirred at room
temperature for 30 minutes. The diphenylurea formed was
separated by filtration and washed with 2 ml of dioxane.
The filtrate and the washing were combined, and 200 mg of
L-alanyl-D-isoglutamine methyl ester trifluoroacetate
and 5 drops of triethylamine were added thereto while
stirring to effect a reaction at room temperature for
40 minutes. After completion of the reaction was confirmed
by thin layer chromatography, the reaction mixture was
concentrated under reduced pressure, and the resulting
syrup was purified by silica gel (20 g) column chromatograpy
- 18 -

(developing agent (a): chloroform; (b): chloroform-methanol=
100:1; (c): chloroform-metAanol=50:1). From the ef~luent
(c), there was obtained 235 mg (yield: 95%) of th~ desired
compounds, 2-acetamido-1-S-acetyl-2-deoxy-4,6-O-isopropylidene-
3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine methyl ester)-
l-thio-~-D-glucopyranose (compound I). Recrystallization
from diethyl ether gave a product having a melting point
of 148 - 151C.
~a]D +11 (c=0.2, chlorofoxm)
Elemental Analysis for C25H40N4OllS:
Calcd. (%): C, 49.6~; H, 6.67; N, 9.27
Found (%): C, 49.76; H, 6.65; N, 9.30
(6) 100 mg of the~compound (I) was dissolved in 5 ml
; of a 80% aqueous acetic acid solu-tion and heated at 45C
for 45 minutes while stirring. The reaction mixture was
concentrated under reduced pressure at below 40C. The acetic
acid was completely removed as an azeotropic mixture with
toluene to obtain 2-acetamido-1-S-acetyl~2-deoxy-3-O-
(D-2-propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-
~-D-glucopyranose (Compound 2) that showed a single spot
on the thin layer chromatogram. ~ecrys~allization from
diethyl ether gave a pure product having a melting point
of 158 - 162C.
-- 19 --

[o~]D5 +62 (c=0.2, chloroform-methanol=l:l)
Elemental Analysis for C22H36N4OllS:
Calcd. (%): C, 46.80; H, 6.43; N, 9.92
Found (%): C, 46.51; H, 6.65; N, 9.85
Example 2
20 mg of Compound 2 as prepared in Example 1-(6)
was dissolved in 2 ml of absolute methanol, and a small
amount of metal sodium was added thereto. Three minutes
later, disappearance of any unreacted substances was
10 conEirmed by thin layer chromatography, and the reaction
mixture was treated with Amberiite IR-120 (H ~ ollowed
by filtration. From the filtrate was obtained 17.6 mg
(yield: 95%) of 2-acetamido-2-deoxy-3-O (D-2-propanoyl-
L-alanyl-D-isoglutamine methyl ester)-l-thio-~-D-glucopyranose
(Compound 3) having a melting point of 117 - 125C
(decomposition).
[a]D5 ~24 (c=0.2, methanol, equilibrium)
Elemental Analysis for C20H34N4OloS
Calcd. (g6): C, 45.97; H, 6.56; N, 10.72
20 Found (~): C, 45.49, H, 6.53, N, 10.88
Example 3
25 mg of Compound 2 as prepared in Example 1-(6)
was dissolved in 2 ml of absolute methanol, and a small
amount of metal sodium was added thereto. Three minutes
,, ~ :
-- 20 --
: :, :",:

later, 1 ml of 0.2M potassium hydroxide was added to
the reaction mixture. After confirming completion of
the reaction by thin layer chromatography, the
reaction mixture was treated with Amberlite [R-120 (H )
to remove the base. The solution was concentrated under
reduced pressure and freeze-dried from 1,4-dioxane
to obtain 22.4 mg (quantitative yield) of 2-acetamido-
2-deoxy-3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine)-
l-thio-~-D-glucopyranose (Compound 4) having a melting
point of 157 -166C (decomposition).
[~]25 -~20 (c=0.2, methanol, e~uilibrium)
Elemental Analysis for ClgH32N4OloS:
Calcd. (%): C, 44.87; H, 6.34; N, 11.02
Found (~): C, 44-.45; H, 6.53; N, 10.99
Example 4
60 mg of Compound 1 as prepared in Example 1-(5)
was dissolved in 5 ml of absolute methanol, and 6 mg of
sodium mekhoxide was added thereto. Ninety minutes later,
a~ter it was confirmed by thin layer chromatoyraphy tha-t
the reactant had completely be converted to 2-acetamido-
l-S-sodium-2-deoxy-4,6-O-isopropylidene-3-O-(D-2-propanoyl-
L-alanyl-D~isogulutamine methyl ester)-1-thio-~-D-glucopyranose
(Compound 5), 15 mg of n-butyl bromide was added to the
reaction mixture, followed by stirring at room temperature
overnight. The reaction mixture was concentrated under
:
- 21 -
~ .

o~
reduced pressure, and the resulting syrup was purified
by silica gel (10 g) column chromatography (developing
agent (a): chloroform; (b): chloroform-methanol=50:1)
to obtain 60 mg (yield: 98%) of l-S-n-butyl 2 acetamido-
2-deoxy-4,6-O-isopropylidene-3-O-(D-2-propanoyl-L-alanyl-
D-isoglutamine methyl ester)-l-thio-~-D-glucopyranoside
(Compound 6) having a melting point of 75 - 78C.
[a]25 +25 ~c=0.5, methanol)
Elemental Analysis for C27H46N4OloS:
Calcd. (%): C, 52.41; H, 7.49; N, 9.06
Found (%): C, 52.25; H, 7.45; N, 9.01
~ Exampl`e 5
Compound 5 was prepared in the same manner as
in Example 4 but using 83 mg of Compound 1. 43 mg of
hexadecanyl bromide was added to the reaction mixture
containing Compound 5, and the resulting mixture was
stirred at room temperature overnight. The reaction
mixture was concentrated under reduced pressure and then
purified by silica gel (lO g) column chromatography
(developing agent (a): chloroform-methanol=100:1,
(b): chloroform-methanol=30:1). From the effluent
(b)~ there was obtained lO0 mg (yield: 93%) of l-S-
hexadecanyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-
(D-2-propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-
thio-3-D-glucopyranoside (Compound 7) having a melting
- 22 -

~.2~
point of 93 - 96C.
[a]D +35 (c=0.3, chloroform)
Elemental Analysis for C39H70N4OloS
Calcd. (%): C, 59.51; H; 8.97; N, 7.17
Found (%): C, 59.33; H, 8.86; N, 7.15
Example 6
50 mg of Compound 6 as prepared in Example 4
was dissolved in 3 ml of a 8% aqueous acetic acid solution,
and hydrolysis was carried out at 45C for 60 minutes.
After confirming by thin layer chromatography, the
reaction mixture was freeze-dried to quantitatively obtain
l-S-n-butyl 2-acetamido-2-deoxy-3-O-(D-2-propanoyl-L-
alanyl-D-isoglutamine methyl ester) l-thio-~-D-glucopyranoside
(Compound 8) having a melting point of 155 - 160C
(decomposition).
[a]D ~14 (c=0.3, methanol)
Elemental Analysis for C24H42N4OllS:
Calcd. (%): C, 49.81; H, 7.32; N, 9.68
Found (~): C, 49.45; H, 7.61; N, 9.55
Example 7
50 mg of Compound 7 as prepared in Example 5
was dissolved in 3 ml of a 80% aqueous acetic acid
solution and heated at 45C for 1 houx. After confirming
disappearance of any unreacted substances by thin layer
- 23 ~

chromatography, the reaction mixture was freeze-dried
to obtain l-S-hexadecanyl 2-acetamido-2-deoxy-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-
~-D-glucopyranoside (Compound 9) having a melting point
of 179 - 182C in a quantitative yield.
- [~]D5 +44 (c=0.2, chloroform-methanol=l:l)
Elemental Analysis for C36H66N4OloS:
Calcd. (~): C, 57.88; H, 8.91; N, 7.51
Found (%): C, 57.65; H, 8.96; N, 7.43
Example 8
20 mg of Compound 8 as prepa~ed in Example 6
was dissolved in 2 ml of methanol, and 2 ml of 0.2M
potassium hydxoxide was added thereto. Twenty minutes
later, the ester was completely hydrolyzed. The reaction
; 15 mixture was treated with Amberlite IR-120 (H ), the
resulting solution was concentrated under reduced pressure
and freeze-dried using 1,4-dioxane to quantitatively
obtain l-S-n-hutyl 2-acetamido-2-deoxy-3-O-(D-2-propanoyl-
L-alanyl-D-isoglutamine)-l-thio-~-D-glucopyranoside
(Compound lO) having a melting point of 108 - 112iC
(decomposition).
[~]D5 -15 (c=0.3, methanol)
Elemental Analysis for C23H40N4OllS:
Calcd. (~): C, 48.92; H, 7.14; N, 9.92
Found (%)~ C, 48.65; H, 7.49; N, 9.65
-
- 24 -
-:

Example 9
17 mg of Compound 9 as prepared in Example 7
was dissolved in a mixture of 3 ml of 1,4-dioxane and
l ml of methanol, and 2 ml of 0.2M potassium hydroxide
was added thereto~ Twenty minutes later, the methyl
ester was completely hydrolyzed. The reaction mixture
was treated with Amberlite IR-120 (H ), the resulting
solution was concentrated and ~reeze-dried from dioxane
to quantitatively give l-S hexadecanyl 2-acetamido-2-
deoxy-3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine)-l-
thio-~-D-glucopyranoside (Compound ll) having a melting
point of 110 - 115C (decomposition).
~]D +24 (c=U.15, methanol)
Elemental Analysis for C35H64N4OloS:
Calcd. (~): C, 57.35; H, 8.80; N, 7.64
Found (~): C, 57.09; H, 8.95; N, 7.55
Example lO
85 mg of Compound l as prepared in Example
1-(5) was dissolved in 5 ml o~ absolute methanol, and
lO mg of sodium methoxide was added thereto. After 1.5
hours, the reaction mixture was treated with Amberlite
IRC-50 (~t~ and then concentrated. The resulting syrup
was purified by silica gel (lO g) column chromatography
(developing agent (a): chloroform; (b): chloro~orm-
methanol=40:1). From the effluent (b) was obtained 76 mg
; (yield: 96~) of 2-acetamido-2-deoxy-4,6-O isopropylidene~
- 25 -
. :

3 0-(D-2-propanoyl-L-alanyl-D-isoglutamine methyl ester)-
l-thio-~-D-glucopyranose (Compound 12). Recrystallization
from diethyl ether gave a product having a melting point
of 105 - 107C (decomposition).
[a]D ~33 (c=0.5, chloroform)
Elemental Analysis for C23~38N401oS:
Calcd. (%): C, 49.10; H, 6.81; N, 9.96
Found (~): C, 49.Zl; H, 6.77; N, 9.83
Example 11
60 mg of Compound 12 as prepared in Example 10
was dissolved in a mixture of 2 ml of anhydrous dichloro-
methane and 1 ml of pyridine, and 1 ml of dichloromethane
containing 40 mg of hexa~decanoyl chlorlde was added
thereto under cooling. The reaction completed in 1 hour.
lS To the reaction mixture was added 1 ml of methanol, and
the mixture was concentrated under reduced pressure.
The resulting syrup was extracted with chloroform, washed
with water and dried over sodiurn sulfate. The chloroform
was distilled off, and the residue was purified by silica
gel -(10 g) column chromatography (developing agent
(a): chlO~Qform-methanol=loo:l; (b): chloroform-methanol=
50:1; (c): chloroform-methanol=30:1). From the effluent
(c), there was obtained 70 mg (yield: 82~) of 2-acetamido-
2-deoxy-1-S-hexadecanQyl-4,6-0-isopropylidene-3-0-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-
-
- 26 -
.,

~f~ 3~
~-D-glucopyranose (Compound 13) having a melting point
of 98 - 100C.
~a]D +30 ~c=0.2, chloroform)
Elemental Analysis for C H N O S:
Calcd. (%): C, 5~8.47; H, 8.56; N, 6.99
Found (%): C, 58.53; H, 8.63; N, 6.75
Example 12
50 mg of Compound 13 as prepared in ~xample 11
was dissolved in 3 ml of a 80% aqueous acetic acid
solution and heated at 45C for 1 hour to hydrolyze the
isopropylidene group. The reaction mixture was concen-
trated under reduced pressure and crystallized from
diethyl ether to give 43 mg (yield: 91%) of 2-acetamido~
2-deoxy-1-S-hexadecanoyl-3-O-~D-2-propanoyl-L-alanyl-
D-isoglutamine methyl ester)-l-thio-~D-glucopyranose
(Compound 14) having a melting point or 181 - 183C which
showed a single spot on thin layer chromatography.
[a~D5 '63 (c=0.2, chloroform-methanol~
Elemental Analysis for C36H6~N~OllS:
Calcd. (%): C, 56.82; H, 8.48; N, 7.36
Found (%): C, 56.77; H, 8.56; N, 7.35
Example 13
80 mg of Compound 1 as o~epared in Example 1 (5)
was dissolved in 5 ml of absolute methanol, and 10 mg of
sodium methoxide was added thereto. After the mixture
- 27 -

was allowed to stand a-t room temperature for 1 hour,
5 ml of 0.2M potassium hydroxide was added thereto.
Five minutes later, the base was removed by treating
with Amberlite IRC-50 (H ) resin, and the resin was washed
with methanol. The filtrate and the washing were combined
and concentrated under reduced pressure at a temperature
below 40C to obtain 2-acetamido-2-deoxy-~,6-0-
isopropylidene-3-G-(D-2-propanoyl ~-alanyl-D-isoglutamine)-
l-thio-~-D-glucopyranose (Compound 15). The product was
dissolved in a mixture of 2 ml of anhydrous dichloro-
methane and l ml of pyridine, and to the resulting solution
was added a solution of 45 mg of hexadecanoyl chloride
in l ml of anhydrous dichloromethane under ice-cooling,
followed by stirring for 1.5 hou~s. The excess of the
chloride was decomposed with methanol, and the de-
composition solution was concentrated under reduced
pres3ure. The residue was extracted with chloroform,
washed with water and dried over sodium sulfate.
The chloroform was distilled off, and the resulting
syrup was purified by silica yel (20 g) column chromato-
graphy (developing agent (a): chloroform-methanol-50:1;
(b) chloroform-methanol=10 1). From the effluent (b),
there was obtained 75 mg (yield: 72~) of 2-acetamido-2-
deoxy-1-S-hexadecanoyl-4,6~0-isopropylidene-3-0-(D-2-
- 28 -

propanoyl-L-alanyl-D-isoglutamine)-l-thio-~-D-glucopyranose
(Compound 16) having a melting point of 103 - 106C.
[~]D +42 (c=0.15, chloroform-methanol=l:l)
Elemental Analysis for C38H66N4OllS:
Calcd. (%): C, 57.99; H, 8.45; N, 7.12
Found (%) C, 57.65; H, 8.69; N, 7.05
Example 14
30 mg of Compound 16 as prepared in Example 13
was dissolved in 2 ml of a 80% aqueous asetic acid solution
and heated at 45C for 1 hour to completely hydrolyze the
isopropylidene group. After confirming that the thin
layer chromatography showed a single spot, the reaction
mixture was freeze-dried to obtain 28 mg of 2-acetamido-
2-deoxy-1 S-hexadecanoyl-3-O-(D-2-propanoyl-L-alanyl-D-
isoglutamine)-l-thio-~-D-glucopyranose (Compound 17) as
a colorless amorphous substance having a melting point
of 125 - 129C.
[a] ~ ~62 ~ 5 (caO ~ 2, chloroform-methanol=l:l)
Elemental Analysis for C35H62N4OllS:
Calcd. (~): C, 56.28; Ht 8.73; N, 7.50
Found (~): C~ 55.95, H, 8.51; N, 7.46
Example 15
To a solution of 85 mg of Compound 1 in 5 ml
of methanol was added 10 mg of sodium methoxide and the
mix-ture was kept for 1~ 5 hours at room temperature, and
!
- 2~ -

treated with Amberlite IRC-50 (H ) resin to remove the
base. The mixture was concentrated and the residue was
chromatographed on a column of silica gel (10 g) with
chloroform and 40:1 chloroform-met~anol. The latter
eluate gave 76 mg of 2-acetamido-2-deoxy-4,6-O-isopropylidene-
3-O-(D-2-propanoyl-L-alanyl-D-isoglutamine methyl ester)-
l-thio-~-D-glucopyranose as crystals having a melting point
of 105 - 107C (decomposition).
~a]25 _33o (c-0.5, chloroform)
Elemental Analysis for C23H38N4OloS
Calcd. (%): C, 49.10; H, 6.81; N, 9.96
Found . (~): C, 48089; H, 6.85; N, 9.83
To an lce-cooled solution of 80 mg of the above
compound in 1 ml of dry pyridine and 2 ml of dichloromethane
was dropwise added, with stirring, a solution of 52 mg of
decanoyl chloride in 1 ml of dichloromethane, and the
mixture wa~ stirred for 40 minutes at 5 to 10. 0.5 ml of
methanol was added to the mixture and the resulting mixture
was concentrated and then extracted with chloroform.
The extract was washed with water, dried over sodium
sulfa~e and concentrated to a syrup which was then chromato-
graphed on a column of silica gel (15 g) with (a) 150:1,
(b) 70:1, and (c) 30:1 chloroform ~ methanol. Eluant (a)
gave 65 mg of amorphous 2-acetamido-2-deoxy-1-S-decanoyl-
4,6-O-isopropylidene-3-O-(D-2-propanoyl-L-alanyl-D-
- 30 -

isoglutamine methyl ester)-l-thio-~-D-glucopyranose.
[~]D5 +6.6 (c=0.33, chloroform)
Elemental Analysis for C33H55N4OllS4
A solution of 50 mg of the above compound in
2 ml of 80 % aqueous acetic acid was heated for 2 hours
at 45C and the mixture was concentrated to give a crystalline
mass. Recrystallization from ether gave 2-acetamido-2-
; deoxy-l-S-decanoyl-3-O-(D-2-propanoyl-L-alanyl-D-iso~lutamine
methyl ester)-l-thio-~-D-glucopyranose ~Compound 18) having
a melting point of 176.5.
[a]D5 ~41.3 (c=0.44, 1:1 chloroform - methanol).
Elemental AnalysiS ~or C30H52N4llS
Calcd. (%): C, 53.23; H, 7.74; N, 8.28
Found (%)~ C, 53.31; H, 7.89; N, 8.33
Examples 16 and 17
___ _ ~ ____
The followiny compounds were prepared ln the same
manner as above.
2-Acetamido-2-deoxy-1-S-octadecanoyl-3-O-(D-2-
propanoyl-L-alanyl-D-isoglutamine methyl ester)-l-thio-~-
D glucopyranose (Compound 19) as an amorphous substance~
[~]D5 +29.0 (c=0.64, 1~1 chloroform - methanol)
Elemental Analysis for C38H68N4OllS
Calcd. (~): C, 57.84; H, 8.69; N, 7.10
Found (%): C, 57.55; H, 8.73; N, 7.01
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2-Acetamido-2-deoxy-1-S-triacontanoyl-3-O-(D-2-
propanoyl~L-alanyl-D-isoglutamine methyl ester)-l--thio-~-
D-glucopyranose (Compound 20).
m.p. 182, [a]25 +83.3 (c=0.6, l:l'chloroform - methanol).
Elemental Analysis for C50H92N4OllS
Calcd. (%): C, 62.73; H, 9.69; N, 5.85
Found (%): C, 62.65; H, 9.83; N, 5~81
While the invention has been described in detail
and with reference to specific embodiments thereof, it
will be apparent to one skilled in the art that various
changes and modifications can be made therein without
departing from the spirit and scope thereof.
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