Biscarbamate analogues of the chemotactic tripeptide fMLF-OMe

Article abstract of DOI:10.1016/S0014-827X(00)00045-8

Based on the sequence of the prototypical chemotactic tripeptide HCO- Met-Leu-Phe-OH (fMLF) and by taking into account the versatility shown by its N-terminal carbamate analogues, the new biscarbamates MeOCO-Met-Leu-gPhe- COOMe (2) and Boc-Met-Leu-gPhe-CO

Full text of DOI:10.1016/S0014-827X(00)00045-8

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 308–313
Biscarbamate analogues of the chemotactic tripeptide fMLFꢀOMe
Giampiero Pagani Zecchini a, Mario Paglialunga Paradisi a, Ines Torrini a,
Marianna Nalli a, Gino Lucente a,*, Susanna Spisani b
a Centro di Studio per la Chimica del Farmaco del CNR, c/o Dipartimento di Studi Farmaceutici,
Uni6ersita` degli Studi di Roma ‘La Sapienza’, 00185 Rome, Italy
b Dipartimento di Biochimica e Biologia Molecolare, Uni6ersita` di Ferrara, 44100 Ferrara, Italy
Received 16 December 1999; accepted 8 March 2000
Abstract
Based on the sequence of the prototypical chemotactic tripeptide HCOꢀMetꢀLeuꢀPheꢀOH (fMLF) and by taking into account
the versatility shown by its N-terminal carbamate analogues, the new biscarbamates MeOCOꢀMetꢀLeuꢀgPheꢀCOOMe (2) and
BocꢀMetꢀLeuꢀgPheꢀCOOMe (4) were synthesized. These two new ligands are characterized by the presence of a gem-diamino
residue (gPhe) replacing the C-terminal Phe and a carbamate functionality positioned at both the ends of the molecule. The
activity of the two new compounds has been determined on human neutrophils and compared to that shown by the corresponding
N-terminal monocarbamates MeOCOꢀMetꢀLeuꢀPheꢀOMe (1) and BocꢀMetꢀLeuꢀPheꢀOMe (3). © 2000 Published by Elsevier
Science S.A. All rights reserved.
Keywords: Biscarbamates; Carbamates; Chemotactic peptides; Human neutrophils
is now well established that the N-formylation is not an
absolute requirement for potent agonist activity and
1. Introduction
that other N-terminal functionalities, different for size,
flexibility, and nature of electrostatic interactions, can
lead to derivatives capable of efficiently binding to the
receptor [8–10]. An interesting class of N-terminal
modified tripeptides, based on the MLF sequence, is
that of the N-alkoxycarbonyl derivatives (amino-termi-
nal carbamates) [6,11,12] recently studied in detail by
Higgins et al. [9]. This functionality maintains the re-
ceptor binding affinity and can impart agonist or antag-
onist activity to the tripeptide depending on the nature
of the alkyl group. In particular, it has been proven
that N-terminal carbamates with unbranched small
alkyl groups generate agonists whereas those with
branched and sterically demanding substituents result
in antagonist peptides. Furthermore, in addition to the
property of modulating the agonist versus antagonist
activity the N-terminal carbamate functionalization
leads to ligands which are significantly and specifically
influenced by the nature of the C-terminal modification
in a manner which is not encountered in the case of the
classic N-formyl–tripeptides. An example is repre-
sented by the relative potency of C-terminal methyl
Neutrophils are involved in host defense mechanisms
against bacterial infections and play a crucial role in
inflammatory processes. Their activation depends upon
different extracellular signals among which those medi-
ated by chemotactic N-formylated peptides of bacterial
origin [1] which are also related to N-terminal frag-
ments of specific mitochondrial proteins [2]. The
chemotactic peptides exert their effects by interacting
with specific receptors located on the neutrophil plasma
membrane [3–5]. The most intensively studied member
of the N-formylated chemotactic peptides is the tri-
peptide N-formyl-L-methionyl-L-leucyl-L-penylalanine
(forꢀMetꢀLeuꢀPhe; fMLF) together with its methyl
ester fMLFꢀOMe [6,7].
Based on the sequence of the prototypical model
fMLF a variety of chemical modifications have been
performed in order to obtain more potent and selective
ligands and to study structure–activity relationships. It
* Corresponding author. Tel.: +39-06-4454218; fax: +39-06-491-
491.
E-mail address: glucente@axrma.uniroma1.it (G. Lucente).
0014-827X/00/$ - see front matter © 2000 Published by Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00045-8

G. Pagani Zecchini et al. / Il Farmaco 55 (2000) 308–313
309
[13–16] and by taking into account the tendency of the
N-terminal carbamates to be highly influenced by the
nature of the C-terminal functionalization, it seemed
interesting to examine the activity of a new type of
tripeptide ligands possessing the carbamate functional-
ity both at the N-terminal and C-terminal position.
These biscarbamate analogues should represent efficient
ligands since the C-terminal carbamate group main-
tains, as compared with acids and esters, the carbonylic
oxygen and contains in addition a NH group. This
observation is in accordance with results of early stud-
ies on chemotactic tripeptides, which put in evidence
that a crucial requirement for the activity was, in
addition to the N-terminal formylation, the presence at
the third position of a carbonylic oxygen as H-bond
acceptor; furthermore, the presence of an additional
NH group, as in the case of tetrapeptides or tripeptide
amides of a primary amine as benzylamine, led to
derivatives more potent than the parent [17]. These data
suggest critical interactions of both the N- and C-termi-
nal end of the tripeptide ligand with the corresponding
receptor areas and a significant role of H-bonding
interactions in correspondence of the C-terminal
residue. Fig. 1 reports a series of C-terminal functional-
izations which have been found compatible with the
activity, together with the here adopted carbamate
modification; it is worth noting that these new ligands,
based on the prototypical MLF sequence, represent the
first examples of analogues in which the carbamate
functionality has been introduced at both ends of the
peptide molecule.
Fig. 1. Functionalities at the C-terminal Phe carboxyl group of MLF
based tripeptides analogues discussed in the text.
esters compared to their corresponding acids; this is
found to be specific for each pair of carbamates and an
ester can be more or less active than the corresponding
acid depending upon the nature of the N-terminal
carbamate [9]. Thus, in addition to the classic and well
known N-formyl tripeptides, the N-alkoxycarbonyl
derivatives of the reference tripeptide MetꢀLeuꢀPhe
represent versatile lead compounds for designing new
ligands and for structure activity studies.
As prosecution of our previous studies in the field
Scheme 1.

310
G. Pagani Zecchini et al. / Il Farmaco 55 (2000) 308–313
Fig. 2. Biological activity of fMLFꢀOMe, methyl carbamate analogues 1, 2, and Boc derivatives 3, 4 towards human neutrophils. (A and B)
Chemotactic activity. (C and D) Release of neutrophil granule enzymes evaluated by determining lysozyme activity.
2. Chemistry
The two
already known N-terminal monocarbamate methyl es-
ters MeOCOꢀMetꢀLeuꢀPheꢀOMe (1) and NBocꢀ
MetꢀLeuꢀPheꢀOMe (3).
biscarbamates
MeOCOꢀMetꢀLeuꢀ
gPheꢀCOOMe (2) and BocꢀMetꢀLeuꢀgPheꢀCOOMe
(4) were synthesized according to Scheme 1. The trans-
formation of BocꢀLeuꢀPheꢀNH2 into the compound
containing the gem-diamino analogue of the Phe
residue, namely BocꢀLeuꢀgPheꢀCOOMe, was per-
formed by adopting a modified procedure of the Hof-
mann rearrangement. Deprotection of the intermediate
biscarbamate with SOCl2/MeOH afforded the key
derivative HCl.LeuꢀgPheꢀCOOMe. Coupling of this
carbamate with MeOCOꢀMetꢀOH or BocꢀMetꢀOH to
give the biscarbamates MeOCOꢀMetꢀLeuꢀgPheꢀ
COOMe (2) and BocꢀMetꢀLeuꢀgPheꢀCOOMe (4), re-
spectively, was performed by the mixed anhydride
method with isobutyl chloroformate. The activity of 2
and 4 was compared to that of the corresponding and
3. Biological results
The biological activity of the carbamate-modified
ligands 1–4 has been determined on human neutrophils
and compared with that of the standard agonist tripep-
tide fMLFꢀOMe; directed migration (chemotaxis), su-
peroxide anion production, and lysozyme release have
been measured. All the tested derivatives were found
unable to elicit superoxide anion production. As shown
in Fig. 2(A), the methyl carbamates 1 is significantly
more active, as chemoattractant, than the dimethoxy-
carbonyl derivative 2 containing the gPhe residue. In
particular the fMLFꢀOMe analogue 1 is, although less

G. Pagani Zecchini et al. / Il Farmaco 55 (2000) 308–313
311
potent, more efficient than the parent, being maximally
effective at the concentration of 10−11 M. As compared
with the methyl carbamate 1, the corresponding Boc
derivative 3 exhibits a lower chemotactic activity (Fig.
2(A) and (B)); on the contrary, the biscarbamate
BocꢀMetꢀLeuꢀgPheꢀCOOMe (4) shows an activity
peak higher than that exhibited by the corresponding
dimethoxycarbonyl derivative 2 at concentration of
10−7 M. When tested as secretagogue agent, the tripep-
tide 1 shows, at low concentration (10−7 M), an activity
higher than the parent (Fig. 2(C)); a significantly lower
potency is found in the case of the three other methyl
carbamates 2, 3, and 4. The Boc derivatives 3–4 show a
homogeneous dose dependent behavior, and a similar
activity as degranulation inducers (Fig. 2(D)). Finally,
no substantial change in lysozyme release was detected
after the substitution in 3 of the Phe with gPhe residue.
were taken at 20°C with a Schmidt–Haensch Polar-
tronic D polarimeter (1 dm cell). IR spectra were
recorded employing a Perkin–Elmer 983 spectrophoto-
meter. 1H NMR spectra were recorded on a Bruker AM
200 spectrometer with Me4Si as internal standard. Thin-
layer (TLC) and preparative chromatographies (PLC)
were performed on silica gel Merck 60 F254 plates. The
drying agent was sodium sulfate. BocꢀMetꢀOH (Fluka
Chemie AG, Switzerland) was employed without purifi-
cation. MeOCOꢀMetꢀOH [9], MeOCOꢀMetꢀLeuꢀ
PheꢀOMe [9], BocꢀLeuꢀPheꢀNH2 [18], BocꢀMetꢀ
LeuꢀPheꢀOMe [19], and parent fMLFꢀOMe [19] were
prepared as described elsewhere. Elemental analyses
were performed in the laboratories of the Servizio Mi-
croanalisi del CNR, Area della Ricerca di Roma, Mon-
telibretti, Italy and were within 90.4% theoretical
values. The abbreviations used are as follows: Boc, t-bu-
toxycarbonyl; DMF, N,N-dimethylformamide; gPhe,
gem-diamino analog of phenylalanine; KRPG, Krebs–
Ringer-phosphate containing 0.1% w/v glucose (pH
7.4); NBS, N-bromosuccinimide; NMM, N-methylmor-
pholine.
4. Discussion
An analysis of the reported biological results indicates
that the introduction of a C-terminal carbamate func-
tion into the N-methoxycarbonyl derivative 1, obtained
by replacing of the Phe with the gPhe residue, causes a
significant decrease of the directed migration activity
and lysozyme release of the resulting modified derivative
2. A different behaviour is exhibited by the N-t-butoxy-
carbonyl derivative 3; in this case, an increase of chemo-
tactic activity, although observed at concentration 10−7
M, is shown by the corresponding biscarbamate 4.
Thus, the same C-terminal modification is beneficial or
detrimental depending upon the nature of the N-termi-
nal protecting group. This result corresponds to the ob-
servation recently made by Higgins et al. [9] when
examining a series of different amino-terminal monocar-
bamate analogues of fMLF.
The results reported in Fig. 2(A) and (B) show that
the methoxycarbonyl derivative 1 exhibits chemotactic
activity on human neutrophils whereas the bulky and
branched analogue 3 is practically inactive. Whereas
these findings could be anticipated on the basis of litera-
ture data, it is worth noting that the chemotactic activity
of these compounds has not been reported before.
Synthesis of analogues of bioactive peptides, obtained
by adopting the here reported approach of a symmetri-
cal functionalization at both ends of the molecule, is
now being further examined in our laboratories.
5.1.1. BocꢀLeuꢀgPheꢀCOOMe
Following the procedure of Huang and Keillor [20],
to a solution of MeONa, prepared by the addition of Na
(0.485 g, 21 mmol) to dry methanol (24 ml),
BocꢀLeuꢀPheꢀNH2 (0.612 g, 1.62 mmol) and NBS
(96.9%; 0.299 g, 1.62 mmol) were added and the reac-
tion mixture was heated to reflux. After time intervals of
3 and 6 min, additional portions of NBS (96.9%; 0.149
g, 0.81mmol) were added. After refluxing for a total of
10 min, the solvent was removed under reduced pressure
and the residue was dissolved in ethyl acetate, washing
with brine to neutrality. The organic phase was dried
and evaporated to give an oily residue which was
purified by PLC [CH2Cl2/EtOAc (95:5) as eluant], af-
fording homogeneous title compound (0.319 g, 48%) as
an oil. [h]D= −28° (c=2.0, CHCl3). IR (CHCl3)
1
cm−1: 3422, 2956, 2933, 1690, 1495, 1162. H NMR
(CDCl3): l (ppm) 0.83 [6H, m, (CH3)2CH], 1.20–1.70
[12H, m, C(CH3)3 (s at 1.40) superimposed on Leu b-
CH2 and g-CH], 2.90 (2H, m, gPhe b-CH2), 3.27 (3H, s,
COOCH3), 4.01 (1H, m, Leu a-CH), 4.97 (1H, d, J=8.2
Hz, Leu NH), 5.34 (1H, m, gPhe a-CH), 6.51 ( 1H, d,
J=9.5 Hz, gPhe NHꢀCO), 7.10–7.42 (5H, m, aro-
matic). Two isomers have been detected and the signals
of the major component are reported. Anal. (C, H, N)
for C21H33N3O5.
5. Experimental
5.1.2. BocꢀMetꢀLeuꢀgPheꢀCOOMe (4)
Thionyl chloride (0.05 ml, 0.633 mmol) was added
dropwise to a solution of the above biscarbamate
(0.258 g, 0.633 mmol) in dry methanol (1.3 ml), cooled
at −15°C. After stirring at −15°C for 30 min and
at 45°C for 2 and a half hours, the solution was evapo-
5.1. Chemistry
Melting points were determined with a Kofler hot
stage apparatus and are uncorrected. Optical rotations

312
G. Pagani Zecchini et al. / Il Farmaco 55 (2000) 308–313
rated under vacuum to give HCl.HꢀLeuꢀgPheꢀCOOMe
as a foam (0.212 g). This salt was used without further
purification. Isobutyl chloroformate (98%, 0.084 ml,
0.633 mmol) was added at −15°C to a stirred solution
of BocꢀMetꢀOH (0.158 g, 0.633 mmol) and NMM
(0.084 ml, 0.76 mmol) in dry CH2Cl2 (3 ml). The
temperature was kept at −15°C for 10 min, then a
solution of the above prepared HCl·HꢀLeuꢀgPheꢀ
COOMe and NMM (0.07 ml, 0.633 mmol) in dry
CH2Cl2 (2.3 ml) was added. The mixture, stirred at
−15°C for 15 min and at room temperature for 1 day,
was then evaporated under vacuum. The residue was
dissolved in ethyl acetate and washed with 2 N HCl,
brine, saturated aqueous NaHCO3, and brine. The or-
ganic phase was dried and evaporated to give a residue
(0.333 g), which was purified by PLC [CH2Cl2/EtOAc
(7:3) as eluant], affording pure BocꢀMetꢀLeuꢀgPheꢀ
COOMe (4) (0.145 g, 42%). M.p. 151–152°C (EtOAc/
n-hexane). [h]D= −35° (c=1.0, CHCl3). IR (KBr)
cm−1: 3314, 2957, 2931, 1690, 1646, 1527, 1170. 1H
NMR (CDCl3): l (ppm) 0.81 [6H, m, (CH3)2CH], 1.15–
1.60 [12H, m, C(CH3)3 (s at 1.44) superimposed on Leu
b-CH2 and g-CH], 1.75–2.02 (2H, m, Met b-CH2), 2.08
(3H, s, S-CH3), 2.52 (2H, t, J=7.1 Hz, CH2ꢀS), 2.87
and 3.03 (2H, A and B of an ABX, J=5.8, 7.0, and
14.0 Hz, gPhe b-CH2), 3.29 (3H, s, COOCH3), 4.31
(1H, m, Met a-CH), 4.38 (1H, m, Leu a-CH), 5.35 (1H,
m, gPhe a-CH), 5.53 (1H, d, J=7.1 Hz, Met NH), 6.96
(2H, apparent d, Leu NH and gPhe NH), 7.12–7.37
(5H, m, aromatic). Anal. (C, H, N) for C26H42N4O6S.
analogues, were prepared in dimethyl sulphoxide and
diluted in KRPG, before use. At the concentration
used, dimethyl sulphoxide did not interfere with any of
the biological assays performed.
5.2.2. Cell preparation
Cells were obtained from the blood of healthy sub-
jects, and human peripheral blood neutrophils were
purified employing the standard techniques of dextran
(Pharmacia, Uppsala, Sweden) sedimentation, centrifu-
gation on Ficoll–Paque (Pharmacia), and hypotonic
lysis of contaminating red cells. The cells were washed
twice and resuspended in KRPG, at a final concentra-
tion of 50×106 cells/ml and kept at room temperature
until used. The percentage of neutrophils was 98–100%
pure and ]99% viable, as determined by the Trypan
blue exclusion test.
5.2.3. Random locomotion
Random locomotion was performed with 48-well mi-
crochemotaxis chamber (Bio Probe, Milan, Italy) and
the migration into the filter was evaluated by the
method of leading-front [21]. The actual control ran-
dom movement is 32 mm93 SE of ten separate experi-
ments performed in duplicate.
5.2.4. Chemotaxis
In order to study the potential chemotactic activity,
each peptide was added to the lower compartment of
the chemotaxis chamber. Peptides were diluted from a
stock solution with KRPG containing 1 mg/ml of
bovine serum albumin (Orha Behringwerke, Germany)
and used at concentrations ranging from 10−12 to 10−5
M. Data were expressed in terms of chemotactic index,
which is the ratio: (migration toward test attractant
minus migration toward the buffer)/migration toward
the buffer; the values are the mean of six separate
experiments performed in duplicate. Standard errors
are in the 0.02–0.09 chemotactic index range.
5.1.3. MeOCOꢀMetꢀLeuꢀgPheꢀCOOMe (2)
Following the procedures above described for the
synthesis of BocꢀMetꢀLeuꢀgPheꢀCOOMe, BocꢀLeuꢀ
gPheꢀCOOMe (0.319 g, 0.782 mmol) and an equimolar
amount of MeOCOꢀMetꢀOH (0.162 g) gave, after final
PLC, pure MeOCOꢀMetꢀLeuꢀgPheꢀCOOMe (2) (0.154
g, 40%). M.p. 162–163°C (EtOAc/n-hexane). [h]D= −
39° (c=1.0, CHCl3). IR (KBr) cm−1: 3295, 2955, 2930,
1
5.2.5. Superoxide anion (O−2 ) production
1694, 1642, 1533. H NMR (CD3)2SO: l (ppm) 0.74
and 0.79 [6H, two d, J=6 Hz, (CH3)2CH], 1.09–1.40
[3H, m, Leu b-CH2 and g-CH], 1.62–1.90 (2H, m, Met
b-CH2), 2.01 (3H, s, SꢀCH3), 2.42 (2H, t, J=7.7 Hz,
CH2ꢀS), 2.75 and 2.93 (2H, A and B of an ABX,
J=4.8, 8.6, and 14.0 Hz, gPhe b-CH2), 3.15 (3H, s,
gPhe NHꢀCOOCH3), 3.52 (3H, s, Met NHꢀCOOCH3),
4.06 (1H, m, Met a-CH), 4.18 (1H, m, Leu a-CH), 5.08
(1H, m, gPhe a-CH), 7.10–7.42 [6H, m, aromatic and
Met NH (d, J=8.0 Hz)], 7.94 (1H, d, J=7.6 Hz, Leu
NH), 8.43 (1H, d, J=9.3 Hz, gPhe NH). Anal. (C, H,
N) for C23H36N4O6S.
The superoxide anion was measured by the superox-
ide dismutase-inhibitable reduction of ferricytochrome
c (Sigma, St. Louis, MO, USA) modified for mi-
croplate-based assays. Tests were carried out in a final
volume of 200 ml containing 4×105 neutrophils, 100
nmol cytochrome c (Sigma) and KRPG. At zero time,
different amounts (10−9−2×10−5 M) of each peptide
were added and the plates were incubated into a mi-
croplate reader (Ceres 900, Bio-TeK Instruments, Inc.)
with the compartment temperature set at 37°C. Ab-
sorbance was recorded at wavelengths of 550 and 465
nm. Differences in absorbance at the two wavelengths
were used to calculate nmol of O−2 produced using an
absorptivity for cytochrome c of 15.5 mM−1/cm. Neu-
trophils were incubated with 5 mg/ml cytochalasin B
(Sigma) for 5 min prior to activation by peptides.
5.2. Biological assay
5.2.1. Peptides
Stock solutions, 10−2 M of fMLFꢀOMe and peptide

G. Pagani Zecchini et al. / Il Farmaco 55 (2000) 308–313
313
structural requirements for synthetic peptide chemoattractants,
Biochemistry 19 (1980) 2404–2410.
5.2.6. Enzyme assay
The release of neutrophil granule enzymes was evalu-
ated by determination of lysozyme activity, modified
for microplate-based assays. Cells, 3×106/well, were
first incubated in triplicate wells of microplates with 5
mg/ml cytochalasin B at 37°C for 15 min and then in
the presence of each peptide in a final concentration of
10−10−2×10−5 M for a further 15 min. The plates
were then centrifuged at 400×g for 5 min and the
lysozyme was quantified nephelometrically by the rate
of lysis of cell wall suspension of Micrococcus lysodeik-
ticus. The reaction rate was measured using a mi-
croplate reader at 465 nm. Enzyme release was
expressed as a net percentage of total enzyme content
released by 0.1% Triton X-100. Total enzyme activity
was 8591 mg/1×107 cells/min.
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[8] J.-L. Gao, E.L. Becker, R.J. Freer, N. Muthukumaraswamy,
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5.2.7. Statistical analysis
The non-parametric Wilcoxon test was used in the
statistical evaluation of differences between groups.
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Acknowledgements
[14] I. Torrini, M. Paglialunga Paradisi, G. Pagani Zecchini, G.
Lucente, E. Gavuzzo, F. Mazza, G. Pochetti, S. Traniello, S.
Spisani, Synthesis, conformation, and biological activity of two
This work was supported in part by the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnologica
(MURST). We are grateful to Banca del Sangue of
Ferrara for providing fresh blood.
fMLPꢀOMe
analogues
containing
the
new
2-[2%-
(methylthio)ethyl]methionine residue, Biopolymers 42 (1997)
415–426.
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