New fMLF-OMe analogues containing constrained mimics of phenylalanine residue

Article abstract of DOI:10.1002/(SICI)1521-4184(199805)331:5<170::AID-ARDP170>3.0.CO;2-B

In the context of a research program aimed at elucidating the HCO-Met- Leu-Phe-OMe (fMLF-OMe) structural features which control interactions with the receptor in correspondence with the C-terminal residue, four different analogues of the native ligand have been synthesized and evaluated. Compounds 1-3 possess the general formula HCO-Met-Leu-Xaa-OMe with Xaa = N- benzylglycine, N-benzylphenylalanine, and α,α-dibenzylglycine, respectively. In the analogue 4 the constraint at the C-terminus has been obtained by incorporating a 2-oxopiperazine ring, made up of two phenylalanine residues, to replace the native C-terminal Phe residue. The consequences of the chemical modifications on the activity of the new analogues are discussed.

Full text of DOI:10.1002/(SICI)1521-4184(199805)331:5<170::AID-ARDP170>3.0.CO;2-B

170
Lucente and co-workers
New fMLF-OMe Analogues Containing Constrained Mimics of
Phenylalanine Residue
a)
a)
a)
a)
a)
Ines Torrini , Gaia Mastropietro , Giampiero Pagani Zecchini , Mario Paglialunga Paradisi , Gino Lucente *, and
b)
Susanna Spisani
a)
Dipartimento di Studi Farmaceutici and Centro di Studio per la Chimica del Farmaco del CNR, Università ”La Sapienza”, 00185 Roma, Italy
b)
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, 44100 Ferrara, Italy
Key Words: Chemotaxis; formylpeptides; peptoids; phenylalanine mimics; 2-piperazinone
Summary
In this paper synthesis and bioactivity of the new fMLF-
OMe tripeptide analogues 1–3 and of the tetrapeptide ana-
logue 4 are reported. Compounds 1–3 contain three different
In the context of a research program aimed at elucidating the
HCO-Met-Leu-Phe-OMe (fMLF-OMe) structural features which
control interactions with the receptor in correspondence with the
C-terminal residue, four different analogues of the native ligand
have been synthesized and evaluated. Compounds 1–3 possess the
general formula HCO-Met-Leu-Xaa-OMe with Xaa = N-benzyl-
glycine, N-benzylphenylalanine, and α,α-dibenzylglycine, respec-
tively. In the analogue 4 the constraint at the C-terminus has been
obtained by incorporating a 2-oxopiperazine ring, made up of two
phenylalanine residues, to replace the native C-terminal Phe resi-
due. The consequences of the chemical modifications on the activ-
ity of the new analogues are discussed.
mimics of the Phe residue. In particular, the aromatic side
[14,15]
chain of 1 is shifted in peptoid fashion
to the adjacent
nitrogen atom, thus giving rise to a “position 3 semipep-
[
16]
toid”
of fMLF-OMe; 2 and 3 contain an additional ben-
zylic side chain bonded at N and at the C atom of the Phe
α
residue, respectively.
Introduction
The tripeptide derivative HCO-Met-Leu-Phe-OH (fMLF)
is the reference agonist of the G protein-coupled N-for-
mylpeptide receptor (FPR). This, together with receptors of
many other chemotactic agents, represents a specific binding
site located on the cell surface of phagocytic leukocytes.
Binding of agonists to FPR triggers chemotaxis and a sub-
sequent cascade of biochemical events which is considered
one of the primary physiological responses to bacterial inva-
sion and tissue injury[ . Information about factors which
control ligand-receptor interactions and comprehension of
structural requirements for optimal and selective activity is
therefore of general interest and pharmaceutical relevance.
In this field we reported previously studies aimed at eluci-
dating fMLF-OMe structure-activity relationships as well as
obtaining potent and selective FPR ligands[ . During this
work several tripeptide and tetrapeptide fMLF-OMe ana-
logues, incorporating conformationally constrained mimics
of the native residues, have been synthesized and evaluated.
1–4]
A further structural modification which appears suitable for
freezing the C-terminal fMLF-OMe backbone flexibility,
limiting at the same time the rotational space of the benzylic
side chain, is the short-range cyclization obtained through the
introduction of a 2-oxopiperazine ring bearing two Phe resi-
dues. This approach has recently been adopted to obtain
5–7]
[
17–20]
conformationally restricted peptidomimetics
and the
NMR and X-ray analyses of selected models indicate that the
Results based on alterations at the C-terminal position N,N′-ethylene bridged backbone fragment tends to maintain
showed the different behaviour of tripeptide and tetrapeptide similar conformations both in solution and in the solid
[
21]
models with regard to the spatial orientation of the aromatic phase . In the specific case of the Phe-Phe dipeptide unit
side chain[
8–13]
; in particular, the positive effect of the intro- the 2-oxopiperazine system presents the N-terminal benzylic
duction of a fourth C-terminal residue, in restoring the activ- side chain folded over the heterocyclic ring in an arrangement
ity of otherwise practically inactive tripeptide models, was resembling that adopted by Phe containing dioxopiperaz-
[
8]
[22,23]
evidenced . Thus, we consider that models characterized by ines
. On the basis of these considerations and in order
local conformational constraint centered at the hydrophobic to gain further information on the structure-activity relation-
C-terminal portion of the reference ligand fMLF-OMe should ships of C-terminal modified tetrapeptide analogues, we syn-
afford valuable additonal information about the structural thesized the ethylene-bridged Phe-Phe unit and inserted this
features which control the interactions in this critical receptor fragment into fMLF-OMe, in place of the native C-terminal
area.
Phe residue, to afford the tetrapeptide analogue 4.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/0505/0170 $17.50 +.50/0

Chemotactic Peptide Analogues
171
Results and Discussion
Chemistry
The N-formyl tripeptides 1–3 were prepared according to
Scheme 1. The mixed anhydride method with isobutyl chlo-
roformate was employed for the synthesis of the intermediate
Boc-protected derivatives Boc-Leu-Xaa-OMe and Boc-Met-
Leu-Xaa-OMe. Deprotection of the dipeptides Boc-Leu-Xaa-
OMe was performed with trifluoroacetic acid (TFA)-CHCl3.
Finally treatment of the Boc-tripeptide methyl esters Boc-
It should be noted here that, although a number of confor-
Met-Leu-Xaa-OMe with HCOOH followed by ethyl 2- mationally restricted chemotactic pseudopeptides have been
[
24]
ethoxy-1,2-dihydro-1-quinolinecarboxylate (EEDQ)
,
designed and evaluated, the incorporation into these ligands
of 2-oxopiperazine building blocks, to enhance backbone
rigidity and limit the space available to the side chains, has
not been described previously. An analogous consideration
applies to the new ligand 1 which represents the first fMLF-
OMe analogue obtained by adopting the topological modifi-
afforded the N-formyltripeptides 1–3.
cation based on the shifting of the side chain from the C atom
α
[
14–16]
to the N atom within the same residue
.
Biological Activity
The new analogues 1–4 were evaluated for their potential
ability to activate chemotaxis, to stimulate superoxide anion
production, and to induce lysozyme release from granules of
human neutrophils. Their activity was compared to that of the
parent tripeptide fMLF-OMe. Compound 1 is unable to elicit
any of the tested biological activities; the observed slight
increments, reported in Figure 1, are not statistically signifi-
cant (p>0.05). Compounds 2, 3, and 4 require high doses to
exert a chemotactic activity (Figure 1A), being maximally
–6
–8
effective at 10 M (2 and 3) and at 10 M (4). In the case of
superoxide anion production, 2 and 3 are less potent than
fMLF-OMe, their activity being about 67% and 46% of the
parent, respectively, at the physiological concentration of
–6
1
0 M (Figure 1B); in contrast, the analogue 4 is more potent
than fMLF-OMe (p>0.05) (Figure 1B). When tested as secre-
tagogue agents, 2, 3, and 4 induce 56–68% of maximal
The synthesis of the N-formyl derivative 4 was performed
according to Scheme 2. The cyclization and esterification of
–5
(
reference) lysozyme release at 10 M (Figure 1C).
The biological results concerning the analogue 1 show that
(2S,7S)-2,7-dibenzyl-3,6-diazaoctane-1,8-dioic acid (N,N′-
the peptoid approach applied at C-terminal fMLF-OMe resi-
ethylene-bridged bis-(S)-phenylalanine, HO-PheePhe-OH)
[
25,26]
due causes the loss of the biological activity towards human
(
5)
monohydrate (Tos-OH H2O) as an acid catalyst
forded methyl (2S,3′S)-2-(3′-benzyl-2′-oxopiperazin-1′-yl)-
in boiling methanol, using p-toluenesulphonic acid
[7]
neutrophils. As previously pointed out , the intrinsic nature
[27,28]
.
, af-
of the residue replacing the phenylalanine, rather than the
absence of the C-terminal NH group, seems responsible of
the observed inactivity of the formyltripeptide 1. This is
confirmed by the activity shown by the analogues 2 and
[
27,28]
3
-phenylpropanoate
(ePhePhe-OMe)
(6)
.
Boc-Met-Leu-ePhePhe-OMe (7) was prepared according the
[29]
carbodiimide method, coupling Boc-Met-Leu-OH and 6. Fi- HCO-Met-Leu-(NMe)Phe-OMe both lacking the H-atom
nal treatment of intermediate N-Boc derivative 7 with formic on the last amide bond; in the superoxide anion production,
acid and EEDQ[ afforded the N-formyl derivative 4.
24]
the latter ligand has been reported to be more potent than the
Arch. Pharm. Pharm. Med. Chem. 331, 170–176 (1998)

172
Lucente and co-workers
parent. The significant decrease of biological activity, in
comparison with the parent, which is observed in the case of
compound 3 contrasts with the favourable effect observed
when the doubling of the side chain was performed at the
[
6,30]
central residue level offMLF-OMe analogues
. Thus, the
low activity of 3, as compared with analogous models modi-
fied at central position, suggests a more congested area in
correspondence of the C-terminal hydrophobic pocket of the
receptor. The biological activity of the ligand 2 which, al-
though less active than the parent, is more potent than the
analogues 1 and 3, is in accordance with the high activity
observed for the above mentioned fMLF-OMe analogue con-
taining the (NMe)phenylalanine residue.
The tetrapeptide analogue 4 shows relevant activity in the
stimulation of superoxide anion production, while it is less
active as chemotactic agent and lysozyme release inductor.
In particular, as shown in Figure 1B, the activity of 4 towards
–
O2 production is higher than that of the reference molecule.
This result can be rationalized by taking into account the
preferred conformation of the benzylic side chain bonded to
[
21]
the 2-oxopiperazine ring
and the activity of previously
studied fMLF-OMe models containing the residue of didehy-
z
drophenylalanine (∆ Phe) in place of the C-terminal Phe.
These tripeptides are characterized by an orientation of the
aromatic ring which is nearly parallel to the adjacent back-
α
[31]
bone atoms (N-C -C′)
and are practically inactive. How-
ever, the introduction in these ligands of an additional
C-terminal Phe, leading to formyltetrapeptides of the general
z
formula HCO-Xaa-Leu-∆ Phe-Phe-OMe, restores the activ-
[
8]
ity . The tetrapeptide analogue4 contains incorrespondence
of the third residue a benzylic side chain folded on the
backbone atoms with reduced tendency to interact with the
corresponding hydrophobic pocket of the receptor. Thus, the
additional binding interaction, established by the benzylic
side chain of the fourth residue, again seems to be the deter-
minant factor for the observed activity.
Acknowledgements
This work was supported in part by the Ministero dell’Università e della
Ricerca Scientifica e Tecnologica (MURST). We are grateful to Banca del
Sangue of Ferrara for providing fresh blood.
Experimental Part
Melting points: Büchi oil bath apparatus, uncorrected.– Optical rotations:
Schmidt-Haensch Polartronic D polarimeter, 1 dm cell, c 1.0, 20 °C.– IR
1
spectra: Perkin-Elmer 983 spectrophotometer.– H NMR spectra (CDCl3):
Varian XL-300 and Bruker AM 400 spectrometers, TMS int. stand.– Column
chromatographies: Merck silica gel 60 (230–400 mesh) (1:40).– TLC and
PLC: silica gel Merck 60 F254 plates.– Drying agent: sodium sulphate.– Light
petroleum: 40–60 °C bp fraction.– Parent fMLF-OMe prepared according to
ref.[ .– Elemental analyses: Servizio Microanalisi del CNR, Area della
Ricerca di Roma, Montelibretti, Italy. The abbreviations used are as follows:
BzlGly, N-benzylglycine; BzlPhe, N-benzylphenylalanine; DbzGly, α,α-
dibenzylglycine; DMF, N,N-dimethylformamide; EDCI, N-ethyl-N′-(di-
methylaminopropyl)carbodiimide hydrochloride; HOBt, 1-hydroxy-
benzotriazole; NMM, N-methylmorpholine; TEA, triethylamine; TFA, tri-
fluoroacetic acid; THF, tetrahydrofuran.
32]
Figure 1. Biological activity of fMLF-OMe and its analogues 1–4 towards
human neutrophils. (A) Chemotactic activity. (B) Superoxide anion produc-
tion. (C) Release of neutrophil granule enzymes evaluated by determining
lysozyme activity.
Arch. Pharm. Pharm. Med. Chem. 331, 170–176 (1998)

Chemotactic Peptide Analogues
173
Boc-Leu-BzlGly-OMe
4.26 (m, 1H, Met α-CH), 4.89 (m, 1H, Leu α-CH), 5.20 (d, J = 8.3 Hz, 1H,
Met NH), 6.72 (d, J = 8.8 Hz, 1H, Leu NH), 7.00–7.36 (m, 10H, aromatic).
Isobutyl chloroformate (0.27 ml, 1.97 mmol) was added at –15 °C to a
stirred solution of Boc-Leu-OH H2O (0.491 g, 1.97 mmol) and NMM
.
(
0.26 ml, 2.36 mmol) in dry CH2Cl2 (9.5 ml). The temperature was kept at
Boc-Leu-DbzGly-OMe
[33]
.
–
1
15 °C for 15 min, then a cold solution of HCl H-BzlGly-OMe (0.425 g,
.97 mmol) and NMM (0.22 ml, 1.97 mmol) in dry CH2Cl2 (7 ml) was
The title dipeptide was obtained following the usual procedure starting
.
from Boc-Leu-OH H2O (0.129 g, 0.52 mmol) and a solution of H-DbzGly-
OMe
added. The mixture was stirred at room temperature for one day and then
evaporated under vacuum. The residue was dissolved in ethyl acetate and
washed with 5% aqueous KHSO4, water, saturated aqueous NaHCO3, and
brine. The organic phase was dried and evaporated to give a residue (0.749 g)
which was chromatographed on a silica column. Elution with CH2Cl2-ether
[36]
(0.139 g, 0.52 mmol) in dry CH2Cl2 (1.8 ml). Usual work up gave
a residue (0.224 g) which was purified by silica column chromatography
eluting with n-hexane-ether (8:2) and PLC [n-hexane-ether (1:1)] to give
Boc-Leu-DbzGly-OMe (0.127 g, 51%), mp 98–100 °C (n-hexane).– [α]D =
–1
1
–
13° (CHCl3).– IR (KBr): 3399, 3312, 1735, 1708, 1661 cm .– H NMR:
(
(
9:1) gave 0.674 g (87%) of the title compound as an oil.– [α]D = –19°
CHCl3).– IR (CHCl3): 3434, 1746, 1703, 1643 cm–1.– H NMR: δ 0.90 [m,
δ0.88 [apparent t, 6H, (CH3)2CH], 1.37 [s, 9H, C(CH3)3], 1.34–1.60 (m, 3H,
Leu β-CH2 and γ-CH), 3.23 and 3.26 (A part of two AX systems, J = 13.5
Hz, 2H, 1H of each CH2-Ph), 3.78 (s, 3H, COOCH3), 3.95 (superimposed X
part of two AX, J = 13.5 Hz, 2H, 1H of each CH2-Ph), 4.07 (m, 1H, Leu
α-CH), 4.75(d, J = 8.5 Hz, 1H, Leu NH), 6.66 (s, 1H, DbzGlyNH), 6.97–7.30
1
6
H, (CH3)2CH], 1.44 [s, 9H, C(CH3)3], 1.48–1.78 (m, 3H, Leu β-CH2 and
γ-CH), 3.71 (s, 3H, COOCH3), 3.75 and 4.25 (A and X of an AX, J = 17 Hz,
H, Ph-CH2-N), 4.48 (m, 1H, Leu α-CH), 4.61 and 4.79 (A and B of an AB,
J = 16 Hz, 2H, BzlGly α-CH2), 5.20 (d, J = 8.8 Hz, 1H, Leu NH), 7.14–7.40
m, 5H, aromatic).
2
(two m, 10H, aromatic).– Anal. (C28H38N2O5) C, H, N.
(
Boc-Met-Leu-DbzGly-OMe
Boc-Met-Leu-BzlGly-OMe
The title tripeptide was synthesized by the usual procedure starting from
Boc-Met-OH (0.095 g, 0.38 mmol) and a solution in dry DMF (0.4 ml) of
The title Boc-protected tripeptide was prepared following the procedure
described above starting from Boc-Met-OH (0.429 g, 1.72 mmol) and a
solution in dry DMF (1.8 ml) of NMM (0.19 ml, 1.72 mmol) and TFA H-
.
NMM (0.04 ml, 0.38 mmol) and TFA H-Leu-DbzGly-OMe (0.38 mmol)
.
[34]
obtained by acidolysis
of the corresponding Boc-dipeptide. In this case
Leu-BzlGly-OMe (1.72 mmol), obtained by acidolysis of Boc-Leu-BzlGly-
OMe with TFA-CHCl3[ . Usual work up gave a residue (0.845 g) which
was chromatographed on a silica column, eluting with CH2Cl2-ether (9:1).
Further purification by PLC [n-hexane-ethyl acetate (7:3) as eluant] of nearly
homogeneous chromatographic fractions afforded pure Boc-Met-Leu-
BzlGly-OMe (0.729 g, 81% overall yield) as an oil.– [α]D = –33° (CHCl3).–
the mixed anhydride was prepared maintaining the temperature at –10 °C for
0 min. Usual work up gave a residue (0.245 g) which was purified by silica
column chromatography eluting with n-hexane-ether (6:4) and PLC [n-hex-
34]
3
ane-ether (1:1)] to afford Boc-Met-Leu-DbzGly-OMe (0.192 g, 83%), as an
–1
1
oil.– [α]D = –16° (CHCl3).– IR (CHCl3): 3395, 1735, 1705, 1670 cm .– H
NMR: δ 0.81 and 0.86 [two d, J = 6 Hz, 6H, (CH3)2CH], 1.35–1.55 [m, 12
H, C(CH3)3 (s at 1.43) superimposed on Leu β-CH2 and γ-CH], 1.70–1.95
–
1
1
IR (CHCl3): 3424, 1746, 1707, 1655 cm .– H NMR: δ 0.88 [m, 6H,
CH3)2CH], 1.44 [s, 9H, C(CH3)3], 1.48–1.74 (m, 3H, Leu β-CH2 and γ-CH),
(
(
3
m, 2H, Met β-CH2), 2.03 (s, 3H, S-CH3), 2.40 (m, 2H, CH2-S), 3.23 and
.28 (A part of two AX, J = 13.5 Hz, 2H, 1H of each CH2-Ph), 3.80 (s, 3H,
1
3
.86–2.09 (m, 2H, Met β-CH2), 2.11 (s, 3H, S-CH3), 2.56 (m, 2H, CH2-S),
.71 (s, 3H, COOCH3), 3.78 and 4.24 (A and X of an AX, J = 17.4 Hz, 2H,
COOCH3), 3.91 and 3.93 (X part of two AX, J = 13.5 Hz, 2H, 1H of each
CH2-Ph), 4.15 (m, 1H, Met α-CH), 4.34 (m, 1H, Leu α-CH), 5.14 (d, J =
Ph-CH2-N), 4.28 (m, 1H, Met α-CH), 4.62 and 4.76 (A and B of an AB, J =
1
1
6.2 Hz, 2H, BzlGly α-CH2), 5.09 (m, 1H, Leu α-CH), 5.25 (d, J = 7 Hz,
H, Met NH), 6.84 (d, J = 8.3 Hz, 1H, Leu NH), 7.14–7.42 (m, 5H, aromatic).
8.2 Hz, 1H, Met NH), 6.51 (m, 2H, Leu and DbzGly NH), 6.91–7.29 (two
m, 10 H, aromatic).
Boc-Leu-BzlPhe-OMe
ePhePhe-OMe (6)
The title dipeptide was prepared following the usual mixed anhydride
Following known procedures[27,28], HO-PheePhe-OH (5)[25,26] (0.178 g,
.
procedure starting from Boc-Leu-OH H2O (0.735 g, 2.95 mmol) and a solu-
.
.5 mmol) was refluxed with Tos-OH H2O (0.19 g, 1 mmol) in dry methanol
0
(
tion of H-BzlPhe-OMe[ (0.794 g, 2.95 mmol) in dry CH2Cl2 (10 ml).
Usual work up gave a residue (1.046 g) which was purified by silica column
chromatography eluting with CH2Cl2-ether (95:5) and PLC [n-hexane-ethyl
acetate (8:2)] to afford pure Boc-Leu-BzlPhe-OMe (0.442 g, 31%), as an
35]
7.5 ml) for 24 h. The salt obtained after evaporation of the solvent was freed
by saturated aqueous NaHCO3, cooling at 0 °C, and the mixture was ex-
tracted with dichloromethane. The organic solution was dried and evaporated
to afford crude oily ePhePhe-OMe (6) (0.171 g), which was used without
further purification. In a separate experiment pure 6 (0.087 g, 0.247 mmol),
obtained by PLC of the crude mixture, using benzene-EtOAc-MeOH (5:4:1)
as eluant, was stirred in dry methanol (1 ml) in the presence of SOCl2
(0.04 ml) at room temperature for 8 min and at 40 °C for 10 min. Evaporation
under vacuum afforded the solid hydrochloride in quantitative yield (mp and
optical rotation as literature[ ).
–1
oil.– [α]D = –125° (CHCl3).– IR (CHCl3): 3432, 1737, 1704, 1642 cm .–
1
H NMR: δ 0.80 and 0.90 [two d, J = 6.5 Hz, 6H, (CH3)2CH], 1.25–1.77 [m,
1
3
4
2 H, C(CH3)3 (s at 1.43) superimposed on Leu β-CH2 and γ-CH], 3.21 and
.33 (A and B of an ABX, J = 5.8, 9.5, and 14 Hz, 2H, Phe β-CH2), 3.82 and
.53 (A and X of an AX, J = 16 Hz, 2H, Ph-CH2-N), 4.20 (X part of an ABX,
25]
J = 5.8 and 9.5 Hz, 1H, Phe α-CH), 4.61 (m, 1H, Leu α-CH), 5.09 (d, J = 9
Hz, 1H, Leu NH), 7.04–7.39 (m, 10H, aromatic).
Boc-Met-Leu-ePhePhe-OMe (7)
Boc-Met-Leu-BzlPhe-OMe
To a solution of crude 6 (obtained from usual treatment of 1 mmol of 5),
Boc-Met-Leu-OH (0.363 g, 1 mmol, Bachem), and HOBt 80% (0.169 g,
1 mmol) in dry DMF (2 ml), EDCI (0.192 g, 1 mmol) and dry DMF (2 ml)
were added. After stirring at room temperature for 3 days, ethyl acetate was
added in excess and the organic layers were washed with water, saturated
aqueous NaHCO3, water, 5% aqueous KHSO4, brine, and dried. The solvent
was evaporated to give a foam (0.539 g) which was purified by PLC
[dichloromethane-ethyl acetate (7:3) and then n-hexane-ethyl acetate (1:1)
as eluants] toaffordthepure titlecompound7 as a foam (0.256 g, 37% overall
yield).– [α]D = –13° (CHCl3).– IR (CHCl3): 3426, 1738, 1707, 1649, 1495,
1437 cm–1.– H NMR: δ 0.86 and 0.88 [two d, J = 6.5 Hz, 6H, (CH3)2CH],
1.20–1.70 [m, 12H, C(CH3)3 (s at 1.45) superimposed on Leu β-CH2 and
γ-CH], 1.85–2.10 (m, 2H, Met β-CH2), 2.12 (s, 3H, SCH3), 2.55 (t, J = 7.5
Hz, 2H, CH2S), 2.65–3.50 (m, 8H, two Phe β-CH2 and N-CH2-CH2-N), 3.78
(s, 3H, COOCH3), 4.24 (m, 1H, Met α-CH), 4.75 (m, 1H, Leu α-CH),
The title N-Boc-protected tripeptide was prepared following the usual
procedure starting from Boc-Met-OH (0.244 g, 0.98 mmol) and a solution in
.
dry DMF (1 ml) of NMM (0.11 ml, 0.98 mmol) and TFA H-Leu-BzlPhe-
OMe (0.98 mmol) obtained by acidolysis[ of Boc-Leu-BzlPhe-OMe.
Usual work up gave a residue (0.516 g) which was purified by silica column
chromatography eluting with n-hexane-ethyl acetate (8:2) and PLC [n-hex-
ane-ethyl acetate (7:3)]to give pure Boc-Met-Leu-BzlPhe-OMe (0.277 g,
34]
4
6%), as an oil.– [α]D = –114° (CHCl3).– IR (CHCl3): 3425, 1737, 1706,
–
1
1
1675, 1644 cm .– H NMR: δ 0.77 and 0.89 [two d, J = 6.3 Hz, 6H,
1
(
CH3)2CH], 1.25–1.70 [m, 12 H, C(CH3)3 (s at 1.44) superimposed on Leu
β-CH2 and γ-CH], 1.83-2.06 (m, 2H, Met β-CH2), 2.12 (s, 3H, S-CH3), 2.53
t, J = 7 Hz, 2H, CH2-S), 3.21 and 3.33 [A and B of an ABX, J = 5.7, 9.6,
and 14 Hz, 2H, Phe β-CH2), 3.81 and 4.50 (A and X of an AX, J = 16 Hz,
H, Ph-CH2-N), 4.20 (X part of an ABX, J = 5.7 and 9.6 Hz, 1H, Phe α-CH),
(
2
Arch. Pharm. Pharm. Med. Chem. 331, 170–176 (1998)

174
Lucente and co-workers
Table 1. Physical, analytical, and spectral data of N-formylpeptides HCO-Met-Leu-Xaa-OMe (1-3) and piperazinone derivative 4..
———————————————————————————————————————————————————
—
Cpd
Yield%
Mp, °C
[α]D
Solvent[a]
Formula[b]
Crystallization
solvent[a]
————————————————————————————————————————————————————
1
80
87
83
84
oil
–22°
C
C22H33N3O5S
C29H39N3O5S
C29H39N3O5S
C33H44N4O6S
2
3
4
102–104
E–LP
–134°
C
182–182.5
B
–13°
DMF
–10°
C
147–148
D–LP
1H NMR (δ)
–1 [c]
IR (cm
)
—
——————————————————————————————————————————
H-CO Met Leu Xaa
α-CH
——————————————————————————————
NH
α-CH
NH
α-CH
NH
—
1
8.17
8.14
8.15
4.79[d]
6.86
5.09
4.92
4.34
7.17
6.98
6.63
4.71[e]
3412, 3317, 1747,
657
1
2
4.74
6.78
4.15
3269, 1748, 1734,
686, 1640
1
3
4.65
6.49[f]
6.47
3388, 3271, 1741,
670, 1643
1
———————————————————————————————
H-CO
Met
Leu
Phe
α-CH
NH
α-CH
NH
two α-CH
———————————————————————————————
4
8.20
4.72
6.86[g] 6.60[g]
4.72
4.92–5.06
3287, 1745, 1641,
493, 1451
———————————————————————————————————————————————————
1
—
[a]B = benzene; C = chloroform; D = dichloromethane; E = ethyl ether; DMF = N,N-dimethylformamide; LP = light petroleum.
[b]C, H, and N analyses were within ± 0.3% of the theoretical values. [c]CHCl3 solution for 1 ; KBr disks for 2, 3 and 4. [d]Superimposed
on BzlGly α-CH2. [e]Middle point of the CH AB q. [f]Superimposed on DbzGly NH. [g]These assignments may be inverted.
2
4
.92-5.04 (m, 2H, two Phe α-CH), 5.19 (d, J = 9.5 Hz, 1H, Met NH), 6.77
tography eluting with CH2Cl2-EtOAc (1:1) to obtain compound 1 and by
PLC, using n-hexane-ethyl acetate (4:6), CH2Cl2-MeOH (95:5), and EtOAc
as eluants, to obtain compounds 2, 3, and 4, respectively.
(
d, J = 8.5 Hz, 1H, Leu NH), 6.92–7.35 (m, 10H, aromatic).
General procedure for the synthesis of formylpeptides 1–4
Biological Assay
Cells
The N-Boc-protected peptide (1 mmol) was dissolved in 98% formic acid
4 ml) and the mixture was stirred at room temperature for 24 h. After
removal of the excess of formic acid in vacuo, the residue was dissolved in
dry chloroform (4 ml) and EEDQ (1.2 mmol) was added. The solution was
stirred at room temperature for 5 h (24 h in the case of 4) and then evaporated
under reduced pressure. The residue was purified by silica column chroma-
(
Human peripheral blood neutrophils were purified employing the standard
techniques of dextran (Pharmacia) sedimentation, centrifugation on Ficoll-
Paque (Pharmacia), and hypotonic lysis of red cells. The cells were washed
Arch. Pharm. Pharm. Med. Chem. 331, 170–176 (1998)

Chemotactic Peptide Analogues
175
twice and resuspended in KRPG (Krebs-Ringer phosphate containing 0.1%
w/v glucose, pH 7.4) at a concentration of 50×10 cells/ml. Neutrophils were
[5] E. Gavuzzo, G. Lucente, F. Mazza, G. Pagani Zecchini, M. Paglialunga
Paradisi, G. Pochetti, I. Torrini, Current Topics in Peptide & Prot. Res.
1994, 1, 275–295.
6
98–100% pure.
[
6] I. Torrini, M. Paglialunga Paradisi, G. Pagani Zecchini, G. Lucente, E.
Random Locomotion
Gavuzzo, F. Mazza, G. Pochetti, S. Traniello, S. Spisani, Biopolymers
1
997, 42, 415–426.
Random locomotion was performed with 48-well microchemotaxis cham-
ber (Bio Probe, Italy) and the migration into the filter was evaluated by the
[
7] G. Pagani Zecchini, M. Paglialunga Paradisi, I. Torrini, G. Lucente, G.
method of leading-front[ .The actual control random movement is 32µm ±
37]
Mastropietro, M. Paci, S. Spisani, Arch. Pharm. Pharm. Med. Chem.
1996, 329, 517–523.
3
SE of ten separate experiments done in duplicate.
[
[
8] I. Torrini, G. Pagani Zecchini, M. Paglialunga Paradisi, S. Spisani,
Arch. Pharm. Pharm. Med. Chem. 1996, 329, 143–148.
Chemotaxis
9] G. Pagani Zecchini, I. Torrini, M. Paglialunga Paradisi, G. Lucente, G.
In order to study the potential chemotactic activity, each peptide was added
to the lower compartment of the chemotaxis chamber. Peptides were diluted
Mastropietro, S. Spisani, Amino Acids, in press.
–
2
from a stock solution (10 M in dimethyl sulphoxide) with KRPG containing
mg/ml of bovine serum albumin (Orha Behringwerke, FRG) and used at
[10] M. Miyazaki, H. Kodama, I. Fujita, Y. Hamasaki, S. Miyazaki, M.
1
Kondo, Pept. Chem. 1994 1995, 32, 293–296.
–
12
–5
concentrations ranging from 10 M to 10 M. Data were expressed in terms
of chemotactic index, which is the ratio: (migration toward test attractant
minus migration toward the buffer)/migration toward the buffer; the values
are the mean of six separate experiments done in duplicate. Standard errors
are in the 0.02–0.09 chemotactic index range.
[
11] C. Toniolo, M. Crisma, S. Pegoraro, G. Valle, G. M. Bonora, E. L.
Becker, S. Polinelli, W. H. J. Boesten, H. E. Shoemaker, E. M. Meijer,
J. Kamphuis, R. Freer, Pept. Res. 1991, 4, 66–71.
[12] C. Toniolo, F. Formaggio, M. Crisma, G. Valle, W. H. J. Boesten, H.
E. Shoemaker, J. Kamphuis, P. A. Temussi, E. L. Becker, G. Précigoux,
Tetrahedron 1993, 49, 3641–3653.
–
Superoxide Anion (O2 ) Production
[
13] A. Bharadwaj, M. Singh, K. Bhandary, E. L. Becker, V. S. Chauhan,
The superoxide anion was measured by the superoxide dismutase-inhibi-
table reduction of ferricytochrome c (Sigma, USA) modified for microplate-
Pept. Res. 1993, 6, 298–307.
based assays. The tests were carried out in a final volume of 200 µl containing
[14] R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A.
Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D.
C. Spellmeyer, R. Tan, A. D. Frankel, D. V. Santi, F. E. Cohen, P. A.
Bartlett, Proc. Natl. Acad. Sci. USA 1992, 89, 9367–9371.
5
4
×10 neutrophils, 100 nmoles cytochrome c and KRPG. At zero time,
–
9
–5
different amounts (10 –2×10 M) of each peptide were added and the plates
were incubated into a microplate reader (Ceres 900, Bio-TeK Instruments,
Inc.) with the compartment T set at 37 °C. Absorbance was recorded at
wavelenghts of 550 and 465 nm. Differences in absorbance at the two
[
15] H. Kessler, Angew. Chem. Int. Ed. Engl. 1993, 32, 543–544.
–
[16] H. Reuveni, A. Gitler, E. Poradosu, C. Gilon, A. Levitzki, Bioorg. Med.
Chem. 1997, 5, 85–92.
wavelenghts were used to calculate nmoles of O2 produced using an absorp-
–
1
–1
tivity for cytochrome c of 15.5 mM cm . Neutrophils were incubated with
5
µg/ml cytochalasin B (Sigma) for 5 min prior to activation by peptides.
[17] C. Toniolo, Int. J. Peptide Protein Res. 1990, 35, 287–300.
–
6
Results were expressed as net nmoles of O2 /1×10 cells/5 min and are the
mean of six separate experiments done in triplicate. Standard errors are in
[
[
[
[
18] A. Pohlmann, V. Schanen, D. Guillaume, J.-C. Quirion, H.-P. Husson,
J. Org. Chem. 1997, 62, 1016–1022.
–
0.1–4 nmoles O2 range.
19] K. Shreder, L. Zhang, M. Goodman, Tetrahedron Lett. 1998, 39,
221–224.
Enzyme Assay
20] A. Pohlmann, D. Guillaume, J.-C. Quirion, H.-P. Husson, J. Peptide
Res. 1998, 51, 116–120.
Release of neutrophil granule enzymes was evaluated by determination of
6
lysozyme activity, modified for microplate-based assays. 3×10 Cells/well
were first incubated in triplicate wells of microplates with 5 µg/ml cytocha-
lasin B at 37 °C for 15 min and then in presence of each peptide in a final
21] C. Czaplewski, B. Lammek, J. Ciarkowski, Polish J. Chem. 1994, 68,
2589–2598.
–
9
–5
concentration of 10 –2×10 M for a further 15 min.The plates were then
centrifugated at 400g for 5 min and the lysozyme wasquantifiednephelomet-
rically by the rate of lysis of cell wall suspension of Micrococcus lysodeik-
ticus. Reaction rate was measured with a microplate 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 µg/1×107
cells/min. The values are the mean of five separate experiments done in
triplicate. Standard errors are in 1–6% range.
[22] M. Paglialunga Paradisi, G. Pagani Zecchini, I. Torrini, G. Lucente, J.
Heterocyclic Chem. 1990, 27, 1661–1664.
[23] A. Liwo, J. Ciarkowski, Tetrahedron Lett. 1985, 1873–1876.
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[
[
25] T. Yamashita, J. Maruo, A. Fujimoto, K. Shibata, Y. Kojima, A.
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Szkróbka, H. I. Trzeciak, J. Peptide Res. 1998, 51, 149–154.
Statistical Analysis
[
[
27] T. Yamashita, H. Takenaka, Y. Kojima, Amino Acids 1993, 4, 187–192.
The nonparametric Wilcoxon test was used in the statistical evaluation of
differences between groups.
28] H. Takenaka, H. Miyake, Y. Kojima, M. Yasuda, M. Gemba, T.
Yamashita, J. Chem. Soc. Perkin Trans. 1 1993, 933–937.
[
[
[
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Cavicchioni, Pept. Res. 1996, 9, 279–282.
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Arch. Pharm. Pharm. Med. Chem. 331, 170–176 (1998)