β-Peptido sulfonamides: For-Met-Leu-Phe-OMe analogues containing taurine and chiral β-amino-ethanesulfonic acid residues

Article abstract of DOI:10.1016/j.farmac.2004.07.014

A series of new β-peptido sulfonamides, related to the chemotactic tripeptide fMLF-OMe, has been synthesized. The examined 1a,b-7a,b models contain the achiral -HN-(CH₂)₂-SO₂- taurine (Tau) residue or the chiral -HN-CH(nBu

Full text of DOI:10.1016/j.farmac.2004.07.014

IL FARMACO 59 (2004) 953–963
http://france.elsevier.com/direct/FARMAC/
b-Peptido sulfonamides: for-Met-Leu-Phe-OMe analogues containing
taurine and chiral b-amino-ethanesulfonic acid residues
Cesare Giordano a, Marianna Nalli b, Mario Paglialunga Paradisi a,b, Anna Sansone a,
Gino Lucente a,b,*, Susanna Spisani c
a Istituto di Chimica Biomolecolare del CNR, Sezione di Roma c/o Dipartimento di Studi Farmaceutici, Università degli Studi di Roma “La Sapienza”,
P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento di Studi Farmaceutici, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy
c Dipartimento di Biochimica e Biologia Molecolare, Via L. Borsari 46, Università di Ferrara, 44100 Ferrara, Italy
Received 22 June 2004; accepted 27 July 2004
Available online 30 September 2004
Abstract
A series of new b-peptido sulfonamides, related to the chemotactic tripeptide fMLF-OMe, has been synthesized. The examined 1a,b–7a,b
models contain the achiral –HN–(CH2)2–SO2– taurine (Tau) residue or the chiral –HN–CH(nBu)–CH2–SO2– and –HN–CH(iBu)–CH2–SO2–
residues, corresponding to the b-aminocarboxylic acid counterparts b3-HNle and b3-HLeu, respectively. The biological activity of the new
analogues has been determined on human neutrophils and compared with that of the reference ligand as well as that of the previously studied
related models. The results are analyzed in terms of structure–activity relationships. The conformational preferences of the new tripeptides 1b
and 2b, containing a central chiral b-amino-ethanesulfonic acid residue, have also been discussed.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Chemotactic peptides; Human neutrophils; b-Peptido sulfonamides; Pseudopeptides; Taurine
1. Introduction
The analysis of the results obtained by examining the
above described models showed that the incorporation of a
b-amino-carboxylic acid residue at the central position of the
parent sequence is the least tolerated structural modification.
Thus, For-Met-bAla-Phe-OMe was found to be completely
inactive and For-Met-b3-HLeu-Phe-OMe, notwithstanding
the presence in the b3-HLeu residue of the central proteino-
genic isobutylic side chain, resulted to be a weak agonist and
the least active among the three mixed a/b3 fMLF-OMe
analogues incorporating a b3-HMet, b3-HLeu or b3-HPhe
residue, respectively.
During our research on the structure–activity relationships
in the field of chemotactic peptides related to For-Met-Leu-
Phe-OMe (fMLF-OMe) we started recently a program cen-
tered on the examination of the biochemical consequences of
incorporating residues of b-amino-carboxylic and b-amino-
ethanesulfonic acids into the molecule of the reference ligand
fMLF-OMe. At present, two types of these new analogues
have been examined. A first type is characterized by the
presence of the achiral residue of b-alanine (H2N–CH2–
CH2–CO2H) or taurine (H2N–CH2–CH2–SO3H) replacing
the central native L-leucine (Leu) [1]. A second type includes
the mixed a-peptide/b-peptide analogues of fMLF-OMe [2]
incorporating the chiral residues of b3-HMet, b3-HLeu and
b3-HPhe [3,4] in place of the native L-methionine (Met),
L-leucine (Leu) and L-phenylalanine (Phe) residues, respec-
tively.
At variance with the above reported findings concerning
b-aminocarboxylic acids, all the pseudopeptidic fMLF-OMe
analogues which incorporate the achiral residue of the
b-amino-ethanesulfonic acid taurine (Tau) at the central po-
sition (namely: For-Met-Tau-Phe-OMe; Boc-Met-Tau-Phe-
OMe and Met-Tau-Phe-OMe) exhibit a significant chemo-
tactic activity, comparable to that shown by the reference
ligand [1]. This despite the absence in the Tau residue of the
isobutylic side chain and the different nature of the amino
* Corresponding author. Tel.: +39-06-445-4218; fax: +39-06-491-491.
E-mail address: gino.lucente@uniroma1.it (G. Lucente).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.07.014

954
C. Giordano et al. / IL FARMACO 59 (2004) 953–963
Table 1
Activity at the optimal peptide concentration of fMLF-OMe derivatives containing a b-aminoacid residue at the central position
–
Compounds
Chemotactic index a
% Lysozyme release a
O2 (nmoles) a
2.2
Boc-Met-Tau-Phe-OMe
0.93
10
(10–9
1.06
(10–9
1
)
)
)
(10–5
10.2
(10–5
5.5
(10–7
4
(10–5
33
)
)
)
)
(10–5
14
HCO-Met-Tau-Phe-OMe
(10–5
17
)
)
)
HCl.Met-Tau-Phe-OMe
(10–9
0.23
(10–7
16
Boc-Met-b3-HLeu-Phe-OMe
(10–10
0.55
)
(10–5
38
HCO-Met-b3-HLeu-Phe-OMe
Boc-Met-Leu-w[CH2SO2]-Phe-OMe (2a)
HCO-Met-Leu-w[CH2SO2)-Phe-OMe (2b)
Boc-Met-Tau-Phe-Phe-OMe (6a)
HCO-Met-Tau-Phe-Phe-OMe (6b)
(10–8
0.47
(10–8
0.58
(10–8
0.84
)
)
)
(5 × 10–5
4
)
(8 × 10–5
3
)
(10–7
41
)
)
)
)
(10–7
56
)
)
)
)
(10–5
7
(10–5
17
(10–10
0.45
)
(10–7
27
(10–7
27
(10–10
)
(10–5
(10–5
a The number in parentheses indicates the peptide molarity.
terminal group in the three models. Particularly unexpected
is the activity shown by the pseudopeptide Boc-Met-Tau-
Phe-OMe (see Table 1) whose bulky protecting group at the
Met amino group is generally associated with antagonistic
activity [5,6].
should be noted here that in compounds 1a,b (which corres-
pond to the analogues of the previously studied Boc- and
For-b3-HMet-Leu-Phe-OMe peptides) the –HN–CH(nBu)–
CH2–SO2– residue related to the b3-norleucine (b3-HNle),
instead of the expected –NH–CH(CH2–CH2–S–CH3)–CH2–
SO2– residue related to b3-methionine (b3-HMet), has been
incorporated. This strategy is based on the observation that
methionine (Met) and norleucine (Nle) show bioisosterism
[5,14,15] and the utilization of this latter residue avoids the
laborious synthesis of the chiral b-amino-ethanesulfonyl de-
rivative possessing the methylthio–ethyl side chain of the
native methionine.
As a continuation of our early studies in the field of linear
and cyclic b-sulfonamido peptides [7–13] and in order to
acquire more information on the role and potentiality of
fMLF-OMe analogues incorporating residues of b-amino
ethanesulfonic acids into the molecule of this potent chemo-
tactic tripeptide, we report here synthesis and activity of a
series of new models.
The second group of here reported peptides is directly
related to the above mentioned remarkable chemotactic acti-
vity previously observed in the case of the pseudotripeptides
containing a residue of taurine at the central position [1].
Thus, in order to get information on this point, the synthesis
of the new series of b-sulfonamido peptides (3–7) containing
the achiral Tau residue in place of a natural amino acid, has
been performed. In particular, in addition to the dipeptides
3a,b and tripeptides 4a,b, characterized by a N-terminal Tau
residue, a positional scan of the sequence -Met-Leu-Phe-,
typical of the native ligand fMLF-OMe, has been performed
by synthesizing the three b-sulfonamido-tetrapeptides 5, 6, 7
in which the Tau residue replaces systematically one of the
first three a-aminoacid residues of the highly active tetrapep-
tide For-Met-Leu-Phe-Phe-OMe (fMLFF-OMe) [14,16].
(1)
(2)
(3)
(4)
(5)
(6)
(7)
R-Nle-w CH SO -Leu-Phe-OMe
͓
͔
2
2
R-Met-Leu-w CH SO -Phe-OMe
͓
͔
2
2
R-Tau-Phe-OMe
R-Tau-Leu-Phe-OMe
R-Tau-Leu-Phe-Phe-OMe
R-Met-Tau-Phe-Phe-OMe
R-Met-Leu-Tau-Phe-OMe
(a) R = tert-Bu-OCO- (Boc-); (b) R = HCO- (For-).
A first group includes the N-Boc- and the N-For-
derivatives 1a,b and 2a,b. These peptidosulfonamide/
peptide hybrids have been synthesized with the aim of com-
paring the consequences of the replacement of the
b-aminocarboxylic acid residues, incorporated into the pre-
viously studied a/b3 hybrid tripeptides [2], with the corres-
ponding chiral b-amino-ethanesulfonic acid residues. It
2. Chemistry
The synthesis of Boc-Nle-w[CH2SO2]-Leu-Phe-OMe
(1a), Boc-Met-Leu-w[CH2SO2]-Phe-OMe (2a) and of the
corresponding N-formyl analogues HCO-Nle-w[CH2SO2]-
Leu-Phe-OMe (1b) and HCO-Met-Leu-w[CH2SO2]-Phe-

C. Giordano et al. / IL FARMACO 59 (2004) 953–963
955
Scheme 1
.
OMe (2b) is reported in Scheme 1. The key synthons along
this route were Cbz-Nle-w[CH2SO2]-Cl and Cbz-Leu-
w[CH2SO2]-Cl, which were prepared by using the mesylate–
thioacetate intermediates starting from Cbz-Nle-OH and
Cbz-Leu-OH, respectively, according to the literature [17].
Coupling of Cbz-Nle-w[CH2SO2]-Cl with HCl. Leu-OMe in
the presence of triethylamine afforded the Cbz-Nle-
w[CH2SO2]-Leu-OMe which, after alkaline hydrolysis of the
methyl ester, gave the corresponding free acid. This latter
was coupled with HCl.Phe-OMe by mixed anhydride method
giving Cbz-Nle-w[CH2SO2]-Leu-Phe-OMe. Removal of the
Cbz group by a solution of HBr in acetic acid and
N-protection of the amino group with di-tert-butyl-
dicarbonate afforded 1a. Coupling of Cbz-Leu-
w[CH2SO2]-Cl with HCl.Phe-OMe by analogous procedure
afforded Cbz-Leu-w[CH2SO2]-Phe-OMe which, after clea-
vage of the Cbz group by HBr in acetic acid solution, was
coupled with Boc-Met-OH by the mixed anhydride method
giving 2a.
Scheme 2
.
The synthesis of the pseudopeptides Boc-Tau-Leu-Phe-
OMe (4a) and Boc-Tau-Leu-Phe-Phe-OMe (5a) and of the
corresponding N-formyl analogues HCO-Tau-Leu-Phe-OMe
(4b) and HCO-Tau-Leu-Phe-Phe-OMe (5b) was performed
according to Scheme 2: alkaline hydrolysis of Cbz-Tau-Leu-
OMe [13] gave the free acid which was coupled with
HCl.Phe-OMe by EDC/HOBT to give Cbz-Tau-Leu-Phe-
OMe. Removal of Cbz group by catalytic hydrogenation in
the presence of trifluoroacetic acid (TFA) furnished
TFA.H2N-Tau-Leu-Phe-OMe which was N-protected with
di-tert-butyldicarbonate to give 4a. Cbz- instead of Boc-
group, for the N-protection of the starting material, is reques-
ted to avoid the instability of the latter in the amino-
ethanesulphonyl chloride preparation step. Alkaline
hydrolysis of 4a gave the free acid which was coupled with
HCl.Phe-OMe to give 5a.
Scheme 3
.
red according to reference [18]. A direct transformation of
the N-Boc derivatives 1a–7a into the corresponding
N-formyl analogues was performed by following the proce-
dure of Lajoie and Kraus [19]. Thus, treatment of the N-Boc
derivatives with formic acid, followed by ethyl 2-ethoxy-1,2-
dihydro-1-quinolinecarboxylate (EEDQ), gave the respec-
tive formyl derivatives 1b–7b.
3. Biological activity
In Scheme 3 the synthesis of the pseudopeptides Boc-Tau-
Phe-OMe (3a), Boc-Met-Tau-Phe-Phe-OMe (6a) and Boc-
Met-Leu-Tau-Phe-OMe (7a) and of the corresponding
N-formyl analogues HCO-Tau-Phe-OMe (3b), HCO-Met-
Tau-Phe-Phe-OMe (6b) and HCO-Met-Leu-Tau-Phe-OMe
(7b) is reported. The starting HBr.Tau-Phe-OMe was prepa-
The biological activity of the new analogues 1–7 has been
determined on human neutrophils and compared with that of
the reference ligand fMLF-OMe. Directed migration (che-
motaxis), superoxide anion production and lysozyme release
have been measured. In Fig. 1 the agonistic activity of the

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C. Giordano et al. / IL FARMACO 59 (2004) 953–963
Fig. 1
.
Biological activity of the two b-sulfonamido tripeptides 1a,b and of
N-terminal chiral b3-
Fig. 2. Biological activity of the two b-sulfonamido tripeptides 2a,b and of
the corresponding analogues containing a central chiral b3-aminocarboxylic
acid residue, compared with the reference ligand fMLF-OMe. (A) Chemo-
tactic activity; (B) superoxide anion production; (C) release of neutrophil
granule enzymes evaluated by determining lysozyme activity.
the corresponding analogues containing
a
aminocarboxylic acid residue, compared with the reference ligand fMLF-
OMe. (A) Chemotactic activity; (B) superoxide anion production; (C) re-
lease of neutrophil granule enzymes evaluated by determining lysozyme
activity.
b3-HLeu-Phe-OMe containing the central b-amino-
carboxylic acid residue and, at slightly higher concentration,
the parent fMLF-OMe.
pseudotripeptides Boc-Nle-w[CH2-SO2]-Leu-Phe-OMe (1a)
and HCO-Nle-w[CH2-SO2]-Leu-Phe-OMe (1b), together
with the biological responses elicited by the previously stu-
died [2] a/b3 mixed tripeptides Boc-b3-HMet-Leu-Phe-OMe
and HCO-b3-HMet-Leu-Phe-OMe, are reported. It can be
seen that the replacement of the N-terminal b3-HMet with the
-Nle-w[CH2-SO2]- residue to give the N-formyl derivative
1b, causes the loss of the activity shown by the corresponding
b3-HMet containing analogue and leaves unaltered the inac-
tivity of the N-Boc b3-HMet containing a/b3 mixed tripep-
tide.
The biological assays were then extended to the pseudo-
peptides Boc-Tau-Phe-OMe (3a), HCO-Tau-Phe-OMe (3b),
Boc-Tau-Leu-Phe-OMe (4a), and HCO-Tau-Leu-Phe-OMe
(4b) containing a N-terminal Tau residue. All these derivati-
ves were found practically inactive in each of the performed
assays (not shown).
Finally, Fig. 3 shows the biological activity of the fMLFF-
OMe pseudotetrapeptide analogues containing the Tau resi-
due in the first (5a,b), second (6a,b), and third (7a,b) posi-
tion, respectively. Inspection of Fig. 3A shows that N-Boc
derivative 6a exhibits the highest chemotactic activity and
this is in agreement with the remarkable activity found for the
previously studied tripeptide Boc-Met-Tau-Phe-OMe [1]
(Table 1). On other hand, the low chemotactic response
elicited by the corresponding formyl derivative HCO-Met-
Tau-Phe-Phe-OMe (6b) is not expected in view of both the
remarkable activity exhibited by the correlated b-peptido-
sulfonamide HCO-Met-Tau-Phe-OMe (Table 1) and of the
expected beneficial effect on the activity induced by the
presence of an additional C-terminal Phe residue [5,14,15].
Concerning the superoxide anion production and the lyso-
zyme release, the formyl derivative 7b, containing the Tau
residue in the third position, was found to be the most active
agonist in both the biological assays. It should be also noted
that the pseudopeptides containing the Tau residue in the first
position (5a,b) were essentially inactive in the three tested
functions. These data, together with the biological inactivity
shown by the derivatives 3a,b and 4a,b, confirm that the
occurrence of N-terminal Tau is not tolerated.
In Fig. 2 the agonistic activity of the pseudotripeptides
Boc-Met-Leu-w[CH2-SO2]-Phe-OMe (2a) and HCO-Met-
Leu-w[CH2-SO2]-Phe-OMe (2b) is compared with that
shown by the previously studied a/b3 mixed tripeptides Boc-
Met-b3-HLeu-Phe-OMe and HCO-Met-b3-HLeu-Phe-OMe
[2]. Concerning the chemotactic activity (Fig. 2A and Ta-
ble 1) it can be seen that the replacement of the central
b3-HLeu with the corresponding b-sulfonyl residue leaves
unaltered the activity of the formylated model while renders
chemoattractant the otherwise inactive Boc-Met-b3-HLeu-
Phe-OMe. This latter result parallels the unexpected agonis-
tic activity previously observed in the case of the tripeptide
Boc-Met-Tau-Phe-OMe as compared with the inactive Boc-
Met-bAla-Phe-OMe. As for the superoxide anion production
(Fig. 2B and Table 1) the tripeptide HCO-Met-Leu-
w[CH2SO2)-Phe-OMe (2b) was found more potent and effi-
cient than the corresponding a/b3 mixed formyl derivative. A
similar trend was also observed in the case of lysozyme
release (Fig. 2C and Table 1). Here, it is worth noting the
considerable activity exhibited by the formyl b-peptido-
sulfonamide 2b which superates both the analogue For-Met-

C. Giordano et al. / IL FARMACO 59 (2004) 953–963
957
1
Fig. 4. Plots of NH proton chemical shifts in the H NMR spectra of the
b-sulfonamido tripeptides 1b (A) and 2b (B) as a function of increasing
amounts of DMSO-d6 (v/v) added to the CDCl3 solution (peptide concen-
tration: 10 mM).
not induce the locally folded structures, centred at the
b-residues through a six membered ring (C6 conformation)
observed for the related a/b3 mixed tripeptides HCO-b3-
HMet-Leu-Phe-OMe and HCO-Met-b3-HLeu-Phe-OMe [2].
Fig. 3. Biological activity of b-sulfonamido tetrapeptides 5a,b–7a,b contai-
ning a taurine residue, compared with the reference ligand fMLF-OMe. (A)
Chemotactic activity; (B) superoxide anion production; compounds 5a and
7a exhibit almost identical values. (C) Release of neutrophil granule enzy-
mes evaluated by determining lysozyme activity.
5. Conclusions
4. Solution conformation
Some significant points of the present study are the fol-
lowings:
Information on the conformational preferences of pseudo-
tripeptides 1 and 2 containing a chiral b-sulphonamido resi-
due have been obtained by examining the involvement of the
NH groups of the formyl derivatives HCO-Nle-w[CH2-SO2]-
Leu-Phe-OMe (1b) and HCO-Met-Leu-w[CH2-SO2]-Phe-
• a b-sulphonamido residue, carrying or not the proteinoge-
nic side chain, when located at the N-terminal position of
fMLF-OMe analogues, leads to derivatives devoid of bio-
logical activity in all the examined models. It should be
noted that the related N-For analogue, containing a b3-
HMet residue in place of the native N-terminal Met, shows
significant chemotactic activity (see Fig. 1A).
• The incorporation of the proteinogenic isobutylic side
chain into the Tau residue of Boc- or For-Met-Tau-Phe-
OMe, leading to compounds 2a,b, causes a reduction of
their remarkable chemotactic activity and the loss of the
selectivity shown by the N-For derivative (see Fig. 2 and
Table 1).
• An unexpected decrease of the remarkable chemotactic
activity exhibited by the previously studied For-Met-Tau-
Phe-OMe [1] is observed on passing from this tripeptide
to the related tetrapeptide For-Met-Tau-Phe-Phe-OMe
(6b). The corresponding N-Boc pseudotetrapeptide 6a
maintains, on other hand, the remarkable chemotactic
activity exhibited by the tripeptide Boc-Met-Tau-Phe-
1
OMe (2b) in intramolecular H-bonds using H NMR. In
Fig. 4 the chemical shift dependence of the NH proton
resonances as a function of increasing DMSO-d6 concentra-
tions in CDCl3 solution (10 mM) is reported. In Table 2 the
solvent exposure of the NH groups of 1b and 2b, expressed
as the difference (Dd, ppm) between the NH chemical shift
values observed in a CDCl3 solution containing 10% DMSO
and in neat CDCl3, is compared with the accessibility of the
corresponding groups of the previously studied [2] a/b3
mixed tripeptides HCO-b3-HMet-Leu-Phe-OMe and HCO-
Met-b3-HLeu-Phe-OMe. It appears that in the two models
under study, all the NH groups interact efficiently with the
solvent, thus indicating the absence of intramolecularly
H-bonded conformations. Thus, the pseudopeptides 1b and
2b do not adopt folded conformations stabilized by intramo-
lecular H-bonds and the chiral b-sulphonamido residue does
Table 2
Solvent accessibility of peptide NH groups: Differences (Dd, ppm) between NH proton chemical shift values observed in a CDCl3 solution containing 10%
DMSO and in neat CDCl3
Compound
N-terminal NH
Central NH
0.95
C-terminal NH
HCO-Nle-w[CH2-SO2]-Leu-Phe-OMe (1b)
HCO-b3-HMet- Leu-Phe-OMe
HCO-Met-Leu-w[CH2-SO2]-Phe-OMe (2b)
HCO-Met-b3-HLeu-Phe-OMe
1.05
0.48
0.93
0.80
1.16
0.67
0.86
1.04
1.25
0.84
0.36

958
C. Giordano et al. / IL FARMACO 59 (2004) 953–963
OMe. Furthermore, it should be noted that these two
dihydro-1-quinolinecarboxylate; EDC, N-(3-dimethylami-
nopropyl)-N′-ethylcarbodiimide hydrochloride; HOBT,
1-hydroxybenzotriazole; KRPG, Krebs–Ringer phosphate
containing 0.1% w/v D-glucose; NMM, N-methylmor-
pholine; DMF, dimethylformamide; TEA, triethylamine.
N-Boc derivatives are selective ligands since show only
chemotactic activity (Fig. 3 and Table 1). An opposite
selectivity is observed in the case of the N-For tetrapeptide
7b which is not chemoattractant but induces superoxide
anion production and lysozyme release (Fig. 3).
6.1.1. Coupling of Cbz-b-aminoethane sulphonylchlorides
with aminoacid methylester hydrochlorides. General
procedure A
• The replacement of the b-amino-carboxylic acid residue
with the corresponding b-amino-sulfonic acid leads to the
models 1b and 2b (see Fig. 4 and Table 2) which do not
maintain the locally folded conformations stabilized by
intramolecular H-bonds found in the case of the corres-
ponding previously studied models [2] containing the b3-
HMet and b3-HLeu residues.
To an ice-cooled mixture containing the Cbz-b-
aminoethane sulphonylchloride (1.0 mmol) and the aminoa-
cid methylester hydrochloride (2.0 mmol) in dry CH2Cl2
(8 ml), TEA (2.0 mmol) in dry CH2Cl2 (8 ml) was added
under nitrogen. The resulting suspension was stirred overni-
ght allowing to warm to room temperature. After dilution
with CH2Cl2 (25 ml), the mixture was washed with 1 M HCl,
saturated NaHCO3 and brine. The organic phase was dried
and evaporated under reduced pressure.
A main indication which emerges from the above reported
results concerns the role of the sequence -Met-Tau-Phe-
which appears associated, both in the tripeptide and in the
tetrapeptide models, with the unusual chemotactic activity
shown by the N-Boc derivatives. Although more data are
necessary to better define this point, the available data sug-
gest that the sulphonamide junction, due to its high polar
character and hydrogen bonding capabilities, can signifi-
cantly alter the usual mode of ligand–receptor interaction and
when centrally located into fMLF-OMe analogues determi-
nes a profound change of the established requirements for an
efficient binding [14,20]. Consistent with this hypothesis is
the observed decrease of activity on passing from the tripep-
tides containing a central Tau to the corresponding models
2a,b possessing the isobutylic proteinogenic Leu side chain
as well as the higher activity shown by the N-Boc derivative
6a as compared with the corresponding N-For analogue 6b.
6.1.2. Cbz-group removal. General procedure B
General procedure B-1 by HBr/AcOH: the N-protected
aminoacid or peptide (1 mmol) was dissolved in a solution of
36% HBr in acetic acid (3.0 ml). After 15 min at room
temperature the reaction mixture was evaporated to dryness
without heating. The residue was repeatedly coevaporated
with anhydrous diethyl ether then dried under high vacuum
overnight.
General procedure B-2 by catalytic hydrogenation: a solu-
tion of the N-protected aminoacid or peptide (1 mmol) and
trifluoroacetic acid (1.2 mmol) in MeOH (10 ml) was flushed
with H2 stream in the presence of 10% palladium on charcoal
at room temperature until the reaction was complete (TLC).
The mixture was filtered on celite and the filtrate evaporated
to dryness under reduced pressure. The oily residue was
repeatedly coevaporated with anhydrous diethyl ether to re-
move the TFA excess and then kept under high vacuum
overnight.
Further studies on the properties of fMLF-OMe analogues
containing b-sulfonic acid residues are in progress in our
laboratories.
6. Experimental section
6.1. Chemistry
6.1.3. Hydrolysis of methylesters. General procedure C
The aminoacid or peptide methylester (1 mmol) was
mixed with 2 N NaOH (1.5 ml) and MeOH (5 ml) and left at
room temperature overnight. After removal of MeOH at
reduced pressure, water (10 ml) was added and the solution
extracted with diethyl ether (2 × 25 ml). The aqueous phase
was then acidified with 1 N HCl and the product extracted
with ethyl acetate (3 × 20 ml). The organic phases were
washed with brine, pooled, dried and evaporated.
Melting points were determined with a Büchi B 540 appa-
ratus and are uncorrected. Optical rotations were taken at
20 °C with a Schmidt-Haensch Polartronic D polarimeter
(1 dm cell, c 1.0 in CHCl3, unless otherwise specified). IR
spectra were recorded in 1% CHCl3 (unless otherwise speci-
fied) solution employing a Perkin-Elmer FT-IR Spectrum
1
1000 spectrometer. H NMR spectra were determined in
CDCl3 solution with a Bruker AM 400 spectrometer and
chemical shifts were indirectly referred to TMS. Thin-layer
and preparative layer chromatographies were performed on
silica gel Merck 60 F254 plates. The drying agent was sodium
sulphate. Elemental analyses were performed in the labora-
tories of the Servizio Microanalisi del CNR, Area della
Ricerca di Roma, Montelibretti, Italy, and were within 0.4%
theoretical values. The abbreviations used are as follows:
Boc, tert-butyloxycarbonyl; EEDQ, ethyl 2-ethoxy-1,2-
6.1.4. Preparation of the N-Boc derivatives. General
procedure D
To a solution of the aminoacid or peptide (as hydrobro-
mide or trifluoroacetate 1 mmol) and TEA (1.5 mmol) in
dioxane (10 ml), di-tert-butyldicarbonate (1.2 mmol) in
dioxane (3 ml) was added and the mixture was allowed at
room temperature overnight. After evaporation at reduced

C. Giordano et al. / IL FARMACO 59 (2004) 953–963
959
pressure, the residue was dissolved in ethyl acetate (25 ml)
and washed with 1 M KHSO4, NaHCO3 saturated solution
and brine. The organic phase was dried and evaporated under
reduced pressure.
procedure A. White solid (0.780 g, 79%). Rf = 0.25
(CHCl3/MeOH 98:2). M.p. 107–108 °C (EtOAc/hexane).
[a]D = +7°. IR: m 3431, 2951, 1743, 1711, 1510, 1331,
1
1144 cm–1. H NMR d: 0.92 [6H, two superimposed d,
CH2CH(CH3)2], 1.25–1.77 [3H, three m, CH2CH(CH3)2],
2.98 (2H, sharp m, CH2SO2), 3.08 and 3.16 (2H, A and B of
an ABX, J = 4.2, 5.2 and 10.5 Hz, Phe b-CH2), 3.75 (s, 3H,
COOCH3), 4.28 (1H, m, CHCH2SO2), 4.45 (1H, m, Phe
a-CH), 4.90 (1H, d, J = 6.9 Hz, Leu-w[CH2SO2] NH), 5.13
(2H, m, PhCH2O), 5.61 (1H, d, J = 6.3 Hz, Phe NH), 7.18–
7.40 (5H, m, aromatics).
6.1.5. Mixed anhydride coupling. General procedure E
Isobutyl chloroformate (1 mmol) was added at –15 °C to a
stirred solution of the N-protected aminoacid or peptide
(1.0 mmol) and NMM (0.153 g, 1.0 mmol) in dry THF
(8.0 ml). The temperature was maintained at –15 °C for
10 min and a freshly prepared mixture of the C-protected
aminoacid hydrochloride (1.0 mmol) and NMM (1.0 mmol)
in dry THF (5.0 ml) was then added. The reaction mixture
was stirred at –15 °C for 30 min and left at 0 °C overnight.
Ethyl acetate was then added and the solution washed with
1 M KHSO4, brine, saturated aqueous NaHCO3 and brine.
The organic phase was dried and evaporated under reduced
pressure.
6.1.10. Cbz-Nle-w[CH2SO2]-Leu-Phe-OMe
The above described Cbz-Nle-w[CH2SO2]-Leu-OMe
(0.76 g, 1.71 mmol) was C-deprotected according to proce-
dure C. The residue was coupled with HCl.Phe-OMe (0.37 g,
1.71 mmol) according to general procedure E. White solid
(0.818 g, 81%). Rf = 0.55 (CHCl3/MeOH 95:5). M.p. 146–
148 °C (EtOAc/hexane). [a]D = +17°. IR: m 3429, 2959, 1740,
1707, 1516, 1330, 1234, 1140 cm–1. 1H NMR d: 0.82–0.98
[9H, m, CH2CH(CH3)2 and CH2CH3], 1.23–1.80 [9H, m,
CH2CH(CH3)2 and CH2CH2CH2CH3], 2.86–3.21 (4H, m,
Phe b-CH2 and CH2SO2), 3.74 (3H, s, COOCH3), 3.97 (1H,
m, Leu a-CH), 4.27 (1H, m, CHCH2SO2), 4.87 (1H, m, Phe
a-CH), 5.11 (2H, s, PhCH2O), 5.69 (1H, d, J = 6.3 Hz,
Nle-w(CH2SO2) NH], 6.65 (1H, d, J = 6.1 Hz, Leu NH),
7.14–7.38 (6H, m, aromatics and Phe NH).
6.1.6. Carbodiimide coupling. General procedure F
A mixture containing the N-protected aminoacid or pep-
tide (1.0 mmol), the C-protected aminoacid or peptide salt
(1.0 mmol), HOBt (1.2 mmol) and TEA (1.2 mmol) in
anhydrous ethyl acetate (6 ml) was prepared at room tempe-
rature. After cooling to 0 °C, EDC (1.0 mmol) was added and
the reaction mixture allowed to warm slowly to room tempe-
rature. After 12 h ethyl acetate was added and the organic
layer washed with 1 M KHSO4, saturated aqueous NaHCO3
and brine. The organic phase was dried and evaporated under
reduced pressure.
6.1.11. Boc-Nle-w[CH2SO2]-Leu-Phe-OMe (1a)
Cbz-Nle-w[CH2SO2]-Leu-Phe-OMe (0.575 g, 0.97 mmol)
underwent to N-deprotection according to general procedure
B-2. The trifluoroacetate salt (white solid) was N-Boc-
protected according to general procedure D. Silica gel flash
chromatography gave the pure product as a colourless oil
(0.367 g, 68%). Rf = 0.78 (CHCl3/MeOH 95:5). [a]D = +14°.
IR: m 3435, 2960, 1741, 1686, 1509, 1368, 1234, 1166 cm–1.
1H NMR d: 0.90–0.96 [9H, m, CH2CH(CH3)2 and CH2CH3],
6.1.7. Preparation of the N-formyl-derivatives. General
procedure G
The N-Boc derivative (1.0 mmol) was dissolved in formic
acid (3 ml) and the mixture allowed to stand at room tempe-
rature overnight. After remotion of the excess of formic acid
under vacuum, the residue was dissolved in dry DMF (2 ml).
EEDQ 97% (1.2 mmol) was added and the solution stirred at
room temperature for 24 h. Evaporation under reduced pres-
sure afforded the crude product.
1.20–1.83
[9H,
three
m,
CH2CH(CH3)2
and
CH2CH2CH2CH3], 1.44 [9H, s, C(CH3)3], 2.94 (2H, d,
J = 5.8 Hz, CH2SO2), 3.08 and 3.19 (2H, A and B of anABX,
J = 5.1, 7.1 and 13.7 Hz, Phe b-CH2) 3.75 (3H, s, COOCH3),
3.99 (1H, m, Leu a-CH), 4.23 (1H, m, CHCH2SO2), 4.70
(1H, d, J = 9.5 Hz, Nle-w[CH2SO2] NH), 4.89 (1H, m, Phe
a-CH), 5.80 (1H, d, J = 6.6 Hz, Leu NH), 6.57 (1H, d,
J = 7.4 Hz, Phe NH), 7.16–7.35 (5H, m, aromatics).
6.1.8. Cbz-Nle-w[CH2SO2]-Leu-OMe
From Cbz-Nle-w[CH2SO2]-Cl (0.920 g, 2.76 mmol) and
HCl.Leu-OMe (0.501 g, 2.76 mmol) according to general
procedureA. White solid (0.760 g, 78%). Rf = 0.40 (CH2Cl2).
M.p. 82–83 °C (Et2O/hexane). [a]D = +2°. IR: m 3434, 2960,
1742, 1709, 1517, 1338, 1139 cm–1. 1H NMR d: 0.83–1.03
[9H, m, CH2CH(CH3)2 and CH2CH3], 1.25–1.87 [9H, m,
CH2CH(CH3)2 and CH2CH2CH2CH3], 3.07–3.17 (2H, m,
CH2SO2), 3.77 (3H, s, COOCH3), 4.20 (1H, m, Leu a-CH),
4.43 (1H, m, CHCH2SO2), 4.97 (1H, d, J = 9.4 Hz, Nle-
w[CH2SO2] NH), 5.12 (2H, s, PhCH2O), 5.77 (1H, d,
J = 9.0 Hz, Leu NH), 7.28–7.40 (5H, m, aromatics).
6.1.12. HCO-Nle-w[CH2SO2]-Leu-Phe-OMe (1b)
From 1a (0.25 g, 0.45 mmol) according to general proce-
dure G. White solid (0.178 g, 82%). Rf = 0.48 (CHCl3/MeOH
95:5). M.p. 112–115 °C (triturated with hexane). [a]D = +17°.
1
IR: m 3423, 2959, 1742, 1682, 1513, 1362, 1143 cm–1. H
NMR d: 0.86–0.96 [9H, m, CH2CH(CH3)2 and CH2CH3],
6.1.9. Cbz-Leu-w[CH2SO2]-Phe-OMe
1.20–1.84
[9H,
three
m,
CH2CH(CH3)2
and
CH2CH2CH2CH3], 2.97–3.25 (4H, m, Phe b-CH2 and
CH2SO2), 3.76 (3H, s, COOCH3), 3.98 (1H, br, Leu a-CH),
From Cbz-Leu-w[CH2SO2]-Cl (0.534 g, 1.6 mmol) and
HCl.Phe-OMe (0.690 g, 3.2 mmol) according to general

960
C. Giordano et al. / IL FARMACO 59 (2004) 953–963
4.61 (1H, m, CHCH2SO2), 4.89 (1H, m, Phe a-CH), 5.68
(1H, br, Leu NH), 6.03 (1H, d, J = 6.9 Hz, Nle-w[CH2SO2]
NH), 6.56 (1H, d, J = 6.2 Hz, Phe NH), 7.16–7.36 (5H, m,
aromatics), 8.18 (1H, s, HCO).
6.1.16. Boc-Tau-Leu-Phe-OMe (4a)
Cbz-Tau-Leu-Phe-OMe (1.31 g, 2.4 mmol) underwent to
N-deprotection according to general procedure B-2. The tri-
fluoroacetate salt (white solid) was N-Boc protected accor-
ding to general procedure D. Silica gel flash chromatography
(CHCl3) gave the pure title compound. White crystals
(0.225 g, 47%). [a]D = +18°. M.p. 98–99 °C (hexane). IR: m
3427, 2959, 1741, 1707, 1509, 1368, 1148 cm–1. 1H NMR d:
0.94 [6H, m, CH2CH(CH3)2], 1.46 [9H, s, C(CH3)3], 1.50
[2H, m, CH2CH(CH3)2], 1.77 [1H, m, CH2CH(CH3)2], 2.96
(2H, m, Tau a-CH2), 3.08 and 3.22 (2H, A and B of an ABX,
J = 5.4, 8.6, and 14.0 Hz, Phe b-CH2), 3.52 (2H, m, Tau
b-CH2), 3.77 (3H, s, COOCH3), 3.92 (1H, m, Leu a-CH),
4.90 (1H, m, Phe a-CH), 5.18 (2H, m, Leu NH and Tau NH),
6.46 (1H, d, J = 8.0 Hz, Phe NH), 7.15–7.35 (5H, m, aroma-
tics).
6.1.13. Boc-Met-Leu-w[CH2SO2]-Phe-OMe (2a)
Cbz-Leu-w[CH2SO2]-Phe-OMe (0.78 g, 1.63 mmol) was
N-deprotected according to general procedure B-2. The tri-
fluoroacetate salt (foam) was coupled with Boc-Met-OH
(0.691 g, 1.63 mmol) according to general procedure E.
Silica gel flash chromatography (CHCl3/MeOH 95:5) gave
the pure title compound. White foam (0.81 g, 86%). Rf = 0.16
(CHCl3/MeOH 98:2). [a]D = –9°. IR: m 3430, 2918, 1743,
1692, 1533, 1332, 1155 cm–1. 1H NMR d: 0.90 [6H, apparent
t, CH2CH(CH3)2], 1.27–1.62 [3H, two m, CH2CH(CH3)2],
1.46 [9H, s, C(CH3)3], 1.85–2.22 (2H, two m, Met b-CH2),
2.13 (3H, s, S–CH3), 2.60 (2H, t, J = 5.4 Hz, Met c-CH2),
2.99 (2H, m, CH2SO2), 3.08 and 3.17 (2H, A and B of an
ABX, J = 4.4, 5.4 and 10.4 Hz, Phe b-CH2) 3.75 (3H, s,
COOCH3), 4.20 (1H, m, Met a-CH), 4.45 (1H, m, Phe
a-CH), 4.53 (1H, m, CHCH2SO2), 5.10 (1H, d, J = 8.4 Hz,
Met NH), 5.96 (1H, d, J = 9.3 Hz, Phe NH), 6.42 (1H, d,
J = 8.8 Hz, Leu-w[CH2SO2] NH), 7.22–7.36 (5H, m, aroma-
tics).
6.1.17. HCO-Tau-Leu-Phe-OMe (4b)
From 4a (0.125 g, 0.25 mmol) according to the general
procedure G. Silica gel flash chromatography (CHCl3/
MeOH 98:2) gave the pure title product. White solid
(0.080 g, 78%). [a]D
= +34°. M.p. 119–120 °C
(EtOAc/hexane). IR: m 3418, 3031, 2958, 1741, 1685, 1517,
1
1438, 1333, 1236,1141, cm–1. H NMR d: 0.94 [6H, m,
CH2CH(CH3)2], 1.51 [2H, m, CH2CH(CH3)2], 1.77 [1H, m,
CH2CH(CH3)2], 2.90 and 2.98 (2H, two m, Tau a-CH2),
3.06 and 3.22 (2H, A and B of an ABX, J = 4.9, 9.1 and
14.0 Hz, Phe b-CH2), 3.66 (2H, m, Tau b-CH2), 3.77 (3H, s,
COOCH3), 3.93 (1H, m, Leu a-CH), 4.90 (1H, m, Phe
a-CH), 5.48 (1H, d, J = 7.3 Hz, Leu NH), 6.62 (1H, d,
J = 7.2 Hz, Phe NH), 6.73 (1H, br, Tau NH), 7.16–7.35 (m,
5H, aromatics), 8.14 (1H, s, HCO).
6.1.14. HCO-Met-Leu-w[CH2SO2]-Phe-OMe (2b)
From 2a (0.438 g, 0.76 mmol) according to general pro-
cedure G. Silica gel flash chromatography (CHCl3/MeOH
98:2) gave the pure product. Pale yellow oil (0.322 g, 84%).
Rf = 0.52 (CHCl3/MeOH 98:2). [a]D = –35°. IR: m 3411,
2959, 1744, 1666, 1557, 1338, 1261, 1151, cm–1. 1H NMR d:
0.91 [6H, two superimposed d, CH2CH(CH3)2], 1.25–1.62
[3H, two m, CH2CH(CH3)2], 1.92–2.25 (2H, two m, Met
b-CH2), 2.13 (3H, s, S–CH3), 2.63 (2H, t, J = 5.3 Hz, Met
c-CH2), 2.98 (2H, m, CH2SO2), 3.09 and 3.18 (2H, A and B
of an ABX, J = 4.2, 5.5 and 10.4 Hz, Phe b-CH2) 3.76 (3H, s,
COOCH3), 4.44 (1H, m, Phe a-CH), 4.49 (1H, m,
CHCH2SO2), 4.64 (1H, m, Met a-CH), 5.85 (1H, d,
J = 6.7 Hz, Phe NH), 6.41 (2H, m, Met NH and Leu-
w[CH2SO2] NH), 7.21–7.37 (5H, m, aromatics), 8.22 (1H, s,
HCO).
6.1.18. Boc-Tau-Leu-Phe-Phe-OMe (5a)
From 4a (0.315 g, 0.63 mmol), which was C-deprotected
according to general procedure C. The free acid (white solid)
was coupled with HCl.Phe-OMe (0.136 g, 0.63 mmol) accor-
ding to general procedure F. Silica gel flash chromatography
(CHCl3/MeOH 98:2) gave the pure product. White solid
(0.28 g, 65%). [a]D = –10°. M.p. 163–164 °C (EtOAc/
hexane). IR: m 3413, 2934, 1743, 1673, 1509, 1368,
1
1157 cm–1. H NMR d: 0.91 [6H, m, CH2CH(CH3)2], 1.44
6.1.15. Cbz-Tau-Leu-Phe-OMe
[9H, s, C(CH3)3], 1.45–1.80 [3H, m, CH2CH(CH3)2], 2.93–
3.11 (6H, two m, Tau a-CH2 and two Phe b-CH2), 3.51 (2H,
m, Tau b-CH2), 3.70 (3H, s, COOCH3), 3.85 (1H, m, Leu
a-CH), 4.64 and 4.75 (2H, two m, two Phe a-CH), 5.18 (1H,
m, Tau NH), 6.26 (1H, d, J = 8,0 Hz, Phe NH), 6.56 (1H, d,
J = 7.9 Hz, Leu NH), 7.02 (1H, d, J = 7.7 Hz, Phe NH),
7.20–7.31 (10H, m, aromatics).
Cbz-Tau-Leu-OMe [2] (1.03 g, 2.45 mmol) was
C-deprotected according to procedure C. The residue was
coupled with HCl.Phe-OMe (0.603 g, 2.8 mmol) according
to the general procedure F. White foam (1.31 g, 88%).
[a]D = –22°. IR: m 3434, 2960, 1720, 1689, 1514, 1352,
1
1146 cm–1. H NMR d: 0.94 [6H, m, CH2CH(CH3)2], 1.47
[2H, m, CH2CH(CH3)2], 1.76 [1 H, m, CH2CH(CH3)2],
2.83–3.03 (2 H, Tau a-CH2), 3.05 and 3.22 (2 H, A and B of
anABX, J = 5.2, 8.8, and 14.0 Hz, Phe b-CH2), 3.57 (2 H, m,
Tau b-CH2), 3.77 (3 H, s, COOCH3), 3.90 (1 H, m, Leu
a-CH), 4.90 (1 H, m, Phe a-CH), 5.11 (3H, m, PhCH2O and
Leu NH), 5.52 (1H, t, J = 6.0 Hz, Tau NH), 6.40 (1H, d,
J = 8.2 Hz, Phe NH), 7.13–7.38 (10H, m, aromatics).
6.1.19. HCO-Tau-Leu-Phe-Phe-OMe (5b)
From 5a (0.216 g, 0.27 mmol) according to general pro-
cedure G. Silica gel flash chromatography (CHCl3/MeOH
98:2) gave the pure product. White solid (0.133 g; 81%).
[a]D = –8°. M.p. 164–165 °C (chloroform/hexane). IR: m

C. Giordano et al. / IL FARMACO 59 (2004) 953–963
961
3412, 3023,1745, 1685, 1510, 1333, 1142 cm–1. 1H NMR d:
0.91 [6H, m, CH2CH(CH3)2], 1.46 [2H, m, CH2CH(CH3)2],
1.72 (1H, m, CH2CH(CH3)2), 2.90–3.11 (6H, two m, Tau
a-CH2 and two Phe b-CH2), 3.63 (2H, m, Tau b-CH2), 3.70
(3H, s, COOCH3), 3.93 (1H, m, Leu a-CH), 4.75 (2H, m, two
Phe a-CH), 5.69 (1H d, J = 8.1 Hz, Leu NH), 6.66 (1H, d,
J = 7.6 Hz, Phe NH), 6.75 (1H, br, Tau NH), 6.98 (1H, d,
J = 7.9 Hz, Phe NH), 7.19–7.29 (10H, m, aromatics), 8.09
(1H, s, HCO).
(0.102 g; 81%). [a]D = –35° (1%, DMF). M.p. 164–165 °C
(hexane). IR (KBr): m 3288, 2965, 1733, 1638, 1550, 1449,
1339 cm–1. 1H NMR (d6-DMSO) d: 0.83 and 0.88 [6H, two d,
J
= 6.5 Hz, CH2CH(CH3)2], 1.42–1.60 [3H, m,
CH2CH(CH3)2], 1.75–1.95 (2H, m, Met b-CH2), 2.03 (3H, s,
S–CH3), 2.43 (2H, t, J = 7.2 Hz, Met c-CH2), 2.81–3.06 (4H,
m, Phe b-CH2 and Tau a-CH2), 3.20 (2H, m, Tau b-CH2),
3.63 (3H, s, COOCH3), 4.15 (1H, m, Phe a-CH), 4.22 (1H,
m, Leu a-CH), 4.41 (1H, m, Met a-CH), 7.20–7.31 (5H, m,
aromatics), 7.90 (1H, poorly resolved t, Tau NH), 8.00–8.07
[3H, m, Phe NH, Leu NH and HCO (s at 8.02)], 8.28 (1H, d,
J = 8.1 Hz, Met NH).
6.1.20. Boc-Tau-Phe-OMe (3a)
From HBr.Tau-Phe-OMe (0.360 g, 1.0 mmol) according
to general procedure D. Silica gel flash chromatography
(CH2Cl2/MeOH 98:2) gave the pure product. White foam
(0.260 g, 67%). [a]D = –20° (2%, CHCl3). IR: m 3366, 3018,
1740, 1709, 1508, 1340, 1150 cm–1. 1H NMR d: 1.46 [9H, s,
C(CH3)3], 2.87 (2H, m, Tau a-CH2), 3.04 and 3.18 (2H, A
and B of an ABX, J = 5.1, 7.5 and 13.8 Hz, Phe b-CH2), 3.42
(2H, m, Tau b-CH2), 3.80 (3H, s, COOCH3), 4.42 (1H, m,
Phe a-CH), 5.08 (2H, m, Tau and Phe NH), 7.19–7.36 (5H,
m, aromatics).
6.1.24. Boc-Met-Tau-Phe-Phe-OMe (6a)
From Boc-Met-Tau-Phe-OMe [5] (0.231 g, 0.44 mmol)
which underwent to C-deprotection according to procedure
C. The residue was coupled with HCl.Phe-OMe (0.095 g,
0.44 mmol) according to general procedure F. Silica gel flash
chromatography (CHCl3/MeOH 98:2) gave the pure title
product as oil which solidified by treatment with hexane.
White solid (0.335 g; 82%). [a]D = +35° (2%, CHCl3). M.p.
98–99 °C. IR: m 1421, 2961, 1718, 1680, 1497, 1289,
1
1145 cm–1. H NMR d: 1.46 [9H, s, C(CH3)3], 1.90–2.15
6.1.21. HCO-Tau-Phe-OMe (3b)
(2H, m, Met b-CH2), 2.11 (3H, s, S–CH3), 2.55 (2H, m, Met
c-CH2), 2.72 (2H, m, Tau a-CH2), 2.92–3.21 (4H, m, two
Phe CH2), 3.32 and 3.68 (2H, two m, Tau b-CH2), 3.72 (3H,
s, COOCH3), 4.19 (2H, m, Met and Phe a-CH), 4.85 (1H, m,
Phe a-CH), 5.32 (1H, d, J = 8.2 Hz, Met NH), 6.00 (1H, m,
Phe NH), 6.55 (1H, d, J = 7.5 Hz, Phe NH), 6.90–7.34 (11H,
m, aromatics and Tau NH).
From 3a (0.120 g, 0.31 mmol) according to general pro-
cedure G. Silica gel flash chromatography (CHCl3/MeOH
98:2) gave the pure title compound. Pale yellow foam
(0.080 g; 83%). [a]D = –25°. IR: m 3367, 3034, 1743, 1686,
1513, 1339, 1144 cm–1. 1H NMR d: 2.87 (2H, m, Tau
a-CH2), 3.01 and 3.19 (2H, A and B of an ABX, J = 4.9, 8.0,
and 13.8 Hz, Phe b-CH2), 3.56 (2H, m, Tau b-CH2), 3.82
(3H, s, COOCH3), 4.39 (1H, m, Phe a-CH), 5.28 (1H, d,
J = 9.1 Hz, Phe NH), 6.37 (1H, br, Tau NH), 7.20–7.38 (5H,
m, aromatics), 8.09 (1H, s, HCO).
6.1.25. HCO-Met-Tau-Phe-Phe-OMe (6b)
From 6a (0.200 g, 0,30 mmol) according to the general
procedure G. Silica gel flash chromatography (CHCl3/
MeOH 98:2) gave the pure title product. White solid
6.1.22. Boc-Met-Leu-Tau-Phe-OMe (7a)
(0.102 g, 57%). [a]D
= +26°. M.p. 168–169 °C
From Boc-Met-Leu-OH (0.218 g, 0.57 mmol) and
HBr.Tau-Phe-OMe (0.228 g, 0.57 mmol) according to gene-
ral procedure E. White solid (0.228 g; 64%). [a]D = –25°.
M.p. 126–127 °C (hexane). IR: m 3420, 2960, 1715, 1673,
(EtOAc/hexane). IR: m 3412, 2955, 1742, 1673, 1521, 1335,
1145 cm–1. 1H NMR d: 1.97–2.18 (2H, m, Met b-CH2), 2.13
(3H, s, S–CH3), 2.55 (2H, t, J = 7.1 Hz, Met c-CH2), 2.68–
3.22 (6H, m, Tau a-CH2 and two Phe b-CH2), 3.30 and 3.60
(2H, two m, Tau b-CH2), 3.73 (3H, s, COOCH3), 4.20 (2H,
m, Phe a-CH), 4.58 (1H, m, Met a-CH), 4.85 (1H, m, Phe
a-CH), 5.96 (1H, d, J = 8.1 Hz, Phe NH), 6.79 (1H, d,
J = 8.0 Hz, Met NH), 7.11 (1H, d, J = 8.1 Hz, Phe NH),
7.21–7.33 (11H, m, aromatics and Tau NH), 8.07 (1H, s,
HCO).
1
1498, 1339, 1219, 1146, 1144 cm–1. H NMR d: 0.93 and
0.96 [6H, two d, J = 6.3 Hz, CH2CH(CH3)2], 1.46 [9H, s,
C(CH3)3], 1.69–1.75 [3H, m, CH2CH(CH3)2], 1.90–2.13
(2H, m, Met b-CH2), 2.12 (3H, s, S–CH3), 2.59 (2H, t,
J = 7.1 Hz, Met c-CH2), 2.94 (2H, m, Tau a-CH2), 3.07 and
3.21 (2H, A and B of an ABX, J = 5.1, 8.8 and 14.0 Hz, Phe
b-CH2), 3.45 and 3.68 (2H, two m, Tau b-CH2), 3.79 (3H, s,
COOCH3), 4.23 (1H, m, Met a-CH), 4.33 (1H, m, Leu
a-CH), 4.47 (1H, m, Phe a-CH), 5.20 (1H, d, J = 7.2 Hz, Met
NH), 5.83 (1H, d, J = 8.9 Hz, Phe NH), 6.69 (1H, d,
J = 8.9 Hz, Leu NH), 7.24–7.34 (5H, m, aromatics).
6.2. Biological assays
6.2.1. Cell preparation
Cells were obtained from the blood of healthy subjects,
and human peripheral blood neutrophils were purified by
using the standard techniques of dextran (Pharmacia,
Uppsala, Sweden) sedimentation, centrifugation on Ficoll-
Paque (Pharmacia), and hypotonic lysis of contaminating red
6.1.23. HCO-Met-Leu-Tau-Phe-OMe (7b)
From 7a (0.150 g, 0.22 mmol) according to the general
procedure G. Silica gel flash chromatography (CHCl3/
MeOH 98:2) gave the pure title product. White solid.

962
C. Giordano et al. / IL FARMACO 59 (2004) 953–963
cells. Cells were washed twice and resuspended in Krebs–
Ringer phosphate (KRPG), pH 7.4, at final concentration of
50 × 106 cells/ml and kept at room temperature until used.
Neutrophils were 98–100% viable, as determined using the
Trypan Blue exclusion test.
plate 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 85 1 mg per 1 ×
107 cells/min. The values are the mean of five separate
experiments done in duplicate. Standard errors are in the
range 1–6%.
6.2.2. Random locomotion
Random locomotion was performed with 48-well micro-
chemotaxis chamber (Bio Probe, Milan, Italy) and migration
into the filter was evaluated by the leading-front method [21].
The actual control random movement is 35 3 µm SE of
10 separate experiments performed in duplicate.
Acknowledgements
We are grateful to Banca del Sangue di Ferrara for provi-
ding fresh blood.
6.2.3. Chemotaxis
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 (BSA; Orha Behringwerke, Germany) and used at
concentrations ranging from 10–12 to 10–5 M. Data were
expressed in terms of chemotactic index (CI), which is the
ratio: (migration toward test attractant minus migration to-
ward 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 CI range.
References
[1] C. Giordano, G. Lucente, M. Nalli, G. Pagani Zecchini,
M. Paglialunga Paradisi, K. Varani, S. Spisani, Synthesis and activity
of HCO-Met-Leu-Phe-OMe analogues containing b-alanine or tau-
rine at the central position, Farmaco 58 (2003) 1121–1130.
[2] C. Giordano, G. Lucente, A. Mollica, M. Nalli, G. Pagani Zecchini,
M. Paglialunga Paradisi, E. Gavuzzo, F. Mazza, S. Spisani, Hybrid
a/b3-peptides with proteinogenic side chains. Monosubstituted ana-
logues of the chemotactic tripeptide for-Met-Leu-Phe-OMe, J. Pep-
tide Sci. 10 (2004) 510–523.
[3] D. Seebach, S. Abele, K. Gademann, G. Guichard, T. Hintermann,
B. Jaun, J.L. Matthews, J.V. Schreiber, L. Oberer, U. Hommel,
H. Widmer, b2- and b3-peptides with proteinaceous side chains. Syn-
thesis and solution structures of constitutional isomers, a novel helical
secondary structure, and the influence of salvation and hydrophobic
interactions on folding, Helv. Chim. Acta 81 (1998) 932–982.
–
6.2.4. Superoxide anion (O2 ) production
This anion was measured by the superoxide dismutase-
inhibitable reduction of ferricytochrome c (Sigma, USA)
modified for microplate-based assays. Tests were carried out
in a final volume of 200 ml containing 4 × 105 neutrophils,
100 nmoles cytochrome c and KRPG. At zero time different
amounts (10–10–8 × 10–5 M) of each peptide were added and
the plates were incubated into a microplate reader (Ceres
900, Bio-TeK Instruments, Inc.) with the compartment tem-
perature set at 37 °C. Absorbance was recorded at 550 and
468 nm. The difference in absorbance at the two wavelengths
[4] S. Sagan, T. Milcent, R. Ponsinet, O. Convert, O. Tasseau, G. Chassa-
ing, S. Lavielle, O. Lequin, Structural and biological effects of a b2- or
b3-amino acid insertion in a peptide, Eur. J. Biochem. 270 (2003)
939–949.
[5] R.J. Freer, A.R. Day, J.A. Radding, E. Schiffmann, S. Aswanikumar,
H.J. Showell, E.L. Becker, Further studies on the structural require-
ments for synthetic peptide chemoattractants, Biochemistry 19 (1980)
2404–2410.
–
was used to calculate nmoles of O2 produced using an
[6] C.K. Derian, H.F. Solomon, J.D. Higgins III, M.J. Beblavy, R.J. San-
tulli, G.J. Bridger, M.C. Pike, D.J. Kroon, A.J. Fischman, Selective
inhibition of N-formylpeptide-induced neutrophil activation by
carbamate-modified peptide analogues, Biochemistry 35 (1996)
1265–1269.
absorptivity for cytochrome c of 18.5 mM–1 cm–1. Neutro-
phils were incubated with 5 µg/ml cytochalasin B (Sigma) for
5 min prior to activation by peptides. Results were expressed
–
as net nmoles of O2 per 1 × 106 cells per 5 min and are the
[7] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, G. Pochetti, Structure
and conformation of peptides containing the sulphonamide junction,
Int. J. Peptide Protein Res. 34 (1989) 319–324.
mean of six separate experiments performed in duplicate.
Standard errors are in 0.1–4 nmoles O2– range.
[8] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, F. Pinnen,
G. Pochetti, D. Rossi, Structure and conformation of peptides contain-
ing the sulphonamide junction. II. Synthesis and conformation of
cyclo[-MeTau-Phe-DPro-], Int. J. Peptide Protein Res. 34 (1989)
471–479.
6.2.5. Enzyme assay
The release of neutrophil granule enzymes was evaluated
by determination of lysozyme activity, modified for
microplate-based assays. Cells, 3 × 106/well, were first incu-
bated in triplicate wells of microplates with 5 µg/ml cytocha-
lasin B at 37 °C for 15 min and then in the presence of each
peptide at a final concentration of 10–10–8 × 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
lysodeikticus. The reaction rate was measured using a micro-
[9] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, F. Pinnen,
G. Pochetti, D. Rossi, Synthesis, crystal and molecular structure of a
taurine containing peptidic oxa-cyclol, Int. J. Peptide Protein Res. 37
(1991) 167–173.
[10] G. Pagani Zecchini, M.Paglialunga Paradisi, I. Torrini, G. Lucente,
E. Gavuzzo, F. Mazza, G. Pochetti, Retrosulfonamido peptide ana-
logues. Synthesis and crystal conformation of Boc-Pro-Leu-w(NH-
SO2)-Gly-NH2, Tetrahedron Lett. 32 (1991) 6779–6782.

C. Giordano et al. / IL FARMACO 59 (2004) 953–963
963
[11] A. Calcagni, D. Rossi, M. Paglialunga Paradisi, G. Lucente, G. Luisi,
E. Gavuzzo, F. Mazza, G. Pochetti, M. Paci, Peptides containing the
sulfonamide junction: synthesis, structure, and conformation of
Z-Tau-Pro-Phe-NHiPr, Biopolymers 41 (1997) 555–567.
[16] I. Torrini, G. Pagani Zecchini, M. Paglialunga Paradisi, S. Spisani,
The role of (Z)-2,3-didehydrophenylalanine C-terminal residue in
determining the chemotactic activity of formylpeptides, Arch. Phar.
Pharm. Med. Chem. 329 (1996) 143–148.
[12] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, E. Morera,
M. Paglialunga Paradisi, D. Rossi, Peptides containing the sulfona-
mide junction: 2. Structure and conformation of Z-Tau-Pro-D-Phe-
NHiPr, Biopolymers 54 (2000) 379–387.
[17] A.J. Brouwer, M.C.F. Monnee, R.M.J. Liskamp, An efficient synthe-
sis of N-protected b-aminoethanesulfonyl chlorides: versatile build-
ing blocks for the synthesis of oligopeptidosulfonamides, Synthesis
11 (2000) 1579–1584.
[13] A. Calcagni, E. Gavuzzo, F. Mazza, F. Pinnen, G. Pochetti, D. Rossi,
Structure and conformation of peptides containing the sulphonamide
junction. IV. Synthesis and crystal conformation of N-benzoyl-L-
phenylalanyl-tauryl-L-leucine methyl ester, Gazz. Chim. Ital. 122
(1992) 17–23.
[18] J. Diaz, C. Cazaubon, H. Demarna, J.P. Gagno, R. Guegan,
Y. Muneaux, P. Perreaut, J.P. Richaud, M. Vedel, R. Roncucci, Eur. J.
Med. Chem. Chim. Ther. 20 (1985) 219–227.
[19] G. Lajoie, J.L. Kraus, Rapid and efficient method for the
[14] R.J. Freer, A.R. Day, N. Muthukumaraswamy, D. Pinon, A. Wu,
H.J. Showell, E.L. Becker, Formyl peptide chemoattractants: a model
of the receptor on rabbit neutrophils, Biochemistry 21 (1982) 257–
263.
N-formylation of C-blocked peptides, Peptides 5 (1984) 653–654.
[20] A.B. Edmundson, K.R. Ely, Binding of N-formylated chemotactic
peptides in crystals of the Mcg light chain dimer: similarities with
neutrophil receptors, Molecular Immunology 22 (1985) 463–475.
[15] I. Torrini, M. Paglialunga Paradisi, G. Pagani Zecchini, G. Lucente,
G. Mastropietro, S. Spisani, Bioactive fMLF-OMe analogs containing
a N-terminal oximic or formylhydrazonic moiety, J. Pept. Res. 55
(2000) 102–109 (references therein).
[21] S.H. Zigmond, J.G. Hirsch, Leukocyte locomotion and chemotaxis.
New methods for evaluation and demonstration of cell-derived
chemotactic factor, J. Exp. Med. 137 (1973) 387–410.