N-{trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl}-L-alanyl-D- glutamic acid: A novel immunologically active carbocyclic muramyl dipeptide analogue

Article abstract of DOI:10.1021/jm970509d

A novel non-pyrogenic carbocyclic muramyl dipeptide (MDP) analogue, N- (trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl}-L-alanyl-D-glutamic acid, was obtained by replacement of the N-acetylmuramic acid part and the D- isoglutamine residue of the MDP mole

Full text of DOI:10.1021/jm970509d

530
J . Med. Chem. 1998, 41, 530-539
N-{tr a n s-2-[[2′-(Acetyla m in o)cycloh exyl]oxy]a cetyl}-L-a la n yl-D-glu ta m ic Acid : A
Novel Im m u n ologica lly Active Ca r bocyclic Mu r a m yl Dip ep tid e An a logu e
Danijel Kikelj,*,† Slavko Pecˇar,† Vladimir Kotnik,§ Anton Sˇtalc,‡ Branka Wraber-Herzog,§ Sasˇa Simcˇicˇ,§
Alojz Ihan,§ Lidija Klamfer,§ Lucˇka Povsˇicˇ,‡ Rok Grahek,‡ Elizabeta Suhadolc,† Mirjam Hocˇevar,†
Helmut Ho¨nig,∇ and Renate Rogi-Kohlenprath∇
Faculty of Pharmacy, University of Ljubljana, Asˇkercˇeva 7, 1000 Ljubljana, Slovenia, Research and Development Division,
Lek d.d., Celovsˇka 135, 1000 Ljubljana, Slovenia, Faculty of Medicine, Institute of Microbiology and Immunology,
Korytkova 2, 1105 Ljubljana, Slovenia, and Institute of Organic Chemistry, Technical University of Graz,
Stremayrgasse 16, A-8010 Graz, Austria
Received August 4, 1997
A novel non-pyrogenic carbocyclic muramyl dipeptide (MDP) analogue, N-{trans-2-[[2′-
(acetylamino)cyclohexyl]oxy]acetyl}-L-alanyl-D-glutamic acid, was obtained by replacement of
the N-acetylmuramic acid part and the D-isoglutamine residue of the MDP molecule by a trans-
2-[[2′-(acetylamino)cyclohexyl]oxy]acetyl moiety and D-glutamic acid, respectively. The title
compound was selected as a promising candidate for further evaluation among several related
analogues on the basis of an immunorestoration test in mice. This novel nor-MDP analogue
protects mice against the immunosuppressive effect of cyclophosphamide and increases the
nonspecific resistance of mice against fungal infection. It is an immunomodulator which
enhances the maturation of lymphocytes B to plasma cells and increases the activity of
lymphocytes B and lymphocytes T as well as that of macrophages but does not alter the number
of these cells.
In tr od u ction
are R-absolute configuration of the first amino acid and
S-absolute configuration of the C-terminal amino acid
of the dipeptide moiety, whereas it has been shown by
numerous examples that the sugar moiety can be
derivatized or replaced with simple acyl groups without
a significant change of immunological activity as exem-
plified by immunologically active lipophilic 6-O-acyl-
MDP derivatives,17-20 FK-156,21-24 pimelautide,25,26
7-(oxoacyl)-L-alanyl-D-isoglutamines,27 N-[2-(2-amino-
alkoxy)propanoyl]-L-alanyl-D-isoglutamine derivatives,28
and carbocyclic MDP analogues in which the polyhy-
droxy-substituted pyranose ring of D-glucosamine in
MDP was replaced by a more lipophilic cyclohexane
ring.29-32 Omission of a methyl group in the lactoyl
moiety of MDP produced nor-MDP which is less active
than MDP but is also less toxic.3 With replacement of
the R-carboxamido group in the D-isoglutamine residue
of MDP by a carboxyl group, the adjuvant and antiin-
fectious activities remain almost unchanged; however,
the resulting derivative is more water-soluble.16
N-(Acetylmuramyl)-L-alanyl-D-isoglutamine (muramyl
dipeptide, MDP, 1; Chart 1) was identified in 1974 as
the minimal immunologically active component of bac-
terial cell wall peptidoglycan.1,2 The main biological
activities of MDP and its derivatives are (a) adjuvant
activity, i.e., stimulation of antibody production when
injected with an antigen and induction of delayed-type
hypersensitivity; (b) stimulation of nonspecific resis-
tance against bacterial, viral, and parasitic infections
and against tumors; and (c) somnogenic activity.3-8
MDP also induces other, mostly undesirable, toxic
responses, such as pyrogenicity, induction of autoim-
mune response, and inflammatory reactions.7-9 The
immunomodulating activity of MDP is based preferen-
tially on stimulation of macrophages as well as on T and
B lymphocyte functions.3-6,8,10-13 Many research groups
have been concerned with the synthesis and immuno-
logical studies of derivatives of this highly active gly-
copeptide in order to obtain molecules with improved
and more defined pharmacological profiles.3,4 These
efforts resulted in the recent introduction of N2-[N-
(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-
L-lysine (romurtide, 2) for the treatment of leukopenia
due to radiotherapy.14,15 Whereas studies of the struc-
ture-activity relationships for this class of compounds
have revealed the importance of the dipeptide moiety
for immunomodulatory activity, it was found that the
intact N-acetyl-D-glucosamine moiety is not essential for
immunostimulant activity of MDP and analogues.3,4,16
Essential prerequisites for immunostimulatory activity
The first carbocyclic analogues of MDP, N-[D-2-
(cyclohexyloxy)propionyl]-L-alanyl-D-isoglutamine (3) and
two isomers of N-[2-[[2′-(acetylamino)-D-6′-hydroxycy-
clohexyl]oxy]propionyl]-L-alanyl-D-isoglutamine (4), were
synthesized and tested by Hasegawa et al. to clarify the
structural requirements in the carbohydrate moiety
which are necessary for adjuvant activity.29 These
compounds in which the carbohydrate moiety of MDP
was replaced with cyclohexanol derivatives were inac-
tive as adjuvants for the induction of delayed-type
hypersensitivity to azobenzenearsonate N-acetyl-L-ty-
rosine in guinea pigs.30 This finding led the authors
to the speculation that the carbohydrate moiety is
essential for the adjuvant activity of MDP and its
analogues.30 Barton et al. reported the synthesis of
† University of Ljubljana.
‡ Lek d.d.
§ Institute of Microbiology and Immunology.
∇ Technical University of Graz.
S0022-2623(97)00509-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/28/1998

Immunologically Active Carbocyclic Muramyl Analogue
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 531
Ch a r t 1
Sch em e 1a
(1′R,2′S,3′S,5′S,6′R)-N-[D-2-[[2′-(acetylamino)-3′,5′,6′-tri-
hydroxycyclohexyl]oxy]propionyl]-L-alanyl-D-isoglu-
tamine (5) and some derivatives thereof31 which exhib-
ited the common activity of MDP, especially the stimu-
lation of unspecific resistance against bacterial and viral
infections, liberation of colony-stimulating factors, in-
duction of IL-1 production in macrophages, and antitu-
mor activity.32
To clarify the importance of the N-acetylmuramyl
moiety of MDP for immunomodulatory activity, we
prepared and studied in different immunological models
novel carbocyclic MDP analogues of general structure
6, obtained by replacement of the N-acetylmuramic acid
part of the MDP molecule by a trans-2-[[2′-(acylamino)-
cyclohexyl]oxy]acyl moiety, and 21, resulting from fur-
a
(i) HCl‚L-Ala-D-Glu(OCH2Ph)2, DPPA, Et3N, DMF; (ii) H2, Pd/
C, MeOH.
of sugar hydroxyl groups for immunoadjuvant activity.
The target molecules comprising several asymmetric
centers in the acyl part were generally prepared as
mixtures of diastereomers and, in some cases, to study
the influence of chirality on their immunological activ-
ity, also in the form of pure diastereomers.
Resu lts a n d Discu ssion
Ch em istr y. N-{trans-2-[[2′-(Acetylamino)cyclohexyl]-
oxy]acetyl}-L-alanyl-D-glutamic acid (9) was obtained as
a 1:1 mixture of 1′R,2′R- and 1′S,2′S-diastereomers by
condensation of racemic trans-2-[[2′-(acetylamino)cyclo-
hexyl]oxy]acetic acid (7)33 with L-alanyl-D-glutamic acid
dibenzyl ester34 in dry N,N-dimethylformamide in the
presence of diphenyl phosphorazidate (DPPA)35-38 and
triethylamine followed by hydrogenolytic cleavage of the
benzyl-protecting groups in the resulting dipeptide 8
(Scheme 1). Pure diastereomers of 9, (1′R,2′R)-N-
{trans-2-[[2′-(acetylamino)cyclohexyl]oxy]acetyl}-L-ala-
nyl-D-glutamic acid (9a ) and (1′S,2′S)-N-{trans-2-[[2′-
(acetylamino)cyclohexyl]oxy]acetyl}-L-alanyl-D-glu-
tamic acid (9b) (Chart 2), as well as the respective
isoglutamine analogues (1′R,2′R)-N-{trans-2-[[2′-(acety-
lamino)cyclohexyl]oxy]acetyl}-L-alanyl-D-isoglutamine
(10a ) and (1′S,2′S)-N-{trans-2-[[2′-(acetylamino)cyclo-
hexyl]oxy]acetyl}-L-alanyl-D-isoglutamine (10b) (Chart
2) were prepared by analogy from enantiomers of
carboxylic acid 7 or by chromatographic separation of
the corresponding diastereomeric mixtures of benzyl-
protected dipeptides as described by us previously.39a
Four diastereomers of N-{trans-2-[[2′-(acetylamino)-
cyclohexyl]oxy]propionyl}-L-alanyl-D-isoglutamine (11a-
d ) (Chart 2) were obtained similarly from respective
ther conformational restriction of the parent carbocyclic
analogue N-{trans-2-[[2′-(acetylamino)cyclohexyl]oxy]-
acetyl}-L-alanyl-D-glutamic acid (9) by incorporation of
the lactoyl fragment and acetamido group into a cyclo-
hexane-fused 1,4-morpholin-3-one ring. In the target
compounds the trans orientation of the two substituents
bound to a cyclohexane ring is similar to the orientation
of the acetamido group and D-lactic acid moiety with
respect to the glucopyranose ring in MDP. The replace-
ment of a polyhydroxy-substituted pyranose ring of
D-glucosamine in MDP by a more lipophilic cyclohexane
ring should provide information about the importance

532 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Kikelj et al.
Ch a r t 2
Sch em e 2a
a
(i) HCl‚L-Ala-D-iGln(OCH2Ph), DPPA, Et3N, DMF; (ii) HCl (g), HOAc; (iii) CH3(CH2)nCOOH, DPPA, Et3N, DMF; (iv) H2, Pd/C, MeOH.
2R,1′R,2′R-, 2S,1′R,2′R-, 2R,1′S,2′S-, and 2S,1′S,2′S-
isomers of 2-[[2′-(acetylamino)cyclohexyl]oxy]propionic
acid.39b
trans-2-azidocyclohexanol (16)42 with diethyl 2-bromo-
2-methylmalonate using sodium hydride in anhydrous
dioxane as a base afforded trans-diethyl 2-[(2′-azidocy-
clohexyl)oxy]-2-methylmalonate (17) which was con-
verted to trans-ethyl 2-methyl-3-oxooctahydro-2H-1,4-
benzoxazine-2-carboxylate (18) on reduction of the azido
group followed by spontaneous cyclization. Alkaline
hydrolysis of 18 gave trans-2-methyl-3-oxooctahydro-
2H-1,4-benzoxazine-2-carboxylic acid (19) which was
coupled with dibenzyl L-alanyl-D-glutamate34 in the
presence of DPPA, and the resulting dipeptide deriva-
tive 20 was subsequently hydrogenolytically cleaved to
give the target compound 21.
Biologica l Da ta . 1. In Vivo Tests. Im m u n or es-
tor a tion Test in Mice. The ability of novel carbocyclic
MDP analogues to enhance nonspecific resistance to
experimental fungal infection in immunosuppressed
animals was examined in an immunorestoration test43
in order to select a promising compound for further
evaluation. It has already been reported that synthetic
analogues of MDP increase nonspecific resistance to
microbial infections mainly by exerting a priming effect
on phagocytic cells leading to an enhancement in
responses to the microbial challenge, such as microbi-
For the preparation of N-{trans-2-[[2′-(hexanoylami-
no)cyclohexyl]oxy]acetyl}-L-alanyl-D-isoglutamine (15a )
and N-{trans-2-[[2′-(dodecanoylamino)cyclohexyl]oxy]-
acetyl}-L-alanyl-D-isoglutamine (15b), a different syn-
thetic strategy was employed (Scheme 2). Thus, racemic
trans-2-[[2′-(tert-butyloxycarbonylamino)cyclohexyl]oxy]-
acetic acid (12)33 was coupled with the benzyl ester of
L-alanyl-D-isoglutamine40,41 in the presence of DPPA to
give benzyl N-{trans-2-[[2′-(tert-butyloxycarbonylami-
no)cyclohexyl]oxy]acetyl}-L-alanyl-D-isoglutaminate (13),
which upon deprotection with hydrogen chloride in
glacial acetic acid and condensation of the resulting
benzyl N-[trans-2-[(2′-aminocyclohexyl)oxy]acetyl]-L-ala-
nyl-D-isoglutaminate hydrochloride with hexanoic and
dodecanoic acid, respectively afforded benzyl-protected
dipeptides 14a ,b. Finally, hydrogenolysis of 14a ,b over
10% palladium on charcoal in methanol gave compounds
15a ,b, respectively, in almost quantitative yields.
The preparation of N-[(trans-2-methyl-3-oxooctahy-
dro-2H-1,4-benzoxazin-2-yl)carbonyl]-L-alanyl-D-glutam-
ic acid (21) is illustrated in Scheme 3. O-Alkylation of

Immunologically Active Carbocyclic Muramyl Analogue
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 533
Sch em e 3a
a
(i) Br(CH3)C(COOC2H5)2, NaH, dioxane; (ii) SnCl2‚2H2O, MeOH; (iii) 1 N NaOH, dioxane; (iv) HCl‚L-Ala-D-Glu(OCH2Ph)2, DPPA,
Et3N, DMF; (v) H2, Pd/C, MeOH.
Ta ble 1. Immunorestoration Test in Mice
survival increasea [dose (mg/kg)]
compd
9a
9b
10a
10b
15a
15b
21
configuration
3 × 0.1
3 × 1
3 × 10
other
formulab
C18H29N3O8
C18H29N3O8
C18H30N4O7
1′R,2′R
1′S,2′S
1′R,2′R
1′S,2′S
2/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
1/6
1/6
1/6
0/6
0/6
2/6
0/6
3/6
3/6
2/6
0/6
0/6
1/6
3/6
0/6
2/6
3/6c
2/6c
0/6c
1/6c
1/6d
3/6d
2/6c
4/6c
3/6f
C18H30N4O7‚1.5H2O
C22H38N4O7
C28H50N4O7
e
C18H27N3O8
MDP
azimexon
a
nactual/npotential, where nactual ) (survival number for a group treated with test substance and cyclophosphamide) - (survival number
for cyclophosphamide-treated group) and npotential ) (survival number for blank control group) - (survival number for cyclophosphamide-
treated group). Satisfactory microanalyses for C,H,N obtained ((0.40%) except where indicated. c Dose: 3 × 100 mg/kg. Dose: 3 × 30
b
d
mg/kg. e Calcd: C, 60.62; H, 9.09; N, 10.10. Found: C, 59.85; H, 9.17; N, 10.61. f The same value obtained in four independent experiments.
cidal activity and release of cytokines.47 In the immu-
norestoration test the carbocyclic MDP analogues were
tested in four different doses (0.1, 1.0, 10, and 30 or 100
mg/kg/day) in mice immunosuppressed with cyclophos-
phamide and infected with Candida albicans. Under
these circumstances a 70-90% mortality was observed
in the untreated immunosuppressed control group
within 10 days. In the vehicle-treated control group in
which the mice were not immunosuppressed with cy-
clophosphamide the mortality was 10-30% within 10
days. The immunorestorative activity was estimated
as the survival increase in groups treated with the
tested substances. Under the described experimental
conditions, in a group of 10 mice treated with a test
substance, the potential survival increase (npotential) in
all experiments was 6 animals (Table 1), and conse-
quently, the standard statistical analysis (chi-square
test) was unreliable. Therefore, the survival increase
of 3 animals in any group treated with a test substance,
which was also usually obtained by azimexon as the
positive control compound, was considered significant
and possibly a result of immunorestorant activity. The
results of the immunorestoration test for compounds
9a ,b, 10a ,b, 15a ,b, and 21 as well as azimexon43,50 and
MDP as reference compounds are presented in Table 1.
2′-Acetylamino analogues 9a ,b, 10a ,b, and 11a -d
which were available in diastereomerically pure forms
were tested as single diastereomers, whereas other
compounds were assessed as equimolar mixtures of
two diastereomers (15a ,b) or four diastereomers (21),
respectively. The 1′R,2′R-isomer of the D-glutamic acid
derivative 9 displayed significant immunorestorant
activity at doses of 10 and 100 mg/kg/day. Interestingly,
both diastereomers of the corresponding D-isoglutamine
derivative (10a ,b) were markedly less active, although
in the isoglutamine MDP analogues the adjuvant and
antiinfectious activities are generally higher as com-
pared to the glutamate series of analogues.16 All four
diastereomers of N-{trans-2-[[2′-(acetylamino)cyclohexy-
l]oxy]propionyl}-L-alanyl-D-isoglutamine (11a -d ) were
also inactive in the immunorestoration test at the dose
of 10 mg/kg/day (results not shown), thus supporting a
hypothesis that in this carbocyclic series of MDP
analogues D-glutamic acid is a prerequisite for immu-
nostimulating activity. Analogues with increased lipo-
philicity (15a ,b) displayed immunostimulation at higher
doses, and the immunostimulating activity was the most
expressed in dodecanoylamino derivative 15b where a
dose-dependent response was achieved. The results
obtained for a partially rigidified analogue (21) revealed
that rigidification in the muramyl part of 9 is not
beneficial for immunorestorative activity. In contrast
to the solution conformation of MDP and some ana-
logues,51,52,53 compound 21 with fixed orientation of the
acetamido carbonyl cannot form a â-turn by hydrogen
bonding between the oxygen atom of the acetamido
group and the amino group of the L-alanyl moiety. It
has been proposed that any structural change performed
in MDP modifying the â-turn can produce measurable

534 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Kikelj et al.
Ta ble 2. Effect of Compound 9 on the Number of Total Spleen Cells, B Cells, T Cells, and Peritoneal Macrophages (in Millions)
total spleen cells B cells T cells peritoneal macrophages
daily dose/
compd
MDP
mouse (µg)
X ( SEM
p
X ( SEM
p
X ( SEM
p
X ( SEM
p
25
12.4 ( 6.32
9 ( 5.66
10.6 ( 6.13
9.93 ( 6.02
8.14 ( 3.38
5.66 ( 3.31
11.42 ( 4.99
0.549
0.348
0.843
0.694
0.361
0.07
24.18 ( 4.69
20.94 ( 2.71
19.34 ( 6.05
20.5 ( 11.06
23.76 ( 10.21
18.42 ( 9.57
24.08 ( 7
0.979
0.358
0.231
0.48
0.945
0.234
13.64 ( 4.49
11.92 ( 1.67
13.32 ( 4.59
9.72 ( 2.57
12.88 ( 6.47
11.7 ( 9.98
14.05 ( 8.32
0.913
0.636
0.709
0.295
0.848
0.401
2.11 ( 0.64
3.67 ( 2.34
1.88 ( 1.09
0.92 ( 0.61
2.87 ( 2.03
2.58 ( 2.23
1.6 ( 2.2
0.47
2.5
0.25
25
0.076
0.923
0.114
0.418
0.976
9
2.5
0.25
control
A modified hemolytic plaque-forming cell assay48 for
the determination of maturation of lymphocytes B
revealed that B lymphocytes may be important target
cells for compound 9. A significant increase (p < 0.001)
of the number of plaques from 136.2 ( 17.0 (n ) 12) to
331.6 ( 25.3 (n ) 13) was observed at the dose of 1 µg
of 9/mouse. The stimulation of B cell maturation to
plasma cells by 9 was comparable to the effect of
MDP.27a
Ta ble 3. Immunomodulatory Effect of MDP and Compound 9
on Blast Transformation Ability of Lymphocytes, Stimulated in
Vitro with Concanavalin A
amount of [3H]thymidine incorporated
in lymphocyte DNAa
compd daily dose (µg)b
X ( SEM (n)
p
MDP
25
2.5
0.25
7460 ( 1980 (5)
12440 ( 3540 (5)
19840 ( 9400 (5)
25990 ( 7850 (5)
16830 ( 6610 (5)
10780 ( 2680 (5)
10130 ( 476 (5)
>0.05
>0.05
>0.05
<0.05
>0.05
>0.05
9
25
2.5
0.25
Studies of concanavalin A (Con A)-induced blast
transformation of lymphocytes led to the conclusion that
MDP did not affect the blastogenic activity of lympho-
cytes T, whereas compound 9 at the dose of 25 µg
increased this function by almost 150% (p < 0.05). This
effect was dose-dependent. In comparison with MDP,
compound 9 gave in this test a statistically significantly
greater (p < 0.05) immunomodulatory effect in lympho-
cytes T at the dose of 25 µg (Table 3).
control
a
cpm. b Said doses were administered to animals for 3 consecu-
tive days.
variation of the biological activity.53 Therefore, reduced
activity of the rigidified analogue 21 could be explained
by its different conformation in aqueous solution. From
the results obtained it can be concluded that compounds
9a ,b and 15b protect mice against the immunosuppres-
sive effect of cyclophosphamide. Because cyclophospha-
mide also impairs the function of lymphocytes44 and
accessory immune system cells45,46 the beneficial effect
of compounds 9 and 15 can result from their effect on
these cells. A possible direct antifungal activity of the
tested substances was excluded by in vitro testing of
compound 15b which was one of the most active
substances in the immunorestoration test against C.
albicans. The negative result of this test ruled out a
possible direct antimicrobial activity of this compound.
On the basis of results of the in vivo testing and
considering the synthetic availability of various tested
carbocyclic MDP analogues, we decided to investigate
in detail in several immunopharmacological models the
analogue 9 as a diastereomeric mixture since no sig-
nificant difference in the immunorestorative activity of
1′R,2′R- and 1′S,2′S-diastereomers could be established.
2. In Vivo/in Vitr o Tests. Neither the number of
splenic lymphocytes B and lymphocytes T nor the
number of peritoneal macrophages was statistically
significantly altered by either MDP or compound 9
(Table 2).
In studies of macrophage activation, the production
-
of O2 demonstrated an increased effectiveness of all
three doses of MDP by about 120% (p < 0.001), whereas
compound 9 increased this activity at the dose of 2.5 µg
by 55% (p < 0.06) and at the dose of 25 µg by 70% (p <
0.005). For compound 9 the effects were dose-dependent
(Table 4). Similar results were found after additional
macrophage activation with phorbol myristate acetate
(PMA). In this case muramyl dipeptide at all doses
increased the activity by about 150% (p < 0.001),
whereas compound 9 increased the activity at the dose
of 2.5 µg by 50% (p < 0.05) and at the dose of 25 µg by
70%. Also in this case the effects were dose-dependent.
Determination of pyrogenicity of compound 9 accord-
ing to the method of USP XXII showed that in contrast
to MDP, compound 9 does not display any pyrogenic
activity. This result may suggest that in comparison
to MDP, the absence of pyrogenicity in 9 is probably a
result of a missing carbohydrate moiety. The average
lethal dose (LD50) for iv application of compound 9 in
male mice was found to be higher than 500 mg/kg.
Ta ble 4. Immunomodulatory Effect of MDP and Compound 9 Upon Macrophage Activation Without or With Costimulation with
PMA
difference in absorbancies of the test sample and blank per mg of cell protein
without costimulation with PMA
with costimulation with PMA
substance
MDP
daily dose (µg)a
X ( SEM (n)
p
X ( SEM (n)
p
25
578 ( 81 (5)
578 ( 42 (5)
614 ( 35 (5)
489 ( 97 (5)
431 ( 85 (5)
237 ( 94 (5)
275 ( 26 (5)
<0.01
<0.001
<0.001
<0.05
<0.06
>0.05
2138 ( 165 (5)
2207 ( 213 (5)
2657 ( 595 (5)
1709 ( 374 (5)
1494 ( 199 (5)
1298 ( 343 (5)
1004 ( 38 (5)
<0.001
<0.001
<0.05
<0.05
<0.05
>0.05
2.5
0.25
25
9
2.5
0.25
control
a
Said doses were administered to animals for 3 consecutive days.

Immunologically Active Carbocyclic Muramyl Analogue
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 535
N-{tr a n s-2-[[2′-(Acetyla m in o)cycloh exyl]oxy]a cetyl}-L-
a la n yl-D-glu ta m ic Acid (9). Dibenzyl ester 8 (1.18 g, 1.98
mmol) was dissolved in MeOH (20 mL) and hydrogenated over
10% Pd/C (160 mg) for 1 h at room temperature and normal
pressure. The catalyst was removed by filtration, and the
filtrate was evaporated in vacuo. There was obtained 820 mg
(99%) of 9 in the form of a white amorphous solid foam: IR
(KBr) 3340, 3085, 2940, 2900-2300 br, 1734, 1654, 1542, 1449,
1375, 1213, 1115, 970, 856, 670, 590 cm-1; UV (CH3OH) λmax
In conclusion, a carbocyclic nor-MDP analogue (9) in
which the N-acetylmuramyl moiety is replaced by a
trans-2-[[2′-(acetylamino)cyclohexyl]oxy]acetyl group and
D-isoglutamine is exchanged for D-glutamic acid retains
the immunostimulating properties of MDP.39c On the
basis of the performed immunobiological tests, we
conclude that compound 9 protects mice against the
immunosuppressive effect of cyclophosphamide and
increases the nonspecific resistance of mice against
fungal infection. Compound 9 is an immunomodulator
which enhances the maturation of lymphocytes B to
plasma cells and increases the activity of lymphocytes
B and lymphocytes T as well as that of macrophages
but does not alter the number of these cells. In the
tested range the effects are dose-dependent. It has been
already reported that as an immunomodulator com-
pound 9 also displays antitumor activity; when com-
bined with the tumor necrosis factor, it increases the
activity and decreases the side effects of the latter.49
Similar effects were also demonstrated for muramyl
dipeptide,3-8 but in contrast to MDP compound 9 is
neither pyrogenic nor toxic.
1
(log ꢀ) ) 202 nm (3.79); H NMR (DMSO-d6) δ 1.0-1.24 (m,
4H, 4Hax), 1.19 (d, 3H, J ) 6.9 Hz, CH3-Ala), 1.55-1.70 (m,
2H, 2Heq), 1.70-1.90 (m, 2H, 1Heq, H-âGlu), 1.80 (1.83) (s, 3H,
COCH3), 1.95-2.15 (m, 2H, 1Heq, H-âGlu), 2.24 (t, 2H, J )
7.1 Hz, CH2-γGlu), 3.10-3.20 (m, 1H, 1′-H), 3.50-3.70 (m, 1H,
2′-H), 3.87 (3.93) (AB system, 2H, J ) 15.3 Hz, OCH2), 4.18-
4.30 (m, 1H, CH-Glu), 4.35-4.42 (m, 1H, CH-Ala), 7.54 (7.56)
(d, 1H, J ) 7.8 Hz, NH), 7.94 (d, 1H, J ) 7.8 Hz, NH), 8.33
(8.35) (d, 1H, J ) 7.9 Hz, NH), 12.4 (s br, 2H, 2 COOH); MS
m/z 415 (M+, <0.01), 84 (100). Anal. (C18H29N3O8×0.5H2O)
C, H, N.
Ben zyl N-{tr a n s-2-[[2′-(ter t-Bu tyloxyca r bon yla m in o)-
cycloh exyl]oxy]a cet yl}-L-a la n yl-D-isoglu t a m in a t e (13).
Using standard procedure A, condensation of racemic carboxy-
lic acid 1233 (0.900 g, 3.29 mmol) and the hydrochloride of
L-alanyl-D-isoglutamine benzyl ester40,41 (1.131 g, 3.29 mmol)
afforded 13 as a solid white amorphous foam: yield 1.60 g
(86%); IR (KBr) 3700-3100 br, 3066, 2937, 2861, 1730, 1684,
1534, 1452, 1366, 1321, 1256, 1169, 1114, 1014, 698 cm-1; 1H
NMR (DMSO-d6) δ 1.00-1.50 (m, 4H, 4Hax), 1.23 (1.24) (d, 3H,
J ) 6.8 Hz, CH3-Ala), 1.36 (s, 9H, C(CH3)3), 1.50-1.68 (m, 2H,
2Heq), 1.70-1.90 (m, 2H, CH2-âiGln), 1.90-2.10 (m, 2H, 2Heq),
2.36 (t, 2H, J ) 7.6 Hz, CH2-γiGln), 3.30-3.32 (m, 2H, 1′-H,
2′-H), 3.93 (AB system, 2H, J ) 15.6 Hz, OCH2), 4.15-4.28
(m, 1H, CH-iGln), 4.28-4.40 (m, 1H, CH-Ala), 5.08 (s, 2H, CH2-
benzyl), 6.85 (6.93) (d, 1H, J ) 7.5 Hz, NH), 7.13 (s, 1H, NH),
7.22-7.50 (m, 6H, 5Harom, NH), 7.55 (7.61) (d, 1H, J ) 7.0 Hz,
NH), 8.23 (8.27) (d, 1H, J ) 8.2 Hz, NH). Anal. (C28H42N4O8)
C, H, N.
Ben zyl N-{tr a n s-2-[[2′-(H exa n oyla m in o)cycloh exyl]-
oxy]acetyl}-L-alan yl-D-isoglu tam in ate (14a). A slow stream
of dry hydrogen chloride gas was passed for 30 min through a
solution of 13 (1.51 g, 2.68 mmol) in glacial acetic acid (10 mL).
HOAc was removed in vacuo and the residue was triturated
with dry diethyl ether to give benzyl N-[trans-2-[(2′-aminocy-
clohexyl)oxy]acetyl]-L-alanyl-D-isoglutaminate hydrochloride
as a white amorphous hygroscopic solid which was used in the
next step without further purification: yield 1.20 g (90%); IR
(KBr) 3700-3100 br, 3052, 2928, 2857, 1730, 1655, 1540, 1451
cm-1; 1H NMR (DMSO-d6) δ 1.00-1.50 (m, 4H, 4Hax), 1.28 (d,
3H, J ) 6.89 Hz, CH3-Ala), 1.50-1.70 (m, 2H, 2Heq), 1.70-
1.90 (m, 1H, CH2-âiGln), 1.95-2.20 (m, 3H, 2H eq, CH2-âiGln),
2.37 (t, 2H, J ) 7.76 Hz, CH2-γiGln), 2.85-3.00 (m, 1H, 1′-H/
2′-H), 3.30-3.45 (m, 1H, 1′-H/ 2′-H), 4.00 (4.01) [AB system,
2H, J A,B ) 15.1 Hz (J AB ) 15.2 Hz), OCH2], 4.15-4.26 (m, 1H,
CH-iGln), 4.25-4.42 (m, 1H, CH-Ala), 5.09 (s, 2H, CH2-benzyl),
7.12 (s br, 1H, CONH2), 7.30-7.45 (m, 6H, 5Harom, CONH2),
8.21 (8.38) (d, 1H, J ) 7.13 Hz, NH), 8.28 (d, 1H, J ) 7.62 Hz,
NH), 8.37 (s br, 3H, NH3+). Anal. (C23H35ClN4O6) Calcd: C,
55.36; H, 7.07; N, 11.23. Found: C, 54.96; H, 7.10; N, 10.88.
Exp er im en ta l Section
Ch em istr y. All reagents and solvents were of commercial
grade and used as such unless specified. MDP was purchased
from Bachem, Switzerland. Melting points were determined
using a Reichert hot-stage microscope and are uncorrected.
IR spectra were recorded on a Perkin-Elmer FTIR 1600
instrument. NMR spectra were recorded on a Varian VXR-
300 spectrometer with tetramethylsilane as internal standard.
Mass spectra were recorded on a VG-Analytical Autospec Q
mass spectrometer. Microanalyses were carried out at the
Department of Organic Chemistry, Faculty of Chemistry and
Chemical Technology, Ljubljana, on a Perkin-Elmer elemental
analyzer 240 C. Optical rotations were determined on a
Perkin-Elmer polarimeter 1241 MC using a 1-dm cell. HPLC
analysis was performed on a Hewlet-Packard 1090 system with
a Licrospher 100 RP18 5-µm (150 × 4.6-mm) column. The
mobile phase was a linear gradient from 0.1% trifluoroacetic
acid in water to 60% acetonitrile and 0.1% trifluoroacetic acid
in water. For preparative purification a semipreparative
column, Microsorb C18 3 µm (50 × 22 mm), was used.
Dib en zyl N-{tr a n s-2-[[2′-(Acet yla m in o)cycloh exyl]-
oxy]a cetyl}-L-a la n yl-D-glu ta m a te (8). Sta n d a r d P r oce-
d u r e A: To a stirred solution of dibenzyl L-alanyl-D-glutamate
hydrochloride (869 mg, 2 mmol) and the acid 7 (430 mg, 2
mmol) in dry DMF (10 mL) were added at 0-2 °C DPPA (550
mg, 2 mmol) and Et3N (0.56 mL, 4 mmol). The mixture was
stirred for 1 h at 0-2 °C and then for 60 h at room
temperature. Ethyl acetate (40 mL) was added, and the
mixture was extracted successively with 10% citric acid (3 ×
5 mL), H2O (3 × 5 mL), saturated NaCl solution (3 × 5 mL),
saturated NaHCO3 solution (3 × 5 mL), H2O (3 × 5 mL), and
saturated NaCl solution (3 × 5 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated in
vacuo. The crude product was purified by column chromatog-
raphy on silica gel using CHCl3/MeOH (9/1) as eluent to give
8 as a colorless viscous oil: yield 1.0 g (84%); IR (film) 3285,
3066, 2934, 2858, 1744, 1649, 1548, 1446, 1374, 1271, 1169,
966, 753, 696, 578 cm-1; UV (CH3OH) λmax (log ꢀ) ) 202 nm
(4.42); 1H NMR (CDCl3) δ 1.00-1.38 (m, 4H, 4Hax), 1.38 (1.39)
(d, 3H, J ) 7.0 Hz, CH3-Ala), 1.60-1.80 (m, 2H, 2Heq), 1.90-
2.15 (m, 4H, 2Heq, CH2-âGlu), 1.95 (1.96) (s, 3H, COCH3), 2.41
(t, 2H, CH2-γGlu), 3.0-3.10 (m, 1H, 1′-H), 3.70-3.90 (m, 1H,
2′-H), 3.97 (3.99) (AB system, 2H, J ) 15.1 Hz, OCH2), 4.40-
4.63 (m, 2H, CH-Glu, CH-Ala), 5.09 (s, 2H, CH2-benzyl), 5.14
(s, 2H, CH2-benzyl), 6.15 (6.62) (d, 1H, J ) 8.0 Hz, NH), 7.20-
7.44 (m, 12H, 10Harom, 2NH); MS m/z 595 (M+, 6), 241 (100);
Anal. (C32H41N3O8) C, H, N.
Using standard procedure A, hexanoic acid (139 mg, 1.2
mmol) and benzyl N-[trans-2-[(2′-aminocyclohexyl)oxy]acetyl]-
L-alanyl-D-isoglutaminate hydrochloride (599 mg, 1.2 mmol)
were condensed to give 14a as a white solid amorphous foam:
yield 504 mg (75%); IR (KBr) 3700-3350 br, 3284, 2919, 2861,
1731, 1654, 1631, 1543, 1449, 1167, 1096 cm-1 1H NMR
;
(DMSO-d6) δ 0.78 (0.79) (t, 3H, J ) 6.64 Hz, CH3), 1.18 (d,
3H, J ) 7.03 Hz, CH3-Ala), 0.95-1.35 (m, 8H, 4CH2), 1.35-
1.85 (m, 6H, 3CH2), 1.85-2.10 (m, 4H, 2CH2), 2.32 (t, 2H, J )
7.69 Hz, CH2-γiGln), 3.02-3.16 (m, 1H, 1′-H/ 2′-H), 3.45-3.60
(m, 1H, 1′-H/ 2′-H), 3.86 (3.87) [AB system, 2H, J A,B ) 15.17
Hz (J AB ) 15.16 Hz), OCH2], 4.05-4.20 (m, 1H, CH-iGln),
4.20-4.32 (m, 1H, CH-Ala), 5.03 (s, 2H, CH2-benzyl), 7.08 (s
br, 1H, CONH2), 7.21-7.42 (m, 6H, 5Harom, CONH2), 7.52 (7.56)
[d, 1H, J ) 6.78 Hz (J ) 7.08 Hz), NH], 7.77 (7.78) [d, 1H, J

536 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Kikelj et al.
) 7.68 Hz (J ) 7.62 Hz), NH], 8.17 (8.21) [d, 1H, J ) 8.08 Hz
(J ) 8.11 Hz), NH]. Anal. (C29H44N4O7) C, H, N.
fraction which distilled at 120-150 °C/0.20-0.67 mbar was
further purified by column chromatography (silica gel, CHCl3/
MeOH ) 100/1) to give 6.72 g (28%) of 17 as a pale-yellow
viscous liquid: IR (film) 2980, 2940, 2860, 2110, 1740, 1450,
Ben zyl N-{tr a n s-2-[[2′-(Dod eca n oyla m in o)cycloh exyl]-
oxy]a cetyl}-L-a la n yl-D-isoglu ta m in a te (14b). Using stan-
dard procedure A, dodecanoic acid (220 mg, 1.1 mmol) and
benzyl N-[trans-2-[(2′-aminocyclohexyl)oxy]acetyl]-L-alanyl-D-
isoglutaminate hydrochloride (549 mg, 1.1 mmol) were con-
densed to give 14b as a white solid amorphous foam: yield
496 mg (70%); IR (KBr) 3700-3350 br, 3285, 2924, 2853, 1735,
1654, 1543, 1450, 1268, 1169 cm-1; 1H NMR (DMSO-d6) δ 0.83
(t, 3H, J ) 6.89 Hz, CH3), 1.00-1.35 (m, 23H, 10CH2, CH3-
Ala), 1.35-1.50 (m, 2H, CH2), 1.55-1.66 (m, 2H, CH2), 1.68-
1.85 (m, 2H, CH2), 1.90-2.15 (m, 4H, 2CH2), 2.35 (t, 2H, J )
7.74 Hz, CH2-γiGln), 3.05-3.18 (m, 1H, 1′-H/ 2′-H), 3.45-3.62
(m, 1H, 1′-H/ 2′-H), 3.89 (3.90) [AB system, 2H, J A,B ) 15.23
Hz (J A,B ) 15.25 Hz), OCH2], 4.12-4.22 (m, 1H, CH-iGln),
4.24-4.36 (m, 1H, CH-Ala), 5.07 (s, 2H, CH2-benzyl), 7.12 (s
br, 1H, CONH2), 7.27-7.41 (m, 6H, 5Harom, CONH2), 7.55 (7.59)
[d, 1H, J ) 7.08 Hz (J ) 7.03 Hz), NH], 7.79 (7.80) [d, 1H, J
) 7.87 Hz (J ) 7.81 Hz), NH], 8.19 (8.25) [d, 1H, J ) 8.06 Hz
(J ) 8.25 Hz), NH]. Anal. (C35H56N4O7) C, H, N.
N-{tr a n s-2-[[2′-(Hexan oylam in o)cycloh exyl]oxy]acetyl}-
L-a la n yl-D-isoglu ta m in e (15a ). Benzyl ester 14a (356 mg,
0.64 mmol) was hydrogenated in tetrahydrofuran (20 mL) over
10% Pd/C (54 mg) for 1 h at room temperature. The catalyst
was removed by filtration, and the filtrate was evaporated in
vacuo to give 258 mg (85%) of 15a as a white amorphous solid
foam: mp 177-181 °C; IR (KBr) 3436, 3295, 2931, 2861, 1725,
1649, 1543, 1449, 1120 cm-1; UV (CH3OH) λmax (log ꢀ) ) 203
nm (3.89), 270 nm (3.55); 1H NMR (DMSO-d6) δ 0.84 (t, 3H, J
) 6.93 Hz, CH3), 1.04-1.32 (m, 9H, 3CH2, CH3-Ala), 1.40-
1.86 (m, 8H, 4CH2), 1.88-2.12 (m, 4H, 2CH2), 2.21 (t, 2H, J )
7.69 Hz, CH2-γiGln), 3.08-3.20 (m, 1H, 1′-H/ 2′-H), 3.50-3.64
(m, 1H, 1′-H/ 2′-H), 3.91 (3.92) [AB system, 2H, J A,B ) 15.14
Hz (J A,B ) 15.4 Hz), OCH2], 4.12-4.24 (m, 1H, CH-iGln), 4.26-
4.40 (m, 1H, CH-Ala), 7.11 (s br, 1H, CONH2), 7.34 (s br, 1H,
CONH2), 7.56 (7.60) [d, 1H, J ) 7.03 Hz (J ) 7.08 Hz), NH],
7.81 (7.82) [d, 1H, J ) 7.57 Hz (J ) 7.57 Hz), NH], 8.20 (8.22)
[d, 1H, J ) 8.30 Hz (J ) 8.11 Hz), NH], 12.16 (s br, 1H,
COOH). Anal. (C22H38N4O7) C, H, N.
N-{tr a n s-2-[[2′-(Dod eca n oyla m in o)cycloh exyl]oxy]-
a cetyl}-L-a la n yl-D-isoglu ta m in e (15b). Benzyl ester 14b
(216 mg, 0.335 mmol) was hydrogenated in tetrahydrofuran
(15 mL) over 10% Pd/C (32 mg) for 1 h at room temperature.
The catalyst was removed by filtration, and the filtrate was
evaporated in vacuo to give 179 mg (96%) of 15b as a white
amorphous solid: mp 171-175 °C; IR (KBr) 3438, 3292, 2926,
2853, 1730, 1648, 1552, 1448, 1274, 1196, 1123 cm-1; UV (CH3-
OH) λmax (log ꢀ) ) 203 nm (3.91), 270 nm (3.38); 1H NMR
(DMSO-d6) δ 0.85 (t, 3H, J ) 6.62 Hz, CH3), 1.00-1.35 (m,
23H, 10CH2, CH3-Ala), 1.35-1.85 (m, 6H, 3CH2), 1.90-2.12
(m, 4H, 2CH2), 2.20 (t, 2H, J ) 7.76 Hz, CH2-γiGln), 3.12-
3.20 (m, 1H, 1′-H/2′-H), 3.45-3.65 (m, 1H, 1′-H/2′-H), 3.90
(3.92) [AB system, 2H, J A,B ) 15.16 Hz (J AB ) 15.35 Hz),
OCH2], 4.10-4.22 (m, 1H, CH-iGln), 4.24-4.38 (m, 1H, CH-
Ala), 7.11 (s br, 1H, CONH2), 7.34 (s br, 1H, CONH2), 7.56
(7.60) [d, 1H, J ) 7.08 Hz (J ) 6.89 Hz), NH], 7.80 (7.82) [d,
1H, J ) 7.43 Hz (J ) 7.57 Hz), NH], 8.19 (8.24) [d, 1H, J )
8.30 Hz (J ) 8.30 Hz), NH], 12.10 (s br, 1H, COOH). Anal.
(C28H50N4O7) Calcd: C, 60.62; H, 9.09; N, 10.10. Found: C,
59.85; H, 9.17; N, 10.61.
tr a n s-Dieth yl 2-[(2′-Azid ocycloh exyl)oxy]-2-m eth ylm a -
lon a te (17). trans-2-Azidocyclohexanol (10.6 g, 75 mmol) was
added dropwise at 70 °C to a magnetically stirred suspension
of NaH (1.8 g, 75 mmol) in anhydrous dioxane (75 mL) in a
three-necked flask equipped with a water-cooled reflux con-
denser. The reaction mixture was stirred at 70 °C for 1.5 h
whereupon diethyl 2-bromo-2-methylmalonate (19 g, 75 mmol)
was added dropwise, and the resulting mixture was stirred
for an additional 4 h at 70-80 °C. After evaporation of the
solvent, water (200 mL) was added and the resulting mixture
was extracted with ethyl acetate (5 × 50 mL). The combined
organic phases were dried over MgSO4, filtered, and evapo-
rated in vacuo. The crude product was distilled, and the
1
1375, 1270, 1150, 1115, 1060, 1025, 860 cm-1; H NMR (300
MHz, CDCl3) δ 1.289 (1.287) (t, 6H, J ) 7.1 Hz, 2COOCH2CH3),
1.20-1.50 (m, 4H, 4Hax), 1.68 (s, 3H, 2-CH3), 1.62-2.20 (m,
4H, 4Heq), 3.36-3.48 (m, 1H, 1′-H/ 2′-H), 3.54-3.68 (m, 1H,
1′-H/ 2′-H), 4.25 (4.24) (q, 4H, J ) 7.1 Hz, 2COOCH2CH3).
Anal. (C14H23N3O5) C, H, N.
tr a n s-Eth yl 2-Meth yl-3-oxoocta h yd r o-2H-1,4-ben zox-
a zin e-2-ca r boxyla te (18). To a stirred solution of SnCl2‚-
2H2O (4.06 g, 18 mmol) in methanol (12 mL) was added a
solution of 17 (3.76 g, 12 mmol) in methanol (12 mL) dropwise
at 0-5 °C. The mixture was stirred overnight at room
temperature, the solvent was evaporated, and the residue was
stirred for 0.25 h with saturated Na2CO3 solution (75 mL). The
mixture was extracted with diethyl ether (5 × 50 mL), and
the combined organic extracts were dried over MgSO4, filtered,
and evaporated in vacuo. The residue was recrystallized from
ethyl acetate to give 18 in the form of white crystals: yield
1.88 g (65%); mp 92-94 °C; IR (KBr) 3190, 3080, 2980, 2940,
2880, 1740, 1670, 1465, 1450, 1420, 1385, 1360, 1340, 1240,
1130, 1080, 1020, 965, 865, 810, 780, 730 cm-1; 1H NMR (300
MHz, CDCl3) δ 1.29 (t, 3H, J ) 7.1 Hz, COOCH2CH3), 1.20-
2.00 (m, 8H, 4CH2), 1.68 (s, 3H, 2-CH3), 3.22-3.42 (m, 2H,
4a-H, 8a-H), 4.23 (m, 2H, J ) 7.1 Hz, COOCH2CH3), 7.15 (s
br, 1H, NH); 13C NMR (75.44 MHz, CDCl3) δ 13.87, 20.21,
23.23, 24.13, 29.74, 30.33, 55.76, 61.91, 75.07, 81.46, 169.11,
169.34; MS m/z (EI) 241 (M+). Anal. (C12H19NO4) C, H, N.
tr a n s-2-Meth yl-3-oxoocta h yd r o-2H-1,4-ben zoxa zin e-2-
ca r boxylic Acid (19). A solution of 18 (4.80 g, 19.9 mmol)
and 1 N NaOH (20.7 mL, 20.7 mmol) in dioxane (80 mL) was
stirred for 20 h at room temperature. The solvent was
removed in vacuo, 1 N HCl (90 mL, 90 mmol) was added to
the residue, and the mixture was extracted with ethyl acetate
(4 × 60 mL). The organic phase was dried over MgSO4, the
solvent was evaporated, and the crude product was recrystal-
lized from ethyl acetate: yield 2.3 g (54%), white crystals; mp
142-144 °C; IR (KBr) 3280, 2940, 2870, 2500, 1930, 1730,
1630, 1460, 1450, 1420, 1380, 1350, 1315, 1300, 1275, 1180,
1165, 1150, 1115, 1080, 975, 930, 880, 850, 810, 760, 710 cm-1
;
1H NMR (300 MHz, CDCl3) δ 1.20-2.15 (m, 8H, 4CH2), 1.70
(1.68) (s, 3H, 2-CH3), 3.10-3.15 (3.15-3.20) (m, 1H, 4a-H/8a-
H), 3.50-3.62 (3.40-3.50) (m, 1H, 4a-H/8a-H), 7.65 (7.70) (s
br, 1H, NH), 11.2 (s br, 1H, COOH); 13C NMR (75.44 MHz,
CDCl3) δ 21.56, 23.36, 24.00, 29.60, 30.22, 56.40, 75.87, 80.11,
171.26, 171.75; MS m/z (EI) 213 (M+). Anal. (C10H15NO4) C,
H, N.
tr a n s-Diben zyl N-[(2-Meth yl-3-oxoocta h yd r o-2H-1,4-
ben zoxa zin -2-yl)ca r bon yl]-L-a la n yl-D-glu ta m a te (20). 20
was prepared from 19 (319 mg, 1.5 mmol) and dibenzyl
L-alanyl-D-glutamate hydrochloride34 (652 mg, 1.5 mmol) using
standard procedure A; yield 770 mg (87%) of crude 20. The
crude product was purified by column chromatography (silica
gel, CHCl3/MeOH ) 9:1) to give 720 mg (81%) of pure 20 as a
white amorphous foam: IR (film) 3289, 2937, 2864, 1737, 1676,
1508, 1454, 1376, 1168, 739, 698, 533 cm-1; 1H NMR (300 MHz,
DMSO-d6) δ 1.00-1.38 (m, 4H, 4Hax), 1.22 (1.23) (d, 3H, J )
6.8 Hz, CH3-Ala), 1.46 (1.47) (s, 3H, 2-CH3), 1.50-1.75 (m, 4H,
4Heq), 1.75-1.98 (m, 1H, CH2-âGlu), 1.98-2.16 (m, 1H, CH2-
âGlu), 2.36-2.48 (m, 2H, CH2-γGlu), 3.00-3.25 (m, 1H, 4a-
H/8a-H), 3.30-3.50 (m, 1H, 4a-H/8a-H), 4.26-4.44 (m, 2H, CH-
Glu, CH-Ala), 5.08 (s, 2H, CH2-benzyl), 5.13 (s, 2H, CH2-
benzyl), 7.28-7.44 (m, 10H, Harom), 7.65-8.50 (m, 3H, 3NH);
MS m/z (FAB) 594 (MH+). Anal. (C32H39N3O8) C, H, N.
tr a n s-N-[(2-Meth yl-3-oxooctah ydr o-2H-1,4-ben zoxazin -
2-yl)ca r bon yl]-L-a la n yl-D-glu ta m ic Acid (21). Hydrogena-
tion of dibenzyl ester 20 (700 mg, 1.18 mmol) over 10% Pd/C
(110 mg) in methanol (20 mL) for 1 h at normal pressure and
room temperature afforded 21 as a white amorphous solid:
yield 453 mg (93%); IR (KBr) 3318, 2843, 1737, 1668, 1521,
1
1450, 1375, 1216, 1169, 1110, 1069, 957, 634 cm-1; H NMR
(300 MHz, DMSO-d6) δ 1.00-1.40 (m, 7H, 4Hax, CH3-Ala), 1.44

Immunologically Active Carbocyclic Muramyl Analogue
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 537
(1.46) (s, 3H, 2-CH3), 1.50-2.10 (m, 6H, 4Heq, CH2-âGlu), 2.10-
2.22 (m, 2H, CH2-γGlu), 3.00-3.24 (m, 1H, 4a-H/8a-H), 3.24-
3.55 (m, 1H, 4a-H/8a-H), 4.26-4.40 (m, 2H, CH-Glu, CH-Ala),
7.62-8.50 (m, 3H, 3NH), 12.50 (s br, 2H, 2COOH). Anal.
(C18H27N3O8) Calcd: C, 52.29; H, 6.58; N, 10.16. Found: C,
51.83; H, 6.87; N, 9.99.
medium only. The cells were then incubated at 37 °C in the
presence of 5% CO2 and at 95% humidity for 2 days followed
by the addition of tritium-labeled thymidine (Amersham, Great
Britain). After 16 h the samples were prepared for measure-
ment in a â-counter (Beta Rach 1450, LKB Sweden). The
results are expressed as incorporation indexes with respect
to the control lymphocyte cultures.
P h a r m a cology. 1. In Vivo Tests. Im m u n or estor a tion
Test . For the immunorestoration test, ICR-derived female
mice, aged 6 weeks, 23-27 g in weight, were used. Animals
were housed in polycarbonate cages containing wood shavings
and covered with a stainless steel wire mesh. Groups of 10
mice were housed in cages measuring 45 cm in length by 23
cm in width and 15 cm in height. Animals were maintained
in a positive pressure room with air filtration, controlled
temperature (22-24 °C), controlled humidity (60-80%), and
a 12-h light-dark cycle for 1 week before and during the
experiments. The animals were allowed free access to stan-
dard laboratory chow and tap water. The test substance in
four different doses (0.1, 1, 10, and 30 or 100 mg/kg) or vehicle
(distilled water) alone was administered ip to groups of 10 mice
on days 1, 3, and 5 with cyclophosphamide (CP) (30 mg/kg,
po) administered on days 2, 4, and 6. One hour after the last
immunosuppressant dose, the mice were challenged with a
suspension of C. albicans sufficient to result in 70-90%
mortality within 10 days in the vehicle (distilled water)-treated
control group. In all experiments 1-[1-(2-cyano-1-aziridinyl)-
1-methylethyl]-2-aziridinecarboxamide (azimexon)43,50 was used
as a control immunostimulating compound. A survival of 3
mice more in any group treated with test substance and CP
than in the group treated with CP and distilled water was
considered significant and possibly a result of immunorestorant
activity.
2. In Vivo/in Vitr o Tests. The in vivo/in vitro tests were
performed on 3-4-week-old female HAN-NmRi strain mice,
weighing 17-20 g. Before initiation of the experiment mice
were veterinarily examined for any detectable disease. On the
day of the experiment onset, 1 mL of Brewer’s thioglycolate
medium was administered to the animals by an ip injection.
One hour later test substances were administered by the ip
injection of 0.5 mL of a solution containing 25, 2.5, and 0.25
µg of compound 9 or MDP, respectively. In the same way the
animals were treated on days 2 and 3. On day 4 the animals
were sacrificed. Spleen was removed, and the peritoneal
macrophages were washed out of the peritoneal cavity with
ice-cold medium (RPMI 1640; Gibco, Great Britain).
Deter m in a tion of Hem olytic P la qu es for th e Assess-
m en t of th e Ma tu r a tion of Lym p h ocytes B. A 1% suspen-
sion of sheep erythrocytes (Institute of Microbiology and
Immunology, Ljubljana, Slovenia) in physiological saline was
used for the ip immunization of mice. Individual mice were
administered 0.2 mL of erythrocyte suspension followed the
next day by 0.1 mL (1 µg/mouse) of the test substance. The
immunization was completed on the fifth day when mice were
sacrificed. Their spleens were removed and gently homog-
enized by squeezing between two sintered glass plates, and
cells were suspended in a Parker 199 medium with added
amino acids, streptomycin, and sodium carbonate. The lym-
phocytes were separated from other cells on Ficoll-Paque
separating medium (Pharmacia, Uppsala, Sweden). After
repeated rinsing with Parker 199 medium, the cells were
resuspended in RPMI 1640 nutrient medium supplemented
with 10% fetal calf serum and streptomycin (100 µg/mL). To
50 µL of the cell suspension was added 450 µL of the 1% trypan
blue solution, and the cells were counted in Neubauer’s
chamber. The number of lymphocytes/mL of the cell suspen-
sion was calculated. To 100 µL of the diluted cell suspension
were added 200 µL of RPMI 1640 nutrient medium, 50 µL of
a 10% suspension of sheep erythrocytes, and 50 µL of guinea
pig complement (Institute of Microbiology and Immunology,
Ljubljana, Slovenia). Mixture of cells (MC) was pipetted into
the testing chambers constructed on the glass slide. The
chambers were sealed with white wax and cells incubated at
37 °C for 60 min. After completion of the incubation, the
plaques were counted under a microscope. The number of
plaques/1 × 106 cells was calculated according to the following
equations:
no. of plaques ) (1 × 106 cells × no. of plaques/chamber)/
(A cells/chamber)
A cells/chamber ) [(amount of MC/chamber (µL)) ×
(no. of cells in MC)]/[amount of MC (µL)]
Activa tion of P er iton ea l Ma cr op h a ges. The peritoneal
cavity of the animals was rinsed with 4 mL of ice-cold RPMI
1640 medium. The erythrocytes were lysed with 0.2% NaCl
solution performing hypoosmotic shock for 1 min and followed
by immediate reconstitution of the osmolarity with 1.2% NaCl
solution. The remaining cells were washed twice in a cooled
centrifuge at 4 °C and 1500 rpm for 5 min with ice-cold RPMI
1640 medium. The cells were resuspended, and the concen-
tration was adjusted to 1.5 × 106/mL; 100 µL of the cell
suspension was distributed to the flat bottom wells of a
microtitration plate (Nunc, Denmark). After 2 h at 37 °C, 5%
CO2, and 95% humidity, the wells were rinsed with warm
RPMI 1640 medium and the adhering macrophages were used
for testing. The macrophages were covered either with 100
µL of a 160 µM ferricytochrome c solution in HBSS (Hank’s
balanced salt solution) without phenol red or with 100 µL of
160 µM ferricytochrome c and 200 nM phorbol myristate
acetate (PMA; Sigma, St. Louis, MO) solution or with 100 µL
of ferricytochrome c, PMA, and 340 U/mL superoxide dismu-
tase (SOD; Sigma, St. Louis, MO) which specifically inhibited
the reduction of cytochrome c with the superoxide ion (blank),
respectively. After the samples were incubated for 90 min at
37 °C, 5% CO2, and 95% humidity, the absorbance was
measured at a wavelength of 570 nm.
Deter m in a tion of th e Nu m ber of Lym p h ocytes B a n d
Lym p h ocytes T. For the determination of lymphocytes B,
membrane-bound immunoglobulin G molecules on isolated
splenic lymphocytes were marked with fluorescein isothiocy-
anate-labeled polyclonal goat anti-mouse Ig G antibodies (Sera-
lab, Great Britain). For the determination of lymphocytes T,
rat anti-Thy-1 monoclonal antibodies (Sera-lab, Great Britain)
were used as primary antibodies and FITC-labeled polyclonal
goat anti-rat Ig antibodies (Sera-lab, Great Britain) as the
second antibody. After incubation and rinsing with RPMI
1640 medium, the lymphocytes were counted by fluorescence
microscopy.
Deter m in a tion of th e Nu m ber of Ma cr op h a ges. The
number of macrophages in the peritoneal cavity washings was
determined after trypan blue addition to the counting medium
in Neubauer’s blood cells counting glass chamber. Contami-
nating cells were excluded by morphology.
Bla st Tr a n sfor m a tion of Lym p h ocytes by Mitogen s.27
Isolated spleen lymphocytes were prepared in the concentra-
tion of 1 × 106/mL in the nutrient RPMI 1640 medium (88
mL of RPMI 1640 supplemented with 10% fetal calf serum
(FCS) (Sera-Lab, Great Britain), 1 mL of 200 mM l-glutamine
solution, and 1 mL of an antibiotic solution (1×106 IU/mL
penicillin and 100 µg/mL streptomycin)). To each flat bottom
well of a microtitration plate (Nunc, Denmark) was distributed
100 µL of the cell suspension. The cells were stimulated in
vitro by adding concanavalin A (Con A) (Pharmacia, Sweden)
at respective concentrations of 16, 8, and 4 µg/mL. Control
lymphocyte cultures were grown in nutrient RPMI 1640
Ack n ow led gm en t. This study was partially sup-
ported by the Ministry of Science and Technology of
Slovenia. The antifungal tests against C. albicans were
performed at the Institute of Microbiology and Im-
munology of the University of Ljubljana. The authors

538 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
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