Novel fluoropeptidomimetics: Synthesis, stability studies and protease inhibition

Article abstract of DOI:10.1016/j.bmc.2005.02.011

Designer fluoropeptidomimetics as protease inhibitors are revealed. The key peptidomimetic region in the inhibitors contains a '-CHF-S-' moiety and is designed to mimic the tetrahedral oxyanion species during the hydrolysis of a peptide bond. Designed flu

Full text of DOI:10.1016/j.bmc.2005.02.011

Bioorganic & Medicinal Chemistry 13 (2005) 2943–2958
Novel fluoropeptidomimetics: synthesis, stability studies
and protease inhibition
Subhash C. Annedi, Kanchana Majumder, Lianhu Wei, Catherine E. Oyiliagu,
Sheeba Samson and Lakshmi P. Kotra*
Molecular Design and Information Technology Center, Leslie Dan Faculty of Pharmacy, University of Toronto,
19 Russell Street, Toronto, Ontario, Canada M5S 2S2
Received 19 January 2005; revised 3 February 2005; accepted 4 February 2005
Abstract—Designer fluoropeptidomimetics as protease inhibitors are revealed. The key peptidomimetic region in the inhibitors con-
tains a Ô–CHF–S–Õ moiety and is designed to mimic the tetrahedral oxyanion species during the hydrolysis of a peptide bond.
Designed fluoropeptidomimetics in aqueous methanol slowly (in several hours to days) yielded the corresponding methyl ether
and/or the oxazole derivatives after cyclization. Alkyl substitutions at the C-2 position exhibited enhanced aqueous stability. Nature
of Ô–CHF–S–Õ moiety and the stabilities of various fluoropeptidomimetics in aqueous solution are disclosed in detail. Fluoropepti-
domimetics containing bulky substitutions at P1 such as compounds 15 and 16 exhibited time-dependent loss of activities against
chymotrypsin, up to 67% and 79% with a Ki of 63 and 120 lM, respectively. Fluoropeptidomimetics are a novel class of protease
inhibitors and the next generation of fluoropeptidomimetics should incorporate enhanced stability.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tidomimetics as a novel strategy where the scissile pep-
tide bond Ô–CO–NH–Õ is substituted with Ô–CHF–S–Õ.4
It is hypothesized that the latter moiety mimics the tet-
rahedral transition-state oxyanion species during the
hydrolysis of a peptide bond (species A and B, Fig. 1).
Peptidomimetics mimicking the transition-state species
during the hydrolysis of a peptide bond (i.e., oxyanion)
and molecules that bind to serine/cysteine proteases via
covalent bond formation have been investigated for a
number of years.1–3 Some of these molecules include
hydroxyethylene-bearing inhibitors, haloalkylketones
such as trifluoromethyl derivatives, boronic acid deriva-
tives, phosphonate derivatives, and b-lactam containing
molecules. Most of the covalent inhibitors take advan-
tage of the catalytic mechanism of the proteases and
covalently bind to the catalytic serine/cysteine residue
or the catalytic histidine residue. These protease inhibi-
tion strategies are used to probe biochemical mecha-
nisms of proteases and protease-like enzymes, as well
as for the development of therapeutic agents against sev-
eral viral, bacterial, fungal, and human proteases.
I
.
F
H
O
F
O
S
N
H
A
B
I I .
H
H
N
N
S
+
S
R
:Nu
R
C
D
R = Side chain
Our laboratory has been investigating the design and
development of new strategies to inhibit proteases and
to selectively inhibit proteases. We proposed fluoropep-
R = Side chain
Figure 1. Possible modes of inhibition of a protease by the fluoro-
peptidomimetic: (I) transition-state mimicry of the tetrahedral oxyan-
ion species (species A) by the fluorine atom in the key moiety of the
fluoropeptidomimetic (species B); (II) elimination of fluorine assisted
by the lone pair of electrons on the adjacent sulfur atom (species C) to
generate the reactive species D in the active site of a protease
predisposing the species for an attack by a nucleophilic residue.
Keywords: Fluoropeptidomimetics; Protease inhibitors; Chemistry and
aqueous stability.
*
Corresponding author. Tel.: +1 416 978 2882; fax: +1 416 978
8511; e-mail: p.kotra@utoronto.ca
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.011

2944
S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
These peptidomimetics are designed to bind to the active
site of a protease and inhibit the protease via one of the
following modes: as transition-state mimics, or slow,
tight-binding inhibitors, or covalent irreversible inhibi-
tors via the formation of sulfenium species (species C
and D, Fig. 1).
Here, for the first time we disclose the synthesis, reactiv-
ities and protease inhibition studies of the above
designer tripeptide derivatives of the fluoropep-
tidomimetics against chymotrypsin, and reveal the sta-
bilities of these molecules.
Interestingly, bacterial haloacid dehalogenases, specifi-
cally 2-fluoroacetate dehalogenase defluorinates fluoro-
acetate to produce glycolate and a fluoride ion.5,6 An
aspartate residue in these enzymes serves as a nucleo-
philic residue and the reaction mechanism appears to in-
volve a covalent complex (an ester species) between the
dehalogenase and its substrate.5 It is also proposed that
the fluorine atom may be involved in hydrogen bonding
in the active site of fluoroacetate dehalogenase.5,6 The
design strategy for fluoropeptidomimetics incorporates
such potential hydrogen bonding of the fluorine atom
with the protease oxyanion hole and proposes to mimic
the transition-state oxyanion formed during the hydro-
lysis of a peptide to function as an inhibitor of a
protease.
2. Synthesis
Compounds 1–20 were designed and/or selected to study
the chemistry, understand their stabilities and evaluate
protease inhibition activities. Compounds 1–14 are
Ôdipeptide mimicsÕ, and compounds 15–20 are Ôtripeptide
mimicsÕ. Here, we describe the nuances involved in the
synthesis of fluoropeptidomimetics, and successful syn-
thesis of tripeptide derivatives providing the ability to
synthesize various derivatives for medicinal chemistry
investigations. Compounds 1–7 were prepared using an
earlier reported procedure by our group.4 In the case
of compounds 5 and 7, where there is a relatively large
substitution on C-2, deprotection of the phthaloyl group
was not complete under standard conditions. Several
deprotection conditions were investigated including
hydrazine hydrate and ethylenediamine to remove the
phthaloyl moiety.9 Deprotection with ethylenediamine10
at room temperature successfully yielded amines 21–25
(Scheme 1) as inseparable mixtures of diastereomers in
moderate yields.4 Increased reaction times or higher
temperatures decreased the product yields. The coupling
Molecules containing the moiety Ô–CHF–S–Õ are little
known in the literature and molecules with Ôside chainÕ
substitutions vicinal to fluorine are rare as well.7 Synthe-
ses of such molecules is not trivial and typically, they
served as intermediates during the synthesis of other
derivatives.8 Recently, we reported the chemistry of sev-
eral dipeptide analogues using the fluoropeptidomimet-
ics strategy, for the first time.4 During our
investigations to design peptidomimetics and develop
synthetic procedures to prepare fluoropeptidomimetics,
we discovered a complex pattern of stabilities and reac-
tivities for these molecules. The comprehensive set of
fluoropeptidomimetics 1–20 were synthesized and evalu-
ated for their aqueous stability to investigate their char-
acteristic profiles. Tripeptide derivatives 15–19 were also
evaluated for their protease inhibition activity against
chymotrypsin. We show here that alkyl substitutions
provide enhanced chemical stability to the fluoropepti-
domimetics, giving rise to an opportunity for biological
activity evaluations for protease inhibition.
F
H2N
i
5, 7-10
SPh
R
21 R = CH2Ph
22 R = CH(CH3)2
23 R = CH(CH3)C2H5
24 R = CH2CH(CH3)2
25 R = CH2(cyclohexyl)
Scheme 1. Reagents: i. MeCO2HN-L-Ala-OH, EDAC, HOBt,
CH2Cl2.
F
O
7
F
1
H
N
H
H2N
N
3
6
4
8
2
5
R2R1N
SPh-R4
N
SPh
H
O
R3
O
R
15 R = CH2Ph
1 R1 = R2 = R3 = R4 = H
16 R = CH2(cyclohexyl)
17 R = CH(CH3)2
2 R1 = R2 = Phth, R3 = R4 = H
3 R1 = R3 = R4 = H, R2 = CO2Me
18 R = CH2CH(CH3)2
19 R = CH(CH3)C2H5
4 R1 = R3 = R4 = H, R2 = Boc
5 R1 = CO2Me, R2 = R4 = H, R3 = CH2Ph
6 R1 = CO2Me, R2 = R4 = H, R3 = CH3
7 R1 = CO2Me, R2 = R4 = H, R3 = CH(CH3)2
8 R1 = CO2Me, R2 = R4 = H, R3 = CH(CH3)C2H5
9 R1 = CO2Me, R2 = R4 = H, R3 = CH2CH(CH3)2
10 R1 = CO2Me, R2 = R4 = H, R3 = CH2(cyclohexyl)
11 R1 = CO2Me, R2 = H, R3 = CH(CH3)2, R4 = p-Cl
12 R1 = CO2Me, R2 = H, R3 = CH(CH3)2, R4 = p-F
13 R1 = CO2Me, R2 = H, R3 = CH(CH3)2, R4 = p-CH3
14 R1 = CO2Me, R2 = H, R3 = CH(CH3)2, R4 = p-OMe
F
O
H
N
H2N
SPh
N
H
Ph
20

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2945
of the second amino acid to monomers 21–25 at the ami-
PhthN
PhthN
i
ii
SPh
OH
no terminal was carried out using EDAC–HOBt condi-
tions to obtain fluorodipeptides 5, 7–10 as inseparable
mixtures of diastereomers. These compounds represent
one set of fluoropeptidomimetics with various side
chains at the C-2 position.
R
R
55 R = CH2Ph
50 R = CH2Ph
56 R = CH2(cyclohexyl)
57 R = CH(CH3)2
51 R = CH2(cyclohexyl)
52 R = CH(CH3)2
58 R = CH2CH(CH3)2
59 R = CH(CH3)C2H5
53 R = CH2CH(CH3)2
54 R = CH(CH3)C2H5
Compounds 11–14, carrying various p-substitutions on
the thiophenyl moiety were prepared from tosylate
2611 and alcohol 27 (Scheme 2). Initially, the monomeric
precursors 28–31 were prepared starting from 26 under
alkaline conditions (Method A, Scheme 2).12 In most
cases most of the starting material was recovered and
the yields were low. Alternatively, the substituted thi-
ophenol derivatives (R-PhSH) were converted into their
corresponding dimeric forms [(R-PhS)2]13 and coupled
to alcohol 27 according to our earlier reported proce-
dure (Method B).4 This method proved to be better than
the earlier one, with reasonable yields. These monomers
28–31 were then fluorinated with SelectfluorTM to give
the fluoro derivatives 32–35 as inseparable mixtures of
diastereomers. The diastereomeric mixture is a result
of the fluorination at C-2 where no stereocontrol was
imposed. Deprotection of the phthaloyl group in 32–
35 with ethylenediamine and coupling of these amines
36–39 to MeCO2HN-L-Ala-OH using EDAC–HOBt
conditions yielded the target dipeptides 11–14 as a mix-
ture of diastereomers.
F
F
iv
H2N
iii
PhthN
SPh
SPh
R
R
65 R = CH2Ph
60 R = CH2Ph
66 R = CH2(cyclohexyl)
67 R = CH(CH3)2
61 R = CH2(cyclohexyl)
62 R = CH(CH3)2
68 R = CH2CH(CH3)2
69 R = CH(CH3)C2H5
63 R = CH2CH(CH3)2
64 R = CH(CH3)C2H5
F
H
N
iii
PhthN-Gly-L-Val
15-19
SPh
R
70 R = CH2Ph
71 R = CH2(cyclohexyl)
72 R = CH(CH3)2
73 R = CH2CH(CH3)2
74 R = CH(CH3)C2H5
Scheme 3. Reagents: (i) PhSSPh, Bu3P, DMF; (ii) SelectfluorTM, Et3N,
CH3CN; (iii) Ethylenediamine, MeOH; (iv) PhthN-Gly-L-Val-OH,
EDAC, HOBt, DMF, 0 ꢁC.
The strategy for the synthesis of target tripeptides 15–19
involved the preparation of fluoro monomers 60–64
(Scheme 3) followed by deprotection and coupling with
appropriate amino acids. Briefly, N-protected amino
alcohols 50–54 were prepared from their corresponding
a-amino acids or esters by reduction with sodium boro-
hydride.14 Compounds 50–54 were then converted into
the phenylthio derivatives 55–59 by reacting with diph-
enyldisulfide in the presence of tributylphosphine
(Scheme 3).15 Compounds 55–59 were subjected to elec-
trophilic fluorination using SelectfluorTM to obtain com-
pounds 60–64, respectively.4,16
Deprotection of the phthaloyl group on the terminal
amino moiety of monomers 60–64 was achieved by
treatment with ethylenediamine at rt for 12–16 h. Com-
pounds 65–69 were coupled to Phthaloyl-N-Gly-L-Val-
OH using standard amino acid coupling conditions to
obtain compounds 70–74 in good yields. Deprotection
of the phthaloyl protecting groups in 70–74 was carried
out with ethylenediamine at room temperature to obtain
the target fluoropeptidomimetics 15–19 in 50–88%
yields. Compounds 15–19 were chromatographically
pure and were used as such for biological activity evalu-
ations. Their purity and the structures were also con-
firmed through various NMR spectral data, as well as
high-resolution mass spectral analyses.
PhthN
i
ii
PhthN
OR
SPh-R
During the synthesis of compounds 15–19, after the
fluorination reactions, all compounds were diastereo-
meric mixtures with no stereospecificity at position 1.
Chromatographically, we could not see any separation
either on TLC or by column chromatography even after
repeated attempts with various solvent systems. Using
NMR, we were able to observe a distinct peak for each
of the two diastereomers in some fluorinated com-
pounds, but not in all. Such mixtures and ratios were
distinctly identified in the spectral data. For all practical
purposes and biological activity evaluations the diaste-
reomers could not be separated, and hence were used
as such.
28 R = p-Cl
26 R = Ts
27 R = H
29 R = p-F
30 R = p-Me
31 R = p-OMe
F
F
iii
iv
H2N
PhthN
11-14
SPh-R
SPh-R
32 R = p-Cl
36 R = p-Cl
37 R = p-F
38 R = p-Me
39 R = p-OMe
33 R = p-F
34 R = p-Me
35 R = p-OMe
Scheme 2. Reagents: i. R-PhSH, KOH, EtOH, Reflux (Method A);
(R-PhS)2, Bu3P, DMF (Method B); ii. SelectfluorTM, Et3N, CH3CN; iii.
Ethylenediamine, MeOH, iv. MeCO2HN-L-Ala-OH, EDAC, HOBt,
CH2Cl2.
Compound 20 was envisioned for synthesis through an
imine formation followed by reduction into the corre-
sponding amine (Scheme 4). Accordingly, aldehyde 40

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S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
Table 1. % Decomposition of compounds 1–14 in CD3OD–D2O (3:2)
after 24 h at rt
F
H
N
i
SPh
RHN
Compound
% Decomposition
FmocHN
CHO
RHN
1
42
68
86
85
63
34
41
55
40
35
46
43
83
98
Ph
2
40
41 R = Fmoc
42 R = H
ii
3
4
F
5
O
H
6
iii
N
SPh
Ph
N
H
7
8
9
10
11
12
13
14
43 R = Fmoc
20 R = H
ii
Scheme 4. Reagents: i. (a) Compound 21, Na2SO4, CH2Cl2; (b)
NaBH4, MeOH, 0 ꢁC; ii. 20% Morpholine in DMF; iii. FmocHN-Gly-
OH, EDAC, HOBt, CH2Cl2–DMF (7:1), 0 ꢁC.
was prepared and coupled to the free amine 21 using
Na2SO4 in CH2Cl2.17,18 The crude imine was reduced
under NaBH4 conditions to obtain the corresponding
deoxy dipeptide 41 (Scheme 4). During the reaction,
racemization at Ca of valine was observed and the prod-
uct was isolated as an inseparable mixture of four diaste-
reomers. The deprotection of the Fmoc-protecting
group using 20% morpholine in DMF gave amine
42.19 Further coupling with FmocHN-Gly-OH, fol-
lowed by deprotection of the Fmoc group was carried
out using EDAC–HOBt conditions and 20% morphol-
ine in DMF, respectively, to obtain the final tripeptide
20 as an inseparable mixture of four diastereomers.
Table 2. % Decomposition of compounds 15–20 and 49 in CD3OD–
D2O (3:2) after 18 h at rt
Compound
% Decomposition
15
16
17
18
19
20
49
12
12
24
27
6
100
0
OMe
SPh
H
N
i
3. Stability studies
4
BocHN
The stability studies for dipeptides 1–14, tripeptides 15–
20 and N-phthaloyl monomer 49 in aqueous methanol
were carried out to understand the chemical behavior
and the effect of various substitutions and/or functional
groups around the core Ô–CHF–S–Õ moiety. Compounds
1–20 and 49 were studied for their stability in 40% aque-
ous methanol by 19F NMR spectral analyses (deuterated
solvents). Methanol was used due to the poor solubilities
of these compounds in water. Qualitative analysis was
carried out using CF3CO2H as an external standard in
these 19F NMR experiments. Complete elimination of
fluorine was observed in 4 days at room temperature
as evidenced by the absence of characteristic –CHF–
peaks in the 19F NMR spectrum. For quantitative inter-
pretations, CFBr3 served as a suitable nonreactive
internal standard for 19F NMR analyses. The ratios of
decomposition (%) for compounds 1–14, 15–20, and
49 are shown in Figures 3 and 4, and their % decompo-
sitions are summarized in Tables 1 and 2.
O
44
Scheme 5. Reagents: i. 50% aq MeOH, 4 days.
(Scheme 5). Three additional minor compounds were
also observed, but they could not be isolated; hence no
further characterization was performed. Compound 44
was fully characterized by 1H, 13C NMR and mass spec-
tral analyses. Similar products were reported with a-flu-
oro-a-amino derivatives under strong alkaline
conditions using additional nucleophiles and their corre-
sponding ether/amide derivatives were isolated.20
When compounds 15, 16, or 18 were stirred in 50%
aqueous methanol, after 4 days 5-substituted-2-thiophen-
yloxazoles 45–47 were obtained as the major com-
pounds, respectively (Scheme 6).4 Product 47 obtained
from compound 18, was characterized by 1H, 13C,
DEPT and HSQC NMR experiments as well as high-
resolution mass spectral analyses to confirm its structure
(Supporting information). Based on spectral compari-
sons and analogous characterization, the products from
compounds 15 and 16 were assigned as 45 and 46.21 Sim-
ilar conditions were also used for the synthesis of oxa-
zole derivatives under alkaline or metal catalyzed
In an effort to identify the products of decomposition,
we undertook a detailed investigation to understand
the transformations involved in the above described
decompositions. Thus, compound 4 was stirred for
4 days in 50% aqueous methanol to isolate any major
products of decomposition (Scheme 5). The single major
product was identified as methyl ether derivative 44

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2947
R
N
H
N
i
15, 16 and 18
H2N
SPh
O
O
45 R = CH2Ph
46 R= CH2(cyclohexyl)
47 R = CH2CH(CH3)2
Scheme 6. Reagents: i. 50% aq MeOH, 4 days.
OMe
SPh
O
H
N
i
H2N
20
N
H
Ph
48
F
PhthN
i
No decomposition
observed
SPh
Ph
49
Scheme 7. Reagents: i. 50% aq MeOH, 4 days.
Figure 2. Models of energy-minimized complexes of the oxyanion
species (A) and compound 16 (B) bound in the active site of
chymotrypsin, covalently and noncovalently, respectively. Residues
of chymotrypsin are shown in capped-stick representation and those of
the transition-state species and compound 16, in ball-and-stick
representation (C: gray, N: blue, O: red, S: yellow, F: green). Broken
line in blue shows hydrogen bonds and corresponding distances.
reaction conditions.22,23 a-Chlorothio derivatives were
converted into the corresponding cyclic oxazoles using
a base.22 Similar cyclizations for the formation of cyclic
oxazoles were developed using SnCl4 as a catalyst.
In order to overcome the cyclic oxazole formation, we
designed compound 20 (vide infra). In the case of deoxy
derivative 20 bearing no amidyl ketone for the internal
cyclization, the methyl ether derivative 48 was obtained
as a major product upon treatment with aqueous meth-
anol (Scheme 7). In the case of compound 49, no chem-
ical transformation was observed and the starting
material 49 was recovered intact after 4 days (Scheme 7).
phobic side chain (P1 side chain) vicinal to the fluorine
substitution to fit into the S1 pocket of chymotrypsin
(Fig. 2B). Compounds 15 and 16 carry benzyl and cyclo-
hexylmethyl side chains. Compounds 17–19 contain
alkyl substitutions, which also render greater stability
to the fluoropeptidomimetics.
Due to the moderate stability of fluoropeptidomimetics
toward acidic and strong basic conditions, standard
peptide chemistry using Boc- and methyl carbamate
protecting groups (3 and 4) was not feasible. Phthaloyl-
and Fmoc- (compounds 2 and 40, respectively) groups
served as suitable protecting groups for the synthesis
of these compounds with good yields. Based on the unu-
sual stability trends exhibited by the fluoropeptidomi-
metics, we undertook a comprehensive investigation of
the stability studies in aqueous media. Tables 1 and 2
and Figures 3 and 4 summarize the stability profiles of
these compounds.
From these observations, it can be concluded that the
derivatives containing the moiety Ô–CHF–S–Õ, although
undergoing defluorination over several days, are rela-
tively stable under aqueous conditions (especially as sug-
gested by the studies on compound 49), and an external
or internal nucleophile could displace or eliminate the
fluorine atom in an appropriate environment. In fact,
this chemical displacement phenomenon provides sup-
port toward the hypothesis that fluorine may be dis-
placed from the fluoropeptidomimetic upon binding in
the active site of a protease, preferably by the nucleo-
philic serine residue, Ser-195 (Fig. 2B).
Compounds 1–14 are dipeptide mimics, where the R3 as-
sumed the side chain of the key fluoropeptidomimetic
monomer and the second amino acid is an alanine.
The protecting group on the second amino acid had a
considerable influence on the stability of these dipep-
tides. When there is no protection on the adjacent amino
acid residue, such as in compound 1, there is a greater
stability compared to those that carried a protecting
group (compound 1 vs compounds 2–4, Table 1). In
these cases, the transformations of compounds 2–4
4. Results and discussion
Compounds 15–19 were designed specifically against
chymotrypsin, a model protease, to establish the mode
of inhibition of proteases by these novel fluoropepti-
domimetics. Design of the tripeptide derivatives 15–19
was based on the large hydrophobic pocket at the S1
pocket in chymotrypsin. These molecules carry a hydro-

2948
S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
tutions were used (compounds 11 and 12, respectively).
Peptide length had a clear effect on the stability of these
molecules under aqueous conditions. The tripeptides
were more stable compared to their corresponding
dipeptides. The phenylalanine derivatives, compounds
5 and 15 (Figs. 3 and 4, respectively) clearly demonstrate
the effect of the peptide length on the stability of these
molecules in aqueous media. Compound 15 is about five
times more stable than compound 5 based on the
decomposition data (12% vs 63% decomposition).
100
80
60
40
20
0
0
1
2
4
Compound 20 was specifically designed to avoid an
internal cyclization to form an oxazole derivative such
as in Scheme 6 and to increase the aqueous stability.
Compound 20 completely decomposed over a period
of 14 h (Table 2). One possibility is the anchimeric effect
of the secondary amine in b-position to fluorine atom
and could be responsible for such a decomposition. In
the case of monomeric mimic 49 carrying the N-phthal-
oyl group, no decomposition was observed even after
4 days and the compound was isolated intact. From
the above experiments, it appears that the stability of
fluoropeptidomimetics depends on the substitution/
functional group modifications, possibly the ability to
induce decomposition via anchimeric effect (100%
decomposition for 20 vs 0% decomposition for 49, Table
2) around the core moiety, and also on modulating the
functional groups that would reduce the neighboring
group effects, such as those proposed for oxazole forma-
tion (47). Thus, the results demonstrate that fluoropep-
tidomimetics with a core moiety Ô–CHF–S–Õ could be
stabilized under aqueous conditions. The above studies
set a basis for the design of stable, next generation
fluoropeptidomimetics.
16
24
14
13
12
11
10
9
8
7
6
5
4
3
2
1
Compound
Figure 3. Stability profiles for compounds 1–14 in 40% deuterated
aqueous methanol over a period of 24 h at rt.
100
80
60
0
40
20
0
1
2
4
14
18
49
20
19
18
17
16
15
Compound
Figure 4. Stability profiles for compounds 15–20 and 49 in 40%
deuterated aqueous methanol over a period of 18 h at rt.
Models of the complexes of fluoropeptidomimetic 16
and the tetrahedral transition-state oxyanion species in
the active site of chymotrypsin were compared to under-
stand their interactions in the active site of the enzyme.
Compound 16 and the transition-state oxyanion species
were docked into the active site of chymotrypsin and
were subjected to energy minimization. Structures of
the transition-state tetrahedral oxyanion species were re-
ported recently suggesting the structural arrangement
for the catalytic residues and atoms near the catalytic
site.24 The computational model of the tetrahedral spe-
cies bound covalently in the active site of chymotrypsin
(Fig. 2A) is similar in the arrangement of residues to
that reported earlier.24 In the energy-minimized complex
of compound 16 and chymotrypsin, the fluorine atom is
may involve the oxazole formation, but it is not
clear why compound 1 is stable toward such a
transformation.
From the stability profiles shown in Figure 3 and Table
1, it is evident that alkyl substitutions on C-2 (6–10) en-
hanced the stabilities. Alanine derivative (6) and cyclo-
hexyl alanine derivative (10) were the most stable
compounds among the dipeptides. The remaining alkyl
derivatives (7–9) also showed greater enhancement in
their aqueous stability as well. This may be due to the
enhanced steric effects due the alkyl substitution on C-
2, and it is also possible that the electronic effects of
these alkyl substitutions may have played a role. In
the case of dipeptides 11 and 12, electron-withdrawing
groups on the phenyl ring did not influence the stability
when compared to the unsubstituted compound 7 (Ta-
ble 1). In the case of electron donating groups such as
p-Me and p-OMe, the effect was clear and decomposi-
tion was rapid (Table 1). This may be due to the induc-
tive character of the electron-rich para substitutions on
the thiophenyl ring facilitating the elimination of fluo-
rine from the molecule via the lone pairs on the sulfur
atom. Such inductive electronic effect, although may
be small, could also explain the slightly decreased or
no change in stability when p-chloro or p-fluoro substi-
˚
2.98 A away from the backbone N on Gly193, interact-
ing via a hydrogen bond (Fig. 2B). Additionally, the
fluorine atom is in close proximity to the catalytic resi-
due Ser195-Oc. These modeling experiments suggest a
possible mimicry of the tetrahedral species by
fluoropeptidomimetics.
With the above models and mode of action in mind, we
assayed compounds 15–19 against a-chymotrypsin for
their inhibitory activity using time-dependent enzyme
activity assays. Each compound was incubated with
the enzyme at 25 ꢁC and the enzyme activity was assayed
after 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h of incubation. Com-

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2949
in 30 min was due to the inhibition by the fluoropepti-
domimetics, but not the oxazole derivatives.
0.4
0.3
0.2
0.1
0
(A)
In conclusion, a new concept of fluoropeptidomimetics
was introduced as a mechanism-based strategy against
serine and cysteine proteases. Various fluoropeptidomi-
metic analogues were designed and their aqueous stabil-
ities were examined. From these findings, it is evident
that the stability of the core moiety Ô–CHF–S–Õ can be
modulated under aqueous conditions. Protecting
groups, side chain substitutions and the length of the flu-
oropeptidomimetic appear to play a strong role in deter-
mining the aqueous stability. The alkyl substitutions and
functional group modifications explored around the
core moiety showed a greater enhancement in their
aqueous stability. Although there is no general rule to
enhance the stabilities of these molecules, our findings
do provide leads toward the preparation of stable flu-
oropeptidomimetics. Thus, a rational approach may be
possible towards designing biologically suitable
fluoropeptidomimetics.
-0.03
-0.02
-0.01
0
0.01
0.02
0.03
1/[I]
-0.1
2
(B)
1.5
1
0.5
0
-1
-0.5
0
0.5
1
1.5
-0.5
-1
1/[I]
Here, for the first time, designer fluoropeptidomimetics
are disclosed as reasonably potent protease inhibitors.
The use of a fluorine moiety as a potential mimic of
the oxyanion group to interact with the oxyanion hole
in proteases, and its potential to function as a protease
inhibitor has merit based on the inhibition of chymo-
trypsin by these molecules. The principles outlined here
form a basis for the further development of this new
class of inhibitors as potential therapeutic agents against
proteases.
Figure 5. Dixon plot of the half-life (t1/2) vs reciprocal of inhibitor
concentration (1/[15]) (panel A), and vs reciprocal of inhibitor 16
concentration (1/[16]) (panel B), against chymotrypsin.
pounds 15 and 16 showed time-dependent loss of activ-
ities, with a maximum inhibition of 67% (after 2 h incu-
bation) and 79% (after 1 h incubation), respectively. The
dissociation constants (Ki) were 63 and 120 lM for com-
pounds 15 and 16, respectively (Fig. 5).25 Compounds
17–19 did not show any significant inhibition in the
time-dependent enzyme assay. After 2 h of incubation,
chymotrypsin recovered its enzyme activity indicating
that the enzyme is turning over the inhibitor molecules
that are bound in the active site, or some changes to
the inhibitor molecule are occurring in solution or en-
zyme active site.
5. Experimental
5.1. General
All anhydrous reactions were performed under an argon
atmosphere. All solvents and reagents were obtained
from commercial sources and were used as received.
Chromatographic purification was performed using sil-
Based on the chemical stabilities of the target fluoropep-
tidomimetics, we were interested in exploring the en-
zyme activity inhibition profiles of oxazoles, 45 and
46. Experiments with 45 and 46 were conducted to delin-
eate any contributions of the oxazoles derivatives to the
time-dependent loss of activity of chymotrypsin ob-
served with compounds 15 and 16. In these experiments,
a modest loss of enzyme activity was observed. Initially,
after 2 h of incubation at [I] = 200 lM, a maximum of
30% and 40% loss of chymotrypsin activities were re-
corded with compounds 45 and 46, respectively. After
4 h of incubation, enzyme activity was recovered and
only a 20% loss of activity was observed independent
of the period of incubation indicating that oxazole
derivatives 45 and 46 do not mimic the inhibition pro-
files of their parent compounds 15 and 16. The recovery
of activity only up to 80% after 4 h of incubation with
compounds 15 and 16, can thus be explained at least
partially by the presence of the corresponding oxazole
derivatives in the enzyme incubation mixture. However,
a steady time-dependent loss of enzyme activity, when
compounds 15 and 16 were incubated with the enzyme,
˚
ica gel (60 A, 70–230 mesh). Due to the sensitivity of
these fluoropeptidomimetics toward the acidic silica
gel, to avoid any decomposition over silica gel column
purification, it was pre-neutralized with 5% Et3N in hex-
anes before loading the compound onto the column.
NMR spectra were recorded at 300 MHz for 1H,
75 MHz for 13C, and 282 MHz for 19F. Chemical shifts
were reported in d ppm using TMS standard for 1H and
13C NMR spectra, and external CF3CO2H standard for
19F NMR spectra. In some of the 13C NMR spectra, due
to the diastereomeric mixtures the spectral values were
reported in a range of chemical shifts (particularly for
aromatic carbons) rather than a particular value.
5.2. 2-(2-Cyclohexyl-1-hydroxymethyl-ethyl)-isoindole-
1,3-dione (51)
Prepared according to the reported procedure.4 Yield:
61% (from 3-cyclohexyl-L-alanine hydrochloride salt);
1
syrup; H NMR (CDCl3) d 0.81–1.01 (m, 2H), 1.12–
1.22 (m, 4H), 1.54–1.68 (m, 5H), 1.84–1.98 (m, 2H),

2950
S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2.60 (br s, 1H), 3.84 (dd, 1H, J = 3.8, 11.8 Hz), 4.00–4.06
(m, 1H), 4.45–4.53 (m, 1H), 7.69–7.75 (m, 2H), 7.80–
7.86 (m, 2H); 13C NMR (CDCl3) d 26.21, 26.34, 26.63,
32.91, 33.75, 34.54, 36.05, 51.56, 63.95, 123.53, 132.03,
134.25, 169.35.
5.9. 2-(1-Cyclohexylmethyl-2-fluoro-2-phenylsulfanyl-
ethyl)-isoindole-1,3-dione (61)
Prepared according to the reported procedure;4 isolated
as a mixture of diastereomers in a 1.4:1 ratio; yield 29%;
syrup; 1H NMR (CDCl3) d 0.72–1.20 (m, 6H), 1.57–1.89
(m, 6H), 2.19–2.29 (m, 1H), 4.52–4.68 (m, 1H), 6.19,
6.33 (2dd, 1H, J = 9.6, 54.0, 9.3, 54.0 Hz), 7.26–7.30
(m, 2H), 7.34–7.41 (m, 2H), 7.53–7.56 (m, 1H), 7.71–
7.77 (m, 2H), 7.82–7.89 (m, 2H); 13C NMR (CDCl3) d
25.97, 26.25, 26.48, 31.88, 32.05, 34.07, 34.41, 34.65,
35.86, 36.23, 51.07 (d, J = 30.2 Hz), 51.45 (d,
J = 19.9 Hz), 100.00 (d, J = 223.2 Hz), 102.16 (d, J =
220.6 Hz), 123.53–134.38 (aromatic), 167.97, 168.29;
19F NMR (CDCl3) d ꢀ74.56, ꢀ72.09 (2dd, J = 7.0,
54.1, 4.5, 57.1 Hz).
5.3. Synthesis of 2-(1-benzyl-2-phenylsulfanyl-ethyl)-iso-
indole-1,3-dione (55)
Synthetic procedure and data were reported earlier.4
5.4. 2-(2-Cyclohexyl-1-phenylsulfanylmethyl-ethyl)-isoin-
dole-1,3-dione (56)
Prepared according the earlier reported procedure.4
Yield 63%; syrup; 1H NMR (CDCl3) d 0.80–0.95
(m, 2H), 1.10–1.17 (m, 4H), 1.55–1.64 (m, 5H), 1.78–
1.82 (m, 1H), 2.04–2.14 (m, 1H), 3.15 (dd, 1H, J = 4.5,
13.8 Hz), 3.67 (dd, 1H, J = 10.2, 13.8 Hz), 4.45–4.54
(m, 1H), 7.02–7.06 (m, 1H), 7.11–7.16 (m, 2H), 7.26–
7.32 (m, 2H), 7.66–7.76 (m, 4H); 13C NMR (CDCl3) d
26.17, 26.32, 26.60, 32.64, 33.79, 34.92, 37.35, 39.71,
49.74, 123.33, 126.74, 129.00, 130.92, 131.97, 133.95,
135.18, 168.63.
5.10. 2-[1-(Fluoro-phenylsulfanyl-methyl)-2-methyl-prop-
yl]-isoindole-1,3-dione (62)
The synthetic procedure and data were reported earlier.4
Isolated as a mixture of diastereomers in a 2.2:1 ratio.
5.11. 2-[1-(Fluoro-phenylsulfanyl-methyl)-3-methyl-
butyl]-isoindole-1,3-dione (63)
5.5. 2-(2-Methyl-1-phenylsulfanylmethyl-propyl)-isoin-
dole-1,3-dione (57)
Prepared according to the earlier reported procedure;4
isolated as a mixture of diastereomers in 1.1:1 ratio;
yield: 48%; syrup; H NMR (CDCl3) d 0.81–0.94 (m,
1
Synthetic procedure and data were reported earlier.4
6H), 1.37–1.50 (m, 1H), 1.64–1.78 (m, 1H), 2.25–2.36
(m, 1H), 4.49–4.66 (m, 1H), 6.19, 6.34 (2dd, 1H,
J = 9.6, 53.7, 9.3, 54.0 Hz), 7.26–7.42 (m, 4H), 7.54–
7.57 (m, 1H), 7.69–7.76 (m, 2H), 7.81–7.88 (m, 2H);
13C NMR (CDCl3) d 21.23, 21.43, 23.68, 25.19, 25.41,
37.24, 37.62, 51.81 (d, J = 30.8 Hz), 52.19 (d,
J = 19.9 Hz), 99.92 (d, J = 222.9 Hz), 102.13 (d,
J = 220.7 Hz), 123.57–134.42 (aromatic), 168.03,
168.36; 19F NMR (CDCl3) d ꢀ75.67, ꢀ73.09 (2dd,
J = 5.0, 53.7, 6.7, 54.1 Hz).
5.6. 2-(3-Methyl-1-phenylsulfanylmethyl-butyl)-isoindole-
1,3-dione (58)
Prepared according our earlier reported procedure.4
1
Yield: 77%; syrup; H NMR (CDCl3) d 0.85 (d, 3H,
J = 6.3 Hz), 0.88 (d, 3H, J = 6.3 Hz), 1.40–1.59 (m,
2H), 2.10–2.20 (m, 1H), 3.15 (dd, 1H, J = 4.5,
13.9 Hz), 3.68 (dd, 1H, J = 10.2, 13.9 Hz), 4.44–4.54
(m, 1H), 7.01–7.07 (m, 1H), 7.11–7.17 (m, 2H), 7.29–
7.33 (m, 2H), 7.64–7.69 (m, 2H), 7.71–7.77 (m, 2H);
13C NMR (CDCl3) d 21.87, 23.22, 25.50, 37.24, 40.97,
50.34, 123.24, 126.71, 128.95, 130.90, 131.89, 133.91,
135.10, 168.56.
5.12. 2-[1-(Fluoro-phenylsulfanyl-methyl)-2-methyl-
butyl]-isoindole-1,3-dione (64)
Prepared according to the reported procedure;4 isolated
as a mixture of diastereomers in 2.4:1 ratio; yield: 60%;
syrup; 1H NMR (CDCl3) d 0.86 (t, 3H, J = 7.2 Hz),
1.03–1.19 (m, 4H), 1.33–1.70 (m, 1H), 2.35–2.68 (m,
1H), 4.37–4.49 (m, 1H), 6.33, 6.56 (2dd, 1H, J = 5.7,
54.7, 8.7, 54.0 Hz), 7.27–7.33 (m, 3H), 7.42–7.53 (m,
2H), 7.72–7.76 (m, 2H), 7.85–7.89 (m, 2H); 13C NMR
(CDCl3) d 10.95, 11.23, 16.46, 16.50, 25.59, 25.86,
34.02, 35.83, 57.87 (d, J = 27.3 Hz), 59.14 (d,
J = 20.8 Hz), 100.46 (d, J = 227.0 Hz), 101.73 (d, J =
220.1 Hz), 123.70–134.39 (aromatic), 168.47; 19F NMR
(CDCl3) d ꢀ75.32, ꢀ73.61 (2dd, J = 19.1, 54.5, 7.6,
53.7 Hz).
5.7. 2-(2-Methyl-1-phenylsulfanylmethyl-butyl)-isoindole-
1,3-dione (59)
Prepared according to the reported procedure.4 Yield:
81%; syrup; 1H NMR (CDCl3)
d 0.80 (t, 3H,
J = 7.2 Hz), 1.00–1.09 (m, 4H), 1.29–1.43 (m, 1H),
2.18–2.27 (m, 1H), 3.33 (dd, 1H, J = 3.3, 13.9 Hz),
3.74 (dd, 1H, J = 11.7, 13.9 Hz), 4.07–4.15 (m, 1H),
7.00–7.06 (m, 1H), 7.10–7.15 (m, 2H), 7.26–7.30 (m,
2H), 7.65–7.69 (m, 2H), 7.72–7.76 (m, 2H); 13C NMR
(CDCl3) d 10.75, 16.49, 26.25, 34.95, 36.50, 57.06,
123.31, 126.74, 128.94, 131.04, 131.48, 133.93, 135.21,
168.77.
5.13. Typical procedure for deprotection of the phthaloyl
group: synthesis of 1-benzyl-2-fluoro-2-phenylsulfanyl-
ethylamine (65)
5.8. Synthesis of 2-(1-benzyl-2-fluoro-2-phenylsulfanyl-
ethyl)-isoindole-1,3-dione (60)
Isolated as a mixture of diastereomers in a 1:1 ratio; syn-
thetic procedure and data were reported earlier.4
A solution of compound 60 (0.05 g, 0.12 mmol) in dry
MeOH (3 mL) was treated with ethylenediamine

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2951
(0.016 mL, 0.24 mmol) under argon. After stirring for
12 h, the reaction mixture was concentrated and the
crude was purified by column chromatography
(EtOAc–hexanes, 1:3) to obtain compound 65 as a syrup
(0.024 g, 75%): Isolated as a mixture of diastereomers in
1:1 ratio; 1H NMR (CDCl3) d 1.38 (br s, 2H), 2.65–2.78
(m, 1H), 2.97–3.09 (m, 1H), 3.30–3.54 (m, 1H), 5.62,
5.68 (2dd, 1H, J = 4.8, 54.0, 4.2, 54.9 Hz), 7.18–7.53
(m, 10H); 13C NMR (CDCl3) d 39.48, 40.71, 56.15 (d,
J = 22.5 Hz), 56.88 (d, J = 21.9 Hz), 105.57 (d,
J = 220.1 Hz), 106.98 (d, J = 219.6 Hz), 126.85–138.01
(aromatic); 19F NMR (CDCl3) d ꢀ82.14, ꢀ72.76 (2dd,
J = 16.0, 54.8, 9.8, 54.1 Hz).
(2dd, 1H, J = 5.1, 54.6, 3.6, 55.6 Hz), 7.19–7.26 (m,
3H), 7.47–7.55 (m, 2H); 19F NMR (CDCl3) d ꢀ84.66,
ꢀ71.81 (2dd, J = 21.4, 55.5, 9.0, 54.4 Hz).
5.18. Typical procedure for amino acid coupling: synthesis
of N-(1-benzyl-2-fluoro-2-phenylsulfanyl-ethyl)-2-[2-(1,3-
dioxo-1,3-dihydro-isoindol-2-yl)acetylamino]-3-methyl-
butyramide (70)
A
solution of PhthN-Gly-L-Val-OH (0.023 g,
0.07 mmol) in anhydrous DMF (1.0 mL) was treated
with EDAC (0.017 g, 0.09 mmol), HOBt (0.01 g,
0.07 mmol), and compound 65 (0.02 g, 0.07 mmol) at
0 ꢁC and stirring was continued for 4 h. The reaction
mixture was concentrated and dissolved into ethyl ace-
tate. The organic layer was washed with 10% citric acid
solution, 10% NaHCO3 solution, brine, and dried
(Na2SO4). The organic layer was evaporated and the
crude was purified by column chromatography
(CHCl3–MeOH 95:5) to obtain compound 70 as a foam
(0.042 g, 88%); isolated as a mixture of diastereomers in
5.14. 1-Cyclohexylmethyl-2-fluoro-2-phenylsulfanyl-
ethylamine (66)
Isolated as a mixture of diastereomers in 1.4:1 ratio;
1
yield: 46%; syrup; H NMR (CDCl3) d 0.79–1.03 (m,
2H), 1.12–1.57 (m, 8H), 1.68–1.72 (m, 5H), 3.14–3.38
(m, 1H), 5.53, 5.72 (2t, 1H, J = 4.2, 3.6, 55.2 Hz),
7.27–7.36 (m, 3H), 7.49–7.53 (m, 2H); 13C NMR
(CDCl3) d 26.25, 26.46, 26.68, 32.62, 32.70, 34.28,
34.42, 40.80, 41.71, 52.43 (d, J = 21.9 Hz), 52.64 (d,
J = 21.9 Hz), 107.02 (d, J = 220.6 Hz), 108.41 (d,
J = 219.5 Hz), 128.00, 129.27, 132.16, 133.38; 19F
NMR (CDCl3) d ꢀ79.41, ꢀ71.33 (2dd, J = 15.5, 55.6,
12.1, 54.7 Hz).
1
a 1:1 ratio; H NMR (DMSO-d6) d 0.80–0.89 (m, 6H),
1.15–1.23 (m, 2H), 1.88–2.05 (m, 1H), 2.72–2.83 (m,
1H), 2.97–3.13 (m, 1H), 4.14–4.30 (m, 3H), 4.35–4.47
(m, 1H), 5.89, 6.00 (2dd, 1H, J = 5.7, 54.9, 3.3,
54.6 Hz), 7.19–7.50 (m, 10H), 7.84–7.93 (m, 4H), 8.19,
8.26 (2d, 1H, J = 9.3, 9.3 Hz), 8.38, 8.44 (2d, 1H,
J = 8.7, 9.3 Hz); 19F NMR (DMSO-d6) d ꢀ78.44,
ꢀ74.45 (2dd, J = 19.7, 54.5, 12.1, 54.9 Hz).
5.15. 1-(Fluoro-phenylsulfanyl-methyl)-2-methyl-propyl-
amine (67)
5.19. N-(1-Cyclohexylmethyl-2-fluoro-2-phenylsulfanyl-
ethyl)-2-[2-(1,3-dioxo-1, 3-dihydro-isoindol-2-yl) acetyl-
amino]-3-methyl-butyramide (71)
Isolated as a mixture of diastereomers in a 2.2:1 ratio;
1
yield: 24%; syrup; H NMR (CDCl3) d 0.88–1.00 (m,
6H), 1.33 (br s, 2H), 1.87–2.06 (m, 1H), 2.69–3.01 (m,
1H), 5.73, 5.84 (2dd, 1H, J = 5.4, 54.4, 3.9, 55.8 Hz),
7.26–7.36 (m, 3H), 7.49–7.54 (m, 2H); 19F NMR
(CDCl3) d ꢀ83.81, ꢀ72.73 (2dd, J = 20.5, 55.5, 9.0,
54.5 Hz).
Isolated as a mixture of diastereomers in a 1.4:1 ratio;
yield: 83%; solid; mp: 218–220 ꢁC; H NMR (DMSO-
1
d6) d 0.75–0.94 (m, 8H), 1.01–1.36 (m, 5H), 1.41–1.70
(m, 6H), 1.92–2.02 (m, 1H), 4.13–4.29 (m, 4H), 5.83,
5.91 (2dd, 1H, J = 5.1, 55.5, 4.2, 54.4 Hz), 7.33–7.48
(m, 5H), 7.84–7.91 (m, 4H), 8.16, 8.18 (2d, 1H,
J = 6.9, 6.9 Hz), 8.35, 8.39 (2d, 1H, J = 9.0, 9.3 Hz);
19F NMR (DMSO-d6) d ꢀ76.91, ꢀ76.39 (2dd, J =
16.6, 54.1, 14.6, 55.2 Hz).
5.16. 1-(Fluoro-phenylsulfanyl-methyl)-3-methyl-butyl-
amine (68)
Isolated as a mixture of diastereomers in a 1.1:1 ratio;
1
yield: 37%; syrup; H NMR (CDCl3) d 0.84–1.00 (m,
5.20. 2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetyl-
amino]-N-[1-(fluoro-phenylsulfanyl-methyl)-2-methyl-
prop- yl]-3-methyl-butyramide (72)
6H), 1.32–1.52 (m, 4H), 1.70–1.84 (m, 1H), 3.09–3.34
(m, 1H), 5.53, 5.72 (2t, 1H, J = 3.9, 3.3, 55.5 Hz),
7.25–7.35 (m, 3H), 7.48–7.52 (m, 2H); 13C NMR
(CDCl3) d 21.74, 21.86, 23.61, 24.72, 24.79, 42.13,
42.17, 43.01, 53.06 (d, J = 17.4 Hz), 53.35 (d,
J = 17.4 Hz), 106.90 (d, J = 220.4 Hz), 108.27 (d,
J = 219.3 Hz), 127.97, 129.22, 132.03, 132.05, 132.12,
132.14, 133.35, 133.45; 19F NMR (CDCl3) d ꢀ80.05,
ꢀ72.11 (2dd, J = 12.1, 54.7, 15.2, 55.5 Hz).
Isolated as a mixture of diastereomers in a 2.2:1 ratio;
yield: 84%; foam; 1H NMR (DMSO-d6) d 0.76–0.99
(m, 12H), 1.92–2.08 (m, 2H), 4.09–4.39 (m, 4H), 5.80–
6.18 (m, 1H), 7.28–7.52 (m, 5H), 7.85–7.92 (m, 4H),
8.03, 8.19 (2d, 1H, J = 9.6, 9.3 Hz), 8.34, 8.40 (2d, 1H,
J = 9.3, 8.7 Hz); 19F NMR (DMSO-d6) d ꢀ80.07,
ꢀ74.46 (2dd, J = 23.6, 54.9, 6.7, 54.1 Hz).
5.17. 1-(Fluoro-phenylsulfanyl-methyl)-2-methyl-butyl-
amine (69)
5.21. 2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetyl-
amino]-N-[1-(fluoro-phenylsulfanyl-methyl)-3-methyl-
butyl]-3-methyl-butyramide (73)
Isolated as a mixture of diastereomers in a 2.4:1 ratio;
1
yield: 27%; syrup; H NMR (CDCl3) d 0.81–0.99 (m,
6H), 1.14–1.30 (m, 1H), 1.37 (br s, 2H), 1.58–1.71 (m,
1H), 2.75–2.83 (m, 1H), 2.91–3.07 (m, 1H), 5.78, 5.89
Isolated as a mixture of diastereomers in a 1.1:1 ratio;
yield: 89%; solid, mp: 202–204 ꢁC; 1H NMR

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S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
(DMSO-d6) d 0.80–0.87 (m, 12H), 1.23–1.61 (m, 3H),
1.96–2.26 (m, 1H), 4.19–4.29 (m, 4H), 5.85, 5.93 (2dd,
1H, J = 4.8, 55.3, 3.9, 54.3 Hz), 7.31–7.49 (m, 5H),
7.84–7.89 (m, 4H), 8.16, 8.19 (2d, 1H, J = 6.3, 6.0 Hz),
8.35, 8.38 (2d, 1H, J = 9.3, 9.0 Hz); 19F NMR
(DMSO-d6) d ꢀ76.94, ꢀ76.32 (2dd, J = 16.6, 57.6,
14.3, 55.6 Hz).
5.25. 2-(2-Amino-acetylamino)-N-[1-(fluoro-phenylsulfa-
nyl-methyl)-2-methyl-propyl]-3-methyl-butyramide (17)
Isolated as a mixture of diastereomers in a 2.2:1 ratio;
1
yield: 65%, foam; H NMR (CD3OD) d 0.83–1.06 (m,
12H), 2.02–2.19 (m, 2H), 3.34 (br s, 2H), 4.05–4.30 (m,
2H), 5.84, 5.98 (2dd, 1H, J = 6.6, 54.0, 3.3, 54.6 Hz),
7.27–7.39 (m, 3H), 7.47–7.53 (m, 2H); 19F NMR
(CD3OD) d ꢀ81.20, ꢀ75.10 (2dd, J = 21.9, 54.1, 8.1,
54.0 Hz); EI-MS m/z 369 (37), 272 (21), 240 (33), 196
(32), 129 (83), 72 (100); EI-HRMS calculated for
C18H28N3O2FS, calculated: 369.188883; observed:
369.188627.
5.22. 2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetyl-
amino]-N-[1-(fluoro-phenylsulfanyl-methyl)-2-methyl-
butyl]-3-methyl-butyramide (74)
Isolated as a mixture of diastereomers in a 2.4:1 ratio;
yield: 70%; foam; 1H NMR (DMSO-d6) d 0.76–0.92
(m, 6H), 1.05–1.19 (m, 1H), 1.40–1.52 (m, 1H), 1.64–
1.76 (m, 1H), 2.01–2.10 (m, 1H), 4.08–4.40 (m, 4H),
5.97, 6.11 (2dd, 1H, J = 6.9, 54.3, 2.7, 55.0 Hz), 7.29–
7.42 (m, 3H), 7.45–7.51 (m, 2H), 7.83–7.92 (m, 4H),
8.03, 8.21 (2d, 1H, J = 9.6, 9.3 Hz), 8.34, 8.39 (2d, 1H,
J = 9.0, 8.7 Hz); 19F NMR (DMSO-d6) d ꢀ79.32,
ꢀ72.52 (2dd, J = 23.4, 54.1, 7.6, 54.1 Hz).
5.26. 2-(2-Amino-acetylamino)-N-[1-(fluoro-phenylsulfa-
nyl-methyl)-3-methyl-butyl]-3-methyl-butyramide (18)
Isolated as a mixture of diastereomers in a 1.1:1 ratio;
1
yield: 88%; foam, H NMR (CDCl3) d 0.83–1.00 (m,
12H), 1.40–1.72 (m, 3H), 2.02–2.18 (m, 1H), 3.32 (br s,
2H), 4.24, 4.29 (2d, 1H, J = 6.9, 6.9 Hz), 4.32–4.50 (m,
1H), 5.76, 5.79 (2dd, 1H, J = 4.2, 55.5, 3.9, 54.0 Hz),
7.28–7.38 (m, 3H), 7.47–7.50 (m, 2H); 13C NMR
(CDCl3) d 18.62, 18.79, 19.95, 20.07, 21.76, 22.01,
23.89, 24.05, 25.82, 32.43, 39.28, 40.25, 45.13, 52.02 (d,
J = 16.2 Hz), 52.33 (d, J = 15.6 Hz), 59.87, 59.96,
105.37 (d, J = 222.3 Hz), 105.93 (d, J = 222.6 Hz),
129.29, 129.34, 130.42, 130.45, 133.10, 133.13, 133.79,
134.47, 173.80, 173.83, 175.40; 19F NMR (CDCl3) d
ꢀ78.75, ꢀ77.86 (2dd, J = 15.7, 55.6, 15.2, 54.1 Hz); EI-
MS m/z 383 (25), 364 (7), 254 (36), 129 (100), 86 (65),
72 (92); EI-HRMS calculated for C19H30N3O2FS, calcu-
lated: 383.203254; observed: 383.204278.
5.23. 2-(2-Amino-acetylamino)-N-(1-benzyl-2-fluoro-2-
phenylsulfanyl-ethyl)-3-methyl-butyramide (15)
Isolated as a mixture of diastereomers in a 1:1 ratio;
1
yield: 50%; solid; mp: 160–162 ꢁC; H NMR (CD3OD)
d 0.88–0.98 (m, 6H), 1.98–2.09 (m, 1H), 2.84, 2.88 (2d,
1H, J = 9.6, 9.9 Hz), 3.04–3.13 (m, 1H), 3.35 (br s,
2H), 4.19, 4.26 (2d, 1H, J = 7.2, 7.2 Hz), 4.52–4.63 (m,
1H), 5.77, 5.81 (2dd, 1H, J = 5.1, 54.3, 3.3, 54.1 Hz),
7.15–7.27 (m, 5H), 7.32–7.38 (m, 3H), 7.45–7.52 (m,
2H); 13C NMR (CD3OD) d 18.51, 18.54, 19.81, 19.92,
32.26, 32.31, 36.86, 38.03, 44.91, 55.10 (d,
J = 23.9 Hz), 55.56 (d, J = 22.2 Hz), 59.79, 59.84,
104.34 (d, J = 222.0 Hz), 105.10 (d, J = 222.1 Hz),
127.68–138.54 (aromatic), 173.41, 173.55, 174.90; 19F
NMR (CD3OD) d ꢀ81.11, ꢀ77.36 (2dd, J = 19.1, 54.4,
12.9, 54.7 Hz); EI-MS m/z 418 (20), 398 (6), 288 (30),
259 (34), 226 (100), 129 (45), 72 (54); EI-HRMS calcu-
lated for C22H29NO2FS (MH+), calculated: 418.19782;
observed: 418.196453.
5.27. 2-(2-Amino-acetylamino)-N-[1-(fluoro-phenylsulfa-
nyl-methyl)-2-methyl-butyl]-3-methyl-butyramide (19)
Isolated as a mixture of diastereomers in a 2.4:1 ratio;
1
yield: 67%; foam, H NMR (CD3OD) d 0.79–1.02 (m,
12H), 1.05–1.25 (m, 1H), 1.46–1.61 (m, 1H), 1.72–1.88
(m, 1H), 2.04–2.17 (m, 1H), 3.31 (br s, 2H), 4.21–4.29
(m, 2H), 5.86, 5.97 (2dd, 1H, J = 6.0, 53.8, 2.7,
54.6 Hz), 7.24–7.36 (m, 3H), 7.45–7.50 (m, 2H); 19F
NMR (CD3OD) d ꢀ81.46, ꢀ74.24 (2dd, J = 23.6, 54.5,
8.1, 53.4 Hz); EI-MS m/z 383 (78), 254 (61), 210 (29),
129 (100), 86 (48), 72 (88); EI-HRMS calculated for
C19H30N3O2FS, calculated: 383.204374; observed:
383.204278.
5.24. 2-(2-Amino-acetylamino)-N-(1-cyclohexylmethyl-2-
fluoro-2-phenylsulfanyl-ethyl)-3-methyl-butyramide (16)
Isolated as a mixture of diastereomers in a 1.4:1 ratio;
1
yield: 77%; foam; H NMR (CD3OD) d 0.75–1.03 (m,
8H), 1.15–1.42 (m, 5H), 1.50–1.78 (m, 6H), 2.03–2.15
(m, 1H), 3.32 (br s, 2H), 4.23, 4.27 (2d, 1H, J = 7.2,
6.6 Hz), 4.36–4.49 (m, 1H), 5.75, 5.78 (2dd, 1H,
J = 4.2, 55.6, 4.2, 54.1 Hz), 7.31–7.36 (m, 3H), 7.46–
7.50 (m, 2H); 13C NMR (CD3OD) d 18.52, 18.66,
19.82, 19.93, 27.09, 27.13, 27.40, 27.57, 32.22, 33.03,
33.26, 35.06, 35.13, 35.27, 37.67, 38.68, 44.97, 51.24 (d,
J = 23.7 Hz), 51.43 (d, J = 23.1 Hz), 59.86, 59.93,
105.28 (d, J = 222.3 Hz), 105.85 (d, J = 222.3 Hz),
129.15, 129.21, 130.29, 130.32, 133.01, 133.05, 133.70;
19F NMR (CD3OD) d ꢀ79.02, ꢀ77.89 (2dd, J = 16.0,
54.8, 15.2, 54.1 Hz); EI-MS m/z 424 (4), 404 (23), 294
(42), 232 (39), 129 (100), 72 (86); EI-HRMS calculated
for C22H35N3O2FS (MH+), calculated: 424.243499; ob-
served: 424.243403.
5.28. 2-Amino-N-[1-(4-benzyl-5-phenylsulfanyl-4,5-dihy-
dro-oxazol-2-yl)-2-methyl-propyl]-acetamide (45)
Compound 15 (0.009 g, 0.02 mmol) was treated with
50% aqueous MeOH (10 mL) at room temperature
and stirred for 4 days. Solvent was evaporated under re-
duced pressure and the crude compound was purified by
column chromatography (CHCl3– MeOH, 95:5) to ob-
tain compound 45 (0.005 g, 58%) as a syrup; isolated
as a mixture of diastereomers in a 2.1:1 ratio; 1H
NMR (CD3OD) 0.86–0.97 (m, 6H), 1.96–2.04 (m, 1H),
2.74–2.83 (m, 1H), 2.99–3.13 (m, 1H), 3.45 (br s, 2H),
4.16, 4.23 (2d, 1H, J = 6.9, 6.9 Hz), 4.36–4.47 (m, 1H),
4.59, 4.67 (2d, 1H, J = 4.2, 5.4 Hz), 7.12–7.55 (m, 10H).

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2953
5.29. 2-Amino-N-[1-(4-cyclohexylmethyl-5-phenylsulfan-
yl-4,5-dihydro-oxazol-2-yl)-2-methyl-propyl]-acetamide
(46)
J = 222.6 Hz), 127.94, 128.26, 128.46, 129.23, 129.35,
129.40, 132.11, 132.54, 132.56, 133.49, 172.31, 172.70;
19F NMR (CDCl3) d ꢀ82.46 (dd, J = 25.0, 55.4 Hz),
ꢀ75.53 (dd, J = 11.5, 54.4 Hz).
Isolated as a mixture of diastereomers in a 2.6:1 ratio;
1
yield: 63%; syrup; H NMR (CD3OD) d 0.76–1.05 (m,
5.34. {1-[1-(Fluoro-phenylsulfanyl-methyl)-3-methyl-
butylcarbamoyl]-ethyl}-carbamic acid methyl ester (9)
8H), 1.15–1.33 (m, 5H), 1.52–1.79 (m, 6H), 2.02–2.09
(m, 1H), 3.36 (br s, 2H), 4.19–4.34 (m, 2H), 4.62, 4.70
(2d, 1H, J = 3.9, 4.8 Hz), 7.23–7.34 (m, 3H), 7.49–7.53
(m, 2H).
Isolated as an inseparable mixture of diastereomers (1:1
ratio); yield: 87%; solid, mp 85–87 ꢁC; 1H NMR
(CDCl3) d 0.90–0.96 (m, 6H), 1.35–1.67 (m, 6H), 3.68
(s, 3H), 4.20–4.38 (m, 1H), 4.44–4.62 (m, 1H), 5.34–
5.44 (m, 1H), 5.69, 5.88 (dd and t, 1H, J = 3.3, 55.3,
3.0, 55.3 Hz), 6.34–6.45 (m, 1H), 7.27–7.36 (m, 3H),
7.47–7.51 (m, 2H); 13C NMR (CDCl3) d 18.79, 19.18,
21.52, 22.08, 23.30, 23.69, 24.90, 38.11, 40.13, 51.07 (t,
J = 22.2, 24.5 Hz), 52.64, 104.63 (d, J = 222.3 Hz),
104.68 (d, J = 222.1 Hz), 128.32, 128.44, 129.42,
132.17, 132.45, 172.35, 172.50; 19F NMR (CDCl3) d
ꢀ82.41 (dd, J = 19.7, 56.4 Hz), ꢀ78.98 (dd, J = 54.1,
17.4 Hz).
5.30. 2-Amino-N-[1-(4-isobutyl-5-phenylsulfanyl-4,5-
dihydro-oxazol-2-yl)-2-methyl-propyl]-acetamide (47)
Isolated as a mixture of diastereomers in a 2.6:1 ratio;
1
yield: 55%; syrup; H NMR (CD3OD) d 0.78–1.04 (m,
12H), 1.46–1.64 (m, 3H), 2.02–2.09 (m, 1H), 3.34–3.44
(m, 2H), 4.19–4.32 (m, 2H), 4.63, 4.70 (2d, 1H,
J = 3.9, 4.8 Hz), 7.24–7.34 (m, 3H), 7.51–7.53 (m, 2H);
13C NMR (CD3OD) d 18.72, 19.93, 20.07, 21.87,
22.18, 23.98, 24.26, 25.98, 30.95, 32.40, 38.77, 40.92,
52.98, 53.77, 57.64, 60.17, 96.70, 97.03, 128.86, 130.33,
133.83, 134.31, 136.59, 173.44; EI-MS m/z 364 (MH+,
13), 289 (53), 133 (68), 129 (76), 72 (100); EI-HRMS cal-
culated for C19H30N3O2S (MH+), calculated:
364.208182; observed: 364.205874.
5.35. [1-(1-Cyclohexylmethyl-2-fluoro-2-phenylsulfanyl-
ethylcarbamoyl)-ethyl]-carbamic acid methyl ester (10)
Isolated as an inseparable mixture of diastereomers
(1.2:1 ratio); yield: 68%; foam; 1H NMR (CDCl3) d
0.83–1.80 (m, 16H), 3.38 (s, 3H), 4.22–4.40 (m, 1H),
4.44–4.64 (m, 1H), 5.52–5.89 (m, 2H), 6.59 (br s, 1H),
7.27–7.35 (m, 3H), 7.46–7.50 (m, 2H); 13C NMR
(CDCl3) d 18.72, 19.16, 26.11, 26.15, 26.41, 26.53,
32.10, 32.65, 33.86, 34.15, 34.24, 36.53, 38.52, 49.99,
50.47 (d, J = 24.5 Hz), 52.57, 104.54 (d, J = 222.3 Hz),
104.75 (d, J = 220.0 Hz), 128.21, 128.34, 129.33,
129.35, 132.05, 132.33, 133.05, 133.24, 156.78, 172.40,
172.55; 19F NMR (CDCl3) d ꢀ82.55 (dd, J = 20.5,
56.2 Hz), ꢀ79.09 (dd, J = 17.7, 54.8 Hz).
5.31. Synthesis of [1-(1-Benzyl-2-fluoro-2-phenylsulfanyl-
ethyl carbamoyl)-ethyl]-carbamic acid methyl ester (5)
A solution of MeO2CHN-L-Ala-OH (0.01 g, 0.07 mmol)
in CH2Cl2 (3 mL) was treated with HOBt (0.009 g,
0.07 mmol), EDAC (0.016 g, 0.08 mmol), and com-
pound 21 (0.02 g, 0.07 mmol) at 0 ꢁC and the reaction
mixture was brought to rt and stirred for 4 h. Solvent
was evaporated under reduced pressure, taken into ethyl
acetate (20 mL), washed with 10% citric acid solution
(10 mL), 10% NaHCO3 solution (10 mL), brine
(10 mL), and dried (Na2SO4). The solvent was evapo-
rated under reduced pressure and the crude was purified
by column chromatography (EtOAc–hexanes, 1:3) to
obtain compound 5 (0.025 g, 89%) as a solid. Spectral
data were reported earlier.4
5.36. 2-[1-(4-Chloro-phenylsulfanylmethyl)-2-methyl-prop-
yl]-isoindole-1,3-dione (28)
Method A: A suspension of KOH (0.58 g, 10.33 mmol)
in EtOH (10 mL) was treated with p-chloro benzenethiol
(1.49 g, 10.33 mmol) at rt and stirred for 10 min. The
resulting mixture was added to a pre-refluxed solution
of compound 26 (2.0 g, 5.16 mmol) in anhydrous EtOH
(10 mL) over a period of 15 min and refluxed for an
additional 3 h. The reaction mixture was brought to rt
and the solvent was evaporated under reduced pressure.
The crude was dissolved into ethyl acetate (50 mL),
washed with water (20 mL), brine (20 mL), and dried
(Na2SO4). The organic layer was evaporated under re-
duced pressure and the crude was purified by column
chromatography (EtOAc–hexanes, 1:9) to obtain com-
pound 28 (0.68 g, 37%) as a solid. mp 56–58 ꢁC; 1H
NMR (CDCl3): d 0.83 (d, 3H, J = 6.6 Hz), 1.04 (d,
3H, J = 6.9 Hz), 2.34–2.46 (m, 1H), 3.27 (dd, 1H,
J = 3.3, 14.1 Hz), 3.73 (dd, 1H, J = 11.7, 14.1 Hz), 4.00
(dt, 1H, J = 3.3, 11.7 Hz), 7.04–7.08 (m, 2H), 7.18–7.22
(m, 2H), 7.67–7.76 (m, 4H); 13C NMR (CDCl3) d
20.19, 20.50, 30.56, 35.00, 58.75, 123.29, 129.06,
131.68, 132.57, 132.98, 133.57, 134.07, 168.71.
Following compounds 7–10 were synthesized using the
procedure described for the synthesis of compound 5.
5.32. {1-[1-(Fluoro-phenylsulfanyl-methyl)-2-methyl-
propyl carbamoyl]-ethyl}-carbamic acid methyl ester (7)
Spectral data were reported earlier.4
5.33. {1-[1-(Fluoro-phenylsulfanyl-methyl)-2-methyl-
butylcabamoyl]-ethyl}-carbamic acid methyl ester (8)
Isolated as an inseparable mixture of diastereomers (3:1
ratio); yield: 83%; syrup; 1H NMR (CDCl3) d 0.83–1.17
(m, 7H), 1.36–1.48 (m, 3H), 1.58–1.88 (m, 2H), 3.68 (s,
3H), 4.17–4.43 (m, 2H), 5.46–6.08 (m, 2H), 6.47–6.56
(m, 1H), 7.28–7.33 (m, 3H), 7.47–7.52 (m, 2H); 13C
NMR (CDCl3) d 11.09, 11.48, 15.64, 16.14, 16.44,
18.76, 19.32, 24.36, 25.47, 35.99, 36.89, 50.77, 52.62,
56.49 (d, J = 21.9 Hz), 56.71, 57.23, 103.61 (d,

2954
S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
5.37. 2-[1-(4-Fluoro-phenylsulfanylmethyl)-2-methyl-
propyl]-isoindole-1,3-dione (29)
J = 28.2 Hz), 99.88 (d, J = 226.6 Hz), 101.34 (d,
J = 220.1 Hz), 123.01–135.08 (aromatic), 168.48; 19F
NMR (CDCl3) d ꢀ75.77 (dd, J = 16.9, 54.4 Hz),
ꢀ75.12 (dd, J = 6.7, 53.4 Hz).
Yield: Method A: 10%, Method B:4 38%, solid, mp 66–
68 ꢁC; 1H NMR (CDCl3) d 0.83 (d, 3H, J = 6.6 Hz), 1.01
(d, 3H, J = 6.6 Hz), 2.32–2.44 (m, 1H), 3.24 (dd, 1H,
J = 3.3, 14.1 Hz), 3.70 (dd, 1H, J = 11.7, 14.1 Hz), 3.98
(dt, 1H, J = 3.3, 11.7 Hz), 6.79–6.85 (m, 2H), 7.27–7.32
(m, 2H), 7.67–7.78 (m, 4H); 13C NMR (CDCl3) d
20.18, 20.44, 30.55, 35.81, 58.63, 115.86, 116.15,
129.98, 130.02, 131.74, 133.93, 134.02, 134.04, 160.54,
163.82, 168.72; 19F NMR (CDCl3) d ꢀ37.01 to ꢀ36.93
(m).
5.41. 2-{1-[Fluoro-(4-fluoro-phenylsulfanyl)-methyl]-2-
methyl-propyl}-isoindole-1,3-dione (33)
Product was isolated as an inseparable mixture of dia-
stereomers (ratio 2.8:1); yield: 45%; syrup; 1H NMR
(CDCl3) d 0.94, 0.96 (2d, 3H, J = 6.9, 6.9 Hz), 1.03–
1.18 (m, 3H), 2.52–2.83 (m, 1H), 4.28–4.39 (m, 1H),
6.28, 6.50 (2dd, 1H, J = 6.6, 53.7 Hz and 9.0, 54.4 Hz),
6.97–7.05 (m, 2H), 7.40–7.45 (m, 1H), 7.50–7.55 (m,
1H), 7.72–7.78 (m, 2H), 7.85–7.90 (m, 2H); 13C NMR
(CDCl3) d 19.28, 19.44, 20.57, 20.64, 28.37, 29.77,
58.55 (d, J = 20.2 Hz), 59.88 (d, J = 28.5 Hz), 100.18
(d, J = 226.4 Hz), 101.59 (d, J = 220.1 Hz), 116.29–
136.13 (aromatic), 161.77, 165.07, 168.52; 19F NMR
(CDCl3) d ꢀ76.00 (dd, J = 16.6, 54.0 Hz), ꢀ75.40 (dd,
J = 6.7, 54.1 Hz), ꢀ34.64 to ꢀ34.51 (m).
5.38. 2-(2-Methyl-1-p-tolylsulfanylmethyl-propyl)-
isoindole-1,3-dione (30)
1
Yield: Method A: 34%; solid, mp 57–59 ꢁC; H NMR
(CDCl3) d 0.82 (d, 3H, J = 6.9 Hz), 1.02 (d, 3H,
J = 6.6 Hz), 2.16 (s, 3H), 2.32–2.45 (m, 1H), 3.25 (dd,
1H, J = 3.3, 13.9 Hz), 3.71 (dd, 1H, J = 11.5, 13.9 Hz),
4.00 (ddd, 1H, J = 3.3, 9.6, 11.5 Hz), 6.91 (d, 2H,
J = 7.8 Hz), 7.14 (d, 2H, J = 8.1 Hz), 7.65–7.69 (m,
2H), 7.73–7.75 (m, 2H); 13C NMR (CDCl3) d 20.20,
20.48, 28.12, 30.59, 35.21, 58.85, 123.23, 129.71,
131.31, 131.73, 131.85, 133.83, 136.92, 168.75.
5.42. 2-[1-(Fluoro-p-tolylsulfanyl-methyl)-2-methyl-prop-
yl]-isoindole-1,3-dione (34)
Product was isolated as an inseparable mixture of dia-
stereomers (ratio 1.8:1); yield: 47%; syrup; 1H NMR
(CDCl3) d 0.93, 0.96 (2d, 3H, J = 2.1, 2.4 Hz), 1.03–
1.07 (m, 3H), 2.33 (s, 3H), 2.53–2.83 (m, 1H), 4.29–
4.40 (m, 1H), 6.30, 6.51 (2dd, 1H, J = 6.3, 54.1 Hz and
9.0, 53.8 Hz), 7.12 (t, 2H, J = 6.6, 7.2 Hz), 7.32 (d, 1H,
J = 8.1 Hz), 7.42 (d, 1H, J = 8.1 Hz), 7.71–7.76 (m,
2H), 7.84–7.89 (m, 2H); 13C NMR (CDCl3) d 19.29,
19.51, 20.59, 20.63, 21.34, 21.36, 28.42, 29.72, 58.64 (d,
J = 20.8 Hz), 60.02 (d, J = 28.2 Hz), 100.26 (d,
J = 219.5 Hz), 101.74 (d, J = 225.8 Hz), 123.65, 123.72,
128.17, 130.06, 130.14, 131.87, 133.40, 133.77, 134.31,
134.38, 138.97, 168.57; 19F NMR (CDCl3) d ꢀ75.54
(dd, J = 7.0, 54.1 Hz), ꢀ74.97 (dd, J = 16.6, 54.1 Hz).
5.39. 2-[1-(4-Methoxy-phenylsulfanylmethyl)-2-methyl-
propyl]-isoindole-1,3-dione (31)
Yield: Method A: 9%; Method B:4 44%, syrup; 1H
NMR (CDCl3) d 0.82 (d, 3H, J = 6.6 Hz), 0.99 (d, 3H,
J = 6.6 Hz), 2.30–2.40 (m, 1H), 3.71 (dd, 1H, J = 3.3,
14.1 Hz), 3.67 (dd, 1H, J = 11.7, 14.1 Hz), 3.68 (s, 3H),
3.98 (dt, 1H, J = 3.3, 11.7 Hz), 6.64–6.67 (m, 2H),
7.25–7.28 (m, 2H), 7.66–7.69 (m, 2H), 7.74–7.77 (m,
2H); 13C NMR (CDCl3) d 20.22, 20.48, 30.61, 36.20,
55.32, 58.89, 114.54, 123.27, 125.34, 131.91, 133.88,
134.36, 159.26, 168.83.
5.40. 2-{1-[(4-Chloro-phenylsulfanyl)-fluoro-methyl]-
2-methyl-propyl}-isoindole-1,3-dione (32)
5.43. 2-{1-[Fluoro-(4-methoxy-phenylsulfanyl)-methyl]-2-
methyl-propyl}-isoindole-1,3-dione (35)
A suspension of SelectfluorTM (1.23 g, 3.48 mmol) in
anhydrous CH3CN (10 mL) was treated with compound
28 (0.62 g, 1.74 mmol) in anhydrous CH3CN (5 mL).
After stirring for 30 min at rt, Et3N (0.24 mL,
1.74 mmol) was added and stirred for another 10 min.
The reaction mixture was treated with water (50 mL)
and extracted into ether (3 · 25 mL). The combined or-
ganic layer was washed with satd NaHCO3 solution
(25 mL), brine (20 mL), and dried (Na2SO4). The organ-
ic layer was evaporated under reduced pressure and the
crude was purified by column chromatography (EtOAc–
hexanes, 1:4) to obtain compound 32 (0.45 g, 69%) as a
syrup; the product was isolated as an inseparable mix-
Isolated as an inseparable mixture of diastereomers (ratio
1.4:1); yield: 53%; syrup; 1H NMR (CDCl3) d 0.94 (d, 3H,
J = 6.9 Hz), 1.02–1.06 (m, 3H), 2.52–2.82 (m, 1H), 3.79
(s, 3H), 4.27–4.38 (m, 1H), 6.24, 6.45 (2dd, 1H, J = 9.0,
53.5 Hz and 6.9, 54.3 Hz), 6.82–6.87 (m, 2H), 7.33–7.46
(m, 1H), 7.47 (d, 1H, J = 8.4 Hz), 7.71–7.82 (m, 2H),
7.84–7.91 (m, 2H); 13C NMR (CDCl3) d 19.25, 19.46,
20.59, 20.67, 28.42, 29.75, 55.51, 58.60 (d, J = 28.8 Hz),
59.89 (d, J = 20.5 Hz), 101.47 (d, J = 225.5 Hz), 101.90
(d, J = 219.2 Hz), 114.83–136.10 (aromatic), 160.55,
168.57; 19F NMR (CDCl3) d ꢀ76.13 (dd, J = 16.0,
54.1 Hz), ꢀ75.64 (dd, J = 7.6, 53.8 Hz).
1
ture of diastereomers (ratio 2.7:1); H NMR (CDCl3)
5.44. Typical procedure for deprotection of phthaloyl
group: synthesis of 1-[(4-chloro-phenylsulfanyl)-fluoro-
methyl-]-2-methyl-propylamine (36)
d 0.93–1.81 (m, 6H), 2.53–2.85 (m, 1H), 4.29–4.40 (m,
1H), 6.31, 6.53 (2dd, 1H, J = 6.3, 54.4 Hz and 9.3,
53.8 Hz), 7.26–7.31 (m, 2H), 7.35–7.38 (m, 1H), 7.43–
7.47 (m, 1H), 7.73–7.79 (m, 2H), 7.85–7.92 (m, 2H);
13C NMR (CDCl3) 19.33, 19.47, 20.30, 20.55, 20.65,
20.69, 28.32, 29.74, 58.59 (d, J = 20.2 Hz), 59.98 (d,
A solution of compound 32 (0.43 g, 1.14 mmol) in dry
MeOH (5 mL) was treated with ethylenediamine
(0.15 mL, 2.28 mmol) under argon. After stirring for

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2955
12 h, reaction mixture was evaporated under reduced
5.49. (1-{1-[Fluoro-(4-fluoro-phenylsulfanyl)-methyl]-2-
methyl-propylcarbamoyl}-ethyl)-carbamic acid methyl
ester (12)
pressure and the crude was purified by column chroma-
tography (EtOAc–hexanes, 1:3) to obtain compound 36
(0.067 g, 24%) as a syrup; Isolated as an inseparable
mixture of diastereomers in 2.7:1 ratio; 1H NMR
(CDCl3) d 0.88–1.11 (m, 6 Hz), 1.33 (br s, 2H), 1.85–
1.98 (m, 1H), 2.69–2.99 (m, 1H), 5.68, 5.79 (2dd, 1H,
J = 5.0, 54.4 Hz and 3.9, 55.8 Hz), 7.25–7.32 (m, 2H),
7.42–7.48 (m, 2H); 19F NMR (CDCl3) d ꢀ84.55 (dd,
J = 20.5, 55.6 Hz), ꢀ73.32 (dd, J = 8.4, 54.4 Hz).
Isolated as an inseparable mixture of diastereomers (ra-
1
tio 2.8:1); yield: 90%; solid, mp 110–112 ꢁC; H NMR
(CDCl3) d 0.85–1.01 (m, 6H), 1.42, 1.47 (2d, 3H,
J = 6.9, 6.9 Hz), 1.88–2.13 (m, 1H), 3.68 (s, 3H), 4.08–
4.36 (m, 2H), 5.45–5.96 (m, 2H), 6.56 (m, 1H), 7.03 (t,
2H, J = 8.4 Hz), 7.46–7.52 (m, 2H); 13C NMR (CDCl3)
d 17.37, 18.57, 19.18, 19.66, 20.27, 20.46, 29.04, 29.88,
30.72, 31.10, 50.83, 52.67, 56.39 (d, J = 21.9 Hz),
56.89, 58.12 (d, J = 20.2 Hz), 93.90, 103.81 (d,
J = 222.9 Hz), 116.12, 116.38, 116.67, 134.96, 135.07,
135.40, 135.53, 136.34, 136.45, 161.65, 164.95, 172.75,
172.89; 19F NMR (CDCl3) d ꢀ82.75 (dd, J = 24.2,
55.1 Hz), ꢀ77.77 (dd, J = 8.4, 55.8 Hz), ꢀ35.50 to
ꢀ34.70 (m).
5.45. 1-[Fluoro-(4-fluoro-phenylsulfanyl)-methyl]-2-
methyl-propylamine (37)
Isolated as an inseparable mixture of diastereomers (ra-
1
tio 2.8:1); yield: 14%; syrup; H NMR (CDCl3) d 0.87–
1.00 (m, 6H), 1.36 (br s, 2H), 1.86–2.08 (m, 1H), 2.63–
2.97 (m, 1H), 5.63, 5.73 (2dd, 1H, J = 5.4, 54.1 Hz and
3.9, 55.8 Hz), 6.99–7.07 (m, 2H), 7.45–7.54 (m, 2H);
19F NMR (CDCl3) d ꢀ84.47 (dd, J = 19.7, 55.5 Hz),
ꢀ73.55 (dd, J = 8.4, 54.4 Hz), ꢀ35.64 to ꢀ35.36 (m).
5.50. {1-[1-(Fluoro-p-tolylsulfanyl-methyl)-2-methyl-prop-
ylcarbamoyl]-ethyl}-carbamic acid methyl ester (13)
Isolated as an inseparable mixture of diastereomers
(ratio 1.8:1); yield: 60%; syrup; 1H NMR (CDCl3) d
0.86–1.01 (m, 6H), 1.38, 1.41 (2d, 3H, J = 6.9, 6.9 Hz),
1.88–2.18 (m, 1H), 2.33, 2.34 (2s, 3H), 3.67, 3.68 (2s,
3H), 4.10–4.36 (m, 2H), 5.39–6.43 (m, 3H), 7.12 (t,
2H, J = 7.5 Hz), 7.38, 7.40 (2d, 2H, J = 2.7 Hz and
2.7 Hz); 13C NMR (CDCl3) d 17.46, 17.54, 18.67,
18.85, 19.19, 19.22, 19.68, 20.30, 20.54, 21.32, 29.18,
29.88, 30.76, 50.89, 52.63, 56.61 (d, J = 21.9 Hz),
56.85, 57.51, 58.25 (d, J = 20.8 Hz), 94.14, 104.01 (d,
J = 222.9 Hz), 130.04, 130.14, 130.17, 132.75, 133.12,
133.15, 134.09, 138.25, 138.64, 138.85, 172.31, 172.65,
172.76; 19F NMR (CDCl3) d ꢀ82.32 (dd, J = 24.2,
54.8 Hz), ꢀ77.27 (dd, J = 14.6, 54.8 Hz).
5.46. 1-(Fluoro-p-tolylsulfanyl-methyl)-2-methyl-propyl-
amine (38)
Isolated as an inseparable mixture of diastereomers (ra-
tio 1.8:1); yield: 25%; syrup; 1H NMR (CDCl3) d 0.90 (t,
3H, J = 7.0, 7.2 Hz), 0.96–0.99 (m, 3H), 1.32 (br s, 2H),
1.87–2.10 (m, 1H), 2.34 (s, 3H), 2.66–2.99 (m, 1H), 5.66,
5.76 (2dd, 1H, J = 5.7, 54.3 Hz and 3.9, 55.6 Hz), 7.09–
7.15 (m, 2H), 7.37–7.44 (m, 2H); 19F NMR (CDCl3) d
ꢀ84.01 (dd, J = 20.0, 55.6 Hz), ꢀ73.06 (dd, J = 8.4,
54.9 Hz).
5.47. 1-[Fluoro-(4-methoxy-phenylsulfanyl)-methyl]-2-
methyl-propylamine (39)
5.51. (1-{1-[Fluoro-(4-methoxy-phenylsulfanyl)-methyl]-
2-methyl-propylcarbamoyl}-ethyl)-carbamic acid methyl
ester (14)
Product was isolated as an inseparable mixture of dia-
stereomers (ratio 1.4:1); yield: 13%; syrup; 1H NMR
(CDCl3) d 0.87–1.13 (m, 6H), 1.43 (br s, 2H), 1.88–
2.07 (m, 1H), 2.60–2.93 (m, 1H), 3.79, 3.80 (2s, 3H),
5.57, 5.66 (2dd, 1H, J = 5.7, 54.1 Hz and 4.2, 55.8 Hz),
6.81–6.89 (m, 2H), 7.41–7.49 (m, 2H); 19F NMR
(CDCl3) d ꢀ84.24 (dd, J = 19.1, 55.5 Hz), ꢀ73.60 (dd,
J = 8.4, 53.7 Hz).
Isolated as an inseparable mixture of diastereomers (ra-
tio 1.4:1); yield: quant.; syrup; 1H NMR (CDCl3) d
0.88–1.03 (m, 6H), 1.41–1.51 (m, 3H), 1.94–2.20 (m,
1H), 3.71 (s, 3H), 3.82, 3.83 (2s, 3H), 4.10–4.37 (m,
2H), 5.43–5.93 (m, 2H), 6.15–6.45 (m, 1H), 6.86–6.90
(m, 2H), 7.45–7.49 (m, 2H); 13C NMR (CDCl3) d
17.32, 17.54, 18.86, 19.16, 19.22, 19.67, 20.29, 20.50,
29.14, 29.88, 30.74, 50.88, 52.58, 55.49, 55.55, 56.53 (d,
J = 22.8 Hz), 56.94, 57.33, 58.12 (d, J = 20.2 Hz),
94.16, 104.29 (d, J = 223.2 Hz), 114.80, 114.96, 135.22,
135.62, 136.45, 160.05, 172.30; 19F NMR (CDCl3) d
ꢀ82.70 (dd, J = 23.6, 55.9 Hz); ꢀ77.76 (dd, J = 13.8,
54.7 Hz).
5.48. (1-{1-[(4-Chloro-phenylsulfanyl)-fluoro-methyl]-2-
methyl-propylcarbamoyl}-ethyl)-carbamic acid methyl
ester (11)
Isolated as an inseparable mixture of diastereomers (ra-
1
tio 2.7:1); yield: 79%; solid, mp 143–145 ꢁC; H NMR
(CDCl3) d 0.85–1.02 (m, 6H), 1.41, 1.46 (2d, 3H,
J = 6.9, 6.9 Hz), 1.89–2.17 (m, 1H), 3.68 (s, 3H), 4.15–
4.37 (m, 2H), 5.45–5.82 (m, 2H), 6.59 (br d, 1H), 7.27–
7.31 (m, 2H), 7.40–7.45 (m, 2H); 13C NMR (CDCl3) d
17.37, 18.61, 19.19, 19.23, 19.68, 20.27, 29.04, 30.72,
50.81, 52.68, 56.40 (d, 22.2 Hz), 58.16 (d, J = 20.5 Hz),
103.40 (d, J = 223.8 Hz), 129.32, 129.56, 133.57,
134.01, 134.04, 134.93, 135.06, 172.75, 172.88; 19F
NMR (CDCl3) d ꢀ82.70, ꢀ77.59 (2dd, J = 24.5,
55.2 Hz and J = 19.9, 54.9 Hz).
5.52. {1-[(1-Benzyl-2-fluoro-2-phenylsulfanyl-ethyl-
amino)-methyl]-2-methyl-propylamino}-(9H-fluren-9-
ylmethoxy)-methanol (41)
A solution of aldehyde 40 (0.34 g, 1.05 mmol), amine 21
(0.27 g, 1.05 mmol), and Na2SO4 (0.05 g) in dry CH2Cl2
(10 mL) was stirred for 1 h at 0 ꢁC, brought to rt and
stirred for an additional 1 h. Solid was filtered off,

2956
S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
washed with CH2Cl2 (3 · 15 mL). The combined organ-
ic layer was evaporated under reduced pressure to ob-
tain the crude imine.
ture of diastereomers in a 57:38:3:2 ratio; 1H NMR
(CDCl3) d 0.74–0.90 (m, 6H), 1.25 (br s, 1H), 1.58–
1.75 (m, 1H), 2.58–2.96 (m, 4H), 3.07–3.34 (m, 1H),
3.65–3.95 (m, 3H), 4.19 (t, 1H, J = 6.9 Hz), 4.37 (t,
2H, J = 6.9 Hz), 5.36 (br s, 1H), 5.64, 5.82 (2dd, 1H,
J = 2.7, 55.0 Hz and 2.7, 55.0 Hz), 6.20, 6.33 (2br d,
1H, J = 8.7 Hz), 7.15–7.57 (m, 16H), 7.75 (d, 2H,
J = 7.5 Hz); 19F NMR (CDCl3) d ꢀ81.78 (dd, J = 19.7,
55.5 Hz), ꢀ80.08 (dd, J = 18.3, 54.2 Hz), ꢀ70.99 (dd,
J = 18.2, 52.4 Hz), ꢀ68.57 (dd, J = 13.8, 54.8 Hz).
A solution of the above crude imine in dry MeOH
(10 mL) was treated with NaBH4 (0.04 g, 1.26 mmol)
at 0 ꢁC, and continued to stir for an additional 2 h at
0 ꢁC. The reaction mixture was diluted with water
(25 mL) and the product was extracted into ethyl acetate
(3 · 15 mL). The combined ethyl acetate layer was
washed with brine (15 mL) and dried (Na2SO4). The
ethyl acetate layer was evaporated under reduced pres-
sure and the crude was purified by column chromatogra-
phy (EtOAc–hexanes, 1:3) to obtain compound 41
(0.42 g, 71% over two steps) as a syrup; isolated as an
inseparable mixture of diastereomers in a 30:20:1.8:1
5.55. 2-Amino-1-{1-[(1-benzyl-2-fluoro-2-phenylsulfanyl-
ethylamino)-methyl]-2-methyl-propylamino}-ethanol (20)
Isolated as an inseparable mixture of diastereomers in
1
57:38:3:2 ratio; yield: 91%, syrup; H NMR (CDCl3) d
1
ratio; H NMR (CDCl3) d 0.75–0.91 (m, 6H), 1.25 (br
0.78–0.93 (m, 6H), 1.25 (br s, 1H), 1.55 (br s, 2H),
1.67–1.79 (m, 1H), 2.59–2.96 (m, 5H), 3.13–3.40 (m,
4H), 3.60–3.90 (m, 1H), 5.74, 5.77 (2dd, 1H, J =3.3,
54.9 Hz and 2.4, 55.2 Hz), 7.14–7.38 (m, 8H), 7.43–
7.51 (m, 2H); 19F NMR (CDCl3) d ꢀ80.83 (dd,
J = 18.8, 54.8 Hz), ꢀ80.53 (d, J = 17.4 Hz), ꢀ69.88
(dd, J = 14.3, 55.1 Hz), ꢀ69.57 (dd, J = 14.3, 55.6 Hz);
EI-MS m/z 405 (MH2+, 25), 404 (MH+, 87), 384 (M-F,
100), 262 (56), 91 (33).
s, 1H), 1.62–1.78 (m, 1H), 2.63–2.98 (m, 4H), 3.10–
3.42 (m, 2H), 4.21 (t, 1H, J = 6.6 Hz), 4.32–4.44 (m,
2H), 5.03, 5.25 (2d, 1H, J = 8.7 Hz and 8.7 Hz), 5.74
(dd, 1H, J = 2.7, 55.2 Hz), 7.17–7.49 (m, 14H), 7.60 (d,
2H, J = 7.5 Hz), 7.74 (d, 2H, J = 7.5 Hz); 19F NMR
(CDCl3) ꢀ159.43 (dd, J = 19.7, 54.8 Hz), ꢀ158.34 (dd,
J = 18.0, 54.4 Hz), ꢀ148.28 (dd, J = 15.2, 55.2 Hz),
ꢀ147.34 (dd, J = 14.3, 55.8 Hz).
5.53. N1-(1-Benzyl-2-fluoro-2-phenylsulfanyl-ethyl)-3-
methyl-butane-1,2-diamine (42)
5.56. 2-[(t-Butoxy-hydroxy-methyl)-amino]-1-(2-meth-
oxy-2-phenylsulfanyl-ethylamino)-propan-1-ol (44)
A solution of compound 41 (0.05 g, 0.08 mmol) in dry
DMF (1.35 mL) was treated with morpholine
(0.15 mL) at 0 ꢁC, brought to rt over a period of 1 h
and stirred for an additional 1 h. The reaction mixture
was diluted with water (20 mL) and the compound
was extracted into ethyl acetate (3 · 15 mL). The com-
bined ethyl acetate layer was washed with brine
(15 mL) and dried (Na2SO4). The organic layer was
evaporated under reduced pressure and the crude was
purified by column chromatography (CHCl3–MeOH,
95:5) to obtain compound 42 (0.025 g, 82%) as a syrup;
isolated as an inseparable mixture of diastereomers in a
A solution of compound 4 (0.12 g, 0.35 mmol) was trea-
ted with MeOH–H2O (20 mL, 1:1) and stirred for 4 days
at rt. Solvent was removed under reduced pressure and
the crude was purified by column chromatography
(EtOAc–hexanes, 3:7) to obtain compound 44 (0.035 g,
30%) as a syrup as an inseparable mixture of diastereo-
1
mers in a 1:1 ratio; H NMR (CDCl3) d 1.32, 1.34 (2d,
3H, J = 6.9, 6.9 Hz), 1.44 (s, 9H), 3.28–3.41 (m, 1H),
3.51, 3.52 (2s, 3H), 3.56–3.66 (m, 1H), 4.06–4.20 (m,
1H), 4.57–4.62 (m, 1H), 4.97 (br d, 1H), 6.40–6.50 (m,
1H), 7.29–7.31 (m, 3H), 7.45–7.49 (m, 2H); 13C NMR
(CDCl3) d 18.60, 28.52, 43.58, 50.35, 56.65, 80.25,
88.53, 88.57, 95.65, 128.37, 129.17, 131.60, 134.49,
155.68, 172.83; EI-MS m/z 355 (MH+, 2), 245 (54), 189
(100), 145 (53), 74 (44).
1
57:38:3:2 ratio; H NMR (CDCl3) d 0.76–0.90 (m, 6H),
1.25 (br s, 1H), 1.51–1.62 (m, 1H), 1.80 (br s, 2H), 2.29–
2.38 (m, 1H), 2.46–2.58 (m, 1H), 2.72–3.01 (m, 3H),
3.10–3.42 (m, 1H), 5.73, 5.74 (2d, 1H, J = 55.2 Hz and
55.2 Hz), 7.18–7.35 (m, 8H), 7.44–7.52 (m, 2H); 19F
NMR (CDCl3) d ꢀ81.57 (dd, J = 18.3, 55.4 Hz),
ꢀ81.23 (dd, J = 18.8, 54.8 Hz), ꢀ71.76 (dd, J = 14.6,
55.2 Hz), ꢀ71.47 (dd, J = 15.2, 55.1 Hz).
5.57. 2-Amino-1-{1-[(1-benzyl-2-methoxy-2-phenylsulfan-
yl-ethylamino)-methyl]-2-methyl-propylamino}-ethanol
(48)
Isolated as an inseparable mixture of diastereomers in a
1
5.54. 1-{1-[(1-Benzyl-2-fluoro-2-phenylsulfanyl-ethyl-
amino)-methyl]-2-methyl-propylamino}-2-{[(9H-fluoren-
9-ylmethoxy)-hydroxymethyl]-amino}-ethanol (43)
1:1 ratio; yield: 73%; syrup; H NMR (CDCl3) d 0.74–
0.87 (m, 6H), 1.25 (br s, 1H), 1.62–1.70 (m, 3H), 1.80–
2.05 (m, 2H), 2.54–2.83 (m, 2H), 2.98–3.71 (m, 5H),
4.43–4.65 (m, 1H), 7.08–7.50 (m, 10H); EI-MS m/z 417
(MH2+, 22), 416 (MH+, 63), 384 (MꢀOMe, 9), 262
(100), 143 (27), 91 (35).
A solution of FmocNH-Gly-OH (0.2 g, 0.69 mmol) in
dry CH2Cl2–DMF (5 mL, 7:1) was treated with EDAC
(0.15 g, 0.83 mmol), HOBt (0.09 g, 0.69 mmol) at 0 ꢁC.
Amine 42 (0.24 g, 0.69 mmol) was added after stirring
for 30 min at 0 ꢁC. The reaction was brought to rt over
a period of 1 h and stirred for an additional 1 h. The
reaction mixture was worked up and purified as de-
scribed for compound 5 to obtain compound 43
(0.35 g, 81%) as a foam; isolated as an inseparable mix-
5.58. Stability studies
The percent decomposition studies for the compounds
1–20 and 49 were conducted using 19F NMR spectro-
scopy. The peak integrals were calculated based on 19F
NMR spectral data obtained at either 282.3320 MHz

S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
2957
(for compounds 1–14 and 49) or 470.3286 MHz (for
compounds 15–20) at a constant temperature of 25 ꢁC.
The NMR spectra were recorded in CD3OD:D2O (3:2)
using CFBr3 as an internal standard. The test compound
was first dissolved in CD3OD (0.6 mL), then D2O
(0.4 mL) was added and the spectra were recorded at
various time intervals. The following quantities of com-
pounds were used: 1 (8.0 mg), 2 (7.1 mg), 3 (2.0 mg), 4
(7.6 mg), 5 (5.9 mg), 6 (6.2 mg), 7 (8.2 mg), 8 (6.5 mg),
9 (8.1 mg), 10 (8.4 mg), 11 (7.3 mg), 12 (6.8 mg), 13
(5.0 mg), 14 (5.5 mg), 15 (6.2 mg), 16 (5.5 mg), 17
(4.6 mg), 18 (7.7 mg), 19 (5.8 mg), and 20 (10.2 mg). In
each case, the concentration of the internal standard
(CFBr3) was 1 mg/mL.
concentration of substrate N-Succ-Ala-Ala-Pro-Phe-p-
nitroanilide versus absorbance at 315 nm. The standard
curve showed linearity till 150 lM concentration. Stock
solutions of the substrate and a-chymotrypsin were pre-
pared in Tris buffer at pH 7.8 (0.1 M Tris base, 0.01 M
CaCl2, 1 N HCl) and the stock solution of the inhibitor
was prepared in methanol. In order to study the time-
dependent loss of activity of chymotrypsin, enzyme,
and inhibitor were incubated at different time periods
at 25 ꢁC and then the substrate was added and the
time-course curves were plotted for 4 min at 410 nm at
0, 0.25, 0.5, 1, 2, 4, 8, and 24 h after incubation with
the respective inhibitors at concentrations of 1, 10, 50,
100 and 200 lM. The control set of reactions did not
contain the inhibitor. The data was analyzed using
MS Excel software. The time-dependent constants and
Ki were derived from the plots of the absorbance vs
time.
Each experiment was performed once, and the data was
recorded at the following time intervals: 0 h (after the
addition of D2O), 1, 2, 3, 4, 8, 14 h for compounds
15–20, 16 h for compounds 1–14, 49, 18 h for com-
pounds 15–20, and 24 h for compounds 1–14, 49. Where
there were diastereomeric mixtures present, more than
one peak was observed in the 19F NMR spectra. The
percent decomposition was calculated based on the inte-
gral of the most prominent peak (as illustrated in the
spectra enclosed in the Supplementary data, Figs. S2
and S3). The integration of each signal started at the
baseline of the peak of interest on an expanded spec-
trum. The peak integral values for the –CHF–S– peak
were measured with reference to the internal standard
(CFBr3). Percent decomposition was calculated as a ra-
tio between the peak integral value at a specific time
interval (24 or 18 h) and the peak integral value at
t = 0 h. During the acquisition, there was no decoupling
turned on. The spectra for compounds 14 and 20 are in-
cluded in the Supplementary data, as an illustration.
Acknowledgements
S.C.A. is a recipient of Rx&D Health Research Founda-
tion and Canadian Institutes for Health Research
(Rx&D HRF-CIHR) post-doctoral fellowship and
L.P.K. is a recipient of Rx&D HRF-CIHR research ca-
reer award. This work was supported by the grants from
Canadian Institutes of Health Research (MOP49414),
Canadian Foundation for Innovation, Ontario Innova-
tion Trust and PremierÕs Research Excellence award.
We acknowledge the use of resources at the Molecular
Design and Information Technology Center at the Uni-
versity of Toronto.
Supplementary data
5.59. Computer modeling
Time-dependent enzyme activity studies of compounds
45 and 46 against chymotrypsin; Illustration of 19F
spectra at various time points for compounds 14 and
All computations were performed on a Silicon Graphics
Onyx 3800 supercomputer. The structures of compound
16 and the transition-state species were drawn in the Sy-
byl molecular modeling program26 and the geometry
was optimized using DFT method (B3LYP/6-31+G*)
for compound 16 and PM3MM for the transition-state
species in the Gaussian98 package.27 ESP charges for
both molecules were obtained from the Gaussian98 un-
der B3LYP/6-31+G* level or default charges in the Am-
ber 7 package.28 After the fluoropeptidomimetic and
intermediate state mimic were docked into the active site
of chymotrypsin, enzyme complexes were immersed into
1
20; H NMR spectra for compounds 8–20, 28–39, 41–
48, 51, 56, 58, 59, 61, 63–74; and 13C NMR spectra
for compounds 8–19, 28–36, 44, 47, 51, 56, 58, 59, 61,
63–66, and 68; and 19F NMR spectra for compounds
8–14, 20, 29, 32–39, and 41–43 are given as the Supple-
mentary information and are available online. COSY
and HSQC NMR Spectra for 47; HRMS data for com-
pounds 15–19 and 45–47. Supplementary data associ-
ated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2005.02.011.
˚
a pre-equilibrated TIP3P water box with 8 A thickness
from the surface of the protein. The cut-off distance
˚
for nonbonded interactions was set at 12 A and the com-
plexes were subjected to energy minimization.
References and notes
1. Hedstrom, L. Serine protease mechanism and specificity.
Chem. Rev. 2002, 102, 4501–4523, and references cited
therein.
5.60. Enzyme activity evaluations
2. Ripka, A. S.; Rich, D. H. Peptidomimetic design. Curr.
Opin. Chem. Biol. 1998, 2, 441–452.
3. Leung, D.; Abbenante, G.; Fairlie, D. P. Protease inhib-
itors: current status and future prospects. J. Med. Chem.
2000, 43, 305–341.
The enzyme activity of chymotrypsin was measured
using the kinetics module on a Shimadzu UV-2401 PC
spectrophotometer. The absorbance of the reaction mix-
ture at various concentrations of the inhibitor and/or
substrate was measured at 410 nm to record p-nitroani-
line. The standard curve was obtained by plotting the
4. Annedi, S. C.; Li, W.; Samson, S.; Kotra, L. P. Design and
synthesis of novel fluoropeptidomimetics as potential

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S. C. Annedi et al. / Bioorg. Med. Chem. 13 (2005) 2943–2958
nating reagent (SelectfluorTM) and a reaction time of
approximately 45 min.
mimics of the transition state during peptide hydrolysis. J.
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