Synthesis of sansalvamide A peptidomimetics: Triazole, oxazole, thiazole, and pseudoproline containing compounds

Article abstract of DOI:10.1016/j.tet.2011.11.089

Peptidomimetic-based macrocycles typically have improved pharmacokinetic properties over those observed with peptide analogs. Described are the syntheses of 13 peptidomimetic derivatives that are based on active sansalvamide A structures, where these analogs incorporate heterocycles (triazoles, oxazoles, thiazoles, or pseudoprolines) along the macrocyclic backbone. The syntheses of these derivatives employ several approaches that can be applied to convert a macrocyclic peptide into its peptidomimetic counterpart. These approaches include peptide modifications to generate the alkyne and azide for click chemistry, a serine conversion into an oxazole, a Hantzsch reaction to generate the thiazole, and protected threonine to generate the pseudoproline derivatives. Furthermore, we show that two different peptidomimetic moieties, triazoles, and thiazoles, can be incorporated into the macrocyclic backbone without reducing cytotoxicity: triazole and thiazole.

Full text of DOI:10.1016/j.tet.2011.11.089

Tetrahedron 68 (2012) 1029e1051
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of sansalvamide A peptidomimetics: triazole, oxazole, thiazole,
and pseudoproline containing compounds
Melinda R. Davis b, Erinprit K. Singh b, Hendra Wahyudi a, Leslie D. Alexander b, Joseph B. Kunicki b,
,
Lidia A. Nazarova b, Kelly A. Fairweather c, Andrew M. Giltrap c, Katrina A. Jolliffe c, Shelli R. McAlpine a
*
a School of Chemistry, 219 Dalton, Gate 2 High Street, University of New South Wales, Kensington, NSW 2052, Australia
b Department of Chemistry and Biochemistry, 5500 Campanile Drive, San Diego State University, San Diego, CA 92182-1030, USA
c School of Chemistry, Building F11, The University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptidomimetic-based macrocycles typically have improved pharmacokinetic properties over those
observed with peptide analogs. Described are the syntheses of 13 peptidomimetic derivatives that are
based on active sansalvamide A structures, where these analogs incorporate heterocycles (triazoles,
oxazoles, thiazoles, or pseudoprolines) along the macrocyclic backbone. The syntheses of these de-
rivatives employ several approaches that can be applied to convert a macrocyclic peptide into its pep-
tidomimetic counterpart. These approaches include peptide modifications to generate the alkyne and
azide for click chemistry, a serine conversion into an oxazole, a Hantzsch reaction to generate the thi-
azole, and protected threonine to generate the pseudoproline derivatives. Furthermore, we show that
two different peptidomimetic moieties, triazoles, and thiazoles, can be incorporated into the macrocyclic
backbone without reducing cytotoxicity: triazole and thiazole.
Received 3 September 2011
Received in revised form 11 November 2011
Accepted 28 November 2011
Available online 6 December 2011
Keywords:
Peptide
Macrocycle
Peptidomimetic
Sansalvamide A
Triazole
Ó 2011 Elsevier Ltd. All rights reserved.
Oxazole
Thiazole
Pseudoproline
1. Introduction
III
O
O
O
O
O
IV
O
N
H
N
H
Recent work describing the synthesis and biological activity of
pentapeptide derivatives that are based on the natural product
sansalvamide A (San A), has brought attention to this compound
class.1e4 San A was isolated from a marine fungus of the genus
Fusarium by Fenical and co-workers.3 The pentapeptide structure
(San A-amide, compound A, Fig. 1) has been used extensively as
a template for the synthesis of compounds, where a number of
these molecules exhibit cytotoxicity.1,2,5,6 We have discovered that
in addition to San A-amide, three derivatives (compounds B, C, and
D, Fig. 1) are cytotoxic against numerous cancer cell lines, and these
compounds inhibit a key protein that enables many proteins in-
volved in tumor progression: Heat shock protein 90 (Hsp90).1,7
Hsp90 is a well-established chemotherapeutic target that
modulates client proteins involved in cellular growth, angiogen-
esis, and apoptosis.8e13 The redundancy of pathways involved in
cancer cell growth means targeting multiple mechanisms simul-
taneously improves it’s chances as a successful therapy. Hsp90
HN
HN
HN
NH
NH
O
NH HN
O
II
N
O
O
V
I
Compound A
Compound B
Sansalvamide A-amide
O
O
N
O
N
O
O
H
N
HN
NH
NH
O
O
NH HN
NH HN
O
O
O
O
(Cbz)HN
Compound C
Compound D
* Corresponding author. Tel.: þ61 4 1672 8896; fax: þ61 4 9385 6141; e-mail
address: s.mcalpine@UNSW.edu.au (S.R. McAlpine).
Fig. 1. San A compounds.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.11.089

1030
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
controls approximately 200 client proteins and co-chaperones,
many of which are involved in multiple cancer-related cell sig-
naling pathways.14e16 There are currently fifteen Hsp90 inhibitors
in development, with two of these in phase III clinical trials.17e22
We have previously reported that San A-amide (A, Fig. 1) is a cy-
totoxic molecule that modulates the activity of Hsp90. This
modulation of Hsp90 acts via an allosteric effect, where San A-
amide binds to the N-middle region and inhibits C-terminal client
proteins.7 This mechanism of action is unique to Hsp90 inhibitors,
making San A and its derivatives valuable molecular tools and
potential lead structures for future chemotherapeutic studies.
In order to prepare these compounds to move into the next
stage of development (mice models) the pharmacokinetic (PK)
properties of these molecules should be improved as they are
relatively poor.1 One mechanism for improving PK is to in-
troduce peptidomimetic features, or structural motifs that
mimic the peptide backbone, where these mimics often improve
the solubility and stability of the molecule without impacting
the cytotoxicity.23,24 Introduction of these motifs into the mac-
rocyclic backbone has been shown to rigidify the macrocycles, as
well as improve the absorption, distribution, metabolism, and
excretion (ADME) properties.25e28 Some common heterocycles
that are known to improve stability of the peptide backbone
include: triazoles, oxazoles, thiazoles, and pseudoprolines.29e32
The inclusion of a triazole, particularly in cyclic peptide back-
bone, has demonstrated an improvement in biological activity.26,31
Further, triazoles induce a rigid conformation by mimicking trans
two fragments, a tri- and dipeptide; conversion of the amine
moiety of the tripeptide to an azide yielded fragment 1, and for-
mation of an alkyne on the dipeptide yielded fragment 2. Both
fragments were coupled via a peptide bond to form the linear
molecule between residues I and V, and the macrocycle was then
clicked shut to generate
a
single 1,4-disubstituted triazole
analog.39
ring closing
III
N3
O
N
N
N
O
HN
IV
NH
HN
NH
NHR
O
HN
HO
II
O
O
HN
NR
O
O
O
V
Fragment 2
I
Fragment 1
N
N
N
N
N
N
O
O
NH
NH
HN
HN
O
O
HN
HN
N
NH
O
O
O
O
amide bonds.26,29 Studies have shown that a single N-methyl,
or N-methyl -aa play a critical role in locking the San A-amide
D-aa
D
Compound 1:
A-Tri-III
Compound 2:
B-Tri-III
macrocyclic analogs into a single conformation.1,2,4e6,33e35 If this
conformation induces an advantageous presentation of the side
chains to their biological target, locking it into place via one of these
structural features will likely improve binding between the com-
pound and the protein target. Likewise, there is also precedence for
oxazole and thiazole pepidomimetic moieties improving biological
stability when substituted within peptide backbones.26 Similarly to
triazoles, pseudoprolines induce a rigid conformation by mimick-
ing cis amide bonds, and thus make structurally interesting com-
parisons to triazoles.25,36,37
Herein we describe the synthesis of 13 peptidomimetics that are
based on San A-amide and the potent analogs BeD (Fig. 1). These
compounds were chosen because they have demonstrated appro-
priate cytotoxicity, and they inhibit Hsp90.1,7 The synthesis of
compounds that incorporate a triazole, oxazole, thiazole, or pseu-
doproline involved both solution and solid-phase approaches.
These peptidomimetic residues are substituted for different fea-
tures within the San A structure. The triazole replaces an amide
bond, whereas the oxazole and thiazole replace both an amide
bond as well as the adjacent amino acid side chain, and the pseu-
doproline replaces only the amino acid side chain. We discuss
a series of synthetic strategies for these four unique classes of
sansalvamide peptidomimetics, where these methods can be ap-
plied as general approaches for the conversion of macrocyclic
peptides into peptidomimetic compounds. Further, biological
testing of our peptidomimetic compounds allowed us to evaluate,
which of these heterocyclic features are ideal for incorporation into
future potent analogs and how their position in the macrocycle
affects their cytotoxicity.
Fig. 2. Triazole synthetic strategy and compounds synthesized.
We made six oxazole peptidomimetics (Ox) (Fig. 3), which were
templated from molecules AeD (Fig. 1). Compounds 3e6 were
designed with the inclusion of an oxazole at position III based on
the macrocycles AeD, whereas compounds 6e8 were only based
on molecule D with the oxazole moiety placed at positions I, II, and
III. These six oxazole-derived molecules were made using two dif-
ferent approaches, which utilized both a solid and solution phase
approach. Initially our synthetic strategy involved oxazole forma-
tion after cyclization, whereupon the cyclic peptide was synthe-
sized, and the serine was cyclized and oxidized after the formation
of the macrocycle. However, the yield for this approach was ex-
tremely low for the oxazole formation (3% final yield for 8) versus
an average of 74% yield for oxazole formation prior to cyclization.
Presumably this was due to the rigidity of the macrocycle inhibiting
the formation of an inflexible heterocycles within the backbone.
Thus, the synthetic strategy for the oxazole derivatives 3e7 in-
volved the synthesis of two fragments; fragment 1 consisted of
a tripeptide, while fragment 2 incorporated the oxazole moiety. The
oxazole was synthesized by coupling a serine to a leucine or valine
(compounds 3e6 and 7, respectively). The oxazole was then formed
via cyclodehydration upon treatment with DAST and potassium
carbonate, then subsequent oxidation using bromochloroform and
DBU.25,31,35 Coupling fragments 1 and 2, followed by peptide
macrocyclization furnished the desired oxazole peptidomimetic
derivatives.
2. Results and discussion
Three thiazole (Th) derivatives were synthesized using two
different approaches (Fig. 4). The first approach involved the
synthesis of fragment 1, which incorporated a bromoketone
moiety, while fragment 2 included a thioamide (Fig. 4, approach
4a). A linear precursor was formed via the Hantzsch-thiazole re-
action40 (Compound 9, A-Th-III), and macrocyclization was per-
formed via peptide bond formation to yield compound 9. Given
Two triazole peptidomimetics (Tri), compounds 1 and 2 (Fig. 2),
were synthesized via a convergent solution phase approach.
Forming the triazole at the cyclization step has been reported as
a
successful
strategy
to
synthesize
cyclic
triazole
peptidomimetics.32,38e40 The synthetic strategy involved making

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1031
Fig. 3. Oxazole peptidomimetic compounds.
that the thiazole linear precursor had an extremely low yield (15%
for thiazole formation), an alternative synthetic route was used for
compounds 10 and 11 (Fig. 4, approach 4b). The alternative route
involved the same general synthetic approach as the oxazoles,
where a solution-phase procedure was used to form tripeptide
fragment 1. The thiazole moiety was synthesized from two pre-
cursors, ethyl-bromopyruvate and a thioamide, via the Hantzsch-
thiazole reaction to yield fragment 2.41 This proved to be a suc-
cessful means of synthesizing the thiazole moiety with an average
of 62% yield for this reaction.
Upon synthesis of these two fragments, amide bond formation
between residues IV and V furnished the linear precursor fol-
lowed by deprotection of the acid and amine and subsequent
peptide cyclization between residues II and III generated com-
pounds 10 and 11.
furnished alkyne 15 in a 43% yield over two steps. Alkyne 15 was
coupled to amino acid 16 and the Boc group was removed to yield
17, fragment 2 (74% yield over two steps). Standard coupling of
amino acids 18 and 19 and deprotection methods produced 20 (87%
yield over two steps). Dipeptide 20 was then coupled to azide 21
and subsequently deprotected using lithium hydroxide to yield free
acid 22, fragment 1 (62% yield over two steps). Amine 17 and acid
22 were coupled using peptide coupling conditions to produce
linear precursor 23 (52% yield). Treatment of 23 with catalytic
amounts of copper produced 1 in 7.5% purified yield of cyclized
compound.
3.2. Synthesis of triazole compound 2
We synthesized two pseudoproline (PP) derivatives based on
the compound A scaffold, where the pseudoproline was placed at
positions II and III, respectively (12 and 13, Fig. 5). These com-
pounds were made via Fmoc solid-phase synthesis through se-
quential peptide coupling from chlorotritylchloride resin loaded
with the appropriate pseudoproline dipeptides.40 Upon formation
of the linear pentapeptide, the compounds were cleaved, cyclized,
and purified.
Compound 2 was synthesized using a linear approach. The
yield from this linear approach was then compared to the yield
using the convergent approach used for compound 1 (Scheme 2).
Aldehyde 24 was converted into an alkyne with the treatment of
p-toluenesulfonyl azide and di-methyl (2-oxopropyl) phosphonate
in acetonitrile and methanol. The Boc protecting group was re-
moved to produce 25 (53% yield for two steps). Alkyne 25 was
coupled to amino acid 26 upon treatment with O-(Benzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU) and
N,N0-diisopropylethylamine (DIPEA). The Boc protecting group
was removed to furnish 27 (70% yield for two steps). This was
coupled to amino acid 28, and deprotected to generate alkyne 29
(60% yield over two steps). Coupling 29 to 30 and subsequent
deprotection furnished tetrapeptide 31 in good yields (80% yield
over two steps). Linear precursor 33 was produced by coupling
tetrapeptide 31 to 32 (29% yield). Finally, the macrocyclization
step used a copper catalyst to click the 1,4-disubstituted triazole
3. Synthesis
3.1. Synthesis of triazole compound 1
Aldehyde 14 was converted to an alkyne by treatment with p-
toluenesulfonyl azide and di-methyl (2-oxopropyl) phosphonate
(Scheme 1). Removal of the Boc protecting group with TFA

1032
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
O
BocHN
OH
O
NH
O
H
16
1. (a) 43%
1. (c) 74%
NH2
NH2
NHBoc
2. (b) 100%
2. (b) 100%
14
15
17, Fragment 2
O
N3
O
N3
O
H2N
BocHN
OH
HN
HO
21
18
MeO
HN
(c) 87%
(c) 66%
(d) 94%
HO
HN
O
O
O
(b) 100%
O
O
20
H2N
OMe
19
22, Fragment 1
N
N
N
N3
17
O
O
(c) 52%
(e) 7.5%
NH
NH
O
HN
HN
O
22
NH
HN
HN
NH
O
O
O
O
23
Compound 1:
A-Tri-III
Linear Precursor
Scheme 1. Synthesis of 1. (a) p-Toluenesulfonyl azide (3 equiv), di-methyl (2-
oxopropyl) phosphonate (3 equiv), potassium carbonate (3 equiv), 0.25 M in acetoni-
trile/methanol (1:1); (b) 20% trifluoroacetic acid/dichloromethane (0.1 M); (c) TBTU
(1.2 equiv), DIPEA (8 equiv), methylene chloride (0.1 M); (d) LiOH (2 equiv), H2O2
(3.4 equiv), methanol (0.1 M), 0 ꢀC; (e) L-ascorbic acid (9 equiv), NaHCO3 (9 equiv),
CuSO4$H2O (0.3 equiv), 0.007 M in methanol/water (1:1).
O
Fig. 4. Thiazole peptidomimetic compounds: 2 approaches.
Boc(Me)N
OH
O
H
O
NH
26
1. (a) 53%
1. (c) 70%
NH2
NHBoc
N
H
2. (b) 100%
2. (b) 100%
III
O
25
24
27
III
O
O
O
IV
O
V
N
O
O
IV
N
H
ring closing
II
O
N
NH
30
HN
NH
O
O
II
O
NH
O
BocHN
28
NH
H2N
HN
NH HN
O
NH HN
O
BocHN
OH
O
O
OH
N
V
N
1. (c) 60%
1. (c) 80%
NH2
O
ring closing
O
I
O
I
2. (b) 100%
2. (b) 100%
29
Compound 12:
A-PP-II
Compound 13:
A-PP-III
31
O
N3
Fig. 5. Pseudoproline compounds.
N
N
N3
N
HO
32
O
O
NH
NH
HN
HN
(d) 7%
heterocycle and close the macrocyclic ring (7% yield for com-
pound 2).
(c) 29%
O
O
N
HN
HN
N
O
O
O
O
3.3. Optimal synthesis strategy for oxazole derivatives
The most optimal synthesis for the oxazole derivatives is de-
scribed. This approach was used on compounds 3e7 and described
is the synthesis of analog 3 where the oxazole is formed prior to
closing the macrocycle (Scheme 3). Amino acid 34 was coupled to
35 and then underwent methyl ester hydrolysis to furnish 36 (82%
yield over two steps). Coupling 36 and 37 generated a tripeptide,
whereupon treatment with lithium hydroxide and hydrogen
Compound 2
B-Tri-III
33, Linear Precursor
Scheme 2. Synthesis of 2. (a) p-Toluenesulfonyl azide (3 equiv), di-methyl (2-
oxopropyl) phosphonate (3 equiv), potassium carbonate (3 equiv), 0.25 M in acetoni-
trile/methanol (1:1); (b) 20% trifluoroacetic acid/dichloromethane (0.1 M); (c) TBTU
(1.2 equiv), DIPEA (8 equiv), methylene chloride (0.1 M); (d) L-ascorbic acid (9 equiv),
NaHCO3 (9 equiv), CuSO4$H2O (0.3 equiv), 0.007 M in methanol/water (1:1).

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1033
MeO
peroxide formed 38, fragment 1 (66% yield over two steps). Amino
OMe
O
Br
O
acids 39 and 40 were coupled and subjected to hydrogenation
to remove the benzyl protecting group, yielding 41 (87% yield).
The oxazole was formed on the dipeptide 41 by treatment of DAST
and K2CO3, which generated the oxazoline, whereupon treatment
with DBU and bromotrichloromethane produced the desired oxa-
zole (in 77% overall yield for two steps). Deprotection of the amine
furnished 42 (100% yield), fragment 2. Fragments 1 and 2 were
coupled together to form 43, the linear precursor in 72% yield. The
Br
O
O
O
OH
47
HN
HN
BocHN
OH
H2N
44
1. (a) 99%
1. (a) 82%
2. (c) 92%
O
MeO
O
HN
2. (b) 100%
O
MeO
O
H2N
OMe
46
45
48, Fragment 1
O
NHBoc
S
O
NH2
O
HO
S
O
BocHN
HN
NH2
1. (d) 100%
51
BocHN
MeO
OMe
O
NH
O
NH2
BocHN
O
OH
2. (e) 68%
3. (b) 100%
(a) 61%
37
O
NHBoc
1. (a) 97%
2. (b) 85%
34
HN
NH2
HO
1. (a) 88%
2. (b) 82%
NHBoc
OMe
49
HO HN
O
50
O
O
52, Fragment 2
NH2
O
36
S
S
N
35
O
HN
38, Fragment 1
N
1. (h) 88%
NH
O
HN
1. (f)
48
52
NH
HN
2. (b) 100%
100%
O
O
O
OH
O
NHBoc
MeO
NH HN
O
O
O
OMe
3. (i) 6%
2. (g)
15%
OMe
O
1. (d) 100%
2. (e) 77%
O
39
O
1. (a) 98%
2. (c) 89%
NH
N
H2N
OMe
O
53
Linear Precursor
3. (f) 100%
NHBoc
NH2
O
41
Compound 9
A-Th-III
BocHN
OH
42, Fragment 2
40
O
Scheme 4. Synthesis of 9. (a) TBTU (1.1 equiv), DIPEA (4 equiv), methylene chloride
(0.1 M); (b) 20% trifluoroacetic acid/methylene chloride (0.1 M); (c) formic acid (0.1 M)
(d) (1:1) ammonium hydroxide/methanol (0.1 M); (e) Lawesson’s reagent (1 equiv),
1,2-dimethoxyethane (.15 M); (f) KHCO3 (8 equiv), 1,2-dimethoxyethane (0.1 M); (g)
pyridine (9 equiv), TFAA (4 equiv), TEA (2 equiv), 1,2-dimethoxyethane (0.1 M) 0 ꢀC; (h)
LiOH (2 equiv), H2O2 (3.4 equiv), methanol (0.1 M), 0 ꢀC; (i) TBTU (0.7 equiv), HATU
(0.7 equiv), DEPBT (0.7 equiv), DIPEA (6 equiv), methylene chloride (0.007 M).
O
O
OMe
N
O
N
BocHN
NH
HN
38
NH
O
1. (b) 90%
2. (f) 100%
NH HN
(a) 72%
O
H
N
HN
O
O
3. (g) 0.4%
O
O
42
furnished the desired thiazole moiety (15% yield over two steps).
Finally, compound 9 was generated by deprotection of the acid and
amine of 53, followed by subsequent peptide macrocyclization
(5.3% overall yield).
Compound 3
A-Ox-III
43, Linear Precursor
Scheme 3. Synthesis of 3. (a) TBTU (1.2 equiv), DIPEA (8 equiv), methylene chloride
(0.1 M); (b) LiOH (2 equiv), H2O2 (3.4 equiv), methanol (0.1 M), 0 ꢀC; (c) H2/Pd
(0.03 equiv), ethanol (0.1 equiv); (d) DAST (1.1 equiv), K2CO3 (2 equiv), methylene
chloride (0.01 M), ꢁ78 ꢀC; (e) DBU (2 equiv), BrCCl3 (2 equiv), methylene chloride
(0.2 M), ꢁ47 ꢀC; (f) 20% trifluoroacetic acid/methylene chloride (0.1 M); (g) TBTU
3.5. Synthesis of thiazole compounds 10 and 11
The synthesis of compounds 10,11 was accomplished using the
approach described in Scheme 5. Compound 54 was converted to
an amide by the treatment of ammonium hydroxide and methanol
(100% yield). Thioamide 55 was generated using Lawesson’s re-
agent (70% yield), whereupon 55 was reacted with 56 (100% yield)
using Hantzsch-thiazole conditions (47% overall yield for thiazole
formation). Subsequent amine deprotection furnished 57, fragment
1. Amino acid 58 was coupled to 59, and the amine was deprotected
to produce dipeptide 60 (94% over two steps). The free amine on 60
was coupled to acid 61, and the dipeptide underwent methyl ester
hydrolysis to furnish 62, fragment 2 (79% yield over two steps).
Fragments 1 and 2 (57 and 62, respectively) were coupled together
to yield the linear precursor 63 (82% yield). The amine and acid
were subsequently deprotected and macrocylization produced
compound 10 (3.5% yield for three steps). Synthesis of compound
11 utilizes this strategy and is described in the Supplementary data.
(0.7 equiv),
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexa-
fluorophosphate (HATU) (0.6 equiv), 3-(diethylphosphoryloxy)-1,2,3-benzotriazin-
4(3H)-one (DEPBT) (0.7 equiv), DIPEA (10 equiv), 0.007 M in methylene chloride/ace-
tonitrile (1:1).
acid and amine deprotection and cyclization generated compound
3. Synthesis of compound 8 is described in the Supplementary data
(page 46e48).
3.4. Synthesis of thiazole compound 9
Following the synthetic approach described in 4a (Fig. 4), we
outline the synthetic method used to form thiazole compound 9
(Scheme 4). Dipeptide 46 was produced by the coupling 44 and 45,
followed by a Boc removal reaction (99% yield over two steps).
Further, coupling 46 to 3-bromo-2,2-dimethyoxypropanoic acid 47
and treatment with formic acid furnished ketone 48, fragment 1
(75% yield over two steps). Amino acid 49 was converted to thio-
amide 50 (68% yield over three steps), which was subsequently
coupled to amino acid 51 to yield fragment 2, 52 (61% yield). Linear
precursor 53 was formed using Hantzsch-thiazole conditions,
where potassium bicarbonate generated the thiazoline in-
termediate, followed by an elimination reaction in the presence of
pyridine, trifluoroacetic anhydride, and triethylamine, which
3.6. Synthesis of pseudoprolines 12 and 13
The synthesis of compound 13 involved loading commercially
available compound 64 onto 2-chlorotrityl-chloride resin, followed
bysubsequentFmocremovalfromleucine, generating65(Scheme6).
Subsequent coupling and deprotection of three additional amino
acids generated compound 66. The linear pentapeptide was cleaved
from the resin by treatment with 2,2,2-trifluoroethanol (TFE)

1034
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
O
providing the double deprotected peptide in an overall yield of 95%.
O
Br
56
O
The linear precursor was cyclized to produce the final compound 13
in 25% overall yield from the starting compound 64. Synthesis of
compound 12 utilizes this strategy and is described in the
Supplementary data.
OEt
S
OEt
N
1. (a) 100%
2. (b) 70%
1. (c) 100%
O
S
BocHN
54
BocHN
NH2
OH
NH2
2. (d) 47%
3. (e) 100%
55
57, Fragment 1
4. Biological and modeling data
O
H
N
MeO
OMe
N
O
In order to evaluate, which of the heterocycles would be ideal
for incorporation into the macrocyclic backbone, cytotoxicity data
for molecule a peptidomimetic functional group was generated
and compared to parent compound A (Fig. 6). All four classes of
peptidomimetic features and compound A were evaluated for
their ability to inhibit growth in HeLa cervical cancer cell lines.
The bar graph indicates that potency is maintained with the in-
clusion of a triazole or thiazole at position III. Interestingly, in-
clusion of an oxazole or pseudoproline in the peptide backbone
decreases cytotoxicity compared to the parent compound. These
data likely represent the consequence of altering the macrocyclic
backbone, whereby the trans conformation, that is, induced by the
triazole is favorable, and the cis conformation induced by the
pseudoproline is not. Further, oxaozles are known to be more rigid
than thiazoles, and the additional flexibility of the thiazole must
be critical for the macrocycle to maintain its binding affinity with
Hsp90.1,20,42
BocHN
BocHN
NH2
O
O
61 OH
58
HN
1. (f) 94%
2. (e) 100%
1. (f) 79%
O
HO
O
N
2. (g) 100%
O
60
O
OH
NHBoc
59
62, Fragment 2
S
O
S
O
N
N
OEt
BocHN
NH
HN
HN
NH
O
O
1. (g) 89%
2. (e) 100%
57
62
(f) 82%
N
O
N
HN
O
O
3. (h) 4%
O
Compound 10
B-Th-III
63, Linear Precursor
Cytotoxicity data
Scheme 5. Synthesis of 10. (a) (1:1) Ammonium hydroxide/methanol (0.1 M); (b)
Lawesson’s reagent (1 equiv), 1,2-dimethoxyethane (0.15 M); (c) KHCO3 (8 equiv), 1,2-
dimethoxyethane (0.1 M); (d) pyridine (9 equiv), TFAA (4 equiv), TEA (2 equiv), 1,2-
A
100
Compound 1= A-Tri-III
dimethoxyethane (0.1 M)
0
ꢀC; (e) 20% trifluoroacetic acid/methylene chloride
80
Compound 3= A-Ox-III
(0.1 M); (f) TBTU (1.1 equiv), DIPEA (4 equiv), methylene chloride (0.1 M); (g) LiOH
(2 equiv), H2O2 (3.4 equiv), methanol (0.1 M), 0 ꢀC; (h) TBTU (0.7 equiv), HATU
(0.7 equiv), DEPBT (0.7 equiv), DIPEA (6 equiv), methylene chloride (0.007 M).
Compound 9= A-Th-III
60
Compound 13= A-PP-III
40
20
O
O
O
O
Cl
1. (a)
2. (b)
0
Cl
FmocHN
N
N
O
Compound A and derivatives
O
H2N
O
OH
Fig. 6. Peptidomimetic compounds run at 25
lines. Each data point is an average of four wells run in three separate assays using
HeLa cancer cell lines. Inhibition is relative to 1% DMSO control.
mM against HeLa cervical cancer cell
65
CTC resin
O
64
O
O
O
N
N
1. (d)
2. (b)
Given the unique biological data, models of the four molecules
were completed using ChemBio3D Ultra. The lowest energy struc-
tures for each peptidomimetic molecule are shown in Fig. 7. Al-
though caution should be employed when drawing conclusions
from relatively simple model systems, these models are useful tools
in developing a hypothesis to explain the highly divergent cyto-
toxicity data we have generated. The inclusion of the triazole pro-
vides an additional atom to the macrocycle, and this appears to
allow a large ring, that is, significantly less puckered than the
pseudoproline large macrocycle structure, compare 1 versus 13.
This is logical given that the triazole induces a trans conformation,
while the pseudoproline induces a cis conformation. Although
structurally similar, the oxazole places the side chains at orienta-
tions that are different from the thiazole, perhaps allowing a com-
poundeprotein interaction with residue II, that is, unique to the
thiazole compound (9) versus oxazole analog (3). Indeed, the
modeling data below suggestion that compounds 1 and 9 have
a conformation that places residues I and II in a similar orientations,
whereas 3 and 13 have very different presentations of both resi-
dues. Although difficult to visualize, it is also possible that residues
IV and V play a role in the improved cytotoxicity observed for
compounds 1 and 9 by allowing an improved binding affinity for
1. (c)
2. (b)
O
NH
NH
O
O
NH
O
O
O
NH2
NH2
67
O
66
O
O
O
O
O
N
N
O
O
O
NH
NH
O
O
1. (e) 95%
2. (f) 26%
HN
NH
1. (c)
2. (b)
O
NH HN
O
NH2
H
N
O
68
Compound 13
A-PP-III
(25% Overall Yield)
Scheme 6. Synthesis of 13. (a) 3 mL/g resin of N-Methylpyrrolidone (NMP), DIPEA
(6 equiv), methylene chloride (10 mL/g AA); (b) 20% piperidine/dimethylformamide
(0.2 M); (c) Fmoc-Leu-OH (1.2 equiv), HOBt (3 equiv), DIC (6 equiv), dimethylforma-
mide (0.2 M); (d) Fmoc-Phe-OH (1.2 equiv), HOBt (3 equiv), DIC (6 equiv), dime-
thylformamide (0.2 M); (e) TFE/methylene chloride (1:1) (0.2 M); (f) HATU (0.7 equiv),
TBTU (0.7 equiv), DEPBT (0.6 equiv), DIPEA (8 equiv), methylene chloride/acetonitrile
(1:1) (0.007 M).

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1035
Fig. 7. Molecular models of peptidomimetic compounds. Energy was minimized using the Merck Molecular Force Field 94 (MMFF94) with ChemBio3D Ultra (version 12.0) available
form CambridgeSoft. Convergence criteria: atomic root mean square force 0.01 kcal/mol; static energy 82.250e115.726 kcal/mol; 500 iterations.
Hsp90 over that of compounds 3 and 13. Docking studies involving
these molecules and Hsp90’s crystal structure are on going and will
be published in due course.
of 2000 cells/well in DMEM (Gibco) supplemented with L-gluta-
mine, 10% fetal bovine serum, and 1% penicillin-streptomycin an-
tibiotic. After overnight incubation, the compounds were added.
The compounds were dissolved in DMSO and tested at the con-
centrations indicated in the manuscript with a final DMSO con-
centration of 1.0%. The DMSO control was also at 1.0%. The cells
were incubated with compound or DMSO for 24 h upon which
5. Conclusion
In summary, we have outlined the synthesis of thirteen pep-
tidomimetics, all of which are related to a class of sansalvamide
cytotoxic agents, with the anticipation that several moieties will
have potency equal to that observed with the natural product. Two
derivatives A-Tri-III (1) and A-Th-III (9) exhibited cytotoxicity at
the same level as the parent San A-amide A. Although it is hard to
predict how these peptidomimetic moieties affect the overall
macrocyclic conformation, it appears that the triazole, which in-
corporates an additional carbon into the macrocyclic backbone as
well as maintaining the side chain at position III is a favorable
option. This is likely due to its ability to induce a trans-amide
conformation, versus a cis amide conformation induced by the
pseudoproline.23,26,30,35 The thiazole moiety is also promising,
presumably because it is a more flexible heterocycle relative to the
oxazole and thus able to accommodate a favorable conformation
for binding to Hsp90. Based on modeling, both compounds 1 and 9
may have improved cytotoxicity because of their similar orienta-
tion of residues I and II. Future work involves the development of
compounds with triazoles and thiazoles in the backbone, with the
anticipation that they will maintain their biological activity, while
having enhanced PK and ADME properties. Further, docking
studies using these two moieties in their backbone will provide
insight into the induced conformation, and will be reported in due
course.
10 mL of CCK-8 solution was added to each well and allowed to
incubate at 37 ꢀC for an additional 2.5 h. The absorbance at 450 nm
was measured using a S6 Genios Fluorimeter (Tecan). Percent
growth inhibition was calculated as 1 minus the absorbance of
compound-treated cells over DMSO-treated cells. All calculations
including mean and SEM were performed using Prism software.
Each data point is an average of four wells run in three separate
assays.
6.2. General solution-phase peptide synthesis
All peptide coupling reactions were carried out under argon
with dry solvent, using methylene chloride and/or acetonitrile for
dipeptide, tripeptide, and pentapeptide couplings. The amine
(1.1 equiv) and acid (1 equiv) were weighed into a dry flask along
with 4e8 equiv of DIPEA and 1.1 equiv of TBTU. *Anhydrous
methylene chloride and/or acetonitrile were added to generate
a 0.1 M solution. The solution was stirred at room temperature and
reactions were monitored by TLC. Reactions were run for 1 h before
checking via TLC. If reaction was not complete an additional
0.25 equiv of TBTU was added. If reaction was complete then work-
up was done by washing with 10% aqueous hydrochloric acid and
saturated sodium bicarbonate. After back extraction of aqueous
layers with methylene chloride, organic layers were combined,
dried over sodium sulfate, filtered, and concentrated. Flash column
chromatography using a gradient of ethyl acetate/hexane gave our
desired peptide.
6. Experimental section
6.1. Cytotoxicity assays
*Some coupling reactions would not go to completion using only
TBTU and therefore 0.2e0.5 equiv of HATU, and/or DEPBT were
used. In a few cases up to 0.7 equiv of all three coupling reagents
were used.
Proliferation of HeLa cells was tested in the presence and ab-
sence of the compounds using CCK-8 assays (Dojindo, catalog#
CK04e13). Cells were cultured in 96 well plates at a concentration

1036
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
6.3. General solution-phase amine deprotection
2-propanol wash (3ꢂ1 min), methanol (3ꢂ1 min). The fully-
assembled peptideeresin was then drained and dried in vacuo
Amines were deprotected using 20% trifluoroacetic acid in
methylene chloride (0.1 M) with 2 equiv of anisole. The reactions
were monitored by TLC. Reactions were allowed to run for 1e2 h
and then concentrated in vacuo.
overnight.
6.9. Cleavage of linear peptide from solid support
The full-length, linear peptide was cleaved from the resin by
swelling and shaking the peptideeresin for 24 h in a 1:1 (v:v) TFE/
methylene chloride (10 vol/g of dried resin). The cleavage solution
was filtered through a Buchner filter, and the drained resin was
washed with additional methylene chloride (5 vol/g of initial dried
peptideeresin) to fully extract the cleaved peptide from the resin.
Solvents in the combined filtrates were evaporated by rotary
evaporation and the solids dried in vacuo overnight. The solids
were then reconstituted in methylene chloride, evaporated by ro-
tary evaporation and dried in vacuo overnight again to remove
residual entrapped TFE.
6.4. General solution-phase acid deprotection
Acids were deprotected using 2 equiv of lithium hydroxide with
3.4 equiv of hydrogen peroxide in methanol (0.1 M). The peptide
was dissolved in methanol and cooled to 0 ꢀC. Hydrogen peroxide
was added followed by lithium hydroxide. The reaction was mon-
itored by TLC and usually done in 1e2 h. Sodium thiosulfate
(3.8 equiv) was added to neutralize the peroxide and 5% hydro-
chloric acid was added till the solution pH was 1. The aqueous so-
lution was extracted five times with methylene chloride, and the
combined organic layer was dried, filtered, and concentrated in
vacuo.
6.10. Macrocyclization procedure (with syringe pump)
6.5. General solid-phase synthesis remarks
Three coupling agents (DEPBT, HATU, and TBTU) were used at
w0.5 to 0.75 equiv each. These coupling agents were dissolved in 3/4
of a calculated volume of dry methylene chloride that would give
a 0.001 Me0.0007 M overall concentration when included in the
volume used for the deprotected peptide. The crude, dry, double
deprotected peptide (free acid and free amine) was dissolved in the
other ¼ solvent volume of methylene chloride. DIPEA (8 equiv) was
then added to the solution containing coupling reagents dissolved
in methylene chloride. The double deprotected peptide was then
added to the bulk solution dropwise using a syringe pump at a rate
of 30 mL/h. The reaction was monitored via LCMS and generally
complete in 1e2 h. Upon completion, the reaction was worked up
by washing with aqueous hydrochloric acid (pH 1) and saturated
sodium bicarbonate. After back extraction of aqueous layers with
large quantities of methylene chloride, the organic layers were
combined, dried, filtered, and concentrated. All macrocycles were
first purified by flash column chromatography using an ethyl ace-
tate/hexane gradient on silica gel. Finally, when necessary, reverse
phase-HPLC was used for additional purification using a gradient of
acetonitrile and deionized water with 0.1% TFA.
Stepwise solid-phase peptide synthesis was performed in
a
polypropylene solid-phase extraction cartridge fitted with
a 20 M polyethylene frit purchased from Applied Separations
(Allentown, PA). 2-chlorotrityl resins were purchased in pre-loaded
form with -Leu, -Leu, or -Phe. In the case of compounds 12 and
m
L
D
D
13, a commercially available pseudoproline moiety was loaded onto
a 2-chlorotrityl resin. Resins were swelled in dimethylformamide
for 30 min prior to assembly of the linear five-residue peptide se-
quence. Solid-phase syntheses were performed on a 0.5 mmol scale
based on resin-loading. All operations were performed at room
temperature under open atmosphere unless stated otherwise.
6.6. General solid-phase peptide synthesis
Fmoc-protected amino acids were coupled using 3 equiv of
amino acid, 3 equiv of HOBt, and 6 equiv of DIC. Couplings were
performed in dimethylformamide at 0.2 M with respect to the in-
coming Fmoc-protected amino acid. Couplings were allowed to
proceed for a minimum of 2 h, and were assayed via ninhydrin test
to verify competition. Once complete, the coupling reaction solu-
tion was drained, and the resin subjected to Fmoc deprotection.
(Note: Fmoc and N-methyl amino acids are coupled according to
the cycle above, however for subsequent coupling onto the sec-
ondary amino terminus, HOBt was substituted with HOAt and the
coupling was allowed to proceed overnight).
6.11. Alkyne formation (SeyfertheGilbert)43,44
Dry K2CO3 (3.0 equiv) was weighed into the flask under argon
atmosphere. Calculated volume of acetonitrile was added to bring
the final concentration of 0.125 M p-tosyl azide (3.0 equiv) and
dimethyl (2-oxypropyl) phosphonate (3.0 equiv) were added to the
reaction mixture to generate the BestmanneOhira reagent. The
reaction mixture stirred at room temperature and was monitored
via TLC. After 2 h aldehyde (1.0 equiv dissolved in calculated
amount of dry methanol to bring total reaction concentration to
0.25 M) was added. The reaction mixture was left stirring at room
temperature overnight. The reaction was usually complete on the
next day. Upon completion, confirmed by TLC, the reaction was
concentrated in vacuo. The crude dried product was dissolved in
200 mL of ethyl acetate and washed with 150 mL of saturated so-
dium bicarbonate (ꢂ two times) and then by 100 mL of saturated
sodium chloride (one time). The organic layer was collected, dried
over sodium sulfate, and concentrated in vacuo. Flash chromatog-
raphy with a gradient of ethyl acetate/hexane was performed to
purify the desired alkyne.
6.7. General solid-phase amine deprotection
Following coupling completion, the peptideeresin was treated
as follows for removal of the Fmoc protecting group: dime-
thylformamide wash (3ꢂ1 min), 20% piperdine/dimethylforma-
mide (1ꢂ5 min), 20% piperdine/dimethylformamide (1ꢂ10 min),
dimethylformamide wash (2ꢂ1 min), 2-propanol wash (1ꢂ1 min),
dimethylformamide wash (1ꢂ1 min), 2-propanol (1ꢂ1 min),
dimethylformamide (3ꢂ1 min). A ninhydrin test was performed to
verify completion.
6.8. General N-terminal solid-phase deprotection
Once the final N-terminal amino acid residue had been coupled,
the peptideeresin was treated as follows for removal of the Fmoc
protecting group: dimethylformamide wash (3ꢂ1 min), 20%
piperdine/dimethylformamide (1ꢂ5 min), 20% piperdine/dime-
thylformamide (1ꢂ10 min), dimethylformamide wash (3ꢂ1 min),
6.12. Cu(I)-catalyzed alkyneeazide cycloaddition
Sodium ascorbate was dissolved in 0.5 mL of water and put into
round bottom flask. Copper sulfate was dissolved in 0.5 mL of water

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1037
and added to the flask. The peptide (with azide and alkyne) was
6.16. General bromoketal acid formation
dissolved in a mixture of solvents methanol/water (1:1) at a con-
centration of 0.005 M 10% of this solvent mixture was added to the
flask. The remaining solvent mixture was added dropwise via sy-
ringe pump to the reaction flask mixture overnight. The concen-
tration of copper was 1.5 mM and concentration of sodium
ascorbate was 45 mM for the overall reaction. Upon completion of
the reaction, methanol was removed under reduced pressure and
the reaction mixture was diluted with 100 mL of methylene chlo-
ride. The organic layer was collected and concentrated in vacuo.
Flash chromatography with a gradient of ethyl acetate/hexane was
performed to purify the desired derivative. Finally, when necessary
reverse phase-HPLC was used for additional purification using
a gradient of Acetonitrile and distilled water with 0.1% trifluoro-
acetic acid.
Trimethyl orthoformate (3 equiv) and sulfuric acid (0.25 equiv)
were used to dissolve bromopyruvic acid (1 equiv) under argon.
The mixture is stirred overnight for less than 24 h. Acid work-up
was done by extracting with 10% aqueous hydrochloric acid. After
back extraction of aqueous layers with methylene chloride and/or
ethyl acetate, the organic layers were combined, dried over sodium
sulfate, filtered, and concentrated in vacuo.
6.17. General ketone deprotection
Ketones were deprotected using formic acid (0.1 M), heated to
60 ꢀC. The reaction was monitored by TLC and usually done within
30 min. Upon completion, the reaction was washed with saturated
aqueous sodium bicarbonate. After back extraction of aqueous
layers with methylene chloride, organic layers were combined,
dried over sodium sulfate, filtered, and concentrated in vacuo.
6.13. General oxazole synthesis
DAST (1.1 equiv) was added (0.1 mL/min) to a solution of
peptidyl-Ser or peptidyl-Phenylserine (1.0 equiv) in methylene
chloride (0.1 M) cooled to ꢁ78 ꢀC under argon atmosphere. The
reaction mixture was stirred for 1 h and anhydrous K2CO3
(2.0 equiv) was added to the reaction mixture to stir at ꢁ78 ꢀC for
1 h. The reaction mixture warmed to room temperature and stirred
for an additional 1.5 h. Upon reaction completion, confirmed by
TLC, the organic solution was poured into saturated aqueous so-
dium bicarbonate and extracted with methylene chloride. After
back extraction of the aqueous layer with methylene chloride and/
or ethyl acetate, the organic layers were combined, dried over so-
dium sulfate, filtered, and concentrated in vacuo to give the oxa-
zoline as an oil. The oxazoline was used without further purification
for the oxidation of the oxazoline to yield the desired oxazole. DBU
(2.0 equiv) was added (0.1 mL/min) to a solution of oxazoline
(1.0 equiv) in methylene chloride (0.1 M) at ꢁ47 ꢀC under argon.
The reaction was stirred for 20 min and BrCCl3 (2.0 equiv) was
added to the reaction mixture (0.1 mL/min). The reaction continued
to stir at ꢁ47 ꢀC for an additional 2 h and then warmed to room
temperature to stir an additional 12 h, or until complete by TLC.
Upon reaction completion, a work-up was done by extracting with
10% aqueous hydrochloric acid. After back extraction of aqueous
layers with large quantities of methylene chloride and/or ethyl
acetate, organic layers were combined, dried over sodium sulfate,
filtered, and concentrated in vacuo. Flash column chromatography
using a gradient of ethyl acetate/hexane gave our desired peptidyl-
oxazole. Finally, when necessary, reversed phase-HPLC was used
for additional purification using a gradient of acetonitrile and
deionized water with 0.1% trifluoroacetic acid.
6.18. General thiazole synthesis (modified Hantzsch)
Thiazole synthesis reaction was carried out under argon with
anhydrous 1,2-dimethoxyethane. KHCO3 (8 equiv) was added to
the dry flask containing peptidyl thioamide (1.0 equiv). Anhydrous
1,2-dimethoxyethane (0.15 M) was added to the reaction, and it
was stirred at room temperature for 15 min. a-Bromo ketone res-
idue (3.0 equiv) was added (0.1 mL/min) and the reaction mixture
was stirred overnight. Upon reaction completion, confirmed by
TLC, the organic solution was poured into pre-prime Celite with
ethyl acetate. The filtrate was concentrated in vacuo to give the
thiazoline intermediate as an oil to be used without further puri-
fication. Next, pyridine (9.0 equiv) was added (0.1 mL/min) to
a solution of thiazoline in 1,2-dimethoxyethane (0.05 M) at 0 ꢀC
under argon for the dehydration of the thiazoline to yield the de-
sired thiazole. The reaction was stirred for 15 min and then TFAA
(4.0 equiv) was added to the reaction mixture (0.1 mL/min). After
3 h, TEA (2.0 equiv) was added to the reaction mixture (0.1 mL/
min) and the reaction continued to stir at room temperature for an
additional 2e3 h, or until complete by TLC. Upon completion, the
reaction was extracted with 10% aqueous hydrochloric acid. After
back extraction of aqueous layers with methylene chloride and/or
ethyl acetate, the organic layers were combined, dried over sodium
sulfate, filtered, and concentrated in vacuo. Flash column chro-
matography using a gradient of ethyl acetate/methylene chloride
gave our desired peptidyl-thiazole. Finally, when necessary, re-
verse phase-HPLC was used for additional purification using
a gradient of acetonitrile and deionized water with 0.1% trifluoro-
acetic acid.
6.14. General amide formation
Boc-protected amino ester (1 equiv) was dissolved in 50% am-
monium hydroxide and 50% methanol (0.05 M). The reaction
mixture was stirred overnight or until complete by TLC. Upon
completion, the solvent was concentrated in vacuo.
6.19. Benzylation procedure
The cyclized peptide was dissolved in 50% tetrahydrofuran and
50% dimethylformamide to make a 0.1 M solution. The 60% NaH
was used at 1.1 equiv and dissolved in the 0.1 M solution. Benzyl
bromide (2 equiv) was then added to the reaction. After 2 h, LC/MS
indicated the reaction was developing. The reaction was com-
pleted in about 5 h and then worked up by washing with deionized
water. After that, the organic layer was collected, dried, and pre-
liminarily purified by flash column chromatography. Finally, re-
verse phase-HPLC was used for further purification by using
a gradient of acetonitrile and deionized water with 0.1% tri-
fluoroacetic acid.
6.15. General thioamide formation
Boc-protected amide (1 equiv) was converted into Boc-
protected thioamide using Lawesson’s reagent (0.8 equiv) in
0.4 M 1,2-dimethoxyethane at room temperature under argon. The
mixture was stirred overnight or until complete by TLC. Upon
completion, the solvent was concentrated in vacuo. Boc-protected
thioamide was purified by flash column chromatography using an
ethyl acetate/methylene chloride gradient on silica gel.

1038
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
6.20. Synthesis of compound 1 (A-tri-III)
peptide synthesis’ procedure. Utilizing 83.0 mg (0.743 mmol) of
amine 15 alkyne-Leu-NH2, 204 mg (0.817 mmol) of acid 16 HO-Leu-
NHBoc, 1.00 mL (6.53 mmol) of DIPEA, 286 mg (0.896 mmol) of
TBTU, in 7.40 mL of methylene chloride. The crude reaction was by
column chromatography (silica gel, EtOAc/Hex) to yield the di-
peptide (241 mg, 74% yield). Rf: 0.6 (EtOAc/Hex 1:4); 1H NMR
6.20.1. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-Leu-
NHBoc was synthesized following the ‘General solution-phase pep-
tide synthesis’ procedure. Utilizing 1.50 g (6.96 mmol) of amine 19
MeO-Phe-NH2$HCl, 1.46 g (6.32 mmol) of acid 18 HO-Leu-NHBoc,
3.95 mL (27.8 mmol) of DIPEA, 2.44 g (7.58 mmol) of TBTU, in
66.0 mL of methylene chloride. The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the di-
peptide (2.26 g, 87% yield). Rf: 0.6 (EtOAc/Hex 1:1). Physical and
spectroscopic data are consistent with those reported in the
literature.29
(400 MHz, CDCl3):
d 0.85e0.90 (12H, m, CH(CH3)2), 1.37 (9H, s,
OC(CH3)3), 1.45e1.50 (1H, m, CH2CH(CH3)2), 1.57e1.64 (4H, m,
CHCH2CH), 1.64e1.74 (1H, m, CH2CH(CH3)2), 2.17 (1H, s, C^CH),
3.91e4.02 (1H, br m,
aCH), 4.65e4.78 (1H, m, aCH), 4.72 (1H, br s,
CHNHCOOtBu), 7.72 (1H, br d, J 6.19 Hz, NH).
6.20.9. Dipeptide alkyne-Leu-Leu-NH2 (17). Dipeptide alkyne-Leu-
Leu-NH2 was synthesized following the ‘General solution-phase
amine deprotection’. This dipeptide was taken on to the next re-
action without further purification or characterization (145 mg,
100% yield).
6.20.2. Dipeptide MeO-Phe-Leu-NH2 (20). Dipeptide MeO-Phe-Leu-
NH2 was synthesized following the ‘General solution-phase amine
deprotection’. This dipeptide was taken on to the next reaction
without further purification or characterization (445 mg, 100%
yield).
6.20.10. Pentapeptide alkyne-Leu-Leu-Phe-Leu-Val-N3 (23). Penta-
peptide alkyne-Leu-Leu-Phe-Leu-Val-N3 was synthesized follow-
ing the ‘General solution-phase peptide synthesis’ procedure. Uti-
lizing 145 mg (0.649 mmol) of amine 17 alkyne-Leu-Leu-NH2,
237 mg (0.591 mmol) of acid 22 HO-Phe-Leu-Val-N3, 0.82 mL
(4.73 mmol) of DIPEA, 189 mg (0.591 mmol) of TBTU, 67.0 mg
(0.177 mmol) of HATU, and 34.2 mg (0.118 mmol) of DEPBT, in 5 mL
of methylene chloride and 2 mL acetonitrile. The crude reaction
was purified by column chromatography (silica gel, EtOAc/Hex) to
yield the pentapeptide (186 mg, 52% yield). Rf: 0.5 (EtOAc/Hex
6.20.3. Monomer HO-Val-N3 (21). Monomer 21 HO-Val-N3 was
synthesized utilizing 500 mg (4.27 mmol) of HO-Val-NH2, 2.82 g
(8.54 mmol) of triflic anhydride, 2.78 g (42.7 mmol) of sodium
azide, 885 mg (6.41 mmol) of potassium carbonate, 10.6 mg
(42.5 mmol) of CuSO4$5H2O, in 48 mL of DCM/MeOH/H2O
(2:1:1) solvent system. The crude reaction was purified using an
aqueous acidic wash to yield the pure monomer (600 mg, 97%
yield).
6.20.4. Tripeptide MeO-Phe-Leu-Val-N3. Tripeptide MeO-Phe-Leu-
Val-N3 was synthesized following the ‘General solution-phase pep-
tide synthesis’. Utilizing 444 mg (1.53 mmol) of amine 20 MeO-Phe-
Leu-NH2, 200 mg (1.39 mmol) of acid 21 HO-Leu-N3, 1.80 mL of
DIPEA, 534 mg (1.66 mmol) of TBTU, in 66.0 mL of methylene
chloride. The crude reaction was purified by column chromatog-
raphy (silica gel, EtOAc/Hex) to yield the tripeptide (420 mg, 66%
yield). Rf: 0.5 (EtOAc/Hex 1:2); 1H NMR (400 MHz, CDCl3):
1:1); 1H NMR (400 MHz, CD3OD):
d 0.88e0.96 (24H, m, CH(CH3)2),
1.26e1.36 (1H, m, CHCH(CH3)2), 1.47e1.65 (6H, m, CHCH2CH),
1.72e1.83 (1H, m, CH2CH(CH3)2), 2.05e2.19 (1H, m, CH2CH(CH3)2),
2.63 (1H, s, C^CH), 2.89e316 (2H, dq, J 80.6, 8.3, 5.6 Hz, CHCH2Ph),
3.55 (1H, d,
J
6.9 Hz, N3CHC]O), 4.32e4.44 (2H, m,
aCH),
4.57e4.70 (2H, m,
a
CH), 7.17e7.26 (5H, m, Ph), 8.01 (1H, d, J
8.05 Hz, NH), 8.07 (1H, d, J 7.7 Hz, NH), 8.11 (1H, d, J 7.8 Hz, NH),
8.21 (1H, d, J 8.4 Hz, NH). LCMS: m/z calcd for C33H51N7O4 (Mþ1)¼
610.8, found 611.6.
d
0.83e0.91 (9H, m, CH(CH3)2), 1.08 (3H, d, J 6.6 Hz, CH(CH3)2),
1.47e1.51 (2H, m, CHCH2(CH3)2), 2.24e2.33 (1H, m, CH(CH3)2),
3.02e3.20 (2H, dq, J 86.5, 14.2, 5.5 Hz, CHCH2Ph), 3.72 (3H, s, OCH3),
6.20.11. Macrocycle Phe-Leu-Val-Triazole-Leu-Leu (1). Macrocycle
Phe-Leu-Val-Triazole-Leu-Leu was synthesized following the
‘Cu(I)-catalyzed alkyne-azide cycloaddition’. Utilizing 150 mg
(0.252 mmol) of linear pentapeptide 23, 389 mg (2.21 mmol) of
3.73 (1H, d, J 4.1 Hz,
m, CH), 6.43 (1H, d, J 7.7 Hz, NH), 6.62 (1H, d, J 7.7 Hz, NH),
7.09e7.32 (5H, m, Ph).
aCH), 4.37e4.42 (1H, m, aCH), 4.90e5.02 (1H,
a
L
-ascorbic acid, 185 mg (2.21 mmol) of NaHCO3, and 18.4 mg
6.20.5. Tripeptide HO-Phe-Leu-Val-N3 (22). Tripeptide HO-Phe-Leu-
Val-N3 was synthesized following the ‘General solution-phase acid
deprotection’. This tripeptide was taken on to the next reaction
without further purification or characterization (382 mg, 94%
yield).
(74.2 mol) CuSO4$5H2O in 35.0 mL MeOH/H2O (1:1). The crude
m
reaction was purified by reverse phase-HPLC to yield the mac-
rocycle (12.3 mg, 7.5% yield). Rf: 0.25 (EtOAc/Hex 1:1); 1H NMR
(400 MHz, CD3OD):
d 0.89e1.0 (24H, m, CH(CH3)2), 1.49e1.54
(2H, m, CHCH2CH), 1.58e1.63 (2H, m, CHCH2CH), 1.65e1.69 (1H,
m, CH2CH(CH3)2), 1.75e1.80 (2H, m, CHCH2CH), 1.98e2.06 (1H,
m, CH2CH(CH3)2), 2.15e2.22 (1H, m, CH2CH(CH3)2), 3.02e3.16
6.20.6. Monomer alkyne-Leu-NHBoc. Monomer alkyne-Leu-NHBoc
was synthesized following ‘Alkyne formation’. Utilizing 165 mg
(0.787 mmol) of 14 Leucinial-NHBoc, 0.353 mL (2.30 mmol) of
pTsN3, 0.310 mL (2.30 mmol) of di-methyl (2-oxopropyl) phos-
phonate, and 317 mg (2.30 mmol) K2CO3 in 3.22 mL of ACN/MeOH
(1:1). The crude reaction was purified by column chromatography
(silica gel, EtOAc/Hex) to yield the monomer (70.0 mg, 43% yield).
Rf: 0.5 (EtOAc/Hex 1:9).
(2H, m, CH2Ph), 3.60e3.69 (1H, m,
4.02e4.09 (1H, m, CH), 4.19e4.27 (1H, m,
m, CH), 7.19e7.32 (5H, m, Ph), 8.04 (1H, s, NCH]C). LCMS: m/z
a
CH), 3.80e3.87 (1H, m,
aCH),
a
a
CH), 5.18e5.24 (1H,
a
calcd for C33H51N7O4 (Mþ1)¼611.8, found 611.9; HRMS (ESI-
TOF): MHþ, found 610.4078, requires 610.4075 >95% pure by
HPLC.
6.20.7. Monomer alkyne-Leu-NH2 (15). Monomer 15 alkyne-Leu-
NH2 was synthesized following the ‘General solution-phase amine
deprotection’. This monomer was taken on to the next reaction
without further purification or characterization (83 mg, 100%
yield).
6.21. Synthesis of compound 2 (B-tri-III)
6.21.1. Monomer HO-Val-N3 (32). Monomer HO-Val-N3 was syn-
thesized utilizing 500 mg (4.27 mmol) of HO-Val-NH2, 2.82 g
(8.54 mmol) of triflic anhydride, 2.78 g (42.7 mmol) of sodium
azide, 885 mg (6.41 mmol) of potassium carbonate, 10.6 mg
6.20.8. Dipeptide alkyne-Leu-Leu-NHBoc. Dipeptide alkyne-Leu-
Leu-NHBoc was synthesized following the ‘General solution-phase
(42.5
mmol) of CuSO4$5H2O, in 48.0 mL of DCM/MeOH/H2O
(2:1:1) solvent system. The crude reaction was purified using an

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1039
aqueous acidic wash to yield the pure monomer (600 mg, 97%
yield).
(4.89) of DIPEA, 235 mg (0.734 mmol) of TBTU, in 6.70 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the tetrapeptide
(332 mg, 80% yield). Rf: 0.4 (EtOAc/Hex 1:3); 1H NMR (400 MHz,
6.21.2. Monomer alkyne-Leu-NHBoc. Monomer alkyne-Leu-NHBoc
was synthesized following ‘Alkyne formation’. Utilizing 310 mg
(1.39 mmol) of 24 Leucinial-NHBoc, 0.635 mL (4.18 mmol) of pTsN3,
0.572 mL (4.18 mmol) of dimethyl (2-oxopropyl) phosphonate, and
577 mg (4.18 mmol) of K2CO3, in 5.50 mL of ACN/MeOH (1:1). The
crude reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the monomer (160 mg, 53% yield). Rf: 0.5
(EtOAc/Hex 1:9).
CDCl3):
d 0.86e0.95 (18H, m, CH(CH3)2), 1.42 (9H, s, COOC(CH3)3),
1.46e1.56 (2H, m, CHCH2CH), 1.56e1.68 (2H, m, CHCH2CH),
1.69e1.80 (1H, m, CH(CH3)2), 2.84e2.93 (2H, m, CHCH2Ph), 2.99
(3H, s, NCH3), 3.04e3.12 (1H, m, CH(CH3)2), 3.28 (1H, d, C^CH),
4.02e4.16 (1H, m,
aCH), 4.59e4.69 (1H, m, aCH), 4.92 (1H, m, aCH),
5.09 (1H, m, CH), 7.17e7.30 (5H, m, Ph), 7.94 (1H, d, J 8.2 Hz, NH),
a
(1H, d, J 8.13 Hz, NH).
6.21.3. Monomer alkyne-Leu-NH2 (25). Monomer alkyne-Leu-NH2
was synthesized following the ‘General solution-phase amine
deprotection’. This monomer was taken on to the next reaction
without further purification or characterization (83.2 mg, 100%
yield).
6.21.9. Tetrapeptide alkyne-Leu-Leu-N(Me)-Phe-Leu-NH2 (31).
Tetrapeptide alkyne-Leu-Leu-N(Me)-Phe-Leu-NH2 was synthesized
following the ‘General solution-phase amine deprotection’. This tri-
peptide was taken on to the next reaction without further purifi-
cation or characterization (233 mg, 100% yield).
6.21.4. Dipeptide alkyne-Leu-Leu-N(Me)Boc. Dipeptide alkyne-Leu-
Leu-N(Me)Boc was synthesized following the ‘General solution-
phase peptide synthesis’ procedure. Utilizing 83.2 mg (0.748 mmol)
of amine 25 alkyne-Leu-NH2, 169 mg (0.689 mmol) of acid 26
HO-Leu-N(Me)Boc, 0.902 mL (5.51 mmol) of DIPEA, 265 mg
(0.823 mmol) of TBTU, in 7.05 mL of methylene chloride. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the dipeptide (245 mg, 70% yield). Rf: 0.6
6.21.10. Pentapeptide alkyne-Leu-Leu-N(Me)-Phe-Leu-Val-N3 (33).
Pentapeptide alkyne-Leu-Leu-N(Me)-Phe-Leu-Val-N3 was synthe-
sized following the ‘General solution-phase peptide synthesis’ pro-
cedure. Utilizing 326 mg (0.655 mmol) of amine Alkyne-Leu-Leu-
N(Me)-Phe-Leu-NH2 (31), 237 mg (0.594 mmol) of acid HO-Val-N3
(32), 0.852 mL (4.75 mmol) of DIPEA, 199 mg (0.623 mmol) of
TBTU, and 71.2 mg (0.193) of HATU in 6 mL of methylene chloride.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the pentapeptide (110 mg, 29% yield). Rf:
(EtOAc/Hex 1:4); 1H NMR (400 MHz, CDCl3):
d 0.77e0.84 (12H, m,
CH(CH3)2), 1.36 (9H, s, COOC(CH3)3), 1.37 (2H, br m, CH(CH3)2),
1.50e1.66 (4H, br m, CHCH2CH), 2.15 (1H, s, C^CH), 2.61 (3H, s,
0.5 (EtOAc/Hex 1:1); 1H NMR (400 MHz, CD3OD):
d 0.88e0.98 (24H,
m, CH(CH3)2), 1.40e1.49 (1H, m, CH(CH3)2), 1.51e1.61 (6H, br m,
CHCH2CH), 1.70e1.81 (1H, m, CH(CH3)2), 2.10e2.14 (1H, m,
CH(CH3)2), 2.64 (1H, s, C^CH), 2.91 (2H, m, CH2Ph), 3.31 (3H, s,
MeNCH), 4.40e4.52 (1H, br s,
CH).
aCH), 4.55e4.67 (1H, q, J 7.3 Hz,
a
MeNC]O), 3.50e3.57 (1H, m,
4.60e4.69 (1H, m, CH), 4.80e4.89 (1H, m,
Ph). LCMS: m/z calcd for C34H53N7O4 (Mþ1)¼624.9, found 646.5.
a
CH), 4.42e4.48 (1H, m,
aCH),
6.21.5. Dipeptide alkyne-Leu-Leu-N(Me)H (27). Dipeptide alkyne-
Leu-Leu-N(Me)H was synthesized following the ‘General solution-
phase amine deprotection’. This dipeptide was taken on to the next
reaction without further purification or characterization (173 mg,
100% yield).
a
a
CH), 7.18e7.31 (5H, m,
6.21.11. Macrocycle Phe-Leu-Val-Triazole-Leu-Leu-N(Me) (2).
Macrocycle Phe-Leu-Val-Triazole-Leu-Leu-N(Me) was synthesized
following the ‘Cu(I)-catalyzed alkyne-azide cycloaddition’. Utilizing
110 mg (0.176 mmol) of linear pentapeptide (33), 1.75 g
6.21.6. Tripeptide alkyne-Leu-Leu-N(Me)-Phe-NHBoc. Tripeptide al-
kyne-Leu-Leu-N(Me)-Phe-NHBoc was synthesized following the
‘General solution-phase peptide synthesis’. Utilizing 302 mg
(1.13 mmol) of acid 28 HO-Phe-NHBoc, 299 mg (1.25 mmol) of
amine 27 Alkyne-Leu-Leu-N(Me)H, 1.54 mL (9.04 mmol) of DIPEA,
362 mg (1.13 mmol) of TBTU, and 128 mg (0.339 mmol) of HATU in
11.0 mL of methylene chloride. The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the trii-
peptide (328 mg, 60% yield). Rf: 0.5 (EtOAc/Hex 1:3); 1H NMR
(8.79 mmol) of L-ascorbic acid, 730 mg (8.79 mmol) of NaHCO3, and
124 mg (0.502 mmol) CuSO4$H2O in 35 mL MeOH/H2O (1:1). The
crude reaction was purified by reverse phase-HPLC to yield the
macrocycle (8.20 mg, 7% yield). Rf: 0.4 (EtOAc/Hex 1:1) 1H NMR
(400 MHz, CD3OD): d 0.71e0.80 (3H, m, CH(CH3)2), 0.83e1.12 (18H,
m, CH(CH3)2), 1.19e2.01 (1H, m, CH(CH3)2), 1.30e1.35 (1H, m,
CH(CH3)2), 1.50e1.57 (2H, m, CHCH2CH), 1.64e1.68 (2H, m,
CHCH2CH), 1.69e1.73 (2H, m, CHCH2CH), 1.84e1.88 (1H, m,
CH(CH3)2), 2.52e2.56 (1H, CH(CH3)2), 2.89e2.95 (2H, m, CH2Ph),
(400 MHz, CDCl3):
d 0.80e0.92 (12H, m, CH(CH3)2), 1.39 (9H, s,
COOC(CH3)3), 1.48e1.58 (2H, m, CHCH2CH), 1.63e1.72 (2H, m,
CHCH2CH), 1.81e1.90 (1H, m, CH2CH(CH2)3), 1.81 (1H, s, C^CH),
2.71 (3H, s, CH3N), 2.84e2.96 (2H, m, CHCH2Ph), 4.64e4.74 (1H, m,
2.99 (s, 3H, MeNC]O), 3.45e3.50 (1H, m,
a
CH), 3.70e3.80 (1H, m,
CH),
a
CH), 4.90e4.98 (1H, m,1H, m, CH), 5.01e5.12 (1H, m, 1H, m, a
a
7.15e7.31 (5H, m, Ph), 7.99 (1H, d, J 7.7 Hz, O]CNHCH), 8.06 (1H, s,
NCH]C), 8.37 (1H, d J 8.1 Hz, O]CNHCH). LCMS: m/z calcd for
C34H53N7O4 (Mþ1)¼624.8, found 625.3; HRMS (ESI-TOF): MHþ,
found 624.4257, C34H53N7O4 requires 624.4232 >95% pure by HPLC.
a
a
CH), 5.05e5.11 (1H, m,
CH), 5.95 (1H, d, J 8.17 Hz, NH), 7.23e7.32 (5H, m, Ph), 7.92 (1H, d, J
aCH), 5.31e5.51 (1H, dd, J 50.8, 7.7 Hz,
8.4 Hz, NH).
6.21.7. Tripeptide alkyne-Leu-Leu-N(Me)-Phe-NH2 (29). Tripeptide
alkyne-Leu-Leu-N(Me)-Phe-NH2 was synthesized following the
‘General solution-phase amine deprotection’. This tripeptide was
taken on to the next reaction without further purification or char-
acterization (262 mg, 100% yield).
6.22. Synthesis of compound 3 (A-Ox-III)
6.22.1. Dipeptide
MeO-Ser(Bzl)-Leu-NHBoc. Dipeptide
MeO-
Ser(Bzl)-Leu-NHBoc was synthesized following the ‘General solu-
tion-phase peptide synthesis’ procedure utilizing 756 mg
(3.39 mmol) of amine MeO-Ser(Bzl)-NH2 (39), 713 mg (3.08 mmol)
of acid HO-Leu-NHBoc (40), 2.10 mL (12.3 mmol) of DIPEA, 1.19 g
(3.70 mmol) of TBTU, in 31.0 mL of methylene chloride. This di-
peptide was taken on to the next reaction without further purifi-
cation (1.31 g, 98% yield). Rf: 0.73 (EtOAc/Hex 1:1); 1H NMR
6.21.8. Tetrapeptide alkyne-Leu-Leu-N(Me)-Phe-Leu-NHBoc. Tetra-
peptide alkyne-Leu-Leu-N(Me)-Phe-Leu-NHBoc was synthesized
following the ‘General solution-phase peptide synthesis’. Utilizing
262 mg (0.674 mmol) of amine 29 Alkyne-Leu-Leu-N(Me)-Phe-
NH2, 152 mg (0.612 mmol) of acid 30 HO-Leu-NHBoc, 0.805 mL

1040
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
(400 MHz, CDCl3):
1.41e1.48 (1H, m, CHCH3), 1.55e1.65 (2H, m, CH2CH), 3.56 (1H, d, J
4.0 Hz, CH2O), 3.65 (3H, s, OCH3), 3.76 (1H, dd, J 4.0, 7.8 Hz, CH2O),
4.22 (1H, br s, NH), 4.38 (2H, m, PhCH2O), 4.66e4.72 (1H, m,
5.49 (1H, d, J 7.8 Hz, CH), 7.14e7.21 (5H, m, Ph).
d
0.85 (6H, d, J 8.0 Hz, CHCH3), 1.36 (9H, s, CCH3),
procedure utilizing 469 mg (0.952 mmol) of acid HO-Leu-Phe-Leu-
NHBoc (38), 223 mg (1.05 mmol) of amine MeO-Oxazole-Leu-NH2
(42), 0.700 mL (3.81 mmol) of DIPEA, 366 mg (1.08 mmol) of TBTU,
in 11.0 mL of methylene chloride. The crude reaction was purified
by column chromatography (silica gel, EtOAc/Hex) to yield the
pentapeptide (468 mg, 72% yield). Rf: 0.35 (EtOAc/Hex 1:1); 1H
aCH),
a
6.22.2. Dipeptide MeO-Ser-Leu-NHBoc (41). Dipeptide MeO-Ser-
Leu-NHBoc was synthesized by dissolving 1.31 g (3.01 mmol) of
dipeptide MeO-Ser(OBn)-Leu-NHBoc in 30 mL of ethyl alcohol, af-
ter purging the reaction vessel several times with hydrogen gas, the
reaction was run overnight. Upon completion by TLC, this dipeptide
was taken on to the next reaction without further purification or
characterization (902 mg, 89% yield).
NMR (400 MHz, CDCl3):
CCH3), 1.47e1.68 (9H, m, CHCH3, CH2eCH), 2.98e3.24 (2H, m,
CH2O), 3.84 (3H, s, OCH3), 4.50e4.78 (2H, m, CH), 4.96e5.02 (1H,
m, CH), 5.17e5.31 (1H, m, CH), 7.07e7.28 (5H, m, Ph), 8.09 (1H, s,
OCH]C).
d 0.70e0.97 (18H, m, CHCH3), 1.40 (9H, s,
a
a
a
6.22.10. Macrocycle Phe-Leu-Oxazole-Leu-Leu (3). Macrocycle Phe-
Leu-Oxazole-Leu-Leu was synthesized following the ‘Macro-
cyclization procedure’ utilizing 349 mg (0.611 mmol) of linear
pentapeptide (43), 1.10 mL (6.11 mmol) of DIPEA, 138 mg
(0.431 mmol) of TBTU, 141 mg (0.375 mmol) of HATU, and 129 mg
(0.432 mmol) of DEPBT, in 9.20 mL methylene chloride and
9.00 mL acetonitrile. The crude reaction was purified by reverse
phase-HPLC to yield the macrocycle (1.3 mg, 0.4% yield). Rf: 0.21
6.22.3. Dipeptide
MeO-Oxazole-Leu-NHBoc. Dipeptide
MeO-
Oxazole-Leu-NHBoc was synthesized following the ‘General oxa-
zole synthesis’ procedure utilizing 0.902 g (2.70 mmol) of dipeptide
MeO-Ser-Leu-NHBoc, 0.390 mL (2.97 mmol) of DAST, 746 mg
(5.40 mmol) of K2CO3, in 40 mL of methylene chloride. The in-
termediate was oxidized into product by using 0.810 mL of DBU
(5.40 mmol), 0.53 mL of CBrCl3 (5.40 mmol), in 14 mL of methylene
chloride. This dipeptide was taken on to the next reaction without
further purification (649 mg, 77% yield). Rf: 0.55 (EtOAc/Hex 1:1).
Physical and spectroscopic data are consistent with those reported
in the literature.45
(EtOAc/Hex 1:1); 1H NMR (600 MHz, CD3OD):
m, CHCH3), 1.18e1.65 (9H, m, CHCH3, CH2CH), 2.97e3.27 (2H, m,
CH2O), 3.52e3.77 (2H, m, CH), 4.03e4.19 (1H, m, CH), 4.38e4.57
(1H, m, CH), 6.63 (1H, br s, NH), 7.03 (1H, br s, NH), 7.17e7.23 (5H,
d 0.69e0.96 (18H,
a
a
a
m, Ph), 7.79 (1H, s, OCH]C). LCMS: m/z calcd for C30H43N5O5
(Mþ1)¼554.6, found 554.3; HRMS (ESI-TOF): MHþ, found
554.3313, requires 554.3337 >90% pure by HPLC.
6.22.4. Dipeptide MeO-Oxazole-Leu-NH2 (42). Dipeptide MeO-
Oxazole-Leu-NH2 was synthesized following the ‘General solution-
phase amine deprotection’. This dipeptide was taken on to the next
reaction without further purification or characterization (210 mg,
100% yield).
6.23. Synthesis of compound 4 (B-Ox-III)
6.23.1. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-Leu-
NHBoc was synthesized following the ‘General solution-phase pep-
tide synthesis’ procedure. Utilizing 850 mg (4.80 mmol) of amine
MeO-Phe-NH2, 1.00 g (4.30 mmol) of acid HO-Leu-NHBoc, 2.90 mL
(17.2 mmol) of DIPEA, 1.66 g (5.20 mmol) of TBTU, in 43.2 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(1.64 g, 97% yield). Rf: 0.3 (EtOAc/Hex 1:3); 1H NMR (400 MHz,
6.22.5. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-Leu-
NHBoc was synthesized following the ‘General solution-phase pep-
tide synthesis’ procedure utilizing 850 mg (4.76 mmol) of amine
MeO-Phe-NH2 (35), 1.00 g (4.32 mmol) of acid HO-Leu-NHBoc (34),
2.90 mL (17.3 mmol) of DIPEA, 1.66 g (5.18 mmol) of TBTU, in
43.0 mL of methylene chloride. This dipeptide was taken on to the
next reaction without further purification (1.64 g, 97% yield). Rf:
0.85 (EtOAc/Hex 1:1). Physical and spectroscopic data are consis-
tent with those reported in the literature.45
CDCl3):
1.35e1.40 (1H, m, CH(CH3)2), 1.45e1.60 (2H, m,
(2H, m, CH2), 3.59 (3H, s, OCH3), 4.00e4.10 (1H, br,
4.70e4.78 (1H, m, CH), 5.25e5.35 (1H, br, NH), 6.86e6.96 (1H, d, J
d
0.78e0.84 (6H, m, CH(CH3)2), 1.33 (9H, s, C(CH3)3),
CH2), 2.90e3.05
CH),
b
b
a
a
6.22.6. Dipeptide HO-Phe-Leu-NHBoc (36). Dipeptide HO-Leu-
D
-
7.8 Hz, NH), 7.00e7.20 (5H, m, Ph).
Leu-NHBoc was synthesized following the ‘General solution-phase
acid deprotection’. This tripeptide was taken on to the next reaction
without further purification or characterization (1.40 g, 85% yield).
6.23.2. Dipeptide HO-Phe-Leu-NHBoc. Dipeptide HO-Phe-Leu-
NHBoc was synthesized following the ‘General solution-phase acid
deprotection’. This dipeptide was taken on to the next reaction
without further purification or characterization (1.35 g, 85% yield).
6.22.7. Tripeptide MeO-Leu-Phe-Leu-NHBoc. Tripeptide MeO-Leu-
Phe-Leu-NHBoc was synthesized following the ‘General solution-
phase peptide synthesis’ procedure utilizing 757 mg (2.00 mmol) of
acid HO-Phe-Leu-NHBoc, 320 mg (2.20 mmol) of amine MeO-Leu-
NH2 (37), 1.40 mL (8.00 mmol) of DIPEA, 706 mg (2.40 mmol) of
TBTU, in 20 mL of methylene chloride. The crude reaction was pu-
rified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (890 mg, 88% yield). Rf: 0.55 (EtOAc/Hex 1:1). Phys-
ical and spectroscopic data are consistent with those reported in
the literature.46
6.23.3. Tripeptide
MeO-Leu-N(Me)-Phe-Leu-NHBoc. Tripeptide
MeO-Leu-N(Me)-Phe-Leu-NHBoc was synthesized following the
‘General solution-phase peptide synthesis’ procedure. Utilizing
338 mg (2.10 mmol) of amine MeO-Leu-N(Me)H, 731 mg
(1.90 mmol) of acid HO-Phe-Leu-NHBoc, 1.35 mL (5.70 mmol) of
DIPEA, 744 mg (2.30 mmol) of TBTU, in 19.3 mL of methylene
chloride. The crude reaction was purified by column chromatog-
raphy (silica gel, EtOAc/Hex) to yield the tripeptide (602 mg, 60%
yield). Rf: 0.22 (EtOAc/Hex 1:1); 1H NMR (400 MHz, CDCl3):
6.22.8. Tripeptide HO-Leu-Phe-Leu-NHBoc (38). Tripeptide HO-Leu-
d
0.84e0.98 (12H, m, CH(CH3)2), 1.34e1.42 (2H, m, CH(CH3)2), 1.47
(9H, s, C(CH3)3), 1.54e1.75 (4H, m, CH2), 2.80 (3H, s, NCH3),
2.94e3.20 (2H, m, CH2), 3.68 (3H, s, OCH3), 4.05e4.15 (1H, br, NH),
4.76e4.84 (1H, br, NH), 4.84e4.88 (1H, m, CH), 5.10e5.20 (1H, m,
CH), 5.25e5.32 (1H, m, CH), 7.10e7.33 (5H, m, Ph).
D-Leu-NHBoc was synthesized following the ‘General solution-phase
b
acid deprotection’. This tripeptide was taken on to the next reaction
without further purification or characterization (709 mg, 82% yield).
b
a
a
a
6.22.9. Pentapeptide MeO-Oxazole-Leu-Leu-Phe-Leu-NHBoc
(43). Pentapeptide MeO-Oxazole-Leu-Leu-Phe-Leu-NHBoc was
synthesized following the ‘General solution-phase peptide synthesis’
6.23.4. Tripeptide HO-Leu-N(Me)-Phe-Leu-NHBoc. Tripeptide HO-
Leu-N(Me)-Phe-Leu-NHBoc was synthesized following the ‘General

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1041
solution-phase acid deprotection’. This tripeptide was taken on to the
next reaction without further purification or characterization
(470 mg, 80% yield).
8.08e8.20 (1H, m, OCH]C). LCMS: m/z calcd for C37H57N5O8
(Mþ1)¼699.88, found 699.8.
6.23.10. Macrocycle Phe-Leu-Oxazole-Leu-Leu-N(Me) (4). Macro-
cycle Phe-Leu-Oxazole-Leu-Leu-N(Me) (4) was synthesized fol-
lowing the ‘Macrocyclization procedure’. Utilizing 281 mg
(0.400 mmol) of linear pentapeptide, 0.420 mL (2.4 mmol) of
DIPEA, 90.0 mg (0.280 mmol) of TBTU, 107 mg (0.280 mmol) HATU,
and 83.8 mg (0.280 mmol) of DEPBT in 571 mL methylene chloride.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) and reverse phase-HPLC to yield the macrocycle
(17.3 mg, 6.4% yield). Rf: 0.42 (EtOAc/Hex 1:1) 1H NMR (400 MHz,
6.23.5. Dipeptide
MeO-Ser(Bzl)-Leu-NHBoc. Dipeptide
MeO-
Ser(Bzl)-Leu-NHBoc was synthesized following the ‘General solu-
tion-phase peptide synthesis’ procedure. Utilizing 756 mg
(3.40 mmol) of amine MeO-Ser(Bzl)-NH2, 713 mg (3.10 mmol) of
acid HO-Leu-NHBoc, 2.10 mL (12.4 mmol) of DIPEA, 1.19 g
(3.70 mmol) of TBTU, in 30.8 mL of methylene chloride. The crude
reaction was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the dipeptide (1.31 g, 97% yield). Rf: 0.76 (EtOAc/Hex
1:1); 1H NMR (400 MHz, CDCl3):
(CHCH3)2), 0.96e0.97 (3H, d, J 3.5 Hz, (CHCH3)2), 1.43 (9H, s,
C(CH3)2), 1.61e1.63 (1H, m, CH(CH3)2), 1.63e1.78 (2H, m, CH2),
3.66e3.70 (1H, dd, J 6.9, 1.5 Hz, CH), 3.75 (3H, s, OCH3), 3.88e3.92
(1H, dd, J 7.1, 1.5 Hz, CH), 4.14e4.21 (1H, br, CH), 4.46e4.59 (2H, q,
CH2OBn), 4.72e4.76 (1H, m, CH), 4.84e4.91 (1H, br, NH),
d
0.95e0.96 (3H, d, J 3.5 Hz,
CDCl3): d 0.75e0.80 (3H, d, J 3.5 Hz, CH(CH3)2), 0.84e0.89 (3H, d, J
3.5 Hz, CH(CH3)2), 0.90e1.00 (21H, br m, C(CH3)3 and CH(CH3)2),
1.08e1.20 (1H, m, CH(CH3)2), 1.43e1.50 (2H, m, CH(CH3)2),
b
b
1.50e1.64 (2H, m,
m, NCH3), 2.98e3.15 (2H, m, b
b
CH2), 1.65e1.80 (4H, m,
CH2), 4.15e4.45 (3H, br, NH and
CH), 4.88e4.98 (1H, br, NH), 4.99e5.10 (2H, m, CH Leu-Ox and
CH Phe), 6.93e7.03 (1H, m, Ph), 7.33e7.38 (4H, m, Ph), 8.15 (1H, s,
bCH2), 2.56e2.66 (3H,
b
a
a
2a
a
6.76e6.80 (1H, br, NH), 7.26e7.40 (5H, m, Ph).
a
OCH]C). LCMS: m/z calcd for C31H45N5O5 (M)¼567.72, found
567.8; HRMS (ESI-TOF): MHþ, found 568.3520, C31H45N5O5 requires
568.3493 >90% pure by HPLC.
6.23.6. Dipeptide MeO-Ser-Leu-NHBoc. Dipeptide MeO-Ser-Leu-
NHBoc was synthesized by dissolving 1.28 g (3.03 mmol) of di-
peptide MeO-Ser(Bzl)-Leu-NHBoc in 30.0 mL EtOH (0.1 M). The
reaction mixture was hydrogenated using a catalytic amount of
Pd/C and excess H2 for 24 h. The reaction was filtered over Celite
to yield pure dipeptide (900 mg, 89% yield). Rf: 0.28 (EtOAc/Hex
1:1).
6.24. Synthesis of compound 5 (C-Ox-III)
6.24.1. Dipeptide
MeO-Lys(Cbz)-Phe-NHBoc. Dipeptide
MeO-
Lys(Cbz)-Phe-NHBoc was synthesized following the ‘General solu-
tion-phase peptide synthesis’ procedure. Utilizing 294 mg
(1.00 mmol) of amine MeO-Lys(Cbz)eNH2, 241 mg (0.910 mmol) of
acid HO-Phe-NHBoc, 1.30 mL (7.28 mmol) of DIPEA, 235 mg
(0.730 mmol) of TBTU, 209 mg (0.550 mmol) of HATU, in 9.00 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(380 mg, 77% yield). Rf: 0.37 (EtOAc/Hex 1:1) 1H NMR (400 MHz,
6.23.7. Dipeptide
MeO-Oxazole-Leu-NHBoc. Dipeptide
MeO-
Oxazole-Leu-NHBoc was synthesized following the ‘General oxa-
zole synthesis’ procedure. The oxazoline intermediate was synthe-
sized utilizing 900 mg (2.72 mmol) of dipeptide MeO-Ser-Leu-
NHBoc, 0.390 mL (2.97 mmol) of DAST, 746 mg (5.40 mmol) of
K2CO3 in 40.0 mL of methylene chloride. The resulting oxazoline
was oxidized utilizing 0.810 mL (5.40 mmol) of DBU, 0.530 mL
(5.40 mmol) of CBrCl3 in 40.0 mL of methylene chloride. The crude
reaction was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the desired peptidyl-oxazole (649 mg, 77% yield over
two steps). Rf: 0.5 (EtOAc/Hex 7:13); 1H NMR (400 MHz, CDCl3):
CDCl3):
1.36e1.64 (2H, m,
(2H, m, CH2 Lys), 3.03e3.16 (2H, m,
4.21e4.35 (1H, m, CH), 4.42e4.52 (1H, m,
d
1.11e1.49 (2H, m,
CH2 Lys), 1.51e1.81 (2H, m,
CH2), 3.60e3.65 (3H, s, OCH3),
CH), 4.85e4.93 (1H, br,
g
CH2 Lys), 1.30e1.36 (9H, s, C(CH3)3),
d
bCH2 Lys), 2.93e3.03
3
b
a
a
NH), 5.00e5.05 (2H, s, CH2Ph), 6.39e6.47 (1H, br, NH), 7.08e7.32
(10H, m, Ph).
d
0.94e0.96 (3H, d, J 3.5 Hz, CHCH3), 0.96e0.98 (3H, d, J 3.5 Hz,
CHCH3), 1.43 (9H, s, C(CH3)3), 1.58e1.64 (1H, m, CH(CH3)2),
1.64e1.80 (2H, m, CH2), 3.93 (3H, s, OCH3), 4.94e5.04 (1H, br, CH),
5.04e5.10 (1H, br, NH), 8.18 (1H, s, OCH]C).
6.24.2. Dipeptide MeO-Lys-Cbz-Phe-NH2. Dipeptide MeO-Lys(Cbz)-
Phe-NH2 was synthesized following the ‘General solution-phase
amine deprotection’. This dipeptide was taken on to the next re-
action without further purification or characterization (309 mg,
100% yield).
b
a
6.23.8. Dipeptide MeO-Oxazole-Leu-NH2. Dipeptide MeO-Oxazole-
Leu-NH2 was synthesized following the ‘General solution-phase
amine deprotection’. This dipeptide was taken on to the next re-
action without further purification or characterization (212 mg,
100% yield).
6.24.3. Tripeptide MeO-Lys(Cbz)-Phe-D-Phe-N(Me)Boc. Tripeptide
MeO-Lys(Cbz)-Phe- -Phe-N(Me)Boc was synthesized following the
D
‘General solution-phase peptide synthesis’ procedure. Utilizing
309 mg (0.700 mmol) of amine MeO-Lys(Cbz)-Phe-NH2, 215 mg
(0.770 mmol) of acid HO-D-Phe-N(Me)Boc, 0.980 mL (4.9 mmol) of
DIPEA, 180 mg (0.560 mmol) of TBTU, 133 mg (0.350 mmol) of
HATU, in 7.00 mL of methylene chloride. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (454 mg, 93% yield). Rf: 0.38 (EtOAc/Hex 7:13); 1H
6.23.9. Pentapeptide MeO-Oxazole-Leu-Leu-N(Me)-Phe-Leu-NHBoc.
Pentapeptide MeO-Oxazole-Leu-Leu-N(Me)-Phe-Leu-NHBoc was
synthesized following the ‘General solution-phase peptide synthesis’
procedure. Utilizing 210 mg (0.990 mmol) of amine MeO-Oxazole-
Leu-NH2, 470 mg (0.930 mmol) of acid HO-Leu-N(Me)-Phe-Leu-
NHBoc, 0.650 mL (3.72 mmol) of DIPEA, 353 mg (1.10 mmol) of
TBTU, in 10.0 mL of methylene chloride. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the pentapeptide (281 mg, 43% yield). Rf: 0.47 (EtOAc/Hex 1:1); 1H
NMR (400 MHz, CDCl3):
(9H, s, C(CH3)3), 1.35e1.48 (2H, m,
Lys), 2.51e2.73 (3H, s, NCH3), 2.82e3.02 (2H, m,
(2H, m, CH2 Lys), 3.16e3.34 (2H, m, CH2), 3.59e3.70 (3H, s, OCH3),
4.34e4.46 (1H, br, CH), 4.47e4.68 (2H, m, 2 CH), 4.98e5.08 (2H, s,
CH2Ph), 6.22e6.61 (2H, br m, NH), 6.98e7.34 (15H, m, Ph).
d
1.10e1.22 (2H, m,
CH2 Lys), 1.51e1.83 (2H, m,
CH2), 3.02e3.15
g
CH2 Lys), 1.22e1.30
d
bCH2
b
3
b
NMR (400 MHz, CDCl3):
(6H, m, CH2), 1.35e1.44 (9H, m, C(CH3)3), 1.57e1.73 (2H, m,
CH(CH3)2), 1.73e1.94 (1H, m, CH(CH3)2), 2.69 (3H, m, NCH3),
2.84e3.23 (2H, m, CH2), 3.91 (3H, m, OCH3), 4.04e4.15 (2H, br,
NH), 4.60e4.70 (1H, br, NH), 4.78e4.96 (2H, m, CH), 5.14e5.28
(1H, m, CH), 5.28e5.38 (1H, m, CH), 7.08e7.30 (5H, m, Ph),
d 0.80e1.02 (18H, m, CH(CH3)2), 1.22e1.57
a
a
b
b
6.24.4. Tripeptide
HO-Lys(Cbz)-Phe-
D-Phe-N(Me)Boc. Tripeptide
a
HO-Lys(Cbz)-Phe-
D-Phe-N(Me)Boc was synthesized following the
a
a
‘General solution-phase acid deprotection’. This tripeptide was taken

1042
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
on to the next reaction without further purification or character-
ization (448 mg, 100% yield).
6.91e7.44 (15H, m, Ph), 8.09 (1H, s, OCH]C). LCMS: m/z calcd for
48H62N6O10 (M)¼883.04, found 883.
C
6.24.5. Dipeptide
MeO-Ser(Bzl)-Leu-NHBoc. Dipeptide
MeO-
6.24.10. Macrocycle Phe-D-Phe-N(Me)-Oxazole-Leu-Lys(Cbz) (5).
Ser(Bzl)-Leu-NHBoc was synthesized following the ‘General solu-
tion-phase peptide synthesis’ procedure. Utilizing 383 mg
(1.83 mmol) of amine MeO-Ser(Bzl)-NH2, 414 mg (1.66 mmol) of
acid HO-Leu-NHBoc, 1.20 mL (6.64 mmol) of DIPEA, 588 mg
(1.83 mmol) of TBTU, in 17.0 mL of methylene chloride. The crude
reaction was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the dipeptide (701 mg, 100% yield). Rf: 0.76 (EtOAc/
Macrocycle Phe- -Phe-N(Me)-Oxazole-Leu-Lys(Cbz) (5) was syn-
D
thesized following the ‘Macrocyclization procedure’. Utilizing
238 mg (0.310 mmol) of linear pentapeptide, 0.430 mL (2.48 mmol)
of DIPEA, 80.0 mg (0.250 mmol) of TBTU, 95.0 mg (0.250 mmol)
HATU, and 75.0 mg (0.250 mmol) of DEPBT in 44.3 mL methylene
chloride. The crude reaction was purified by column chromatog-
raphy (silica gel, EtOAc/Hex) and reverse phase-HPLC to yield the
macrocycle (19.8 mg, 8.5% yield). Rf: 0.18 (EtOAc/Hex 1:1); 1H NMR
Hex 1:1). 1H NMR (400 MHz, CDCl3):
CHCH3), 0.96e0.97 (3H, d, J 3.5 Hz, CHCH3), 1.42e1.46 (9H, s, C(CH3)
3), 1.61e1.63 (1H, m, CH(CH3)2), 1.63e1.78 (2H, m, CH2), 3.66e3.70
CH Ser), 3.73e3.76 (3H, s, OCH3), 3.88e3.92
CH), 4.14e4.21 (1H, br, CH), 4.46e4.59 (2H,
CH), 4.84e4.91 (1H, br, NH),
d 0.95e0.96 (3H, d, J 3.5 Hz,
(400 MHz, CDCl3):
0.89e0.94 (3H, d, J 15.7 Hz, CH(CH3)2), 1.03e1.19 (2H, br,
1.33e1.58 (2H, m, CH2 Lys), 1.58e1.70 (2H, m, CH2 Lys), 1.86e2.03
(2H, m, CH2), 2.17e2.33 (1H, m, CH(CH3)2), 2.62e2.67 (3H, s,
NCH3), 2.82e3.21 (2H, m, CH2), 3.01e3.11 (2H, m, CH2 Lys),
3.35e3.44 (1H, dd, J 8.85, 2.92 Hz, CHaHbb Phe), 3.46e3.56 (1H, br,
CH), 4.58e4.65 (1H, dd, J 8.85, 2.94 Hz, CHaHb Phe), 4.65e4.78
(2H, m, 2 CH), 5.01e5.06 (2H, m, CH2Ph), 5.14e5.24 (1H, m, CH),
d
0.82e0.88 (3H, d, J 15.7 Hz, CH(CH3)2),
b
g
CH2 Lys),
(1H, dd, J 7.2, 1.5 Hz,
(1H, dd, J 7.9 1.5 Hz,
b
b
d
b
a
b
m, CH2OBn), 4.72e4.76 (1H, m,
a
b
3
6.76e6.80 (1H, br, NH), 7.26e7.40 (5H, m, Ph).
a
b
6.24.6. Dipeptide MeO-Ser-Leu-NHBoc. Dipeptide MeO-Ser-Leu-
NHBoc was synthesized by dissolving 701 mg (1.66 mmol) of di-
peptide MeO-Ser(Bzl)-Leu-NHBoc in 4.00 mL EtOH (0.48 M). The
reaction mixture was hydrogenated using a catalytic amount of Pd/
C and excess H2 for 24 h. The reaction was filtered over Celite to
yield pure dipeptide (521 mg, 95% yield). Rf: 0.28 (EtOAc/Hex 1:1).
a
a
6.76e6.93 (3H, m, NH), 7.07e7.33 (15H, m, Ph), 8.00e8.08 (1H, s,
OCH]C). LCMS: m/z calcd for C42H50N6O7 (M)¼750.88, found 751;
HRMS (ESI-TOF): MHþ, found 751.3821, C42H50N6O7 requires
751.3814 >95% pure by HPLC.
6.25. Synthesis of compound 6 (D-Ox-III)
6.24.7. Dipeptide
MeO-Oxazole-Leu-NHBoc. Dipeptide
MeO-
Oxazole-Leu-NHBoc was synthesized following the ‘General oxa-
zole synthesis’ procedure. The oxazoline intermediate was syn-
thesized utilizing 421 mg (1.27 mmol) of dipeptide MeO-Ser-Leu-
NHBoc, 0.170 mL (1.40 mmol) of DAST, 351 mg (2.54 mmol) of
K2CO3 in 12.7 mL of methylene chloride. The resulting oxazoline
was oxidized utilizing 0.380 mL (2.54 mmol) of DBU, 0.250 mL
(2.54 mmol) of CBrCl3 in 12.7 mL of methylene chloride. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the desired peptidyl-oxazole (298 mg,
76% yield over two steps). Rf: 0.5 (EtOAc/Hex 7:13); 1H NMR
6.25.1. Dipeptide NH2-(2R,3R)/(2S,3S)-
mixture of NH2- -Phe-O-Resin (2.00 g, 1.44 mmol), (2R,3R)/
(2S,3S)-racemic Fmoc- -OH-Phe-OH (1.74 g, 4.32 mmol), HOBt
b-OH-Phe-D-Phe-O-Resin. A
D
b
(661 mg, 4.32 mmol), and DIC (1.34 mL, 8.64 mmol) were stirred
at room temperature for 3 h following the ‘General solid-phase
peptide synthesis’ procedure. Completion of the coupling reaction
was verified by a negative ninhydrin test. The reaction mixture
was then drained to leave the amine-protected resin-bound di-
peptide. Deprotection of amine was performed following the
‘General solid-phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal and gave the title
compound.
(400 MHz, CDCl3):
0.96e0.98 (3H, d, J 3.5 Hz, CHCH3), 1.42e1.46 (9H, s, C(CH3)3),
1.58e1.64 (1H, m, CH(CH3)2), 1.64e1.80 (2H, m, CH2), 3.93 (3H,
CH), 5.04e5.10 (1H, br, NH), 8.18
d 0.94e0.96 (3H, d, J 3.5 Hz, CHCH3),
b
s, OCH3), 4.94e5.04 (1H, br,
(1H, s, OCH]C).
a
6.25.2. Tripeptide Boc-Leu-(2R,3R)/(2S,3S)-
b-OH-Phe-D-Phe-OH. A
mixture of NH2-(2R,3R)/(2S,3S)- -OH- -Phe-O-Resin (1.44 mmol),
b
D
6.24.8. Dipeptide MeO-Oxazole-Leu-NH2. Dipeptide MeO-Oxazole-
Leu-NH2 was synthesized following the ‘General solution-phase
amine deprotection’. This dipeptide was taken on to the next re-
action without further purification or characterization (202 mg,
100% yield).
Boc-Leu-OH residue (1.00 g, 4.32 mmol), HOBt (661 mg,
4.32 mmol), and DIC (1.34 mL, 8.64 mmol) were stirred at room
temperature for 3 h following the ‘General solid-phase peptide syn-
thesis’ procedure. Completion of the coupling reaction was verified
by a negative ninhydrin test. The reaction mixture was then drained
and dried in vacuo overnight to leave the amine-protected resin-
bound tripeptide. The tripeptide was cleaved from the resin fol-
lowing the ‘Cleavage of linear peptide from solid support’ procedure.
6.24.9. Pentapeptide MeO-Oxazole-Leu-Lys(Cbz)-Phe-
D
-Phe-N(Me)
Boc. Pentapeptide MeO-Oxazole-Leu-Lys(Cbz)-Phe-
D-Phe-N(Me)
Boc was synthesized following the ‘General solution-phase peptide
synthesis’ procedure. Utilizing 152 mg (0.720 mmol) of amine MeO-
Oxazole-Leu-NH2, 447 mg (0.650 mmol) of acid HO-Lys(Cbz)-Phe-
A resin slurry of Boc-Leu-(2R,3R)/(2S,3S)-b-OH-Phe-D-Phe-O-Resin
(2.50 g), 2,2,2-trifluoroethanol (12.5 mL) and of methylene chloride
(12.5) was stirred for 24 h, after which it was filtered, washed with
additional methylene chloride, and dried in vacuo for 24 h (596 mg,
76% yield).
D-Phe-N(Me)Boc, 0.910 mL (5.2 mmol) of DIPEA, 167 mg
(0.520 mmol) of TBTU, 148 mg (0.390 mmol) of HATU in 7.00 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide
(274 mg, 48% yield). Rf: 0.2 (EtOAc/Hex 1:1) 1H NMR (400 MHz,
6.25.3. Dipeptide MeO-Ser(Bzl)-
D-Leu-NHBoc. A mixture of amine
MeO-Ser(Bzl)eNH2 (709 mg, 3.39 mmol), acid HO-
D
-Leu-NHBoc
CDCl3):
1.23e1.27 (9H, s, C(CH3)3), 1.33e1.48 (2H, m,
(2H, m, CH2 Lys), 1.64e1.87 (3H, m, CH(CH3)2, CH2b Leu), 2.35 (3H,
m, NCH3), 2.85e3.05 (2H, m, CH2), 3.05e3.19 (2H, m, CH2 Lys),
CH2), 3.75 (3H, s, OCH3), 4.21e4.43 (1H, br, CH),
CH), 4.46e4.70 (1H, m, CH), 4.95e5.06 (2H, m,
CH, NH), 6.28e6.78 (2H, m, NH),
d
0.78e0.92 (6H, m, CH(CH3)2), 1.10e1.31 (2H, m,
g
CH2 Lys),
(767 mg, 3.08 mmol), DIPEA (4.30 mL, 2.46 mmol), TBTU (1.19 g,
3.94 mmol) in methylene chloride (30.8 mL) was stirred at room
temperature under an argon atmosphere for 2.5 h following the
‘General solution-phase peptide synthesis’ procedure. The crude
reaction was purified by column chromatography (20% EtOAc/
Hex) to yield the title compound (1.24 g, 96% yield). Rf: 0.78 (50%
dCH2 Lys), 1.48e1.60
b
b
3
3.19e3.32 (2H, m,
4.33e4.55 (1H, br,
CH2Ph), 5.06e5.26 (2H, m,
b
a
a
a
a
EtOAc/Hex); 1H NMR (200 MHz, CDCl3):
d 0.9e0.95 (6H, m,

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1043
CH(CH3)2), 1.43 (9H, s, C(CH3)3), 1.63e1.73 (2H, m, CHCH2CH),
3.65e3.70 (1H, dd, J 2.8, 8.9 Hz, OCHaHb), 3.73 (3H, s, OCH3),
3.86e3.91 (1H, dd, J 3.0, 9.0 Hz, OCHaHb), 4.18e4.26 (1H, br,
8.8 Hz, NH), 6.95 (2H, m, 2NH), 7.01 (1H, d, J 8.5 Hz, NH), 7.07 (1H, d,
J 6.1 Hz, NH), 7.12e7.18 (6H, m, 4CCHCH, 2CHCHCH), 7.22e7.34
(10H, m, Ph), 7.4 (4H, m, 4CHCHCH), 7.60 (2H, d, J 7.2 Hz, NH), 8.19
(2H, s, CCHO). LCMS (ESI): m/z called for C39H53N5O9 (Mþ)¼735.4,
found 735.8.
a
CH), 4.45e4.54 (2H, q, J 11.9 Hz, CH2Ph), 4.70e4.75 (1H, m,
CH), 4.95e5.07 (1H, br, NH), 7.01 (1H, br, NH), 7.23e7.36 (5H,
a
m, Ph).
6.25.8. Deprotected pentapeptide HO-Oxazole-
(2S,3S)- -OH-Leu-NH2. A mixture of pentapeptide MeO-Oxazole-
Leu- -Phe-(2R,3R)/(2S,3S)- -OH-Leu-NHBoc (100 mg, 0.136 mmol),
lithium hydroxide (17.0 mg, 0.408 mmol), and 30% hydrogen per-
oxide (14.0 L) in methanol (340 L) was stirred at room temper-
ature under open atmosphere for 12 h following the ‘General
solution-phase acid deprotection’. Upon completion, the reaction
was diluted with methylene chloride (50 mL) and quenched with
sodium thiosulfate (154 mg) in pH 1 hydrochloric acid solution
(200 mL). The organic layer was separated and the aqueous layer
was back extracted with ethyl acetate. The combined organic layers
were dried (Na2SO4) and concentrated in vacuo to yield the
D-Leu-D-Phe-(2R,3R)/
6.25.4. Dipeptide MeO-Ser- -Leu-NHBoc. A mixture of dipeptide
MeO-Ser(Bzl)-D-Leu-NHBoc (1.24 g, 2.94 mmol) and a catalytic
D
b
D-
D
b
amount of Pd/C in ethanol (29.4 mL) was stirred under a hydrogen
atmosphere for 24 h. Upon completion, confirmed by TLC, the re-
action mixture was filtered over Celite to yield the title compound
(839 mg, 86% yield). Rf: 0.44 (50% EtOAc/Hex). Physical and spec-
troscopic data are consistent with those reported in the literature.47
m
m
6.25.5. Dipeptide MeO-Oxazole-
Oxazole- -Leu-NHBoc was synthesized following the ‘General
oxazole synthesis’ procedure. The oxazoline intermediate was
synthesized utilizing dipeptide MeO-Ser- -Leu-NHBoc (819 mg,
2.46 mmol), DAST (357 L, 2.71 mmol), K2CO3 (664 mg,
4.92 mmol) in methylene chloride (24.6 mL). The resulting oxa-
zoline was oxidized utilizing DBU (746 L, 4.92 mmol), CBrCl3
(489 L, 4.92 mmol) in methylene chloride (12.3 mL). The crude
D
-Leu-NHBoc. Dipeptide MeO-
D
D
deprotected acid HO-Oxazole-
Leu-NHBoc. Deprotection of the amine resulted from a mixture of
acid HO-Oxazole- -Leu- -Phe-(2R,3R)/(2S,3S)- -OH-Leu-NHBoc
(76.0 mg, 0.105 mmol), anisole (23.0 L, 0.211 mmol) in TFA
(211 L) and methylene chloride (842 L) stirred at room temper-
D-Leu-D-Phe-(2R,3R)/(2S,3S)-b-OH-
m
D
D
b
m
m
m
m
m
reaction was purified by column chromatography (40% EtOAc/
Hex) to yield the desired title compound (516 mg, 70% yield over
two steps). Rf: 0.80 (50% EtOAc/Hex); 1H NMR (200 MHz, CDCl3):
ature for 40 min under open atmosphere following the ‘General
solution-phase amine deprotection’. Reaction completion was con-
firmed by TLC and the mixture was concentrated in vacuo with
several washes with methylene chloride. This deprotected penta-
d
0.86 (3H, d, J 1.6 Hz, CHCH3), 0.87 (3H, d, J 1.8 Hz, CHCH3), 1.35
(9H, s, C(CH3)3), 1.53e1.61 (1H, m, CH(CH3)2), 1.63e1.71 (2H, m,
peptide HO-Oxazole-D-Leu-D-Phe-(2R,3R)/(2S,3S)-b-OH-Leu-NH2
CHCH2CH), 3.83 (3H, s, OCH3), 4.87e4.95 (1H, br,
(1H, br, NH), 8.11 (1H, s, CCHO).
aCH), 5.08e5.14
was taken on to the next reaction without further purification or
characterization (65.0 mg, 77% yield over two steps). LCMS (ESI): m/
z called for C33H43N5O7 (Mþ2Hþ)¼623.7, found 623.8.
6.25.6. Dipeptide MeO-Oxazole-D-Leu-NH2. A mixture of peptidyl
MeO-Oxazole- -Leu-NHBoc (516 mg, 1.65 mmol) in TFA (3.30 mL)
D
6.25.9. Macrocycle (2R,3R)-
(6). A mixture of TBTU (41.0 mg, 0.126 mmol), HATU (36.0 mg,
0.948 mol), and DIPEA (147 L, 0.842 mmol) in methylene chlo-
ride (6.77 mL) and acetonitrile (4.53 mL) stirred at room tempera-
ture in a round bottom flask under an argon atmosphere following
the ‘Macrocyclization procedure’. The deprotected pentapeptide HO-
b-benzoxy-Phe-Leu-Oxazole-D-Leu-D-Phe
and methylene chloride (13.2 mL) was stirred at room temperature
under open atmosphere for 30 min following the ‘General solution-
phase amine deprotection’. Reaction completion was confirmed by
TLC and the mixture was concentrated in vacuo with several
washes with methylene chloride. This dipeptide was taken on to
the next reaction without further purification or characterization
(350 mg, quantitative yield).
m
m
Oxazole-D-Leu-
D
-Phe-(2R,3R)/(2S,3S)-
b-OH-Leu-NH2
(65.0 mg,
0.105 mmol) was dissolved in methylene chloride (2.26 mL) and
acetonitrile (1.49 mL) and added to the reaction flask via syringe
pump at the rate of 15 mL/h. After 12 h, the reaction was diluted
with methylene chloride (200 mL) and quenched with ammonium
chloride solution (200 mL, satd aq). The organic layer was sepa-
rated, dried (Na2SO4), and concentrated in vacuo. The crude re-
action was semi-purified by reverse phase-HPLC to yield the
6.25.7. Pentapeptide MeO-Oxazole-
OH-Leu-NHBoc. A mixture of amine MeO-Oxazole-
(257 mg, 1.21 mmol), acid Boc-Leu-(2R,3R)/(2S,3S)- -OH-Phe-
D
-Leu-
D
-Phe-(2R,3R)/(2S,3S)-
b
-
D-Leu-NH2
b
D
-
Phe-OH (596 mg, 1.1 mmol), TBTU (424 mg, 1.32 mmol), HATU
(301 mg, 7.92 mmol), and DIPEA (2.11 mL, 12.1 mmol) in methylene
chloride (12.1 mL) was stirred at room temperature under an argon
atmosphere for 3 h following the ‘General solution-phase peptide
synthesis’ procedure. Upon completion, confirmed by TLC, the re-
action mixture was diluted with methylene chloride (200 mL),
quenched by the addition of 10% hydrochloric acid solution
(200 mL), and further washed with sodium bicarbonate solution
(500 mL, satd aq) and then brine (200 mL). The organic layer was
dried (Na2SO4) and the solvent was evaporated in vacuo. The crude
reaction was purified by column chromatography (90% EtOAc/Hex)
to yield the title compound (490 mg, 61% yield). Rf: 0.40 (50% EtOAc/
macrocycle
(2R,3R)/(2S,3S)-b-OH-Phe-Leu-Oxazole-D-Leu-D-Phe
(19 mg, 29% yield). Rf: 0.2 (65% EtOAc/Hex); LCMS: m/z called for
C33H41N5O6 (Mþ1)¼604.7, found 603.9 Finally, a mixture of mac-
rocycle (2R,3R)-
0.298
(14.0
b-benzoxy-Phe-Leu-Oxazole-D-Leu-D-Phe (18.0 mg,
m
mol), NaH (1.50 mg, 0.596
mmol), and benzyl bromide
m
L, 0.0119 mmol) in THF (1.19 mL) stirred at room tempera-
ture for 12 h under an atmosphere of argon following the ‘Benzy-
lation procedure’. The crude reaction was purified by reverse phase-
HPLC to yield the title compound 6 (1.0 mg, 0.4%); stereochemistry
was assigned previously.1 Rf: 0.40 (65% EtOAc/Hex); >90% pure by
Hex); 1H NMR (400 MHz, CDCl3):
d 0.76e0.81 (9H, m, 3CHCH3), 0.84
HPLC 1H NMR (600 MHz, CD3OD):
d 0.79 (3H, d, J 6.6 Hz, CHCH3),
(3H, d, J 4.0 Hz, CHCH3), 0.89 (6H, d, J 3.4 Hz, 2CHCH3), 0.91 (3H, d, J
3.5 Hz, CHCH3), 1.38e1.42 (4H, m, buried, 2CH(CH3)2), 1.39 (9H, s,
C(CH3)3), 1.40 (9H, s, C(CH3)3), 1.63 (2H, m, CHCH2CH), 1.74 (2H, m,
CHCH2CH), 2.75 (1H, dd, J 6.6, 13.9 Hz, PhCHaHb), 3.10 (2H, m,
PhCHaHb), 3.21 (1H, dd, J 7.7, 13.8 Hz, PhCHaHb), 3.63 (3H, s, OCH3),
0.82 (3H, d, J 6.5 Hz, CHCH3), 0.90 (3H, d, J 6.4 Hz, CHCH3), 1.05 (3H,
d, J 6.6 Hz, CHCH3), 1.27e1.34 (2H, m, 2CH(CH3)2), 1.57e1.63 (4H, m,
2CH2CH), 3.19 (1H, m, CHaHbPh), 3.43 (1H, m, CHaHbPh), 3.75e3.80
(2H, m, OCH2Ph), 4.34e4.38 (2H, m, 2aCH), 4.70 (1H, d, J 5.3 Hz,
a
CH), 4.83 (1H, m, CH), 5.06 (1H, d, J 5.2 Hz, PhCHO), 6.81 (1H, d, J
a
3.87 (3H, s, OCH3), 4.53 (1H, m,
CH), 4.88 (1H, m, CH), 4.99 (2H, m, 2
CHOH), 5.30 (1H, q, J 7.9 Hz, CHOH), 6.55 (1H, br, NH), 6.64 (1H, d, J
aCH), 4.63 (2H, m, 2aH), 4.80 (1H, m,
8.7 Hz, NH), 7.14e7.34 (10H, m, 2Ph), 7.37e7.47 (5H, m, Ph), 7.58
(1H, d, J 4.3 Hz, NH), 7.59 (1H, d, J 4.7 Hz, NH), 8.42 (1H, s, CCHO),
8.52 (1H, m, NH), 8.77 (1H, m, NH). LCMS: m/z called for C40H47N5O6
a
a
a
CH), 5.20 (1H, q, J 7.8 Hz,

1044
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
(Mþ1)¼694.8, found 694.4; HRMS (ESI-TOF): MHþ, found
the desired title compound (494 mg, 75% yield over two steps). Rf:
0.82 (EtOAc/Hex 1:1); 1H NMR (400 MHz, CDCl3):
0.87 (3H, d, J
694.3592, C40H47N5O6 requires 694.3599.
d
6.7 Hz, CHCH3), 0.93 (3H, d, J 5.6 Hz, CHCH3), 1.43 (9H, s, C(CH3)3),
2.41e2.52 (1H, m, CH(CH3)2), 2.76 (3H, s, NCH3), 3.40 (1H, s, OH),
6.26. Synthesis of compound 7 (D-Ox-II)
3.84 (3 h, s, OCH3), 4.86 (1H, d, J 10.5 Hz,
NH), 8.19 (1H, s, CCHO).
aCH), 5.12 (1H, d, J 10.9 Hz,
6.26.1. Dipeptide NH2-
D
-Phe-
D
-Leu-O-Resin. A mixture of NH2-
D-
Leu-O-Resin (1.50 g, 1.10 mmol), Fmoc-
D-Phe-OH (1.27 g,
3.29 mmol), HOBt (503 mg, 3.29 mmol), and DIC (1.02 mL,
6.57 mmol) were stirred at room temperature for 3 h following the
‘General solid-phase peptide synthesis’ procedure. Completion of the
coupling reaction was verified by a negative ninhydrin test. The
reaction mixture was then drained to leave the amine-protected
resin-bound dipeptide. Deprotection of amine was performed fol-
lowing the ‘General solid-phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal and gave the
title compound.
6.26.6. Dipeptide MeO-Oxazole-Val-N(Me)H. A mixture of peptidyl
MeO-Oxazole-Val-N(Me)-NBoc (494 mg, 1.58 mmol) in TFA
(3.16 mL) and methylene chloride (12.6 mL) was stirred at room
temperature under open atmosphere for 30 min following the
‘General solution-phase amine deprotection’. Reaction completion
was confirmed by TLC and the mixture was concentrated in vacuo
with several washes with methylene chloride. This dipeptide was
taken on to the next reaction without further purification or char-
acterization (335 mg, quantitative yield).
6.26.2. Tripeptide Boc-(2R,3R)/(2S,3S)-
mixture of NH2- -Phe- -Leu-O-Resin (1.10 mmol), (2R,3R)/
(2S,3S)-racemic Boc- -OH-Phe-OH residue (923 mg, 3.29 mmol),
b
-OH-Phe-
D
-Phe-
D
-Leu-OH. A
6.26.7. Pentapeptide MeO-Oxazole-Val-N(Me)-
(2S,3S)- -OH-Phe-NHBoc. A mixture of amine MeO-Oxazole-Val-
N(Me)H (230 mg, 1.09 mmol), acid Boc-(2R,3R)/(2S,3S)-
-Phe- -Leu-OH (534 mg, 0.986 mmol), TBTU
D-Leu-D-Phe-(2R,3R)/
D
D
b
b
b-OH-Phe-
HOBt (503 mg, 3.29 mmol), and DIC (1.02 mL, 6.57 mmol) were
stirred at room temperature for 3 h following the ‘General solid-
phase peptide synthesis’ procedure. Completion of the coupling
reaction was verified by a negative ninhydrin test. The reaction
mixture was then drained and dried in vacuo overnight to leave
the amine-protected resin-bound tripeptide. The tripeptide was
cleaved from the resin following the ‘Cleavage of linear peptide
from solid support’ procedure. A resin slurry of Boc-(2R,3R)/
D
D
(189 mg,
0.592 mmol), HATU (375 mg, 0.986 mmol), and DIPEA (1.38 mL,
7.89 mmol) in acetonitrile (9.86 mL) was stirred at room tempera-
ture under an argon atmosphere for 1.5 h following the ‘General
solution-phase peptide synthesis’ procedure. Upon completion,
confirmed by TLC, the reaction mixture was diluted with ethyl ac-
etate (300 mL), quenched by the addition of 10% hydrochloric acid
solution (200 mL), and further washed with sodium bicarbonate
solution (500 mL, satd aq) and then brine (200 mL). The organic
layer was dried (Na2SO4) and the solvent was evaporated in vacuo.
The crude reaction was purified by column chromatography (85%
EtOAc/Hex) to yield the title compound (158 mg, 22% yield). Rf: 0.30
(2S,3S)-
b
-OH-Phe-
D
-Phe-
D
-Leu-O-Resin
(2.07 g),
2,2,2-
trifluoroethanol (10.3 mL), and of methylene chloride (10.3) was
stirred for 24 h, after which it was filtered, washed with additional
methylene chloride, and dried in vacuo for 24 h to yield the title
compound (534 mg, 90% yield).
(EtOAc/Hex 1:1); 1H NMR (400 MHz, CDCl3):
d 0.76 (6H, J 2.7 Hz,
CH(CH3)2), 0.78 (6H, J 1.7 Hz, CH(CH3)2), 0.81e0.79 (6H, J 3.5 Hz,
CH(CH3)2), 0.87e0.83 (6H, m, 2CH(CH3)2), 1,14 (9H, s, C(CH3)3), 1.22
(9H, s, C(CH3)3), 1.32e1.44 (2H, br, CH(CH3)2), 2.32e2.44 (4H, m,
2CHCH2CH), 2.73 (2H, m, CH(CH3)2), 2.84e2.90 (6H, m, 2NCH3),
3.78 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.82 (4H, br, 2CH2Ph),
6.26.3. Dipeptide MeO-Ser(Bzl)-Val-N(Me)Boc. A mixture of amine
MeO-Ser(Bzl)-NH2 (709 mg, 3.39 mmol), acid HO-Val-N(Me)-Boc
(712 mg, 3.09 mmol), DIPEA (5.16 mL, 2.46 mmol), TBTU (1.19 g,
3.69 mmol) in methylene chloride (30.8 mL) was stirred at room
temperature under an argon atmosphere for 2.5 h following the
‘General solution-phase peptide synthesis’ procedure. The crude re-
action was purified by column chromatography (20% EtOAc/Hex) to
yield the title compound (1.22 g, 97% yield). Rf: 0.80 (EtOAc/Hex
4.09e4.21 (2H, m, 2
4.51e4.65 (1H, m, CH), 4.78 (2H, br, 2
(1H, m, CH), 5.32 (1H, d, J 8.6 Hz, CHOH), 5.42 (1H, t, J 12.3 Hz,
a
CH), 4.44e4.52 (1H, q, J 6.4 Hz,
aCH),
a
a
CH), 4.86 (1H, m, CH), 4.90
a
a
CHOH), 6.00 (2H, d, J 6.7 Hz, 2NH), 6.59 (2H, d, J 8.4 Hz, 2NH),
6.83e6.87 (2H, m, 2NH), 7.02e7.32 (20H, m, 4Ph), 8.05 (1H, s,
CCHO), 8.10 (1H, s, CCHO).
1:1); 1H NMR (400 MHz, CDCl3):
d 0.82 (3H, d, J 6.7 Hz, CHCH3), 0.95
(3H, d, J 6.1 Hz, CHCH3), 1.41 (9H, s, C(CH3)3), 2.19e2.30 (1H, m,
CH(CH3)2), 2.74 (3H, s, NCH3), 3.58e3.62 (1H, dd, J 3.6, 9.4 Hz,
CHCHaHbO), 3.70 (3H, s, OCH3), 4.10 (1H, br,
J 7.7 Hz, CHCHaHbO), 4.40e4.56 (2H, q, J 13.0 Hz, CH2Ph), 4.67 (1H,
m, CH), 6.93 (1H, br, NH), 7.20e7.30 (5H, m, Ph).
a
CH), 3.77e3.82 (1H, d,
6.26.8. Deprotected pentapeptide HO-Oxazole-Val-N(Me)-
Phe-(2R,3R)/(2S,3S)- -OH-Phe-NH2. A mixture of pentapeptide
MeO-Oxazole-Val-N(Me)- -Leu- -Phe-(2R,3R)/(2S,3S)- -OH-Phe-
NHBoc (158 mg, 0.215 mmol), lithium hydroxide (18.0 mg,
0.429 mmol), and 30% hydrogen peroxide (70.0 L) in water
(134 L) and THF (403 L) was stirred at room temperature under
open atmosphere for 3 h following the ‘General solution-phase acid
deprotection’. Upon completion, the reaction was diluted with ether
(100 mL) and quenched with sodium thiosulfate (175 mg) in pH 1
hydrochloric acid solution (200 mL). The organic layer was sepa-
rated and the aqueous layer was back extracted with ether
(240 mL). The combined organic layers were dried (Na2SO4) and
D-Leu-D-
b
a
D
D
b
6.26.4. Dipeptide MeO-Ser-Val-N(Me)Boc. A mixture of dipeptide
MeO-Ser(Bzl)-Val-N(Me)-Boc (1.22 g, 3.01 mmol) and a catalytic
amount of Pd/C in ethanol (30.1 mL) was stirred under a hydrogen
atmosphere for 24 h. Upon completion, confirmed by TLC, the re-
action mixture was filtered over Celite to yield the title compound
(723 mg, 75% yield). Rf: 0.5 (EtOAc/Hex 1:1). Physical and spectro-
scopic data are consistent with those reported in the literature.48
m
m
m
6.26.5. Dipeptide MeO-Oxazole-Val-N(Me)Boc. Dipeptide MeO-
Oxazole-Val-N(Me)-Boc was synthesized following the ‘General
oxazole synthesis’ procedure. The oxazoline intermediate was syn-
thesized utilizing dipeptide MeO-Ser-Val-N(Me)-Boc (700 mg,
concentrated in vacuo to yield the deprotected acid HO-Oxazole-
Leu- -Phe-(2R,3R)/(2S,3S)- -OH-Leu-NHBoc. Deprotection of the
amine resulted from a mixture of acid HO-Oxazole- -Leu- -Phe-
(2R,3R)/(2S,3S)- -OH-Leu-NHBoc (155 mg, 0.215 mmol), anisole
(50.0 L, 0.429 mmol) in TFA (537 L) and methylene chloride
(1.61 mL) stirred at room temperature for 40 min under open at-
mosphere following the ‘General solution-phase amine deprotection’.
Reaction completion was confirmed by TLC and the mixture was
concentrated in vacuo with several washes with methylene
D-
D
b
D
D
b
2.11 mmol), DAST (306
in methylene chloride (21.1 mL). The resulting oxazoline was oxi-
dized utilizing DBU (420 L, 4.21 mmol), CBrCl3 (420 L,
mL, 2.32 mmol), K2CO3 (569 mg, 4.21 mmol)
m
m
m
m
4.21 mmol) in methylene chloride (21.1 mL). The crude reaction
was purified by column chromatography (40% EtOAc/Hex) to yield

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1045
chloride. This deprotected pentapeptide HO-Oxazole-Val-N(Me)-
D
-
The reaction mixture was then drained to leave the amine-
protected resin-bound tripeptide. Deprotection of amine was per-
formed following the ‘General solid-phase amine deprotection’ pro-
cedure. A positive ninhydrin test served to verify Fmoc removal and
gave the title compound.
Leu- -Phe-(2R,3R)/(2S,3S)- -OH-Phe-NH2 was taken on to the next
D
b
reaction without further purification or characterization (135 mg,
quantitative yield). LCMS (ESI): m/z called for C33H43N5O7
(MþHþ)¼621.7, found 621.8.
6.26.9. Macrocycle
Leu- -Phe (7). A mixture of TBTU (69.0 mg, 0.215 mmol), HATU
(82.0 mg, 0.215 mol), and DIPEA (375 L, 2.15 mmol) in methylene
chloride (7.70 mL) and acetonitrile (7.70 mL) stirred at room tem-
perature in a round bottom flask under an argon atmosphere fol-
lowing the ‘Macrocyclization procedure’. The deprotected
(2R,3R)-
b
-benzoxy-Phe-Oxazole-Val-N(Me)-
D
-
6.27.3. Tetrapeptide
Tetrapeptide Fmoc-
NH2-
D-Phe-D-Leu-N(Me)-Val-Leu-O-Resin.-
D
D
-Phe- -Leu-N(Me)-Val-Leu-O-Resin was syn-
D
m
m
thesized following the ‘General solid-phase peptide synthesis’
procedure. Using the NH2- -Leu-N(Me)-Val-Leu-O-Resin prepared
above, the -Phe residue was incorporated using 947 mg
(2.44 mmol) of Fmoc- -Phe-OH, 374 mg (2.44 mmol) of HOBt, and
D
D
D
pentapeptide HO-Oxazole-Val-N(Me)-
D
-Leu-
D
-Phe-(2R,3R)/(2S,3S)-
0.756 mL of DIC (4.88 mmol). Completion of the coupling reaction
was verified by a negative ninhydrin test. The reaction mixture was
then drained to leave the amine-protected resin-bound tetrapep-
tide. Deprotection of amine was performed following the ‘General
solid-phase amine deprotection’ procedure. A positive ninhydrin test
served to verify Fmoc removal and gave the title compound.
b
-OH-Phe-NH2 (134 mg, 0.215 mmol) was dissolved in methylene
chloride (7.7 mL) and acetonitrile (7.7 mL) and added to the re-
action flask via syringe pump at the rate of 15 mL/h. After 12 h, the
reaction was diluted with ethyl acetate (500 mL), quenched with
pH 1 hydrochloric acid solution (200 mL, satd aq), washed with
sodium bicarbonate solution (200 mL, satd aq), and then brine
(200 mL). The organic layer was separated, dried (Na2SO4), and
concentrate in vacuo. The crude reaction was semi-purified by re-
6.27.4. Pentapeptide
N(Me)-Val-Leu-O-Resin. Pentapeptide Fmoc-
Phe- -Phe- -Leu-N(Me)-Val-Leu-O-Resin was synthesized follow-
ing the ‘General solid-phase peptide synthesis’ procedure. Using the
NH2- -Phe- -Leu-N(Me)-Val-Leu-O-Resin prepared above, the
OH(2R,3R)(2S,3S)-Phe residue was incorporated using 657 mg
(1.63 mmol) of Fmoc- -OH(2R,3R)(2S,3S)-Phe-OH, 374 mg
NH2-
b
-OH(2R,3R)(2S,3S)-Phe-D-Phe-D-Leu-
b-OH(2R,3R)(2S,3S)-
verse phase-HPLC to yield the macrocycle (2R,3R)-
zole-Val-N(Me)- -Leu- -Phe (29 mg, 23% yield). Rf: 0.21 (65%
EtOAc/Hex); LCMS: m/z called for C33H41N5O6 (Mþ1)¼604.7, found
604.3. Finally, a mixture of macrocycle (2R,3R)- -OH-Phe-Oxazole-
Val-N(Me)- -Leu- -Phe (18.0 mg, 0.298 mol), NaH (1.50 mg,
0.596 mol), and benzyl bromide (14.0 L, 0.0119 mmol) in THF
b
-OH-Phe-Oxa-
D
D
D
D
D
D
b-
b
D
D
m
b
m
m
(2.44 mmol) of HOBt, 0.756 mL of DIC (4.88 mmol). Completion of
the coupling reaction was verified by a negative ninhydrin test. The
reaction mixture was then drained to leave the amine-protected
resin-bound pentapeptide. Deprotection of amine was performed
following the ‘General solid-phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal and gave the
title compound.
(1.19 mL) stirred at room temperature for 12 h under an atmo-
sphere of argon following the ‘Benzylation procedure’. The crude
reaction was purified by reverse phase-HPLC to yield the title
compound 7 (1.3 mg, 0.6%); stereochemistry was assigned pre-
viously.1 Rf: 0.37 (65% EtOAc/Hex); >90% pure by HPLC 1H NMR
(600 MHz, CD3OD):
d 0.79 (3H, d, J 6.7 Hz, CHCH3), 0.82 (3H, d, J
6.4 Hz, CHCH3), 1.04 (3H, d, J 6.8 Hz, CHCH3), 1.37 (3H, d, J 7.3 Hz,
CHCH3), 1.49 (1H, m, CH(CH3)2), 1.93 (2H, m, CH2CH), 2.92 (1H, m,
CH(CH3)2), 3.18 (1H, m, CHaHbPh), 3.32 (3H, s, NCH3), 3.42 (1H, m,
CHaHbPh), 3.74 (2H, m, OCH2Ph), 4.69 (1H, d, J 5.6 Hz, OCH), 4.90
6.27.5. Double deprotected pentapeptide NH2-
Phe- -Phe- -Leu-N(Me)-Val-Leu-OH. Double deprotected pentapep-
tide NH2- -OH(2R,3R)(2S,3S)-Phe- -Phe- -Leu-N(Me)-Val-Leu-OH
was synthesized following the ‘Cleavage of linear peptide from solid
support’ procedure. Utilizing the 1.32 g of dried NH2-
OH(2R,3R)(2S,3S)-Phe- -Phe- -Leu-N(Me)-Val-Leu-O-Resin, 6.59 mL
of2,2,2-trifluoroethanoland6.59 mLofmethylenechloride. Theresin
slurry was stirred for 24 h, after which it was filtered, washed with
additional methylene chloride, and dried in vacuo for 24 h (242 mg,
45% yield).
b-OH(2R,3R)(2S,3S)-
D
D
b
D
D
(1H, m,
aCH), 5.04 (1H, m, aCH), 6.64 (1H, m, aCH), 6.81 (1H, m,
a
CH), 7.12e7.20 (5H, m, CH2Ph), 7.23e7.31 (10H, m, 2Ph), 7.43 (1H,
b-
d, J 8.8 Hz, NH), 7.71 (1H, d, J 8.8 Hz,1H, NH), 8.41 (1H, s, CCHO), 8.70
(1H, d, J 10.3 Hz, NH). LCMS: m/z called for C40H47N5O6 (Mþ1)¼
694.8, found 694.0; HRMS (ESI-TOF): MHþ, found 694.3570,
C40H47N5O6 requires 694.3599.
D
D
6.27. Synthesis of compound 8 (D-Ox-I)
6.27.6. Macrocycle
b
-OH(2R,3R)(2S,3S)-Phe-D-Phe-
D-Leu-N(Me)-Val-
6.27.1. Dipeptide NH(Me)-Val-Leu-O-Resin. Dipeptide Fmoc-N(Me)-
Val-Leu-O-Resin was synthesized following the ‘General solid-phase
peptide synthesis’ procedure. Using 1.01 g (0.815 mmol) of NH2-Leu-
O-Resin, the residue N(Me)-Val residue was incorporated using
864 mg of Fmoc-N(Me)-Val-OH (2.44 mmol), 374 mg (2.44 mmol,
3 equiv) of HOBt, and 0.756 mL (4.88 mmol) of DIC. Completion of
the coupling reaction was verified by a negative ninhydrin test. The
reaction mixture was then drained to leave the amine-protected
resin-bound dipeptide. Deprotection of amine was performed fol-
lowing the ‘General solid-phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal and gave the
title compound.
Leu (8). Macrocycle
b
-OH(2R,3R)(2S,3S)-Phe-
D
-Phe-D
-Leu-N(Me)-
Val-Leu was synthesized following the ‘Macrocyclization procedure’.
Utilizing 242 mg (0.363 mmol) of linear pentapeptide, 0.580 mL
(3.63 mmol) of DIPEA, 109 mg (0.338 mmol) of TBTU, 128 mg
(0.338 mmol) HATU, and 58.0 mg (0.193 mmol) of DEPBT. The crude
reaction was purified by reverse phase-HPLC to yield the macro-
cycle (69.0 mg, 29% yield). 1H NMR (600 MHz, CD3OD):
d 0.84 (3H,
d, J 6.7 Hz, CHCH3), 0.85 (12H, dd, J 8.4, 6.7 Hz CHCH3), 0.93 (3H, d, J
6.7 Hz, CHCH3), 1.42e1.51 (2H, m, CH2CH(CH3)2), 1.74 (2H, m,
CHCH2CH), 1.81 (2H, m, CHCH2CH), 2.05 (1H, m, CHCH(CH3)2), 2.83
(3H, s, NCH3), 3.02 (1H, m, CHCH2C), 3.11 (1H, m, CHCH2C), 3.85 (1H,
m,
aCH), 4.08 (1H, m,
aCH), 4.15 (1H, m, aCH), 4.56 (1H, m, 1H, m,
aCH), 4.65 (1H, m,
a
CH), 4.91 (1H, d, J 4.6 Hz, HOCHCH), 7.01e7.45
6.27.2. Tripeptide
Fmoc- -Leu-N(Me)-Val-Leu-O-resin was synthesized following the
‘General solid-phase peptide synthesis’ procedure. Using the
NH(Me)-Val-Leu-O-Resin prepared above, the -Leu residue was
incorporated using 864 mg (2.44 mmol) of Fmoc- -Leu-OH, 374 mg
NH2-
D
-Leu-N(Me)-Val-Leu-O-Resin. Tripeptide
(10H, m, Ph). LCMS: m/z called for C36H51N5O6 (Mþ1)¼649.82,
D
found 650.1.
D
6.27.7. Macrocycle Phe-Oxazole-
Macrocycle Phe-Oxazole- -Phe- -Leu-N(Me)-Val-Leu was synthe-
sized following the ‘General oxazole synthesis’ procedure. The oxa-
zoline intermediate was synthesized utilizing 42.7 mg (66.5 mol)
D-Phe-D-Leu-N(Me)-Val-Leu (8).
D
D
D
(2.44 mmol) of HOAt, and 0.756 mL (4.88 mmol) of DIC. Completion
of the coupling reaction was verified by a negative ninhydrin test.
m

1046
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
of
11.2
macrocycle
b
-OH(2R,3R)-Phe-
D
-Phe-
D
-Leu-N(Me)-Val-Leu,
3.05e3.20 (2H, dq, J 86.5, 14.2, 5.5 Hz, CH2Phe), 3.78 (3H, s, OCH3),
4.35e4.45 (1H, m, CH), 4.39e4.50 (2H, m, CH2Br), 4.82e4.90 (1H,
m, CH ), 6.32e6.38 (1H, m, NH), 7.04e7.10 (2H, m, Ph), 7.20e7.30
m
L (72.0 mol) of DAST, 11.3
m
mL (0.131 mmol) of pyridine in
a
0.660 mL of methylene chloride. The resulting oxazoline was oxi-
dized utilizing 20.1 L (0.130 mmol) of DBU, 0.130 mL (0.130 mmol)
of CBrCl3 in 0.331 mL of methylene chloride. The crude reaction was
purified by HPLC to yield the desired Phe-Oxazole- -Phe- -Leu-
N(Me)-Val-Leu macrocycle (1.30 mg, 3.1% yield over two steps). 1H
NMR (600 MHz, CDCl3): 0.64 (3H, d, J 7.3 Hz CHCH3), 0.79 (3H, d, J
6.7 Hz CHCH3), 0.82 (3H, d, J 7.3 Hz CHCH3), 0.88 (3H, d, J 4.9 Hz
CHCH3), 0.91 (3H, d, J 6.5 Hz CHCH3), 0.92 (3H, d, J 6.4 Hz CHCH3),
1.41e1.50 (2H,m, CHCH2CH), 1.75 (2H,m, CHCH2CH), 1.86 (1H, m,
CHCH(CH3)2), 2.40 (1H, m, CHCH(CH3)2), 2.53 (1H, m, CHCH(CH3)2),
3.04 (1H, m, CHCH2C), 3.69 (3H, s, NCH3), 4.09 (1H, m, CHCH2C), 4.15
a
a
m
(3H, m, Ph).
D
D
6.28.6. Monomer amide-Leu-NHBoc. Monomer Amide-Leu-NHBoc
was synthesized following the ‘General amide formation’ procedure.
Utilizing 471 mg (1.79 mmol, 1.0 equiv) of MeO-Leu-NHBoc (49) in
36.0 mL of ammonium hydroxide and 36.0 mL of methanol. The
title compound was taken on to the next reaction without further
purification (444 mg, 100% yield). Rf: 0.05 (EtOAc/Hex 1:1).
d
6.28.7. Monomer thioamide-Leu-NHBoc. Monomer thioamide-Leu-
NHBoc was synthesized following the ‘General thioamide formation’
procedure. Utilizing 444 mg (1.79 mmol) of amide-Leu-NHBoc and
724 mg (1.79 mmol) of Lawesson’s reagent in 12.0 mL of dime-
thoxyethane. This thioamide was purified by column chromatog-
raphy (silica gel, EtOAc/DCM) to yield the thioamide-Leu-NHBoc
(322 mg, 68% yield). Rf: 0.74 (EtOAc/Hex 1:1).
(1H, m,
aCH), 4.22 (1H, m, aCH), 4.35 (1H, m, aCH), 5.31 (1H, m,
a
CH), 6.78 (1H, d, J 8.8 Hz, NH), 6.83 (1H, m, NH), 6.95 (1H, d, J
8.8 Hz, NH) 7.01e7.38 (10H, m, Ph) LCMS: m/z called for C36H47N5O5
(Mþ1)¼629.79, found 630.7; HRMS (ESI-TOF): MHþ, found
630.3646, C36H47N5O5 requires 630.3650 >75% pure by HPLC.
6.28. Synthesis of compound 9 (A-Th-III)
6.28.8. Monomer thioamide-Leu-NH2 (50). Monomer thioamide-
Leu-NH2 was synthesized following the ‘General solution-phase
amine deprotection’. This thioamide-Leu-NH2 was taken on to the
next reaction without further purification or characterization
(200 mg, 100% yield).
6.28.1. HO-Pyruvic-Ketal-Br (47). HO-Pyruvic-Ketal-Br was syn-
thesized following the ‘General bromoketal acid formation’ pro-
cedure. Utilizing bromopyruvic acid 806 mg (4.86 mmol) and
0.120 mL (0.122 mmol) of sulfuric acid in 1.60 mL (14.6 mmol) of
trimethyl orthoformate. This title compound 47 was taken on to the
next reaction without further purification or characterization
(700 mg, 67% yield).
6.28.9. Dipeptide thioamide-Leu-Leu-NHBoc (52). Dipeptide thio-
amide-Leu-Leu-NBoc was synthesized following the ‘General solu-
tion-phase peptide synthesis’ procedure. Utilizing 199 mg
(1.22 mmol) of amine thioamide-Leu-NH2, 274 mg (1.10 mmol) of
acid HO-Leu-NHBoc, 0.801 mL (4.40 mmol) of DIPEA, 391 mg
(1.22 mmol) of TBTU, in 11.0 mL methylene chloride. The crude
reaction was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the dipeptide (265 mg, 61% yield). Rf: 0.6 (EtOAc/Hex
6.28.2. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-Leu-
NHBoc was synthesized following the ‘General solution-phase pep-
tide synthesis’ procedure. Utilizing 700 mg (3.90 mmol) of amine
MeO-Phe-NH2 (45), 886 mg (3.55 mmol) of acid HO-Leu-NHBoc
(44), 2.50 mL (14.2 mmol) of DIPEA, 1.26 g (3.90 mmol) of TBTU,
in 35.5 mL of methylene chloride. The crude reaction was purified
by column chromatography (silica gel, EtOAc/Hex) to yield the di-
peptide (1.44 g, 99% yield). Rf: 0.3 (EtOAc/Hex 1:3); 1H NMR
1:1); 1H NMR (400 MHz, CDCl3):
d 0.90e0.95 (6H, d, J 3.5 Hz,
CH(CH3)2), 0.95e1.00 (6H, d, J 3.5 Hz, CH(CH3)2), 1.46 (9H, s,
C(CH3)3), 1.62e1.76 (4H, m, CHCH2CH), 1.76e1.92 (2H, m, CH(CH3)2),
(400 MHz, CDCl3):
d
0.78e0.84 (6H, m, CH(CH3)2), 1.30e1.35 (9H, s,
4.02e4.12 (1H, m, aCH), 4.75e4.83 (1H, m, aCH), 4.90e4.98 (1H, d, J
C(CH3)3),1.35e1.40 (1H, m, CH(CH3)2),1.45e1.60 (2H, m, CHCH2CH),
2.90e3.05 (2H, m, CHCH2CH), 3.58e3.60 (3H, d, J 1.8 Hz, OCH3),
13.3 Hz, NH), 6.70e6.78 (1H, d, J 13.3 Hz, NH), 7.48e7.56 (1H, br,
CSNH), 8.04e8.12 (1H, br, CSNH).
4.00e4.10 (1H, br, aCH), 4.70e4.78 (1H, m, aCH), 5.25e5.35 (1H, br,
NH), 6.86e6.96 (1H, m, NH), 7.00e7.20 (5H, m, Ph).
6.28.10. Pentapeptide MeO-Phe-Leu-Thiazole-Leu-Leu-NHBoc
(53). Pentapeptide MeO-Phe-Leu-Thiazole-Leu-Leu-NHBoc was
synthesized following the ‘General thiazole synthesis’ procedure.
Thioamide-Leu-Leu-NHBoc (52) 265 mg (0.670 mmol) and
537 mg (5.36 mmol) of potassium bicarbonate were dissolved in
6.00 mL dimethoxyethane. MeO-Phe-Leu-Ketone-Br (48) 413 mg
(0.940 mmol) was dissolved in 7.50 mL dimethoxyethane and
added dropwise to the thioamide mixture to yield thiazoline
intermediate. Thiazoline was dehydrated using 0.400 mL
(2.68 mmol) of trifluoroacetic acid, 0.490 mL (6.03 mmol) of
pyridine, 0.190 mL (1.34 mmol) triethylamine in 13.5 mL dime-
thoxyethane. The crude reaction was purified by column chro-
matography (silica gel, EtOAc/Hex) to yield the pentapeptide
(71.0 mg, 15% yield). Rf: 0.37 (EtOAc/Hex 2:3); 1H NMR (400 MHz,
6.28.3. Dipeptide MeO-Phe-Leu-NH2 (46). Dipeptide MeO-Phe-
Leu-NH2 was synthesized following the ‘General solution-phase
amine deprotection’. This dipeptide was taken on to the next re-
action without further purification or characterization (1.09 g,
100% yield).
6.28.4. Tripeptide MeO-Phe-Leu-Ketal-Br. Tripeptide MeO-Phe-Leu-
Ketal-Br was synthesized following the ‘General solution-phase
peptide synthesis’ procedure. Utilizing 1.09 g (3.52 mmol) of amine
MeO-Phe-Leu-NH2 (46), 688 mg (3.20 mmol) of acid HO-Pyruvic-
Ketal-Br (47), 2.46 mL (12.8 mmol) of DIPEA, 1.13 g (3.52 mmol) of
TBTU, in 35.0 mL of methylene chloride. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (1.33 g, 82% yield). Rf: 0.55 (EtOAc/Hex 1:1).
CDCl3):
d 0.86e1.02 (18H, m, CH(CH3)2), 1.46 (9H, s, C(CH3)3),
1.60e1.82 (8H, br m, 3CHCH2CH and 2CH(CH3)2), 1.88e2.00 (1H,
m, CH(CH3)2), 3.02e3.18 (2H, m, CH2Phe), 3.74 (3H, s, OCH3),
6.28.5. Tripeptide MeO-Phe-Leu-Ketone-Br (48). Tripeptide MeO-
D
-
4.05e4.15 (1H, m,
a
CH), 4.55e4.62 (1H, m,
aCH), 4.80e4.88 (1H,
Phe-Leu-Ketone-Br was synthesized following the ‘General ketone
deprotection’ procedure. Utilizing 507 mg (1.00 mmol) of MeO-Phe-
Leu-Ketal-Br in 26.0 mL of formic acid. The reaction mixture was
refluxed. This tripeptide was taken on to the next reaction without
further purification (405 mg, 92% yield). Rf: 0.56 (EtOAc/Hex 1:1);
m, CH), 5.30e5.38 (1H, m, a
a
CH), 6.66e6.70 (1H, d, J 13.3 Hz,
NH), 6.74e6.82 (1H, br, NH), 7.05e7.32 (5H, m, Ph), 7.45e7.52
(1H, d, J 13.3 Hz, NH), 8.00 (1H, s, SCH]C). LCMS: m/z calcd for
C36H55N5O7S (Mþ23)¼724.92, found 724.3.
1H NMR (200 MHz, CDCl3):
d
0.88e0.96 (6H, m, CH(CH3)2),
6.28.11. Macrocycle Phe-Leu-Thiazole-Leu-Leu (9). Macrocycle Phe-
Leu-Thiazole-Leu-Leu (9) was synthesized following the
1.55e1.65 (2H, m, CHCH2CH), 1.55e1.76 (1H, m, CH(CH3)2),

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1047
‘Macrocyclization procedure’. Utilizing 40.0 mg (70.1
ear pentapeptide (53), 0.070 L (0.420 mmol) of DIPEA, 15.5 mg
(50.4 mol, 0.7 equiv) of TBTU, 18.2 mg (50.4 mol) HATU, and
15.0 mg (50.2 mol) of DEPBT in 97.6 mL methylene chloride. The
crude reaction was purified by column chromatography (silica gel,
EtOAc/Hex) and reverse phase-HPLC to yield the macrocycle
(2.30 mg, 6% yield). Rf: 0.1 (EtOAc/Hex 1:1); 1H NMR (400 MHz,
m
mol) of lin-
taken on to the next reaction without further purification (437 mg,
100% yield). Rf: 0.05 (EtOAc/Hex 1:1).
m
m
m
m
6.29.6. Monomer thioamide-Leu-NHBoc (55). Monomer thioamide-
Leu-NHBoc was synthesized following the ‘General thioamide for-
mation’ procedure. Utilizing 437 mg (1.76 mmol) of amide-Leu-
NHBoc and 712 mg (1.76 mmol) of Lawesson’s reagent in 12.0 mL of
dimethoxyethane. The title compound 55 was purified by column
chromatography (silica gel, EtOAc/DCM) to yield the thioamide-
Leu-NHBoc (324 mg, 70% yield). Rf: 0.74 (EtOAc/Hex 1:1).
CD3OD):
d 0.84e1.05 (18H, m, CH(CH3)2), 1.26e1.42 (9H, m,
C(CH3)3), 1.46e1.85 (8H, m, 3CHCH2CH and 2CH(CH3)2), 1.95e2.05
(1H, m, CH(CH3)2), 3.02e3.18 (2H, m, CHCH2CH), 4.38e4.45 (1H,
m,
a
CH), 4.52e4.55 (1H, m,
aCH), 4.80e4.88 (1H, m, aCH),
5.23e5.27 (1H, m,
a
CH), 7.05e7.32 (5H, m, Ph), 8.03 (1H, s, SCH]
6.29.7. Dipeptide EtO-Thiazole-Leu-NHBoc. Dipeptide EtO-Thiazole-
Leu-NHBoc was synthesized following the ‘General thiazole syn-
thesis’ procedure. Thioamide-Leu-NHBoc (55) 324 mg (1.23 mmol)
and 981 mg (9.80 mmol) of potassium bicarbonate were dissolved
in 24.6 mL dimethoxyethane. Ethyl-Br-pyruvate (56) 0.310 mL
(2.46 mmol) was added dropwise to the thioamide mixture to yield
thiazoline intermediate. Thiazoline was dehydrated using 0.680 mL
(4.92 mmol) of trifluoroacetic acid, 0.890 mL (11.1 mmol) of
pyridine, 0.340 mL (2.46 mmol) triethylamine in 24.6 mL dime-
thoxyethane. The crude reaction was purified by column chroma-
tography (silica gel, EtOAc/DCM) to yield the pentapeptide (198 mg,
47% yield). Rf: 0.47 (EtOAc/Hex 1:3); 1H NMR (400 MHz, CDCl3):
C). LCMS: m/z calcd for C30H43N5O4S (Mþ1)¼570.76, found
570.30; HRMS (ESI-TOF): MHþ, found 570.3102, requires 570.3108
>95% pure by HPLC.
6.29. Synthesis of compound 10 (B-Th-III)
6.29.1. Dipeptide MeO-Leu-N(Me)-Phe-NHBoc. Dipeptide MeO-Leu-
N(Me)-Phe-NHBoc was synthesized following the ‘General solution-
phase peptide synthesis’ procedure. Utilizing 325 mg (2.04 mmol) of
amine MeO-Leu-N(Me)H (58), 494 mg (1.86 mmol) of acid HO-Phe-
NHBoc (59), 1.30 mL (7.44 mmol) of DIPEA, 655 mg (2.04 mmol) of
TBTU, in 18.6 mL of methylene chloride. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the dipeptide (711 mg, 94% yield). Rf: 0.79 (EtOAc/Hex 1:1); 1H NMR
d
0.88e0.90 (3H, d, J 4.3 Hz, CHCH3), 0.90e0.92 (3H, d, J 4.3 Hz,
CHCH3), 1.29e1.35 (3H, t, OCH2CH3), 1.34e1.39 (9H, s, C(CH3)3),
1.60e1.74 (1H, m, CH(CH3)2), 1.77e1.98 (2H, m, CH2CH(CH3)2),
4.29e4.39 (2H, q, OCH2CH3), 4.85e5.14 (1H, br, CONHCH),
7.98e8.00 (1H, s, SCH]C).
(400 MHz, CDCl3): d 0.88e0.92 (6H, m, CH(CH3)2),1.22e1.25 (1H, m,
CH(CH3)2), 1.34e1.50 (9H, s, C(CH3)3), 1.58e1.74 (3H, m, CHCH2CH),
2.79e2.82 (3H, s, NCH3), 2.94e3.14 (2H, m, CH2Phe), 3.65e3.72 (3H,
6.29.8. Dipeptide EtO-Thiazole-Leu-NH2 (57). Dipeptide EtO-Thia-
zole-Leu-NH2 was synthesized following the ‘General amine
deprotection’. This dipeptide was taken on to the next reaction
without further purification or characterization (140 mg, 100%
yield).
d, J 2.5 Hz, OCH3), 4.55e4.62 (1H, br, NH), 4.80e4.88 (1H, m,
a
CH),
4.94e5.00 (1H, br, NH), 5.25e5.32 (1H, m,
m, Ph).
aCH), 7.10e7.33 (5H,
6.29.2. Dipeptide MeO-Leu-N(Me)-Phe-NH2 (60). Dipeptide MeO-
Leu-N(Me)-Phe-NH2 was synthesized following the ‘General solu-
tion-phase amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(536 mg, 100% yield).
6.29.9. Pentapeptide EtO-Thiazole-Leu-Leu-N(Me)-Phe-Leu-NHBoc
(63). Pentapeptide EtO-Thiazole-Leu-Leu-N(Me)-Phe-Leu-NHBoc
was synthesized following the ‘General solution-phase peptide syn-
thesis’ procedure. Utilizing 140 mg (0.580 mmol) of amine EtO-
Thiazole-Leu-NH2 (57), 276 mg (0.530 mmol) of acid HO-Leu-
N(Me)-Phe-Leu-NHBoc (62), 0.370 mL (2.12 mmol) of DIPEA,
186 mg (0.580 mmol) of TBTU, in 5.30 mL of methylene chloride.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the pentapeptide (317 mg, 82% yield). Rf:
6.29.3. Tripeptide
MeO-Leu-N(Me)-Phe-Leu-NHBoc. Tripeptide
MeO-Leu-N(Me)-Phe-Leu-NHBoc was synthesized following the
‘General solution-phase peptide synthesis’ procedure. Utilizing
536 mg (1.75 mmol) of amine MeO-Leu-N(Me)-Phe-NH2 (60),
400 mg (1.60 mmol) of acid HO-Leu-NHBoc (61), 1.10 mL
(6.40 mmol) of DIPEA, 562 mg (1.75 mmol) of TBTU, in 16.0 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the tripeptide
(683 mg, 79% yield). Rf: 0.22 (EtOAc/Hex 1:1); 1H NMR (400 MHz,
0.63 (EtOAc/Hex 1:1); 1H NMR (400 MHz, CDCl3):
d 0.74e1.00 (18H,
m, CH(CH3)2), 1.24e1.30 (3H, t, J 4.2 Hz, OCH2CH3), 1.35e1.44 (13H,
m, C(CH3)3, 2CHCH2CH), 1.46e1.66 (2H, m, CHCH2CH), 1.66e1.88
(2H, m, CH(CH3)2), 1.90e2.04 (1H, m, CH(CH3)2), 2.64e2.90 (3H, m,
NCH3), 2.90e3.23 (2H, m, CH2Phe), 4.04e4.15 (1H, br, NH),
CDCl3):
d
0.84e0.98 (12H, m, 2CH(CH3)2), 1.34e1.42 (2H, m,
4.09e4.15 (2H, m, OCH2CH3), 4.30e4.44 (2H, m,
4.60e4.70 (1H, br, NH), 4.85e5.01 (1H, m, CH), 5.01e5.21 (1H, m,
CH), 5.25e5.40 (1H, br, CH), 7.08e7.30 (5H, m, Ph), 7.95e8.05
aCH, NH),
2CH(CH3)2), 1.42e1.50 (9H, s, C(CH3)3), 1.54e1.75 (4H, m,
2CHCH2CH), 2.79e2.82 (3H, s, NCH3), 2.94e3.20 (2H, m, CH2Phe),
3.65e3.72 (3H, s, OCH3), 4.05e4.15 (1H, br, NH), 4.76e4.84 (1H, br,
a
a
a
(1H, m, SCH]C). LCMS: m/z calcd for C38H59N5O7S (Mþ1)¼730.41,
NH), 4.84e4.88 (1H, m,
a
CH), 5.10e5.20 (1H, m,
a
CH), 5.25e5.32
found 730.10.
(1H, m, CH), 7.10e7.33 (5H, m, Ph).
a
6.29.10. Macrocycle Phe-Leu-Thiazole-Leu-Leu-N(Me) (10). Macro-
cycle Phe-Leu-Thiazole-Leu-Leu-N(Me) (10) was synthesized fol-
lowing the ‘Macrocyclization procedure’. Utilizing 170 mg
(0.280 mmol) of linear pentapeptide (63), 0.290 mL (1.68 mmol) of
6.29.4. Tripeptide HO-Leu-N(Me)-Phe-Leu-NHBoc (62). Tripeptide
HO-Leu-N(Me)-Phe-Leu-NHBoc was synthesized following the
‘General solution-phase acid deprotection’. This tripeptide was taken
on to the next reaction without further purification or character-
ization (665 mg, 100% yield).
DIPEA, 64.2 mg (50.2
mmol) of TBTU, 76.0 mg (50.5 mmol) HATU,
and 60.0 mg (50.2 mol) of DEPBT in 300 mL methylene chloride
m
and 100 mL acetonitrile. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) and reverse phase-HPLC to
yield the macrocycle (6.50 mg, 4% yield). Rf: 0.35 (EtOAc/Hex 1:1);
6.29.5. Monomer amide-Leu-NHBoc. Monomer amide-Leu-NHBoc
was synthesized following the ‘General amide formation’ procedure.
Utilizing 463 mg (1.76 mmol) of MeO-Leu-NHBoc (54) in 35.0 mL of
ammonium hydroxide and 35.0 mL of methanol. This amide was
1H NMR (400 MHz, CDCl3):
d
0.81e0.84 (3H, d, J 3.5 Hz, CH(CH3)2),
0.86e0.89 (3H, d, J 3.5 Hz, CH(CH3)2), 0.98e1.08 (12H, m, CH(CH3)2),

1048
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1.25e1.36 (2H, m, CH(CH3)2), 1.58e1.80 (6H, m, 3CHCH2CH),
2.10e2.30 (1H, m, CH(CH3)2), 2.53e2.63 (3H, m, NCH3), 3.03e3.18
dimethoxyethane. This thioamide was purified by column chro-
matography (silica gel, EtOAc/DCM) to yield the thioamide-Leu-
NHBoc (240 mg, 68% yield). Rf: 0.74 (EtOAc/Hex 1:1).
(2H, m, CH2Phe), 4.65e4.79 (1H, m,
a
CH), 4.90e4.98 (1H, m,
aCH),
5.10e5.20 (1H, m, CH), 5.23e5.32 (1H, m,
a
aCH), 6.42e6.50 (1H, m,
NH), 6.92e7.00 (1H, m, NH), 7.20e7.33 (5H, m, Ph), 7.42e7.50 (1H,
m, NH), 8.15 (1H, s, SCH]C). LCMS: m/z calcd for C31H45N5O4S (M)¼
584.3265, found 583.80; HRMS (ESI-TOF): MHþ, found 584.3253,
C31H45N5O4S requires 610.4075 >95% pure by HPLC.
6.30.7. Dipeptide EtO-Thiazole-Leu-NHBoc. Dipeptide EtO-Thiazole-
Leu-NHBoc was synthesized following the ‘General thiazole syn-
thesis’ procedure. Thioamide-Leu-NHBoc 240 mg (0.910 mmol) and
729 mg (7.30 mmol) of potassium bicarbonate were dissolved in
18.2 mL dimethoxyethane. Ethyl-Br-pyruvate 0.230 mL (2 equiv)
was added dropwise to the thioamide mixture to yield thiazoline
intermediate. Thiazoline was dehydrated using 0.510 mL
(3.64 mmol) of trifluoroacetic acid, 0.660 mL (8.19 mmol) of pyri-
dine, 0.250 mL (1.82 mmol) triethylamine in 18.2 mL dimethoxy-
ethane. The crude reaction was purified by column
chromatography (silica gel, EtOAc/DCM) to yield the pentapeptide
(404 mg, 77% yield). Rf: 0.81 (EtOAc/Hex 1:1); 1H NMR (400 MHz,
6.30. Synthesis of compound 11 (C-Th-III)
6.30.1. Dipeptide
MeO-Lys-Cbz-Phe-NHBoc. Dipeptide
MeO-
Lys(Cbz)-Phe-NHBoc was synthesized following the ‘General solu-
tion-phase peptide synthesis’ procedure. Utilizing 392 mg
(1.30 mmol) of amine MeO-Lys(Cbz)-NH2, 379 mg (1.43 mmol) of
acid HO-Phe-NHBoc, 1.80 mL (10.4 mmol) of DIPEA, 334 mg
(1.04 mmol) of TBTU, 297 mg (0.780 mmol) of HATU, in 13.0 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(578 mg, 80% yield). Rf: 0.37 (EtOAc/Hex 1:1); 1H NMR (400 MHz,
CDCl3):
d 0.88e0.90 (3H, d, J 4.3 Hz, CHCH3), 0.90e0.92 (3H, d, J
4.3 Hz, CHCH3), 1.29e1.35 (3H, t, J 4.3 Hz, OCH2CH3), 1.36 (9H, s,
C(CH3)3), 1.60e1.74 (1H, m, CH(CH3)2), 1.77e1.98 (2H, m,
CH2CH(CH3)2), 4.29e4.39 (2H, q, J 2.1 Hz, OCH2CH3), 4.85e5.14 (1H,
br, CONHCH), 7.99 (1H, s, SCH]C).
CDCl3):
1.36e1.64 (2H, m,
CH2), 3.03e3.16 (2H, m, CH2Phe), 3.60e3.65 (3H, s, OCH3),
4.21e4.35 (1H, m, CH ), 4.42e4.52 (1H, m, CH), 4.85e4.93 (1H,
d
1.11e1.49 (2H, m,
gCH2), 1.30e1.36 (9H, s, C(CH3)3),
d
CH2), 1.51e1.81 (2H, m,
bCH2), 2.93e3.03 (2H, m,
3
6.30.8. Dipeptide EtO-Thiazole-Leu-NH2. Dipeptide EtO-Thiazole-
Leu-NH2 was synthesized following the ‘General amine depro-
tection’. This dipeptide was taken on to the next reaction without
further purification or characterization (287 mg, 100% yield).
a
a
a
br, NH), 5.03 (2H, s, CH2Ph), 6.39e6.47 (1H, br, NH), 7.08e7.32 (10H,
m, Ph).
6.30.2. Dipeptide
MeO-Lys(Cbz)-Phe-NH2. Dipeptide
MeO-
6.30.9. Pentapeptide
EtO-Thiazole-Leu-Lys(Cbz)-Phe- -Phe-N(Me)
D
Lys(Cbz)-Phe-NH2 was synthesized following the ‘General solution-
phase amine deprotection’. This dipeptide was taken on to the next
reaction without further purification or characterization (459 mg,
100% yield).
Boc. Pentapeptide
Boc was synthesized following the ‘General solution-phase peptide
synthesis’ procedure. Utilizing 266 mg (1.10 mmol) of amine EtO-
EtO-Thiazole-Leu-Lys(Cbz)-Phe-D-Phe-N(Me)
Thiazole-Leu-NH2, 688 mg (1.00 mmol) of acid HO-Lys(Cbz)Phe-D-
Phe-N(Me)Boc, 1.05 mL (6 mmol) of DIPEA, 257 mg (0.802 mmol) of
TBTU, 190 mg (0.501 mmol) of HATU in 11.0 mL of methylene
chloride. The crude reaction was purified by column chromatog-
raphy (silica gel, EtOAc/Hex) to yield the pentapeptide (497 mg,
49.5% yield). Rf: 0.16 (EtOAc/Hex 1:1); 1H NMR (400 MHz, CDCl3):
6.30.3. Tripeptide MeO-Lys(Cbz)-Phe-
D
-Phe-N(Me)Boc. Tripeptide
MeO-Lys(Cbz)-Phe- -Phe-N(Me)Boc was synthesized following the
D
‘General solution-phase peptide synthesis’ procedure. Utilizing
459 mg (1.04 mmol) of amine MeO-Lys(Cbz)-Phe-NH2, 320 mg
(1.14 mmol) of acid HO-
D
-Phe-N(Me)Boc, 1.50 mL (8.32 mmol) of
d
0.79e0.91 (6H, m, CH(CH3)2), 1.09e1.30 (12H, m, C(CH3)3 and
OCH2CH3), 1.34e1.60 (2H, m, CH2), 1.66e1.87 (6H, m, 2 CH2 and
CH2), 1.88e2.01 (1H, m, CH(CH3)2), 2.26e2.44 (3H, m, NCH3),
DIPEA, 267 mg (0.830 mmol) of TBTU, 198 mg (0.520 mmol) of
HATU, in 10.4 mL of methylene chloride. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (731 mg, 100% yield). Rf: 0.38 (EtOAc/Hex 7:13); 1H
g
b
d
2.86e3.04 (2H, m,
3.19e3.33 (1H, m,
3
b
NMR (400 MHz, CDCl3):
C(CH3)3), 1.35e1.48 (2H, m,
2.51e2.73 (3H, s, NCH3), 2.82e3.02 (2H, m, CH2Phe), 3.02e3.15 (2H,
m, CH2), 3.16e3.34 (2H, m, CH2Phe), 3.59e3.70 (3H, s, OCH3),
4.34e4.46 (1H, br, CH), 4.47e4.68 (2H, m, 2 CH), 4.98e5.08 (2H, s,
CH2Ph), 6.22e6.61 (2H, br m, NH), 6.98e7.34 (15H, m, Ph).
d
1.10e1.22 (2H, m,
g
CH2), 1.22e1.30 (9H, s,
(2H, m, 2aCH), 4.96e5.04 (2H, m, CH2Ph), 5.06e5.23 (2H, m, 2aCH),
d
CH2), 1.51e1.83 (2H, m,
b
CH2),
6.29e6.79 (2H, br m, 2NH), 6.89e7.36 (15H, m, Ph), 8.02e8.13 (1H,
m, SCH]C). LCMS: m/z calcd for C49H64N6O9S (M)¼913.13, found
913.00.
3
a
a
6.30.10. Macrocycle Phe-
D-Phe-N(Me)-Thiazole-Leu-Lys(Cbz) (11).
Macrocycle Phe- -Phe-N(Me)-Thiazole-Leu-Lys(Cbz) (11) was syn-
D
6.30.4. Tripeptide
HO-Lys(Cbz)-Phe-
‘General solution-phase acid deprotection’. This tripeptide was taken
on to the next reaction without further purification or character-
ization (716 mg, 100% yield).
HO-Lys(Cbz)-Phe-
D-Phe-N(Me)Boc was synthesized following the
D
-Phe-N(Me)Boc. Tripeptide
thesized following the ‘Macrocyclization procedure’. Utilizing
418 mg (0.530 mmol) of linear pentapeptide, 0.750 mL (4.24 mmol)
of DIPEA, 138 mg (0.430 mmol) of TBTU, 164 mg (0.430 mmol)
HATU, and 129 mg (0.430 mmol) of DEPBT in 533 mL methylene
chloride. The crude reaction was purified by column chromatog-
raphy (silica gel, EtOAc/Hex) and reverse phase-HPLC to yield the
macrocycle (50.3 mg, 12.3% yield). Rf: 0.53 (EtOAc/Hex 9:1); 1H
6.30.5. Monomer amide-Leu-NHBoc. Monomer amide-Leu-NHBoc
was synthesized following the ‘General amide formation’ procedure.
Utilizing 353 mg (1.34 mmol) of MeO-Leu-NHBoc in 27.0 mL of
ammonium hydroxide and 27.0 mL of methanol. This amide was
taken on to the next reaction without further purification (333 mg,
100% yield). Rf: 0.05 (EtOAc/Hex 1:1).
NMR (400 MHz, CDCl3):
0.87e0.92 (3H, d, J 11.4 Hz, CH(CH3)2), 1.08e1.30 (4H, m,
CH2), 1.34e1.68 (3H, m, CHCH2CH and CHCHaHbCH), 1.83e2.00
(1H, m, CHaHbCH), 2.21e2.36 (1H, m, CH(CH3)2), 2.49e2.72 (3H, m,
NCH3), 2.87e3.24 (2H, m, CH2Phe), 3.00e3.12 (2H, m, CH2),
3.29e3.39 (1H, d, J 9.75 Hz, CHaHbPhe), 3.50e3.64 (1H, br, NH),
3.98e4.10 (1H, m, CH), 4.35e4.42 (1H, d, J 9.75 Hz, CHaHbPhe),
4.76e4.87 (2H, m, 2 CH ), 4.97e5.08 (2H, m, CH2Ph), 5.24e5.34
(1H, m, CH), 6.66e6.85 (2H, m, NH), 6.97e7.34 (15H, m, Ph),
7.52e7.64 (1H, m, NH), 7.94e8.02 (1H, m, SCH]C). LCMS: m/z calcd
d
0.79e0.87 (3H, d, J 11.4 Hz, CH(CH3)2),
gCH2 and
d
3
6.30.6. Monomer thioamide-Leu-NHBoc. Monomer thioamide-Leu-
NHBoc was synthesized following the ‘General thioamide formation’
procedure. Utilizing 333 mg (1.34 mmol) of amide-Leu-NHBoc and
542 mg (1.34 mmol) of Lawesson’s reagent in 9.00 mL of
a
a
a
a

M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
1049
for C42H50N6O6S (M)¼766.95, found 767; HRMS (ESI-TOF): MHþ,
found 767.3583, C42H50N6O6S requires 767.3585 >95% pure by
HPLC.
‘Cleavage of linear peptide from solid support’ procedure. Utilizing
the 250 mg of dried Resin-O-Thr( Me,Me-Pro)-Val-Leu-Leu-Phe-
J
NH2, 1 mL of hexafluoroisopropanol and 4 mL of methylene chlo-
ride. The resin slurry was stirred for 30 min, after which it was
filtered, washed with additional methylene chloride, and dried in
vacuo to give the double deprotected pentapeptide (150 mg, 95%
yield).
6.31. Synthesis of compound 12 (A-PP-II)
6.31.1. Fmoc-Val-Thr(
chlorotrityl-chloride resin (100 mg, 0.100 mmol), dipeptide Fmoc-
Val-Thr( Me,Me-Pro)-OH (96.0 mg, 0.200 mmol), and DIPEA
JMe,Me-Pro)-O-Resin. A mixture of 2-
J
6.31.7. Macrocycle Phe-Thr(
cycle Phe-Thr( Me,Me-Pro)-Val-Leu-Leu was synthesized follow-
ing the ‘Macrocyclization procedure’. Utilizing linear pentapeptide
(59.0 mg, 90.0 mol), DIPEA (80.0
L), and DMTMM BFꢁ4 (87.0 mg,
JMe,Me-Pro)-Val-Leu-Leu (12). Macro-
(3.00 mL) in anhydrous methylene chloride (0.400 M) was stirred
under argon at room temperature for 12 h. The reaction mixture
was filtered and the collected resin was washed with a solution of
CH2Cl2/CH3OH/DIPEA (30.0 mL 17:4:1, v:v:v), followed by methy-
lene chloride (20.0 mL), dimethylformamide (20.0 mL), and finally
methylene chloride (30 mL). The resin was drained well and dried
in vacuo overnight to give the resin-bound dipeptide. Resin loading
was determined via RP-HPLC to be 0.250 mmol/g.
J
m
m
0.270 mmol), the linear peptide and coupling reagent were dis-
solved in dimethylformamide (10 mL) and added dropwise via sy-
ringe pump (0.5 mL/h) to a vigorously stirred solution of DIPEA and
dimethylformamide (1 mM) under N2. The reaction mixture was
stirred for 3 days, after which time the solvent was removed under
reduced pressure and the crude reaction was purified by reverse
phase-HPLC to yield the macrocycle 12 (14.5 mg, 26% yield). 1H
6.31.2. Dipeptide
NH2-Val-Thr( Me,Me-Pro)-O-Resin was synthesized using Fmoc-
Val-Thr( Me,Me-Pro)-O-Resin synthesized above and followed the
NH2-Val-Thr(JMe,Me-Pro)-O-Resin. Dipeptide
J
NMR (CD3CN, 400 MHz):
d, J 5.6 Hz, CHCH3), 1.39e1.78 (12H, m, CHaCHbCH3), 2.88 (1H, m,
CHMe2), 3.30e3.40 (2H, m, CH2Ph), 3.67 (1H, m, CH), 3.85 (1H, m,1
CH), 3.91e4.04 (2H, m, 2 CH), 4.21e4.46 (2H, m, 2 CH), 6.59 (1H,
d 0.80e1.03 (18H, m, 3CH(CH3)2), 1.19 (3H,
J
‘General solid-phase amine deprotection’ procedure. A positive nin-
hydrin test served to verify Fmoc removal.
a
a
a
a
m, NH), 6.69 (1H, m, NH), 6.87 (1H, m, NH), 7.12e7.41 (5 h, m, Ph),
7.48 (1H, d, J 8.0 Hz, NH) >95% pure by HPLC. HRMS (ESI-TOF):
MHþ, found 614.3887, C33H51N5O6 requires 614.3912.
6.31.3. Tripeptide
wing the ‘General solid-phase peptide synthesis’ procedure, a mix-
ture of NH2-Val-Thr( Me,Me-Pro)-O-Resin (0.250 mmol), Fmoc-
NH2-Leu-Val-Thr(JMe,Me-Pro)-O-Resin. Follo-
J
Leu-OH (177 mg, 0.500 mmol), HBTU (190 mg, 0.500 mmol), and
DIPEA (3.00 mL, 0.400 M) in DMF was stirred at room temperature
for 2 h. Completion of the coupling reaction was verified by a neg-
ative ninhydrin test. The reaction mixture was drained and washed
with DMF (30 mL) to give the resin-bound tripeptide. The Fmoc was
then removed following the ‘General solid-phase amine depro-
tection’ procedure to give the title tripeptide. A positive ninhydrin
test served to verify Fmoc removal.
6.32. Synthesis of compound 13 (A-PP-III)
6.32.1. Fmoc-Leu-Thr(
chlorotrityl-chloride resin (3.00 g, 4.74 mmol), dipeptide 64
Fmoc-Leu-Thr( Me,Me-Pro)-OH (4.68 g, 9.48 mmol), N-methyl-2-
JMe,Me-Pro)-O-Resin. A mixture of 2-
J
pyrrolidone (14.04 mL or 3.00 mL per gram of dipeptide), and
DIPEA (4.90 mL, 28.4 mmol) in anhydrous methylene chloride
(46.8 mL) was stirred under argon at room temperature for 12 h.
The reaction mixture was filtered and the collected resin was
washed with a solution of CH2Cl2/CH3OH/DIPEA (30.0 mL 17:4:1,
v:v:v), followed by methylene chloride (20.0 mL), dimethylforma-
mide (20.0 mL), and finally methylene chloride (30 mL). The resin
was drained well and dried in vacuo overnight to give the resin-
bound dipeptide. Resin loading was determined via RP-HPLC to
be 0.400 mmol/g.
6.31.4. Tetrapeptide NH2-Leu-Leu-Val-Thr(
in. Following the ‘General solid-phase peptide synthesis’ pro-
cedure, a mixture of NH2-Leu-Val-Thr( Me,Me-Pro)-O-Resin
prepared above (0.250 mmol), Fmoc-Leu-OH (177 mg,
JMe, Me-Pro)-O-Res-
J
0.500 mmol), HBTU (190 mg, 0.500 mmol), and DIPEA (3 mL,
0.400 M) in dimethylformamide was stirred at room temperature
for 12 h. Completion of the coupling reaction was verified by
a negative ninhydrin test. The reaction mixture was drained and
washed with DMF (30 mL) to give the resin-bound tetrapeptide.
The Fmoc was then removed following the ‘General solid-phase
amine deprotection’ procedure to give title tetrapeptide. A positive
ninhydrin test served to verify Fmoc removal.
6.32.2. Dipeptide NH2-Leu-Thr(
JMe,Me-Pro)-O-Resin (65). Title
compound 65 was synthesized utilizing Fmoc-Leu-Thr(
J
Me,Me-
Pro)-O-Resin from above and followed the ‘General solid-phase
amine deprotection’ procedure. A positive ninhydrin test served to
verify Fmoc removal.
6.31.5. Pentapeptide NH2-Phe-Leu-Leu-Val-Thr(
in. Following the ‘General solid-phase peptide synthesis’ procedure,
a mixture of NH2-Leu-Leu-Val-Thr( Me,Me-Pro)-O-Resin prepared
J
Me,Me-Pro)-O-Res-
6.32.3. Tripeptide NH2-Leu-Leu-Thr(
Following the ‘General solid-phase peptide synthesis’ procedure,
mixture of NH2-Leu-Thr( Me,Me-Pro)-O-Resin 65 (1.00 g,
JMe,Me-Pro)-O-Resin (66).
J
a
J
above (0.250 mmol), Fmoc-Phe-OH (194 mg, 0.500 mmol), HBTU
(190 mg, 0.500 mmol), and DIPEA (3.00 mL, 0.400 M) in dime-
thylformamide was stirred at room temperature for 12 h. Com-
pletion of the coupling reaction was verified by a negative
ninhydrin test. The reaction mixture was drained and washed with
DMF (30 mL) to give the resin-bound pentapeptide. The Fmoc was
then removed following the ‘General solid-phase amine depro-
tection’ procedure to give the title pentapeptide. A positive ninhy-
drin test served to verify Fmoc removal. The resin was then dried in
vacuo for 24 h in a vacuum desiccator.
0.400 mmol), Fmoc-Leu-OH (420 mg, 1.20 mmol), HOBt (180 mg,
1.20 mmol), and DIPEA (0.370 mL, 2.40 mmol) in DMF was stirred
at room temperature for 2 h. Completion of the coupling reaction
was verified by a negative ninhydrin test. The reaction mixture was
drained and washed with DMF (30 mL) to give the resin-bound
tripeptide. The Fmoc was then removed following the ‘General
solid-phase amine deprotection’ procedure to give title compound 66.
A positive ninhydrin test served to verify Fmoc removal.
6.32.4. Tetrapeptide NH2-Phe-Leu-Leu-Thr(
(67). Following the ‘General solid-phase peptide synthesis’ pro-
cedure, a mixture of NH2-Leu-Leu-Thr( Me,Me-Pro)-O-Resin 66
JMe, Me-Pro)-O-Resin
6.31.6. Double deprotected pentapeptide NH2-Phe-Leu-Leu-Val-
J
Thr(
J
Me,Me-Pro)-OH. Double deprotected pentapeptide NH2-Phe-
prepared above, Fmoc-Phe-OH (460 mg, 0.120 mmol), HOBt
(180 mg, 1.20 mmol), and DIPEA (0.370 mL, 2.40 mmol) in DMF was
Leu-Leu-Val-Thr(
J
Me,Me-Pro)-OH was synthesized following the

1050
M.R. Davis et al. / Tetrahedron 68 (2012) 1029e1051
stirred at room temperature for 12 h. Completion of the coupling
reaction was verified by a negative ninhydrin test. The reaction
mixture was drained and washed with DMF (30 mL) to give the
resin-bound tetrapeptide. The Fmoc was then removed following
the ‘General solid-phase amine deprotection’ procedure to give title
compound 67. A positive ninhydrin test served to verify Fmoc
removal.
Supplementary data
Supplementary data can be associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2011.11.089.
References and notes
1. Sellers, R. P.; Alexander, L. D.; Johnson, V. A.; Lin, C.-C.; Savage, J.; Corral, R.;
Moss, J.; Slugocki, T. S.; Singh, E. K.; Davis, M. R.; Ravula, S.; Spicer, J. E.; Oelrich,
J. L.; Thornquist, A.; Pan, C.-M.; McAlpine, S. R. Bioorg. Med. Chem. 2010, 18,
6822e6856.
6.32.5. Pentapeptide
Resin (68). Following the ‘General solid-phase peptide synthesis’
procedure, a mixture of NH2-Phe-Leu-Leu-Thr( Me,Me-Pro)-O-
NH2-Leu-Phe-Leu-Leu-Thr(JMe,Me-Pro)-O-
J
2. Otrubova, K.; Lushington, G. H.; Vander Velde, D.; McGuire, K. L.; McAlpine, S. R.
J. Med. Chem. 2008, 51, 530e544.
Resin 67 prepared above, Fmoc-Leu-OH (420 mg, 1.20 mmol), HOBt
(180 mg, 1.20 mmol), and DIPEA (0.370 mL, 2.40 mmol) in DMF was
stirred at room temperature for 12 h. Completion of the coupling
reaction was verified by a negative ninhydrin test. The reaction
mixture was drained and washed with DMF (30 mL) to give the
resin-bound pentapeptide. The Fmoc was then removed following
the ‘General solid-phase amine deprotection’ procedure to give title
compound 68. A positive ninhydrin test served to verify Fmoc re-
moval. The resin was then dried in vacuo for 24 h in a vacuum
desiccator.
3. Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1999, 40, 2913e2916.
4. Pan, P.-S.; Vasko, R. C.; Lapera, S. A.; Johnson, V. A.; Sellers, R. P.; Lin, C.-C.; Pan,
C.-M.; Davis, M. R.; Ardi, V. C.; McAlpine, S. R. Bioorg. Med. Chem. 2009, 17,
5806e5825.
5. Otrubova, K.; McGuire, K. L.; McAlpine, S. R. J. Med. Chem. 2007, 50, 1999e2002.
6. Pan, P. S.; McGuire, K.; McAlpine, S. R. Bioorg. Med. Chem. Lett. 2007, 17,
5072e5077.
7. Vasko, R. C.; Rodriguez, R. A.; Cunningham, C. N.; Ardi, V. C.; Agard, D. A.;
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brine (200 mL), dried over sodium sulfate, and the solvent evapo-
rated in vacuo to give the crude product, which was then purified
by flash chromatography (75% ethyl acetate/hexanes) and reverse
phase-HPLC to yield macrocycle 13 (1.01 mg, 1.60% yield). Rf: 0.48
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a
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a
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HRMS (ESI-TOF): MHþ, found 628.4040, C34H53N5O6 requires
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We thank the University of New South Wales for support of
S.R.M., the University of Sydney for a Visiting International Fel-
lowship to S.R.M., the Frasch Foundation (658-HF07) for support of
M.R.D., E.K.S., L.D.A., and L.A.N., NIH 1R01CA137873 for support of
E.K.S., L.D.A., and S.R.M., and NIH MIRT for support of E.K.S., L.D.A.,
and J.B.K.
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