Novel chemotactic For-Met-Leu-Phe-OMe (fMLF-OMe) analogues based on Met residue replacement by 4-amino-proline scaffold: Synthesis and bioactivity

Article abstract of DOI:10.1016/j.bmc.2008.11.010

cis-(2S,4S) 4-Amino-proline (cAmp) and trans-(2S,4R) 4-amino-proline (tAmp) residues, bearing N-For or N-Boc substituents at the two amino groups, have been incorporated into the potent chemotactic agent fMLF-OMe in place of the N-terminal native (S)-methionine to give the analogues 17a-19a and 17b-19b. The new ligands have been examined for their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils as agonists and antagonists. Compounds 19a and 19b, bearing two N-For groups at the proline scaffold, are active and selective chemoattractants. The ligand 18b, containing N-For at the 4-amino group of the N-Boc-tAmp residue, exhibits significant chemotactic antagonism. The influence of the different substitution at the N-terminal position of the new analogues is discussed.

Full text of DOI:10.1016/j.bmc.2008.11.010

Bioorganic & Medicinal Chemistry 17 (2009) 251–259
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel chemotactic For-Met-Leu-Phe-OMe (fMLF-OMe) analogues based on Met
residue replacement by 4-amino-proline scaffold: Synthesis and bioactivity
Domenica Torino a, Adriano Mollica b, Francesco Pinnen b, Federica Feliciani b, Susanna Spisani c,
Gino Lucente a,
*
a Dipartimento di Chimica e Tecnologie del Farmaco and Istituto di Chimica Biomolecolare, CNR Sezione di Roma, ‘‘Sapienza”, Università di Roma, P.le A.Moro, 00185 Roma, Italy
b Dipartimento di Scienze del Farmaco, Università di Chieti-Pescara ‘‘G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy
c Dipartimento di Biochimica e Biologia Molecolare, Via L. Borsari 46, Università di Ferrara, 44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2008
Revised 30 October 2008
Accepted 3 November 2008
Available online 12 November 2008
cis-(2S,4S) 4-Amino-proline (cAmp) and trans-(2S,4R) 4-amino-proline (tAmp) residues, bearing N-For or
N-Boc substituents at the two amino groups, have been incorporated into the potent chemotactic agent
fMLF-OMe in place of the N-terminal native (S)-methionine to give the analogues 17a–19a and 17b–19b.
The new ligands have been examined for their activity (chemotaxis, superoxide anion production and
lysozyme release) on human neutrophils as agonists and antagonists. Compounds 19a and 19b, bearing
two N-For groups at the proline scaffold, are active and selective chemoattractants. The ligand 18b, con-
taining N-For at the 4-amino group of the N-Boc-tAmp residue, exhibits significant chemotactic antago-
nism. The influence of the different substitution at the N-terminal position of the new analogues is
discussed.
Keywords:
4(S)- and 4(R)-Amino-(S)-proline
Chemotaxis
fMLF-OMe analogues
Human neutrophils
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
highly sensible to alterations centred at the N-terminal formyl
group and/or at the sulphur-containing Met side chain. However,
Due to their key role in the body’s physiological defence against
bacterial infections chemotactic N-formyl peptides, which are
products of bacterial metabolism, continue to stimulate the inter-
est of chemists and biochemists. They act by binding at highly spe-
cific G-protein-coupled formylpeptide receptors (FPRs) located on
the membrane of neutrophils. The interaction triggers, in addition
to a rapid movement of the phagocytic cells towards the sites of
infections (chemotaxis), a series of intracellular responses, relevant
among which are lysosomal enzyme release and superoxide anion
production.1–4 Despite extensive studies, both the essential cellular
mechanisms controlling the neutrophil migration and the struc-
ture-activity relationships regulating the N-formylpeptide-recep-
tor interactions, are not completely understood and still under
study.4–6 However, since the discovery of N-formyl-methionyl pep-
tides as agonists at the neutrophil receptors, the potent chemotac-
tic agent N-formyl-Met-Leu-Phe-OH (fMLF) and its methyl ester
(fMLF-OMe) have been chosen as the reference molecules and a
variety of structural modifications have been performed on these
tripeptides for systematic studies on biochemical mechanism and
structure activity relationships.7–11
both the aromatic C-terminal residue and the central Leu isobutyl
side chain are more tolerant towards replacement or modification
and several analogues of fMLF-OMe modified at these two residues
have been examined. Thus, a remarkable degree of specificity is
postulated for the region of FPRs devoted to the accommodation
of the N-terminus portion of fMLF-OMe ligand and its analogues.1,8
This property has been related to the presence in the involved
receptor region of (i) a hydrophobic pocket containing an area of
positive charge suitable to interact with the electron rich methyl-
thio group of Met side chain and (ii) a small cavity capable to bind
through a hydrogen bonding system the formyl HACOANH group.
It is also relevant to note in this context that the replacement of the
small N-formyl group with the bulkier substituents such as t-butyl-
oxycarbonyl (Boc) transforms fMLF-OMe and several of its active
analogues into inactive or weakly active agonists, generally en-
dowed with antagonistic activity.2,8
In order to get information on the factors responsible for the
critical role exerted by the receptor area interacting with the N-ter-
minal elements of the chemotactic N-formyl tripeptide ligands, we
reported recently results obtained by studying new fMLF-OMe
analogues incorporating the two epimeric proline-methionine
chimera residues cis-4(S)-methylthio-(S)-proline (cMtp) and
trans-4(R)-methylthio-(S)-proline (tMtp) at N-terminal position
(Fig. 1).12 In these tripeptide analogues the presence of the pyrrol-
idine scaffold, combined with the different stereochemistry at
Examination of the above cited studies indicate that both recep-
tor recognition and agonistic activity of fMLF-OMe analogues are
* Corresponding author. Tel.: +39 06 4454218; fax: +39 06 491491.
E-mail address: gino.lucente@uniroma1.it (G. Lucente).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.010

252
D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
point concerning the ligand–receptor interaction. Based on this
Me
S
Me
S
consideration we decided to examine a group of new and unusual
fMLF-OMe derivatives in which the sulfurated Met side chain is
omitted and the 4(S) and 4(R) 4-amino-(S)-proline (cAmp and
tAmp, respectively) (Fig. 1) are used as a quite rigid skeleton on
which the N-for and N-Boc acylating groups can be attached in dif-
ferent combinations.
N
N
O
O
2. Chemistry
R
R
I (cMtp-)
II (tMtp-)
The synthesis of the N-protected 4-amino prolines 6 and 14 and
the target tripeptides 17–19 are reported in Schemes 1–3. Exami-
nation of the literature shows that efficient approaches can be fol-
lowed to obtain different 4-substituted prolines starting from the
Residues of I): cis-(2S,4S)-4-methylthio- and II):
trans-(2S,4R)-4-methylthio-proline (R = For, Boc)
commercially available trans-4-hydroxy-L-proline. cis-isomers can
be easily formed by direct Mitsunobu reaction13,14 or by nucleo-
philic SN2 displacement performed on trans-4-O-tosyl or 4-O-me-
syl N-protected derivatives.12,15–17 An alternative synthetic
H2N
H2N
strategy, based on N-protected 4-hydroxy-L-prolines, involves the
OH
OH
oxidation of the 4-OH group and the stereoselective reduction of
the resultant ketone with NaBH4. This useful stereoselective reduc-
tion, originally observed by Patchett and Witkop,18 gives exclu-
sively the cis isomer and is frequently used in the synthesis of
peptides containing 4-substituted proline analogues.13,19
N
H
N
H
O
O
According to the above reported literature data, commercially
available N-Boc-trans-(2S,4R)-4-hydroxyproline was used as start-
ing material for the synthesis of the N-Boc-cis-(2S,4S)-4-benzyl-
cis-(2S,4S)-4-amino-proline
trans-(2S,4R)-4-amino-proline
(cAmp)
(tAmp)
oxycarbonylamino-proline [Boc-cAmp(Cbz)-OH]
6 (Scheme 1).
Figure 1. Chemical structure of proline scaffolds cited in the text.
Mesylation of the 4-OH, followed by inversion of the absolute con-
17
figuration by nucleophilic substitution with NaN3 gave, after
position 4 of the ring, allowed the examination of ligands with re-
stricted spatial orientation of the N-protecting groups (N-formyl or
N-Boc) with respect to the S-methyl at position 4 and the amide
junction at position 2 of the ring. No other analogues of this type,
involving the Met residue, have been reported so far. Examination
of these new models showed, quite unexpectedly, that the two N-
For tripeptides are practically inactive while the N-Boc derivatives
show significant chemotactic activity with cMtp containing ana-
logue almost equipotent with the reference ligand fMLF-OMe. It
seems that in these models the interaction of the N-terminal func-
tional groups with the involved receptor area leads to a different
type of accommodation as compared with usual ligands. This
strongly suggests that studies based on conformationally restricted
analogues of this type may shed light on a critical and still unclear
reduction and amino protection, the desired precursor aminoacid
derivative 6. A more complex strategy was adopted for the synthe-
sis of N-Boc-trans-(2S,4R)-4-benzyloxycarbonylamino-proline
[Boc-tAmp(Cbz)-OH] 14, the diastereoisomeric analogue of 6. In
this case (Scheme 2) the N-Boc-trans-(2S,4R)-4-hydroxyproline
was initially oxidized to give the 4-oxo derivative 7. This was ste-
reoselectively reduced18,19 to N-Boc-cis-(2S,4S)-4-hydroxyproline 8
and esterified with ethereal diazomethane to give the methyl ester
9 as a single product on TLC. The same sequence of reactions, al-
ready followed to obtain compound 6, led to the final N-Boc-
tAmp(Cbz)-OH 14.
The above described precursors 6 and 14 were used for the syn-
thesis of two series of fMLF-OMe analogues. Each series contains,
as reported in Scheme 3, cAmp (compounds 17a–19a) or tAmp
HO
HO
N3
Ms-O
OH
a
d
c
b
O
N
O
O
N
N
N
O
Boc
O
O
O
Boc
Boc
Boc
1
2
3
H2N
Cbz-HN
Cbz-HN
e
f
O
O
OH
N
N
N
O
O
O
Boc
Boc
Boc
6
4
5
Scheme 1. Synthesis of compound 6. Reagents and conditions: (a) ethereal diazomethane/MeOH, 100%; (b) MsCl, TEA, CH2Cl2, 0 °C 3 h, 96%; (c) NaN3/DMF, 55 °C overnight,
73%; (d) H2/Pd–C, MeOH, rt 6 h, 76%; (e) Cbz-Cl/TEA/CH2Cl2 55%.; (f) NaOH 1 N/MeOH, 75%.

D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
253
HO
O
HO
HO
OH
b
c
OH
d
a
OH
N
N
O
N
N
O
O
O
Boc
Boc
Boc
O
Boc
7
8
9
Ms-O
N3
H2N
Cbz-HN
O
f
e
g
O
O
O
N
N
N
N
Boc
13
O
O
O
O
Boc
Boc
11
Boc
12
10
Cbz-HN
h
OH
N
O
Boc
14
Scheme 2. Synthesis of compound 14. Reagents and conditions: (a) CrO3/Py, 31%; (b) NaBH4/H2O, MeOH, 80%; (c) ethereal diazomethane/MeOH, 100%; (d) MsCl, TEA, CH2Cl2,
0 °C 30 min, 95%; (e) NaN3/DMF, 55 °C, overnight, 80%; (f) H2/Pd–C, MeOH, rt, 6 h, 52%; (g) Cbz-Cl/TEA/CH2Cl2, 33%; (h) NaOH 1 N/MeOH, 95%.
(compounds 17b–19b) as N-terminal residue. The tripeptides were
obtained by using the Leu-Phe-OMe dipeptide as common C-termi-
nal fragment to provide the orthogonally protected key precursors
15a–b. Hydrogenolytic deprotection of the Cbz-NH- bound at the
4-amino group of the proline residue gave the two N-Boc tripep-
tides 16a–b from which the two couples of target substrates
17a–b and 18a–b were obtained by N-Boc protection or N-formy-
lation, respectively, of the free proline 4-amino group.
In order to synthesize the last couple of substrates 19a–b, char-
acterized by the presence of a formyl group bound at each of the
two amino functions of the prolinic scaffold, Boc deprotection fol-
lowed by N-formylation of 16a–b is required. When the deprotec-
tion was performed with trifluoroacetic acid and the resulting
tripeptidic bis-trifluoroacetates were used for the subsequent
formylation reaction low yields and rather complex reaction mix-
tures were obtained. This reaction sequence was sensibly im-
proved when HCOOH, instead of the usual trifluoroacetic acid,
was used for N-Boc deprotection and the resulting bis-formates
were formylated by adopting the mixed anhydride strategy.
chemotactic agonists are the two peptides 19a and 19b, both char-
acterized by the presence of a N-For acylating group at both the
amino groups of the proline scaffold. At very low concentration
(about 10À12 M) these models equal the activity of the reference
molecule and reach maximal activity (0.75 C.I.) at a concentration
(10À10 M), lower than that where the fMLF-OMe peak (10À9 M) ap-
pears (Table 1).
All the models examined are practically inactive as superoxide
anion producers (Fig. 2B) and only very weak secretagogue agents
(Fig. 2C). The highest value for this latter function (24% of lysozyme
release at concentration 10À6 M) is shown by the peptide 19a con-
taining two N-For acylating group at the cAmp residue.
3.2. Antagonism
All models under study were tested for their ability to inhibit
the three biological functions, except for the chemotaxis of com-
pounds 19a–b which are good agonists in this function. The antag-
onism was determined by measuring the ability to inhibit the
activity stimulated by fMLF-OMe on human neutrophils. The influ-
ence of an increasing concentration of 17a–b and 18a–b on chemo-
taxis induced by 10 nM fMLF-OMe, which is the optimal dose for
chemotaxis activation, is shown in Figure 3A. A significant dose-
dependent inhibition of the chemotactic index, which reaches the
value of 73% at the highest concentration, is observed for com-
pound 18b containing the 4-NH For substituent at the N-Boc-tAmp
residue. Interestingly, the epimeric model 18a, containing the
cAmp residue, is only a very modest antagonist. Good chemotactic
antagonism and almost identical values (about 60% inhibition at
the maximum concentration) are also shown by 17a and 17b con-
taining two N-Boc groups at the cAmp and tAmp residue, respec-
tively. As can be deduced by Figure 3B and C, which refer to the
superoxide anion production and inhibition of lysozyme release,
only the two latter mentioned analogues (17a and 17b) exhibit a
modest activity in the superoxide anion production inhibition,
with a maximum value of 30% at 10À5 M concentration (Fig. 3B).
None of the other tripeptides show significant antagonism towards
3. Results and discussion
3.1. Agonism
The biological activity of the six models under study has been
determined on human neutrophils and compared with that of
the parent tripeptide fMLF-OMe.20,21 The analogues of the two ser-
ies (17–19a and 17–19b) were evaluated for their ability to acti-
vate directed migration (chemotaxis), stimulate production of
superoxide anion radicals (O2À) and induce lysozyme release from
granules of human neutrophils. Results are plotted in Figure 2
and the activity values exhibited at the optimal peptide concentra-
tion are summarized in Table 1. All the four models containing the
bulky N-Boc protecting group (i.e: compounds 17a, 18a and 17b,
18b) show moderate chemotactic activity with the highest value
(0.45 C.I.) reached by the tAmp containing peptide 17b at the opti-
mal concentration of 10À10 M (Table 1 and Fig. 2A). Highly active

254
D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
O
TFA. H2N
O
N
H
O
b
a
Cbz-HN
Cbz-HN
O
O
H
H
N
O
O
N
O
N
N
H
N
N
H
O
c
O
O
Boc
Boc
15a
15b
c
H2N
H2N
O
O
H
H
N
N
O
O
N
N
N
N
H
H
O
O
O
O
Boc
Boc
16a
16b
d
e
f
f
d
e
17a
R'-HN
18a
19a
17b
19b
18b
R'-HN
O
O
H
H
N
O
N
O
N
R
N
H
N
R
N
H
O
O
O
O
17a
: R = R' = Boc
17b: R = R' = Boc
18b: R = Boc; R' = For
19b
18a: R =Boc; R' = For
19a: R = R' = For
: R = R' = For
Scheme 3. Synthesis of compounds 17a,b–19a,b. Reagents and conditions: (a) Boc-cAmp(Cbz)-OH (6), TFAÁLeu-Phe-OMe, IBCF, NMM, CH2Cl2, 64%; (b) Boc-tAmp(Cbz)-OH,
TFAÁLeu-Phe-OMe, IBCF, NMM, CH2Cl2, 72%; (c) H2/Pd–C, MeOH, rt, 3 h, 100%; (d) Boc2O/EtOAc/TEA, rt, 1 h, (17a: 62%; 17b: 55%); (e) HCOOH, IBCF, NMM, CH2Cl2 (18a 59%;
18b 73%); (f) HCOOH, IBCF, NMM, CH2Cl2, (19a 36%; 19b 26%).
both superoxide anion production and lysozyme release (Figs. 3B
and C).
which also supports the bulky N-Boc protecting group. Contrary
to our expectation, suggested in part by results obtained with
fMLF-OMe analogues containing the N-Boc-4-methylthio-proline
at the N-terminal position, all the synthesized tripeptides charac-
terized by the presence of the Boc group, including the two models
18a–b bearing a N-formylic group at the 4-amino-proline scaffold,
display only modest chemotactic activity, with a maximum value
of 0.45 of the chemotactic index (Table 1) shown by tripeptide
17b. Conversely, the derivatives 19a and 19b, in which both the
N-terminal amino groups are formylated, are potent agonists with
comparable potency. These latter models are among the first fMLF
analogues which, although devoid of the sulfur-containing Met
3.3. Conclusion
In the present study, we tried to evaluate the functional group
requirements of the FPRs in the critical region devoted to the inter-
action with the N-terminal residue of fMLF and related N-formyl-
tripeptide ligands. Peptide models used in this effort are of an
unusual type and contain the pyrrolidine ring of 4-amino L-proline
in place of the native methionine. The crucial formyl group has
been bound at either of the amino groups of the proline scaffold

D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
255
70
60
50
40
30
20
10
0
1.2
1
60
C
A
B
50
40
30
20
10
0
0.8
0.6
0.4
0.2
0
-5 5*10- 5
-13 -12 -11 -10 -9
-8
-7
-6
-5
-10
-9
-8
-7
-6
-5 5*10- 5
-10
-9
-8
-7
-6
Log[M]
Log[M]
Log[M]
fMLF-OMe
18a
19a
19b
17b
17a
18b
Figure 2. Biological activities of tripeptide derivatives 17a,b–19a,b and fMLF-OMe. (A) Chemotactic index (C.I.); (B) superoxide anion production; (C) release of neutrophil
granule enzymes evaluated by determining lysozyme activity.
Table 1
fication at the N-terminal residue of fMLF-OMe may represent an
Chemotactic activity, superoxide anion production and lysozyme release of fMLF-
OMe derivatives
useful basis for the design of ‘‘pure” chemotactic agonists. As it is
well known,5,22,23 agonists of this type capable to select or modu-
OÀ2 (nmol)
% Lysozyme releasea
a
Peptides
Chemotactic indexa
late the different neutrophil responses (distinct from ‘‘full” ago-
nists), are of great utility as probes to gain information on the
complex biochemical mechanisms regulating the signal transduc-
tion pathways.4
fMLF-OMe
17a
18a
19a
17b
1.10 0.08 (10À9 M)
0.40 0.02 (10À9 M)
0.34 0.02 (10À9 M)
0.70 0.01 (10À10 M)
0.45 0.01 (10À10 M)
0.40 0.02 (10À9 M)
0.75 0.02 (10À10 M)
52 4 (10À6 M)
14.6 1 (10À5ÀM5 )
2.0 0.5 (10 M)
4.0 0.5 (10À6 M)
2.0 0.5 (10À5 M)
1.0 0.5 (10À5 M)
5.0 0.5 (10À6 M)
60 4 (10À6 M)
12 1 (10À6 M)
12 1 (10À6 M)
24 2 (10À6 M)
18 1 (10À6 M)
13 1 (10À6 M)
15 1 (10À6 M)
In conclusion, in this study a new type of fMLF-OMe tripeptide
analogues, obtained by replacing the critical native Met residue
with a 4-amino-proline scaffold bearing different substituents,
have been described. This synthetic strategy leads to molecules
which, depending upon the nature of the acylating groups at the
N-terminal position, exhibit significant chemotactic agonism or
antagonism with selectivity towards the neutrophil biological
functions. Based on these results and taking into account the re-
cent identification of novel FPRs agonists which broadens the spec-
trum of functional significance of such receptors,24,25 further
models possessing the basic structural feature characterizing the
here described analogues are in progress in our laboratories.
18b
19b
, Standard errors of the mean (SEM); optimal concentration values in parentheses.
a
Values indicate the efficacy (activity at the optimal peptide concentrations).
side chain, exhibit good chemotactic activity. It seems then that the
absence of bulky groups at the N-terminal position, combined with
the presence of the N-For groups at the rigid scaffold, may lead to a
right fitting with the receptor even if the methylthio group of the
Met side chain is absent. Connected with the chemotactic activity
of 19a and 19b is the antagonism displayed by the N-Boc-tripep-
tide 18b and, with reduced potency, by 17a–b (Fig. 3A). It can be
seen here that the replacement of the formylic group with a bulky
substituent leads to the same change of activity from agonism to
antagonism, primarily observed on passing from fMLF-OMe to
Boc-MLF-OMe. This behaviour is not observed in the case of the
fMLF-OMe analogues containing proline-methionine chimera resi-
dues at the N-terminal position. Finally, concerning the different
stereochemistry at the 4-amino-proline residue, characterizing
the three couples of the examined tripeptides, this feature does
not influence appreciably the biological functions tested, regard-
less of the presence of N-Boc or N-For substituents. However, as
can be seen by the values summarized inTable 1 the trans models
are in any case slightly more active than the epimeric cis models.
A final observation concerns the agonistic activity of the tripep-
tides under study toward the superoxide anion production and
lysozyme release (Fig. 2B and C). With the exception of the low val-
ues observed for the secretagogue activity in the case of 19a (24%
as maximum value at 10À6 M concentration; Table 1 and Fig. 2B)
all the other compounds are practically inactive toward these
two biological functions. Thus, the here reported structural modi-
4. Experimental
4.1. Chemistry
4.1.1. General procedures
Optical rotations were taken at 20 °C with a Schmidt–Haensch
Polartronic D polarimeter (1 dm cell, in CHCl3 except otherwise
stated). Anhydrous solvents were prepared by distillation over so-
dium metal or calcium hydride prior to use. Thin-layer and pre-
parative layer chromatographies were performed on Merck 60
F254 silica gel plates. The drying agent was sodium sulphate. 1H
NMR spectra were determined in CDCl3 solution with a Bruker
AM 400 spectrometer and chemical shifts were indirectly referred
to TMS. Coupling constants are in Hz. Most of the reactions were
carried out under inert atmosphere of nitrogen. The reported yields
are for purified materials but were not optimized.
4.1.1.1 Catalytic hydrogenolysis. A solution of the compound in
MeOH and 10% Pd on C (10%) was vigorously stirred at room tem-
perature and atmospheric pressure for 2–6 h (TLC control) under a

256
D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
120
100
80
60
40
20
0
120
120
100
80
60
40
20
0
A
C
B
100
80
60
40
20
0
-10
-9
-8
-7
-6
-5
-12 -11 -10 -9
-8
-7
-6
-5
-10
-9
-8
-7
-6
-5
Log[M]
Log[M]
Log[M]
17a
17b
18a
18b
19a
19b
Figure 3. Effect of N-Boc-protected tripeptides 17a,b–19a,b on the neutrophil activities triggered by fMLF-OMe. (A) Chemotactic index (C.I.); (B) superoxide anion
production; (C) release of neutrophil granule enzymes evaluated by determining the lysozyme activity.
slow stream of hydrogen. The catalyst was removed by filtration
and washed with MeOH. The filtrate was evaporated under re-
duced pressure and used for the subsequent reaction.
Pro
44.57; H, 6.55; N, 4.33. Found: C, 44.67; H, 6.58; N, 4.31.
a
-CH), 5.24 (1H, m, Pro C4H). Anal. Calcd for C12H21NO7S: C,
4.1.2.3. N-Boc-cis-(4-azido)-L-proline methyl ester (3). A solu-
4.1.1.2. Coupling with the mixed anhydride method. To a mix-
ture cooled at À15 °C containing the required N-protected amino
acid (1.0 mmol) in CH2Cl2 were added isobutyl chloroformate IBCF
(1.0 mmol) and NMM (1.1 mmol). The mixture was stirred for
10 min at the same temperature and then the required amino-
derivative salt (1.0 mmol) and NMM (1.1 mmol) were added. After
15 min at À15 °C the mixture was kept at room temperature over-
night. After removal of the solvent under reduced pressure, the res-
idue was dissolved in EtOAc and washed with 5% citric acid
(2 Â 15 mL), NaHCO3 ss (2 Â 15 mL) and NaCl ss (15 mL). The or-
ganic layer was dried over Na2SO4 and evaporated under reduced
pressure.
tion of 2 (0.806 g, 2.49 mmol) and NaN3 (0.194 mg, 2.98 mmol)
in dry DMF (5 mL) was stirred at 55 °C overnight. After removal
of the solvent under reduced pressure, the residue was dissolved
in EtOAc and washed with water and NaCl ss. The organic layer
was dried and evaporated under reduced pressure. The residue
was purified by silica gel chromatography (petroleum ether/EtOAc
5:1) to give pure 3 as a colourless oil (0.496 g, 73%).
4.1.2.4. N-Boc-cAmp-OMe (4). Reduction of the azido group was
performed by adopting the general procedure above reported for
the catalytic hydrogenolysis:
3 (0.496 g, 1.84 mmol), MeOH
(16.5 mL) and Pd–C (0.0496 g) gave 4 (0.34 g, 76%) after 6 h at
room temperature.
4.1.2. Synthesis
4.1.2.1. N-Boc-trans-(4-hydroxy)-L-proline methyl ester (1). To
4.1.2.5. N-Boc-cAmp(Cbz)-OMe (5). To an ice-cooled solution of
4 (0.359 g, 1.47 mmol) in CH2Cl2/DMF 9:1 (20 mL) TEA (0.62 mL,
4.41 mmol) and Cbz-Cl (0.29 mL, 1.76 mmol) were added. The
resulting mixture was stirred for 2 h at 0 °C and 1 h at room tem-
perature. After removal of the solvent under reduced pressure, the
residue was dissolved in EtOAc and washed with 5% citric acid,
NaHCO3 ss and NaCl ss. The organic layer was dried and evaporated
under reduced pressure. The residue was purified by silica gel
chromatography (CHCl3/EtOAc 7:3) to give pure 5 as a colourless
a solution of N-Boc-trans-4-hydroxy-L-proline (1 g, 4,33 mmol) in
methanol (10 mL) an ethereal diazomethane solution was added
until a yellow colour persisted. The solution was evaporated under
reduced pressure to give title compound, pure on TLC (quantitative
yield); [
a
]
D À65° (c 0.6); 1H NMR d: 1.49 [9H, s, C(CH3)3], 2.05–2.31
(2H, m, Pro C3H2), 3.48–3.67 (2H, m, Pro d-C5H2), 3.75 (3H, s,
COOCH3), 4.39–4.51 (2H, m, Pro
a
-CH and Pro C4H). Anal. Calcd
for C11H19NO5: C, 53.87; H, 7.81; N, 5.71. Found: C, 53.81; H,
7.82; N, 5.75.
oil (0.308 g, 55%); [
1698 cmÀ1 1H NMR d: 1.45 [9H, s, C(CH3)3], 1.89–2.03 and 2.39–
2.6 (2H, m, Pro C3H2), 3.4–3.81 (2H, m, Pro C5H2), 3.71 (3H, s,
COOCH3), 4.21–4.5 (2H, m, Pro
-CH, Pro C4H), 5.18 (2H, s, C6H5-
a]
À13.9° (c 0.3); IR (CHCl3)
m: 3434, 1743,
D
;
4.1.2.2.
N-Boc-trans-(4-mesyloxy)-L-proline methyl ester
(2). To an ice-cooled solution of the methyl ester 1 (1.06 g,
4.33 mmol) in dry CH2Cl2 (10 mL), TEA (0.73 mL, 5.2 mmol) and
MsCl (0.4 mL, 5.2 mmol) were added. The resulting mixture was
stirred for 3 h at 0 °C. The mixture was diluted with CH2Cl2 and
washed with 1 N HCl, NaHCO3 ss and brine. The organic layer
was dried and evaporated under reduced pressure to yield 1.34 g
a
CH2), 5.75 (1H, d, J = 8, Z-NH), 7.03–7.45 (5H, m, aromatic). Anal.
Calcd for C19H26N2O6: C, 60.30; H, 6.93; N, 7.40. Found: C, 60.47;
H, 6.90; N, 7.43.
(96%) of pure 2 as a pale yellow oil; [
a
]
À52° (c 1.6); 1H NMR d:
4.1.2.6. N-Boc-cAmp(Cbz)-OH (6). To an ice-cooled solution of
D
1.41 [9H, s, C(CH3)3], 2.21–2.67 (2H, m, Pro C3H2), 2.98 (3H, s,
CH3SO2), 3.70–3.85 (5H, m, Pro C5H2 and COOCH3), 4.44 (1H, m,
methyl ester
5 (0.145 g, 0.38 mmol) in MeOH 1 N NaOH
(1.14 mL) was added dropwise and the mixture was stirred at

D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
257
0 °C for 15 min and at room temperature overnight. The MeOH was
evaporated under reduced pressure, water was added and the solu-
tion extracted with diethyl ether (2 Â 25 mL). The aqueous phase
was then acidified with 5% citric acid and the product extracted
with EtOAc (3 Â 20 mL). The organic layer was washed with brine,
pooled, dried and evaporated to give the TLC pure title product as
white foam (0.105 g, 75%).
was dried and evaporated under reduced pressure. The residue
was purified by silica gel chromatography (CH2Cl2/MeOH
99.8:0.2) to give pure 11 as a colourless oil (0.434 g, 80%).
4.1.2.12. N-Boc-tAmp-OMe (12). Reduction of the azido group
was performed by adopting the general procedure above reported
for the catalytic hydrogenolysis: 11 (0.469 g, 1.74 mmol), MeOH
(15.6 mL) and Pd–C (0.0469 g) gave 12 (0.22 g, 52%) after 6 h at
room temperature.
4.1.2.7. N-Boc-(4-oxo)-L-proline (7). To a mixture of pyridine
(8.8 mL) and dry CH2Cl2 (20 mL) cooled at 0 °C, CrO3 (5.2 g,
52 mmol) was added. Stirring was continued at 0 °C for 30 min
and the mixture was allowed to warm at room temperature over
4.1.2.13. N-Boc-tAmp(Cbz)-OMe (13). To an ice-cooled solution
of
4 (0.22 g, 0.902 mmol) in CH2Cl2/DMF 9:1 (15 mL) TEA
a period of 30 min. A solution of N-Boc-4-trans-hydroxy-
L
-proline
(0.33 mL, 2.706 mmol) and Cbz-Cl (0.179 mL, 1.082 mmol) were
added. The resulting mixture was stirred for 2 h at 0 °C and 1 h
at room temperature. After removal of the solvent under reduced
pressure, the residue was dissolved in EtOAc and washed with
5% citric acid, NaHCO3 ss and NaCl ss. The organic layer was dried
and evaporated under reduced pressure. The residue was purified
by silica gel chromatography (CHCl3/EtOAc 9:1) to give pure 13
(2 g, 8.66 mmol) in CH2Cl2 (30 mL) was added over 5 min and stir-
ring was continued for 1 h at room temperature. The mixture was
taken up in diethyl ether and washed with 5% citric acid and brine.
The organic layer was dried, filtered and evaporated under reduced
pressure to give crude compound 7 (0.610 g, 31%), as a pale yellow
solid; [
2.75 (2H, m, Pro C3H2), 3.50–3.90 (2H, m, Pro C5H2), 4.75 (1H, m,
Pro -CH). Anal. Calcd for C10H15NO5: C, 52.40; H, 6.60; N, 6.11.
Found: C, 52.27; H, 6.61; N, 6.09.
a]
+18° (c 0.5, acetone); 1H NMR d: 1.45 [9H, s, C(CH3)3],
D
as a colourless oil (0.11 g, 33%); [
3439, 1742, 1697 cmÀ1 1H NMR d: 1.42 [9 H, s, C(CH3)3], 2.18–
2.26 (2H, m, Pro C3H2), 3.36–3.43 and 3.76–3.81 (2H, m, Pro
a]
À23.4° (c 0.4); IR (CHCl3)
m:
D
a
;
C5H2), 3.75 (3H, s, COOCH3), 4.27–4.40 (2H, m, Pro
a-CH, Pro
4.1.2.8. N-Boc-cis-(4-hydroxy)-L-proline
(8). N-Boc-4-oxo-
L
-
C4H), 4.92 (1H, m, Cbz-NH), 5.18 (2H, s, C6H5-CH2-O-), 7.1–7.47
(5H, m, aromatic). Anal. Calcd for C19H26N2O6: C, 60.30; H, 6.93;
N, 7.40. Found: C, 60.42; H, 6.91; N, 7.41.
proline 7 (0.610 g, 2.66 mmol) was dissolved in MeOH (12 mL)
and the reaction flask was cooled to 0 °C. A solution of NaBH4
(0.328 g, 8.67 mmol) in water (1.45 mL) was added dropwise; after
10 min the reaction was allowed to stand at 4 °C for 15 min and
concentrated under reduced pressure. The residue was diluted
with water, acidified to pH 3 with 10% citric acid and extracted
with EtOAc. The organic layer was washed with brine, dried and
concentrated to yield 0.490 g of TLC pure derivative 8 (80%) as a
4.1.2.14. N-Boc-tAmp(Cbz)-OH (14). To an ice-cooled solution of
methyl ester 13 (0.1 g, 0.264 mmol) in MeOH (5 mL) 1 N NaOH
(0.792 mL) was added dropwise and the mixture was stirred at
0 °C for 15 min and at room temperature overnight. The MeOH
was evaporated under reduced pressure, water was added and
the solution extracted with diethyl ether (2 Â 25 mL). The aqueous
phase was then acidified with 5% citric acid and the product ex-
tracted with EtOAc. The organic layer was washed with brine,
pooled, dried and evaporated to give the TLC pure title product
14 as white foam (0.09 g, 95%).
foam; [
a
]
À39° (c 0.66, MeOH); 1H NMR d: 1.45 [9H, s, C(CH3)3],
D
2.19–2.59 (2H, m, Pro C3H2), 3.44–3.56 (2H, m, Pro C5H2), 4.53–
4.65 (2H, m, Pro
C, 51.94; H, 7.41; N, 6.06. Found: C, 52.09; H, 7.42; N, 6.04.
a
-CH and Pro C4H). Anal. Calcd for C10H17NO5:
4.1.2.9. N-Boc-cis-(4-hydroxy)-
L-proline methyl ester (9). To a
solution of N-Boc-cis-4-hydroxy-L-proline (0.490 g, 2.12 mmol) in
4.1.2.15. N-Boc-cAmp(Cbz)-Leu-Phe-OMe (15a). Compound 6
methanol (4.9 mL) an ethereal diazomethane solution was added
until a yellow colour persisted. The solution was evaporated under
reduced pressure to give the title compound pure on TLC (quanti-
(0.105 g, 0.288 mmol) in CH2Cl2 (5 mL) was treated with
TFAÁLeu-Phe-OMe (0.117 g, 0.29 mmol) according to the general
procedure B. Silica gel chromatography (CHCl3/EtOAc 3:1) gave a
tative yield); [
a
]
À63.9° (c 2.3, EtOH); 1H NMR d: 1.45 [9 H, s,
pure product 15a as a white foam (0.119 g, 64%); [
a]
D
À65.9° (c
D
C(CH3)3], 2.07–2.49 (2H, m, Pro C3H2), 3.52–3.65 (2H, m, Pro
0.3,); IR (CHCl3)
m
: 3421, 1741, 1681 cmÀ1 1H NMR d: 0.69–1.0
;
C5H2), 3.77 (3H, s, COOCH3), 4.27–4.40 (2H, m, Pro
a-CH and Pro
[6H, m, Leu CH(CH3)2], 1.45 [9H, s, C(CH3)3], 1.59–1.68 [3H, m,
Leu CH2-CH(CH3)2], 2.0–2.4 (2H, m, Pro C3H2), 3.0–3.21 (2H, m,
Phe b-CH2) 3.38–3.6 (2H, m, Pro C5H2), 3.71 (3H, s, COOCH3), 4.2–
C4H). Anal. Calcd for C11H19NO5: C, 53.87; H, 7.81; N, 5.71. Found:
C, 53.92; H, 7.79; N, 5.72.
4.42 (3H, m, Pro
a-CH), 5.18 (2H, s, C6H5-CH2-O-), 6.5 (1H, d, J = 7.6, Cbz-NH), 6.67
a a-CH), 4.84 (1H, m, Phe
-CH, Pro C4H and Leu
4.1.2.10.
N-Boc-cis-(4-mesyloxy)-
L
-proline
methyl
ester
(10). To an ice-cooled solution of the methyl ester 9 (0.519 g,
2.12 mmol) in dry CH2Cl2 (4.9 mL) TEA (0.385 mL, 2.76 mmol)
and MsCl (0.215 mL, 2.76 mmol) were added. The resulting mix-
ture was stirred for 30 min at 0 °C. The mixture was diluted with
CH2Cl2 and washed with 1 N HCl, Na2CO3 ss and brine. The organic
layer was dried and evaporated under reduced pressure to yield
0.65 g (95%) of pure 10 as a pale yellow oil. 1H NMR d: 1.44 [9H,
s, C(CH3)3], 2.48–2.60 (2H, m, Pro C3H2), 3.03 (3H, s, CH3SO2),
3.75 and 4.45 (2H, m, Pro C5H2), 3.77 (3H, s, COOCH3), 4.44 and
(1H, d, J = 7.6, Phe-NH), 7.03 (10H, m, aromatic), 7.63 (1H, d,
J = 7.6, Leu NH). Anal. Calcd for C34H46N4O8: C, 63.93; H, 7.26; N,
8.77. Found: C, 63.73; H, 7.23; N, 8.74.
4.1.2.16. N-Boc-tAmp(Cbz)-Leu-Phe-OMe (15b). Compound 14
(0.091 g, 0.250 mmol) in CH2Cl2 (5 mL) was treated with TFAÁLeu-
Phe-OMe (0.101 g, 0.250 mmol) according to the general procedure
B. Silica gel chromatography (CHCl3/EtOAc 4:1) gave a pure prod-
uct 15b as a white foam (0.115 g, 72%); [
a]
D À36° (c 1.0); IR (CHCl3)
5.29 (2H, m, Pro
a
-CH and Pro C4H). Anal. Calcd for C12H21NO7S:
m
: 436, 1722, 1682 cmÀ1 1H NMR d: 0.90 [6H, m, Leu CH(CH3)2],
;
C, 44.57; H, 6.55; N, 4.33; S, 9.92. Found: C, 44.43; H, 6.53; N, 4.31.
1.47 [9H, s, C(CH3)3], 1.57 [3H, m, Leu CH2-CH(CH3)2], 2.1–2.4
(2H, m, Pro C3H2), 3.1–3.18 (2H, m, Phe b-CH2), 3.25 (2H, m, Pro
4.1.2.11. N-Boc-trans-(4-azido)-L-proline methyl ester (11).
A
C5H2), 3.74 (3H, s, COOCH3), 4.29–4.33 (3H, m, Pro -CH, Pro C4H
a
solution of 10 (0.65 g, 2.01 mmol) and NaN3 (0.157 g, 2.41 mmol)
in dry DMF (5 mL) was stirred at 55 °C overnight. After removal
of the solvent under reduced pressure, the residue was dissolved
in EtOAc and washed with water and NaCl ss. The organic layer
and Leu a-CH), 4.84 (1H, m, Phe a-CH), 5.18 (2H, s, C6H5-CH2-O-),
6.55 (1H, m, Cbz-NH), 6.71 (1H, m, Phe-NH), 7.04–7.42 (11H, m,
aromatic and Leu NH). Anal. Calcd for C34H46N4O8: C, 63.93; H,
7.26; N, 8.77. Found: C, 63.71; H, 7.21; N, 8.72.

258
D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
4.1.2.17. N-Boc-cAmp-Leu-Phe-OMe (16a). The title compound
16a was prepared starting from compound 15a by following the
general procedure A: 15a (0.108 g, 0.169 mmol), MeOH (10 mL)
and Pd–C (0.047 g), 3 h at room temperature, quantitative yield.
4.1.2.22. N-Boc-tAmp(For)-Leu-Phe-OMe (18b). To a solution of
HCOOH (0.029 g, 0.63 mmol) in CH2Cl2 (5 mL) cooled at À15 °C, IBCF
(0.082 mL, 0.63 mmol) and NMM (0.069 mL, 0.63 mmol) were
added. The mixture was stirred for 10 min at the same temperature
and then a solution in CH2Cl2 of 16b (0.032 g, 0.063 mmol) and NMM
(0.069 mL, 0.63 mmol) was added. Work up according to general
procedure B was then followed. Silica gel chromatography (CHCl3/
EtOAc 1:2) gave a pure product 18b as a white foam (0.024 g,
4.1.2.18. N-Boc-tAmp-Leu-Phe-OMe (16b). The title compound
16b was prepared starting from compound 15b by following the
general procedure A: 15b (0.040 g, 0.063 mmol), MeOH (5 mL),
Pd–C (0.004 g), 3 h at room temperature, quantitative yield.
73%); [
a
]
D À32.1° (c 0.65); IR (CHCl3)
m
: 3429, 1742, 1686 cmÀ1 1H
;
NMR d: 0.91 [6H, m, Leu CH(CH3)2] 1.47 [9H, s, C(CH3)3], 1.62 [3H,
m, Leu CH2-CH(CH3)2], 2.0–2.3 (2H, m, Pro C3H2), 3.07–3.15 (2H, m,
Phe b-CH2) 3.25 and 3.77 (2H, m, Pro C5H2), 3.74 (3H, s, COOCH3),
4.35 (2H, m, Leu
(1H, m, Phe -CH), 7.06-7.32 (8H, m, Leu NH, Phe NH, For NH and aro-
4.1.2.19. N-Boc-cAmp(Boc)-Leu-Phe-OMe (17a). To a solution
of compound 16a (0.025 g, 0.05 mmol) in EtOAc (5 mL) Boc2O
(0.022 g, 0.10 mmol) and TEA (0.014 mL, 0.10 mmol) were added
and the mixture was stirred 1 h at room temperature. The mixture
was diluted with EtOAc and washed with 5% citric acid, NaHCO3 ss
and brine. The organic layer was dried and evaporated under re-
duced pressure. The residue was purified by silica gel chromatog-
raphy (CHCl3/EtOAc 1:1) to give pure 17a as a white foam
a-CH and Pro a
-CH), 4.53 (1H, m, Pro C4H), 4.83
a
matic), 8.15 (1H, s, HCO). Anal. Calcd for C27H40N4O7: C, 60.88; H,
7.57; N, 10.52. Found: C, 61.01; H, 7.56; N, 10.56.
4.1.2.23. N-For-cAmp(For)-Leu-Phe-OMe (19a). HCOOH (0.066 g,
1.44 mmol) in CH2Cl2 (5 mL) was treated with 2HCOOHÁ cAmp-Leu-
Phe-OMe (0.024 g, 0.048 mmol) according to the general procedure
B. Preparative layer chromatography (EtOAc) gave a pure product
(0.0185 g, 62%); [
a]
À76.3° (c 0.35); IR (CHCl3)
m: 3417, 1741,
D
1681 cmÀ1 1H NMR d: 0.9 [6 H, m, Leu CH(CH3)2], 1.47 [18H, s, 2
;
C(CH3)3], 1.59 [3H, m, Leu CH2-CH(CH3)2], 2.0–2.38 (2H, m, Pro
19a as a white foam (0.008 g, 36%); [
a
]
À71.9° (c 0.5); IR (CHCl3)
C3H2), 3.0–3.11 (2H, m, Phe b-CH2), 3.37–3.57 (2H, m, Pro C5H2),
D
m
: 3427, 1740, 1687 cmÀ1 1H NMR d: 0.91 [6H, m, Leu CH(CH3)2],
;
3.76 (3H, s, COOCH3), 4.19–4.26 (3H, m, Leu
a-CH, Pro a-CH, Pro
1.62 [3H, m, Leu CH2-CH(CH3)2], 2.16–2.31 (2H, m, Pro C3H2), 3.10–
3.24 (2H, m, Phe b-CH2), 3.6 and 3.77 (2H, m, Pro C5H2), 3.73 (3H, s,
C4H), 4.83 (1H, m, Phe
a
-CH), 6.25 (1H, d, J = 7.6, Boc-NH), 6.61
(1H, d, J = 7.8, Phe NH), 7.12–7.40 (5H, m, aromatic), 7.76 (1H, d,
J = 7.6, Leu NH). Anal. Calcd for C31H48N4O8: C, 61.57; H, 8.00; N,
9.26. Found: C, 61.69; H, 7.98; N, 9.28.
COOCH3), 4.29 (1H, m, Leu
a
-CH), 4.52 (1H, m, Pro
a-CH), 4.55 (1H,
m, Pro C4H), 4.86 (1H, m, Phe
a-CH), 6.52 (1H, d, J = 7.6, Phe NH),
7.12–7.4 (5H, m, aromatic), 7.5 (1H, d, J = 7.4, Leu NH), 7.75 (1H, d,
J = 7.8, For-NH), 8.15 (1H, s, HCO), 8.25 (1H, s, HCO). Anal. Calcd for
C23H32N4O6: C, 59.99; H, 7.00; N, 12.17. Found: C, 59.75; H, 6.97;
N, 12.21.
4.1.2.20. N-Boc-tAmp(Boc)-Leu-Phe-OMe (17b). To a solution
of compound 16b (0.032 g, 0.063 mmol) in EtOAc (6.3 mL) Boc2O
(0.027 g, 0.126 mmol) and TEA (0.017 mL, 0.126 mmol) were
added and the mixture was stirred 1 h at room temperature. The
mixture was diluted with EtOAc and washed with 5% citric acid,
NaHCO3 ss and brine. The organic layer was dried and evaporated
under reduced pressure. The residue was purified by silica gel
chromatography (CHCl3/EtOAc 4:1) to give pure 17b as a white
4.1.2.24. N-For-tAmp(For)-Leu-Phe-OMe (19b). HCOOH (0.0566 g,
1.23 mmol) in CH2Cl2 (5 mL) was treated with 2HCOOH tAmp-Leu-
Phe-OMe (0.029 g, 0.059 mmol) according to the general procedure
B. Preparative layer chromatography (EtOAc/MeOH 9:1) gave the
pure product 19b as a white foam (0.005 g, 26%); [a D
]
À35° (c
foam (0.021 g, 55%); [
a]
D
À34.2° (c 0.6); IR (CHCl3)
m: 3439, 1741,
0.5); IR (CHCl3)
m
: 3425, 1742, 1685 cmÀ1 1H NMR d: 0.89 [6H, m,
;
1683 cmÀ1 1H NMR d: 0.89 [6H, m, Leu CH(CH3)2], 1.47 [18H, s, 2
;
Leu CH(CH3)2], 1.27 [3H, m, Leu CH2-CH(CH3)2], 2.10–2.4 (2H, m,
C(CH3)3], 1.60 [3H, m, Leu CH2-CH(CH3)2], 2.03–2.35 (2H, m, Pro
Pro C3H2), 3.11–3.27 (2H, m, Phe b-CH2), 3.6 and 3.77 (2H, m, Pro
C3H2), 3.08–3.14 (2H, m, Phe b-CH2), 3.31 and 3.53 (2H, m, Pro
C5H2), 3.72 (3H, s, COOCH3), 4.36 (1H, m, Leu
Pro a a-CH), 6.53 (1H, d, J = 7.6,
a
-CH), 4.58 (2H, m,
C5H2), 3.70 (3H, s, COOCH3), 4.19–4.26 (3H, m, Leu
a
-CH, Pro
a-
-CH and Pro C4H), 4.80 (1H, m, Phe
CH, Pro C4H), 4.83 (1H, m, Phe
a-CH), 6.3 (1H, m, Boc-NH), 7.08–
Phe NH), 7.0-7.5 (7H, m, Leu NH, For NH and aromatic), 8.13 (1H,
s, HCO), 8.21 (1H, s, HCO). Anal. Calcd for C23H32N4O6: C, 59.99; H,
7.00; N, 12.17. Found: C, 59.76; H, 6.98; N, 12.20.
7.35 (7H, m, Phe NH, Leu NH and aromatic). Anal. Calcd for
C31H48N4O8: C, 61.57; H, 8.00; N, 9.26. Found: C, 61.63; H, 8.01;
N, 9.25.
4.2. Biological assays
4.1.2.21. N-Boc-cAmp(For)-Leu-Phe-OMe (18a). To a solution of
HCOOH (0.018 g, 0.40 mmol) in CH2Cl2 (5 mL) cooled at À15 °C,
IBCF (0.052 mL, 0.40 mmol) and NMM (0.044 mL, 0.40 mmol) were
added. The mixture was stirred for 10 min at the same temperature
and then a solution in CH2Cl2 of 16a (0.02 g, 0.0396 mmol) and
NMM (0.044 mL, 0.40 mmol) was added. Work up according to
general procedure B was then followed. Preparative layer chroma-
tography (CHCl3/EtOAc 1:2) gave a pure product 18a as a amor-
4.2.1. Cell preparation
Cells were obtained from the blood of healthy subjects, and hu-
man peripheral blood neutrophils were purified by using the stan-
dard techniques of dextran (Pharmacia, Uppsala, Sweden)
sedimentation, centrifugation on Ficoll-Paque (Pharmacia), and
hypotonic lysis of contaminating red cells. Cells were washed twice
and resuspended in Krebs–Ringer phosphate (KRPG), pH 7.4, at fi-
nal concentration of 50 Â 106 cells/mL and kept at room tempera-
ture until used. Neutrophils were 98–100% viable, as determined
using the Trypan Blue exclusion test. The study was approved by
the local Ethics Committee and informed consent was obtained
from all participants.
phous solid (0.0125 g, 59%); [
a
]
D
À75.9° (c 0.5); IR (CHCl3)
m:
3419, 1741, 1675 cmÀ1 1H NMR d: 0.86 [6H, m, Leu CH(CH3)2],
;
1.47 [9H, s, C(CH3)3], 1.58 [3H, m, Leu CH2-CH(CH3)2], 2.06–2.32
(2H, m, Pro C3H2), 3.07–3.23 (2H, m, Phe b-CH2), 3.42–3.57 (2H,
m, Pro C5H2), 3.76 (3H, s, COOCH3), 4.32 (1H, m, Leu
a
-CH), 4.40
(1H, m, Pro
a
-CH), 4.57 (1H, m, Pro C4H), 4.86 (1H, m, Phe
a
-CH),
6.58 (1H, d, J = 8, Phe-NH), 7.09–7.31 (5H, m, aromatic), 7.76 (2H,
m, Leu NH and For NH), 8.11 (1H, s, HCO). Anal. Calcd for
C27H40N4O7: C, 60.88: H, 7.57; N, 10.52. Found: C, 61.00; H, 7.55;
N, 10.54.
4.2.2. Random locomotion
Random locomotion was performed with 48-well microchemo-
taxis chamber (Bio Probe, Milan, Italy) and migration into the filter
was evaluated by the leading-front method.26 The actual control

D. Torino et al. / Bioorg. Med. Chem. 17 (2009) 251–259
259
random movement is 35
performed in duplicate.
3 lm SE of 10 separate experiments
Acknowledgments
This work was supported by grants from Fondazione Cassa di
Risparmio di Ferrara. We are grateful to Banca del Sangue of Ferr-
ara for providing fresh blood.
4.2.3. Chemotaxis
Each peptide was added to the lower compartment of the che-
motaxis 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
10–13 to 10–5 M. Data were expressed in terms of chemotactic in-
dex (C.I.), 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 C.I. range.
References and notes
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microplate-based assays. Tests were carried out in a final volume
of 200
l
L containing 4Á105 neutrophils, 100 nmol cytochrome c
and KRPG. At zero time different amounts (10–10–5Á10–5M) of each
peptide were added and the plates were incubated into a micro-
plate reader (Ceres 900, Bio-TeK Instruments, Inc.) with the com-
partment temperature set at 37 °C. Absorbance was recorded at
550 and 468 nm. The difference in absorbance at the two wave-
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absorptivity for cytochrome c of 18.5 mmol–1 cm–1. Neutrophils
were incubated with 5 lg/mL cytochalasin B (Sigma) for 5 min
prior to activation by peptides. Results were expressed as net nmol
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experiments performed in duplicate. Standard errors are in 0.1 -
4 nmol OÀ2 range.
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lysodeikticus. The reaction rate was measured using a microplate
reader at 465 nm. Enzyme release was expressed as a net percent-
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enzyme activity was 85
are the mean of five separate experiments done in duplicate. Stan-
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1
l
g per 1 Â 107 cells/min. The values