Modulation of cytokine production by some phthalimido-desmuramyl dipeptides and their cytotoxicity

Article abstract of DOI:10.1016/j.farmac.2004.01.003

Muramyl dipeptide (MDP) is the smallest bacterial cell wall peptidoglycan component having immunomodulatory activity. In an attempt to obtain MDP derivatives with improved and better defined pharmacological profiles we synthesized a new lipophilic phthali

Full text of DOI:10.1016/j.farmac.2004.01.003

IL FARMACO 59 (2004) 345–352
www.elsevier.com/locate/farmac
Modulation of cytokine production by some phthalimido-desmuramyl
dipeptides and their cytotoxicity
Stanislav Gobec a,*, Marija Sollner-Dolenc a, Uroš Urleb a,b, Branka Wraber c,
Saša Simcˇicˇ c, Metka Filipicˇ d
a Faculty of Pharmacy, University of Ljubljana, Aškercˇeva 7, 1000 Ljubljana, Slovenia
b Lek d.d., Verovškova 57, 1000 Ljubljana, Slovenia
c Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia
d Department of Genetic Toxicology and Tumor Biology, National Institute of Biology, Vecˇna pot 111, 1000 Ljubljana, Slovenia
Received 12 December 2003; accepted 12 January 2004
Abstract
Muramyl dipeptide (MDP) is the smallest bacterial cell wall peptidoglycan component having immunomodulatory activity. In an attempt
to obtain MDP derivatives with improved and better defined pharmacological profiles we synthesized a new lipophilic phthalimido-
desmuramyl dipeptide, LK 508. This novel MDP analogue and three structurally related phthalimido-desmuramyl dipeptides (LK 413, LK
511 and LK 512) were evaluated immunologically. Their ability to modulate the production of cytokines was measured in vitro by their
inclusion in cultures of human peripheral blood mononuclear cells (PBMC) activated by ionomycin and phorbol-12-myristate-13-acetate
(PMA). The results were compared with the analogous activity of MDP. All compounds tested are strong up-regulators of IL-12 synthesis. All
compounds except LK 512 also stimulated IFNc synthesis. LK 508, LK 511 and LK 512 are effective in up-regulating IL-2 production. LK
508 and LK 512 considerably up-regulate the synthesis of IL-4 and IL-10. LK 413 and MDP stimulated the production of Th1 promoting and
Th1 (IFNc and IL-12) cytokines, while LK 508, LK 511 and LK 512 non-selectively up-regulated the production of both Th1 and Th2-types
of (IL-4 and IL-10) cytokines. None of the phthalimido-desmuramyl dipeptides was cytotoxic in vitro against the normal cell line HUVEC
(human endothelial cells) thereby indicating their potential for use in vivo.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Muramyl dipeptide analogues; Phthalimido-desmuramyldipeptides; Synthesis; Cytokines; Cytotoxic activity
1. Introduction
[5]. Recently, a
lipophilic MDP derivative N2-[N-
(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-
lysine (romurtide) was marketed for the treatment of
radiotherapy-induced leukopenia [6,7]. Most of the MDP
analogues known so far possess an intact dipeptide moiety,
L-Ala-D-Glu-NH2 or L-Ala-D-Glu. On the other hand, it has
been generally accepted that the N-acetyl-D-glucosamine
fragment is not essential for the immunomodulating activity
of this class of compounds [5,8-10]. Its derivatization or
replacement with various acyl groups is thus an important
approach in the design and synthesis of new immunologi-
cally active MDP analogues, as demonstrated by FK-156
[11], pimelautide [12], 7-(oxoacyl)-L-alanyl-D-iso-
glutamines [13], some carbocyclic MDP analogues [5,14],
and by the adamantyl-desmuramyl dipeptides [15].
N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyl
dipeptide, MDP, Figure 1) has been known since 1974 as the
minimal fragment of the Mycobacterium cell wall which still
retains most of the biological activity, in particular the immu-
noadjuvant activity [1]. MDP also induces side effects, such
as pyrogenic and somnogenic activity, induction of autoim-
mune responses, and inflammatory reactions [2-4]. In order
to obtain molecules with improved and more specific phar-
macological profiles, many MDP derivatives and analogues
have been designed, synthesized and evaluated biologically
In the search for a suitable N-acetylmuramyl surrogate we
focused our attention on phthalimido substituted alkanoic
* Corresponding author.
E-mail address: gobecs@ffa.uni-lj.si (S. Gobec).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.003

346
S. Gobec et al. / IL FARMACO 59 (2004) 345–352
OH
O
O
HO
N
O
OH
NHCOCH3
O
O
O
O
O
R
CH3
O
CH3
H
O
N
O
CH3
H
N
D
D
N
H
N
H
N
N
H
L
OH
L
OH
O
O
O
O
O
O
HO
NH2
LK 423
MDP
thalidomide
O
O
O
O
N
N
N
N
O
O
O
O
O
H
N
O
H
D
N
CH3
L
O
CH3
D
O
O
H
N
O
H
N
CH3
L
O
P
CH3
O
P
R,S
Me
D
OEt
OEt
N
H
L,D
N
H
D
N
H
N
H
OH
OH
OH
L
O
O
O
O
O
O
O
O
H2N
H2N
HO
H2N
LK 413
LK 512
LK 508
LK 511
Fig. 1.
acids. The phthalimide containing sedative thalidomide (N-
phthaloglutarimide, Figure 1) was shown to have immuno-
suppressive activity [16,17]. It also reduces agonist-induced
tumour necrosis factor (TNF) production by accelerating the
decay of TNF mRNA [18]. Further investigations have re-
vealed that thalidomide can both enhance and inhibit TNF
production, depending on the cell type and the TNF produc-
tion inducer [19,20]. Certain other compounds bearing a
substituted phthalimide fragment can also influence the im-
mune system. Immunosupressive activities were observed
for the N-phthaloylated derivative of 2-amino-4-chloro-
phenylthialzole-5-acetic acid [21] and phthalimido substi-
tuted 1-aziridinecarboxylic acid derivatives [22]. We postu-
lated that connecting the phthalimide ring to the essential
dipeptide part of MDP (L-Ala-D-Glu-NH2 or its bioisos-
teres) could produce new immunomodulators with improved
pharmacological properties [23]. The most promising com-
pound in the first series of phthalimido-desmuramyl dipep-
tides was LK 423 (Figure 1), which exhibited very interesting
activities in different immunological models. It was found to
augment the capacity to produce interleukin-10 in the spleen
cells of cyclophosphamide-treated mice [24], and it allevi-
ated dextran sulfate sodium-induced colitis in rodents [25].
LK 423 is thus a candidate substance for development as an
anti-inflammatory pharmaceutical agent [25]. The com-
pound was also able to stimulate the production of TNF in in
vitro phorbol 12-myristate 13-acetate (PMA) and
ionomycin-stimulated cultures of human peripheral blood
mononuclear cells [26]. To obtain more information about
structure-activity relationships, we recently synthesized
some phthalimido-desmuramyl dipeptides related to LK 423,
where the lead compound was modified bioisosterically at
various positions (side chain, peptide bond, terminal car-
boxyl group) [27-30].
production of cytokines is a key event in the activation and
effector phases of innate and specific immunity and serves to
mediate and regulate these responses. So far, several experi-
mental models have addressed the influence of MDP and
some analogues on the production of cytokines [26,32-34].
In an effort to define further the effect of compounds of the
MDP series, we have determined the ability of four
phthalimido-desmuramyldipeptides, LK 413, LK 508, LK
511, and LK 512 (Figure 1), to modulate cytokine produc-
tion. IL-12, TNFa, IFNc, IL-2, IL-4, and IL-10 were assayed
in in vitro ionomycin and PMA polyclonally activated hu-
man peripheral blood mononuclear cell (PBMC) cultures.
Taking into account its profound immunomodulatory poten-
tial, MDP was used as a comparative substance.
Although MDP has low anticancer activity, some MDP
derivatives have proved to be interesting as potential antican-
cer agents. Acylation of MDP with mycolic acids [35], hy-
droxy fatty acids [36], and quinonylalkanoic acids [37] re-
sulted in compounds with antitumor activity. Dzierzbicka
and coworkers have recently synthesized conjugates of MDP
and nor-MDP with the strong anticancer agents acridine,
hydroxyacridine, and acridone and shown them to be both
immunologically active, and to exhibit potent in vitro cyto-
toxic activity against a panel of human tumour cell lines
[38,39]. Conjugates of MDP and nor-MDP with batracylin
were not cytotoxic, but some of them reduced proliferation of
Ab melanoma cells in vitro [40]. Recently, we demonstrated
weak cytotoxicity of a series of adamantyl-desmuramyl-
dipeptides [41]. In addition, many compounds with the ph-
thalimide motif were either cytotoxic against the growth of
human cancer cultured cell lines [42-45] or active in in vivo
antitumor assays [46].
In this paper we present the results of both in vitro immu-
nological evaluation (modulation of cytokine production)
and determination of cytotoxicity of a series of phthalimido-
desmuramyl dipeptides (Figure 1). The synthesis of one new
MDP analogue (LK 508) is also presented.
Immunological activities of muramyl dipeptides and re-
lated compounds appear to depend on the stimulation or
inhibition of the biosynthesis of many cytokines [31]. The

S. Gobec et al. / IL FARMACO 59 (2004) 345–352
347
2. Experimental
2.2.3. Benzyl
N-(5-phthalimidopentanoyl)-D-alanyl-D-isoglutaminate (3)
To a stirred solution of 477 mg (1.5 mmol) compound 2
and 409 mg (1.5 mmol) D-isoglutamine benzyl ester hydro-
chloride in 10 mL DMF, 495 mg (1,8 mmol) diphenyphos-
phoryl azide (DPPA) and 0,42 mL (3 mmol) triethylamine
were added at 0° C. The reaction mixture was stirred at 0° C
for 3 h and then at room temperature for 48 h. After the usual
work-up procedure (addition of 80 mL ethyl acetate and
extraction with 5 % citric acid (3 x 10 mL), saturated solution
of NaHCO3 (3 x 10 mL) and water (3 x 10 mL)), recrystalli-
zation from DMF/ether yielded 0.6 g (75 %) of pure com-
pound 3, m.p. 194-196° C. 1H-NMR (300 MHz, DMSO-d6):
d= 1.17 (d, 3H, J= 7.14 Hz, CH3), 1.40-1.60 (m, 4H, 2 CH2),
1.70-1.85 in 1.90-2.05 (2m, 1 H each, b- CH2-iGln), 2.14 (t,
2H, J= 6.8 Hz, CH2CONH), 2.34 (t, 2H, J= 7.5 Hz, CH2) 3.55
(t, 2H, J=7.5 Hz, CH2N), 4.15-4.25 (m, 2H, CH-Ala, CH-
iGln), 5.06 (s, 2H, CH2Ph), 7.04 and 7.27 (2s, 1H each, NH2),
7.30-7.40 (m, 5H, Ph), 7.80-7.90 (m, 5H, phthaloyl, NH),
8.03 (d, 1H, J= 6.8 Hz, NH) ppm. MS(FAB): 537 (M+H)+.
2.1. Chemistry
LK 413, LK 511 and LK 512 were synthesized as de-
scribed previously [23,28,29]. MDP was supplied by Sigma.
All reagents and solvents were of commercial grade and used
as such. Melting points were determined using a Reichert hot
stage microscope and are uncorrected. Optical rotation was
measured on a Perkin-Elmer 1241 MC polarimeter. Elemen-
tal C, H, N analyses were performed at the Faculty of Chem-
istry and Chemical Engineering, University of Ljubljana, on
a Perkin-Elmer elemental analyzer 240 C. IR spectra were
measured by a Perkin-Elmer FTIR 1600 instrument on KBr
pelleted samples. Mass spectra were obtained by a Micro-
mass AutospecQ mass spectrometer using FAB ionization.
NMR spectra were obtained on a Bruker Avance DPX
1
300 instrument. H NMR was done at 300.13 MHz with
tetramethylsilane as an internal standard.
2.2. Synthesis of LK 508
␣
20 = 15.6° (0.05; methanol). C28H32N4O7 (536.6) Calcd.
͓ ͔
D
C 62.68 H 6.01 N 10.44 Found C 62.25 H 6.07 N 10.57.
2.2.1. N-(5-Phthalimidopentanoyl)-D-alanine benzyl ester
(1)
2.2.4. N-(5-phthalimidopentanoyl)-D-alanyl-D-isoglutamine
(LK-508)
To a stirred solution of 0.741 g (3 mmol) 5-phthali-
midopentanoic acid [47] and 1,05 g (3 mmol) D-alanine
benzyl ester p-toluene sulfonate in 10 mL DMF, 1 g
(3,6 mmol) diphenyphosphoryl azide (DPPA) and 0,83 mL
(6 mmol) triethylamine were added at 0° C. The reaction
mixture was stirred at 0° C for 3 h and then at room tempera-
ture for 48 h. After the usual work-up procedure (addition of
80 mL ethyl acetate and extraction with 5 % citric acid (3 x
15 mL), saturated solution of NaHCO3 (3 x 15 mL) and water
(3 x 15 mL)), recrystallization from acetone yielded 0.82 g
(67 %) of pure compound 1. M.p. 111° C. 1H-NMR
(300 MHz, DMSO-d6): d= 1.26 (d, 3H, J= 7.5 Hz, CH3),
1.40-1.65 (m, 4H, 2 CH2), 2.13 (t, 2H, J= 6.8 Hz,
CH2CONH), 3.55 (t, 2H, J=6.8 Hz, CH2N), 4.20-4.35 (m,
1H, CH), 5.09 (s, 2H, CH2Ph), 7.25-7.45 (m, 5H, Ph), 7.80-
90 mg (0.17 mmol) compound 3 was dissolved in 20 mL
glacial acid. The solution was then treated with 50 mg 10%
palladium/carbon and hydrogen for 45 min. The catalyst was
removed and the filtrate evaporated. The yield was 60 mg
(79 %). M.p. 200-203° C. 1H-NMR (300 MHz, DMSO-d6):
d= 1.18 (d, 3H, J= 7.16 Hz, CH3), 1.40-1.65 (m, 4H, 2 CH2),
1.65-1.85 in 1.85-2.00 (2m, 1H each, b-CH2-iGln), 2.10-
2.23 (m, 4H, 2 CH2) 3.58 (t, 2H, J= 6.8 Hz, CH2N), 4.05-4.30
(m, 2H, CH-Ala, CH-iGln), 7.00 and 7.23 (2s, 1H each,
NH2), 7.80-8.00 (m, 5H, phthaloyl, NH), 8.04 (d, 1H,
J= 6.8 Hz, NH) ppm. MS(FAB): 447 (M+H)+. ␣ 20 = 33.3°
͓ ͔
(0.3; methanol). C21H26N4O7 (446) Calcd. C 56.50DH 5.83 N
12.56 Found C 56.68 H 6.05 N 12.60.
7.95 (m, 4H, phthaloyl), 8.24 (d, 1H, J= 7.15 Hz, NH) ppm.
20
MS(FAB): 409 (M+H)+.
a
͓ ͔
= 24.9° (0.39; methanol).
2.3. Biological evaluation
C23H24N2O5 (408.4) Calcd. C D67.63 H 5.92 N 6.86 Found C
67.93 H 6.06 N 6.85.
2.3.1. In vitro cytotoxicity
Cytotoxicity of tested compounds was determined with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) according to Mosmann [48], with minor modi-
fications. This assay measures the conversion of MTT to
insoluble formazan by mitochondrial dehydrogenase en-
zymes of living cells. The in vitro cytotoxicity was evaluated
against three human tumour cell lines- MDA-MB231 (breast
cancer), 103H (large cell lung cancer) and HepG2 (human
hepatoma) and normal cell line HUVEC (human endothelial
cells).
2.2.2. N-(5-Phthalimidopentanoyl)-D-alanine (2)
0.6 g (1.47 mmol) compound 1 was dissolved in 25 mL
glacial acid. The solution was then treated with 60 mg 10%
palladium/carbon and hydrogen for 3 h. The catalyst was
removed and the filtrate evaporated. The yield was 406 mg
(87 %). M.p. 142° C. 1H-NMR (300 MHz, DMSO-d6):
d= 1.26 (d, 3H, J= 7.2 Hz, CH3), 1.45-1.70 (m, 4H, 2 CH2),
2.13 (t, 2H, J= 6.8 Hz, CH2CONH), 3.56 (t, 2H, J=6.8 Hz,
CH2N), 4.10-4.25 (m, 1H, CH), 5.09 (s, 2H, CH2Ph), 7.75-
7.95 (m, 4H, phthaloyl), 8.06 (d, 1H, J= 7.54 Hz, NH) ppm.
MDA-MB-231 cells were cultivated in L-15 (Sigma, St.
Louis, USA) medium, 103H cells in RPMI 1640 (Sigma, St.
Louis, USA), HepG2 cells in William’s medium E (Sigma,
St. Louis, USA) and HUVEC in EMEM with nonessential
20
MS(FAB): 319 (M+H)+.
␣
͓ ͔
=14.3° (0.37; methanol).
D
C16H18N2O5 (318) Calcd. C 60.37 H 5.70 N 8.80 Found C
59.96 H 5.69 N 8.70.

348
S. Gobec et al. / IL FARMACO 59 (2004) 345–352
amino acids (Sigma, St. Louis, USA). All the media were
supplemented with 10% foetal bovine serum, 2 mM
L-glutamine and 100 U/ml penicillin/streptomycin. The cells
were incubated 37°C in humidified atmosphere with 5%
CO2.
3. Results and discussion
3.1. Synthesis
The synthesis of LK 508 is presented in Scheme 1. Start-
ing from carboxyl-protected D-alanine and 5-phthalimi-
dopentanoic acid, N-(5-phthalimidopentanoyl)-D-alanine
benzyl ester (1) was obtained after activation of the carboxy-
lic group with diphenylphosphoryl azide (DPPA). Hydro-
genolysis of compound 1 gave N-(5-phthalimidopentanoyl)-
D-alanine (2) which was then coupled with D-isoglutamine
benzyl ester hydrochloride. The DPPA-coupling method was
very useful in both coupling reactions, giving satisfactory
yields and no undesired side products. Finally, the benzyl
protecting group of compound 3 was removed by catalytic
hydrogenation over Pd/C to give the target compound LK
508.
Cells were seeded in 200 µL of growth media in Corning
96-well microtiter plates at 25,000 cells per well (MDA-
MB231), 15,000 cells per well (103H) or 20,000 cells per
well (HepG2 and HUVEC). On the next day the medium was
replaced by complete growth medium containing 10-4 M test
compound and the cells incubated further for 24 hours (con-
trol cells were incubated in complete medium containing
0.1% DMSO). The cells were then washed twice with PBS
and the medium was replaced by fresh complete growth
medium containing MTT (final concentration 0.5 mg/ml).
After 3 h the medium was removed and the resulting forma-
zan crystals dissolved in DMSO. The absorbance (A) of each
well was measured at 570 nm (reference filter 690 nm) using
an ELISA microplate reader. Survival (viability) was deter-
mined by comparing absorbance in wells containing treated
cells with that of untreated cells. Six replicates were mea-
sured for each concentration.
3.2. Cytotoxicity
The in vitro cytotoxicity of phthalimido-desmuramyl-
dipeptides (LK 413, LK 508, LK 511 and LK 512) was
evaluated against three human tumour cell lines, MDA-MB
231 (breast cancer), 103H (large cell lung cancer) and Hep
G2 (hepatoma). 100 µM LK 413 reduced the viability of
103H cells by 21% and 100 µM LK 511 reduced the viability
of MDA-MB 231 and HepG2 cells by 19 and 15%, respec-
tively. To further evaluate the therapeutic potential and safety
of a series of phthalimido-desmuramyl dipeptides, the cyto-
toxicity against normal cell line HUVEC (human endothelial
cells) was determined. None of the compounds at concentra-
tion 100 µM reduced the viability of HUVEC cells, indicat-
ing that the compounds are not cytotoxic to non-tumour cells.
The modest selective cytotoxicity of LK 413 against tumour
cells 103H, and of LK 511 against MDA-MB 231 and HepG2
does not qualify this compound for consideration for antitu-
mour therapeutic use.
2.3.2. Modulation of cytokine production
The desmuramyldipeptide compounds, LK 413, LK 508,
LK 511 and LK 512, and MDP used for in vitro stimulation
of human PBMC were dissolved in dimethyl sulfoxide
(DMSO, Sigma, USA) and further diluted in RPMI 1640
(Sigma, USA) supplemented as described below, so that the
final concentration of DMSO did not exceed 0,1 %.
Human PBMC from buffy coat of two healthy blood
donors were isolated by density gradient centrifugation with
Ficoll-Paque (Pharmacia, Sweden). Cells were cultured in
RPMI 1640 supplemented with 100 U/mL penicillin (Sigma,
USA), 100 µg/mL streptomycin (Sigma, USA), 2 mM
L-glutamine (Sigma, USA), 20 mM Hepes (Sigma, USA)
and 10 % heat-inactivated AB normal human serum (Sigma,
USA) for TNFa production or 5% heat-inactivated foetal calf
serum (Sigma, USA) for the production of other cytokines.
1 x 106 cells (final culture volume 1,5 mL) were plated in
24-well culture plates (Nunc, Denmark) with either a combi-
nation of a test compound (final concentration in cell cultures
was 11 µM) with ionomycin (500 nM) and PMA
(3,33 ng/mL) or medium alone, at 37°C, and cell-free super-
natants were collected at 40 hours and stored at –70°C before
being evaluated for 6 cytokines.
The concentration of cytokines (pg/mL) was measured by
commercially available ELISA kits, TNFa from Innogenet-
ics (Belgium), and IL-2, IL-4, IL-10, IL-12 and IFNc from
Endogen (USA). The ability to modulate cytokine synthesis
by a test substance was evaluated as an index according to the
ionomycin and PMA induced cytokine production
(Stimulation/suppression index = [cytokine concentration in-
duced by ionomycin + PMA)/ (cytokine concentration in-
duced by ionomycin + PMA + test substance]). Cytokine
concentration indices below 0.8 and above 1.2 are considered
as suppression and stimulation indices respectively.) [26].
3.3. Modulation of cytokine production
The immunomodulating activity of test substances LK
413, LK 508, LK 511 and LK 512 was evaluated in terms of
their ability to modify IL-12, TNFa, IFNc, IL-2, IL-4, and
IL-10 synthesis, induced in human PBMC cultures by iono-
mycin and PMA. PBMC were isolated from buffy coat (do-
nor 1). Following ionomycin and PMA stimulation with the
test substance added, supernatants were screened for the
content of the 6 different cytokines. The levels of each cytok-
ine in turn obtained on incubating PBMC with ionomycin
and PMA in the presence and absence of the test substances
were compared. In addition, the response to MDP, a well
established immunomodulating substance, in the same sys-
tem, was measured with PBMC from another buffy coat
(donor 2) (data published previously [15]). In both experi-
ments the baseline production of cytokines was measured in

S. Gobec et al. / IL FARMACO 59 (2004) 345–352
349
O
CH3
COOH
SO3H . H2N
COOCH2Ph
H3C
N
D
O
a
CH3
O
COHN
COOCH2Ph
N
D
1
O
b
CH3
CONH2
O
COHN
COOH
HCl . NH2
COOCH2Ph
D
N
D
2
O
a
CH3
D
CONH2
O
CONH
COOCH2Ph
COHN
D
N
3
O
b
CONH2
CH3
O
CONH
COOH
COHN
D
N
D
LK-508
O
Scheme 1. a) DPPA, Et3N, DMF; b) H2, Pd/C(10 %), CH3COOH
non-stimulated PBMC cultures, with added RPMI 1640 only.
The cytokine concentrations are displayed in Table 1 and 2.
Stimulation/suppression indices were calculated (Table 3) to
evaluate the immunomodulating activity of the substances, as
described previously [26], and to take into account the differ-
ent cytokine concentrations produced in PBMC cultures
from healthy blood donors [49].
All substances tested are strong up-regulators of IL-12
synthesis (stimulation indices 1.74 to 1.98, Table 3). It is well
known that the production of IL-12 by macrophages is in-
duced by various bacteria, bacterial products, intracellular
pathogens, and viruses. A major biological effect of IL-12 is
regulation of adaptive immunity, especially a strong promo-
tion of Th1 responses [50,51]. IL-12 induces the production
of IFNc, which stimulates the microbial killing activity of
macrophages. IFNc also stimulates macrophages, NK cells
and CD8+ T cells to produce higher levels of IL-12, consti-
tuting a powerful positive feedback mechanism [50,51]. In
our experiment the up-regulation of IL-12 is followed by
increased IFNc synthesis when PBMC are incubated with
LK 413, LK 508, LK 511 and MDP. This is not the case with
LK 512, a phthalimido-desmuramyl dipeptide where MDP’s
N-acetylmuramic acid has been replaced by the more lipo-
philic 5-phthalimidopentanoyl fragment and D-isoglutamine
has been replaced by racemic 2-amino-4-(diethoxy-
phosphoryl)butanoic acid. Here the up-regulation of IL-12 is
not followed by increased IFNc production.
IFNc is a well-defined inducer of TNFa synthesis. In the
presence of LK 508, LK 511 and MDP, the synthesis of
TNFa was also stimulated. However, LK 413 shows a ten-
dency to downregulate TNFa secretion, without altering the
ionomycin/PMA-induced augmentation of IL-12 and IFNc
production. LK 413 is a lipophilic MDP analogue where the
L-Ala-D-Glu-NH2 dipeptide part is N-acylated with 5-phtha-
limidopentanoic acid. We observed similar cytokine induc-
ing properties in the case of the adamantane substituted
desmuramyl dipeptide LK 517, which also caused TNFa
suppression despite augmented IL-12 and IFNc production
[15].
LK 511 is a lipophilic phthalimido desmuramyldipeptide
derived from LK 413, where the amide bond between the
dipeptide part and the 5-phthalimidopentanoyl side chain has

350
S. Gobec et al. / IL FARMACO 59 (2004) 345–352
Table 1
Concentrations of cytokines (pg/ml) in culture supernatants of human PBMC activated by ionomycin and PMA in the presence and absence of LK 413, LK 508,
LK 511 and LK 512, together with the baseline production of cytokines in non-stimulated culture (donor 1)
Cytokine Non-stimulated culture Ionomycin/PMA
(RPMI 1640)
Ionomycin/PMA +
LK 413
159
Ionomycin/PMA +
LK 508
161
Ionomycin/PMA +
LK 511
177
Ionomycin/PMA + LK
512
IL-12
TNFaa
IFNc
IL-2
10.7
93.9
3220
2116
1449
15.7
39.6
155
<3.00
<1.00
<1.00
<1.00
3.34
1865
3456
1705
18.4
2554
3389
2162
21.6
4042
3406
2270
22.3
1908
2418
1911
17.3
45.6
IL-4
IL-10
47.0
60.2
65.7
a From reference [26].
Table 2
Concentrations of cytokines (pg/ml) in culture supernatants of human PBMC activated by ionomycin and PMA in the presence and absence of MDP, together
with the baseline production of cytokines in non-stimulated culture (donor 2) [15]
Cytokine
IL-12
TNFa
IFNc
Non-stimulated culture (RPMI 1640)
Ionomycin/PMA
117
Ionomycin/PMA + MDP
1.16
207
5.90
2116
2838
15255
9965
14.4
59.9
<1.00
<1.00
5.56
10940
10480
16.4
IL-2
IL-4
IL-10
7.00
62.0
Table 3
Immunomodulating effects of LK 413, LK 508, LK 511 and LK 512 compared to MDP on cytokine production in ionomycin and PMA activated human PBMC
cultures. Results are expressed in stimulation/suppression indicesa
Cytokine
Stimulation/supression indices
Ionomycin/PMA + LK
Ionomycin/PMA + LK
Ionomycin/PMA+ LK
Ionomycin/PMA+ LK
Ionomycin/PMA+ MDP
413
508
511
512
IL-12
TNFa
IFNc
IL-2
1.78
0.90
1.63
1.18
1.17
1.19
1.80
1.24
1.60
1.49
1.38
1.52
1.98
1.96
1.60
1.57
1.42
1.66
1.74
0.93
1.14
1.32
1.10
1.15
1.77
1.34
1.39
0.95
0.85
0.97
IL-4
IL-10
a Stimulation/suppression index = (cytokine concentration induced by ionomycin + PMA)/ (cytokine concentration induced by ionomycin + PMA + test
substance). Cytokine concentration indices below 0.8 and above 1.2 are considered as suppression and stimulation indices respectively [26].
been replaced by phosphinamide isostere. In order to obtain
more data about structure-activity relationships we also pre-
pared LK 508, a diastereomer of LK 413, in which L-alanine
is replaced by D-alanine. Both LK 508 and LK 511 were
highly active in stimulating IL-4 and IL-10 production in
T-cells. The main physiological function of Th2 cytokine
IL-4 is as a regulator of IgE- and mast cell/eosinophil-
mediated immune reactions [51]. IL-10 is an inhibitor of both
innate and T cell-mediated specific immune inflammation
due to inhibition of cytokine production by macrophages and
the inhibition of the accessory function of macrophages in T
cell activation [51-53]. Although IL-10 is a well known
inhibitor of TNFa and IL-12 production, in the case of
compounds LK 508 and LK 511 high concentrations of
IL-10 did not inhibit the synthesis of TNFa and IL-12.
IL-2 is the major autocrine and paracrine growth factor for
T lymphocytes, which also stimulates NK cells and B cells
[51,53]. We found that the synthesis of IL-2 is not signifi-
cantly affected when PMBC are incubated with LK 413 or
MDP. On the other hand, other phthalimido-desmuramyl-
dipeptides tested were highly active in this compartment with
LK 511 being the most active (stimulation index 1.57). The
introduction of the phosphinamide moiety into phthalimido-
desmuramyldipeptide analogues thus resulted in compound
with the greatest stimulation of IL-2 production in T-cells.
Cytokines play a critical role in regulating the develop-
ment of naïve CD4+ T cells into either T helper 1 (Th1) cells
or Th2 cells. The former produce IFNc exclusively and
favour cell-mediated responses, macrophage activation, and
the production of opsonizing antibodies. The latter produce
IL-4, IL-5, IL-13 exclusively, together with a more pro-
nounced production of IL-10, and favour humoral immunity
and allergic responses [51-53]. From the cytokine
stimulation/suppression pattern exhibited by the compounds
investigated here, we can assume that LK 413 and MDP
could shift the immune system towards the production of Th1
and Th1 promoting cytokines (strong stimulation of IL-12
and IFNc production, weak stimulation or even no stimula-
tion of IL-4 and IL-10 production). Other phthalimido-
desmuramyldipeptides (LK 508, LK 511 and LK 512) non-

S. Gobec et al. / IL FARMACO 59 (2004) 345–352
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