Design and synthesis of broad-based mono- and bi- cyclic inhibitors of FIV and HIV proteases

Article abstract of DOI:10.1016/S0968-0896(03)00054-3

Based on the substrate transition state and our strategy to tackle the problem of drug resistance, a series of HIV/FIV protease (HIV/FIV PR) monocyclic inhibitors incorporating a 15- or 17-membered macrocycle with an equivalent P3 or P3′ group and a uniqu

Full text of DOI:10.1016/S0968-0896(03)00054-3

Bioorganic & Medicinal Chemistry11 (2003) 2025–2040
Design and Synthesis of Broad-Based Mono- and Bi- Cyclic
Inhibitors of FIV and HIV Proteases
Chi Ching Mak,a Ashraf Brik,a Danica L. Lerner,b John H. Elder,b Garrett M. Morris,b
a,
Arthur J. Olsonb and Chi-HueyWong *
aDepartment of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 8 October 2002; accepted 31 December 2002
Abstract—Based on the substrate transition state and our strategyto tackle the problem of drug resistance, a series of HIV/FIV
protease (HIV /FIV PR) monocyclic inhibitors incorporating a 15- or 17-membered macrocycle with an equivalent P3 or P30 group
and a unique unnatural amino acid, (2R, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, have been designed and synthesized. In
addition, based on the structure of TL3 with small P3/P30 group, we have synthesized two conformationally restricted bicyclic
inhibitors containing the macrocycle, which mimic the P1/P10-P3/P30 tripeptide [Phe-Val-Ala] of TL3. We have found that the
contribution of the macrocycle in our monocyclic inhibitors is important to the overall activity, but the ring size does not affect the
activityto a significant extent. Several inhibitors that were developed in this work, exhibit low nanomolar inhibitoryactivityagainst
the wild-type HIV/FIV PR and found to be highly effective against some drug-resistant as well as TL3-resistant mutants of HIV
PRs. Compound 15, in particular, is the most effective cyclic inhibitor in hand to inhibit FIV replication in tissue culture at a
concentration of 1.0 mg/mL (1.2 mM).
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
HIV PR mutants have a shrunken P3 binding site,
which is a structural characteristic of FIV PR, and good
inhibitors of FIV PR are usuallybetter inhibitors of the
wild type and drug-resistant HIV proteases.5 Therefore,
FIV PR maybehave like the drug-resistant phenotype
of HIV protease and could be used as a model for the
development of new broad-based inhibitors to slow
down the development of drug resistance.
Since human immunodeficiencyvirus (HIV-1) was
identified as the causative agent of acquired immuno-
deficiencysnydrome (AIDS), and the virallyencoded
aspartic protease (PR) was proven essential for the
proliferation of HIV, intensive research has been devo-
ted to the development of potent inhibitors of HIV PR.
Despite six competitive inhibitors of this protease have
been approved and several others are in clinical trials,1
manydrug-resistant variants of HIV have been identi-
fied,2 rendering AIDS with no definitive cure. Feline
immunodeficiencyvirus (FIV) causes an immunodefi-
ciencysnydrome in cats comparable to AIDS in
humans.3 Although their active site structures are
superimposable and have an identical mechanism of
catalysis,4 binding of inhibitors to these proteases is
remarkablydifferent. All the approved inhibitors with
Ki values in the low nanomolar range for HIV PR, only
bind to FIV PR in micromolar range. In addition, many
It has been known that the inhibitors of aspartic PRs
commonlybind in an extended b-strand conforma-
tion.6 Restriction of an inhibitor’s conformation to
one recognized bythe enzmye mayincrease the
potencybylowering the entropic barrier to complex
formation. As a result, conformational restriction of
inhibitors through the introduction of macrocyles into
such extended conformation has been shown to be an
6,7
effective wayof increasing affinitytoward HIV PR.
The introduction of cyclic structures in PR inhibitors
have also been demonstrated to have comparable or
better antiviral activitycompared to the acyclic analo-
gues,7b,d suggesting improved cell penetration properties
and resistance to cellular enzymes for the macrocyclic
inhibitors.
*Corresponding author. Tel.: + 1-858-784-2487; fax: + 1-858-784-
2409; e-mail: wong@scripps.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00054-3

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C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
Recently, HIV PR inhibitors containing allophenylnor-
statine [(2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric
acid] have been reported byseveral research groups. 8 In
particular, Kiso et al.8d have demonstrated that inhi-
bitor 1 (JE-2147) (Fig. 1) has verypotent antiviral
activities in vitro and exhibits good oral bioavailability
and plasma pharmacokinetic profiles. Earlier we dis-
closed our approach9 for the design, synthesis and in
PRs and some mutants of HIV PRs and the full
details of the synthesis for all these cyclic inhibitors
are discussed.
Results and Discussion
Chemistry
vitro evaluation of
a series of phenylnorstatine
(Pns),[(2R, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid],
containing macrocyclic HIV/FIV PR inhibitors. The
macrocycle was designed to mimic the conformationally
constrained Phe-Val-Ala motif, which has been used in
the development of FIV PR and drug-resistant HIV PR
inhibitors such as 2 (TL3) (Fig. 1).5 All these results
provided the foundations of the work described in this
paper. Reported herein are additional findings and
examples for our monocyclic PR inhibitors as well as
the bicyclic PR inhibitors (Fig. 2). To the best of our
knowledge, there is onlyone example reported so far for
the preparation of bicyclic inhibitor against HIV PR.7f
All these cyclic inhibitors were found to be effective
against FIV PR, suggesting being also effective against a
broad range of HIV variants. The evaluation of their
inhibitoryactivities against both wild-tpye HIV/FIV
Our group previouslydisclosed that inhibitors with a
small P3 residue, particularly 2 (TL3) (Fig. 1), are
effective against HIV and its drug-resistant variants, as
well as SIV and FIV.5 The X-raystructure of FIV PR
complexed with 2 reveals that the P1 and P3 residues are
positioned verycloselyin the neighboring hydrophobic
pockets.10 This prompted us the design of the bicyclic
peptidomimetic inhibitors 3 and 4, in which the P1/P10
and P3/P30 residues are connected with the aliphatic
chain in a macrocycle, which may render the solution
conformation similar to the bound conformation in the
active site of the protease.
The synthesis of 3 with P1 and P3 residues connected
together with an amide linkage in the meta-position of
the Phe residue is illustrated in Scheme 1. The free acid
in 3-nitro-tyrosine was protected as a methyl ester fol-
lowed bycoupling with Boc-Val-OH. The resulting
dipeptide Boc-Val-Tyr(3-NO2)-OMe11 was reacted with
5-chlorophenyl tetrazole and the hydroxyl and nitro
groups in 17 were simultaneouslyremoved and reduced,
respectivelyafter refluxing in EtOH/cyclohexene in the
presence of Pd/C. The free amine was then protected
with Cbz group and the protected dipeptide 18 was
subjected to subsequent LiBH4 reduction and oxidation
using TEMPO/BAIB.12 Vanadium catalyzed coupling13
of aldehyde 19 followed byprotection and chromato-
graphic separation of the diastereomeric mixture affor-
ded 20 in 48% overall yield. The Cbz groups in 20 were
removed and coupled with Z-Glu-OtBu to obtain the
bicyclic precursor 21 in 66% overall yield. Removal of
all Boc and t-Bu groups in 21 with TFA followed by
intramolecular amide bond formation in CH3CN
Figure 1.
Figure 2.

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2027
Scheme 1. Reagents and conditions: (a) AcCl, MeOH; (b) Boc-Val-OH, HBTU, DIPEA; (c) 5-chlorophenyl tetrazole, NaHCO3, DMAC; (d) Pd/C,
cyclohexene, reflux; (e) CbzCl, py; (f) LiBH4, THF; (g) PhI(OAc)2, TEMPO; (h) Zn, VCl3(thf)3; (i) 2,2-dimethoxypropane, TsOH; (j) sep. diaster-
eomers; (k) Pd/C, H2; (l) Z-Glu-OtBu, HBTU, DIPEA; (m) TFA/CH2Cl2; (n) BOP, DIPEA, CH3CN; (o) TFA/H2O.
(5 mM) and unmasking the diol core with TFA/H2O
provided 3 in 60% overall yield.
Compounds 5–16 are all pentapeptide mimetics consist-
ing of a 15- or 17-membered C-terminal macrocycle
designed to mimic the P10-P30 tripeptide (Phe-Val-Ala)
of a substrate for HIV/FIV PRs. The hydroxyl-
methylcarbonyl moiety among these phenylnorstatine-
based inhibitors mimics the transition state of peptide
In a similar manner to 3, compound 4 was synthesized
(Scheme 2). The crucial steps in the synthesis of 4 with
P1 and P3 residues joined together with an ether linkage
in the para-position of the Phe residue, included the
pinacol coupling and the intramolecular cyclization
reactions. The dipeptide aldehyde 22 was derived from
H-Tyr(Bzl)-OMe after coupling with Boc-Val-OH and
subsequent reduction and oxidation. Pinacol coupling
of 22, protection and separation of the mixture provided
23 in 60% overall yield. Deprotection of the hydroxyl
groups (Pd/C, H2) in 23 followed byMitsunobu reac-
tion with 2414 afforded the bicyclic precursor 25 in 47%
overall yield. Finally, deprotection of the Boc groups
with TFA followed byhigh dilution ccylization and
removal of the isopropylidine group (TFA/H2O) affor-
ded 4 in 46% overall yield.
hydrolysis and the hydroxyl configuration is known to
8
be detrimental to the inhibitoryactivity.
The R con-
figuration was adopted in all these compounds since
such stereochemistryaround this carbinol function was
previouslyreported to have better inhibitoryactivityfor
HIV/FIV PRs than that of the S isomer.9 Since potent
peptidomimetic inhibitors for HIV/FIV PRs have been
achieved either bycapping the N-terminus of the basic
core with a large protecting group together with a rela-
tivelysmall P3 residue (e.g., Ala) 5 or vice versa,15 all our
mono-cyclic inhibitors except 12 (with small P3 residue
and N-terminal group) have either of these two patterns
(Fig. 2). Also HIV PR inhibitors with paracyclophanes,
Scheme 2. Reagents and conditions: (a) Boc-Val-OH, HBTU, DIPEA; (b) LiBH4, THF; (c) PhI(OAc)2, TEMPO; (d) Zn, VCl3(thf)3; (e) 2,2-dime-
thoxypropane, TsOH; (f) sep. diastereomers; (g) Pd/C, H 2; (h) DIAD, PPh3; (i) TFA/CH2Cl2; (j) BOP, DIPEA, CH3CN; (k) TFA/H2O.

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C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
containing oxyethylene substructures, were reported to
be water soluble at physiological PH and have potent
antiviral activities.7g Therefore, we reasoned that the
incorporation of either a 15-(28) or 17-(32) membered
macrocycle into our mono-cyclic inhibitors (Scheme 3),
could enhance the binding and the solubilitypropertied
of these inhibitors
appropriate acyclic components 40–46 and 48 after Boc
removal and ester hydrolysis (Scheme 4). Macrocycliza-
tion of 27 and 31 were achieved efficientlyusing Cs 2CO3
in CH3CN (10 mM) in the presence of tetra-
butylammonium iodide (1 mol equiv) at room tempera-
ture (Scheme 3). The NMR spectra indicate the
aromatic ring in 32 is less constrained than that in 28,
where four distinct unequivalent aromatic protons can
be observed. The tripeptide 40–45 were synthesized by
coupling the core unit 3316 after Boc removal and the
The mono-cyclic inhibitors 5–12, 15 and 16 were
assembled byappending the macrocycles 28 or 32 to the
Scheme 3. Reagents and conditions: (a) Boc-Val-OH, HBTU, DIPEA; (b) Boc-Tyr-OH, HBTU, DIPEA; (c) CsCO3, TBAI, CH3CN; (d) HBTU,
DIPEA; (e) LiOH, MeOH/H2O; (f) 2-(2-aminoethoxy)ethanol, HBTU, DIPEA; (g) CBr4, PPh3; (h) CsCO3, TBAI, CH3CN.
Scheme 4. Reagents and conditions: (a) (i) LiOH, MeOH/H2O, (ii) 33, TFA/CH2Cl2, (iii) HBTU, DIPEA; (b) (i) LiOH, MeOH/H2O, (ii) 28 or 32,
TFA/CH2Cl2, (iii) HBTU, DIPEA; (c) (i) Pd/C, H2, (ii) p-toluene sulfonyl chloride, Et3N; (d) (i) LiOH, MeOH/H2O, (ii) 28, TFA/CH2Cl2, (iii)
HBTU, DIPEA; (e) (i) TFA/CH2Cl2, (ii) Boc-Val-OH, HBTU, DIPEA; (f) (i) TFA/CH2Cl2, (ii) Ac-Ala-OH, HBTU, DIPEA.

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2029
corresponding dipeptide methyl ester 34–39 after
hydrolysis (Scheme 4). Compound 46 was derived from
40 after Cbz removal and reaction with p-toluene sulfo-
nyl chloride, while 48 was obtained bycoupling of Boc-
removed 33 with Boc-Val-OH followed bycondensation
with Ac-Ala-OH (Scheme 4). In fact, 44 and 45 were
prepared from the racemate of Ac-Phe(4-F)-OH and
their structural identities were defined bycomparing the
NMR signals of the same tripeptide 53. Compound 53
was derived from the coupling product of opticallypure
Boc-l-Phe (4-F)-OH and H-Val-OMe, followed by
deprotection of the Boc group. The product then was
coupled to AcOH followed bycoupling to Boc-removed
33 to give the tripeptide 53 (Scheme 5), which has the
mutant HIV and TL3-resistant mutant HIV PRs. All
the mutant enzymes tested here have their mutated
residues within the S3 and S30 subsites, which have been
identified as the important area, associated with the
development of drug resistance. The beneficial effect for
the conformationallyconstrainted structures in the
bicyclic inhibitors 3 and 4 was not observed to a sig-
nificant extent although theyhave also small P3 resi-
dues. As compared to TL3 compound 3 was less
effective for both HIV/FIV PRs, while compound 4 only
retained similar potencies for HIV/FIV PRs as well as
the drug-resistant HIV PR mutants, though it has better
activities against the TL3-resistant HIV PR mutants.
The cyclic inhibitors 7–10, 15 and 16 have similar
potencycompared to TL3 against the mutant V82F, but
less active against the drug-resistant mutant G48V.
However, these inhibitors have higher potencythan that
of TL3 against V82A and hexa-mutant.
1
same H and 13C NMR signals as 44.
Boc removal of 28 or 32 and coupling with tripeptide
40–46 and 48 after saponification afforded mono-cyclic
inhibitors 5–12, 15 and 16 (30–70%). Compound 13 and
14 were prepared in a slightlydifferent procedure and
were shown in Scheme 6. Coupling of Boc removed 28
with 33 after hydrolysis provided 49, which was then
reacted subsequentlywith Boc-Val-OH and Ac-His(1-
Trt)-OH under the same deprotection and coupling
conditions to afford 13. The trityl group was then
removed with TFA to provide 14. All inhibitors 5–16
were precipitated out during the work up procedure and
the filtered products were pure enough for the char-
acterization byNMR spectroscopyand high-resolution
mass spectrometry.
Amongst all the mono-cyclic inhibitors, compounds 7–
10, 15 and 16 containing a relativelylarge P3 residues
and a small N-terminal protecting group still showed
significant activitycomparable to TL3, with IC values
50
in the range of 6–10 nM, against HIV PR. Theyalso
exhibited remarkable activities toward FIV PR with
IC50 values 2–3-fold lower compared to TL3. However,
when we introduced to the mono-cyclic inhibitors a
small residue (e.g., 12) or a verylarge P3 residue (e.g.,
13), their activities against FIV PR decreased, while
theyremained effective against HIV PR. In addition, we
found that the incorporation of a P3 ligand with
d-configuration (e.g., 11) lead to about 100-fold reduc-
tion in the activityagainst FIV PR, but did not affect
Finally, compounds 55, 56 were prepared through cou-
pling of 43 after saponification with Boc removed 29 to
give 55, and Boc removed 54 to give 56 (Scheme 7).
the high activityagainst HIV PR (IC
9 nM). These
50
results indicate that, while the P3 binding subsites in
HIV PR are highlyflexible and can accommodate vari-
etyof P3 ligands, however, in FIV PR theyare more
restricted and the balance between the size of the P3
residue and the N-terminal protecting group seems to be
important in order to achieve potent inhibitors.
Biological evaluation and discussion
Table 1 summarizes the inhibitoryactivities of each
cyclic compound against FIV, HIV, drug-resistant
To understand the contribution of the macrocycle on
the overall activityof the inhibitors, we also tested the
inhibitorypotencyof pentapeptide 55 and 56, which are
the acyclic analogues of 9. Compared to 9, theydis-
played about 20- to 200-fold less potency against HIV/
FIV PRs. Such results maybe attributed to the struc-
tural mimics of tripeptide (Phe-Val-Ala) in 9 fixed in the
extended strand conformation, resulting in an entropic
energygained over the acyclic compounds. Increasing
Scheme 5. Reagents and conditions: (a) H-Val-OMe, HBTU, DIPEA;
(b) (i) TFA/CH2Cl2, (ii) AcOH, HBTU, DIPEA; (c) (i) LiOH, MeOH/
H2O, (ii) 33, TFA/CH2Cl2, (iii) HBTU, DIPEA.
Scheme 6. Reagents and conditions: (a) (i) LiOH, MeOH/H2O, (ii) 28, TFA/CH2Cl2, (iii) HBTU, DIPEA; (b) (i) TFA/CH2Cl2, (ii) Boc-Val-OH,
HBTU, DIPEA; (c) (i) TFA/CH2Cl2, (ii) Ac-His(1-Trt)-OH, HBTU, DIPEA; (d) TFA/H2O.

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C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
ing core structure for the design of HIV/FIV PR inhi-
bitors. Several of these inhibitors that were developed in
this work, exhibited verypotent inhibitoryactivity
against HIV/FIV PR, some drug-resistant and TL3-
resistant mutants of HIV PRs. Compound 15, in parti-
cular, is effective against FIV replication in tissue cul-
ture and work is continue to studyits effectiveness
against other FIV/HIV variants.
Experimental
In general, reagents and solvents were used as pur-
chased without further purification. Methylene chloride
(CH2Cl2) and acetonitrile (CH3CN) were distilled over
calcium hydride and tetrahydrofuran (THF) was freshly
distilled from sodium/benzophenone ketyl under argon.
Analytical TLC was performed using silica gel 60 F254
glass plates (Merck). Flash column chromatography
was performed on silica gel 60 Geduran (35–75 mm, EM
Science). NMR (1H, 13C) spectra were recorded either
on a Bruker AMX-400, AMX-500 or AMX-600 MHz
spectrometer. Coupling constants (J) are reported in
hertz, and chemical shifts are reported in parts per mil-
lion (d) relative to tetramethylsilane (TMS, 0.0 ppm) or
DMSO (2.49 ppm for 1H and 39.5 ppm for 13C) or
CD3OD(3.30 ppm for 1H and 49.0 ppm for 13C) as
internal reference. Dipeptide 34 was provided byDr.
Taekyu Lee.
Scheme 7. Reagents and conditions: (a) (i) LiOH, MeOH/H2O, (ii)
TFA/CH2Cl2, (iii) HBTU, DIPEA.
the ring size from 15- to 17-membered macrocycle (7/
10–15/16) did not affect the IC50 values to a significant
extent, which was consistent with the results reported
earlier.7g The linkers between P10 and P30 maybe situ-
ated outside the cleft of the active site and that the
overall conformation of the P10 and P20 residues are not
altered bythe size of the rings. Finally, Compound 15
was found to be most effective to inhibit FIV replication
in tissue culture at 1.0 mg/mL (1.2 mM), which is com-
parable to TL3 at the same concentration.
Method A: general procedure for peptide bond formation
To a solution of a carboxylic acid (1.0 mol equiv) and a
free amine (1.0 mol equiv) in dryTHF, CH 3CN or
DMF (0.2–0.5 M) was added HBTU (1.1 mol equiv)
followed byDIPEA (1.1 mol equiv or 2.2 mol equiv if
the free amine is in the form of anyacid salt). The
reaction mixture was stirred at room temperature for 2–
4 h and then diluted with EtOAc. The organic layer was
washed with 1 M HCl, saturated NaHCO3, brine, dried
Conclusion
In summary, we have described the strategies to intro-
duce the rigid structures in the form of a 15 to 17-
membered ring into our mono- and bi-cyclic inhibitors,
using the hydroxymethylcarbonyl isostere as a promis-
Table 1. Biological activity(IC nM) of TL3 and cyclic inhibitors 3–16 against FIV-WT, HIV-WT, drug-resistant HIV and TL3-resistant HIV
50
proteases
Drug-resistant HIV mutants
TL3-resistant HIV mutants
Inhibitors
FIV PR
HIV PR
HIV (G48V)
HIV (V82F)
IC50 (nM)
HIV (V82A)
HIV (L24I, M46I, F53L, L63P, V77I, V82A)
TL3
3
4
5
6
7
8
9
10
11
12
13
14
15
16
72
31
88
95
147
49
35
27
4
389
6
10
13
10
7
6
3
9
6
20.5 (5X)a
ndb
34 (6X)
nd
nd
71 (7X)
94 (13X)
57 (9.5X)
27 (9X)
nd
nd
nd
nd
33 (7X)
45 (9X)
14.9 (4X)
nd
23 (4X)
nd
nd
30 (3X)
32 (4X)
26 (4X)
20 (7X)
nd
16 (4X)
nd
5 (1X)
nd
nd
10 (1X)
12 (2X)
11 (2X)
7 (2X)
nd
144 (36X)
nd
23 (4X)
nd
nd
49 (5X)
79 (11X)
70 (12X)
33 (11X)
nd
nd
nd
nd
29 (6X)
45 (9X)
20
2000
121
138
51
45
37
nd
nd
nd
nd
nd
nd
nd
nd
17
18
5
5
nd
nd
aNumber in parentheses denotes number of fold higher IC50 values in inhibition compared to that of the wild-type HIV PR.
bNot determined.

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2031
over MgSO4 and filtered. The filtrate was concentrated
in vacuo and the crude product purified as described in
the text below.
4.93 (1H, m), 5.12–5.23 (2H, ABq, J=12.1 Hz), 5.37
(1H, d, J=9.2 Hz), 6.47 (1H, d, J=8.0 Hz), 6.71 (1H, d,
J=7.6 Hz), 7.05 (1H, s), 7.19 (1H, t, J=8.0 Hz), 7.29–
7.40 (5H, m), 7.55 (1H, d, J=7.8 Hz), 7.82 (1H, s); 13C
NMR (100 MHz, CDCl3) 17.8, 19.2, 28.2, 30.5, 37.0,
52.3, 52.7, 60.1, 66.8, 80.0, 117.2, 120.1, 123.4, 128.2,
128.4, 129.2, 135.9, 136.1, 138.4, 153.5, 156.1, 171.2,
171.4; HRMS (MALDI), calcd for MH+ C28H38N3O7
m/e 528.2710, found m/e 528.2714.
Method B: general procedure for Boc removal or hydro-
lysis of t-butyl ester. The protected peptide dissolved in
50% TFA in dryCH Cl2 (ꢀ0.3 M) or in 4 M HCl in
2
1,4-dioxane (ꢀ0.5 M) was stirred at room temperature
for 1–3 h. The solvent was removed in vacuo and the
remaining TFA/HCl was removed byrepeated eva-
poration from toluene in vacuo to give the TFA/HCl
salt of the amine or the free acid.
Aldehyde 19. To a solution of 18 (1.0 g, 1.9 mmol) in
THF (15 mL) was added LiBH4 (85 mg, 3.9 mmol) and
MeOH (1 mL). The mixture was stirred at room tem-
perature for 3 h. The reaction was quenched slowlywith
1 M HCl (10 mL) and most of the THF removed. The
residue was diluted with EtOAc and the aqueous layer
separated. The organic layer was washed with saturated
NaHCO3, brine, dried over MgSO4, filtered and the fil-
trate concentrated in vacuo. To a solution of the above
crude alcohol in CH2Cl2 (10 mL) was added TEMPO
(60 mg, 0.38 mmol) and BAIB (0.70 g, 2.2 mmol) and the
mixture stirred at room temperature for 2 h. The mix-
ture was diluted with EtOAc, washed with saturated
Na2S2O3, saturated NaHCO3, brine, dried over MgSO4
and filtered. The filtrate was concentrated in vacuo and
the crude aldehyde 19 used in the next step without
purification.
Method C: general procedure for the hydrolysis of
methyl ester. To the methyl ester of any peptide dis-
solved in a mixture of 40% MeOH in THF (ꢀ0.5 M)
was added LiOH (4.0 mol equiv) and water (0.5–2 mL).
The reaction was monitored byTLC and usuallytook
about 2–4 h to complete. Most solvents were removed in
vacuo and the residue was diluted in EtOAc. The
organic layer was washed with 10% citric acid, brine,
dried over MgSO4 and filtered. The free acid was
obtained after removal of the solvent in vacuo.
Compound 17. A mixture of Boc-Val-Tyr(3-Nitro)-
OMe11 (6.0 g, 14 mmol), 5-chloro-1-phenyl tetrazole
(3.0 g, 17 mmol), and sodium bicarbonate (2.5 g,
30 mmol) in DMAC (70 mL) was heated to 90 ꢁC for 2
days. The reaction mixture was cooled to 0 ꢁC and
poured into the iced cold water (450 mL). The resulting
solid was filtered and washed thoroughlywith water.
The solid was further purified byrepeated washing with
ether–hexane mixture (1:1 v/v) and then dried to give 17
(7.0 g, 88%) as a bright yellow solid: 1H NMR
(500 MHz, CDCl3) 0.91 (3H, d, J=6.5 Hz), 0.95 (3H, d,
J=6.8 Hz), 1.43 (9H, s), 2.09–2.16 (1H, m), 3.21 (1H,
dd, J=14.0, 6.0 Hz), 3.33 (1H, dd, J=14.0, 5.8 Hz), 3.87
(1H, dd, J=8.4, 6.3 Hz), 4.93 (1H, dd, J=13.1, 6.1 Hz),
5.01 (1H, d, J=7.0 Hz), 6.60 (1H, d, J=6.9 Hz), 7.52–
7.62 (5H, m), 7.82 (2H, d, J=8.6 Hz), 7.99 (1H, s); 13C
NMR (125 MHz, CDCl3) 17.8, 19.2, 28.2, 30.4, 37.1,
52.7, 52.8, 60.1, 80.0, 122.6, 123.9, 127.2, 129.7, 129.8,
132.6, 136.6, 136.9, 140.0, 144.9, 155.8, 159.2, 170.9,
171.8; HRMS (MALDI), calcd for MNa+
C27H33N7O8Na m/e 606.2288, found m/e 606.2273.
Compound 20. Under argon, Zn (84 mg, 1.3 mmol) was
added to a solution of VCl3(thf)3 (0.90 g, 2.4 mmol) in
CH2Cl2 (5 mL). The mixture was stirred at room tem-
perature until a color change from red to green. A
solution of 19 (0.60 g, 1.2 mmol) in CH2Cl2 (5 mL) was
added and the resulting mixture stirred for 5 h. An
aqueous 1 M HCl (5 mL) was added and stirred for
15 min. The mixture was extracted with EtOAc and the
combined organic layers washed with saturated
NaHCO3, brine, dried over MgSO4 and concentrated in
vacuo. To a solution of the crude product in CH2Cl2
(10 mL) was added 2,2-dimethoxypropane (10 mL) and
TsOH (40 mg) and the mixture stirred for 1 h. The solvent
was removed and the desired diastereomer separated by
flash chromatographyon silica gel (hexane–EtOAc=1:1)
1
to afforded 20 (0.30 g, 48%) as a white foam: H NMR
(600 MHz, MeOH-d4) 0.60 (6H, d, J=6.5 Hz), 0.69 (6H,
d, J=6.6 Hz), 1.39 (6H, s), 1.42 (18H, s), 1.80–1.90 (2H,
m), 2.74–2.85 (4H, m), 3.64 (2H, s), 3.80 (2H, d,
J=6.6 Hz), 4.37 (2H, dd, J=9.2, 5.7 Hz), 5.12–5.21 (4H,
ABq, J=12.5 Hz), 6.91 (2H, d, J=7.4 Hz), 7.14 (2H, t,
J=7.7 Hz), 7.22–7.42 (14H, m); 13C NMR (150 MHz,
MeOH-d4) 18.0, 19.8, 27.8, 28.7, 31.7, 40.0, 50.2, 61.8,
67.5, 80.1, 80.7, 109.9, 118.0, 120.9, 125.1, 129.1, 129.5,
129.9, 138.2, 140.1, 155.8, 157.9, 174.2; HRMS
(MALDI), calcd for MCs+ C57H76N6O12Cs m/e
1169.4576, found m/e 1169.4524.
Compound 18. Cyclohexene (30 mL) was added to the
mixture of Pd/C (10%, 0.50 g) and 17 (2.0 g, 3.4 mmol)
in EtOH (40 mL) and the mixture heated to reflux under
an argon atmosphere for 1 day. The solution was fil-
tered through a pad of Celite and the solvent removed
in vacuo. The crude amine was treated with CbzCl
(0.80 mL, 5.6 mmol) and DIPEA (1.0 mL, 5.7 mmol)
and stirred in CH2Cl2 (30 mL) for 1.5 h. The solvent was
diluted with EtOAc and washed with saturated
NaHCO3, brine, dried over MgSO4 and filtered. The
solvent was removed and the crude product purified by
flash chromatographyon silica gel (hexane–
EtOAc=3:1) to afford 18 (1.0 g, 55%) as a white foam:
1H NMR (400 MHz, CDCl3) 0.88 (3H, d, J=6.8 Hz),
0.93 (3H, d, J=6.8 Hz), 1.37 (9H, s), 2.02–2.13 (1H, m),
3.06 (1H, dd, J=13.7, 4.4 Hz), 3.13 (1H, dd, J=13.8,
5.3 Hz), 3.71 (3H, s), 3.95 (1H, dd, J=9.3, 6.3 Hz), 4.87–
Bicyclic precursor 21. Compound 20 (0.20 g, 0.19 mmol)
was hydrogenated (2 h) with 10% Pd/C in MeOH to
give the diamine of 20. The crude diamine was then
coupled with Z-Glu-OtBu (0.2 g, 0.68 mmol) in CH3CN
according to the method A and purified byflash chro-
matographyon silica gel (hexane–EtOAc=1:1) to
afford 21 (0.18 g, 66%) as a pale brown foam: 1H NMR

2032
C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
(500 MHz, MeOH-d4) 0.64 (6H, d, J=6.5 Hz), 0.69 (6H,
d, J=6.5 Hz), 1.40 (6H, s), 1.42 (18H, s), 1.45 (18H, s),
1.80–1.90 (2H, m), 1.91–2.01 (2H, m), 2.15–2.25 (2H,
m), 2.46 (4H, t, J=7.5 Hz), 2.73–2.90 (4H, m), 3.64 (2H,
s), 3.80 (2H, t, J=6.3 Hz), 4.11 (2H, dd, J=9.4, 4.8 Hz),
4.35–4.47 (2H, m), 5.00–5.15 (4H, ABq, J=12.4 Hz),
6.53 (2H, d, J=9.3 Hz), 6.97 (2H, d, J=7.3 Hz), 7.16
(2H, t, J=7.7 Hz), 7.23–7.45 (12H, m), 7.53 (2H, d,
J=9.3 Hz),; 13C NMR (125 MHz, MeOH-d4) 18.1, 19.8,
27.8, 28.3, 28.4, 28.8, 31.8, 34.2, 40.0, 50.2, 55.9, 61.9,
67.7, 80.1, 80.7, 82.9, 110.0, 119.4, 122.2, 126.1, 128.9,
129.0, 129.5, 129.9, 138.2, 139.7, 140.1, 157.8, 158.7,
173.0, 174.3, 174.4; HRMS (MALDI), calcd for MCs+
C75H106N8O18Cs m/e 1539.6679, found m/e 1539.6600.
afford 25 (0.38 g, 47%) as a white foam: 1H NMR
(500 MHz, MeOH-d4) 0.71 (12H, d, J=6.2 Hz), 1.39
(6H, s), 1.43 (18H, s), 1.44 (18H, s), 1.72–1.89 (8H, m),
1.90–1.97 (2H, m), 2.74 (4H, d, J=7.4 Hz), 3.62 (2H, s),
3.75–3.82 (2H, m), 3.86–3.96 (4H, m), 4.06–4.13 (2H,
m), 4.24–4.35 (2H, m), 5.03–5.14 (4H, ABq,
J=12.5 Hz), 6.39 (2H, d, J=9.2 Hz), 6.76 (4H, d,
J=8.1 Hz), 7.09 (4H, d, J=8.4 Hz), 7.24–7.37 (10H, m),
7.52 (2H, d, J=9.2 Hz); 13C NMR (150 MHz, MeOH-
d4) 18.3, 19.9, 26.8, 27.7, 28.3, 28.8, 29.6, 31.9, 39.4,
50.6, 56.0, 61.7, 67.6, 68.3, 79.7, 80.6, 82.8, 109.8, 115.6,
128.8, 129.0, 129.4, 131.2, 138.3, 157.8, 158.6, 159.0,
173.3, 174.0; HRMS (MALDI), calcd for MNa+
C75H108N6O18Na m/e 1403.7612, found m/e 1403.7599.
Bicyclic inhibitor 3. Boc removal and hydrolysis of t-Bu
ester in 21 (88 mg, 0.063 mmol) were performed accord-
ing to the method B. To the crude deprotected product
in CH3CN (3 mM) was added BOP (0.10 g, 0.23 mmol)
and DIPEA (0.06 mL, 0.34 mmol) and the solution stir-
red for 38 h. Most of the solvent was removed, the solid
filtered and washed with CH3CN and Et2O. The solid
was redissolved in TFA (2 mL) and H2O (0.2 mL) added
at 0 ꢁC. The mixture was stirred for 2 h and the solvent
removed in vacuo. The residue was triturated in Et2O
and the solid filtered. The solid was washed with Et2O
to afford 3 (40 mg, 60%) as a pale brown solid: 1H
NMR (500 MHz, DMSO-d6) 0.60–0.90 (12H, m), 1.60–
2.10 (10H, m), 2.58 (2H, d, J=15.4 Hz), 2.86 (2H, t,
J=13.4 Hz), 3.97–4.27 (6H, m), 4.69 (2H, s), 4.97 (4H,
br s), 6.80–7.40 (24H, m), 7.83 (2H, br s); HRMS
(MALDI), calcd for MNa+ C54H66N8O12 m/e
1041.4692, found m/e 1041.4689.
Bicyclic inhibitor 4. Preparation was as described for 3
1
and afforded 4 (11 mg, 46%) as a off-white solid: H
NMR (600 MHz, DMSO-d6) 0.78 (6H, d, J=7.0 Hz),
0.81 (6H, d, J=6.5 Hz), 1.27–1.60 (8H, m), 1.69–1.78
(2H, m), 2.46 (2H, d, J=10.9 Hz), 2.64 (2H, t,
J=12.3 Hz), 3.95–4.24 (10H, m), 4.58 (2H, t,
J=8.3 Hz), 4.97 (4H, s), 6.62 (2H, d, J=8.4 Hz), 6.74
(2H, d, J=8.0 Hz), 6.92–7.00 (4H, m), 7.06 (2H, d,
J=7.9 Hz), 7.24–7.36 (10H, m), 7.39 (2H, d,
J=10.1 Hz), 7.48 (2H, d, J=8.8 Hz); 13C NMR
(125 MHz, DMSO-d6) 18.2, 19.1, 22.0, 28.3, 31.9, 37.9,
51.1, 53.4, 56.7, 65.1, 67.5, 73.6, 116.0, 118.4, 127.7,
128.2, 128.3, 129.8, 130.6, 131.7, 137.2, 154.3, 155.2,
169.6, 170.3; HRMS (MALDI), calcd for MH+
C54H69N6O12 m/e 993.4968, found m/e 993.4936.
Compound 26. This was prepared from 3-bromopropyl
amine (2.8 g, 13 mmol) and Boc-Val-OH (2.5 g,
12 mmol) in THF according to the method A and pur-
ified byflash chromatographyon silica gel (hexane–
EtOAc=2:1) to afford 26 (3.5 g, 90%) as a white solid:
1H NMR (500 MHz, CDCl3) 0.92 (3H, d, J=7.0 Hz),
0.96 (3H, d, J=6.6 Hz), 1.45 (9H, s), 2.05–2.19 (3H, m),
3.38–3.46 (4H, m), 3.84 (1H, dd, J=8.5, 6.3 Hz), 5.05
(1H, br s), 6.27 (1H, br s); 13C NMR (125 MHz, CDCl3)
17.9, 19.3, 28.3, 30.5, 30.7, 32.0, 37.9, 60.2, 80.0, 156.0,
172.0; MS (MALDI): m/z=237 [M–Boc]+.
Aldehyde 22. This was prepared from Boc-Val-Tyr
(OBz)-OMe according to the procedure as described for
19 and the crude aldehyde 22 was used in the next step
without purification.
Compound 23. This was prepared from 22 according to
the procedure as described for 20. Separation of the
desired diastereomer byflash chromatographyon silica
gel (hexane–EtOAc=2:1) afforded 23 (0.22 g, 60%) as a
1
white foam: H NMR (500 MHz, MeOH-d4) 0.70 (6H,
Cyclic precursor 27. This was prepared from 26 (2.9 g,
8.6 mmol) and Boc-Tyr-OH (2.3 g, 8.2 mmol) in THF
according to the method A and purified byflash chro-
matographyon silica gel (hexane–EtOAc=1:1) to
afford 27 (3.9 g, 95%) as a white foam: 1H NMR
(500 MHz, CDCl3) 0.86 (3H, d, J=6.5 Hz), 0.90 (3H, d,
J=6.7 Hz), 1.41 (9H, s), 1.98–2.08 (2H, m), 2.10–2.28
(1H, m), 2.85–3.05 (2H, m), 3.24–3.33 (1H, m), 3.35–
3.45 (3H, m), 4.21 (1H, t, J=7.5 Hz), 4.32–4.40 (1H, m),
5.29 (1H, d, J=5.8 Hz), 6.76 (2H, d, J=8.1 Hz), 6.88
(1H, d, J=8.5 Hz), 6.98 (2H, d, J=8.2 Hz), 7.71 (1H, s);
13C NMR (125 MHz, CDCl3) 17.7, 19.2, 28.2, 30.2,
30.7, 32.1, 37.0, 38.0, 56.2, 58.9, 80.7, 115.7, 127.2,
130.2, 155.5, 155.9, 171.3, 172.1; HRMS (MALDI),
calcd for MNa+ C22H34BrN3O5Na m/e 522.1574, found
m/e 522.1582.
d, J=5.5 Hz), 0.71 (6H, d, J=5.5 Hz), 1.39 (6H, s), 1.43
(18H, s), 1.81–1.91 (2H, m), 2.75 (4H, d, J=7.3 Hz),
3.63 (2H, s), 3.79 (2H, d, J=6.6 Hz), 4.29 (2H, t,
J=7.2 Hz), 5.01 (4H, s), 6.85 (4H, d, J=8.4 Hz), 7.11
(4H, d, J=8.1 Hz), 7.25–7.42 (10H, m); 13C NMR
(150 MHz, MeOH-d4) 18.2, 19.9, 27.7, 28.8, 31.9, 39.3,
50.5, 61.8, 71.1, 79.8, 80.7, 109.9, 116.0, 128.5, 128.8,
129.4, 131.3, 131.6, 138.9, 157.8, 159.0, 174.0; HRMS
(MALDI), calcd for MNa+ C55H74N4O10Na m/e
973.5297, found m/e 973.5312.
Bicyclic precursor 25. Compound 23 (0.36 g, 0.47 mmol)
was hydrogenated (2 h) with 10% Pd/C in MeOH to
give the diamine of 25. To a solution of the crude dia-
mine, 24 (0.38 g, 1.2 mmol) and PPh3 (0.50 g, 1.9 mmol)
in CH2Cl2 (15 mL) was added DIAD (0.4 mL,
2.0 mmol). The mixture stirred for 17 h and the solvent
removed. The crude product was purified byflash chro-
matographyon silica gel (hexane–EtOAc=2:1) to
Macrocycle 28. A solution of 27 (0.71 g, 1.4 mmol), ces-
cium carbonate (0.55 g, 1.7 mmol) and tetra-
butylammonium iodide (0.55 g, 1.5 mmol) in CH3CN

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2033
(150 mL) was stirred at room temperature for 20 h.
Most of the solvent was removed and then diluted with
EtOAc. The organic layer was washed with 1 M HCl,
saturated NaHCO3, brine, dried over MgSO4 and fil-
tered. The crude product was purified byflash chroma-
tographyon silica gel (hexane–EtOAc=1:3) to afford 28
(0.26 g, 44%) as a white solid: 1H NMR (400 MHz,
CDCl3) 0.79 (3H, d, J=6.7 Hz), 0.82 (3H, d, J=6.6 Hz),
1.46 (9H, s), 1.70–1.85 (1H, m), 1.94–2.08 (1H, m),
2.31–2.46 (1H, m), 2.63 (1H, t, J=11.7 Hz), 2.86–3.00
(1H, m), 3.23 (1H, dd, J=12.6, 6.8 Hz), 3.50 (1H, t,
J=7.6 Hz), 3.60–3.74 (1H, m), 4.11–4.30 (2H, m), 4.42
(1H, dt, J=12.6, 4.8 Hz), 5.15–5.24 (1H, m), 5.40 (1H,
d, J=8.5 Hz), 5.80 (1H, d, J=7.3 Hz), 6.79 (1H, dd,
J=8.5, 2.6 Hz), 6.86 (1H, dd, J=8.2, 2.3 Hz), 7.00 (1H,
dd, J=8.2, 2.1 Hz), 7.17 (1H, d, J=8.5 Hz); 13C NMR
(125 MHz, DMSO-d6) 18.4, 18.9, 26.6, 28.1, 31.8, 35.5,
37.0, 56.9, 57.8, 67.9, 77.8, 117.7, 118.2, 129.5, 130.9,
154.9, 157.4, 169.8, 170.7; HRMS (MALDI), calcd for
MNa+ C22H33N3O5Na m/e 442.2318, found m/e
442.2308.
(CHCl3–EtOAc=1:1) afforded 31 (0.70 g, 66%) as a
1
white solid: H NMR (500 MHz, MeOH-d4) 0.92 (3H,
d, J=6.3 Hz), 0.93 (3H, d, J=6.6 Hz), 1.37 (9H, s),
2.00–2.10 (1H, m), 2.73 (1H, dd, J=14.0, 8.8 Hz), 2.98
(1H, dd, J=13.6, 5.5 Hz), 3.29–3.32 (1H, m), 3.35–3.42
(1H, m), 3.48 (2H, t, J=5.9 Hz), 3.54 (2H, t, J=5.5 Hz),
3.75 (2H, t, J=6.2 Hz), 4.13 (1H, d, J=7.3 Hz), 4.25
(1H, dd, J=8.8, 5.9 Hz), 6.68 (2H, d, J=8.5 Hz), 7.03
(2H, d, J=8.1 Hz); 13C NMR (150 MHz, MeOH-d4)
18.6, 19.7, 28.7, 31.3, 32.2, 38.1, 40.3, 57.7, 60.0, 70.2,
72.1, 80.7, 116.2, 129.2, 131.3, 157.2, 157.7, 173.3, 174.4;
HRMS (MALDI), calcd for MNa+ C23H36BrN3O6Na
m/e 552.1680, found m/e 552.1669.
Macrocycle 32. Preparation was as described for 28 and
the crude product was purified byflash chromatography
on silica gel (hexane–EtOAc=1:3) to afford 32 (75 mg,
1
36%) as a white solid: H NMR (500 MHz, MeOH-d4)
0.80 (3H, d, J=6.6 Hz), 0.82 (3H, d, J=6.6 Hz), 1.44
(9H, s), 2.00–2.13 (1H, m), 2.77 (1H, t, J=12.5 Hz), 2.89
(1H, dd, J=12.9, 4.8 Hz), 2.59–3.06 (1H, m), 3.65–3.81
(2H, m), 4.00–4.08 (1H, m), 418-4.29 (2H, m), 4.30–4.39
(1H, m), 6.86 (1H, d, J=7.4 Hz), 6.90 (2H, d,
J=8.8 Hz), 7.00 (1H, br s), 7.42 (1H, d, J=8.1 Hz); 13C
NMR (150 MHz, MeOH-d4) 17.9, 19.4, 28.7, 32.9, 38.4,
41.1, 58.2, 59.5, 68.4, 71.2, 73.3, 80.5, 117.0, 129.7,
131.0, 157.4, 159.6, 172.7, 174.2; HRMS (MALDI),
calcd for MNa+ C23H35N3O6Na m/e 472.2418, found
m/e 472.2418.
Compound 29. This was prepared from Boc-Tyr-OH
.
(0.50 g, 1.8 mmol) and H-Val-OMe HCl (1.33 g,
2.0 mmol) in THF according to the method A. The
crude product was triturated in EtOAc–Et2O (1:3 v/v)
and the solid filtered to afford 29 (0.67 g, 95%) as a
1
white solid: H NMR (600 MHz, MeOH-d4) 0.91 (3H,
d, J=6.6 Hz), 0.92 (3H, d, J=7.0 Hz), 1.37 (9H, s),
2.05–2.15 (1H, m), 2.73 (1H, dd, J=13.6, 8.8 Hz), 2.80
(3H, s), 2.96 (1H, dd, J=14.0, 5.9 Hz), 3.67 (3H, s), 4.27
(1H, dd, J=8.5, 5.9 Hz), 4.30 (1H, d, J=6.3 Hz), 6.68
(2H, d, J=8.1 Hz), 7.03 (2H, d, J=8.5 Hz); 13C NMR
(150 MHz, MeOH-d4) 18.4, 19.4, 28.7, 32.1, 38.3, 52.5,
57.5, 59.1, 80.6, 116.1, 129.1, 131.4, 157.2, 157.6, 173.2,
174.6; HRMS (MALDI), calcd for MNa+
C20H30N2O6Na m/e 417.1996, found m/e 417.2004.
Dipeptide 35. This was prepared from Ac-Trp-OH
.
(2.0 g, 8.1 mmol) and H-Val-OMe HCl (1.5 g, 9.0 mmol)
in THF according to the method A and purified byflash
chromatographyon silica gel (hexane–EtOAc=1:2 gra-
dient to EtOAc) to afford 35 (2.6 g, 89%) as a white
foam: 1H NMR (400 MHz, MeOH-d4) 0.88 (6H, d,
J=6.8 Hz), 1.91 (3H, s), 2.01–2.10 (1H, m), 3.08 (1H,
dd, J=14.4, 7.6 Hz), 3.22 (1H, dd, J=14.7, 6.8 Hz), 3.61
(3H, s), 4.27 (1H, d, J=6.5 Hz), 4.70 (1H, t, J=7.2 Hz),
6.96–7.10 (3H, m), 7.31 (1H, d, J=7.9 Hz), 7.58 (1H, d,
J=7.9 Hz); 13C NMR (150 MHz, MeOH-d4) 18.5, 19.4,
22.4, 28.9, 32.0, 38.9, 52.4, 55.6, 59.2, 110.8, 112.2,
119.3, 119.7, 122.3, 124.5,128.8, 138.0, 173.1, 173.2,
174.3; HRMS (MALDI), calcd for MNa+
C19H25N3O4Na m/e 382.1737, found m/e 382.1749.
Compound 30. 29 (0.73 g, 1.9 mmol) was deprotected
(method C) to give the corresponding free acid followed
bycoupling with 2-(2-aminoethox)yethanol (0.20 g,
1.9 mmol) in CH3CN-DMF (25 mL, 4:1 v/v) according
to the method A. After stirring at room temperature for
2.5 h and normal aqueous work up, the crude product
was purified byflash chromatographyon silica gel
1
(EtOAc) to afford 30 (0.34 g, 39%) as a white foam: H
NMR (400 MHz, MeOH-d4) 0.92 (6H, d, J=6.8 Hz),
1.37 (9H, s), 1.96–2.09 (1H, m), 2.73 (1H, dd, J=13.5,
8.8 Hz), 2.97 (1H, dd, J=12.6, 5.9 Hz), 3.32–3.39 (1H,
m), 3.48–3.56 (4H, m), 3.62–3.69 (2H, m), 4.13 (1H, d,
J=7.3 Hz), 4.24 (1H, dd, J=8.8, 5.9 Hz), 6.68 (2H, d,
J=8.5 Hz), 7.03 (2H, d, J=8.2 Hz); 13C NMR
(150 MHz, MeOH-d4) 18.5, 19.6, 28.6, 32.2, 38.1, 40.3,
57.7, 60.0, 62.2, 70.4, 73.4, 80.7, 116.2, 129.2, 131.3,
157.2, 173.3, 174.4; HRMS (MALDI), calcd for MNa+
C23H37N3O7Na m/e 490.2524, found m/e 490.2508.
Dipeptide 36. This was prepared from Ac-Phe-OH
.
(1.0 g, 4.9 mmol) and H-Val-OMe HCl (0.81 g,
4.9 mmol) in THF according to the method A and pur-
ified byflash chromatographyon silica gel (hexane–
EtOAc=1:1) to afford 36 (1.3 g, 83%) as a white solid:
1H NMR (400 MHz, MeOH-d4) 0.92 (3H, d, J=6.6 Hz),
0.93 (3H, d, J=7.0 Hz), 1.88 (3H, s), 2.06–2.14 (1H, m),
2.86 (1H, dd, J=13.9, 9.2 Hz), 3.09 (1H, dd, J=14.0,
5.9 Hz), 3.66 (3H, s), 4.30 (1H, d, J=6.3 Hz), 4.68 (1H,
dd, J=8.8, 5.9 Hz), 7.17–7.21 (1H, m), 7.22–7.28 (4H,
m); 13C NMR (100 MHz, MeOH-d4) 18.5, 19.4, 22.3,
31.9, 38.8, 52.5, 55.9, 59.2, 127.7, 129.4, 130.3, 138.4,
173.1, 173.2, 173.9; HRMS (MALDI), calcd for MNa+
C17H24N2O4Na m/e 343.1628, found m/e 343.1629.
Cyclic precursor 31. To a solution of 30 (1.0 g,
2.2 mmol) and triphenylphosphine (0.64 g, 2.4 mmol) in
CH2Cl2 (20 mL) was added carbon tetrabromide (0.80 g,
2.4 mmol). The reaction mixture was stirred at room
temperature for 2 h and the solvent removed in vacuo.
Purification byflash chromatographyon silica gel
Dipeptide 37. This was prepared from Ac-Tyr-OH
.
(2.0 g, 9.0 mmol) and H-Val-OMe HCl (1.5 g, 9.0 mmol)

2034
C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
in THF according to the method A and purified byflash
chromatographyon silica gel (hexane–EtOAc=1:2 gra-
dient to EtOAc) to afford 37 (3.0 g, 98%) as a white
foam: 1H NMR (600 MHz, MeOH-d4) 0.91 (3H, d,
J=6.5 Hz), 0.92 (3H, d, J=6.6 Hz), 1.90 (3H, s), 2.04–
2.11 (1H, m), 2.77 (1H, dd, J=14.0, 8.8 Hz), 2.96 (1H,
dd, J=14.0, 6.6 Hz), 3.66 (3H, s), 4.27 (1H, d,
J=6.1 Hz), 4.58 (1H, dd, J=8.8, 6.6 Hz), 6.67 (2H, d,
J=8.3 Hz), 7.04 (2H, d, J=8.3 Hz); 13C NMR
(125 MHz, MeOH-d4) 18.5, 19.4, 22.3, 32.0, 38.1, 38.9,
52.5, 56.3, 59.2, 116.1, 128.9, 131.3, 157.3, 173.1, 173.2,
174.0; HRMS (MALDI), calcd for MNa+
C17H24N2O5Na m/e 359.1577, found m/e 359.1570.
(1H, dd, J=13.2, 8.6 Hz), 3.06 (1H, dd, J=15.0,
8.8 Hz), 3.23 (1H, dd, J=14.9, 5.7 Hz), 3.64 (3H, s), 4.08
(1H, d, J=2.2 Hz), 4.16 (1H, d, J=7.0 Hz), 4.44–4.49
(1H, m), 4.71 (1H, dd, J=8.8, 5.3 Hz), 6.98–7.02 (1H,
m), 7.04–7.08 (2H, m), 7.13–7.18 (1H, m), 7.23–7.28
(4H, m), 7.29 (1H, d, J=8.3 Hz), 7.61 (1H, d,
J=7.9 Hz); 13C NMR (150 MHz, MeOH-d4) 18.4, 19.7,
22.4, 28.7, 32.1, 38.5, 52.6, 54.9, 55.5, 60.1, 71.3, 111.1,
112.3, 119.4, 119.8, 122.4, 124.4, 127.6, 128.8, 129.5,
130.4, 138.0, 139.2, 172.9, 173.3, 174.0, 174.8; HRMS
(MALDI), calcd for MNa+ C29H36N4O6Na m/e
559.2527, found m/e 559.2529.
Tripeptide 42. This was prepared from 33 and 36
according to the procedure as described for 40. The
crude product was triturated in EtOAc–Et2O (1:5 v/v)
and the solid filtered to afford 42 (0.15 g, 94%) as a
Dipeptides 38 and 39. These were prepared from a race-
mate of Ac-p-F-Phe-OH (1.0 g, 4.4 mmol) and H-Val-
.
OMe HCl (0.75 g, 4.5 mmol) in THF according to the
1
method A. The crude product was triturated in Et2O
and the solid filtered to afford the diastereomeric mix-
ture of 38 and 39 (1.4 g, 95%) as a white solid: 1H NMR
(500 MHz, MeOH-d4) 0.78 (3H, d, J=7.0 Hz), 0.79 (3H,
d, J=7.0 Hz), 0.91 (3H, d, J=6.6 Hz), 0.92 (3H, d,
J=6.6 Hz), 1.89 (3H, s), 1.90 (3H, s), 1.97–2.05 (1H, m),
2.05–2.14 (1H, m), 2.85 (1H, dd, J=14.3, 8.8 Hz), 2.88
(1H, dd, J=13.9, 8.1 Hz), 3.03 (1H, dd, J=13.6,
7.0 Hz), 3.05 (1H, dd, J=14.0, 6.3 Hz), 3.67 (3H, s), 3.69
(3H, s), 4.20 (1H, d, J=5.9 Hz), 4.29 (1H, d, J=6.2 Hz),
4.65 (1H, dd, J=8.5, 6.3 Hz), 4.70 (1H, t, J 7.7 Hz),
6.95–7.01 (4H, m), 7.21–7.27 (4H, m),; 13C NMR
(125 MHz, MeOH-d4) 18.5, 19.4, 22.3, 22.4, 31.7, 32.0,
38.0, 38.5, 52.4, 55.9, 59.2, 59.3, 115.9 (2JFꢂC=21.9 Hz),
116.0 (2JFꢂC=21.9 Hz), 132.1 (3JFꢂC=6.7 Hz), 132.2
(3JFꢂC=6.7 Hz), 134.3, 163.2 (1JFꢂC=242.3 Hz), 163.3
(1JFꢂC=242.3 Hz), 172.9, 173.0, 173.1, 173.2, 173.4,
173.7; HRMS (MALDI), calcd for MNa+
C17H23FN2O4Na m/e 361.1534, found m/e 361.1523.
white solid: H NMR (500 MHz, MeOH-d4) 0.86 (3H,
d, J=6.6 Hz), 0.87 (3H, d, J=6.6 Hz), 1.87 (3H, s),
1.93–2.01 (1H, m), 2.78–2.87 (2H, m), 2.94 (1H, dd,
J=13.6, 8.5 Hz), 3.09 (1H, dd, J=14.3, 5.2 Hz), 3.65
(3H, s), 4.09 (1H, d, J=2.2 Hz), 4.14 (1H, d, J=7.0 Hz),
4.44–4.50 (1H, m), 4.63 (1H, dd, J=9.5, 5.1 Hz), 7.15–
7.20 (2H, m), 7.22–7.29 (8H, m); 13C NMR (125 MHz,
MeOH-d4) 18.5, 19.5, 22.3, 32.1, 38.6, 52.6, 54.9, 55.9,
59.2, 60.1, 71.3, 127.6, 127.7, 129.4, 129.6, 130.2, 130.4,
138.6, 139.2, 172.8, 173.2, 173.6, 174.8; HRMS
(MALDI), calcd for MNa+ C27H35N3O6Na m/e
520.2418, found m/e 520.2433.
Tripeptide 43. This was prepared from 33 and 37
according to the procedure as described for 40. The
crude product was triturated in EtOAc–Et2O (1:2 v/v)
and the solid filtered to afford 43 (0.67 g, 82%) as a
1
white solid: H NMR (400 MHz, MeOH-d4) 0.85 (3H,
d, J=6.8 Hz), 0.86 (3H, d, J=6.8 Hz), 1.89 (3H, s),
1.93–2.01 (1H, m), 2.73 (1H, dd, J=14.1, 9.7 Hz), 2.82
(1H, dd, J=13.5, 7.0 Hz), 2.95 (1H, dd, J=13.5,
9.5 Hz), 2.99 (1H, dd, J=14.4, 5.0 Hz), 3.64 (3H, s), 4.08
(1H, d, J=2.4 Hz), 4.17 (1H, d, J=7.0 Hz), 4.45–4.51
(1H, m), 4.58 (1H, dd, J=9.4, 5.0 Hz), 6.68 (2H, d,
J=8.5 Hz), ), 7.05 (2H, d, J=8.5 Hz), 7.14–7.21 (1H,
m), 7.23–7.32 (4H, m); 13C NMR (100 MHz, MeOH-d4)
18.5, 19.8, 22.3, 32.1, 37.9, 38.6, 52.6, 54.9, 56.3, 60.1,
71.2, 116.2, 127.6, 129.2, 129.6, 130.4, 131.2, 139.2,
157.2, 172.8, 173.2, 173.8, 174.9; HRMS (MALDI),
calcd for MNa+ C27H35N3O7Na m/e 536.2367, found
m/e 536.2341.
Tripeptide 40. 33 (0.47 g, 1.5 mmol) was deprotected
(method B) to give the corresponding TFA salt and 34
(0.50 g, 1.6 mmol) deprotected (method C) to give the
corresponding free acid. The TFA salt and the free acid
were then coupled in THF according to the method A.
The crude product was purified byflash chromato-
graphyon silica gel (hexane–EtOAc=1:1 gradient to 1/
1
2) to afford 40 (0.60 g, 77%) as a white foam: H NMR
(400 MHz, MeOH-d4) 0.84 (6H, d, J=6.8 Hz), 1.30 (3H,
d, J=7.3 Hz), 1.91–2.00 (1H, m), 2.81 (1H, dd, J=13.5,
7.3 Hz), 2.94 (1H, dd, J=13.2, 8.2 Hz), 3.64 (3H, s), 4.09
(1H, d, J=1.8 Hz), 4.11–4.21 (2H, m), 4.45–4.54 (1H,
m), 5.08 (2H, s), 7.15–7.40 (10H, m); 13C NMR
(150 MHz, MeOH-d4) 18.2, 18.3, 19.8, 32.1, 38.5, 52.6,
54.9, 55.8, 60.0, 67.6, 71.3, 127.6, 128.9, 129.0, 129.5,
130.4, 138.2, 139.2, 158.3, 172.9, 174.8, 175.4; HRMS
(MALDI), calcd for MNa+ C27H35N3O7Na m/e
536.2367, found m/e 536.2359.
Tripeptide 44 and 45. These were prepared from 33 and
a diastereomeric mixture of 38 and 39 according to the
procedure as described for 40. The crude product was
purified byflash chromatographyon silica gel (hexane–
EtOAc=1:4) to afford 44 and 45 (0.39 g, 51%) as white
solids. Spectral data for 44: 1H NMR (500 MHz,
MeOH-d4) 0.85 (3H, d, J=7.0 Hz), 0.86 (3H, d,
J=7.0 Hz), 1.88 (3H, s), 1.95–2.05 (1H, m), 2.77–2.86
(2H, m), 2.94 (1H, dd, J=13.2, 8.5 Hz), 3.06 (1H, dd,
J=14.0, 5.2 Hz), 3.65 (3H, s), 4.09 (1H, d, J=2.2 Hz),
4.13 (1H, d, J=7.0 Hz), 4.44–4.51 (1H, m), 4.61 (1H,
dd, J=9.5, 5.1 Hz), 6.93–7.00 (2H, m), 7.14–7.20 (1H,
m), 7.20–7.30 (6H, m); 13C NMR (150 MHz, MeOH-d4)
18.4, 19.8, 22.3, 32.1, 37.8, 38.5, 52.7, 54.9, 56.0, 60.1,
Tripeptide 41. This was prepared from 33 and 35
according to the procedure as described for 40. The
crude product was triturated in EtOAc–Et2O (1:1 v/v)
and the solid filtered to afford 41 (0.54 g, 87%) as a
1
white solid: H NMR (600 MHz, MeOH-d4) 0.81 (3H,
d, J=6.6 Hz), 0.82 (3H, d, J=6.5 Hz), 1.89 (3H, s),
1.92–1.99 (1H, m), 2.78 (1H, dd, J=13.7, 7.5 Hz), 2.92

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2035
71.3, 116.0 (2JFꢂC=21.8 Hz), 127.7, 129.6, 130.4, 132.0
(3JFꢂC=6.9 Hz), 134.5, 139.2, 163.1 (1JFꢂC=269.0 Hz),
172.8, 173.3, 173.4, 174.9; HRMS (MALDI), calcd for
MNa+ C27H34FN3O6Na m/e 538.2324, found m/e
538.2301.
Tripeptide 48. This was prepared from 47 and Ac-Ala-
OH according to the procedure as described for 47. The
crude product was triturated in Et2O and the solid fil-
1
tered to afford 48 (0.15 g, 63%) as a white solid: H
NMR (600 MHz, MeOH-d4) 0.85 (3H, d, J=6.6 Hz),
0.86 (3H, d, J=7.0 Hz), 1.30 (3H, d, J=7.0 Hz), 1.96
(3H, s), 1.95–2.05 (1H, m), 2.81 (1H, dd, J=13.6,
7.5 Hz), 2.94 (1H, dd, J=13.6, 8.3 Hz), 4.09 (1H, d,
J=2.2 Hz), 4.13–4.16 (1H, m), 4.35 (1H, q, J=7.4 Hz),
4.45–4.51 (1H, m), 7.10–7.20 (1H, m), 7.20–7.30 (4H,
m); 13C NMR (150 MHz, MeOH-d4) 17.8, 18.3, 19.8,
22.3, 32.1, 38.5, 50.4, 52.6, 54.9, 60.0, 71.4, 127.6, 129.5,
130.4, 139.2, 173.0, 173.1, 174.8, 175.0; HRMS
(MALDI), calcd for MNa+ C21H31N3O6Na m/e
444.2105, found m/e 444.2123.
1
Spectral data for 45. H NMR (500 MHz, MeOH-d4)
0.50 (3H, d, J=7.0 Hz), 0.57 (3H, d, J=6.6 Hz), 1.91–
1.99 (1H, m), 2.03 (3H, s), 2.81 (1H, dd, J=13.6,
7.4 Hz), 2.90–3.02 (3H, m), 3.63 (3H, s), 3.98 (1H, d,
J=5.9 Hz), 4.08 (1H, d, J=2.6 Hz), 4.50–4.58 (2H, m),
6.95–7.02 (2H, m), 7.15–7.20 (1H, m), 7.20–7.30 (6H,
m); 13C NMR (150 MHz, MeOH-d4) 17.6, 19.5, 22.3,
30.8, 37.6, 38.6, 52.6, 55.0, 57.0, 60.1, 72.1, 116.2
(2JFꢂC=21.8 Hz),
127.6,
129.5,
130.5,
132.2
(3JFꢂC=8.0 Hz), 133.8, 139.2, 163.4 (1JFꢂC=241.6 Hz),
172.9, 173.8, 174.2, 174.3; HRMS (MALDI), calcd for
MNa+ C27H34FN3O6Na m/e 538.2324, found m/e
538.2329.
Cyclic inhibitor 5. 28 (35 mg, 0.084 mmol) was depro-
tected (method B) to give the corresponding HCl salt
and 40 (42 mg, 0.084 mmol) deprotected (method C) to
give the corresponding free acid. The HCl salt and the
free acid were then coupled in DMF (3 mL) according
to the method A. After stirring at room temperature for
19 h, water (2 mL) was added and the solid filtered. The
solid was washed with Et2O to afford 5 (35 mg, 52%) as
a white solid: 1H NMR (400 MHz, DMSO-d6) 0.67 (6H,
d, J=6.7 Hz), 0.70 (6H, d, J=6.8 Hz), 1.16 (3H, d,
J=7.0 Hz), 1.57–1.73 (2H, m), 1.76–1.86 (1H, m), 1.94–
2.06 (1H, m), 2.34 (1H, t, J=10.8 Hz), 2.54–2.69 (2H,
m), 2.77 (1H, dd, J=13.5, 7.4 Hz), 3.10 (1H, dd,
J=12.3, 6.8 Hz), 3.42 (1H, t, J=7.6 Hz), 3.83 (1H, dd,
J=5.8, 2.3 Hz), 4.03–4.18 (3H, m), 4.24–4.37 (2H, m),
4.41–4.49 (1H, m), 5.00 (2H, s), 5.94 (1H, d, J=5.8 Hz),
6.72 (1H, dd, J=8.2, 2.1 Hz), 6.76–7.84 (2H, m), 6.94
(1H, d, J=7.9 Hz), 7.06 (1H, dd, J=8.5, 1.8 Hz), 7.12–
7.40 (10H, m), 7.49 (1H, d, J=7.6 Hz), 7.53 (1H, d,
J=9.1 Hz), 7.68 (1H, d, J=9.1 Hz), 7.76 (1H, d,
J=7.6 Hz); 13C NMR (125 MHz, DMSO-d6) 17.7, 18.1,
18.7, 18.8, 19.1, 26.5, 30.9, 31.8, 35.6, 37.0, 50.1, 52.8,
54.7, 57.2, 57.9, 65.3, 67.7, 70.9, 117.6, 118.0, 126.0,
127.7, 128.1, 128.3, 129.1, 129.3, 130.8, 137.0, 138.6,
155.6, 157.4, 169.2, 169.6, 170.1, 171.0, 172.1; HRMS
(MALDI), calcd for MNa+ C43H56N6O9Na m/e
823.4001, found m/e 823.4027.
Tripeptide 46. To a solution of 40 (0.10 g, 0.20 mmol) in
MeOH (5 mL) was added Pd/C (30 mg) and the mixture
stirred under a balloon of H2 for 1 h. Filtration through
Celite followed byconcentration in vacuo yield the free
amine. To the crude free amine in CH2Cl2 (4 mL) was
added p-toluenesulfonyl chloride (40 mg, 0.21 mmol)
followed byEt 3N (50 mL, 0.36 mmol). The reaction
mixture was stirred at room temperature for 6 h and
then diluted with EtOAc. The organic layer was washed
with 1 M HCl, saturated NaHCO3, brine, dried over
MgSO4 and filtered. Purification byflash chromato-
graphyon silica gel (hexane–EtOAc=1:3) afforded 46
(83 mg, 78%) as a white solid: 1H NMR (600 MHz,
MeOH-d4) 0.74 (3H, d, J=6.5 Hz), 0.77 (3H, d,
J=7.0 Hz), 1.15 (3H, d, J=7.0 Hz), 1.88–1.96 (1H, m),
2.41 (3H, s), 2.81 (1H, dd, J=13.6, 7.5 Hz), 2.93 (1H,
dd, J=13.6, 8.1 Hz), 3.64 (3H, s), 3.81 (1H, q,
J=7.1 Hz), 4.04 (1H, d, J=6.5 Hz), 4.09 (1H, d,
J=2.2 Hz), 4.44–4.49 (1H, m), 7.15–7.20 (1H, m), 7.23–
7.29 (4H, m), 7.36 (2H, d, J=7.9 Hz), 7.76 (2H, d,
J=8.3 Hz); 13C NMR (150 MHz, MeOH-d4) 18.1, 19.3,
19.7, 21.5, 31.9, 38.3, 52.7, 53.5, 54.9, 59.9, 71.4, 127.6,
128.3, 129.5, 130.4, 130.8, 138.6, 139.2, 145.0, 172.7,
174.2, 174.7; HRMS (MALDI), calcd for MNa+
C26H35N3O7SNa m/e 556.2088, found m/e 556.2104.
Cyclic inhibitor 6. This was prepared from 28 and 46
according to the procedure as described for 5. After
stirring at room temperature for 17 h, water (2 mL) was
added and the solid filtered. The solid was washed with
Et2O to afford 6 (35 mg, 59%) as a white solid: 1H
NMR (600 MHz, DMSO-d6) 0.60 (3H, d, J=6.5 Hz),
0.61 (3H, d, J=6.6 Hz), 0.66 (3H, d, J=7.0 Hz), 0.69
(3H, d, J=7.0 Hz), 0.99 (3H, d, J=7.0 Hz), 1.58–1.70
(2H, m), 1.70–1.77 (1H, m), 1.95–2.04 (1H, m), 2.30–
2.37 (1H, m), 2.33 (3H, s), 2.57 (1H, dd, J=13.6,
7.4 Hz), 2.61–2.68 (1H, m), 2.76 (1H, dd, J=13.1,
7.4 Hz), 3.09 (1H, dd, J=12.3, 6.6 Hz), 3.41 (1H, t,
J=7.0 Hz), 3.79–3.85 (2H, m), 4.01 (1H, dd, J=8.8,
6.1 Hz), 4.11 (1H, ddd, J=11.8, 7.9, 5.7 Hz), 4.25–4.34
(2H, m), 4.43 (1H, dt, J=10.1, 7.2 Hz), 5.94 (1H, d,
J=6.1 Hz), 6.73 (1H, dd, J=8.3, 2.2 Hz), 6.79 (1H, dd,
J=8.3, 1.7 Hz), 6.81 (1H, dd, J=8.6, 2.4 Hz), 6.91 (1H,
d, J=7.9 Hz), 7.05 (1H, dd, J=8.3, 1.7 Hz), 7.13–7.26
(5H, m), 7.31 (1H, d, J=8.3 Hz), ), 7.36–7.40 (1H, m),
Dipeptide 47. 33 (0.55 g, 1.8 mmol) was deprotected
(method B) to give the corresponding TFA salt followed
bycoupling with Boc-Val-OH (0.40 g, 1.8 mmol) in
CH3CN (15 mL) according to the method A. After stir-
ring at room temperature for 2 h and normal aqueous
work up, the crude product was purified byflash chro-
matographyon silica gel (hexane–EtOAc=2:1) to
afford 47 (0.65 g, 90%) as a white solid: 1H NMR
(500 MHz, MeOH-d4) 0.82 (3H, d, J=7.0 Hz), 0.84 (3H,
d, J=6.6 Hz), 1.44 (9H, s), 1.85–1.95 (1H, m), 2.82 (1H,
dd, J=13.2, 7.4 Hz), 2.95 (1H, dd, J=13.6, 8.5 Hz), 3.64
(3H, s), 3.79–3.85 (1H, m), 4.09 (1H, d, J=2.2 Hz),
4.44–4.52 (1H, m), 7.10–7.35 (5H, m); 13C NMR
(150 MHz, MeOH-d4) 18.2, 19.8, 28.7, 32.0, 38.5, 52.6,
54.9, 61.5, 71.4, 80.5, 127.6, 129.5, 130.4, 139.3, 157.9,
173.9, 174.7; HRMS (MALDI), calcd for MNa+
C21H32N2O6Na m/e 431.2152, found m/e 431.2160.

2036
C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
7.61 (1H, d, J=10.1 Hz), 7.63 (1H, d, J=9.2 Hz), 7.65
(2H, d, J=7.9 Hz), 7.74 (1H, d, J=7.4 Hz), 7.86 (1H, d,
J=8.8 Hz); 13C NMR (125 MHz, DMSO-d6) 17.6, 18.7,
18.8, 19.0, 19.3, 20.9, 26.5, 30.7, 31.8, 35.6, 37.0, 38.8,
51.5, 52.7, 54.6, 57.2, 57.9, 67.7, 70.8, 117.6, 118.0,
126.0, 126.6, 128.1, 129.1, 129.3, 129.4, 130.8, 138.3,
138.6, 142.4, 157.5, 169.2, 169.6, 169.9, 171.0; HRMS
(MALDI), calcd for MNa+ C42H56N6O9SNa m/e
843.3722, found m/e 843.3753.
171.0, 171.1; HRMS (MALDI), calcd for MNa+
C43H56N6O8Na m/e 807.4052, found m/e 807.4046.
Cyclic inhibitor 9. This was prepared from 28 and 43
according to the procedure as described for 5. After
stirring at room temperature for 14 h and normal aqu-
eous work up, the crude product was triturated in
MeOH–H2O (3:1 v/v) and the solid filtered. The solid
was washed with Et2O to afford 9 (65 mg, 68%) as a
1
white solid: H NMR (500 MHz, DMSO-d6) 0.67 (3H,
Cyclic inhibitor 7. This was prepared from 28 and 41
according to the procedure as described for 5. After
stirring at room temperature for 17 h and normal aqu-
eous work up, the crude product was triturated in
MeOH–H2O (5:1 v/v) and the solid filtered. The solid
was washed with Et2O to afford 7 (0.10 g, 64%) as a
d, J=7.0 Hz), 0.70 (3H, d, J=7.0 Hz), 0.71 (6H, d,
J=7.4 Hz), 1.59–1.71 (2H, m), 1.73 (3H, s), 1.80–1.88
(1H, m), 1.95–2.05 (1H, m), 2.35 (1H, dd, J=12.5,
10.6 Hz), 2.55–2.68 (3H, m), 2.79 (1H, dd, J=13.6,
7.4 Hz), 2.86 (1H, dd, J=14.0, 3.7 Hz), 3.09 (1H, dd,
J=12.5, 6.6 Hz), 3.43 (1H, t, J=7.7 Hz), 3.85 (1H, dd,
J=6.3, 2.8 Hz), 4.07–4.16 (2H, m), 4.25–4.36 (2H, m),
4.40–4.48 (2H, m), 5.92 (1H, d, J=6.3 Hz), 6.61 (2H, d,
J=8.4 Hz), 6.73 (1H, dd, J=8.3, 2.4 Hz), 6.79 (1H, dd,
J=8.5, 2.0 Hz), 6.82 (1H, dd, J=8.4, 2.2 Hz), 6.90 (1H,
d, J=8.1 Hz), 7.03 (2H, d, J=8.5 Hz), 7.04 (1H, dd,
J=8.4, 2.0 Hz), 7.13–7.18 (1H, m), 7.20–7.27 (4H, m),
7.35 (1H, dd, J=5.6, 4.0 Hz), 7.67 (2H, d, J=8.8 Hz),
7.76 (1H, d, J=7.4 Hz), 7.98 (1H, d, J=8.5 Hz); 13C
NMR (100 MHz, DMSO-d6) 17.9, 18.6, 18.9, 19.2, 22.5,
26.5, 30.7, 31.8, 35.6, 36.4, 37.0, 52.8, 54.2, 54.6, 57.4, 57.9,
67.8, 70.9, 114.5, 117.6, 118.0, 126.1, 128.2, 128.3, 129.2,
129.3, 130.1, 130.8, 138.7, 155.7, 157.5, 169.1, 169.2, 169.6,
170.2, 171.0, 171.3; HRMS (MALDI), calcd for MNa+
C43H56N6O9Na m/e 823.4001, found m/e 823.3978.
1
white solid: H NMR (500 MHz, DMSO-d6) 0.67 (3H,
d, J=6.6 Hz), 0.70 (3H, d, J=6.8 Hz), 0.71 (6H, d,
J=7.0 Hz), 1.59–1.71 (2H, m), 1.74 (3H, s), 1.80–1.89
(1H, m), 1.95–2.05 (1H, m), 2.35 (1H, t, J=11.4 Hz),
2.58–2.69 (2H, m), 2.79 (1H, dd, J=14.0, 7.5 Hz), 2.88
(1H, dd, J=14.5, 10.5 Hz), 3.05–3.13 (2H, m), 3.43 (1H,
t, J=7.5 Hz), 3.86 (1H, s), 4.07–4.18 (2H, m), 4.25–4.38
(2H, m), 4.40–4.48 (1H, m), 4.56 (1H, ddd, J=9.5, 8.5,
4.3 Hz), 6.72 (1H, dd, J=8.1, 2.2 Hz), 6.79 (1H, dd,
J=8.5, 2.0 Hz), 6.81 (1H, dd, J=8.4, 2.2 Hz), 6.88 (1H,
d, J=7.7 Hz), 6.96 (1H, t, J=7.2 Hz), 6.99–7.06 (2H,
m), 7.10 (2H, d, J=2.2 Hz), 7.11–7.16 (1H, m), 7.20–
7.27 (3H, m), 7.35 (1H, dd, J=5.5, 4.1 Hz), 7.62 (1H, d,
J=8.1 Hz), 7.67 (1H, d, J=5.1 Hz), 7.69 (1H, d,
J=5.5 Hz), 7.77 (1H, d, J=7.4 Hz), 8.05 (1H, d,
J=8.5 Hz); 13C NMR (100 MHz, DMSO-d6) 17.9, 18.7,
18.9, 19.2, 22.6, 26.5, 27.4, 30.8, 31.9, 35.6, 37.1, 52.9, 53.3,
54.7, 57.5, 57.9, 67.8, 70.9, 110.5, 111.3, 117.6, 118.0,
118.2, 118.6, 120.8, 123.5, 126.1, 127.4, 128.2, 129.2, 129.3,
130.8, 136.1, 138.7, 157.5, 169.2, 169.3, 169.6, 170.2, 171.0,
171.6; HRMS (MALDI), calcd for MNa+
C45H57N7O8Na m/e 846.4161, found m/e 846.4136.
Cyclic inhibitor 10. This was prepared from 28 and 44
according to the procedure as described for 5. After
stirring at room temperature for 12 h, water (3 mL) was
added and the solid filtered. The solid was washed with
1
Et2O to afford 10 (10 mg, 30%) as a white solid: H
NMR (600 MHz, DMSO-d6) 0.67 (3H, d, J=6.5 Hz),
0.68–0.74 (9H, m), 1.58–1.70 (2H, m), 1.72 (3H, s),
1.80–1.88 (1H, m), 1.95–2.05 (1H, m), 2.34 (1H, t,
J=11.4 Hz), 2.56–2.72 (3H, m), 2.79 (1H, dd, J=13.1,
7.0 Hz), 2.95 (1H, dd, J=14.0, 3.3 Hz), 3.08 (1H, dd,
J=12.3, 6.6 Hz), 3.42 (1H, t, J=7.4 Hz), 3.85 (1H, dd,
J=5.6, 2.2 Hz), 4.07–4.16 (2H, m), 4.25–4.37 (2H, m),
4.41–4.48 (1H, m), 4.48–4.54 (1H, m), 5.93 (1H, d,
J=6.1 Hz), 6.69–6.75 (1H, m), 6.76–6.84 (2H, m), 6.90
(1H, d, J=7.9 Hz), 7.00–7.09 (3H, m), 7.12–7.18 (1H,
m), 7.19–7.32 (6H, m), 7.36–7.42 (1H, m), 7.69 (1H, d,
J=9.2 Hz), 7.77 (1H, d, J=7.0 Hz), 8.07 (1H, d,
J=8.3 Hz); 13C NMR (125 MHz, DMSO-d6) 17.9, 18.8,
18.9, 19.2, 22.4, 26.5, 30.7, 31.9, 35.6, 36.4, 37.0, 38.8,
52.9, 53.9, 54.7, 57.6, 57.9, 67.8, 70.9, 114.7
(2JFꢂC=21.0 Hz), 117.6, 118.0, 126.1, 128.2, 129.2,
129.3, 130.8, 131.0 (3JFꢂC=7.6 Hz), 134.4, 138.7, 157.5,
160.9 (1JFꢂC=240.4 Hz), 169.2, 169.3, 169.6, 170.2,
171.1; HRMS (MALDI), calcd for MNa+
C43H55FN6O8Na m/e 825.3957, found m/e 825.3923.
Cyclic inhibitor 8. This was prepared from 28 and 42
according to the procedure as described for 5. After
stirring at room temperature for 14 h and normal aqu-
eous work up, the crude product was triturated in
MeOH–Et2O (1:3 v/v) and the solid filtered. The solid
was washed with Et2O to afford 8 (60 mg, 70%) as a
1
white solid: H NMR (500 MHz, DMSO-d6) 0.67 (3H,
d, J=6.6 Hz), 0.70 (3H, d, J=6.6 Hz), 0.72 (6H, d,
J=7.0 Hz), 1.50–1.70 (2H, m), 1.72 (3H, s), 1.80–1.89
(1H, m), 1.96–2.05 (1H, m), 2.35 (1H, dd, J=11.7,
10.6 Hz), 2.58–2.74 (3H, m), 2.79 (1H, dd, J=13.6,
7.4 Hz), 2.98 (1H, dd, J=14.0, 3.7 Hz), 3.09 (1H, dd,
J=12.5, 7.0 Hz), 3.43 (1H, t, J=7.4 Hz), 3.85 (1H, d,
J=2.6 Hz), 4.07–4.16 (2H, m), 4.25–4.37 (2H, m), 4.41–
4.48 (1H, m), 4.53 (1H, ddd, J=10.1, 8.5, 4.0 Hz), 6.73
(1H, dd, J=8.5, 2.6 Hz), 6.79 (1H, dd, J=8.5, 1.9 Hz),
6.82 (1H, dd, J=8.4, 2.2 Hz), 6.90 (1H, d, J=8.1 Hz),
7.03 (1H, dd, J=8.1, 1.9 Hz), 7.12–7.18 (2H, m), 7.19–
7.28 (7H, m), 7.33–7.38 (1H, m), 7.67–7.79 (3H, m), 8.07
(1H, d, J=8.5 Hz); 13C NMR (125 MHz, DMSO-d6)
17.9, 18.7, 18.8, 19.1, 22.4, 26.5, 30.7, 31.8, 35.5, 36.9,
37.1, 38.6, 52.8, 53.8, 54.7, 57.5, 57.9, 67.7, 70.8, 117.6,
118.0, 126.0, 126.1, 127.9, 127.7, 128.1, 129.1, 129.2,
129.3, 130.7, 138.2, 157.4, 169.1, 169.2, 169.5, 170.1,
Cyclic inhibitor 11. This was prepared from 28 and 45
according to the procedure as described for 5. After
stirring at room temperature for 12 h, water (3 mL) was
added and the solid filtered. The solid was washed with
Et2O to afford 11 (15 mg, 43%) as a pale brown solid:
1H NMR (600 MHz, DMSO-d6) 0.62 (6H, d,

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2037
J=6.6 Hz), 0.66 (3H, d, J=7.0 Hz), 0.69 (3H, d,
J=7.0 Hz), 1.58–1.70 (2H, m), 1.75 (3H, s), 1.78–1.84
(1H, m), 1.95–2.04 (1H, m), 2.34 (1H, t, J=11.0 Hz),
2.57–2.71 (3H, m), 2.77 (1H, dd, J=13.1, 7.4 Hz), 2.90
(1H, dd, J=13.6, 5.7 Hz), 3.09 (1H, dd, J=12.7,
7.0 Hz), 3.83 (1H, dd, J=6.6, 2.6 Hz), 4.07–4.15 (2H,
m), 4.25–4.34 (2H, m), 4.39–4.46 (1H, m), 4.60–4.66
(1H, m), 5.90 (1H, d, J=6.5 Hz), 6.73 (1H, dd, J=7.9,
2.2 Hz), 6.78 (1H, dd, J=8.8, 2.2 Hz), 6.81 (1H, dd,
J=8.3, 2.2 Hz), 6.91 (1H, d, J=7.9 Hz), 7.01–7.07 (3H,
m), 7.12–7.17 (1H, m), 7.19–7.29 (5H, m), 7.35–7.39
(1H, m), 7.67 (1H, d, J=9.2 Hz), 7.76 (1H, d,
J=7.4 Hz), 7.92 (1H, d, J=9.2 Hz), 8.12 (1H, d,
J=8.3 Hz); 13C NMR (100 MHz, DMSO-d6) 17.9, 18.8,
18.9, 19.1, 22.5, 26.5, 30.6, 31.9, 35.7, 35.9, 37.3, 37.5,
53.0, 53.9, 54.8, 57.5, 56.0, 67.8, 70.9, 114.7
(2JFꢂC=19.7 Hz), 117.7, 118.1, 126.2, 128.3, 129.2,
129.3, 129.4, 130.8, 131.1 (3JFꢂC=7.6 Hz), 134.0, 138.7,
157.5, 161.0 (1JFꢂC=239.6 Hz), 169.2, 169.3, 169.7,
170.2, 171.0, 171.1; HRMS (MALDI), calcd for MH+
C43H56FN6O8 m/e 803.4138, found m/e 803.4151.
J=7.9 Hz), 7.67 (2H, d, J=8.3 Hz), 7.89 (1H, d,
J=7.4 Hz), 7.94 (1H, s), 8.04 (1H, d, J=8.3 Hz); 13C
NMR (125 MHz, DMSO-d6) 17.7, 18.2, 18.9, 19.1, 22.5,
27.4, 30.7, 31.3, 35.8, 37.1, 37.7, 52.7, 53.3, 53.6, 57.4,
57.5, 66.6, 69.5, 70.8, 71.4, 110.4, 111.2, 115.2, 118.1,
118.5, 120.8, 123.4, 126.0, 127.3, 128.0, 128.1, 129.2,
129.6, 136.0, 138.6, 157.3, 169.1, 169.6, 169.7, 170.1,
170.9, 171.5; HRMS (MALDI), calcd for MNa+
C46H59N7O9Na m/e 876.4266, found m/e 876.4240.
Cyclic inhibitor 16. This was prepared from 28 and 44
according to the procedure as described for 5. After stir-
ring at room temperature for 2 h, water (2 mL) was added
and the solid filtered. The solid was washed with Et2O to
afford 16 (25mg, 45%) as a white solid: 1H NMR
(600 MHz, DMSO-d6, 60ꢁC) 0.70–0.76 (12H, m), 1.75
(3H, s), 1.80–2.00 (2H, m), 2.60–2.70 (2H, m), 2.79–2.96
(4H, m), 3.00 (1H, dd, J=14.0, 4.4 Hz), 3.30–3.43 (3H,
m), 3.60–3.70 (2H, m), 3.87–3.97 (2H, m), 4.11 (1H, dd,
J=8.3, 6.6 Hz), 4.20–4.30 (2H, m), 4.33–4.41 (1H, m),
4.47–4.57 (2H, m), 5.76 (1H, d, J=6.1 Hz), 6.81–6.89 (3H,
m), 6.92–7.05 (4H, m), 7.11–7.17 (1H, m), 7.20–7.30 (5H,
m), 7.40 (1H, d, J=8.3 Hz), 7.46 (1H, d, J=9.2 Hz),
7.57 (1H, d, J=8.3 Hz), 7.79 (1H, d, J=7.5 Hz), 7.90
(1H, d, J=8.3 Hz); 13C NMR (150 MHz, DMSO-d6,
60 ꢁC) 17.5, 17.7, 18.6, 18.8, 22.1, 30.2, 30.9, 36.1, 36.5,
37.6, 52.6, 53.6, 53.9, 57.4, 57.6, 66.7, 69.3, 70.9, 71.1, 114.3
(2JFꢂC=19.7 Hz), 115.3, 115.4, 125.7, 127.8, 128.0, 128.1,
128.8, 129.3, 130.6 (3JFꢂC=7.6 Hz), 134.0, 138.4, 157.2,
161.5, 168.9, 169.5, 169.6, 169.7, 170.7, 170.8; HRMS
(MALDI), calcd for MNa+ C44H57FN6O9Na m/e
855.4063, found m/e 855.4039.
Cyclic inhibitor 12. This was prepared from 28 and 48
according to the procedure as described for 5. After
stirring at room temperature for 2 h, water (2 mL) was
added and the solid filtered. The solid was washed with
1
Et2O to afford 12 (30 mg, 47%) as a white solid: H
NMR (600 MHz, DMSO-d6) 0.67 (3H, d, J=6.6 Hz),
0.68–0.72 (9H, m), 1.14 (3H, d, J=7.0 Hz), 1.60–1.71
(2H, m), 1.81 (3H, s), 1.80–1.87 (1H, m), 1.95–2.04 (1H,
m), 2.36 (1H, dd, J=12.2, 10.6 Hz), 2.59 (1H, dd,
J=13.1, 7.4 Hz), 2.62–2.68 (1H, m), 2.74–2.81 (1H, m),
3.09 (1H, dd, J=12.3, 6.6 Hz), 3.42 (1H, t, J=7.4 Hz),
3.84 (1H, dd, J=6.2, 2.6 Hz), 4.06–4.16 (2H, m), 4.24–
4.36 (3H, m), 4.40–4.48 (1H, m), 6.73 (1H, dd, J=8.3,
2.2 Hz), 6.79 (1H, dd, J=8.3, 1.7 Hz), 6.82 (1H, dd,
J=8.8, 2.6 Hz), 6.89 (1H, d, J=8.3 Hz), 7.06 (1H, dd,
J=8.3, 1.8 Hz), 7.13–7.18 (1H, m), 7.18–7.26 (4H, m),
7.32–7.38 (1H, m) 7.55 (1H, d, J=9.2 Hz), 7.60 (1H, d,
J=9.2 Hz), 7.75 (1H, d, J=7.5 Hz), 8.02 (1H, d,
J=7.9 Hz); 13C NMR (150 MHz, DMSO-d6) 17.7, 18.0,
18.7, 18.8, 19.1, 22.4, 26.5, 30.6, 31.8, 35.6, 36.9, 38.7, 48.0,
52.8, 54.7, 57.3, 57.9, 67.7, 70.9, 117.6, 118.0, 126.0, 128.1,
129.2, 129.3, 130.8, 138.6, 157.4, 169.0, 169.3, 169.6, 170.1,
171.0, 172.1; HRMS (MALDI), calcd for MNa+
C37H52N6O8Na m/e 731.3739, found m/e 731.3709.
Compound 49. 28 (0.11 g, 0.26 mmol) was deprotected
(method B) to give the corresponding TFA salt and 33
(81 mg, 0.26 mmol) deprotected (method C) to give the
corresponding free acid. The TFA salt and the free acid
were then coupled in THF (6 mL) according to the
method A. After stirring at room temperature for 2 h
and normal aqueous work up, the crude product was
triturated in hexane–EtOAc (1:2 v/v) and the solid fil-
tered. The solid was washed with Et2O to afford 49
(0.10 g, 64%) as a white solid: 1H NMR (500 MHz,
MeOH-d4) 0.76 (3H, d, J=7.0 Hz), 0.77 (3H, d,
J=7.0 Hz), 1.31 (9H, s), 1.71–1.82 (2H, m), 2.13–2.22
(1H, m), 2.61 (1H, t, J=11.8 Hz), 2.75 (1H, dd, J=13.2,
8.5 Hz), 2.81 (1H, ddd, J=14.0, 7.3, 3.0 Hz), 2.89 (1H,
dd, J=13.6, 6.6 Hz), 3.16 (1H, dd, J=12.5, 6.6 Hz), 3.45
(1H, d, J=7.4 Hz), 3.49 (1H, ddd, J=13.9, 9.2, 2.9 Hz),
4.03 (1H, d, J=2.2 Hz), 4.11–4.16 (1H, m), 4.20 (1H,
ddd, J=12.5, 8.8, 5.2 Hz), 4.35 (1H, dt, J=12.5,
5.2 Hz), 4.50 (1H, dd, J=11.0, 6.6 Hz), 6.80–6.87 (2H,
m), 6.94 (1H, dd, J=8.1, 2.2 Hz), 7.13 (1H, dd, J=8.5,
1.8 Hz), 7.15–7.29 (5H, m); 13C NMR (100 MHz,
MeOH-d4) 19.3, 19.4, 27.9, 28.7, 33.5, 37.3, 38.9, 39.3,
56.2, 56.9, 60.3, 68.9, 72.9, 80.1, 118.8, 119.0, 127.3,
129.4, 130.3, 130.5, 130.6, 132.4, 139.8, 157.6, 159.6,
172.0, 172.3, 174.6; HRMS (MALDI), calcd for MNa+
C32H44N4O7Na m/e 619.3102, found m/e 619.3085.
Cyclic inhibitor 15. This was prepared from 28 and 41
according to the procedure as described for 5. After
stirring at room temperature for 2 h, water (2 mL) was
added and the solid filtered. The solid was washed with
1
Et2O to afford 15 (35 mg, 61%) as a white solid: H
NMR (600 MHz, DMSO-d6) 0.69–0.74 (12H, m), 1.74
(3H, s), 1.80–2.00 (2H, m), 2.60–2.68 (2H, m), 2.77–2.85
(2H, m), 2.88–2.94 (2H, m), 3.08 (1H, dd, J=14.9,
4.0 Hz), 3.40–3.47 (1H, m), 3.60–3.67 (2H, m), 3.88 (1H,
dd, J=6.1, 2.2 Hz), 3.92 (1H, dd, J=8.8, 6.6 Hz), 4.17
(1H, dd, J=8.8, 6.5 Hz), 4.18–4.30 (2H, m), 4.34–4.41
(1H, m), 4.46–4.52 (1H, m), 4.53–4.59 (1H, m), 5.95
(1H, d, J=6.4 Hz), 6.83 (2H, d, J=8.3 Hz), 6.91 (2H, d,
J=8.3 Hz), 6.96 (1H, t, J=7.4 Hz), 7.04 (1H, t,
J=7.4 Hz), 7.10 (1H, d, J=2.0 Hz), 7.12–7.17 (1H, m),
7.18–7.26 (4H, m), 7.30 (1H, d, J=7.9 Hz), 7.61 (1H, d,
Compound 50. 49 (60 mg, 0.10 mmol) was deprotected
(method B) to give the corresponding HCl salt followed
bycoupling with Boc-Val-OH (25 mg, 0.12 mmol) in

2038
C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
DMF (3 mL) according to the method A. After stirring
at room temperature for 18 h and normal aqueous work
up, the crude product was triturated in EtOAc–Et2O
(1:2 v/v) and the solid filtered. The solid was washed
with Et2O to afford 50 (40 mg, 58%) as a white solid: 1H
NMR (500 MHz, DMSO-d6) 0.66 (6H, d, J=6.6 Hz),
0.69 (6H, d, J=6.6 Hz), 1.37 (9H, s), 1.59–1.72 (2H, m),
1.74–1.84 (1H, m), 1.94–2.05 (1H, m), 2.36 (1H, t,
J=11.4 Hz), 2.57 (1H, dd, J=13.4, 7.9 Hz), 2.62–2.70
(1H, m), 2.79 (1H, dd, J=13.6, 7.2, 3.0 Hz), 3.09 (1H,
dd, J=12.5, 7.0 Hz), 3.42 (1H, d, J=7.4 Hz), 3.74 (1H,
d, J=8.1 Hz), 3.85 (1H, dd, J=5.5, 2.6 Hz), 4.06–4.15
(1H, m), 4.24–4.35 (2H, m), 4.43–4.49 (1H, m), 5.98
(1H, d, J=5.9 Hz), 6.42 (1H, d, J=8.5 Hz), 6.72 (1H,
dd, J=8.5, 2.2 Hz), 6.77–6.84 (2H, m), 6.88 (1H, d,
J=8.1 Hz), 7.06 (1H, dd, J=8.5, 1.9 Hz), 7.13–7.29 (5H,
m), 7.31–7.37 (1H, m), 7.54 (1H, d, J=9.2 Hz), 7.72
(1H, d, J=7.7Hz); 13C NMR (125MHz, DMSO-d6) 17.8,
18.7, 18.8, 19.2, 26.5, 28.2, 30.4, 31.8, 35.6, 36.8, 38.7, 52.9,
54.7, 57.9, 59.5, 67.8, 71.0, 77.9, 117.6, 118.1, 126.0, 128.1,
129.2, 129.3, 130.8, 138.7, 155.4, 157.5, 169.3, 169.6, 170.7,
171.0; HRMS (MALDI), calcd for MNa+
C37H53N5O8Na m/e 718.3786, found m/e 718.3804.
6.72 (1H, d, J=7.4 Hz), 6.76–6.86 (2H, m), 7.04 (1H, d,
J=7.9 Hz), 7.12–7.37 (9H, m), 7.38–7.45 (1H, m), 7.73–
7.82 (1H, m), 8.15–8.25 (1H, m), 8.91 (1H, s); 13C NMR
(125MHz, DMSO-d6) 17.6, 18.7, 18.8, 19.1, 22.4, 26.5,
27.2, 30.6, 31.8, 35.6, 37.1, 38.8, 51.4, 52.9, 54.7, 57.6, 57.9,
67.7, 70.7, 117.6, 118.0, 126.1, 127.0, 127.5, 127.9, 128.2,
129.1, 129.2, 129.6, 130.7, 138.6, 157.5, 169.2, 169.4, 169.5,
169.9, 170.4, 171.0; HRMS (MALDI), calcd for MNa+
C40H54N8O8Na m/e 797.3957, found m/e 797.3978.
Dipeptide 51. This was prepared from Boc-p-F-Phe-OH
.
(0.50 g, 1.8 mmol) and H-Val-OMe HCl (0.30 g,
1.8 mmol) in CH3CN according to the method A. The
crude product was triturated in Et2O–hexane (1:2 v/v)
and the solid filtered. The filtrate was concentrated to
afford 51 (0.68 g, 97%) as a white solid: 1H NMR
(400 MHz, MeOH-d4) 0.92 (3H, d, J=6.8 Hz), 0.93 (3H,
d, J=6.7 Hz), 1.37 (3H, s), 2.05–2.20 (1H, m), 2.81 (1H,
dd, J=13.9, 9.0 Hz), 3.04 (1H, dd, J=13.8, 5.9 Hz), 3.68
(3H, s), 4.31 (1H, d, J=5.9 Hz), 4.33 (1H, dd, J=9.1,
5.8 Hz), 6.93–7.04 (2H, m), 7.18–7.30 (2H, m); 13C
NMR (150 MHz, MeOH-d4) 18.4, 19.4, 28.6, 32.0, 38.3,
52.5, 57.2, 59.1, 80.6, 115.9 (2JFꢂC=21.8 Hz), 132.1
(3JFꢂC=6.9 Hz), 134.5, 157.6, 163.2 (1JFꢂC=241.6 Hz),
173.2, 174.3; HRMS (MALDI), calcd for MNa+
C20H29FN2O5Na m/e 419.1953, found m/e 419.1939.
Cyclic inhibitor 13. This was prepared from 50 and Ac-
His(1-Trt)–OH according to the procedure as described
for 50. After stirring at room temperature for 4 h, water
(5 mL) was added and the solid filtered. The solid was
washed with Et2O to afford 13 (43 mg, 73%) as a white
solid: 1H NMR (500 MHz, DMSO-d6) 0.66 (6H, d,
J=7.0 Hz), 0.69 (6H, d, J=6.3 Hz), 1.59–1.69 (1H, m),
1.72 (3H, s), 1.78–1.88 (1H, m), 1.92–2.04 (1H, m), 2.37
(1H, t, J=11.4 Hz), 2.55 (1H, dd, J=13.2, 7.7 Hz),
2.59–2.69 (2H, m), 2.78 (1H, dd, J=14.0, 7.7 Hz), 2.82–
2.87 (1H, m), 3.08 (1H, dd, J=13.2, 7.7 Hz), 3.43 (1H, t,
J=7.3 Hz), 3.77–3.87 (1H, m), 4.02 (1H, s), 4.05–4.15
(2H, m), 4.22–4.35 (2H, m), 4.41–4.52 (2H, m), 5.92
(1H, d, J=5.9 Hz), 6.62–6.70 (2H, m), 6.76–6.85 (2H,
m), 6.95 (1H, d, J=8.1 Hz), 6.99–7.10 (7H, m), 7.10–
7.27 (6H, m), 7.27–7.42 (10H, m), 7.56 (1H, d,
J=8.1 Hz), 7.69–7.79 (2H, m), 8.00 (1H, d, J=8.4 Hz);
13C NMR (125 MHz, DMSO-d6) 17.5, 18.7, 19.1, 22.4,
26.5, 30.5, 30.7, 31.7, 35.5, 35.8, 36.6, 52.5, 52.8, 54.7,
57.5, 57.9, 67.7, 70.9, 74.4, 117.6, 118.0, 118.9, 126.0,
127.9, 128.1, 129.1, 129.2, 129.3, 130.8, 137.3, 137.5,
138.6, 142.3, 157.4, 162.3, 169.0, 169.2, 169.6, 170.1,
171.1, 171.2; HRMS (MALDI), calcd for MNa+
C59H68N8O8Na m/e 1039.5052, found m/e 1039.5013.
Dipeptide 52. 51 (0.70 g, 1.8 mmol) was deprotected to
the corresponding TFA salt (method B) followed by
coupling with acetic acid (0.20 mL, 3.5 mmol) (method
A). The crude product was triturated in Et2O–MeOH
(10:1 v/v) and the solid filtered to afford 52 (0.38 g,
1
64%) as a white solid: H NMR (500 MHz, MeOH-d4)
0.91 (3H, d, J=6.6 Hz), 0.92 (3H, d, J=7.0 Hz), 1.89
(3H, s), 2.05–2.15 (1H, m), 2.85 (1H, dd, J=14.0,
8.8 Hz), 3.06 (1H, dd, J=13.6, 6.3 Hz), 3.67 (3H, s), 4.28
(1H, d, J=6.3 Hz), 4.64 (1H, dd, J=8.8, 6.3 Hz), 6.95–
7.01 (2H, m), 7.21–7.27 (2H, m); 13C NMR (150 MHz,
MeOH-d4) 18.5, 19.4, 22.3, 32.0, 38.3, 52.5, 55.9, 59.2,
115.9 (2JFꢂC=21.8 Hz), 132.1 (3JFꢂC=8.0 Hz), 134.3,
163.3 (1JFꢂC=241.5 Hz), 173.1, 173.2, 173.7; HRMS
(MALDI), calcd for MNa+ C17H23FN2O4Na m/e
361.1534, found m/e 361.1538.
Tripeptide 53. 33 (0.27 g, 0.87 mmol) was deprotected
(method B) to give the corresponding TFA salt and 52
(0.30 g, 0.88 mmol) deprotected (method C) to give the
corresponding free acid. The TFA salt and the free acid
were then coupled in THF (8 mL) according to the
method A. The crude product was purified byflash
chromatographyon silica gel (CHCl –EtOAc=1:3 gra-
3
dient to EtOAc) to afford 53 (0.20 g, 45%) as a white
solid. Spectral data of 53 are the same as those of 44.
Cyclic inhibitor 14. 13 (32 mg, 0.032 mmol) was dis-
solved in 10% H2O in TFA (1 mL) and stirred at room
temperature for 1 h. The solvent was removed in vacuo
and the remaining TFA and water were removed by
repeated evaporation from toluene in vacuo. The crude
product was triturated in E2O and the solid filtered. The
solid was washed with Et2O to afford 14 (24 mg, 97%)
Dipeptide 54. This was prepared from Boc-Tyr-OH
.
(0.50 g, 1.8 mmol) and H-Val-NH2 HCl (0.28 g,
1
as a white solid: H NMR (600 MHz, DMSO-d6) 0.60–
1.8 mmol) in THF according to the method A. The
crude product was triturated in Et2O and the solid fil-
0.75 (12H, m), 1.57–1.72 (2H, m), 1.81 (3H, s), 1.77–
1.88 (1H, m), 1.96–2.06 (1H, m), 2.33 (1H, t,
J=10.7 Hz), 2.57–2.69 (2H, m), 2.74–2.82 (1H, m),
2.85–2.93 (1H, m), 2.96–3.05 (1H, m), 3.06–3.13 (1H,
m), 3.85 (1H, s), 4.03–4.21 (2H, m), 4.24–4.38 (2H, m),
4.40–4.48 (1H, m), 4.59–4.67 (1H, m), 6.02 (1H, br s),
1
tered to afford 54 (0.60 g, 90%) as a white solid: H
NMR (400 MHz, MeOH-d4) 0.93 (3H, d, J=7.0 Hz),
0.95 (3H, d, J=7.0 Hz), 1.37 (9H, s), 2.02–2.12 (1H, m),
2.73 (1H, dd, J=13.8, 9.1 Hz), 3.00 (1H, dd, J=13.8,
5.3 Hz), 4.20 (1H, d, J=6.5 Hz), 4.25 (1H, dd, J=8.8,

C. C. Mak et al. / Bioorg. Med. Chem. 11 (2003) 2025–2040
2039
5.3 Hz), 6.68 (2H, d, J=8.2 Hz), 7.04 (2H, d, J=8.5 Hz);
References and Notes
13C NMR (150 MHz, MeOH-d4) 18.2, 19.7, 28.7, 32.0,
38.0, 57.7, 59.5, 80.7, 116.2, 129.2, 131.3, 157.2, 157.7,
174.5, 175.8; HRMS (MALDI), calcd for MNa+
C19H29N3O5Na m/e 402.1999, found m/e 402.2013.
1. (a) Erickson, J. W.; Neidhart, D. J.; VanDrie, J.; Kempf,
D. J.; Wang, X. C.; Norbeck, D. W.; Plattner, J. J.; Ritten-
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Betebenner, D. A.; Erickson, J.; Norbeck, D. W. J. Med.
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Pentapeptide 55. 29 (80 mg, 0.20 mmol) was deprotected
(method B) to give the corresponding HCl salt and 43
(92 mg, 0.18 mmol) deprotected (method C) to give the
corresponding free acid. The HCl salt and the free acid
were then coupled in DMF (3 mL) according to the
method A. The crude product was triturated in MeOH–
Et2O (1:5 v/v) and the solid filtered. The solid was
washed with Et2O to afford 55 (50 mg, 32%) as a white
solid: 1H NMR (600 MHz, DMSO-d6) 0.65 (3H, d,
J=7.0 Hz), 0.66 (3H, d, J=6.5 Hz), 0.85 (3H, d,
J=6.6 Hz), 0.88 (3H, d, J=6.5 Hz), 1.72 (3H, s), 1.77–
1.85 (1H, m), 1.97–2.04 (1H, m), 2.52–2.59 (2H, m),
2.72–2.79 (2H, m), 2.83 (1H, dd, J=14.5, 3.9 Hz), 2.87
(1H, dd, J=14.0, 6.2 Hz), 3.62 (3H, s), 3.75 (1H, dd,
J=6.6, 2.6 Hz), 4.08–4.14 (2H, m), 4.28 (1H, ddd,
J=16.6, 7.4, 2.2 Hz), 4.41 (1H, ddd, J=12.3, 8.3,
4.0 Hz), ), 4.52–4.57 (1H, m), 6.16 (1H, d, J=6.2 Hz),
6.54 (2H, d, J=8.8 Hz), 6.61 (2H, d, J=8.3 Hz), 6.84
(2H, d, J=8.3 Hz), 7.01 (2H, d, J=8.8 Hz), 7.12–7.27
(5H, m), 7.57 (1H, d, J=9.2 Hz), 7.63 (1H, d,
J=8.3 Hz), 7.65 (1H, d, J=8.8 Hz), 7.99 (1H, d,
J=8.3 Hz), 8.39 (1H, d, J=7.9 Hz); 13C NMR
(100 MHz, DMSO-d6) 17.7, 18.4, 19.0, 19.1, 22.5, 30.0,
30.8, 36.4, 36.9, 37.3, 51.8, 52.7, 52.9, 54.2, 57.4, 57.6,
70.7, 114.8, 126.1, 126.8, 128.3, 129.2, 130.1, 130.4,
138.7, 155.7, 155.8, 169.2, 170.1, 170.7, 171.0, 171.4,
171.9; HRMS (MALDI), calcd for MNa+
C41H53N5O10Na m/e 798.3685, found m/e 798.3706.
Pentapeptide 56. This was prepared from 54 and 43
according to the procedure as described for 55. The
crude product was triturated in MeOH–Et2O (1:5 v/v)
and the solid filtered. The solid was washed with
1
Et2O to afford 56 (20 mg, 33%) as a white solid: H
NMR (600 MHz, DMSO-d6) 0.65 (6H, d, J=6.5 Hz),
0.82 (3H, d, J=8.3 Hz), 0.84 (3H, d, J=7.0 Hz), 1.72
(3H, s), 1.77–1.85 (1H, m), 1.90–1.97 (1H, m), 2.53–2.62
(2H, m), 2.74–2.92 (5H, m), 3.78 (1H, d, J=2.2 Hz),
4.04–4.13 (1H, m), 4.26–4.33 (1H, m), 4.36–4.44 (1H,
m), 4.46–4.53 (1H, m), 6.55 (2H, d, J=7.4 Hz), 6.62
(2H, d, J=7.9 Hz), 6.82 (2H, d, J=8.3 Hz), 7.01 (2H, d,
J=8.3 Hz), 7.08 (1H, s), 7.11–7.17 (1H, m), 7.19–7.27
(4H, m), 7.46 (1H, s), 7.60 (1H, d, J=9.2 Hz), 7.64–7.71
(2H, m), 8.07 (1H, d, J=8.3 Hz), 8.14 (1H, d,
J=8.8 Hz); 13C NMR (125 MHz, DMSO-d6) 17.7, 18.3,
19.1, 19.4, 22.5, 30.5, 30.7, 36.4, 36.8, 36.9, 52.9, 53.0,
54.3, 57.5, 57.8, 70.7, 114.8, 126.0, 126.8, 128.2, 129.2,
130.0, 130.4, 138.8, 155.8, 169.2, 170.0, 170.1, 170.7,
171.1, 171.4, 172.9; HRMS (MALDI), calcd for
MNa+ C40H52N6O9Na m/e 783.3688, found m/e
783.3683.
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This work was supported bythe NIH. A.B. thanks the
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