Synthesis and activity of N-Benzyl pseudopeptides HIV protease inhibitors

Article abstract of DOI:10.1016/S0968-0896(02)00563-1

A series of N-benzyl pseudopeptides was designed, synthesized and tested as HIV-1 protease inhibitors. The ability of the new compounds containing N-benzyl hydroxyalkylamino acid core structure to inhibit HIV replication in cell culture is comparable to their capacity to inhibit the isolated enzyme, a result compatible with good pharmacokinetic properties of these derivatives. The pseudotripeptide Fmoc-Leu-N(Bzl)Hse-Met-NH-tBu was the best inhibitor of the series (IC₅₀=170 nM) showing promising inhibition of viral replication (ED₅₀=52 nM). All new compounds exhibit high enzymatic resistance and stability against cell cultures and plasma enzymes.

Full text of DOI:10.1016/S0968-0896(02)00563-1

Bioorganic & Medicinal Chemistry 11 (2003) 2477–2483
Synthesis and Activity of N-Benzyl Pseudopeptides HIV
Protease Inhibitors
Mauro Marastoni,* Martina Bazzaro, Fabrizio Bortolotti and Roberto Tomatis
Dipartimento di Scienze Farmaceutiche e Centro di Biotecnologie, via Fossato di Mortara 17-19,
`
Universita di Ferrara, I-44100 Ferrara, Italy
Received 2 August 2002; accepted 4 November 2002
Abstract—A series of N-benzyl pseudopeptides was designed, synthesized and tested as HIV-1 protease inhibitors. The ability ofthe
new compounds containing N-benzyl hydroxyalkylamino acid core structure to inhibit HIV replication in cell culture is comparable
to their capacity to inhibit the isolated enzyme, a result compatible with good pharmacokinetic properties ofthese derivatives. The
pseudotripeptide Fmoc-Leu-N(Bzl)Hse-Met-NH-tBu was the best inhibitor ofthe series (IC 50=170 nM) showing promising inhib-
ition ofviral replication (ED 50=52 nM). All new compounds exhibit high enzymatic resistance and stability against cell cultures
and plasma enzymes.
# 2002 Published by Elsevier Science Ltd.
Introduction
pseudopeptide inhibitors which incorporate amide bond
modified and hydroxyalkane residue on the core
structure.12ꢀ18 Some previous compounds containing an
hydroxyalkyl gem-diamino or N-hydroxy amide func-
tion (Fig. 1) showed significant activity against the
isolated enzyme (IC50=140–160 nM), satisfactory
inhibition ofHIV replication in cell culture (ED 50=98–
110 nM) and good metabolic stability.
The pandemic spread ofhuman immunodeficiency virus
(HIV), the etiologic agent ofAIDS, has promoted an
unequalled scientific effort to understand and control
this disease. The resultant understanding ofHIV-1 lief
cycle have defined many different targets for potential
drug intervention. The virally encoded homodimeric
aspartyl protease (HIV Pr) is currently one ofthe more
promising therapeutic targets for the treatment of AIDS
due to its critical role in the virus maturation and repli-
cation.1 The HIV-1 Pr has an homodimeric C-2 sym-
metric structure and each monomer contributes one
catalytic aspartic residue and flexible flap, which is able
to bind the substrates and inhibitors.2ꢀ4 In addition, a
characteristic bound water molecule forms an hydrogen
bonding network between the flaps and bond substrates
creating a tetrahedral transition-state intermediate.
Many different classes ofHIV-1 protease inhibitors
have been developed, showing excellent antiviral
profiles.5ꢀ8 On the other hand there is the necessity to
introduce new potent inhibitors that show good
pharmacokinetics and activity against mutant strains of
HIV. On the basis of these features and following the
In this paper, we describe a new series ofHIV-1 inhibi-
tors, which contain a N-benzyl hydroxyalkylamino acid
in the core structure. The introduction ofbenzyl group
on the N near to hydroxy function was predicted to
have the potential to make supplementary favourable
interactions at P1 position (Fig. 1).19
Compounds 1–6 contain N-benzyl serine, while deriva-
tives 7–12 have homoserine (Hse) in the core structure,
to modulate distance and flexibility ofthe hydroxyalkyl
side chain. Bulky protected phenylalanine or leucine
were placed at P1/P2 in pseudotripeptides 3–6 and 9–12
to increase the number ofvan der Waals interactions
with the enzyme. Methionine t-butylamide was selected
in analogy with other sulfur containing residues at P2I
position in potent HIV protease inhibitors.20,21 Pseudo-
dipeptides 1, 2 and 7, 8 were designed to ameliorate
pharmacokinetic properties. In effect, for all analogues,
the N-benzyl amide function can increase metabolic
stability in comparison with the cognate amide bond.
9ꢀ11
Kempfstrategy,
we designed different series of
*Corresponding author. Tel.: +39-053-2-291281; fax: +39-053-2-
291296; e-mail: mrn@dns.unife.it
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
doi:10.1016/S0968-0896(02)00563-1

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M. Marastoni et al. / Bioorg. Med. Chem. 11 (2003) 2477–2483
were obtained by treatment with trifluoroacetic acid
(TFA). The key intermediate protected N-benzyl dipep-
tide was acylated by t-butyloxycarbonyl-phenylalanine
or -leucine via mixed anhydride. t-Butyl deprotection,
followed by coupling of the Fmoc-OSu or Z-OSu, gave
the desired N-benzyl pseudotripeptides 3–6.
Compounds 7–12 containing N-benzyl homoserine core
structure were prepared by the same synthetic strategy
(Scheme 2). The difficulty in incorporating homoserine
into synthetic peptides stem from the well known ten-
dency of g-hydroxyamino acids and their derivatives to
form g-lactone.23ꢀ25 To avoid this drawback we used an
approach where the key ofthe synthesis was the prepa-
ration of N-benzylated aspartyl dipeptides and the sub-
sequent conversion in the corresponding Hse-products
or intermediates by chemoselective reduction via mixed
anhydrides.26ꢀ28
Crude 1–12 were purified by RP-HPLC and their
structure verification was achieved by 1H NMR and
mass-spectrometry (Table 1).
Antiviral activity
IC50 and ED50 values, respectively against HIV-1 pro-
tease and for inhibition of viral replication in cell culture
for pseudopeptides 1–12 are shown in Table 2.
Figure 1. More representative diamino-hydroxyalkane derivative (I),
N-hydroxyamide inhibitor (II) and general structure of N-benzyl
pseudotripeptides (III).
IC50 values ofthe inibitory potencies against purified
recombinant HIV-Pr were determined using synthetic
peptide H-His-Lys-Ala-Arg-Val-Leu-Phe(pNO2)-Glu-
Ala-Nle-Ser-OH (Bachem Bioscience) as a substrate.
The C-terminal pentapeptide cleaved product was mea-
Results and Discussion
Chemistry
sured at 220 nm by reversed-phase HPLC analysis at
29
different times ofincubation.
The HIV Pr inhibition
Synthesis of N-benzyl pseudopeptides is outlined in
Schemes 1 and 2.
results show a moderate activity for mono- and di-
benzylated pseudopeptides 1, 2, 7 and 8, indicating that
the reduction ofmolecular size decreases the number of
interactions between enzyme and inhibitor especially at
P2 site.
Analogues 1–6 containing N-benzyl serine were pre-
pared by initial coupling ofFmoc-protected serine
t-butyl ether with methionine t-butylamide (Scheme 1).
Resulting dipeptide, after Fmoc removal, was benzyl-
ated by reductive alkylation and resulting mixture of
mono- and di-N-benzylated products was separated by
column chromatography.22 Pseudodipeptides 1 and 2
In pseudotripeptides, bulky tricyclic system (Fmoc) is
preferred in P2 position confirming the trend previously
observed.14ꢀ16 In P1, branched aliphatic side chain
(Leu) is slightly better as compared aromatic ring (Phe)
Table 1. Analytical data and physicochemical properties of N-benzyl pseudopeptides
20
D
(C=1, MeOH)
No.
Compd
HPLC
Mp
(ꢁC)
½aŠ
MS
(M+H+
)
KI (A)
KI (B)
1
2
3
4
5
6
7
8
NH(Bzl)Ser-Met-NH-tBu
N(Bzl)2Ser-Met-NH-tBu
5.92
7.25
10.45
11.70
9.42
10.63
6.25
7.21
10.55
12.41
9.73
4.83
6.41
9.28
10.34
8.51
9.14
5.52
6.43
9.11
10.76
8.39
10.22
93–95
87–89
ꢀ33.4
ꢀ31.8
ꢀ20.7
ꢀ16.3
ꢀ12.1
ꢀ9.5
382.6
472.7
662.5
751.8
628.9
716.7
396.6
486.6
676.6
765.7
643.1
730.8
Z-Phe-N(Bzl)Ser-Met-NH-tBu
Fmoc-Phe-N(Bzl)Ser-Met-NH-tBu
Z-Leu-N(Bzl)Ser-Met-NH-tBu
Fmoc-Leu-N(Bzl)Ser-Met-NH-tBu
NH(Bzl)Hse-Met-NH-tBu
132–136
124–127
143–145
138–141
88–91
ꢀ30.3
ꢀ27.1
ꢀ18.5
ꢀ15.4
ꢀ17.7
ꢀ11.2
N(Bzl)2Hse-Met-NH-tBu
82–84
9
Z-Phe-N(Bzl)Hse-Met-NH-tBu
Fmoc-Phe-N(Bzl)Hse-Met-NH-tBu
Z-Leu-N(Bzl)Hse-Met-NH-tBu
Fmoc-Leu-N(Bzl)Hse-Met-NH-tBu
117–120
109–113
140–142
129–133
10
11
12
11.62

M. Marastoni et al. / Bioorg. Med. Chem. 11 (2003) 2477–2483
2479
Scheme 1. Synthesis of N-benzyl pseudopeptides 1–6.
suggesting in this case a non-favourable conformational
perturbation due at presence ofthe adjacent N-benzyl
moiety.
(1–6), promote easier hydrogen bond formation with
catalytic aspartic acid.
In general, methionine t-butylamide is well tolerant in
P1I and P2I sites and the presence ofa supplementary
benzyl group in P1 catalytic position seems to generate
additional van der Waals interactions; this is a favour-
able condition to decrease drug resistance through
enzyme mutation (Fig. 2).
The higher length ofthe hydroxyalkyl chain in N-benzyl
homoserine core residue (7–12) over N-benzyl serine
Table 2. Inhibitory potencies and metabolic degradation of N-benzyl
pseudopeptides
No.
IC50
(nM)a
ED50
(nM)a
Half-life (min)
Better isolate enzyme N-benzyl inhibitors were also tes-
ted for their capacity to inhibit viral replication in the
HIV-1 strain IIIB in CEM cells.30 All compounds dis-
played satisfactory activity quite close to the IC50 values
for HIV-1 Pr inhibition. In particular N-benzyl pseudo-
tripeptide 12 (ED50=52 nM) shows good potency con-
firming its favourable pharmacokinetic properties,
especially an essential cell membrane penetration.
Culture medium
Human plasma
1
2
3
4
5
6
7
8
3700
2220
1170
789
1010
370
2643
1730
620
297
365
NDb
NDb
NDb
543
NDb
310
NDb
NDb
245
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
270
230
>360
310
>360
>360
325
>360
342
>360
9
Metabolic stability
10
11
12
118
295
52
The stability against enzymatic hydrolysis ofinhibitors
1–12 was evaluated in cell culture medium (RPMI) and
in human plasma, after incubation at 37 ꢁC. The time
course ofpseudopeptide degradation was ofllowed by
RP-HPLC analysis at varying periods ofincubation. 31,32
170
aValues are the average ofat least two determinations ( n=2) unless
otherwise noted.
bNot determined.

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M. Marastoni et al. / Bioorg. Med. Chem. 11 (2003) 2477–2483
Scheme 2. Synthesis ofthe N-benzyl analogues 7–12.
All compounds display very high stability in cell culture
medium, in human plasma only pseudotripeptides 3, 4,
6, 9 and 11 exhibit an half-life less to 360 min (Table 2).
Experiments carried out using human plasma showed
essentially identical trend ofinhibitor degradation, with
a very slowly formation of a single catabolite. The
degradation products were isolated by RP-HPLC and
after mass spectrometry analysis were found to be com-
patible with the N-terminal pseudodipeptides.
The data ofthe present study demonstrate, that the
new inhibitors were very weakly cleaved ofthe amide
bond between N-benzyl core residue and methionine
t-butylamide.
Conclusion
Figure 2. Schematic drawing showing the expected interactions
between N-benzyl pseudotripeptide 12 and the HIV-1 protease active
site. Hydrogen bonds are shown as dashed lines with distances in A.
Data were obtained by means ofbinding model previously suggested
by diamino-hydroxyalkane derivatives (I) dynamic simulations.
On the basis ofprevious efforts in our laboratories which
resulted in the development ofHIV-1 protease inhibi-
tors, we designed, synthesized and tested new pseudo-
peptides containing N-benzyl hydroxyalkyl residue in

M. Marastoni et al. / Bioorg. Med. Chem. 11 (2003) 2477–2483
2481
core structure. Our results show that the N-alkylation of
central amide bond confer elevated metabolic stability
and maintain satisfactory antiviral activity. The N-benzyl
homoserine pseudotripeptide 12 is endowed with a good
inhibition against isolated enzyme and HIV replication
in culture cell.
NMM (1 mmol), HOBt (1.1 mmol) and WSC
(1.1 mmol) in this order at 0 ꢁC. The reaction mixture
was stirred for 1 h at 0 ꢁC and 18 h at room temperature;
then the solution was diluted with EtOAc (100 mL) and
washed consecutively with HCl 0.1 N, brine, NaHCO3
and brine. The organic phase was dried (MgSO4), fil-
tered and evaporated to dryness. The residue was trea-
ted with Et2O and resulting solid, separated by
centrifugation.
In summary, our work in this field produced a pseudo-
peptide model which can permit structural changes in
order to ameliorate viral activity and pharmacokinetic
properties.
Fmoc removal (b). The Fmoc group was cleaved with
a solution of 20% piperidine in DMF for 25 min. After
evaporation the residue was triturated with Et2O,
centrifugated and resulting solid was collected and
dried.
Experimental
General
Amino acids, amino acid derivatives, resins and chemi-
cals were purchased from Bachem, Novabiochem, or
Fluka (Switzerland).
N-Benzylation (c). To a solution ofdipeptide H-
Ser(tBu)-Met-NH-tBu or H-Asp(OtBu)-Met-NH-tBu
(2 mmol) in acetonitrile were added benzaldehyde
(2 mmol) and NaCNBH3 (3 mmol). The reaction
mixture was stirred for 15 min and acetic acid was
added dropwise until pH 7; after 45 min, the solution
was evaporated under reduced pressure. The residue
was diluted with EtOAc (50 mL) and washed with
water; the organic phase was dried (MgSO4), filtered
and evaporated. The resulting mixture ofmono and di
N-benzylated dipeptides was separated by column
chromatography.
Crude pseudopeptides were purified by preparative
reversed-phase HPLC using a Water Delta Prep 4000
system with a Waters PrepLC 40 mm Assembly column
C18 (30 Â 4 cm, 300 A, 15 mm spherical particle size col-
umn). The column was perfused at a flow rate of 50 mL/
min with a mobile phase containing solvent A (10%, v/v,
acetonitrile in 0.1% TFA), and a linear gradient from 0
to 50% ofsolvent B (60%, v/v, acetonitrile in 0.1%
TFA) in 25 min was adopted for the elution of com-
pounds. HPLC analysis was performed by a Beckman
System Gold with a Hypersil BDS C18 column (5 mm;
4.6 Â 250 mm). Analytical determination and capacity
factor (K0) ofthe peptides were determined using HPLC
conditions in the above solvent system (solvents A and
B) programmed at flow rates of1 mL/min using the
following linear gradients: (a) from 0 to 100% B in
25 min and (b) from 10 to 70% B in 25 min. All
pseudopeptides showed less than 1% impurities when
monitored at 220 and 254 nm.
TFA deprotection (d). t-Butyl protections were
removed by treating intermediates with aqueous 90%
TFA (1:10, w/v) for 30–40 min. After evaporation, the
residue was worked up as described in (b).
Coupling via mixed anhydride (e). To a stirred solution
ofBoc-amino acid (1 mmol) in DMF (3 mL), triethyl-
amine (TEA, 1 equiv) was added; the mixture was
cooled to ꢀ10 ꢁC, treated with isobutylchloroformate
(IBCF, 1 equiv), and allowed to react for 5 min. A pre-
cooled solution ofthe amino component tri-
fluoroacetate (1.1 mmol) in DMF (3 mL) was added to
the mixture, followed by TEA (1.1 equiv). The reaction
mixture was stirred for 1 h at ꢀ10 ꢁC and 3 h at room
temperature and then evaporated. After evaporation the
residue was worked up as described in (a).
Molecular weight ofcompounds were determined by a
MALDI-TOF analysis using
a Hewlett Packard
G2025A LD-TOF system mass spectrometer and
a-cyano-4-hydroxycinnamic acid as a matrix. The
values are expressed as MH+. TLC was performed in
precoated plates ofsilica gel F254 (Merck, Darmstadt,
Germany) using the following solvent systems: (c)
AcOEt/n-hexane (1:1, v/v), (d) CH2Cl2/methanol
(9.5:0.5, v/v), (e) CH2CL2/methanol (9:1, v/v), (f)
CH2CL2/methanol/toluene (17:2:1, v/v/v). Ninhydrin
(1%) or chlorine iodine spray reagents were employed
to detect the peptides. Melting points were determined
by a Kofler apparatus and are uncorrected. Optical rota-
tions were determined by a Perkin–Elmer 141 polarimeter
with a 10-cm water-jacketed cell. 1H NMR spectroscopy
was obtained on a Bruker AC 200 spectrometer.
Introduction of Z or Fmoc (f). To a solution of
N-benzyl pseudotripeptide (0.5 mmol) in DMF (5 mL)
was added Z-OSu or Fmoc-OSu (0.5 mmol) at 0 ꢁC. The
mixture was stirred for 1 h at 0 ꢁC and 15 h at room
temperature and then evaporated. The resulting target
compounds 3–6 and 9–12 were purified by preparative
HPLC.
Chemistry
General procedures. Coupling with WSC/HOBt (a). To a
solution ofthe carboxy component (1 mmol) in DMF
(10 mL) were added the amino component (1 mmol),
Reduction of b-carboxylic side chain (g). To a stirred
solution ofmono or di N-benzyl aspartyl dipeptide
(1 mmol) in THF (5 mL) at ꢀ15 ꢁC, N-methilmorpholine

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M. Marastoni et al. / Bioorg. Med. Chem. 11 (2003) 2477–2483
(NMM, 1 mmol) was added, followed by isobutyl-
chloroformate (IBCF, 1 mmol). After 20 min, the pre-
cipitate was removed by filtration and the filtrate was
Z-Leu-N(Bzl)-Hse-Met-NH-tBu
(11).
1H
NMR
(CDCl3) 1.07 (d, 6H), 1.39 (s, 9H), 1.79–1.85 (m, 3H),
2.19 (s, 3H), 2.33–2.45 (m, 6H), 3.39 (m, 1H), 4.03 (s, 1H),
4.15 (m, 2H), 4.45–4.70 (m, 4H), 5.25 (s, 2H), 5.67 (sbr,
1H), 6.03 (sbr, 1H), 7.08–7.29 (m, 10H), 7.44 (sbr, 1H).
added dropwise to a cold (ꢀ10 ꢁC) solution ofNaBH
4
(3 mmol) in EtOH (3 mL). The solution was stirred for
10 min at 0 ꢁC and for additional 15 min at room tem-
perature and then evaporated. The residue was worked
up as described in (a).
Fmoc-Leu-N(Bzl)-Hse-Met-NH-tBu (12). 1H NMR
(CDCl3) 1.09 (d, 6H), 1.28 (s, 9H), 1.60–1.74 (m, 3H),
2.06 (s, 3H), 2.25–2.38 (m, 6H), 3.21 (m, 1H), 3.82 (s,
1H), 3.96 (m, 2H), 4.35–4.70 (m, 4H), 5.28 (s, 2H), 5.66
(sbr, 1H), 6.04 (sbr, 1H), 7.14–7.44 (m, 15H).
1
NH(Bzl)-Ser-Met-NH-tBu (1). H NMR (CDCl3) 1.42
(s, 9H), 2.15–2.23 (m, 5H), 2.87 (m, 2H), 3.65 (d, 1H),
3.91–4.02 (m, 4H), 4.53 (m, 1H), 4.90 (sbr, 1H), 6.15
(sbr, 1H), 6.32 (sbr, 1H), 7.21 (m, 5H).
Metabolic stability assay
N(Bzl)2-Ser-Met-NH-tBu (2). 1H NMR (CDCl3) 1.40 (s,
9H), 2.11–2.21 (m, 5H), 2.62 (m, 2H), 3.60 (d, 1H),
3.81–4.05 (m, 6H), 4.86 (sbr, 1H), 6.11 (sbr, 1H), 6.35
(sbr, 1H), 7.15–7.34 (m, 10H).
The kinetics ofnew inhibitors degradation were studied
in culture medium (RPMI) and human plasma. 0.1 mL
ofa solution ofeach compound (10 mg/mL in aceto-
nitrile/H2O 1:1) was added to 1 mL ofRPMI containing
20% fetal calf serum. Alternatively, test compounds
were incubated with plasma (0.6 mL) in a total volume
of1.5 mL of10 mM Tris–HCl buffer, pH 7.5. Incu-
bation was performed at 37 ꢁC for different time: up to
360 min in the case ofhuman plasma and up to 2 days
in the case ofRPMI containing 20% FCS. The incu-
bation was terminated by addition ofethanol (0.2 mL),
the mixture poured at 21 ꢁC and after centrifugation
(5000 rpm for 10 min), aliquots (20 mL) ofthe clear
supernatant were injected into RP-HPLC column.
HPLC was performed as described above (see Experi-
mental procedures, general).
1
Z-Phe-N(Bzl)-Ser-Met-NH-tBu (3). H NMR (CDCl3)
1.32 (s, 9H), 2.04 (s, 3H), 2.27–2.42 (m, 4H), 3.40–3.58
(m, 3H), 4.03 (m, 2H), 4.53–4.86 (m, 5H), 5.34 (S, 2H),
5.86 (sbr, 1H), 6.11 (sbr, 1H), 7.05–7.19 (m, 15H), 7.45
(sbr, 1H).
Fmoc-Phe-N(Bzl)-Ser-Met-NH-tBu (4). 1H NMR
(CDCl3) 1.27 (s, 9H), 1.94 (s, 3H), 2.11–2.28 (m, 4H),
3.40–3.56 (m, 3H), 3.99 (m, 2H), 4.45–4.66 (m, 5H), 5.32
(S, 2H), 5.77 (sbr, 1H), 6.01 (sbr, 1H), 7.02–7.25 (m,
19H), 7.37 (sbr, 1H).
1
Z-Leu-N(Bzl)-Ser-Met-NH-tBu (5). H NMR (CDCl3)
Test for the inhibition of HIV-1 protease
1.03 (d, 6H), 1.32 (s, 9H), 1.74–1.82 (m, 3H), 2.09 (s,
3H), 2.24–2.36 (m, 4H), 3.36 (m, 1H), 3.95 (s, 1H), 4.05
(m, 2H), 4.48–4.79 (m, 4H), 5.30 (s, 2H), 5.76 (sbr, 1H),
6.05 (sbr, 1H), 7.15–7.27 (m, 10H), 7.40 (sbr, 1H).
For determination ofIC 50 values, affinity-purified HIV-1
protease (Bachem Bioscience) 1.1 nM final concen-
tration, was added to a solution (100 mL final volume)
containing inhibitor, 4 mM peptide substrate (His-Lys-
Ala-Arg-Val-Leu-p-nitro-Phe-Glu-Ala-Nle-Ser, Bachem
Bioscience), and 1.0% dimethyl sulfoxide in assay buf-
fer: 1.0 mM dithiothreitol, 0.1% glycerol, 80 mM
sodium acetate, 160 mM sodium chloride, 1.0 mM
EDTA, all at pH 4.7. The solution was mixed and
incubated for 25 min at 37 ꢁC and reaction quenched by
the addition oftrifluoroacetic acid, 2% final concen-
tration. The Leu-Phe(p-NO2) bond ofthe substrate was
cleaved by the enzyme. The cleavage products and sub-
strate were separated by RP-HPLC. Absorbance was
measured at 220 nm, peak areas were determined, and
percent conversion to product was calculated using
relative peak areas. The data were plotted as percent
control (the ratio ofpercent conversion in the presence
and absence ofinhibitor) versus inhibitor concentration
and fitted with the equation Y=100/1+(X/IC50)A,
where IC50 is the inhibitor concentration at 50% inhi-
bition and A is the slope ofthe inhibition curve.
Fmoc-Leu-N(Bzl)-Ser-Met-NH-tBu (6). 1H NMR
(CDCl3) 1.09 (d, 6H), 1.37 (s, 9H), 1.70–1.85 (m, 3H),
2.15 (s, 3H), 2.31–2.44 (m, 4H), 3.29 (m, 1H), 3.80 (s, 1H),
4.10 (m, 2H), 4.42–4.73 (m, 4H), 5.22 (s, 2H), 5.59 (sbr,
1H), 5.98 (sbr, 1H), 7.02–7.26 (m, 14H), 7.51 (sbr, 1H).
1
NH(Bzl)-Hse-Met-NH-tBu (7). H NMR (CDCl3) 1.38
(s, 9H), 2.13–2.28 (m, 7H), 2.80 (m, 2H), 3.55 (d, 1H),
3.90–4.09 (m, 4H), 4.65 (m, 1H), 4.87 (sbr, 1H), 6.11
(sbr, 1H), 6.29 (sbr, 1H), 7.13 (m, 5H).
1
N(Bzl)2-Hse-Met-NH-tBu (8). H NMR (CDCl3) 1.44
(s, 9H), 2.24–2.32 (m, 7H), 2.58 (m, 2H), 3.51 (d, 1H),
3.77–3.94 (m, 6H), 4.81 (sbr, 1H), 5.97 (sbr, 1H), 6.19
(sbr, 1H), 7.03–7.25 (m, 10H).
1
Z-Phe-N(Bzl)-Hse-Met-NH-tBu (9). H NMR (CDCl3)
1.30 (s, 9H), 1.99 (s, 3H), 2.17–2.31 (m, 6H), 3.45–3.59
(m, 3H), 4.21 (m, 2H), 4.63–4.79 (m, 5H), 5.32 (S, 2H),
5.80 (sbr, 1H), 6.22 (sbr, 1H), 7.13–7.30 (m, 15H), 7.50
(sbr, 1H).
Cell culture activity against HIV-1 IIIB
HIV-1 IIIB was obtained from HIV-1 IIIB chronically
infected Molt-4 cells as a supernatant fluid. The 50%
tissue culture infection dose (TC ID50) was determined
by an end point titration procedure.35 CEM cells (5000/
mL) were exposed to HIV-1 IIIB fluid at a multiplicity
Fmoc-Phe-N(Bzl)-Hse-Met-NH-tBu (10). 1H NMR
(CDCl3) 1.39 (s, 9H), 2.03 (s, 3H), 2.20–2.35 (m, 6H),
3.33–3.45 (m, 3H), 3.97 (m, 2H), 4.31–4.47 (m, 5H), 5.19
(S, 2H), 5.70 (sbr, 1H), 5.98 (sbr, 1H), 7.23–7.47 (m, 20H).

M. Marastoni et al. / Bioorg. Med. Chem. 11 (2003) 2477–2483
2483
ofinefction (m.o.i.) 0.001 TC ID
(mL). Aliquots
13. Marastoni, M.; Fantin, G.; Bortolotti, F.; Tomatis, R.
Arzneim.-Forsch./Drug Res. 1996, 46, 1099.
14. Marastoni, M.; Salvadori, S.; Bortolotti, F.; Tomatis, R.
J. Pept. Res. 1997, 37, 538.
15. Marastoni, M.; Bortolotti, F.; Salvadori, S.; Tomatis, R.
Arzneim.-Forsch./Drug Res. 1998, 6, 709.
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50
(0.2 mL) ofcells were placed in 96-well microtitre plates
with 2 mL ofthe appropriate concentrations ofinhibi-
tors dissolved in DMSO. After incubation for 6 days in
RPMI-1640 medium containing 10% fetal calf serum,
the p24 antigen ofHIV in the supernatant was deter-
mined by an ELISA assay kit (RETRO-TEK, Cellular
Products Inc., Buffalo, USA). The ED50 (50% dose)
values were calculated as the dose ofthe inhibitor that
resulted in a 50% reduction in p24 levels as compared to
those in control wells.
19. Bold, G.; Fassler, A.; Capraro, H. G.; Cozens, R.; Klim-
¨
kait, T.; Lazdins, J.; Mestan, J.; Poncioni, B.; Rosel, J.; Stover,
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Acknowledgements
Financial support ofthis work by University ofFerrara
and by Ministero dell’Universita e della Ricerca Scienti-
fica e Tecnologica (MURST) is gratefully acknowledged.
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