Design and synthesis of Hsp90 inhibitors: Exploring the SAR of Sansalvamide A derivatives

Article abstract of DOI:10.1016/j.bmc.2010.07.042

Utilizing the structure-activity relationship we have developed during the synthesis of the first two generations and mechanism of action studies that point to the interaction of these molecules with the key oncogenic protein Hsp90, we report here the design of 32 new Sansalvamide A derivatives and their synthesis. Our new structures, designed from previously reported potent compounds, were tested for cytotoxicity on the HCT116 colon cancer cell line, and their binding to the biological target was analyzed using computational studies involving blind docking of derivatives using Autodock. Further, we show new evidence that our molecules bind directly to Hsp90 and modulate Hsp90's binding with client proteins. Finally, we demonstrate that we have integrated good ADME properties into a new derivative.

Full text of DOI:10.1016/j.bmc.2010.07.042

Bioorganic & Medicinal Chemistry 18 (2010) 6822–6856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of Hsp90 inhibitors: Exploring the SAR of Sansalvamide
A derivatives
Robert P. Sellers, Leslie D. Alexander, Victoria A. Johnson, Chun-Chieh Lin, Jeremiah Savage, Ricardo Corral,
Jason Moss, Tim S. Slugocki, Erinprit K. Singh, Melinda R. Davis, Suchitra Ravula, Jamie E. Spicer,
*
Jenna L. Oelrich, Andrea Thornquist, Chung-Mao Pan, Shelli R. McAlpine
Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Utilizing the structure–activity relationship we have developed during the synthesis of the first two gen-
erations and mechanism of action studies that point to the interaction of these molecules with the key
oncogenic protein Hsp90, we report here the design of 32 new Sansalvamide A derivatives and their syn-
thesis. Our new structures, designed from previously reported potent compounds, were tested for cyto-
toxicity on the HCT116 colon cancer cell line, and their binding to the biological target was analyzed
using computational studies involving blind docking of derivatives using Autodock. Further, we show
new evidence that our molecules bind directly to Hsp90 and modulate Hsp90’s binding with client pro-
teins. Finally, we demonstrate that we have integrated good ADME properties into a new derivative.
Published by Elsevier Ltd.
Received 13 May 2010
Revised 13 July 2010
Accepted 19 July 2010
Available online 22 July 2010
Keywords:
Sansalvamide A
Pancreatic cancer
Macrocyclic peptides
Macrocycles
Cytotoxicity
Growth inhibition
Docking
Hsp90
1. Introduction
position IV (Fig. 1, amino acid IV). Cytotoxicity of San A-amide
derivatives against pancreatic,7–9 colon,3,4,10,11 breast, prostate,
Compounds isolated from natural resources can provide novel
structures that can be used in the development of new small mol-
ecules that have novel mechanisms of action. One such compound
is Sansalvamide A (San A) (Fig. 1). San A, which was isolated from a
marine fungus (Fusarium spp.), exhibits anti-tumor activity against
multiple cancer cell lines.1–3 To date, the synthesis of 89 analogs
have been reported by our lab4–6 and 11 by Silverman and co-
workers.7 The natural product is a depsipeptide (Fig. 1), which is
prone to ring opening at the ester bond by esterases. Given the
depsipeptide’s lability, Silverman and co-workers synthesized the
natural product peptide, and found that the peptide was 10-fold
more active in a cell-based cytotoxicity assay than the natural
product depsipeptide, presumably because the peptide macrocycle
was more stable within cells than the depsipeptide.7,8 Thus, to
avoid degradation via ring opening, all 89 derivatives reported by
our laboratory were synthesized as derivatives of the San A peptide
(San A-amide), where an amino acid replaced the alcohol acid at
and melanoma cancers7 clearly indicate San A-amide’s potential
as a new therapeutic lead structure in the treatment of various
cancers and support further exploration of this class of compounds.
Amino Acid III
Amino Acid IV
Amino Acid III
Hydroxy Acid IV
O
O
N
H
O
N
O
H
NH
NH
Amino
Acid II
O
Amino
Acid II
HN
O
HN
O
NH
HN
HN
O
O
Amino Acid V
Amino Acid V
O
O
Amino Acid I
Amino Acid I
Sansalvamide A amide
"San A-amide"
Sansalvamide A
"San A"
Abbreviations: San A, Sansalvamide A; San A-amide, Sansalvamide A-amide;
Hsp90, Heat shock protein 90.
Compound 1
* Corresponding author. Tel.: +1 619 594 5580; fax: +1 619 594 4634.
E-mail address: mcalpine@chemistry.sdsu.edu (S.R. McAlpine).
Figure 1. Structure of Sansalvamide A (San A) and Sansalvamide A-amide (San A-
amide).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.07.042

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6823
Ten of the San A-amide derivatives prepared by Silverman and co-
ATP
ATP
workers contained an N-methyl within the peptide structure and
three were found to be more potent than San A-amide.7,8,12,13 From
our reported 89 derivatives, we have concluded that the most
important structural motifs are the inclusion of two consecutive
O
N
O
N
N
H
HN
NH
O
NHHN
O
O
D
-amino acids and an N-methyl moiety. This work has demon-
San A-amide
strated that three compounds containing these motifs were
significantly more potent than the natural product peptide, San
A-amide.6 Our work has been validated by several current exam-
ples in the recent literature where cyclic peptides, and specifically
M
M
Client proteins:
FKBP
pentapeptides, with both an N-methyl and D-amino acid lock the
C
C
Apoptosis
IP6K2
macrocycle into a single conformation.14–16 Data from these
studies suggest that the compounds, once locked into a major con-
formation, will be appropriately positioned as a beta or gamma
turn, which is likely to lead to a well-defined, high affinity interac-
tion with the protein target.17,18
C-terminus
client proteins
Geldanamycin
17-AAG
San A-amide
ATP
We report here the synthesis of 32 new Sansalvamide A deriva-
tives. These compounds were designed using the structure–activity
relationship (SAR) that we observed in earlier generations, and
utilized specific features known to play a key role in compound
potency, that is, the incorporation of several aromatic moieties,
ATP
Hsp70
complex
Hsp70
Complex
Immature
protein
Hsp70 complex,
HOP and immature
D
-amino acids, and N-methyl amino acids. Further, San A-amide
derivatives were shown to bind to Heat shock protein 90
(Hsp90).19 Given that Hsp90 is an oncogenic protein of inter-
est,20–23 and that this new series of compounds expound on the
SAR of previously reported potent derivatives by exploring new
avenues for incorporating aromatic moieties, these data describe
an important advance in the development of the San A-amide com-
pound class as a potential drug lead.
protein
p23
ATP
N-domain
ADP
M-domain
C-domain
Precedence has already been set for peptides to be used as
drugs. To date, there are 617 peptide drugs or drug candidates,
24% of these are in clinical trials, 65% are in advanced preclinical
phases, and 11% are on the market.24–26 These peptide drugs are
used to treat a variety of diseases such as prostate and breast can-
cer, HIV infections, osteoporosis, acute coronary syndrome, and
serve as immunosuppressants.27 Several key peptide-based drugs
include: Cyclosporin A (MW = 1185), Caspofungin (MW = 1093),
Vancomycin (MW = 1431), and Fuzeon (MW = 4492). Cyclosporin
A is an 11 amino acid macrocyclic peptide that is used to suppress
the immune system after organ transplants.28 Caspofungin, Vanco-
mycin, and Fuzeon are peptide-based antifungal, antibacterial, and
anti-HIV drugs, respectively. Aplidine (MW = 1067) is an eight ami-
no acid peptide-based cancer agent that is currently in clinical tri-
als.29–31 Thus, peptides are successfully used to treat diseases,
setting excellent precedence for San A-amide drug development
(MW = ꢀ600).32
Pi
ADP
Mature protein
Figure 2. (a) Interaction of San A-amide with Hsp90; (b) mechanism of San
A-amide on Hsp90, inhibition of 2 C-terminal client proteins: IP6K2 and FKBP52
while binding to the N-middle domain.19
investigation that inhibit binding of client proteins to the N-ter-
minal domain. This distinctive mechanism supports the further
investigation of San A-amide compounds as potential new thera-
peutic drugs.
San A-amide derivatives have been tested extensively on
numerous cancer cell lines, including several colon cancer cell
lines.1,3,4,10,61,62 Carcinogenesis in the colon rectum is thought to
occur through two different pathways. The two pathways are usu-
ally referred to as having microsatellite stability (MSS) or microsat-
ellite instability (MSI). Currently, only the MSS colon cancers are
known to respond to chemotherapeutic drugs. Additionally, the
Recently we showed evidence that the target for San A-amide
is heat shock protein 90 (Hsp90).19 Hsp90 functions as a molecu-
lar chaperone for intracellular signaling molecules,33–36 and it
folds, assembles, and stabilizes proteins that regulate the growth
of cells. It is also up-regulated in most cancers.33,37–50 There are
three distinct regions of Hsp90: the N-terminal, C-terminal, and
middle domain, and it exists as a homodimer, connected via the
C-terminal region.51–53 Its ATP binding site (located at the N-ter-
minal domain) is the binding site for the two inhibitors currently
in clinical trials, 17-DMAG and 17-AAG.23,33,39–44,54–60 In our pre-
vious work,19 we show that San A-amide analogs bind to Hsp90
and inhibit its activity via an allosteric mechanism, where it binds
to the N-middle domain, and inhibits, presumably via a confor-
mational change, the binding of two C-terminal client proteins
(Fig. 2). By inhibiting their binding to Hsp90, these two client
proteins are now forced to remain in the cytosol, inducing apop-
tosis via their cytosolic pathways. San A-amide’s mechanism is
unique from inhibitors that are currently in clinical development
because San A-amide interferes with clients that interact with the
C-terminus of Hsp90, as opposed to those currently under
drug of choice for treatment, 5-fluorouracil (5-FU) [IC50 = 5 lM],
has significant side effects, making it desirable to develop a drug
with improved efficacy. Because MSI colon cancers do not respond
to 5-FU, or to other current chemotherapeutic drugs,63,64 finding
new structures that target both cancer pathways is imperative.
The 32 compounds and the derivatives from which they were de-
signed were tested on the HCT116 colon cancer cell line. This can-
cer cell line was chosen not only because it is a commonly used cell
line, found in the NCI 60 cell line panel, but it is also known to be
microsatellite instable (MSI). Although major efforts have been
made, few truly novel classes of compounds have been identified
that have activity against drug-resistant (MSI) colon cancer tu-
mors. This work reports our understanding of the complex struc-
ture–activity relationship of the 32 new compounds in a drug-
resistant colon cancer cell line, establishes a phenotype for cyto-
toxicity in cell-based assays, and models these compounds bound
to their biological target Hsp90.

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R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
tested.4–6 It was noted in our previous work that two consecutive
-amino acids and a single N-methyl were incorporated into the
2. Design and biological activity of new Sansalvamide A-amide
derivatives
D
most potent structures. However, it was not known if the position-
ing of these structural features was important to the conformation,
or if simply molecules containing these features were similarly po-
tent. Thus, a series of derivatives were made that incorporated
these features and they were moved throughout the ring.
Compounds 3–7 were all based on compound 2, where com-
pounds 3–5 the N-methyl and two D-amino acids were rotated
around the positions of the ring. Compound 6 was identical to 2,
except that it did not contain an N-methyl moiety at position IV.
In order to explore the potency of this structural class we de-
signed compounds based on several of the most interesting first
and second-generation structures,4–6,10 which were tested against
HCT-116 cancer cell lines.61,62 Each new derivative was designed
to examine the change in potency by altering the amino acid at
one position relative to it’s ‘‘parent” compound(s). These altera-
tions included a change in the stereochemistry of one of the aa’s
to investigate the effect on potency as it relates to conformation
or the replacement of one aa with another to investigate the effect
as it relates to polarity at that position. Structural differences in the
San A-amide derivatives can be easily identified by the reader in all
Derivative 7 involved the incorporation of an additional
acid at IV, as well as a second N-methyl moiety on the N at III. Com-
pound 10 has two -amino acids, one at position I and one at posi-
tion II based on the structures of 8 and 9. Compound 12 replaced
the -Leu of 11 with a -Phe.
D-amino
D
subsequent figures as all
L-amino acids are shown with wedged
D
D
bonds, and -amino acids are shown with dashed bonds.
D
Noteworthy second-generation compounds chosen as leads in-
cluded: 13, 14, 16, 17, 19, 22, and 25 (Fig. 4).5,6 The structures
resulting from these leads include: 15, 18, 20, 21, 23, 24, 26, and
Several first generation structures were chosen as leads: 2, 8, 9,
and 11 (Fig. 3).9 These structures were chosen as they had reason-
able potency in at least one of several cells lines in which they were
O
III
O
O
O
O
IV
O
N
H
N
O
H
O
O
N
H
HN
HN
NH
HN
N
H
N
O
O
N
NH
HN
N
NH
O
O
N
O
II
NH HN
O
O
O
HN
NH
NH
O
V
O
O
I
Compound 3
Compound 4
Compound 5
Compound 2
O
O
O
N
H
O
HN
HN
N
NH
O
HN
N
O
NH
O
O
NH HN
O
O
Compound 6
Compound 7
O
O
O
O
O
N
H
N
H
O
N
H
HN
NH
HN
NH
O
O
HN
HN
NH
O
NH HN
NH HN
O
O
O
NH
O
O
O
Compound 8
Compound 9
Compound 10
O
O
O
O
N
H
O
O
N
H
HN
NH
O
HN
NH
O
NH HN
O
NH HN
O
Compound 11
Compound 12
Figure 3. The design of new derivatives based on potent first generation molecules: (a) structures based on compound 2; (b) structures based on compounds 8, 9, and 11.

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6825
III
O
O
O
O
IV
N
H
O
N
O
O
N
H
H
HN
HN
NH
II
N
NH
O
N
NH
O
O
N
HN
NH
O
O
N
O
HN
V
O
O
I
Compound 15
Compound 13
Compound 14
O
O
O
O
O
N
N
H
O
H
HN
NH
N
H
HN
NH
O
O
HN
HN
NH
O
HN
NH
O
NH HN
O
O
NH
O
O
O
Compound 16
Compound 17
Compound 18
O
O
O
O
N
O
O
O
N
H
N
H
H
NH
N
HN
NH
O
N
NH
O
O
NH HN
O
O
NH HN
O
NH HN
O
O
O
HO
O
Compound 19
Compound 20
Compound 21
NHCbz
NH2
O
O
O
O
N
O
N
HN
O
N
H
HN
H
NH
H
NH
HN
NH
O
O
O
NH HN
NH HN
NH HN
O
O
O
O
O
O
HO
HO
HO
Compound 23
Compound 24
Compound 22
NH2
NHCbz
O
HN
NH
CbzHN
NH
O
N
O
O
H
O
O
HN
NH
N
H
O
O
O
N
H
HN
NH
HN
O
NH
NH HN
O
O
NH HN
O
NH HN
O
O
HN
N
H
N
H
Compound 25
Compound 26
Compound 27
Figure 4. The design of new derivatives based on the second-generation structures.
27. Compound 15 was derived from 13 and 14, where and N-
methyl -Phe was inserted at position II and an N-methyl -Leu
was inserted at position V. Derivative 18 was designed from 16
and 17, where a -Phe and a -Val were incorporated into positions
II and III, respectively. Compounds 20 and 21 were designed from
19, where an N-methyl moiety located at position II in 19 is not in-
cluded in 20. The serine at V was a free alcohol in 21 as opposed to
benzyl protected in 19. Derivatives 23 and 24 were based on 22,
where a lysine (protected and unprotected, respectively) was in-
cluded at position IV. Similarly 26 and 27 were based on 25, and
included an arginine at position IV (protected and unprotected,
respectively).
D
D
D
D
Compound 14 was seen as an excellent lead structure, demon-
strating potency in both pancreatic (IC50 = 1.4 l
M for PL-45)6,65

6826
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
and colon cancer cell lines (IC50 = 1.9
l
M for HCT-116).5,61 Thus, it
Chloro-Cbz on biological activity. Compound 41 maintains the ste-
reochemistry, but moves the N-methyl inbetween the two -amino
acids. Finally, 42 has an N-methyl at position V rather than at III.
was used as a template to design an additional new series of
molecules to investigate the incorporation of a lysine residue to
the structure. The first of these was compound 28, where the solu-
tion phase synthesis required the use of an orthogonal protecting
group (Cbz) on the lysine side chain. It was found that 28 also
had potent cytotoxic effects on two colon cancer cell lines (Table
1). Thus, we used 28 as a lead to make additional compounds that
would explore the structure activity series of these molecules
(Fig. 5). The structures resulting from compounds 14 and 28 are
compounds 29 and 30. In contrast to 28’s Cbz-protected lysine at
position V, these compounds contain a Boc-protected lysine and
a free lysine at position V, respectively.
Given the success of compound 28, we chose three additional
compounds from which we designed new structures with Boc-pro-
tected lysines and free lysines: 1, 16, and 33 (Fig. 6). Compound 1,
San A-amide, was chosen as a ‘‘control” molecule, where 31 and 32
are related to the natural product peptide via a single change: a
protected or free lysine at V. Compounds 16 and 33 have shown
significant cytotoxicity in several types of cell lines, and as such
were considered interesting leads.10,61,62 The structures that were
designed from these leads were 34–37. In contrast to 28, 29, and
30, none of the six molecules in Figure 6 have the N-methyl
D
In previous work, we had noticed that in addition to the trend
that two consecutive D-amino acids and an N-methyl were key to
potency,6 there was a trend that the potent molecules typically
contained 2–3 phenyl groups within the structure (similar to com-
pounds 38 through 42). Thus, we designed four new compounds
that would incorporate three phenyl rings, while maintaining the
N-methyl and
D-amino acids in the core structure. These com-
pounds are: 43, 44, 45, and 46 (Fig. 8), where these four molecules
are diastereomers of each other. Positions II–V are identical, and at
position I, the alpha and beta carbons of the benzylated beta-hy-
droxy-phenylalanine have alternating stereochemistry.
3. Synthesis of Sansalvamide A-Amide derivatives
All 32 derivatives described here were constructed as the pep-
tide analogs (Figs. 3–8). Two synthetic protocols have been devel-
oped for the creation of these 32 derivatives: a convergent
solution-phase strategy, which we have previously published,4,66
as well as a solid-phase approach, which is described here the for
the first time (Fig. 9). Both routes have provided access to large
milligram quantities of San A-amide derivatives. Ten of these
new compounds were synthesized using solution phase (6, 7, 10,
12, 15, 18, 23, 24, 26, and 27), 21 of these compounds were synthe-
sized using solid-phase 3, 4, 5, 20, 21, 29–32, 34–37, and 39–46,
and one compound, 28, was made using both methods.
D-Phe at II, but they still contain a Boc-protected lysine or a free
lysine at V, respectively. Molecules 34 and 35, similar to 33, con-
tain an N-methyl at V in addition to the Boc-protected lysine and
free lysine at V. Derivatives 36 and 37, similar to 16, incorporate
a D-Val at III, as well as either a Boc-protected lysine or free lysine,
respectively, at V.
For the 10 solution-phase compounds we used our previously
published synthesis.5 The solid-phase synthesis compounds were
synthesized using a pre-loaded chlorotrityl resin (where the first
amino acid was already bound to the resin) (Fig. 9). Sequential cou-
pling then deprotection of four Fmoc protected amino acids using
coupling conditions of hydroxybenzotriazole (HOBT, 3 equiv), and
N,N0-diisopropylcarbodiimide (DIC, 3 equiv) in 0.2 M DMF, and
standard deprotection conditions of piperidine/DMF (20:80 ratio)
yielded a resin-bound linear pentapeptide. Cleavage from the resin
was accomplished using trifluoroethanol (TFE) and DCM in a 1:1
ratio for 24 h. After complete removal of residual TFE (to avoid
A recent publication describing the potency of compound 38
when tested against pancreatic cancer cell lines6 prompted us to
design compounds that were based on the structure of this
molecule. We created four compounds that would mimic 38 and
explore the importance not only of stereochemistry but also of
the protecting group on the lysine side chain. These compounds
are 39, 40, 41, and, 42 (Fig. 7). Compound 39 contains an
Chloro-Cbz-protected Lysine at IV rather than the -2-Chloro-
Cbz-protected lysine seen in 38. Compound 40 maintains the ste-
L-2-
D
D
reochemistry at IV but utilizes a Cbz-protected lysine rather than a
2-chloro-Cbz lysine in order to investigate the effect of the 2-
O
O
N
H
N
NH
O
HN
NH
O
BocHN
O
O
III
O
IV
O
O
O
N
H
Compound 29
N
H
N
NH
N
II
NH
O
O
NH HN
O
O
NH HN
O
O
O
N
CbzHN
V
H
N
NH
I
O
NH HN
O
Compound 28
Compound 14
H2N
O
Compound 30
Figure 5. The design of new derivatives based on compound 14 and 28.

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6827
III
O
O
O
O
O
O
IV
O
N
H
N
H
N
H
II
HN
NH
HN
HN
HN
HN
NH
NH
O
O
HN
NH
O
NH
O
NH
O
O
O
O
BocHN
H2N
V
I
Compound 1
Compound 31
Compound 32
O
O
O
O
O
O
N
H
N
H
N
H
HN
HN
HN
HN
NH
NH
O
HN
NH
O
O
N
N
O
O
O
HN
N
O
O
O
BocHN
H2N
Compound 33
Compound 34
Compound 35
O
O
O
O
N
H
O
O
N
H
N
H
HN
HN
NH
O
HN
HN
NH
HN
HN
NH
O
O
NH
O
NH
NH
O
O
O
O
BocHN
O
H2N
Compound 16
Compound 36
Compound 37
Figure 6. The design of new derivatives based on compound 1, 33, and 16.
trifluoroethyl esterification during the cyclization step) and
confirmation of each linear pentapeptide via NMR and LCMS, cycli-
zation was accomplished using our standard cyclization conditions
employing a cocktail of three coupling agents (2-(1H-7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophos-
phate methanaminium (HATU), 3-(diethoxy-phosphoryloxy)-3H-
benzo[d][1,2,3] triazin-4-one (DEPBT), and O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU) 0.7 equiv
each), N,N-diisopropylethylamine (DIPEA), and run at 0.007–
0.0007 M in ACN and DCM.66 These synthesis conditions generated
a total of 17 compounds, all in moderate yields (5–76%, average
ꢀ40%). This solid-phase route, although slightly more expensive,
proves to be more efficient, quickly generating high purity linear
precursors which result in cyclized compounds with overall signif-
icantly higher yields. The final purity of all compounds was verified
by NMR and LCMS.66
correlated to a decrease in cell proliferation in the presence of the
compound, and hence the more toxic the compound. Data below
is shown as a % growth inhibition, where the greater the % inhibi-
tion, the more cytotoxic the compound. Cytotoxicity data are
shown by sequentially starting with first generation compounds
and those designed from these structures, then the second-genera-
tion molecules and the cytotoxicity of derivatives designed from
these, and finally the toxicity of the de novo compounds.
Table 1 outlines the biological activity of new compounds and
compares their values to the earlier generation structures from
which they were designed. It is important to note that first gener-
ation compounds 1, 2, 8, 9, and 11 all have significantly higher %
growth inhibition values in other cell lines: PL-45, BxPc3 (both
pancreatic cancer cell lines) or SW480 and HT-29 (MSS colon can-
cer cell lines that respond to treatment with 5-FU), which is why
they were initially chosen as lead structures.6,9 However, given
the enormous problems seen in treating drug-resistant colon can-
cer, we have chosen to focus on finding molecules that have a high
percent growth inhibition against the drug-resistant (MSI) colon
cancer cell line HCT-116. As the data shows, there was no signifi-
cant improvement in the new compounds (bold) compared to
the first generation leads (non-bold).
4. Biological data for compounds
All compounds were tested for their cytotoxicity on colon cancer
cell line HCT-116 using 3H-thymidine incorporation assays. These
new third generation compounds were compared to the potency
of compounds from which they were designed, and cell prolifera-
tion was monitored by measuring how much 3H-thymidine was
incorporated into a cell’s DNA. Lower thymidine incorporation is
Next, we examine the biological activity of compounds that
were designed from the second-generation structures. As the data
below shows, there was no significant improvement in the biolog-
ical activity of the new compounds (bold) compared to the second-

6828
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
CbzHN
ClCbzHN
O
O
O
O
O
O
N
N
HN
NH
HN
NH
O
O
NH HN
O
NH HN
O
ClCbzHN
III
O
IV
O
O
N
II
HN
NH
O
Compound 40
Compound 39
HN
NH
O
Cl-ZHN
ClCbzHN
V
O
O
I
O
O
O
O
N
H
N
H
HN
N
HN
NH
O
O
Compound 38
NH HN
O
N
O
HN
Compound 42
Compound 41
Figure 7. The design of new derivatives based on compound 38.
III
O
This conclusion was validated by others who found that cyclic pen-
tapeptides containing both an N-methyl and -amino acids were
O
O
D
IV
N
O
N
fixed into a major or even single conformation.14,15 Further, these
data indicate that the compounds, if appropriately situated once
locked, will have a well-defined, high affinity interaction with
the protein target.17,18 The cytotoxicity data describing compounds
19 and 20 supports this conclusion. Second-generation compounds
22 and 25 were reasonably potent in numerous colon cancer cell
lines (HCT116, HCT15,61,62 and HT-29,5 as well as pancreatic cancer
cell lines6,9) yet it is interesting to see that polar compounds 24 and
27, which were based on 22 and 25 but incorporated a polar resi-
due that would improve solubility in aqueous media, and 23 and
26, with protecting groups on the respective polar residues, were
significantly less potent than their lead structures in colon cancer
cell lines.
HN
NH
HN
NH
O
O
NH HN
II
O
O
NH HN
O
O
R
O
S
S
V
R
O
(R,R)
(S,S)
I
Compound 43
Compound 44
O
O
O
N
O
N
HN
NH
HN
NH
O
O
The following series shows the biological activity of compounds
that were designed based on a second-generation lead structure
14, as well as compounds designed from a new third generation
structure that proved to be relatively cytotoxic, 28 (% growth inhi-
NH HN
HN
NH
O
O
O
O
R
S
O
O
S
R
bition at 10
ing compounds 14 and 19’s potency the inclusion of an N-methyl
and -Phenylalanine is favorable, which explains compound 28’s
lM = 99%). As noted in earlier SAR discussions describ-
(R,S)
Compound 46
(S,R)
Compound 45
D
relative potency. It is interesting to note that structurally similar
compound 29 is not nearly as potent, where there is an exchange
of the Cbz to a Boc moiety. Given the poor cytotoxicity of 24 and
27, which contain a free lysine and free arginine, respectively, it
is not surprising that the free lysine-containing 30 is not active.
Compounds 31, 34, and 36 were designed based on 29, 33, and
16, respectively. Compounds 32, 35, and 37 were then free lysine
derivatives of 31, 34, and 36, respectively, and, not surprisingly,
were inactive.
We then show the biological activity of compounds that were de-
signed based on a lead structure described in our most recent publi-
cation,6 38. Of the three compounds that were based on 38, we
synthesized one compound, 42, with improved cytotoxicity over
the second-generation lead structure. It should be noted that the
Figure 8. The design of new derivatives based on de novo design using SAR.
generation leads (non-bold). However, a very interesting struc-
ture–activity relationship is observed between compound 19 and
20, where 19 is significantly more potent than 20. Perhaps not sur-
prisingly, 19 was also significantly more potent than derivative 21.
This supports the conclusion we have published in a prior manu-
script: an N-methyl is imperative for inducing an appropriate
three-dimensional structure. That is, we have shown that the most
important structural motif is the inclusion of two consecutive D-
amino acids with an N-methyl moiety, and we had demonstrated
that three compounds were significantly more potent than the nat-
ural product peptide, San A-amide, when they followed this motif.6
structure with a Chloro-carboxybenzyl protected L-Lysine that was

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6829
R3
O
O
NHFmoc II
(3 eq)
NRFmoc
(3 eq)
III
O
O
O
HRN
HO
HO
O
R2
R3
R2
R
N
RN
RN
R1
R2
1. HOBT (3 eq),
DIC (6 eq), DMF 0.2M
NH2
1. HOBT (3 eq),
DIC (6 eq), DMF 0.2M
NRH
R1
I-II
O
I
2. 20% piperidine/DMF
2 x 10 min wash
2. 20% piperidine/DMF
2 x 10 min wash
R1
O
R = H or Me
I-II-III
O
R4
IV
(3 eq)
OH
O
R3
O
R3
R4
O
NRFmoc
N
R
O
O
R4
1. HOBT (3 eq),
DIC (6eq), DMF 0.2M
N
R
O
NR
NHR
RN
RN
R2
1. 1:1 (v/v) TFE:CH2Cl2, 24 hr
NR
RN
R2
O
2. 20% piperidine/DMF
2 x 10 min wash
R5
O
2.TBTU (0.7 eq), HATU (0.7 eq)
DEPBT (0.7 eq), DIPEA (6 eq)
10:1 CH2Cl2:CH3CN
NR RN
R1
San A derivative
R1
R5
OH
V
O
(3 eq)
O
(0.007-0.0007M)
O
NHFmoc
R5
Linear Pentapeptide
3. HOBT (3 eq),
DIC (6 eq), DMF 0.2M
4. 20% piperidine/DMF
2 x 10 min wash
Figure 9. Solid-Phase synthesis of San A-amide derivatives.
substituted at position IV generated compound 39, which was less
potent than the parent compound 38 that had the Chloro-carboxyb-
tionships were exponential in nature, although it does appear that
these compounds have limited solubility, which inhibits the com-
pounds from dissolving at concentrations higher than 500 nM
(0.5 lM). This indicates that for future studies we should include
a polar moiety in order to improve solubility on an area of the mol-
enzyl protected
hypothesis that the inclusion of two consecutive
an N-methyl moiety is important for potency. However, interest-
ingly, the molecule where a carboxybenzyl protected -lysine was
placed at position IV (compound 40), which contained two consecu-
tive -amino acids with an N-methyl moiety, was significantly less
D
-Lysine was at this position. This data supports our
D
-amino acids with
D
ecule that will not interfere with binding to their biological target.
D
5. Summary of SAR results
potentthan either 38or 39, suggestingthat theaddition of thechloro
substituent on the carboxybenzyl was crucial for improving binding
to this molecule’s biological target. We noted the movement of the
N-methyl to position V, compound 42, produced a highly potent
compound that is more toxic than its lead structure 38, or its struc-
turally related analog 41. It is very remarkable to note that although
both 41 and 42 contain the chloro-carboxybenyl moiety, an N-
methyl moiety, and two consecutive D-amino acids the placement
of the N-methyl moiety is critical for potency as 41 is not very potent
at all, but 42 shows remarkable cytotoxicity. These data support our
In summary, the most important features to emerge from this
SAR study include the observation that the potent molecules con-
tain two consecutive D-amino acids and an N-methyl moiety. In
addition to understanding the importance of that structural fea-
ture, we also learn that (a) a chloro-Cbz moiety improves cytotox-
icity over a Cbz (38 vs 40), (b) a Cbz generates a molecule with
better cytotoxicity than one protected with a Boc group (28 vs
29), and (c) an N-methyl positioned on the
D-phenylalanine
produces a molecule that is more potent than without the
N-methyl (14 vs 17, 19 vs 20, and 42 vs 38). Finally, we learn that
the benzylated beta-hydroxy-phenylalanine, when in the R,R con-
figuration (43), affords a structure that is relatively potent com-
pared to the other diastereomers (44, 45, and 46). Thus, it would
appear that the ideal structure would have the following features:
the benzylated beta-hydroxy-phenylalanine in with R,R stereo-
hypothesis that two consecutive D-amino acids combined with an
appropriately placed N-methyl moiety are important for inducing
a favorable conformation. However, they also indicate that there
may be a favorable electronic effect on binding induced by the inclu-
sion of a chlorine in the structure.
Finally we look at the biological activity of compounds that
were designed via a de novo process. These four compounds, 43–
chemistry (I), the
in order to enhance solubility (III), a
chloro-Cbz (IV), and an N-methyl- -phenylalanine (V). It is noted
that although structures 14, 19, and 28 incorporate an N-methyl-
-phenylalanine at position II, this moiety is already incorporated
L
-leucine (II), an amino acid with polar moiety
46, included an N-methyl, at least two
phenyl moieties within the core structure. The most potent com-
pound, 43, also has two consecutive -amino acids and an N-
D-amino acids, and three
D
-lysine protected with a
D
D
methyl moiety, and then discretely places the benzyl protected
phenyl threonine below the ring plane. Given that 43 has signifi-
cantly great potency than the other three derivatives, it appears
that this placement plays a key role in potency and demonstrates
that the 3-D shape of the molecule is important for obtaining a
tight binding to its target.
D
at position V of the ideal structure, and therefore it seems unlikely
that it should also be included at position II. Further, it will only in-
crease the hydrophobicity of this molecule, which already lies out-
side the cLog P values that are quoted in Lipinski’s rules for
enhancing drug-like properties.67 Rather, leaving positions II and
III open to modification with polar amino acids or peptidomimetic
structures such as oxazoles or thiazoles seems like a better ap-
proach. These two options would decrease the hydrophobicity
and move the molecule into more reasonable cLog P values. One
pro-drug approach is to place a methyl-protected acid side chain
The most potent compounds, defined as P60% cytotoxicity
against HCT-116 at 10
and the IC50 values were calculated by plotting five concentrations
(10, 3, 1, 0.3, and 0.1 M) and extracting data from the curves
lM, were then run in cytotoxicity assays
l
(Fig. 10). There were eight compounds that exhibited P60% growth
inhibition, these included: 14, 19, 28, 38, 42, 43, 44, and 46. All rela-

6830
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
Table 1
Bolded compounds are new structures, and compounds from which they were designed are non-bolded
Compound
Structure
cLog P
% Growth inhibition
IC50 (lM)
2
3.847
3.847
18
15
—
—
D
-Phe-Leu-Val-N-Me-Leu-
-Phe- -Leu-Val-Leu-N-Me-Leu
-Leu- -Val-Leu-Leu
Phe-N-Me-Leu- -Val- -Leu-Leu
-Phe-Leu-Val-Leu- -Leu
D-Leu
3
D
D
4
5
6
N-Me-Phe-
D
D
3.847
3.847
3.624
30
30
7
—
—
—
D
D
D
D
D
D
7
4.473
3.624
15
26
—
—
-Phe-Leu-N-Me-Val-N-Me-D-Leu-D-Phe
8
-Phe-Leu-Val-Leu-Leu
9
10
Phe-
D
-Leu-Val-Leu-Leu
3.624
3.624
3
14
—
—
D
-Phe-
D
-Leu-Val-Leu-Leu
11
12
Phe-Leu-Val-
Phe-Leu-Val-
D
-Leu-
-Leu-D
D
-Leu
-Phe
3.624
4.026
20
28
—
—
D
13
14
15
16
17
18
19
20
21
22
Phe-Leu-Val-Leu-N-Me-
D
-Leu
3.847
4.249
4.473
3.624
4.026
4.026
5.186
4.962
2.818
3.320
32
99
47
8
21
4
93
10
18
50
—
1.9
—
—
—
—
1.9
—
—
—
Phe-N-Me-
D
-Phe-Val-Leu-Leu
Phe-N-Me-
D
-Phe-Val-Leu-N-Me-D-Leu
Phe-Leu-
D
-Val-Leu-Leu
-Phe-Val-Leu-Leu
-Phe- -Val-Leu-Leu
Phe-
Phe-
D
D
D
Phe-N-Me-
Phe- -Phe-
Phe-N-Me-
D
-Phe-
D
-Val-Cha-Ser(Bn)
D
D-Val-Cha-Ser(Bn)
D
-Phe-
D
-Val-Cha-Ser
D-Tyr-Leu-Val-Leu-Leu
D-Tyr-Leu-Val-Lys(Cbz)-Leu
D-Tyr-Leu-Val-Lys-Leu
D-Trp-Leu-Val-Leu-Leu
D-Trp-Leu-Val-Arg(Cbz)-Leu
D-Trp-Leu-Val-Arg-Leu
23
24
25
26
27
3.652
1.489
3.452
5.443
1.288
0
0
—
—
—
—
—
30
2
0
14
28
29
30
Phe-N-Me-
Phe-N-Me-
Phe-N-Me-
Phe-N-Me-
D
D
D
D
-Phe-Val-Leu-Leu
4.249
5.026
4.356
2.863
99
99
36
0
1.9
3.9
—
-Phe-Val-Leu-Lys(Cbz)
-Phe-Val-Leu-Lys(Boc)
-Phe-Val-Leu-Lys
—
1
31
32
33
34
35
16
36
37
Phe-Leu-Val-Leu-Leu
Phe-Leu-Val-Leu-Lys(Boc)
Phe-Leu-Val-Leu-Lys
Phe-Leu-Val-Leu-N-Me-Leu
Phe-Leu-Val-Leu-N-Me-Lys(Boc)
Phe-Leu-Val-Leu-N-Me-Lys
3.624
3.730
2.238
3.847
3.953
2.461
3.624
3.730
2.238
35
0
0
35
58
0
8
9
0
—
—
—
—
—
—
—
—
—
Phe-Leu-
Phe-Leu-
Phe-Leu-
D
D
D
-Val-Leu-Leu
-Val-Leu-Lys(Boc)
-Val-Leu-Lys
38
39
40
41
42
Phe-Leu-N-Me-Val-
Phe-Leu-N-Me-Val-Lys(Cl-Cbz)-
Phe-Leu-N-Me-Val- -Lys(Cbz)-
Phe-Leu-Val-N-Me- -Lys(Cl-Cbz)-
Phe-Leu-Val- -Lys(Cl-Cbz)-N-Me-
D
-Lys(Cl-Cbz)-
-Phe
-Phe
D
-Phe
5.630
5.630
5.026
5.630
5.630
69
58
36
36
98
7.6
—
—
—
2.9
D
D
D
D
D
-Phe
-Phe
D
D
43
44
45
46
(R,R)b-OH(Bn)-Phe-Leu-N-Me-Val-
D
-Leu- -Phe
D
5.698
5.698
5.698
5.698
94
64
25
63
3.2
5.8
—
(S,S)b-OH(Bn)-Phe-Leu-N-Me-Val-
(S,R)b-OH(Bn)-Phe-Leu-N-Me-Val-
(R,S)b-OH(Bn)-Phe-Leu-N-Me-Val-
D
-Leu-D-Phe
-Leu-
-Leu-
D
D
D-Phe
D-Phe
8.9
Note: specific residues that were altered in the new structures are shown in bold. Cytotoxicity and cLog P data for all newly synthesized derivatives and parent compounds.
IC50 values were determined for eight of the most potent derivatives. cLog P values were calculated using software from ChemAxon.
at II or III (i.e., glutamic acid or aspartic acid with a methyl ester on
the side chain), which would allow the molecule to cross the
hydrophobic cell wall and then be cleaved upon entering the cell.
The other approach is to incorporate thiazoles and oxazoles so as
to increase the molecules hydrophilicity slightly, but still maintain
a peptide-like backbone. Both of these approaches are now being
pursued based on the above SAR.
this tagged derivative was no longer identical to compound 1 we
wanted to confirm our findings. In order to do this, we have run
a number of assays described in published work that shows our
molecule inhibits the binding between several client proteins and
Hsp90. However, we have not show data with a direct binding
interaction between Hsp90 and compound 1-tag. Shown in
Fig. 11 is new, direct evidence that our molecule binds to Hsp90.
This competitive binding assay was completed using compound
1, biotinylated compound 1 at position IV, and Hsp90. Increasing
concentrations of compound 1 were incubated with Hsp90, fol-
lowed by incubation with biotin-SanA1. It was found that com-
6. Hsp90 competitive binding assay
As described in our recent work,19 we identified Hsp90 as the
target of the Sansalvamide A peptide (compound 1) using com-
pound 1 tagged with biotin at position IV. Aware of the fact that
pound
19.7 M (Fig. 11), thus, confirming our findings that Hsp90 is a tar-
get of Sansalvamide A-amide (compound 1).
1 inhibited binding of biotin-SanA1 with an IC50 of
l

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6831
100
80
60
40
20
0
IC50 Values of 8 most potent compounds
10
8
6
4
2
0
IC50=19.7µM
14
19
28
38
42
43
44
46
1.9
1.9
3.9
7.6
2.9
3.2
5.8
8.9
HCT-116
O
O
O
O
N
N
H
H
NH
N
N
NH
O
O
NH HN
O
NH HN
O
O
O
O
0
10
20
30
40
50
Concentration of drug (uM)
Figure 11. Competitive binding affinity of SanA1 with tag at IV to Hsp90.
Compound 19
ClCbzHN
Compound 14
and is a pro-apoptotic protein that is active when not bound to
Hsp90.70 Compound 20 was used as the negative control as it
exhibits little cytotoxic activity and only differs from 19 by a single
N-methyl at position II. Excitingly, we found that compound 19
inhibited the binding of both IP6K2 and Her2 to Hsp90 (Fig. 12).
In contrast, compound 20 did not have any affect on the binding
of IP6K2 orHer2 to Hsp90. These data suggest that compound 19
does bind to and modulate the function of Hsp90 and that the pres-
ence of an N-methyl is crucial for compound 19’s activity.
O
O
N
O
O
O
N
H
HN
N
NH
NH
O
O
NH HN
NH HN
O
O
CbzHN
O
Compound 28
Compound 38
ClCbzNH
San A 19
110
O
O
100
90
80
70
60
50
40
O
O
O
N
N
H
HN
HN
HN
NH
NH
O
O
N
O
NH HN
R
O
O
O
R
(R,R)
Compound 43
Compound 42
30
O
Her2
O
20
O
IP6K2
N
O
N
10
0
HN
NH
HN
NH
O
O
NH HN
0
1
2
3
4
5
6
7
8
9
10 11
O
O
NH HN
O
O
R
San A 19 (
µ
M)
S
O
S
S
O
(R,S)
Compound 46
(S,S)
Compound 44
San A 20
110
100
90
80
70
60
50
40
30
20
10
0
Figure 10. IC50s of potent compounds. Each data point is an average of four wells
run in three assays at 10, 3, 1, 0.3, 0.1 M concentrations. Data represents the
concentration required of each compound to inhibit 50% of viable cell growth in the
assay using HCT-116 colon cancer cell lines. Inhibition is relative to 1% DMSO
control.
l
7. Hsp90 client protein assays
Her2
IP6K2
Our data indicated that the cytotoxicity of compound 1 was
due, at least in part, to its ability to bind to Hsp9019 and inhibit cli-
ent proteins and co-chaperones from binding. Thus, we anticipated
that the cytotoxic effect of compound 19 was due, at least in part,
to its ability to bind to also inhibit client proteins from binding to
Hsp90. In order to test this hypothesis, we performed an in vitro
binding assay, testing 19’s ability to inhibit binding between
Hsp90 and two client proteins: Her2 and IP6K2. Her2 is a client
0
1
2
3
4
5
6
7
8
9
10 11
San A 20 (µM)
Figure 12. In vitro binding assay: (a) San A 19 inhibits the binding of both IP6K2
and Her2 to Hsp90. (b) San A 20 does not affect Her2 and IP6K2 binding to Hsp90.
Percent Hsp90 bound to client protein was quantified by densitometric scanning of
Hsp90 protein on Western blot with normalization to client protein loading using
Image J.
protein that is associated with the
N and M domains of
Hsp90,68,69 while IP6K2 is associated with the C-terminal domain

6832
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
8. Docking to Hsp90 using Autodock
We have shown that the San A-amide, compound 1, binds to
Hsp90 between the N-middle domain.19 Although we cannot as-
sume that all of the potent compounds shown in Fig. 10 will bind
to Hsp90, based on our published work, we investigated their bind-
ing affinity for this target at the N-middle domain. We used Auto-
dock to visualize how our molecules may bind to Hsp90. This
program is well established and is frequently used to dock small
molecules to large protein targets via an automated prediction of
ligand-binding sites.71–74 It generates an efficient docking of pep-
tides and small molecules to proteins,72,73,75 and thus it is a pow-
erful tool for visualizing protein inhibitors. As our recent work
has shown, San A derivatives bind to Hsp90 at the N-middle do-
main,19 thus we focused on binding our molecules to Hsp90 (pdb
file 2CG9.pdb) in this region (Figs. 13–15). Using the Autoligand
program, we identified two potential binding sites on the protein
between these domains. Next, using Autodock 4.2, we docked four
derivatives to Hsp90 and found one of the sites gave a much lower
binding energy to all 4 molecules (ꢁ3.5 kcal/mol vs ꢁ7.5 kcal/mol).
Because San A-amide is a known micromolar inhibitor and thus
reasonably potent, we chose to dock all molecules to the site that
gave the lowest binding energy as it seemed to be an accurate
reflection of the binding energy data obtained from Autodock. To
examine the binding of our San A-amide derivatives, we docked
each compound 250 times with the binding site identified on
19 docked to the Hsp90 N-M domain
(Blue-Green respectively). N-methyl of 19 is shown in
orange.
a
Compound 19 docked to Hsp90 N-M
domain (N domain = Blue, Middle = Green, and C
domain = Red)
b
Compound 20 docked to the Hsp90 N-M domain
Figure 14. Close-up of docking results: (a) compound 19 and (b) compound 20
bound to Hsp90. Compound 19 contains a methyl group (orange) in the cyclic
peptide backbone, which is not present in the peptide backbone of compound 20.
The specific interaction that may be responsible for enhanced binding affinity of 19
is shown in the box, (a), and its absence is also highlighted, (b). Thus, the N-methyl
has an obvious effect on the confirmation of the molecule and it’s binding to Hsp90.
Hsp90. Docking modes that returned similar binding energies
and conformations were clustered together to generate potential
binding orientations for each derivative. The mode or orientation
for each derivative that gave the lowest mean binding energy
was selected and visualized using PyMol. This allowed us to visu-
alize the conformation and relative orientation of each of the 8 po-
tent derivatives bound to Hsp90.
In Figures 13 and 14, we show two structurally similar deriva-
tives, 19 and 20, bound to the yeast variant of Hsp90. Compounds
19 and 20 differ only in the presence of an N-methyl group at posi-
tion II. However, although structurally similar, compounds 19 and
20’s cytotoxicity and ability to inhibit client proteins from binding
20 docked to Hsp90, N-M domain
Figure 13. (a) Full-length Hsp90 monomer with compound 19 in the predicted
binding site between the N-middle domain. Blue, green and red are for the N,
middle and C terminal domains respectively. Compound 19 (space-filling, grey) is
bound to the region between the N and middle domains of full-length yeast Hsp90.
This potent derivative adopts a conformation that fits well inside the binding
pocket. (b) Compound 20 (space-filling, grey) is bound to the region between the N
and Middle domains. Note how the differences in conformation prevent this non-
potent derivative from inserting deep into the pocket, exposing a majority of the
structure to solvent. The proposed binding site on Hsp90 was identified using
AutoLigand correlated with pull-down assay results and the San A derivative
binding mode was determined using AutoDock4.2. Molecular graphics were
prepared using PyMol.

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6833
43 docked to Hsp90 N-M domain
43 docked to Hsp90 N-M domain
44 docked to Hsp90 N-M domain
Figure 15. (a) Full-length Hsp90 with compound 43 (space-filling, grey) docked to
the N-middle domain. Blue, green and red are for the N, middle and C terminal
domains, respectively. The benzylated beta-hydroxy phenylalanine side chain is
highlighted in orange. This derivative is predicted to adopt a conformation that
orients the two aromatic groups of this side chain relatively coplanar with rest of
the macrocycle and allows them to insert into the N–M pocket of the protein. (b)
Compound 44 (space-filling, grey) is bound at the N-middle domain with the
benzylated beta-hydroxy phenylalanine side chain highlighted in orange. This
derivative is predicted to adopt a conformation that orients the aromatic groups of
this side chain relatively perpendicular to the rest of the macrocycle. This prevents
the key moiety from inserting into the pocket and forces the compound to bind in a
much different orientation with the benzylated beta-hydroxy phenylalanine side
chain pointed out into solution. The proposed binding site on Hsp90 was identified
using AutoLigand correlated with pull-down assay results and the San A derivative
binding mode was determined using AutoDock4.2. Molecular graphics were
prepared using PyMol.
44 docked to Hsp90 N-M domain
to Hsp90 differ tremendously, and thus it is anticipated that their
binding modes to Hsp90 may be different. Thus, it is not surprising
that Autodock predicted separate binding modes for these two
compounds. Compound 19’s potency is reflected in its greater
binding affinity as predicted by Autodock. Figures 13a and 14a
show that compound 19 adopts a conformation that allows it to in-
sert into the binding pocket between the N–M domain (the antic-
ipated binding site based on our published data),19 which results in
an interaction between the aromatic side groups of the derivative
and the sixth alpha-helix (blue) of Hsp90 (see boxed aromatic side
chain) that is not present in compound 20 docked to Hsp90 (Fig-
ures 13b and 14b). It could be this contact with the helix that ex-
plains the ability of compound 19 to disrupt the binding of Hsp90
to its client protein Her2 and partially disrupt the binding of client
protein IP6K2. The less potent compound 20, (Figures 13b and 14b)
is shown to adopt a different conformation, preventing it from
inserting into the binding pocket and engaging in the interactions
with the helix that observed with 19.
Figure 16. Close-up images of 43 (R,R) (top) and 44 (S,S) (bottom) as a line-bond
structure with the benzylated beta-hydroxy phenylalanine side chain colored in
orange (oxygen is red). Both molecules show an interaction between a phenyl group
and the same a-helix as observed with 38 and 40. However, 43 is predicted to insert
into the binding pocket in a orientation that allows all three of its aromatic groups
to interact with the protein, whereas 44 adopts a conformation that forces it to bind
with two of these aromatics relatively uninvolved with protein interactions. The
interaction of multiple aromatic moieties with the protein target may explain the
greater cytotoxicity demonstrated by compound 43 over 44.
a conformation that results in the molecule binding with this moi-
ety inserted into the binding pocket between the N–M domains.
This same moiety on 44, with S,S stereochemistry, results in a
conformation that prevents the compound from binding in the
same orientation. The very different binding orientation predicted
for these two compounds reflects the difference in cytotoxicity ob-
served for them. These two models show how we can use the blind
docking approach with Autodock to examine our derivatives bound
to Hsp90 and use these images to help develop more potent
derivatives.
Similarly, we show two homologous derivatives, 43 and 44,
Figures 15 and 16, respectively. 43’s key feature is the benzylated
beta-hydroxy-phenylalanine with R,R stereochemistry and 44 has
this moiety with S,S stereochemistry. The predicted binding modes,
in this case, showed the derivatives binding in much different ori-
entations. It appears that the stereochemistry of the benzylated
beta-hydroxy-phenylalanine of 43 allows the compound to adopt
9. ADME studies
Although the cytotoxic effects of lead compounds are thought to
be primarily due to its ability to bind to Hsp90, other factors such

6834
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
Table 2
Solubility, half-life and efflux data for compounds 19 and 43
Compound
Kinetic solubility (
l
M)
Half-life (min)
Efflux ratio
n1
n2
Average
n1
45
172
n2
Average
19
43
<5
7
<5
7
<5
7
30
>200
38
>172
25
3
as solubility, stability and/or efflux properties within the cell may
also contribute. Therefore, we commissioned Biofocus, an outside
company, to run ADME (Adsorption, Distribution, Metabolism,
and Excretion) experiments. The two potent derivatives that were
discussed in the modeling, compounds 19 and 43, were selected for
ADME experiments. It was found that the de novo designed com-
pound 43 had better overall ADME properties (Table 2). Compound
The compounds were dissolved in DMSO at a final concentration of
1.0% and tested at the concentrations indicated in the manuscript.
The DMSO control was also at 1.0%. After the cells had been incu-
bated with the compounds for 54 h, 1 mCi 3H-thymidine per well
was added and the cells were cultured for an additional 18 h (for
the cells to have a total of 72 h treatment), at which time the cells
were harvested using a PHD cell harvester (Cambridge Technology
Inc.). The samples were then counted (CPM) in a scintillation coun-
ter for 1.0 m. Decreases in 3H-thymidine incorporation, as com-
pared to DMSO controls, are an indication that the cells are no
longer progressing through the cell cycle or synthesizing DNA, as
is shown in the studies presented. Mean growth inhibition
(n = 8–12) is the 1 minus CPM of compound-treated cells over
DMSO-treated cells. IC50 were determined using 0, 0.1, 0.3, 1, 3,
19 is hindered by very low aqueous solubility (<5
lM). In compar-
ison, compound 43 has good solubility, 7 M. Compound 19
l
showed a half-life of 38 min, while compound 43 showed a half-
life of >172 min. Finally, the Caco-2 permeation study showed that
compound 19 had a higher efflux ratio than 43 (25–3, respec-
tively), where it is desirable to have an efflux ratio as close to 1
as possible. These data show that by using the SAR, we have im-
proved the properties of a San A molecule, improving their drug-
like character in this new series of derivatives.
and 10 lM of compound (in 1% DMSO final concentration). All
calculations including mean, SEM, and IC50 were performed on
Excel.
10. Conclusion
11.2. Hsp90 binding constant assays
For the first time, we report here the synthesis of 32 new San-
salvamide A structures and their activity against the drug-resistant
colon cancer cell line HCT-116. We have identified characteristics
that are common to the potent molecules, and provided evidence
that these characteristics play a role in their 3-D conformation.
We have shown that the active molecules have unique docking
interactions with the known biological target, Hsp90, compared
to structurally related compounds that are inactive. We have also
provided evidence that our molecules not only bind to Hsp90 di-
rectly, but that the potent molecule 19 inhibits 2 client proteins
from binding to Hsp90, thus indicating its mode of action may in
part be due to modulating the function of Hsp90 via these two cli-
ent proteins. Finally, we have shown that our most promising lead
structure in this series, 43, has improved ADME properties over
compound 19, which indicates that we have built in some pharma-
cokinetic stability into the compounds. These data indicate that
our molecules are cytotoxic, and act in part by modulating the
activity of Hsp90. The synthesis of these new compounds and their
evaluation in the context of their lead structures as well as their
interactions with their potential protein target, Hsp90, provide in-
sight into how this unique set of molecules induce cytotoxicity.
Studies involving these compounds and their modulation of
Hsp90’s function are ongoing and will be reported in due course.
Purified, native Hsp90 (Stressgen) was incubated in PBS (with-
out Ca/Mg) with or without SanA compounds for 1 h at room temp,
and then incubated with biotin-SanA for 1 h at room temp. Strpta-
vidin beads were added and incubated for 30 min at room temp fol-
lowed by removal of the unbound supernatant. The beads were
washed three times with PBS and heated for 15 min at 100 °C in
SDS–PAGE sample buffer. Samples were analyzed on SDS page pro-
tein gels (Invitrogen), and western blots done using Hsp90 antibod-
ies. Bands in the western blots were quantified using Image J, and
the percentage of Hsp90 still bound to the beads was calculated.
11.3. General solution phase peptide synthesis
All peptide coupling reactions were carried out under argon
with dry solvent, using methylene chloride and acetonitrile (9:1)
for dipeptide, tripeptide, and pentapeptide couplings. The amine
(1.1 equiv) and acid (1 equiv) were weighed into a dry flask along
*
with 4–8 equiv of DIPEA and 1.1 equiv of TBTU. Anhydrous meth-
ylene chloride and acetonitrile was added to generate a 0.1 M solu-
tion. 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 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 methy-
lene chloride, organic layers were combined, dried over sodium
sulfate, filtered and concentrated. Flash column chromatography
using a gradient of ethyl actetate–hexane gave our desired peptide.
11. Experimental procedures
11.1. Thymidine uptake assays
*
Some coupling reactions would not go to completion using
Proliferation of the HCT-116 colon cancer cells was tested in the
presence and absence of the compounds using 3H-thymidine up-
take assays. Cells treated with the compounds were compared to
dimethyl sulfoxide (DMSO) controls for their ability to proliferate
as indicated by the incorporation of 3H-thymidine into their
DNA. Cells were cultured in 96 well plates at a concentration of
only TBTU and therefore 0.2–0.5 equiv of HATU, and/or DEPBT
were used. In a few cases up to 0.7 equiv of all three coupling re-
agents were used.
11.4. General solution phase amine deprotection
4000–5000 cells/well in DMEM (Gibco) supplemented with L-glu-
tamine, 10% fetal bovine serum and 1% penicillin–streptomycin
antibiotic. After overnight incubation, the compounds were added.
Amines were deprotected using 20% TFA in methylene chloride
(0.1 M) with 2 equiv of anisole. The reactions were monitored by

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6835
TLC. Reactions were allowed to run for 1–2 h and then concentrated
11.9. General solid phase amine deprotection
in vacuo.
Following coupling completion, the peptide-resin was treated
as follows for removal of the Fmoc protecting group: DMF wash
(3 ꢂ 1 min), 20% Piperdine/DMF (1 ꢂ 5 min), 20% Piperdine/DMF
(1 ꢂ 10 min), DMF wash (2 ꢂ 1 min), IPA wash (1 ꢂ 1 min), DMF
was (1 ꢂ 1 min), IPA (1 ꢂ 1 min), DMF (3 ꢂ 1 min). A ninhydrin test
was performed to verify completion.
11.5. 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 moni-
tored by TLC and usually done in 1–2 h. Sodium thiosulfate
(3.8 equiv) was added to neutralize the peroxide and 5% hydrochlo-
ric acid was added till the solution pH was 1. The aqueous solution
was extracted five times with methylene chloride, and the
combined organic layer was dried, filtered and concentrated in
vacuo.
11.10. General N-terminal solid phase amine deprotection
Once the final N-terminal amino acid residue had been coupled,
the peptide-resin was treated as follows for removal of the Fmoc
protecting group: DMF wash (3 ꢂ 1 min), 20% Piperdine/DMF (1 ꢂ
5 min), 20% Piperdine/DMF (1 ꢂ 10 min), DMF wash (3 ꢂ 1 min),
IPA wash (3 ꢂ 1 min), MeOH (3 ꢂ 1 min). The fully-assembled
peptide-resin was then drained and dried in vacuo overnight.
11.6. Macrocyclization procedure (in situ)
All pentapeptides were acid and amine deprotected using the
general deprotection methods described above. Three coupling
agents (DEPBT, HATU, and TBTU) were used at ꢀ0.5 to 0.75 equiv
each. The dry double deprotected peptide (free acid and free
amine) and coupling agents were dissolved in acetonitrile and
methylene chloride (1:9 ratio) at a concentration of 0.1–0.007 M.
DIPEA (6–10 equiv in order to neutralize the pH) were then added
to the reaction. TLC (macrocycle Rf similar to protected linear pen-
tapeptide) and LCMS were used to monitor the reaction which was
usually finished in 1–2 h. If reaction was not complete in 2 h, addi-
tional coupling agents were added. If reaction was complete then
work-up was done by extracting with 10% aqueous hydrochloric
acid and saturated sodium bicarbonate. After back extraction of
aqueous layers with large quantities of methylene chloride, organic
layers were combined, dried over sodium sulfate, filtered and con-
centrated. All macrocycles were first purified by flash column chro-
matography using an ethyl acetate/hexane gradient on silica gel.
Finally, when necessary, reversed-phase HPLC was used for addi-
tional purification using a gradient of acetonitrile and deionized
water with 0.1% TFA.
11.11. Cleavage of linear peptide
The full-length, linear peptide was cleaved from the resin by
swelling and shaking the peptide-resin for 24 h in a 1:1 (v/v)
2,2,2-trifluoroethanol/CH2Cl2 (10 volumes/gram of dried resin).
The cleavage solution was filtered through a Buchner filter, and
the drained resin was washed with additional CH2Cl2 (5 volumes/
gram of initial dried peptide-resin) 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 CH2Cl2, evapo-
rated by rotary evaporation and dried in vacuo overnight again
to remove residual entrapped TFE.
11.12. Macrocyclization procedure (syringe pump)
Three coupling agents (DEPBT, HATU, and TBTU) were used at
ꢀ0.5 to 0.75 equiv each. These coupling agents were dissolved in
3
of a calculated volume of dry methylene chloride that would
4
give a 0.001–0.0007 M overall concentration when included in
the volume used for the deprotected peptide. The crude, dry, dou-
ble deprotected peptide (free acid and free amine) was dissolved in
11.7. General solid phase synthesis remarks
the other 1 solvent volume of methylene chloride. DIPEA (8 equiv)
4
Stepwise solid phase peptide synthesis was performed in a poly-
propylene solid-phase extraction cartridge fitted with a 20 lM
polyethylene frit purchased from Applied Separations (Allentown,
PA). 2-Chlorotrityl resins were purchased in pre-loaded form with
was then added to the solution containing coupling reagents dis-
solved 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 1–2 h. Upon completion, the reaction
was worked up by washing with aqueous HCl (pH 1) and saturated
sodium bicarbonate. After back extraction of aqueous layers with
large quantities of CH2Cl2, the organic layers were combined, dried,
filtered and concentrated. All macrocycles were first purified by
flash column chromatography using an ethyl acetate/hexane gradi-
ent on silica gel. Finally, when necessary, reversed-phase HPLC was
used for additional purification using a gradient of acetonitrile and
deionized water with 0.1% TFA.
L
-Phe, D-Phe, or L-Leu. Resins were swelled in DMF for 30 min prior
to assembly of the linear five-residue peptide sequence. Solid-phase
syntheses were performed on a 0.5 mmol scale based on resin-load-
ing. All operations were performed at room temperature under
open atmosphere unless stated otherwise.
11.8. General solid phase peptide synthesis
Fmoc-protected amino acids were coupled using 3 equiv of ami-
no acid, 3 equiv of 1-hydroxybenzotriazole, and 6 equiv of diiso-
propylcarbodiimide. Couplings were performed in DMF at 0.2 M
with respect to the incoming Fmoc-protected amino acid. Cou-
plings were allowed to proceed for a minimum of 2 h, and were as-
sayed via ninhydrin test to verify competition. Once complete, the
coupling reaction solution 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 cou-
pling onto the secondary amino terminus, 1-hydroxybenzotriazole
was substituted with 1-hydroxy-7-azabenzotriazole and the cou-
pling was allowed to proceed overnight).
11.13. Benzylation procedure (for compounds 39–42)
The cyclized peptide was dissolved in 50% THF and 50% DMF to
make a 0.1 M solution. The 60% NaH was used at 1.1 equiv and dis-
solved 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 completed in about 5 h and then
worked up by washing with deionized water. After that, the organ-
ic layer was collected, dried and preliminarily purified by flash col-
umn chromatography. Finally, reverse phase-HPLC was used for

6836
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
further purification by using a gradient of acetonitrile and deion-
ized water with 0.1% TFA.
12.1. Synthesis of compound 3
12.1.1. Dipeptide Fmoc-Val-
Dipeptide Fmoc- -Leu-Val-O-Resin was synthesized following
the Section 11.8 procedure. Using 1025.0 mg (0.830 mmol, 1 equiv)
of NH2- -Leu-O-Resin, the Val residue was incorporated using
D-Leu-O-Resin
D
12. Methods of chromatographic purity
D
Method A
844 mg of Fmoc-Val-OH (2.49 mmol, 3 equiv), 331 mg (2.49 mmol,
3 equiv) of HOBt, and 0.770 mL (6 equiv) 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.
Instrument:
Agilent 1200 Series HPLC
Agilent 62440A LC/MSD Trap
Zorbax SB-C18
2.1 ꢂ 30 mm 3.5-Micron
Column:
Mobile Phase A: 0.1% (v/v) formic acid, 100% (v/v) water
Mobile Phase B: 0.1% (v/v) formic acid, 100% (v/v) acetonitrile
12.1.2. Dipeptide NH2-Val-D-Leu-O-Resin
Dipeptide NH2- -Val- -Phe-O-Resin was synthesized following
the ‘General solid phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal.
D
D
Gradient:
Time (min)
Profile %A
Profile %B
80
12.1.3. Tripeptide Fmoc-Leu-Val-
Tripeptide Fmoc-Leu-Val- -Leu-O-Resin was synthesized fol-
lowing the Section 11.8 procedure. Using the NH2- -Val- -Phe-O-
D-Leu-O-Resin
D
0
20
D
D
Resin prepared above, the Leu residue was incorporated using
879.0 mg (2.49 mmol, 3 equiv) of Fmoc-Leu-OH, 331 mg
(2.49 mmol, 3 equiv) of HOBt, and 0.770 mL of DIC (6 equiv). Com-
pletion of the coupling reaction was verified by a negative ninhy-
drin test. The reaction mixture was then drained to leave the
amine-protected resin-bound tripeptide.
4.5
90
10
10
85
4.6
90
7.0
15
12.1.4. Tripeptide NH2-Leu-Val-D-Leu-O-Resin
Flow Rate: 1.0 mL/min.
Tripeptide NH2-Leu-Val- -Leu-O-Resin was synthesized follow-
D
Injection: 4
Solvent: 100% methanol.
l
L
ing the ‘General solid phase amine deprotection’ procedure. A po-
sitive ninhydrin test served to verify Fmoc removal.
12.1.5. Tetrapeptide Fmoc-NMe-Leu-Leu-Val-
Tetrapeptide Fmoc-NMe-Leu-Leu-Val- -Leu-O-Resin was syn-
thesized following the Section 11.8 procedure. Using the NH2-
Leu-Val- -Leu-O-Resin prepared above, the Ser(Bzl) residue was
incorporated using 915.0 mg (2.49 mmol, 3 equiv) of Fmoc-NMe-
Leu-OH, 381 mg (2.49 mmol, 3 equiv) of HOBt, and 0.770 mL of
DIC (6 equiv). 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 tetrapeptide.
D-Leu-O-Resin
Method B
Instrument:
D
Waters Flex Inject
Waters 2487 Dual k Absorbance Detector
Symmetry C18 3.5
4.6 ꢂ 75 mm Column
D
Column:
lm
Mobile Phase A: 0.1% (v/v) Trifluoroacetic acid, 100% (v/v)
water
Mobile Phase B: 0.1% (v/v) Trifluoroacetic acid, 100% (v/v)
acetonitrile
k1: 215nm
12.1.6. Tetrapeptide NH-Me-Leu-Leu-Val-D-Leu-O-Resin
k2: 222nm
Tetrapeptide NH-Me-Leu-Leu-Val- -Leu-O-Resin was synthe-
D
sized following the ‘General solid phase amine deprotection’ proce-
dure. A positive ninhydrin test served to verify Fmoc removal.
Gradient:
12.1.7. Pentapeptide Fmoc-
D
-Phe-NMe-Leu-Leu-Val-
D
-Leu-O-
Time (min)
Resin
Profile %A
Profile %B
70
Pentapeptide
was synthesized following the Section 11.8 procedure. Using the
NH-Me-Leu-Leu-Val- -Leu-O-Resin prepared above, the -Phe res-
idue was incorporated using 964.7 mg (2.49 mmol, 3 equiv) of
Fmoc- -Phe-OH, 339 mg (2.2 mmol, 3 equiv) of HOAt, 0.770 mL
of DIC (6 equiv). 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.
Fmoc-D-Phe-NMe-Leu-Leu-Val-D-Leu-O-Resin
0
30
D
D
4.00
100
13.00
100
15.00
30
0
0
D
70
70
16.00
30
12.1.8. Pentapeptide NH2-
D-Phe-Nme-Leu-Leu-Val-D-Leu-O-
Resin
Pentapeptide NH2-D-Phe-NMe-Leu-Leu-Val-D-Leu-O-Resin was
Flow rate: 0.50 mL/min
synthesized following the Section 11.10 procedure. A positive nin-
hydrin test served to verify Fmoc removal. The resin was then dried
in vacuo for 24 h in a vacuum desiccator.
Injection: 20
lL
Solvent: 100% methanol

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6837
12.1.9. Double deprotected pentapeptide NH2-
Leu-Val- -Leu-OH
Double deprotected pentapeptide NH2-
Val-
dure. Utilizing the 1264.4 mg of dried NH2-
Val-
D
-Phe-NMe-Leu-
12.2.6. Tetrapeptide NH2-
D-Leu-NMe-Phe-Leu-Leu-O-Resin
D
Tetrapeptide NH2- -Leu-NMe-Phe-Leu-Leu-O-Resin was syn-
D
D
-Phe-NMe-Leu-Leu-
-Phe-NMe-Leu-Leu-
thesized following the ‘General solid phase amine deprotection’
procedure. A positive ninhydrin test served to verify Fmoc removal.
D
-Leu-OH was synthesized following the Section 11.11 proce-
D
D
-Leu-O-Resin, 6.5 mL of 2,2,2-trifluoroethanol and 6.5 mL of
12.2.7. Pentapeptide Fmoc-
D
-Val-D-Leu-NMe-Phe-Leu-Leu-O-
CH2Cl2. The resin slurry was stirred for 24 h, after which it was fil-
tered, washed with additional CH2Cl2, and dried in vacuo for 24 h
(436.3 mg, 85% yield).
Resin
Pentapeptide
was synthesized following the Section 11.8 procedure. Using the
NH2- -Leu-NMe-Phe-Leu-Leu-O-Resinprepared above, the -Val
residue was incorporated using 885 mg (2.61 mmol, 3 equiv) of
Fmoc- -Val-OH, 399 mg (2.61 mmol, 3 equiv) of HOAt, 0.810 mL
Fmoc-
D
-Val-
D
-Leu-NMe-Phe-Leu-Leu-O-Resin
D
D
12.1.10. Macrocycle
D
-Phe-D-Leu-Val-Leu-Leu-NMe
Macrocycle -Phe-
D
D
-Leu-Val-Leu-Leu-NMe was synthesized
D
following the ‘Macrocyclization procedure’. Utilizing 436 mg
(0.706 mmols, 1.0 equiv) of linear pentapeptide, 0.74 mL (6 equiv)
of DIPEA, 113.0 mg (0.353 mmols, 0.6 equiv) of TBTU, 161 mg
(0.424 mmols, 0.6 equiv) HATU, and 129 mg (0.424 mmols,
0.6 equiv) of DEPBT. The crude reaction was purified by reverse
phase-HPLC to yield the macrocycle (16.9 mg, 3.4% yield).
of DIC (6 equiv). 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.
12.2.8. Pentapeptide NH2-D-Val-D-Leu-Nme-Phe-Leu-Leu-O-Resin
Pentapeptide NH2- -Val-
D
D
-Leu-NMe-Phe-Leu-Leu-O-Resin was
1H NMR (400 MHz, CD3OD): d 0.7–0.9 (m, 8H), 1.2–1.9 (m, 9H),
synthesized following the Section 11.10 procedure. A positive nin-
hydrin test served to verify Fmoc removal. The resin was then dried
in vacuo for 24 h in a vacuum desiccator.
2.6 (s, 2H), 2.8–3.2 (m, 6H), 3.7 (m,
aH), 3.8 (m,
aH), 3.9 (m, aH),
4.1 (m,
4H).
aH), 4.2 (m, aH), 5.4 (m, 2H), 7.0–7.3 (m, 5H), 7.6–8.2 (d,
LCMS: m/z called for C33H53N5O5 (M+1) = 599.8, found 600.4.
12.2.9. Double Deprotected Pentapeptide NH2-
Phe-Leu-Leu-OH
D-Val-D-Leu-NMe-
Double Deprotected Pentapeptide NH2-
Phe-Leu-Leu-OHwas synthesized following the Section 11.11
procedure. Utilizing the 1286.4 mg of dried NH2- -Val- -Leu-
D
-Val-
D-Leu-NMe-
12.2. Synthesis of compound 4
D
D
12.2.1. Dipeptide Fmoc-Leu-Leu-O-Resin
NMe-Phe-Leu-Leu-O-Resin, 6.5 mL of 2,2,2-trifluoroethanol and
6.5 mL of CH2Cl2. The resin slurry was stirred for 24 h, after which
it was filtered, washed with additional CH2Cl2, and dried in vacuo
for 24 h (445.0 mg, 83% yield).
Dipeptide Fmoc-Leu-Leu-O-Resin was synthesized following
the Section 11.8 procedure. Using 1072.0 mg (0.870 mmol, 1 equiv)
of NH2-Leu-O-Resin, the Leu residue was incorporated using
921 mg of Fmoc-Leu-OH (2.61 mmol, 3 equiv), 399 mg (2.61 mmol,
3 equiv) of HOBt, and 0.810 mL (6 equiv) 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.
12.2.10. Macrocycle Phe-Nme-D-Leu-D-Val-Leu-Leu
Macrocycle Phe-NMe- -Leu-
D
D-Val-Leu-Leu was synthesized
following the ‘Macrocyclization procedure’. Utilizing 200 mg
(0.324 mmols, 1.0 equiv) of linear pentapeptide, 0.48 mL (6 equiv)
of DIPEA, 52.0 mg (0.162 mmols, 0.6 equiv) of TBTU, 74 mg
(0.194 mmols, 0.6 equiv) HATU, and 58 mg (0.194 mmols, 0.6 equiv)
of DEPBT. The crude reaction was purified by reverse phase-HPLC to
yield the macrocycle (17.2 mg, 8.8% yield).
12.2.2. Dipeptide NH2-Leu-Leu-O-Resin
Dipeptide NH2-Leu-Leu-O-Resin was synthesized following the
‘General solid phase amine deprotection’ procedure. A positive nin-
hydrin test served to verify Fmoc removal.
1H NMR (400 MHz, CD3OD): d0.6–1.0 (m, 11H), 1.4–1.8 (m, 6H),
2.8 (s, 1H), 3.1 (s, 2H), 3.6 (m,
H), 4.4 (m, H), 5.3 (m, 1H), 5.7 (m, 1H) 7.1–7.3 (m, 5H) 7.4–8.2
(m, 4H).
aH), 3.9 (m, aH), 4.0 (m, aH), 4.2 (m,
12.2.3. Tripeptide Fmoc-NMe-Phe-Leu-Leu-O-Resin
a
a
Tripeptide Fmoc-NMe-Phe-Leu-Leu-O-Resin was synthesized
following the Section 11.8 procedure. Using the NH2-Leu-Leu-O-
Resin prepared above, the NMe-Phe residue was incorporated
using 1010.0 mg (2.61 mmol, 3 equiv) of Fmoc-NMe-Phe-OH,
399 mg (2.61 mmol, 3 equiv) of HOBt, and 0.810 mL of DIC
(6 equiv). 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 tripeptide.
LCMS: m/z called for C33H53N5O5 (M+1) = 599.8, found 600.3.
12.3. Synthesis of compound 5
12.3.1. Dipeptide Fmoc-Leu-
Dipeptide Fmoc-Leu- -Leu-O-Resin was synthesized following
the Section 11.8 procedure. Using 1014.0 mg (0.821 mmol, 1 equiv)
of NH2- -Leu-O-Resin, the Leu residue was incorporated using
D-Leu-O-Resin
D
D
12.2.4. Tripeptide NH-NMe-Phe-Leu-Leu-O-Resin
868 mg of Fmoc-Leu-OH (2.45 mmol, 3 equiv), 376 mg (2.45 mmol,
3 equiv) of HOBt, and 0.760 mL (6 equiv) 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.
Tripeptide NH-NMe-Phe-Leu-Leu-O-Resin was synthesized fol-
lowing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal.
12.2.5. Tetrapeptide Fmoc-
Tetrapeptide Fmoc- -Leu-NMe-Phe-Leu-Leu-O-Resin was syn-
thesized following the Section 11.8 procedure. Using the NH-
NMe-Phe-Leu-Leu-O-Resin prepared above, the -Leu residue was
incorporated using 921.3 mg (2.61 mmol, 3 equiv) of Fmoc- -Leu-
D-Leu-NMe-Phe-Leu-Leu-O-Resin
D
12.3.2. Dipeptide NH2-Leu-
D-Leu-O-Resin
Dipeptide NH2-Leu- -Leu-O-Resin was synthesized following
D
D
the ‘General solid phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal.
D
OH, 355 mg (2.61 mmol, 3 equiv) of HOBt, and 0.810 mL of DIC
(6 equiv). 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 tetrapeptide.
12.3.3. Tripeptide Fmoc-Phe-Leu-
Tripeptide Fmoc-Phe-Leu- -Leu-O-Resin was synthesized fol-
lowing the Section 11.8 procedure. Using the NH2-Leu- -Leu-O-Re-
D-Leu-O-Resin
D
D

6838
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
sin prepared above, the Phe residue was incorporated using
952.0 mg (2.46 mmol, 3 equiv) of Fmoc-NMe-Phe-OH, 376 mg
(2.46 mmol, 3 equiv) of HOBt, and 0.760 mL of DIC (6 equiv). Com-
pletion of the coupling reaction was verified by a negative ninhy-
drin test. The reaction mixture was then drained to leave the
amine-protected resin-bound tripeptide.
LCMS: m/z called for C33H53N5O5 (M+1) = 599.7, found 600.3.
12.4. Synthesis of compound 6
12.4.1. Dipeptide MeO-
Dipeptide MeO- -Phe-Leu-NHBoc was synthesized following the
‘Generalpeptidesynthesis’procedure. Utilizing380.7 mg(1.8 mmols,
1.1 equiv) of amine OMe- -Phe-NH2, 400 mg (1.6 mmols, 1.0 equiv)
D-Phe-Leu-NHBoc
D
12.3.4. Tripeptide NH-Phe-Leu-
D
-Leu-O-Resin
D
Tripeptide NH-Phe-Leu- -Leu-O-Resin was synthesized follow-
ing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal.
D
of acid HO-Leu-NHBoc, 3.0 mL (11 equiv) of DIPEA, 566.7 mg
(1.8 mmols, 1.1 equiv) of TBTU, in 16 mL of methylene chloride.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the dipeptide (622.1 mg, 99% yield).
Rf: 0.9 (EtOAc/Hex 1:1).
12.3.5. Tetrapeptide Fmoc-NMe-Leu-Phe-Leu-
Tetrapeptide Fmoc-NMe-Leu-Phe-Leu- -Leu-O-Resin was syn-
thesized following the Section 11.8 procedure. Using the NH-
Phe-Leu- -Leu-O-Resin prepared above, the NMe-Leu residue was
D-Leu-O-Resin
D
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.4–1.7
(m, 3H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br,
4.8–5.0 (q, H), 6.6 (d, 1H), 7.1–7.4 (m, 5H).
aH), 4.8 (d, 1H),
D
a
incorporated using 954 mg (2.46 mmol, 3 equiv) of Fmoc-NMe-
Leu-OH, 376 mg (2.46 mmol, 3 equiv) of HOBt, and 0.760 mL of
DIC (6 equiv). 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 tetrapeptide.
12.4.2. Dipeptide MeO-
D-Phe-Leu-NH2
Dipeptide MeO- -Phe-Leu-NH2 was synthesized following the
D
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(464 mg, 100% yield).
12.3.6. Tetrapeptide NH2-NMe-Leu-Phe-Leu-D-Leu-O-Resin
Tetrapeptide NH2-NMe-Leu-Phe-Leu- -Leu-O-Resin was syn-
thesized following the ‘General solid phase amine deprotection’
procedure. A positive ninhydrin test served to verify Fmoc removal.
D
12.4.3. Tripeptide MeO-
Tripeptide MeO- -Phe-Leu-Val-NHBoc was synthesized follow-
ing the ‘General peptide synthesis’ procedure. Utilizing 464 mg
(1.6 mmols, 1.0 equiv) of amine MeO- -Phe-Leu-NH2, 313 mg
D-Phe-Leu-Val-NHBoc
D
D
12.3.7. Pentapeptide Fmoc-
O-Resin
D
-Val-NMe-Leu-Phe-Leu-
D
-Leu-
(1.4 mmols, 1.0 equiv) of acid HO-Val-NHBoc, 2.6 mL (10 equiv) of
DIPEA, 509 mg (1.6 mmols, 1.1 equiv) of TBTU, in 12 mL of methy-
lene chloride and 3 mL of acetonitrile. The crude reaction was puri-
fied by column chromatography (silica gel, EtOAc/Hex) to yield the
tripeptide (614 mg, 87% yield).
Pentapeptide
was synthesized following the Section 11.8 procedure. Using the
NH2-NMe-Leu-Phe-Leu- -Leu-O-Resin prepared above, the -Val
residue was incorporated using 834 mg (2.46 mmol, 3 equiv) of
Fmoc- -Val-OH, 335 mg (2.61 mmol, 3 equiv) of HOAt, 0.760 mL
Fmoc-D-Val-NMe-Leu-Phe-Leu-D-Leu-O-Resin
D
D
Rf: 0.5 (EtOAc/Hex 1:1).
D
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 12H), 1.4 (s, 9H), 1.4–
1.6 (m, 2H), 1.7 (s, 1H), 2.1 (m, 1H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 3.8
of DIC (6 equiv). 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.
(dd, aH), 4.4 (dd, aH), 4.8 (dd, aH), 5.0 (d, 1H), 6.3 (d, 1H), 6.6 (d,
1H), 7.1–7.3 (m, 5H).
12.3.8. Pentapeptide NH2-
D
-Val-NMe-Leu-Phe-Leu-
D
-Leu-O-
12.4.4. Tripeptide MeO-D-Phe-Leu-Val-NH2
Resin
Dipeptide MeO-D-Phe-Leu-Val-NH2 was synthesized following
Pentapeptide NH2-
D
-Val-NMe-Leu-Phe-Leu-
D
-Leu-O-Resin was
the ‘General amine deprotection’. This dipeptide was taken on to
the next reaction without further purification or characterization
(489 mg, 100% yield).
synthesized following the Section 11.10 procedure. A positive nin-
hydrin test served to verify Fmoc removal. The resin was then dried
in vacuo for 24 h in a vacuum desiccator.
12.4.5. Dipeptide MeO-Leu-D-Leu-NHBoc
12.3.9. Double deprotected pentapeptide NH2-
Phe-Leu- -Leu-OH
Double deprotected
Phe-Leu- -Leu-OH was synthesized following the Section 11.11
procedure. Utilizing the 1956.0 mg of dried NH2- -Val-NMe-
Leu-Phe-Leu- -Leu-O-Resin, 6.0 mL of 2,2,2-trifluoroethanol and
6.0 mL of CH2Cl2. The resin slurry was stirred for 24 h, after which
it was filtered, washed with additional CH2Cl2, and dried in vacuo
for 24 h (450.0 mg, 89% yield).
D
-Val-NMe-Leu-
Dipeptide MeO-Leu- -Leu-NBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 321 mg (1.8 mmols,
1.1 equiv) of amine MeO-Leu-NH2, 400 mg (1.6 mmols, 1.0 equiv) of
D
D
pentapeptide
NH2-D-Val-NMe-Leu-
D
acid HO-D-Leu-NBoc, 2.2 mL (8 equiv) of DIPEA, 567 mg (1.8 mmols,
D
1.1 equiv) of TBTU, in 16 mL methylene chloride and 4 mL acetoni-
trile. The crude reaction was purified by column chromatography
(silica gel, EtOAc/Hex) to yield the dipeptide (530.3 mg, 92% yield).
Rf: 0.8 (EtOAc/Hex 1:1).
D
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (d, 12H), 1.4 (s, 9H),
1.4–1.8 (m, 6H), 3.7 (s, 3H), 4.1 (br,
6.6 (d, 1H).
aH), 4.6 (br, aH), 4.8 (br, 1H),
12.3.10. Macrocycle Phe-Leu-NMe-
D
-Val-D-Leu-Leu
Macrocycle Phe-Leu-NMe- -Val-
D
D-Leu-Leu-Leu was synthesized
following the ‘Macrocyclization procedure’. Utilizing 250 mg
(0.405 mmols, 1.0 equiv) of linear pentapeptide, 0.42 mL (6 equiv)
of DIPEA, 64.0 mg (0.20 mmols, 0.6 equiv) of TBTU, 91 mg
(0.24 mmols, 0.6 equiv) HATU, and 72 mg (0.24 mmols, 0.6 equiv)
of DEPBT. The crude reaction was purified by reverse phase-HPLC
to yield the macrocycle (9.2 mg, 4.7% yield).
12.4.6. Dipeptide HO-Leu-
D-Leu-NHBoc
Dipeptide HO- Leu- -Leu-NHBoc was synthesized following the
D
‘General acid deprotection’. This tripeptide was taken on to the
next reaction without further purification or characterization
(491 mg, 98% yield).
1H NMR (400 MHz, CD3OD): d 0.6–1.0 (m, 8H), 1.2–1.6 (m, 6H),
12.4.7. Pentapeptide MeO-
Pentapeptide MeO- -Phe-Leu-Val-Leu-
thesized following the ‘General peptide synthesis’ procedure. Uti-
D-Phe-Leu-Val-Leu-
D-Leu-NHBoc
-Leu-NHBoc was syn-
2.6 (s, 1H), 3.0 (s, 1H), 3.6 (m,
aH), 3.8 (m,
a
H), 4.1 (m,
a
H), 4.2 (m,
D
D
a
H), 4.4 (m, H), 7.1–7.3 (m, 5H).
a

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6839
lizing 489 mg (1.3 mmols, 1.0 equiv) of amine MeO-
NH2, 491 mg (1.4 mmols, 1.1 equiv) of acid HO-Leu-
D
-Phe-Leu-Val-
-Leu-NHBoc,
12.5.4. Tripeptide MeO-
D-Phe-Leu-N-Me-Val-NH2
D
Tripeptide MeO- -Phe-Leu-N-Me-Val-NH2 was synthesized fol-
D
1.0 mL (5 equiv) of DIPEA, 201 mg (0.63 mmols, 0.5 equiv) of TBTU,
238 mg (0.63 mmols, 0.5 equiv) of HATU, and 75 mg (0.25 mmols,
0.2 equiv) of DEPBT, in 13 mL of methylene chloride and 2 mL ace-
tonitrile. The crude reaction was purified by column chromatogra-
phy (silica gel, EtOAc/Hex) to yield the pentapeptide (201 mg, 22%
yield).
lowing the ‘General amine deprotection’. This tripeptide was taken
on to the next reaction without further purification or character-
ization (861 mg, 100% yield).
12.5.5. Tetrapeptide MeO-
Tetrapeptide MeO- -Phe-Leu-N-Me-Val-N-Me-
synthesized following the ‘General peptide synthesis’ procedure.
Utilizing 562 mg (1.39 mmols, 1.1 equiv) of MeO- -Phe-Leu-N-
Me-Val-NH2, 328 mg (1.26 mmols, 1.0 equiv) of Boc-N-Me- -Leu-
D
-Phe-Leu-N-Me-Val-N-Me-D-Leu-Boc
D
D-Leu-Boc was
Rf: 0.4 (EtOAc/Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 24H),1.3 (m, 2H), 1.4
(s, 9H), 1.5 (m, 3H), 1.6 (m, 2H), 1.7 (m, 2H), 2.2 (m, 1H), 2.9–3.2
D
D
(m, 2H), 3.7 (s, 3H), 4.1 (m,
(m, H), 7.1–7.3 (m, 5H).
a
H), 4.2 (m,
a
H), 4.4 (m, 2
a
H), 4.6
OH, 1.32 mL (6 equiv) of DIPEA, 162 mg (0.5 mmols, 0.4 equiv) of
TBTU, and 479 mg (1.26 mmols, 1.0 equiv) of HATU. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the tetrapeptide (294 mg, 33.5% yield).
Rf: 0.55 (EtOAc/Hex 1:1).
a
12.4.8. Macrocycle
D
-Phe-Leu-Val-Leu-
D-Leu
Macrocycle -Phe-Leu-Val-Leu-
D
D
-Leu was synthesized following
1H NMR (400 MHz, CDCl3): d 0.7–1.0 (m, 18H), 1.2–1.4 (m, 6H),
1.5–1.6 (m, 9H), 2.2 (m, 2H), 3.0 (m, 3H), 3.1–3.2 (m, 2H), 3.7 (s,
3H), 4.4 (m, 2H), 4.7 (m, 2H), 5.0 (m, 1H), 5.2 (m, 1H), 7.1–7.3
(m, 5H).
the ‘Macrocyclization procedure’. Utilizing 169.3 mg (0.28 mmols,
1.0 equiv) of linear pentapeptide, 0.7 mL (15 equiv) of DIPEA,
45 mg (0.14 mmols, 0.5 equiv) of TBTU, 74.6 mg (0.2 mmols,
0.7 equiv) HATU, and 41.9 mg (0.14 mmols, 0.5 equiv) of DEPBT
in 15 mL methylene chloride, 4 mL acetonitrile and 2 mL dimethyl
formamide. The crude reaction was purified by reverse phase-HPLC
to yield the macrocycle (12.3 mg, 7.5% yield).
12.5.6. Tetrapeptide MeO-
D
-Phe-Leu-N-Me-Val-N-Me-D-Leu-NH2
Tetrapeptide MeO- -Phe-Leu-N-Me-Val-N-Me-
D
D
-Leu-NH2 was
synthesized following the ‘General amine deprotection’. This tetra-
peptide was taken on to the next reaction without further purifica-
tion or characterization (247 mg, 100% yield).
Rf: 0.25 (EtOAc/Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.7–1.0 (m, 24H), 1.2–1.8 (m, 9H),
2.0 (m, 1H), 2.9–3.1 (m, 2H), 3.6 (m,
aH), 3.8 (m, aH), 4.2 (m, aH),
4.5 (m, H), 4.6 (m, H), 7.1–7.3 (m, 5H), 6.6 (d, 1H), 7.0 (d, 1H), 7.5
(d, 1H), 7.6 (d, 1H), 8.0 (d, 1H).
a
a
12.5.7. Pentapeptide MeO-
Phe-Boc
D
-Phe-Leu-N-Me-Val-N-Me-
D
-Leu-
D-
LCMS: m/z calcd for C32H51N5O5 (M+1) = 586.4, found 587.5.
Pentapeptide
Boc was synthesized following the ‘General peptide synthesis’ pro-
cedure. Utilizing 294 mg (0.47 mmols, 1.1 equiv) of MeO- -Phe-
Leu-N-Me-Val-N-Me- -Leu-NH2, 112 mg (0.42 mmols, 1.0 equiv)
of Boc- -Phe-OH, 0.6 mL (8 equiv) of DIPEA, 81 mg (0.25 mmols,
MeO-D-Phe-Leu-N-Me-Val-N-Me-
D-Leu- -Phe-
D
12.5. Synthesis of compound 7
D
D
12.5.1. Dipeptide MeO-
Dipeptide MeO- -Phe-Leu-Boc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 951 mg (4.4 mmols,
1.1 equiv) of MeO- -Phe-NH2, 1000 mg (4.0 mmols, 1.0 equiv) of
D-Phe-Leu-Boc
D
D
0.6 equiv) of TBTU, and 129 mg (0.34 mmols, 0.8 equiv) HATU.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the pentapeptide (224 mg, 68% yield).
Rf: 0.4 (EtOAc/Hex 1:1).
D
Boc-Leu-OH, 2.8 mL (4 equiv) of DIPEA, 1545 mg (4.8 mmols,
1.2 equiv) of TBTU. The crude reaction was purified by column chro-
matography (silica gel, EtOAc/Hex) to yield the dipeptide (1381 mg,
88% yield).
1H NMR (400 MHz, CD3OD): d 0.7–1.0 (m, 9H), 1.2–1.4 (m, 12H),
1.6–1.7 (m, 12H), 2.3 (m, 2H), 2.8 (s, 3H), 3.0 (s, 3H), 3.1–3.2 (m,
2H), 3.7 (s, 3H), 4.3–4.4 (m, 2
5.5 (t, 1H), 6.4–6.6 (dd, 2H), 7.0–7.4 (m, 10H).
aH), 4.8–4.9 (m, 2aH), 5.2 (m, 1H),
Rf: 0.35 (EtOAc/Hex 3:7).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m,
2H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.2 (m,
1H), 7.0–7.3 (m, 5H).
aH), 4.7–5.0 (m, 2H), 6.5 (d,
12.5.8. Macrocycle
D
-Phe-Leu-N-Me-Val-N-Me-
D
-Leu-
-Phe was syn-
D-Phe
Macrocycle -Phe-Leu-N-Me-Val-N-Me-
D
D-Leu-D
thesized following the ‘Macrocyclization procedure’. Utilizing
184 mg (0.24 mmols, 1.0 equiv) of double deprotected linear pen-
tapeptide, 0.42 mL (10 equiv) of DIPEA, 62 mg (0.19 mmols,
0.8 equiv) of TBTU, 73 mg (0.19 mmols, 0.8 equiv) of HATU, and
29 mg (0.09 mmols, 0.4 equiv) of DEPBT. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) yield
the macrocycle (1 mg, 0.6% yield).
12.5.2. Dipeptide MeO-
D-Phe-Leu-NH2
Dipeptide MeO- -Phe-Leu-NH2 was synthesized following the
D
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(1029 mg, 100% yield).
1H NMR (400 MHz, CD3OD): d0.7–1.0 (m, 9H), 1.2–1.4 (m, 12H),
1.6–1.7 (m, 6H), 2.2 (m, 2H), 2.7 (s, 3H), 2.9 (s, 3H), 3.1–3.2 (m, 2H),
12.5.3. Tripeptide MeO-
Tripeptide MeO- -Phe-Leu-N-Me-Val-Boc was synthesized
following the ‘General peptide synthesis’ procedure. Utilizing
686 mg (2.34 mmols, 1.1 equiv) of MeO- -Phe-Leu-NH2, 493 mg
D-Phe-Leu-N-Me-Val-Boc
D
4.2–4.4 (m, 2aH), 4.6–4.8 (m, 2aH), 5.0 (m, 1H), 5.4 (t, 1H), 7.0–7.4
D
(m, 10H).
(2.13 mmols, 1.0 equiv) of Boc-N-Me-Val-OH, 1.49 mL (4 equiv) of
DIPEA, 324 mg (0.85 mmols, 0.4 equiv) of HATU. 547 mg
(1.7 mmols, 0.8 equiv) of TBTU. The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the tripep-
tide (1073 mg, 99.6% yield).
LCMS: m/z calcd for C37H53N5O5 = 647.85, found 647.6.
12.6. Synthesis of compound 10
12.6.1. Dipeptide MeO-
Dipeptide MeO- -Phe-
the ‘General peptide synthesis’ procedure. Utilizing 951 mg
(4.4 mmols, 1.1 equiv) of amine OMe- -Phe-NH2, 1.0 g (4.0 mmols,
1.0 equiv) of acid HO- -Leu-NHBoc, 2.8 mL (4 equiv) of DIPEA,
1.54 g (4.8 mmols, 1.2 equiv) of TBTU. The crude reaction was
D
-Phe-D-Leu-NHBoc
Rf: 0.75 (EtOAc/Hex 1:1).
D
D-Leu-NHBoc was synthesized following
1H NMR (200 MHz, CDCl3): d 0.9–1.1 (m, 9H), 1.5 (s, 9H), 1.6–1.8
(m, 3H), 2.3 (m, 2H), 2.8 (s, 3H), 3.1 (m, 3H), 3.7 (s, 3H), 4.0
D
(d,
aH), 4.6 (m, aH), 4.8 (m, aH), 6.4 (d, 1H), 6.7 (d, 1H), 7.1–7.3
D
(m, 5H).

6840
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the dipeptide (1.57 g, 98% yield).
12.6.8. Macrocycle
D
-Phe-
D-Leu-Val-Leu-Leu
Macrocycle -Phe-
D
D
-Leu-Val-Leu-Leu was synthesized following
Rf: 0.5 (EtOAc/Hex 1:4).
the ‘Macrocyclization procedure’. Utilizing 163 mg (0.27 mmols,
1.0 equiv) of linear pentapeptide, 0.38 mL (8 equiv) of DIPEA,
43.1 mg (0.14 mmols, 0.5 equiv) of TBTU, 51.1 mg (0.12 mmols,
0.5 equiv) HATU, and 56.3 mg (0.19 mmols, 0.7 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (8 mg, 5% yield).
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 6H), 1.4 (s, 9H), 1.6–1.8
(m, 3H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.1 (m,
H), 4.8–4.9, 6.6 (d, 1H), 7.1–7.3 (m, 5H).
aH), 4.8–5.0 (m,
a
12.6.2. Dipeptide MeO-
D
-Phe-
D-Leu-NH2
Dipeptide MeO- -Phe-
D
D
-Leu-NH2 was synthesized following the
Rf: 0.5 (EtOAc/Hex 4:1).
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(1.15 g, 100% yield).
1H NMR (600 MHz, CD3OD): d 0.8–1.0 (m, 24H), 1.2–1.4 (m, 3H),
1.5–1.7 (m, 6H), 2.9–3.1 (m, 1H), 3.2–3.4 (m, 1H), 3.6–3.8 (m, 1H),
4.0–4.1 (m, 1
H), 7.2–7.4 (m, 5H).
LCMS: m/z calcd for C32H51N5O5 (M+23) = 608.39, found 608.6.
aH), 4.1–4.2 (m, 2aH), 4.2–4.4 (m, 1aH), 4.6–4.8 (m,
1a
12.6.3. Tripeptide MeO-
Tripeptide MeO- -Phe-
lowing the ‘General peptide synthesis’ procedure. Utilizing 1.15 g
(3.95 mmols, 1.1 equiv) of amine MeO- -Phe- -Leu-NH2, 780 mg
D
-Phe-
D-Leu-Val-NHBoc
D
D
-Leu-Val-NHBoc was synthesized fol-
12.7. Synthesis of compound 12
D
D
(3.59 mmols, 1.0 equiv) of acid HO-Val-NHBoc, 2.5 mL (4 equiv)
of DIPEA, 1.38 g (4.30 mmols, 1.2 equiv) of TBTU. The crude reac-
tion was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the tripeptide (1.72 g, 97% yield).
12.7.1. Dipeptide MeO-Phe-Leu-NHBoc
Dipeptide MeO-Phe-Leu-NHBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 380.7 mg
(1.8 mmols, 1.1 equiv) of amine MeO-Phe-NH2, 400 mg (1.6 mmols,
1.0 equiv) of acid HO-Leu-NHBoc, 3.0 mL (11 equiv) of DIPEA,
566.7 mg (1.8 mmols, 1.1 equiv) of TBTU, in 16 mL of Methylene
Chloride. The crude reaction was purified by column chromatogra-
phy (silica gel, EtOAc/Hex) to yield the dipeptide (622.1 mg, 99%
yield).
Rf: 0.5 (EtOAc/Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.8–1.1 (m, 12H), 1.4 (s, 9H),
1.6–1.8 (m, 3H), 2.1–2.2 (m, 1H), 3.0–3.2 (m, 2H), 3.7 (s, 3H),
3.8–4.0 (m,
aH), 4.4–4.5 (m, aH), 4.7–4.9 (m, aH), 4.9 (br, 1H),
6.1–6.3 (d, 1H), 6.5–6.6 (br, 1H), 7.1–7.4 (m, 5H).
Rf: 0.9 (EtOAc/Hex 1:1).
12.6.4. Dipeptide MeO-Leu-Leu-NHBoc
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.4–1.7
Dipeptide MeO-Leu-Leu-NHBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 801 mg (4.4 mmols,
1.1 equiv) of amine MeO-Leu-NH2, 1.0 g (4.0 mmols, 1.0 equiv) of
acid HO-Leu-NHBoc, 2.8 mL (4 equiv) of DIPEA, 1.54 g (4.8 mmols,
1.2 equiv) of TBTU. The crude reaction was purified by column chro-
matography (silica gel, EtOAc/Hex) to yield the dipeptide (1.43 g,
98% yield).
(m, 3H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br,
4.8–5.0 (q, H), 6.6 (d, 1H), 7.1–7.4 (m, 5H).
aH), 4.8 (d, 1H),
a
12.7.2. Dipeptide MeO-Phe-Leu-NH2
Dipeptide MeO-Phe-Leu-NH2 was synthesized following the
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(464 mg, 100% yield).
Rf: 0.5 (EtOAc/Hex 1:3).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 12H), 1.2–1.3 (m, 4H),
1.4 (m, 9H), 1.6–1.7 (m, 2H), 3.7 (s, 3H), 4.1–4.2 (m,
(m, H), 4.8–4.9 (br, 1H), 6.4–6.5 (br, 1H).
a
H), 4.6–4.7
12.7.3. Tripeptide MeO-Phe-Leu-Val-NHBoc
a
Tripeptide MeO-D-Phe-Leu-Val-NHBoc was synthesized follow-
ing the ‘General peptide synthesis’ procedure. Utilizing 464 mg
(1.6 mmols, 1.0 equiv) of amine MeO-Phe-Leu-NH2, 313 mg
(1.4 mmols, 1.0 equiv) of acid HO-Val-NHBoc, 2.6 mL (10 equiv)
of DIPEA, 509 mg (1.6 mmols, 1.1 equiv) of TBTU, in 12 mL of meth-
ylene chloride and 3 mL of acetonitrile. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (614 mg, 87% yield).
12.6.5. Tripeptide MeO-
D
-Phe-
D-Leu-Val-NH2
Dipeptide MeO- -Phe-
D
D
-Leu-Val-NH2 was synthesized follow-
ing the ‘General amine deprotection’. This tripeptide was taken
on to the next reaction without further purification or character-
ization (1.36 g, 100% yield).
12.6.6. Dipeptide HO-Leu-Leu-NHBoc
Rf: 0.5 (EtOAc/Hex 1:1).
Dipeptide HO-Leu-Leu-NHBoc was synthesized following the
‘General acid deprotection’. This dipeptide was taken on to the next
reaction without further purification or characterization (1.23 g,
90% yield).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 12H), 1.4 (s, 9H), 1.4–
1.6 (m, 2H), 1.7 (s, 1H), 2.1 (m, 1H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 3.8
(dd,
aH), 4.4 (dd, aH), 4.8 (dd, aH), 5.0 (d, 1H), 6.3 (d, 1H), 6.6 (d,
1H), 7.1–7.3 (m, 5H).
12.6.7. Pentapeptide MeO-
Pentapeptide MeO- -Phe-
thesized following the ‘General peptide synthesis’ procedure. Uti-
lizing 1.37 g (3.49 mmols, 1.1 equiv) of amine MeO- -Phe- -Leu-
Val-NH2, 1.09 g (3.17 mmols, 1.0 equiv) of acid HO-Leu-Leu-
NHBoc, 4.43 mL (8 equiv) of DIPEA, 611 mg (1.90 mmols, 0.6 equiv)
of TBTU, 361 mg (0.95 mmols, 0.3 equiv) HATU, and 284 mg
(0.95 mmols, 0.3 equiv) of DEPBT. The crude reaction was purified
by column chromatography (silica gel, EtOAc/Hex) to yield the
pentapeptide (1.323 g, 58% yield).
D
-Phe-
D
-Leu-Val-Leu-Leu-NHBoc
12.7.4. Tripeptide MeO-Phe-Leu-Val-NH2
D
D
-Leu-Val-Leu-Leu-NHBoc was syn-
Tripeptide MeO-Phe-Leu-Val-NH2 was synthesized following
the ‘General amine deprotection’. This tripeptide was taken on to
the next reaction without further purification or characterization
(464 mg, 100% yield).
D
D
12.7.5. Dipeptide MeO-
Dipeptide MeO- -Leu-
the ‘General peptide synthesis’ procedure. Utilizing 377 mg
(2.0 mmols, 1.1 equiv) of amine MeO- -Leu-NH2, 500 mg
(1.8 mmols, 1.0 equiv) of acid HO- -Phe-NBoc, 1.3 mL (4 equiv) of
D
-Leu-D-Phe-NBoc
D
D-Phe-NBoc was synthesized following
D
Rf: 0.5 (EtOAc/Hex 1:1).
D
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 24H), 1.4 (s, 9H), 3.1–
DIPEA, and 724 mg (2.26 mmols, 1.2 equiv) of TBTU. The crude reac-
tion was purified by column chromatography (silica gel, EtOAc/Hex)
to yield the dipeptide (740 mg, 98% yield).
3.2 (m, 2H), 3.7 (s, 3H), 4.2 (m, 2
H), 7.2–7.3 (m, 5H).
aH), 4.4–4.5 (m, 2aH), 4.6–4.7 (m,
a

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6841
Rf: 0.8 (EtOAc/Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 6H), 1.4 (s, 9H), 2.5–2.6
(m, 3H), 3.0–3.1 (d, 2H), 3.7 (s, 3H), 4.3–4.4 (m, 1 H), 4.5–4.6 (m,
H), 6.2–6.3 (d, 1H), 7.2–7.4 (m, 5H).
12.8.3. Tripeptide MeO-Phe-N-Me-
Tripeptide MeO-Phe-N-Me- -Phe-Val-NHBoc was synthesized
following the ‘General peptide synthesis’ procedure. Utilizing
467 mg (1.3 mmols, 1.1 equiv) of amine MeO-Phe-N-Me- -Phe-
D-Phe-Val-NHBoc
D
a
1a
D
NH2, 260 mg (1.2 mmols, 1.0 equiv) of acid HO-Val-NHBoc,
1.2 mL (6 equiv) of DIPEA, 231 mg (0.7 mmols, 0.6 equiv) of TBTU,
319 mg (1.3 mmol, 0.7equiv) in 12 mL of methylene chloride and
3. The crude reaction was purified by column chromatography (sil-
ica gel, EtOAc/Hex) to yield the tripeptide (509 mg, 79% yield).
Rf: 0.5 (EtOAc/Hex 1:1).
12.7.6. Dipeptide HO-
D
-Leu-
D-Phe-NHBoc
Dipeptide HO- -Leu-
D
D-Phe-NHBoc was synthesized following
the ‘General acid deprotection’. This dipeptide was taken on to
the next reaction without further purification or characterization
(379 mg, 98% yield).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 12H), 1.4 (s, 9H), 1.4–
1.6 (m, 2H), 1.7 (s, 1H), 2.1 (m, 1H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 3.8
12.7.7. Pentapeptide MeO-Phe-Leu-Val-
D-Leu-
D-Phe-NHBoc
-Phe-NHBoc was syn-
(dd,
aH), 4.4 (dd, aH), 4.8 (dd, aH), 5.0 (d, 1H), 6.3 (d, 1H), 6.6 (d,
Pentapeptide MeO-Phe-Leu-Val- -Leu-
D
D
1H), 7.1–7.3 (m, 5H).
thesized following the ‘General peptide synthesis’ procedure.
Utilizing 464 mg (1.19 mmols, 1.0 equiv) of amine MeO-Phe-Leu-
Val-NH2, 494 mg (1.30 mmols, 1.1 equiv) of acid HO-D-Leu-D-Phe-
12.8.4. Tripeptide MeO-Phe-N-Me-
D-Phe-Val-NH2
Tripeptide MeO-Phe-N-Me- -Phe-Val-NH2 was synthesized fol-
D
NHBoc, 1.65 mL (8 equiv) of DIPEA, 190 mg (0.59 mmols, 0.5 equiv)
of TBTU, 270 mg (0.71 mmols, 0.6 equiv) HATU, and 141 mg
(0.47 mmols, 0.4 equiv) of DEPBT. The crude reaction was purified
by column chromatography (silica gel, EtOAc/Hex) to yield the
pentapeptide (355 mg, 41% yield).
lowing the ‘General amine deprotection’. This dipeptide was taken
on to the next reaction without further purification or character-
ization (509 mg, 100% yield).
12.8.5. Dipeptide MeO-Leu-N-Me-
D-Leu-Boc
Rf: 0.5 (EtOAc/Hex 1:1).
Dipeptide MeO-Leu-N-Me- -Leu-Boc was synthesized following
D
1H NMR (600 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.4–1.5 (m, 2H),
1.5–1.6 (m, 2H), 2.0–2.1 (m, 2H), 2.9 (m, 1H), 3.0 (m, 2H), 3.1–3.2
the ‘General peptide synthesis’ procedure. Utilizing 326 mg
(2.2 mmols, 1.1 equiv) of amine MeO-Leu-NH2, 500 mg (2.0 mmols,
1.0 equiv) of acid HO-N-Me-D-Leu-Boc, 2.0 mL (6 equiv) of DIPEA,
(m, 2H), 4.0–4.1 (m,
aH), 4.2 (m, aH), 4.3 (m, aH), 4.4 (m, aH),
4.6 (m, H), 7.1–7.3 (m, 10H).
a
722 mg (2.2 mmols, 1.1 equiv) of TBTU, in 20 mL methylene chlo-
ride. The crude reaction was purified by column chromatography
(silica gel, EtOAc/Hex) to yield the dipeptide (709.3 mg, 93% yield).
Rf: 0.8 (EtOAc/Hex 1:1).
12.7.8. Macrocycle Phe-Leu-Val-
D
-Leu-D-Phe
Macrocycle Phe-Leu-Val- -Leu-
D
D-Phe was synthesized following
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (d, 12H), 1.4 (s, 9H), 1.4–1.8
the ‘Macrocyclization procedure’. Utilizing 219 mg (0.34 mmols,
1.0 equiv) of linear pentapeptide, 0.47 mL (8 equiv) of DIPEA,
77 mg (0.20 mmols, 0.6 equiv) of TBTU, 65 mg (0.20 mmols,
0.6 equiv) HATU, and 61 mg (0.20 mmols, 0.6 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (10 mg, 5% yield).
(m, 6H), 3.7 (s, 3H), 4.1 (br, aH), 4.6 (br, aH), 4.8 (br, 1H), 6.6 (d, 1H).
12.8.6. Dipeptide HO-Leu-N-Me-D-Leu-Boc
Dipeptide HO-Leu-N-Me- -Leu-Boc was synthesized following
D
the ‘General acid deprotection’. This tripeptide was taken on to
the next reaction without further purification or characterization
(614 mg, 91% yield).
Rf: 0.5 (EtOAc/Hex 4:1).
1H NMR (400 MHz, (C4D8)O): d 0.8–1.0 (m, 18H), 1.0–1.1 (m,
1H), 1.4–1.6 (m, 2H), 1.9–2.0 (m, 2H), 2.0–2.1 (m, 1H), 2.6–2.7
12.8.7. Pentapeptide MeO-Phe-N-Me-
D
-Phe-Val-Leu-N-Me-
D-
(m, 1H), 2.8–3.0 (m, 4H), 3.9–4.0 (m,
a
H), 4.1–4.2 (m,
aH), 5.3–
Leu-Boc
4.4 (m, H), 4.5–4.6 (m, H), 4.6–4.7 (m, a
a
a
H), 7.0–7.4 (m, 10H),
Pentapeptide MeO-Phe-N-Me-D-Phe-Val-Leu-N-Me-D-Leu-Boc
7.6–7.7 (br, 1H), 8.0–8.1 (br, 1H), 8.1–8.2 (br, 1H).
was synthesized following the ‘General peptide synthesis’ proce-
dure. Utilizing 439 mg (0.94 mmols, 1.1 equiv) of amine MeO-
LCMS: m/z calcd for C35H49N5O5 (M+1) = 620.79, found 620.9.
Phe-N-Me-
HO-Leu-N-Me-
D
-Phe-Val-NH2, 301 mg (0.84 mmols, 1.0 equiv) of acid
-Leu-Boc, 1.2 mL (8 equiv) of DIPEA, 161 mg
12.8. Synthesis of compound 15
D
(0.5 mmols, 0.6 equiv) of TBTU, 223 mg (0.58 mmols, 0.7 equiv) of
HATU, and 100 mg (0.34 mmols, 0.4 equiv) of DEPBT, in 8.4 mL of
methylene chloride. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide
(420 mg, 40% yield).
12.8.1. Dipeptide MeO-Phe-N-Me-
D-Phe-Boc
Dipeptide MeO-Phe-N-Me- -Phe-Boc was synthesized following
D
the ‘General peptide synthesis’ procedure. Utilizing 295.5 mg
(1.4 mmols, 1.1 equiv) of amine OMe-Phe-NH2, 700 mg (1.6 mmols,
1.0 equiv) of acid HO-N-Me-D-Phe-Boc, 0.87 mL (4 equiv) of DIPEA,
321 mg (1.0 mmols, 0.8 equiv) of TBTU, in 12.5 mL of Methylene
Chloride. The crude reaction was purified by column chromatogra-
phy (silica gel, EtOAc/Hex) to yield the dipeptide (606.7 mg, 97%
yield).
Rf: 0.4 (EtOAc/Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 24H),1.3 (m, 2H), 1.4
(s, 9H), 1.5 (m, 3H), 1.6 (m, 2H), 1.7 (m, 2H), 2.2 (m, 1H), 2.9–3.2
(m, 2H), 3.7 (s, 3H), 4.1 (m,
(m, H), 7.s–7.3 (m, 5H).
aH), 4.2 (m, aH), 4.4 (m, 2aH), 4.6
a
Rf: 0.8 (EtOAc/Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.4–1.7
12.8.8. Macrocycle Phe-N-Me-
D
-Phe-Val-Leu-N-Me-D-Leu
(m, 3H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br,
4.8–5.0 (q, H), 6.6 (d, 1H), 7.1–7.4 (m, 5H).
aH), 4.8 (d, 1H),
Macrocycle Phe-N-Me- -Phe-Val-Leu-N-Me-
D
D-Leu was synthe-
a
sized following the ‘Macrocyclization procedure’. Utilizing 179 mg
(0.28 mmols, 1.0 equiv) of linear pentapeptide, 0.4 mL (8 equiv) of
DIPEA, 54 mg (0.17 mmols, 0.6 equiv) of TBTU, 85.1 mg (0.2 mmols,
0.8 equiv) HATU, and 67.0 mg (0.17 mmols, 0.8 equiv) of DEPBT in
2.3 mL methylene chloride, 2.3 mL acetonitrile. The crude reaction
was purified by reverse phase-HPLC to yield the macrocycle
(12.3 mg, 7.5% yield).
12.8.2. Dipeptide MeO-Phe-N-Me-
D-Phe-NH2
Dipeptide MeO-Phe-N-Me- -Phe-NH2 was synthesized follow-
D
ing the ‘General amine deprotection’. This dipeptide was taken
on to the next reaction without further purification or character-
ization (464 mg, 100% yield).

6842
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
Rf: 0.25 (EtOAc/Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.7–1.0 (m, 24H), 1.2–1.8 (m, 9H),
2.0 (m, 1H), 2.9–3.1 (m, 2H), 3.6 (m, H), 3.8 (m, H), 4.2 (m, H),
4.5 (m, H), 4.6 (m, H), 7.1–7.3 (m, 5H), 6.6 (d, 1H), 7.0 (d, 1H), 7.5
12.9.7. Pentapeptide MeO-Phe-
Pentapeptide MeO-Phe- -Phe-
thesized following the ‘General peptide synthesis’ procedure. Uti-
lizing 724 mg (1.7 mmols, 1.1 equiv) of amine MeO-Phe- -Phe-D-
D
-Phe-
D-Val-Leu-Leu-NHBoc
D
D-Val-Leu-Leu-NHBoc was syn-
a
a
a
a
a
D
(d, 1H), 7.6 (d, 1H), 8.0 (d, 1H).
Val-NH2, 532 mg (1.5 mmols, 1.0 equiv) of acid HO-Leu-Leu-
NHBoc, 2.2 mL (8 equiv) of DIPEA, 346 mg (1.1 mmols, 0.7 equiv)
of TBTU, and 505 mg (1.3 mmols, 0.8 equiv) HATU. The crude reac-
tion was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the pentapeptide (931 mg, 80% yield).
LCMS: m/z calcd for C32H51N5O5 (M+1) = 586.4, found 587.5.
12.9. Synthesis of compound 18
Rf: 0.5 (EtOAc/Hex 3:1).
12.9.1. Dipeptide MeO-Phe-
D-Phe-NHBoc
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.4 (s, 9H), 1.5–
1.8 (m, 4H), 1.9–2.0 (m, 1H), 2.0–2.1 (m, 1H), 2.7–2.8 (m, 1H), 2.9–
Dipeptide MeO-Phe- -Phe-NHBoc was synthesized following
D
the ‘General peptide synthesis’ procedure. Utilizing 450 mg
(2.0 mmols, 1.1 equiv) of amine OMe-Phe-NH2, 500 mg (1.9 mmols,
1.0 equiv) of acid HO-D-Phe-NHBoc, 1.3 mL (4 equiv) of DIPEA,
3.0 (m, 2H), 3.0–3.1 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (m, 2
(m, H), 4.6–4.7 (m, 2 H), 7.1–7.3 (m, 10H).
aH), 4.4–4.5
a
a
724 mg (2.3 mmols, 1.2 equiv) of TBTU. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the dipeptide (798 mg, 99% yield).
12.9.8. Macrocycle Phe-
D
-Phe-
D-Val-Leu-Leu
Macrocycle Phe- -Phe-
D
D-Val-Leu-Leu was synthesized following
the ‘Macrocyclization procedure’. Utilizing 163 mg (0.27 mmols,
1.0 equiv) of linear pentapeptide, 0.38 mL (8 equiv) of DIPEA,
43.1 mg (0.14 mmols, 0.5 equiv) of TBTU, 51.1 mg (0.12 mmols,
0.5 equiv) HATU, and 56.3 mg (0.19 mmols, 0.7 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (8 mg, 5% yield).
Rf: 0.5 (EtOAc/Hex 1:4).
1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H), 2.9–3.1 (m, 4H), 3.7 (s,
3H), 4.3–4.4 (m, aH), 4.8–5.0 (m, aH), 6.3–6.4 (m, 1H), 6.9–7.0 (m,
1H), 7.1–7.3 (m, 10H).
12.9.2. Dipeptide MeO-Phe-D-Phe-NH2
Rf: 0.5 (EtOAc/Hex 4:1).
Dipeptide MeO-Phe-D-Phe-NH2was synthesized following the
1H NMR (600 MHz, CD3OD): d 0.8–1.0 (m, 24H), 1.2–1.4 (m, 3H),
1.5–1.7 (m, 6H), 2.9–3.1 (m, 1H), 3.2–3.4 (m, 1H), 3.6–3.8 (m, 1H),
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(610 mg, 100% yield).
4.0–4.1 (m, 1
H), 7.2–7.4 (m, 5H).
LCMS: m/z calcd for C32H51N5O5 (M+23) = 608.39, found 608.6.
aH), 4.1–4.2 (m, 2aH), 4.2–4.4 (m, 1aH), 4.6–4.8 (m,
1a
12.9.3. Tripeptide MeO-Phe-
Tripeptide MeO-Phe- -Phe-
lowing the ‘General peptide synthesis’ procedure. Utilizing 610 mg
(1.87 mmols, 1.1 equiv) of amine MeO-Phe- -Phe-NH2, 369 mg
(1.7 mmols, 1.0 equiv) of acid HO- -Val-NHBoc, 1.5 mL (4 equiv) of
D-Phe-
D-Val-NHBoc
D
D-Val-NHBoc was synthesized fol-
12.10. Synthesis of compound 20
D
12.10.1. Dipeptide Fmoc-
Dipeptide Fmoc- -Phe-Phe-O-Resin was synthesized following
the Section 11.8 procedure. Using 1021.6 mg (0.735 mmol, 1 equiv)
of NH2- -Phe-O-Resin, the -Val residue was incorporated using
748 mg of Fmoc- -Val-OH (2.2 mmol, 3 equiv), 338 mg (2.2 mmol,
D-Val-D-Phe-O-Resin
D
D
DIPEA, 655 mg (4.30 mmols, 1.2 equiv) of TBTU. The crude reaction
was purified by column chromatography (silica gel, EtOAc/Hex) to
yield the tripeptide (890 mg, 98% yield).
D
D
D
Rf: 0.5 (EtOAc/Hex 1:1).
3 equiv) of HOBt, and 0.680 mL (6 equiv) 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.
1H NMR (200 MHz, CDCl3): d 0.7–0.9 (m, 6H), 1.4 (s, 9H), 2.0–2.1
(m, 1H), 2.9–3.1 (m, 4H), 3.7 (s, 3H), 3.8–4.0 (m,
aH), 4.6–4.9 (m,
2aH), 4.8–4.9 (m, 1H), 6.4–6.5 (m, 2H), 7.1–7.3 (m, 10H).
12.9.4. Dipeptide MeO-Leu-Leu-NHBoc
12.10.2. Dipeptide NH2-
D
-Val-D-Phe-O-Resin
Dipeptide MeO-Leu-Leu-NHBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 402 mg (2.2 mmols,
1.1 equiv) of amine MeO-Leu-NH2, 500 mg (2.0 mmols, 1.0 equiv) of
acid HO-Leu-NHBoc, 1.4 mL (4 equiv) of DIPEA, 774 mg (2.4 mmols,
1.2 equiv) of TBTU. The crude reaction was purified by column chro-
matography (silica gel, EtOAc/Hex) to yield the dipeptide (664 mg,
92% yield).
Dipeptide NH2- -Val-
the ‘General solid phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal.
D
D-Phe-O-Resin was synthesized following
12.10.3. Tripeptide Fmoc-Cha-
Tripeptide Fmoc-Cha- -Val-
lowing the Section 11.8 procedure. Using the NH2-
Resin prepared above, the Cha residue was incorporated using
866.9 mg (2.2 mmol, 3 equiv) of Fmoc-Cha-OH, 338 mg (2.2 mmol,
3 equiv) of HOBt, and 0.680 mL of DIC (6 equiv). 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 tripeptide.
D
-Val-
-Phe-O-Resin was synthesized fol-
-Val- -Phe-O-
D-Phe-O-Resin
D
D
D
D
Rf: 0.5 (EtOAc/Hex 1:3).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 12H), 1.2–1.3 (m, 4H),
1.4 (m, 9H), 1.6–1.7 (m, 2H), 3.7 (s, 3H), 4.1–4.2 (m,
(m, H), 4.8–4.9 (br, 1H), 6.4–6.5 (br, 1H).
aH), 4.6–4.7
a
12.9.5. Tripeptide MeO-Phe-
D-Phe-
D-Val-NH2
Dipeptide MeO-Phe- -Phe-
D
D-Val-NH2 was synthesized follow-
ing the ‘General amine deprotection’. This tripeptide was taken
on to the next reaction without further purification or character-
ization (724 mg, 100% yield).
12.10.4. Tripeptide NH2-Cha-
D
-Val-
D-Phe-O-Resin
Tripeptide NH2-Cha- -Val-
D
D
-Phe-O-Resin was synthesized fol-
lowing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal.
12.9.6. Dipeptide HO-Leu-Leu-NHBoc
Dipeptide HO-Leu-Leu-NHBoc was synthesized following the
‘General acid deprotection’. This dipeptide was taken on to the next
reaction without further purification or characterization (573 mg,
90% yield).
12.10.5. Tetrapeptide Fmoc-Ser(Bzl)-Cha-
Tetrapeptide Fmoc-Ser(Bzl)-Cha- -Val-
thesized following the Section 11.8 procedure. Using the NH2-Cha-
-Val- -Phe-O-Resin prepared above, the Ser(Bzl) residue was
D
-Val-
D-Phe-O-Resin
D
D-Phe-O-Resin was syn-
D
D

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6843
incorporated using 920.0 mg (2.2 mmol, 3 equiv) of Fmoc-Ser(Bzl)-
OH, 338 mg (2.3 mmol, 3 equiv) of HOBt, and 0.680 mL of DIC
(6 equiv). Completion of the coupling reaction was verified by a neg-
ative ninhydrin test. The reaction mixture was then drained to leave
the amine-protected resin-bound tetrapeptide.
12.11.2. Dipeptide NH-N-Me-D-Phe-Phe-O-Resin
Dipeptide NH2-Leu-Phe-O-Resin was synthesized following the
‘General solid phase amine deprotection’ procedure. A positive nin-
hydrin test served to verify Fmoc removal.
12.11.3. Tripeptide Fmoc-
Tripeptide Fmoc- -Val-N-Me-
sized following the Section 11.8 procedure. Using the NH-N-
Me- -Phe-Phe-O-Resin prepared above, the -Val residue was
incorporated using 1620 mg (4.8 mmol, 3 equiv) of Fmoc- -Val-
OH, 750 mg (4.8 mmol, 3 equiv) of HOAt, and 1.50 mL of DIC
(6 equiv). Completion of the coupling reaction was verified by a neg-
ative ninhydrin test. The reaction mixture was then drained to leave
the amine-protected resin-bound tripeptide.
D
-Val-N-Me-
D-Phe-Phe-O-Resin
D
D-Phe-Phe-O-Resin was synthe-
12.10.6. Tetrapeptide NH2-Ser(Bzl)-Cha-
D
-Val-
D-Phe-O-Resin
Tetrapeptide NH2-Ser(Bzl)-Cha- -Val-
D
D-Phe-O-Resin was syn-
D
D
thesized following the ‘General solid phase amine deprotection’
procedure. A positive ninhydrin test served to verify Fmoc removal.
D
12.10.7. Pentapeptide Fmoc-Phe-Ser(Bzl)-Cha-
Resin
D-Val-D-Phe-O-
Pentapeptide Fmoc-Phe-Ser(Bzl)-Cha-
synthesized following the Section 11.8 procedure. Using the NH2-
Ser(Bzl)-Cha- -Val- -Phe-O-Resin prepared above, the Phe residue
D-Val-D-Phe-O-Resin was
12.11.4. Tripeptide NH2-
D
-Val-N-Me-
D-Phe-Phe-O-Resin
Tripeptide NH2- -Val-N-Me-
D
D-Phe-Phe-O-Resin was synthe-
D
D
sized following the ‘General solid phase amine deprotection’ proce-
dure. A positive ninhydrin test served to verify Fmoc removal.
was incorporated using 853.7 mg (2.2 mmol, 3 equiv) of Fmoc-Phe-
OH, 338 mg (2.2 mmol, 3 equiv) of HOBt, 0.680 mL of DIC (6 equiv).
Completion of the coupling reaction was verified by a negative nin-
hydrin test. The reaction mixture was then drained to leave the
amine-protected resin-bound pentapeptide.
12.11.5. Tetrapeptide Fmoc-Cha-
Tetrapeptide Fmoc-Cha- -Val-N-Me-
synthesized following the Section 11.8 procedure. Using the NH2-
-Val-N-Me- -Phe-Phe-O-Resin prepared above, the Cha residue
D
-Val-N-Me-
D-Phe-Phe-O-Resin
D
D
-Phe-Phe-O-Resin was
D
D
12.10.8. Pentapeptide NH2-Phe-Ser(Bzl)-Cha-D-Val-D-Phe-O-
was incorporated using 1860 mg (4.8 mmol, 3 equiv) of Fmoc-
Cha-OH, 750 mg (4.8 mmol, 3 equiv) of HOBt, and 1.50 mL of DIC
(6 equiv). 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 tetrapeptide.
Resin
Pentapeptide NH2-Phe-Ser(Bzl)-Cha-D-Val-D-Phe-O-Resin was
synthesized following the Section 11.10 procedure. A positive nin-
hydrin test served to verify Fmoc removal. The resin was then dried
in vacuo for 24 h in a vacuum desiccator.
12.11.6. Tetrapeptide NH2-Cha-
D
-Val-N-Me-
D-Phe-Phe-O-Resin
12.10.9. Double deprotected pentapeptide NH2-Phe-Ser(Bzl)-
Tetrapeptide NH2-Cha- -Val-N-Me-
D
D-Phe-Phe-O-Resin was syn-
Cha-
Double deprotected pentapeptide NH2-Phe-Ser(Bzl)-Cha-
-Phe-OH was synthesized following the Section 11.11 procedure.
Utilizing the 1353.5 mg of dried NH2-Phe-Ser(Bzl)-Cha- -Val-
-Phe-O-Resin, 6.7 mL of 2,2,2-trifluoroethanol and 6.7 mL of
D-Val-D-Phe-OH
thesized following the ‘General solid phase amine deprotection’
procedure. A positive ninhydrin test served to verify Fmoc removal.
D
-Val-
D
D
12.11.7. Pentapeptide Fmoc-Ser(Bzl)-Cha-D-Val-N-Me-D-Phe-
D
Phe-O-Resin
CH2Cl2. The resin slurry was stirred for 24 h, after which it was
filtered, washed with additional CH2Cl2, and dried in vacuo for
24 h (273 mg, 82% yield).
Pentapeptide Fmoc-Ser(Bzl)-Cha-
sin was synthesized following the Section 11.8 procedure. Using
the NH2-Cha- -Val-N-Me- -Phe-Phe-O-Resin prepared above, the
D-Val-N-Me-D-Phe-Phe-O-Re-
D
D
Ser(Bzl) residue was incorporated using 2000 mg (4.8 mmol,
3 equiv) of Fmoc-Ser(Bzl)-OH, 750 mg (4.8 mmol, 3 equiv) of HOBt,
1.50 mL of DIC (6 equiv). 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.
12.10.10. Macrocycle Phe-
D
-Phe-
D-Val-Cha-Ser(Bzl)
Macrocycle Phe- -Phe-
D
D-Val-Cha-Ser(Bzl) was synthesized
following the ‘Macrocyclization procedure’. Utilizing 160 mg
(0.21 mmols, 1.0 equiv) of linear pentapeptide, 0.3 mL (8 equiv) of
DIPEA, 41.5 mg (0.13 mmols, 0.6 equiv) of TBTU, 49.2 mg
(0.13 mmols, 0.6 equiv) HATU, and 38.7 mg (0.13 mmols, 0.6 equiv)
of DEPBT. The crude reaction was purified by reverse phase-HPLC to
yield the macrocycle (7.9 mg, 5.1% yield).
12.11.8. Pentapeptide NH2-Ser(Bzl)-Cha-D-Val-N-Me-D-Phe-Phe-
O-Resin
Pentapeptide NH2-D-Phe-N-Me-D-Lys(2-Cl-Cbz)-Val-Leu-Phe-O-
Resin was synthesized following the Section 11.10 procedure. A
positive ninhydrin test served to verify Fmoc removal. The resin
was then dried in vacuo for 24 h in a vacuum desiccator.
1H NMR (400 MHz, CD3OD): d0.7–0.9 (m, 6H), 1.0–1.4 (m, 11H),
1.9 (s, 2H), 2.6 (s, 1H), 3.2–3.6 (m, 6H), 4.4–4.5 (m,
7.0–7.3 (m, 15H).
aH), 4.6 (m, 2H),
LCMS: m/z called for C42H53N5O6 (M+1) = 724.9, found 727.0.
12.11.9. Double Deprotected Pentapeptide NH2-Ser(Bzl)-Cha-
D
-Val-N-Me-
Double Deprotected Pentapeptide NH2-Ser(Bzl)-Cha-
N-Me- -Phe-Phe-OH was synthesized following the Section 11.11
procedure.Utilizing the 3830.1 mg of dried NH2-Ser(Bzl)-Cha-
-Val-N-Me- -Phe-Phe-O-Resin, 18.5 mL of 2,2,2-trifluoroethanol
D-Phe-Phe-OH
12.11. Synthesis of compound 21
D-Val-
D
12.11.1. Dipeptide Fmoc-N-Me-
Dipeptide Fmoc-N-Me- -Phe-Phe-O-Resin was synthesized fol-
lowing the Section 11.8 procedure. Using 2510 mg (1.62 mmol,
1 equiv) of NH2-Phe-O-Resin, the N-Me- -Phe residue was incorpo-
rated using 1930 mg of Fmoc-N-Me-Phe-OH (4.8 mmol, 3 equiv),
750 mg (4.8 mmol, 3 equiv) of HOBt, and 1.50 mL (6 equiv) 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.
D-Phe-Phe-O-Resin
D
D
D
and 18.5 mL of CH2Cl2. The resin slurry was stirred for 24 h, after
which it was filtered, washed with additional CH2Cl2, and dried
in vacuo for 24 h (894 mg, 82% yield).
D
12.11.10. Macrocycle Phe-N-Me-
Macrocycle Phe-N-Me- -Phe-
sized following the ‘Macrocyclization procedure’. Utilizing 300 mg
D
-Phe-D-Val-Cha-Ser(Bzl)
D
D-Val-Cha-Ser(Bzl) was synthe-

6844
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
(0.397 mmols, 1.0 equiv) of linear pentapeptide, 0.543 mL (8 equiv)
of DIPEA, 67.0 mg (0.21 mmols, 0.7 equiv) of TBTU, 80.0 mg
(0.21 mmols, 0.7 equiv) HATU, and 62.5 mg (0.21 mmols, 0.7 equiv)
of DEPBT. The crude reaction was purified by reverse phase-HPLC to
yield the macrocycle (42.3 mg, 16.2% yield).
(4.41 mmols, 1.1 equiv) of amine MeO-Lys(CBz)-NH2, 1000 mg
(4.01 mmols, 1.0 equiv) of acid HO-Leu-NHBoc, 2.8 mL (4 equiv)
of DIPEA, and 1545 mg (4.8 mmols, 1.2 equiv) of TBTU. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the dipeptide (1182 mg, 97% yield).
Rf: 0.65 (EtOAc/Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.6–1.0 (m, 8H), 1.2 (m, 11H),
1.4–1.6 (m, 3H), 1.7–1.8 (m, 4H), 2.0 (m, 1H), 3.4 (m, 3H), 4.1 (s,
1H NMR (200 MHz, CDCl3): d 1.0–1.2 (d, 6H), 1.6 (s, 9H), 1.7–2.2
aH), 4.2 (s,
aH), 4.5 (s,
aH), 5.2 (m, 2H), 7.0–7.2 (m, 22H).
(m, 9H), 3.3 (m, 2H), 3.7 (s, 3H), 4.3 (dd,
2H), 7.5 (d, 5H).
aH), 4.6 (dd, aH), 5.3 (s,
12.11.11. Macrocycle Phe-N-Me-
Macrocycle Phe-N-Me- -Phe-
utilizing 20 mg (0.08 mmols, 1.0 equiv) of macrocycle Phe-N-Me-
-Phe- -Val-Cha-Ser(Bzl).The compound was dissolved in 0.8 mL
EtOH (0.1 M) and was hydrogenated using a catalytic amount of
Pd/C and excess H2 for 24 h. The reaction was filtered over Celite
D
-Phe-
D-Val-Cha-Ser
D
D-Val-Cha-Ser was synthesized
12.12.6. Dipeptide HO-Lys(CBz)-Leu-NHBoc
Dipeptide HO-Lys(CBz)-Leu-NHBoc was synthesized following
the ‘General acid deprotection’. This dipeptide was taken on to
the next reaction without further purification or characterization
(1089 mg, 95% yield).
D
D
and afforded 18 mg (93% yield) of pure macrocycle Phe-N-Me-D-
Phe-D-Val-Cha-Ser.
12.12.7. Pentapeptide MeO-
Pentapeptide MeO- -Tyr-Leu-Val-Lys(CBz)-Leu-NHBoc was syn-
thesized following the ‘General peptide synthesis’ procedure. Utiliz-
ing 1021 mg (2.5 mmols, 1.1 equiv) of amine MeO- -Tyr-Leu-Val-
NH2, 1089 mg (2.3 mmols, 1.0 equiv) of acid HO-Lys(CBz)-Leu-
NHBoc, 1.6 mL (4 equiv) of DIPEA, and 826 mg (2.76 mmols,
1.2 equiv) of DEPBT. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide
(192 mg, 20% yield).
D-Tyr-Leu-Val-Lys(CBz)-Leu-NHBoc
1H NMR (400 MHz, CD3OD): d 0.7–0.9 (m, 8H), 1.2 (m, 11H),
D
1.4–1.6 (m, 3H), 1.7–1.8 (m, 4H), 2.0 (m, 1H), 3.4 (m, 3H), 4.2–4.3
(s,
a
H), 4.2 (s,
aH), 4.5 (s, aH), 5.2 (m, 2H), 7.2–7.4 (m, 14H).
D
LCMS: m/z called for C36H49N5O6 (M+1) = 648.4, found 648.3.
12.12. Synthesis of compounds 23 and 24
12.12.1. Dipeptide MeO-
Dipeptide MeO- -Tyr-Leu-NHBoc was synthesized following
the ‘General peptide synthesis’ procedure. Utilizing 1026.9 mg
(4.43 mmols, 1.1 equiv) of amine MeO- -Tyr-NH2, 1000.9 mg
D-Tyr-Leu-NHBoc
D
Rf: 0.6 (EtOAc/Hex 3:1).
1H NMR (500 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.3 (m, 2H), 1.5
(s, 9H), 1.6–1.8 (m,6H), 2.0–2.2 (m, 6H), 2.9 (m, 2H), 3.1 (m, 4H),
D
(4.01 mmols, 1.0 equiv) of acid HO-Leu-NHBoc, 2.8 mL (4 equiv)
of DIPEA, and 1440 mg (4.81 mmols, 1.2 equiv) of DEPBT. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the dipeptide (1526 mg, 93% yield).
Rf: 0.65 (EtOAc/Hex 1:1).
3.7 (s, 3H), 4.2 (m, 2
aH), 4.4 (m, 2aH), 4.6 (m, aH), 5.0 (s, 2H),
6.7–7.0 (d, 4H), 7.4 (d, 5H), 7.8 (m, 2H), 8.0–8.2 (m, 4H).
12.12.8. Macrocycle
protected)
D-Tyr-Leu-Val-Lys(CBz)-Leu (Cyclized
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (d, 6H), 1.4 (s, 9H), 1.6 (br,
1H), 2.0 (s, 2H), 3.0 (m, 2H), 3.7 (s, 3H), 4.1 (dd,
aH), 4.8 (dd, aH),
Macrocycle
D-Tyr-Leu-Val-Lys(CBz)-Leu was synthesized fol-
5.0 (s, 1H), 6.6–6.8 (d, 2H), 7.0 (d, 2H), 7.2 (s, 2H).
lowing the ‘Macrocyclization procedure’. Utilizing 169 mg
(0.22 mmols, 1.0 equiv) of linear pentapeptide, 0.3 mL (8 equiv) of
DIPEA, 35 mg (0.11 mmols, 0.5 equiv) of TBTU, 42 mg (0.11 mmols,
0.5 equiv) HATU, and 33 mg (0.11 mmols, 0.5 equiv) of DEPBT. The
crude reaction was purified by reverse phase-HPLC to yield the
macrocycle (30 mg, 18% yield).
12.12.2. Dipeptide MeO-D-Tyr-Leu-NH2
Dipeptide MeO- -Tyr-Leu-NH2 was synthesized following the
D
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(1153 mg, 100% yield).
Rf: 0.6 (EtOAc/Hex 1:0).
1H NMR (500 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.2–1.4 (m, 4H),
12.12.3. Tripeptide MeO-
Tripeptide MeO- -Tyr-Leu-Val-NHBoc was synthesized follow-
ing the ‘General peptide synthesis’ procedure. Utilizing 1153 mg
(3.73 mmols, 1.1 equiv) of amine MeO- -Tyr-Leu-NH2, 750 mg
D-Tyr-Leu-Val-NHBoc
1.5–1.8 (m, 8H), 2.6 (m,
aH), 3.1 (m, 4H), 3.2 (m, 2H), 4.1 (m, 2aH),
D
4.3 (m, 2 H), 4.5 (m, H), 5.1 (s, 2H), 6.7 (dd, 2H), 7.0 (dd, 2H), 7.2–
a
a
7.4 (d, 5H), 7.5 (d, 1H), 7.7 (d, 1H), 8.1 (d, 1H), 8.3 (d, 1H), 8.5 (d,
1H), 8.7 (d, 1H).
D
(3.4 mmols, 1.0 equiv) of acid HO-Val-NHBoc, 2.4 mL (4 equiv) of
DIPEA, and 1240 mg (4.07 mmols, 1.2 equiv) of DEPBT. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the tripeptide (1754 mg, 73% yield).
Rf: 0.65 (EtOAc/Hex, 3:1).
LCMS: m/z calcd for C34H52N6O5 (M+1) = 750.4, found 752.
12.12.9. Removal of Cbz group
12.12.9.1. Macrocycle
D-Tyr-Leu-Val-Lys-Leu. Macrocyclic pen-
1H NMR (200 MHz, CD3OD): d 0.8–0.9 (m, 12H), 1.4 (s, 9H), 1.9
(br, 1H), 2.0 (s, 2H), 2.8 (m, 1H), 3.0 (m, 2H), 3.7 (s, 3H), 3.8 (dd,
tapeptide -Tyr-Leu-Val-Lys(CBz)-Leu was further deprotected to
D
remove Cbz protecting group of the lysine residue. The compound
was synthesized by utilizing 16 mg (0.021 mmols, 1.0 equiv) of
cyclic pentapeptide, 5.0 mL of Ethanol, 8.0 mg 10% wt palladium
on carbon. The crude reaction was purified by reverse phase-HPLC
aH), 4.4 (dd,
aH), 4.6 (dd, 2aH), 5.0 (s, 1H), 6.6–6.8 (d, 2H), 7.0
(d, 2H).
to yield the deprotected macrocycle
(5.0 mg, 38% yield).
D-Tyr-Leu-Val-Lys-Leu
12.12.4. Tripeptide MeO-
D-Tyr-Leu-Val-NH2
Tripeptide MeO- -Tyr-Leu-Val-NH2 was synthesized following
D
Rf: 0.5 (DCM/MeOH 98:2).
the ‘General amine deprotection’. This dipeptide was taken on to
the next reaction without further purification or characterization
(1021 mg, 100% yield).
1H NMR (500 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.2–1.4 (m, 4H),
1.5–1.8 (m, 8H), 2.0 (m, 2H), 2.6 (m, H), 3.0 (m, 4H), 3.2 (m, 2H),
a
4.1 (m, 2 H), 4.3 (m, 2 H), 4.5 (m, aH), 6.7 (dd, 2H), 7.0 (dd, 2H),
a
a
7.5 (d, 1H), 7.7 (d, 1H), 8.1 (d, 1H), 8.3 (d, 1H), 8.5 (d, 1H), 8.7 (d,
1H).
12.12.5. Dipeptide MeO-Lys(CBz)-Leu-NHBoc
Dipeptide MeO-Lys(CBz)-Leu-NHBoc was synthesized following
the ‘General peptide synthesis’ procedure. Utilizing 1459 mg
LCMS: m/z calcd for C34H52N6O5 (M+1) = 617.

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6845
12.13. Synthesis of compounds 26 and 27
chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide
(430 mg, 47% yield).
12.13.1. Dipeptide MeO-
Dipeptide MeO- -Trp-Leu-NHBoc was synthesized following
the ‘General peptide synthesis’ procedure. Utilizing 1126.3 mg
(4.4 mmols, 1.1 equiv) of amine MeO- -Trp-NH2, 1008.9 mg
D
-Trp-Leu-NHBoc
Rf: 0.55 (EtOAc/Hex 3:1).
D
1H NMR (500 MHz, CD3OD): d0.8–1.0 (m, 18H), 1.4 (m, 9H), 1.5–
1.8 (m, 10H), 2.1 (m, 1H), 3.2 (m, 3H), 3.4 (m, 2H), 3.7 (s, 3H), 4.1 (d,
a
D
H), 4.6 (m, 4
aH), 5.1 (s, 2H), 7.0–7.5 (m, 15H), 7.8 (d, 1H), 8.0 (d,
(4.01 mmols, 1.0 equiv) of acid HO-Leu-NHBoc, 2.8 mL (4 equiv) of
DIPEA, and 1446 mg (4.83 mmols, 1.2 equiv) of DEPBT. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the dipeptide (1692 mg, 98% yield).
Rf: 0.65 (EtOAc/Hex 1:1).
1H), 8.1 (d, 1H), 8.3 (d,1H).
12.13.8. Macrocycle
Protected)
D-Trp-Leu-Val-Arg(CBz)-Val (Cyclized
Macrocycle D-Trp-Leu-Val-Arg(CBz)-Val was synthesized follow-
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (d, 6H), 1.4 (s, 9H), 1.5–1.7
ing the ‘Macrocyclization procedure’. Utilizing 430 mg (0.45 mmols,
1.0 equiv) of linear pentapeptide, 0.3 mL (4 equiv) of DIPEA, 72.5 mg
(0.22 mmols, 0.5 equiv) of TBTU, 85.5 mg (0.224 mmols, 0.5 equiv)
HATU, and 67.3 mg (0.225 mmols, 0.5 equiv) of DEPBT. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the pentapeptide (212 mg, 50% yield).
Rf: 0.65 (EtOAc/MeOH 98:2).
(m, 2H), 2.0 (m, 1H), 3.3 (d, 2H), 3.6 (s, 3H), 4.0 (m,
H), 6.6 (br, 1H), 7.0–7.5 (m, 4H), 8.1 (br, 1H).
aH), 4.9 (m,
a
12.13.2. Dipeptide MeO-D-Trp-Leu-NH2
Dipeptide MeO- -Trp-Leu-NH2 was synthesized following the
D
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization
(1298 mg, 100% yield).
1H NMR (500 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.2–1.4 (m, 6H),
1.5–1.7 (m, 4H), 2.0 (m, 1H), 2.8 (m,
H), 4.4 (m, 2 H), 4.8 (m, 2 H), 5.3 (s, 2H), 7.0–7.5 (m, 15H), 7.5
(d, 1H), 8.2 (d, 1H), 8.8 (d, 1H).
aH) 3.1–3.2 (m, 2H), 4.1 (d,
a
a
a
12.13.3. Tripeptide MeO-
Tripeptide MeO- -Trp-Leu-Val-NHBoc was synthesized follow-
ing the ‘General peptide synthesis’ procedure. Utilizing 1298 mg
(3.91 mmols, 1.1 equiv) of amine MeO- -Trp-Leu-NH2, 771 mg
D-Trp-Leu-Val-NHBoc
D
LCMS: m/z calcd for C34H52N6O5 (M+1) = 802, found 802.
D
12.13.9. Removal of Cbz group
(3.55 mmols, 1.0 equiv) of acid HO-Val-NHBoc, 2.5 mL (4 equiv)
of DIPEA, and 1274 mg (4.26 mmols, 1.2 equiv) of DEPBT. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the tripeptide (1465 mg, 78% yield).
Rf: 0.55 (EtOAc/Hex 1:1).
12.13.9.1. Macrocycle
D-Trp-Leu-Val-Arg-Val. Macrocyclic pen-
tapeptide -Trp-Leu-Val-Arg(CBz)-Val was further deprotected to
D
remove Cbz protecting group of the arginine residue. The compound
was synthesized by utilizing 106 mg (0.113 mmols, 1.0 equiv) of
cyclic pentapeptide, 2.5 mL of Ethanol (0.05 M), 45.0 mg 10% wt
palladium on carbon. The crude reaction was purified by reverse
1H NMR (200 MHz, CDCl3): d 0.7–1.0 (dd, 12H), 1.4 (s, 9H), 2.0
(m, 2H), 3.2–3.3 (m, 3H), 3.6 (s, 3H), 3.8 (m,
a
H), 4.4 (m,
aH), 4.8
phase-HPLC to yield the deprotected macrocycle
Arg-Val (6.0 mg, 48% yield).
D-Trp-Leu-Val-
(m, H), 6.5 (br, 1H), 7.0–7.6 (m, 5H), 8.2 (br, 1H).
a
Rf: 0.5 (DCM/MeOH 98:2).
12.13.4. Tripeptide MeO-
D
-Trp-Leu-Val-NH2
1H NMR (500 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.2–1.4 (m, 6H),
Tripeptide MeO- -Trp-Leu-Val-NH2 was synthesized following
D
1.5–1.7 (m, 4H), 2.0 (m, 3H), 2.8 (m,
aH) 3.1–3.2 (m, 2H), 4.2 (d,
the ‘General amine deprotection’. This tripeptide used 503 mg
(0.95 mmols, 1.0 equiv) was taken on to the next reaction without
further purification or characterization (410 mg, 100% yield).
a
H), 4.4 (m, 2 H), 4.8 (m, 2 H), 6.8 (m, 1H), 7.0–7.5 (m, 14H),
a
a
7.8 (d, 1H), 8.2 (d, 1H), 8.3 (d, 1H), 8.5 (d, 1H), 8.8 (d, 1H).
LCMS: m/z calcd for C34H52N6O5 (M+1) = 668.
12.13.5. Dipeptide MeO-Arg(CBz)-Val-NHBoc
12.14. Synthesis of compound 28
Dipeptide MeO-Arg(CBz)-Val-NHBoc was synthesized follow-
ing the ‘General peptide synthesis’ procedure. Utilizing 799 mg
(4.4 mmols, 1.1 equiv) of amine MeO-Arg(CBz)-NH2, 1 g (4.0 mmols,
1.0 equiv) of acid HO-Val-NHBoc, 2.8 mL (4 equiv) of DIPEA, and
1.5 g (1.4 mmols, 1.2 equiv) of TBTU. The crude reaction was purified
by column chromatography (silica gel, EtOAc/Hex) to yield the
dipeptide (660 mg, 98% yield).
12.14.1. Dipeptide MeO-Phe-D-N-Me-Phe-NBoc
Dipeptide MeO-Ia-IIc-NBoc was synthesized using the ‘General
peptide synthesis’ procedure. Utilizing 309 mg (1.43 mmols,
1.1 equiv) of amine Ia, 600 mg (1.30 mmols, 1.0 equiv) of acid IIc,
0.91 mL (4 equiv) of DIPEA, and 502 mg (1.56 mmols, 1.2 equiv)of
TBTU. The crude reaction was purified by an acid-base wash to
yield the dipeptide (574 mg, 99% yield).
Rf: 0.5 (EtOAc/Hex 1:1).
1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H), 1.6–2.0 (m, 10H), 3.4
Rf: 0.5 (EtOAc/Hex 1:3).
(s, 2H), 3.6 (s, 3H), 4.0 (dd,
a
H), 4.6–4.8 (dd, 2
a
H), 5.0 (s, 2H), 5.1
1H NMR (200 MHz, CD3OD): d 1.2–1.4 (s, 9H), 2.7 (s, 3H), 2.8 (m,
(s, 2H), 7.2–7.6 (m, 10H), 7.8 (s, 2H).
1H), 2.9 (m, 1H), 3.0–3.2 (m, 3H), 3.7 (s, 3H), 4.8–5.0 (m, 2
7.3 (m, 10H), 7.8 (d, 1H).
aH), 7.1–
12.13.6. Dipeptide HO-Arg(CBz)-Val-NHBoc
Dipeptide HO-Arg(CBz)-Val-NHBoc was synthesized following
the ‘General acid deprotection’. This dipeptide was taken on to
the next reaction without further purification or characterization
(564 mg, 89% yield).
12.14.2. Dipeptide MeO-Phe-
D-N-Me-Phe-NH
Dipeptide MeO-Phe- -N-Me-Phe-NH was synthesized using the
D
‘General amine deprotection’ procedure. The dipeptide was taken
on to the next reaction without further purification or character-
ization (439 mg, 100% yield).
12.13.7. Pentapeptide MeO-
Pentapeptide MeO- -Trp-Leu-Val-Arg(CBz)-Val-NHBoc was syn-
thesized following the ‘General peptide synthesis’ procedure.
Utilizing 362 mg (0.84 mmols, 1.1 equiv) of amine MeO- -Trp-
Leu-Val-NH2, 501 mg (0.76 mmols, 1.0 equiv) of acid HO-Arg(CBz)-
Val-NHBoc, 0.53 mL (4 equiv) of DIPEA, and 274 mg (0.92 mmols,
1.2 equiv) of DEPBT. The crude reaction was purified by column
D-Trp-Leu-Val-Arg(CBz)-Val-NHBoc
D
12.14.3. Tripeptide MeO-Phe-
D-N-Me-Phe-Val-NHBoc
Tripeptide MeO-Phe- -N-Me-Phe-Val-NHBoc was constructed
D
D
following the procedure outlined ‘General peptide synthesis’. Uti-
lizing 439 mg (1.29 mmols, 1.1 equiv) of amine MeO-Ia-IIc-NH2,
254 mg (1.17 mmols, 1.0 equiv) of acid IIIa, 1.92 mL (8 equiv) of
DIPEA, and 452 mg (1.41 mmols, 1.2 equiv)of TBTU. The crude

6846
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
reaction was purified by an acid-base wash and column chroma-
tography (silica gel, EtOAc/Hex) to yield the desired tripeptide
(667 mg, 96% yield).
12.14.10. Macrocycle Phe-
D-N-Me-Phe-Val-Leu-Lys(CBz)
Macrocycle Phe- -N-Me-Phe-Val-Leu-Lys(CBz) was constructed
D
using the ‘General macrocyclization procedure’. Utilizing 149 mg
(0.19 mmols, 1.0 equiv) of the double deprotected linear pentapep-
tide, 30.0 mg (0.09 mmols, 0.5 equiv) TBTU, 35.2 mg (0.09 mmols,
0.5 equiv) HATU, 28.4 mg (0.09 mmols, 0.5 equiv) DEPBT, and
0.27 mL (8 equiv) of DIPEA. The crude material was purified using
column chromatography (silica gel, EtOAc/Hex) to yield 19.2 mg
(17% yield) of the macrocycle.
Rf: 0.5 (EtOAc/Hex 3:2).
1H NMR (200 MHz, CDCl3): d 0.6 (d, 6H), 1.4 (s, 9H), 1.6–1.8
(m, 4H), 2.9 (m, 3H), 3.0–3.2 (dd, 1H), 3.3 (s, 1H), 3.7 (m, 3H), 4.2
(dd, 1aH), 4.7–4.9 (dd, 1aH), 5.0 (d, 1H), 5.5 (dd, 1H), 6.9 (d, 1H),
7.0–7.4 (m, 10H).
12.14.4. Tripeptide MeO-Phe-
D
-N-Me-Phe-Val-NH
Rf: 0.5 (EtOAc/Hex 4:1).
Tripeptide MeO-Ia-IIc-IIIa-NH was synthesized using the
‘General amine deprotection’ procedure. The tripeptide was taken
on to the next reaction without further purification or character-
ization (620 mg, 100% yield).
1H NMR (400 MHz, CD3OD): d 0.5 (d, 1H), 0.6 (d, 4H), 0.7 (m,
4H), 0.8–1.0 (m, 6H), 1.2–1.4 (m, 6H), 1.5 (m, 2H), 1.7 (m, 2H),
1.8 (m, 2H), 2.9 (s, 3H), 3.0 (m, 2H), 3.1 (m, 2H), 3.9 (t, 1
(t, 1 H), 4.4 (t, 1 H), 4.6 (t, 1 H), 5.1 (s, 2H), 5.3 (s, 1H), 7.1–7.4
(m, 15H).
LCMS: m/z calculated for C44H58N6O7 (M+1) = 784.1, found
784.2.
aH), 4.1
a
a
a
12.14.5. Dipeptide MeO-Leu-Lys(CBz)-NHBoc
Dipeptide MeO- Leu-Lys(CBz)-NHBoc was constructed follow-
ing the procedure outlined ‘General peptide synthesis’. Utilizing
315 mg (1.74 mmols, 1.1 equiv) of amine IVa, 600 mg (1.57 mmols,
1.0 equiv) of acid Vd, 1.10 mL (4 equiv) of DIPEA, and 605 mg
(1.88 mmols, 1.2 equiv)of TBTU. The crude reaction was purified
by an acid-base wash to yield the dipeptide (715 mg, 90% yield).
Rf: 0.4 (EtOAc/Hex 1:1).
12.15. Synthesis of compound 29
12.15.1. Dipeptide Fmoc-N-Me- -Phe-Phe-O-Resin
Dipeptide Fmoc-N-Me-Lys(Boc)-Leu-O-Resin was synthesized
following the Section 11.8 procedure. Using 510.4 mg (0.326 mmol,
1 equiv) of H-Phe-O-Resin, the N-Me-
rated using 392.6 mg of Fmoc-N-Me-
3 equiv), 150 mg (0.978 mmol, 3 equiv) of HOBt, and 0.303 mL
(6 equiv) 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.
D
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.5–1.7
(m, 6H), 1.8 (m, 1H), 3.1 (q, 2H), 3.7 (s, 3H), 4.0–4.2 (m, 1
D
-Phe residue was incorpo-
-Phe-OH (0.978 mmol,
aH), 4.5–
D
4.7 (m, 1 H), 5.0 (m, 1H), 5.1 (s, 2H), 6.4 (d, 1H), 7.2–7.4 (m, 5H).
a
12.14.6. Dipeptide HO-Leu-Lys(CBz)-NHBoc
Dipeptide HO-Leu-Lys(CBz)-NHBoc was synthesized using the
‘General acid deprotection’ procedure. The dipeptide was taken
on to the next reaction without further purification or character-
ization (650 mg, 86% yield).
12.15.2. Dipeptide H-N-Me-
D-Phe-Phe-O-Resin
Dipeptide H-N-Me- -Phe-Phe-O-Resin was synthesized follow-
D
ing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal.
12.14.7. Pentapeptide MeO-Phe-
NHBoc
D-N-Me-Phe-Val-Leu-Lys(CBz)-
Pentapeptide MeO-Phe-D-N-Me-Phe-Val-Leu-Lys(CBz)-NHBoc
was constructed using the ‘General peptide synthesis’ procedure.
Utilizing 283 mg (0.65 mmols, 1.1 equiv) of MeO-Ia-IIc-IIIa-NH2,
289 mg (0.59 mmols, 1.0 equiv) of HO-IVa-Vd-NHBoc, 178 mg
(0.49 mmols, 0.8 equiv) HATU, 70.1 mg (0.23mmols, 0.4 equiv)
DEPBT, and 0.82 mL (8 equiv) of DIPEA. The crude material was
purified using column chromatography (silica gel, EtOAc/Hex) to
yield 415 mg (78% yield) of the pentapeptide.
12.15.3. Tripeptide Fmoc-Val-N-Me-
Tripeptide Fmoc-Val-N-Me- -Phe-Phe-O-Resin was synthesized
following the Section 11.8 procedure. Using the H-N-Me- -Phe-
Phe-O-Resin prepared above, the Val residue was incorporated
using 332 mg (0.978 mmol, 3 equiv) of Fmoc-Val-OH, 133 mg
(0.978 mmol, 3 equiv) of HOAt, and 0.303 mL of DIC (6 equiv).
Completion of the coupling reaction was verified by a negative nin-
hydrin test. The reaction mixture was then drained to leave the
amine-protected resin-bound tripeptide.
D-Phe-Phe-O-Resin
D
D
Rf: 0.5 (EtOAc/Hex 7:3).
1H NMR (500 MHz, CDCl3): d 0.5 (d, 2H), 0.6 (d, 2H), 0.9 (m, 4H),
1.2–1.4 (m, 1H), 1.4 (s, 9H), 1.5 (m, 1H), 1.6 (m, 1H), 1.7 (m, 1H), 2.9
(s, 3H), 3.2 (m, 2H), 3.3 (dd, 1H), 3.7 (s, 3H), 4.1 (s, 1
aH), 4.4 (m,
12.15.4. Tripeptide NH2-Val-N-Me-
D-Phe-Phe-O-Resin
2a
H), 4.8 (dd, 1 H), 5.1 (s, 3H), 5.6 (m, 1H), 6.5 (d, 1H), 6.7 (d,
a
Tripeptide NH2-Val-N-Me- -Phe-Phe-O-Resin was synthesized
D
1H), 7.0 (d, 1H), 7.1–7.4 (m, 15H).
LCMS: m/z calculated for C50H70N5O10 (M+1) = 915.6, found
915.6.
following the ‘General solid phase amine deprotection’ procedure.
A positive ninhydrin test served to verify Fmoc removal.
12.15.5. Tetrapeptide Fmoc-Leu-Val-N-Me-
Tetrapeptide Fmoc-Leu-Val-N-Me- -Phe-Phe-O-Resin was syn-
thesized following the Section 11.8 procedure. Using the NH2-
Val-N-Me- -Phe-Phe-O-Resin prepared above, the Leu residue
was incorporated using 346 mg (0.978 mmol, 3 equiv) of Fmoc-
Leu-OH, 150 mg (0.978 mmol, 3 equiv) of HOBt, and 0.303 mL of
DIC (6 equiv). 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 tetrapeptide.
D-Phe-Phe-O-Resin
12.14.8. Pentapeptide HO-Phe-
NHBoc
D-N-Me-Phe-Val-Leu-Lys(CBz)-
D
Pentapeptide HO-Phe-D-N-Me-Phe-Val-Leu-Lys(CBz)-NHBoc-
D
was synthesized using the ‘General acid deprotection’ procedure.
The pentapeptide was taken on to the next reaction without fur-
ther purification or characterization (346 mg, 87% yield).
12.14.9. Pentapeptide HO-Phe-D-N-Me-Phe-Val-Leu-Lys(CBz)-
NH
Pentapeptide HO-Phe-D-N-Me-Phe-Val-Leu-Lys(CBz)-NH was
12.15.6. Tetrapeptide NH2-Leu-Val-N-Me-
D-Phe-Phe-O-Resin
synthesized using the ‘General amine deprotection’ procedure.
The pentapeptide was taken on to the next reaction without fur-
ther purification (298 mg, 100% yield).
Tetrapeptide NH2-Leu-Val-N-Me- -Phe-Phe-O-Resin was syn-
D
thesized following the ‘General solid phase amine deprotection’
procedure. A positive ninhydrin test served to verify Fmoc removal.
LCMS: m/z calculated for C44H60N6O8 (M+1) = 802, found 801.5.

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6847
12.15.7. Pentapeptide Fmoc-Lys(Boc)-Leu-Val-N-Me-
D
-Phe-Phe-
lizing 439 mg (1.29 mmols, 1.1 equiv) of amine MeO-Phe-D-N-
O-Resin
Me-Phe-NH2, 254 mg (1.17 mmols, 1.0 equiv) of acid HO-Val-
NHBoc, 1.92 mL (8 equiv) of DIPEA, and 452 mg (1.41 mmols,
1.2 equiv) of TBTU. The crude reaction was purified by an acid-base
wash and column chromatography (silica gel, EtOAc/Hex) to yield
the desired tripeptide (667 mg, 96% yield).
Pentapeptide Fmoc-Lys(Boc)-Leu-Val-N-Me-
was synthesized following the Section 11.8 procedure. Using the
H-Leu-Val-N-Me- -Phe-Phe-O-Resin prepared above, the Lys(Boc)
residue was incorporated using 458 mg (0.978 mmol, 3 equiv)
of Fmoc-Lys(Boc)-OH, 150 mg (0.978 mmol, 3 equiv) of HOBt,
0.303 mL of DIC (6 equiv). 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.
D-Phe-Phe-O-Resin
D
Rf: 0.5 (EtOAc/Hex 3:2).
1H NMR (200 MHz, CDCl3): d 0.6 (d, 6H), 1.4 (s, 9H), 1.6–1.8
(m, 4H), 2.9 (m, 3H), 3.0–3.2 (dd, 1H), 3.3 (s, 1H), 3.7 (m, 3H), 4.2
(dd, 1aH), 4.7–4.9 (dd, 1aH), 5.0 (d, 1H), 5.5 (dd, 1H), 6.9 (d, 1H),
7.0–7.4 (m, 10H).
12.15.8. Pentapeptide NH2-Lys(Boc)-Leu-Val-N-Me-D-Phe-Phe-
O-Resin
12.16.4. Tripeptide MeO-Phe-D-N-Me-Phe-Val-NH2
Pentapeptide NH2-Lys(Boc)-Leu-Val-N-Me-
D
-Phe-Phe-O-Resin
Tripeptide MeO-Phe- -N-Me-Phe-Val-NH2 was synthesized
D
was synthesized following the Section 11.10 procedure. A positive
ninhydrin test served to verify Fmoc removal. The resin was then
dried in vacuo for 24 h in a vacuum desiccator.
using the ‘General amine deprotection’ procedure. The tripeptide
was taken on to the next reaction without further purification or
characterization (620 mg, 100% yield).
12.15.9. Double deprotected pentapeptide NH2-Lys(Boc)-Leu-
12.16.5. Dipeptide MeO-Leu-Lys(CBz)-NHBoc
Val-N-Me-
D
-Phe-Phe-OH
Dipeptide MeO-Leu-Lys(CBz)-NHBoc was constructed following
the procedure outlined ‘General peptide synthesis’. Utilizing
315 mg (1.74 mmols, 1.1 equiv) of amine MeO-Leu-NH2, 600 mg
(1.57 mmols, 1.0 equiv) of acid HO-Lys(CBz)-NHBoc, 1.10 mL
(4 equiv) of DIPEA, and 605 mg (1.88 mmols, 1.2 equiv)of TBTU.
The crude reaction was purified by an acid-base wash to yield
the dipeptide (715 mg, 90% yield).
Double deprotected pentapeptide NH2-Lys(Boc)-Leu-Val-N-Me-
D
-Phe-Phe-OH was synthesized following the Section 11.11 proce-
dure.Utilizing the 420 mg of dried NH2-Lys(Boc)-Leu-Val-N-Me-D-
Phe-Phe-O-Resin, 2.5 mL of 2,2,2-trifluoroethanol and 2.5 mL of
CH2Cl2. The resin slurry was stirred for 24 h, after which it was fil-
tered, washed with additional CH2Cl2, and dried in vacuo for 24 h
(203 mg, 92% yield).
Rf: 0.4 (EtOAc/Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.5–1.7
12.15.10. Macrocycle Lys(Boc)-Leu-Val-N-Me-
D
-Phe-Phe
(m, 6H), 1.8 (m, 1H), 3.1 (q, 2H), 3.7 (s, 3H), 4.0–4.2 (m, 1
aH),
Macrocycle Lys(Boc)-Leu-Val-N-Me- -Phe-Phe was synthesized
D
4.5–4.7 (m, 1
5H).
aH), 5.0 (m, 1H), 5.1 (s, 2H), 6.4 (d, 1H), 7.2–7.4 (m,
following the ‘Macrocyclization procedure’. Utilizing 203 mg
(0.30 mmols, 1.0 equiv) of linear pentapeptide, 0.36 mL (8 equiv)
of DIPEA, 66.3 mg (0.206 mmols, 0.8 equiv) of TBTU, 86.1 mg
(0.206 mmols, 0.8 equiv) HATU, and 46.4 mg (0.155 mmols,
0.6 equiv) of DEPBT. The crude reaction was purified by silica based
column to yield the macrocycle (17.5 mg, 7.8% yield).
12.16.6. Dipeptide HO-Leu-Lys(CBz)-NHBoc
Dipeptide HO-Leu-Lys(CBz)-NHBoc was synthesized using the
‘General acid deprotection’ procedure. The dipeptide was taken
on to the next reaction without further purification or character-
ization (650 mg, 86% yield).
Rf: 0.65 (EtOAc/Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.6 (d, 12H), 0.7–1.0 (m, 4H), 1.2
(s, 9H), 1.6 (m, 3H), 1.7 (m 2H), 1.9 (m, 1H), 2.8 (s, 1H), 2.9–3.0 (m,
12.16.7. Pentapeptide MeO-Phe-
D-N-Me-Phe-Val-Leu-Lys(CBz)-
6H), 3.8 (t, aH), 3.9 (t, aH), 4.5 (t, aH), 5.1 (t, aH) 7.0–7.3 (m, 10H).
NHBoc
LCMS: m/z calcd for C41H60N6O7 (M+1) = 749.0, found 750.8.
Pentapeptide MeO-Phe-
was constructed using the ‘General peptide synthesis’ procedure.
Utilizing 283 mg (0.65 mmols, 1.1 equiv) of MeO-Phe- -N-Me-
D-N-Me-Phe-Val-Leu-Lys(CBz)-NHBoc
12.16. Synthesis of compound 30
D
Phe-Val-NH2, 289 mg (0.59 mmols, 1.0 equiv) of HO-Leu-Lys(CBz)-
NHBoc, 178 mg (0.49 mmols, 0.8 equiv) HATU, 70.1 mg (0.23mmols,
0.4 equiv) DEPBT, and 0.82 mL (8 equiv) of DIPEA. The crude mate-
rial was purified using column chromatography (silica gel, EtOAc/
Hex) to yield 415 mg (78% yield) of the pentapeptide.
12.16.1. Dipeptide MeO-Phe-
D-N-Me-Phe-NBoc
Dipeptide MeO-Phe- -N-Me-Phe-NBoc was synthesized using
D
the ‘General peptide synthesis’ procedure. Utilizing 309 mg
(1.43 mmols, 1.1 equiv) of amine MeO-Phe-NH2, 600 mg (1.30
mmols, 1.0 equiv) of acid HO-N-Me-Phe-NBoc, 0.91 mL (4 equiv) of
DIPEA, and 502 mg (1.56 mmols, 1.2 equiv)of TBTU. The crude reac-
tion was purified by an acid-base wash to yield the dipeptide
(574 mg, 99% yield).
Rf: 0.5 (EtOAc/Hex 7:3).
1H NMR (500 MHz, CDCl3): d 0.5 (d, 2H), 0.6 (d, 2H), 0.9 (m, 4H),
1.2–1.4 (m, 1H), 1.4 (s, 9H), 1.5 (m, 1H), 1.6 (m, 1H), 1.7 (m, 1H), 2.9
(s, 3H), 3.2 (m, 2H), 3.3 (dd, 1H), 3.7 (s, 3H), 4.1 (s, 1
aH), 4.4 (m,
Rf: 0.5 (EtOAc/Hex 1:3).
2
a
H), 4.8 (dd, 1 H), 5.1 (s, 3H), 5.6 (m, 1H), 6.5 (d, 1H), 6.7 (d,
a
1H NMR (200 MHz, CD3OD): d 1.2–1.4 (s, 9H), 2.7 (s, 3H), 2.8
(m, 1H), 2.9 (m, 1H), 3.0–3.2 (m, 3H), 3.7 (s, 3H), 4.8–5.0 (m,
1H), 7.0 (d, 1H), 7.1–7.4 (m, 15H).
LCMS: m/z calculated for C50H70N5O10 (M+1) = 915.6, found 915.6.
2aH), 7.1–7.3 (m, 10H), 7.8 (d, 1H).
12.16.8. Pentapeptide HO-Phe-D-N-Me-Phe-Val-Leu-Lys(CBz)-
12.16.2. Dipeptide MeO-Phe-
D-N-Me-Phe-NH
NHBoc
Dipeptide MeO-Phe- -N-Me-Phe-NH was synthesized using the
D
Pentapeptide HO-Phe-D-N-Me-Phe-Val-Leu-Lys(CBz)-NHBoc-
‘General amine deprotection’ procedure. The dipeptide was taken
on to the next reaction without further purification or character-
ization (439 mg, 100% yield).
was synthesized using the ‘General acid deprotection’ procedure.
The pentapeptide was taken on to the next reaction without fur-
ther purification or characterization (346 mg, 87% yield).
12.16.3. Tripeptide MeO-Phe-
Tripeptide MeO-Phe- -N-Me-Phe-Val-NHBoc was constructed
following the procedure outlined ‘General peptide synthesis’. Uti-
D
-N-Me-Phe-Val-NHBoc
12.16.9. Pentapeptide HO-Phe-
Pentapeptide HO-Phe- -N-Me-Phe-Val-Leu-Lys(CBz)-NH was
synthesized using the ‘General amine deprotection’ procedure.
D-N-Me-Phe-Val-Leu-Lys(CBz)-NH2
D
D

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R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
The pentapeptide was taken on to the next reaction without fur-
ther purification (298 mg, 100% yield).
12.17.5. Tetrapeptide Fmoc-Leu-Val-Leu-Phe-O-Resin
Tetrapeptide Fmoc-Leu-Val-Leu-Phe-O-Resin was synthesized
following the Section 11.8 procedure. Using the NH2-Leu-Val-
Leu-Phe-O-Resin prepared above, the Leu residue was incorporated
using 340.0 mg (0.96 mmol, 3 equiv) of Fmoc-Leu-OH, 147.0 mg
(0.96 mmol, 3 equiv) of HOBt, and 0.3 mL of DIC (6 equiv). Comple-
tion of the coupling reaction was verified by a negative ninhydrin
test. The reaction mixture was then drained to leave the amine-
protected resin-bound tetrapeptide.
LCMS: m/z calculated for C44H60N6O8 (M+1) = 802, found 801.5.
12.16.10. Macrocycle Phe-
D-N-Me-Phe-Val-Leu-Lys(CBz)
Macrocycle Phe- -N-Me-Phe-Val-Leu-Lys(CBz) was constructed
D
using the ‘General macrocyclization procedure’. Utilizing 149 mg
(0.19 mmols, 1.0 equiv) of the double deprotected linear pentapep-
tide, 30.0 mg (0.09 mmols, 0.5 equiv) TBTU, 35.2 mg (0.09 mmols,
0.5 equiv) HATU, 28.4 mg (0.09 mmols, 0.5 equiv) DEPBT, and
0.27 mL (8 equiv) of DIPEA. The crude material was purified using
column chromatography (silica gel, EtOAc/Hex) to yield 19.2 mg
(17% yield) of the macrocycle.
12.17.6. Tetrapeptide NH2-Leu-Val-Leu-Phe-O-Resin
Tetrapeptide NH2-Leu-Val-Leu-Phe-O-Resin was synthesized
following the ‘General solid phase amine deprotection’ procedure.
A positive ninhydrin test served to verify Fmoc removal.
Rf: 0.5 (EtOAc/Hex 4:1).
1H NMR (400 MHz, CD3OD): d 0.5 (d, 1H), 0.6 (d, 4H), 0.7 (m,
4H), 0.8–1.0 (m, 6H), 1.2–1.4 (m, 6H), 1.5 (m, 2H), 1.7 (m, 2H),
12.17.7. Pentapeptide Fmoc-Lys(Boc)-Leu-Val-Leu-Phe-O-Resin
Pentapeptide Fmoc-Lys(Boc)-Leu-Val-Leu-Phe-O-Resin was syn-
thesized following the Section 11.8 procedure. Using the NH2-Leu-
Val-Leu-Phe-O-Resin prepared above, the Lys(Boc) residue was
incorporated using 450.0 mg (0.96 mmol, 3 equiv) of Fmoc-Lys(-
Boc)-OH, 147.0 mg (0.96 mmol, 3 equiv) of HOBt, 0.3 mL of DIC
(6 equiv). Completion of the coupling reaction was verified by a neg-
ative ninhydrin test. The reaction mixture was then drained to leave
the amine-protected resin-bound pentapeptide.
1.8 (m, 2H), 2.9 (s, 3H), 3.0 (m, 2H), 3.1 (m, 2H), 3.9 (t, 1
(t, 1 H), 4.4 (t, 1 H), 4.6 (t, 1 H), 5.1 (s, 2H), 5.3 (s, 1H), 7.1–7.4
(m, 15H).
LCMS: m/z calculated for C44H58N6O7 (M+1) = 784.1, found
784.2.
aH), 4.1
a
a
a
12.16.11. Macrocycle Phe-
D-N-Me-Phe-Val-Leu-Lys
Macrocycle Phe- -N-Me-Phe-Val-Leu-Lys was synthesized
D
using ‘General carboxybenzyl removal procedure using HBr’. The
crude product was purified by reverse phase HPLC (15.2 mg, 33%
yield).
12.17.8. Pentapeptide NH2-Lys(Boc)-Leu-Val-Leu-Phe-O-Resin
Pentapeptide NH2-Lys(Boc)-Leu-Val-Leu-Phe-O-Resin was syn-
thesized following the Section 11.10 procedure. A positive ninhy-
drin test served to verify Fmoc removal. The resin was then dried
in vacuo for 24 h in a vacuum desiccator.
Rf: 0.3 (MeOH/Hex 4:1).
1H NMR (500 MHz, CD3OD): d 0.7 (d, 2H), 0.8–1.0 (m, 12H), 1.2
(m, 2H), 1.3 (m, 4H), 1.5 (m, 2H), 1.6 (m, 2H), 1.7 (m, 2H), 1.8 (m,
2H), 1.9 (m, 2H), 2.7 (m, 1H), 2.8–3.1 (m, 2H), 3.7 (m, 1H), 3.9
(m, 1
7.1–7.4 (m, 10H).
a
H), 4.1 (m, 1
aH), 4.4 (m, 1
a
H), 4.6 (m, 1
a
H), 5.2 (m, 1H),
12.17.9. Double deprotected pentapeptide NH2-Lys(Boc)-Leu-
Val-Leu-Phe-OH
LCMS: m/z calculated for C36H52N6O5 (M+1) = 649.8, found 649.5.
Double deprotected pentapeptide NH2-Lys(Boc)-Leu-Val-Leu-
Phe-OH was synthesized following the Section 11.11 procedure. Uti-
lizing the 691.3 mg of dried NH2-Lys(Boc)-Leu-Val-Leu-Phe-O-Re-
sin, 3.5 mL of 2,2,2-trifluoroethanol and 3.5 mL of CH2Cl2. The resin
slurry was stirred for 24 h, after which it was filtered, washed with
additional CH2Cl2, and dried in vacuo for 24 h (186 mg, 66% yield).
12.17. Synthesis of compounds 31 and 32
12.17.1. Dipeptide Fmoc-Leu(Boc)-Phe-O-Resin
Dipeptide Fmoc-Leu(Boc)-Phe-O-Resin was synthesized follow-
ing the Section 11.8 procedure. Using 1005.0 mg (0.40 mmol,
1 equiv) of NH2-Phe-O-Resin, the Leu residue was incorporated
using 679 mg of Fmoc-Leu-OH (1.92 mmol, 3 equiv), 294.0 mg
(1.92 mmol, 3 equiv) of HOBt, and 0.60 mL (6 equiv) of DIC. Com-
pletion of the coupling reaction was verified by a negative ninhy-
drin test. The reaction mixture was then drained to leave the
amine-protected resin-bound dipeptide.
12.17.10. Macrocycle Lys(Boc)-Leu-Val-Leu-Phe
Macrocycle Lys(Boc)-Leu-Val-Leu-Phe was synthesized follow-
ing the ‘Macrocyclization procedure’. Utilizing 40 mg (0.056 mmols,
1.0 equiv) of linear pentapeptide, 0.06 mL (6 equiv) of DIPEA,
12.6 mg (0.04 mmols, 0.7 equiv) of TBTU, 14.9 mg (0.04 mmols,
0.7 equiv) HATU, and 11.7 mg (0.04 mmols, 0.7 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (13 mg, 40% yield).
12.17.2. Dipeptide NH2-Leu-Phe-O-Resin
Dipeptide NH2-Leu-Phe-O-Resin was synthesized following the
‘General solid phase amine deprotection’ procedure. A positive nin-
hydrin test served to verify Fmoc removal.
Rf: 0.8 (100% EtOAc).
1H NMR (400 MHz, CD3OD): d0.8–1.2 (m, 18H), 1.4 (s, 9H), 1.6 (m,
2H), 1.8 (m, 2H), 2.2 (m, 1H), 3.1–3.2 (m, 2H), 3.6 (m, 2H), 3.8 (m,
aH),
4.1 (m, H), 4.4 (m, H), 4.5 (m, H), 4,7 (m, H), 7.1–7.3 (m, 5H).
a
a
a
a
12.17.3. Tripeptide Fmoc-Val-Leu-Phe-O-Resin
LCMS: m/z calcd for C37H60N6O7 (M+Na) = 724.3, found 724.3.
Tripeptide Fmoc-Val-Leu-Phe-O-Resin was synthesized follow-
ing the Section 11.8 procedure. Using the NH2-Leu-Phe-O-Resin pre-
pared above, the Phe residue was incorporated using 640.0 mg
(1.92 mmol, 3 equiv) of Fmoc-Val-OH, 294 mg (1.92 mmol, 3 equiv)
of HOBt, and 0.60 mL of DIC (6 equiv). Completion of the coupling
reaction was verified by a negative ninhydrin test. The reaction mix-
ture was then drained to leave the amine-protected resin-bound
tripeptide.
12.17.11. Macrocycle Lys-Leu-Val-Leu-Phe
Macrocycle Lys-Leu-Val-Leu-Phe was synthesized following the
‘General amine deprotection’. Utilizing 11.0 mg (0.015 mmols,
1.0 equiv) of Macrocycle D-Val-Leu-Phe-Lys(Boc)-Leu, 1.20 mL of
methylene chloride, 0.3 mL of TFA (20% TFA, 0.1 M) to remove
the Boc protecting group on the Lysine. The crude reaction was ta-
ken on to the next reaction without further purification or charac-
terization (68.7 mg, quantitative yield).
12.17.4. Tripeptide NH2-Val-Leu-Phe-O-Resin
1H NMR (400 MHz, CD3OD): d 0.8–1.2 (m, 18H), 1.3 (m, 2H), 1.6
Tripeptide NH2-Val-Leu-Phe-O-Resin was synthesized following
the ‘General solid phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal.
(m, 2H), 1.8 (m, 2H), 2.2 (m, 1H), 2.9–3.3 (m, 4H), 3.9–4.1 (m,
4.1 (m, H), 4.4 (m, 2 H), 4.5 (m, H), 4,7 (m, H), 7.1–7.3 (m, 5H).
LCMS: m/z calcd for C32H52N6O5 (M+1) = 601.79, found 601.7.
aH),
a
a
a
a

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6849
12.18. Synthesis of compounds 34 and 35
12.18.9. Double deprotected pentapeptide NH2-Val-Leu-Phe-N-
Me-Lys(Boc)-Leu-OH
12.18.1. Dipeptide Fmoc-N-Me-Lys(Boc)-Leu-O-Resin
Dipeptide Fmoc-N-Me-Lys(Boc)-Leu-O-Resin was synthesized
following the Section 11.8 procedure. Using 1233.0 mg (0.99 mmol,
1 equiv) of H-Leu-O-Resin, the N-Me-Lys residue was incorporated
using 935 mg of Fmoc-N-Me-Lys(Boc)-OH, 458 mg of HOBt,
0.756 mL of DIC, and 5 mL of DMF. Completion of the coupling reac-
tion was verified by a negative ninhydrin test. The reaction mixture
was then drained to leave the Fmoc-protected resin-bound
dipeptide.
Double deprotected pentapeptide NH2-Val-Leu-Phe-N-Me-Lys(-
Boc)-Leu-OH was synthesized following the Section 11.11 proce-
dure.Utilizing the 1708.3 mg of dried NH2-Val-Leu-Phe-N-Me-
Lys(Boc)-Leu-O-Resin, 10 mL of 2,2,2-trifluoroethanol and 10 mL
of CH2Cl2. The resin slurry was stirred for 24 h, after which it
was filtered, washed with additional CH2Cl2, and dried in vacuo
for 24 h (565 mg, 77% yield).
12.18.10. Macrocycle Val-Leu-Phe-N-Me-Lys(Boc)-Leu
Macrocycle Leu-Phe-N-Me-Lys(Boc)-Leu was synthesized
following the ‘Macrocyclization procedure’. Utilizing 188 mg
(0.25 mmols, 1.0 equiv) of linear pentapeptide, 0.34 mL (8 equiv)
of DIPEA, 57 mg (0.14 mmols, 0.6 equiv) of TBTU, 58 mg
(0.18 mmols, 0.6 equiv) HATU, and 46 mg (0.15 mmols, 0.6 equiv)
of DEPBT. The crude reaction of cyclization was purified by Column
Chromatography to yield the macrocycle Leu-Phe-N-Me-Lys(Boc)-
Leu (65 mg, 36% yield).
12.18.2. Dipeptide NH-N-Me-Lys(Boc)-Leu-O-Resin
Dipeptide NH-N-Me-Lys(Boc)-Leu-O-Resin was synthesized fol-
lowing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc deprotection.
12.18.3. Tripeptide Fmoc-Phe-N-Me-Lys(Boc)-Leu-O-Resin
Tripeptide Fmoc-Phe-N-Me-Lys(Boc)-Leu-O-Resin was synthe-
sized following the Section 11.8 procedure. Using the H-N-Me-
Lys(Boc)-Leu-O-Resin prepared above, the Phe residue was
incorporated using 1150 mg (2.9 mmol, 3 equiv) of Fmoc-Phe-OH,
407 mg (2.9 mmol, 3 equiv) of HOAt, 0.756 mL of DIC, and 5 mL
of DMF. Completion of the coupling reaction was verified by a
negative ninhydrin test. The reaction mixture was then drained
to leave the Fmoc-protected resin-bound tripeptide.
Rf: 0.2 (75% EtOAc/25% hexane).
1H NMR (400 MHz, CD3OD: d 0.8–1 (m, 18H), 1.1–1.2 (s, 9H),
1.3–1.4 (m, 2H), 1.4–1.5 (m, 2H), 1.6–1.7 (m, 2H), 1.8–2.0 (m,
2H), 2.0–2.1 (m, 2H), 2.1–2.2 (m, 2H), 2.8 (m, 1H), 2.9–3.0 (m,
1H), 3.0–3.1 (m, 1H), 3.1–3.2 (m, 1H), 3.2–3.4 (m, 1H), 3.2 (s, 3H)
3.9 (m, 1aH), 4.0–4.1 (m, 1aH), 4.2–4.3 (m, 1aH), 4.6 (m, 1aH),
5.0 (m, 1
a
H), 7.1–7.3 (m, 5H), 7.7 (m, 1H), 8.0 (m, 1H), 8.1
(m1H), 8.3 (m, 1H).
12.18.4. Tripeptide NH2-Phe-N-Me-Lys(Boc)-Leu-O-Resin
Tripeptide NH2-Phe-N-Me-Lys(Boc)-Leu-O-Resin was synthe-
sized following the ‘General solid phase amine deprotection’ proce-
dure. A positive ninhydrin test served to verify Fmoc deprotection.
LCMS: m/z calcd C38H62N6O7 = 714.93, found 716.1.
12.18.11. Macrocycle Val-Leu-Phe-N-Me-Lys-Leu
Macrocycle Val-Leu-Phe-N-Me-Lys-Leu was synthesized follow-
ing the ‘General amine deprotection’. The crude reaction of Boc re-
moval was purified by reverse phase HPLC to yield the macrocycle
Leu-Phe-N-Me-Lys-Leu (1.3 mg, 27% yield).
12.18.5. Tetrapeptide Fmoc-Leu-Phe-N-Me-Lys(Boc)-Leu-O-
Resin
Tetrapeptide Fmoc-Leu-Phe-N-Me-Lys(Boc)-Leu-O-Resin was
synthesized following the Section 11.8 procedure. Using the H-
Phe-N-Me-Lys(Boc)-Leu-O-Resin prepared above, the Leu residue
was incorporated using 1056 mg (2.9 mmol, 3 equiv) of Fmoc-
Leu-OH, 458 mg (2.9 mmol, 3 equiv) of HOBt, 0.756 mL of DIC,
and 5 mL of DMF. Completion of the coupling reaction was verified
by a negative ninhydrin test. The reaction mixture was then
drained to leave the Fmoc-protected resin-bound tetrapeptide.
1H NMR (400 MHz, CD3OD: d 0.8–1 (m, 18H), 1.2–1.4 (m, 4H),
1.5–1.7 (m, 2H), 1.8–2.0 (m, 2H), 2.1–2.2 (m, 2H), 2.1–2.2 (m,
2H), 2.6 (m, 1H), 2.7–2.8 (m, 1H), 2.8–2.9 (m, 1H), 3.0–3.1 (m,
1H), 3.4–3.5 (m, 1H), 3.6 (s, 3H) 3.9 (m, 1
aH), 4.2–4.4 (m, 1aH),
4.4–4.6 (m, 1 H), 5.0 (m, 1 H), 5.3 (m, 1 H), 7.2–7.4 (m, 5H), 7.8
a
a
a
(m, 1H), 8.0 (m, 1H), 8.4 (m1H), 8.6 (m, 1H).
LCMS: m/z calcd C33H54N6O5 = 614.82, found 615.7.
12.19. Synthesis of compounds 36 and 37
12.18.6. Tetrapeptide NH2-Leu-Phe-N-Me-Lys(Boc)-Leu-O-Resin
Tetrapeptide NH2-Leu-Phe-N-Me-Lys(Boc)-Leu-O-Resin was
synthesized following the ‘General solid phase amine deprotection’
12.19.1. Dipeptide Fmoc-Lys(Boc)-Leu-O-Resin
Dipeptide Fmoc-Lys(Boc)-Leu-O-Resin was synthesized follow-
ing the Section 11.8 procedure. Using 1003.0 mg (0.81 mmol,
1 equiv) of NH2-Leu-O-Resin, the Lys residue was incorporated
using 1138.6 mg of Fmoc-Lys(Boc)-OH (2.43 mmol, 3 equiv),
371.8 mg (2.43 mmol, 3 equiv) of HOBt, and 0.756 mL (6 equiv) 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.
procedure.
A positive ninhydrin test served to verify Fmoc
deprotection.
12.18.7. Pentapeptide Fmoc-Val-Leu-Phe-N-Me-Lys(Boc)-Leu-O-
Resin
Pentapeptide Fmoc-Val-Leu-Phe-N-Me-Lys(Boc)-Leu-O-Resin
was synthesized following the Section 11.8 procedure. Using the
H-Leu-Phe-N-Me-Lys(Boc)-Leu-O-Resin prepared above, the Val
residue was incorporated using 1010 mg (2.9 mmol, 3 equiv) of
Fmoc-Val-OH, 458 mg (2.9 mmol, 3 equiv) of HOBt, 0.756 mL of
DIC, and 5 mL of DMF. Completion of the coupling reaction was ver-
ified by a negative ninhydrin test. The reaction mixture was then
drained to leave the Fmoc-protected resin-bound pentapeptide.
12.19.2. Dipeptide NH2-Lys(Boc)-Leu-O-Resin
Dipeptide NH2-Lys(Boc)-Leu-O-Resin was synthesized follow-
ing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal.
12.19.3. Tripeptide Fmoc-Phe-Lys(Boc)-Leu-O-Resin
12.18.8. Pentapeptide H-Val-Leu-Phe-N-Me-Lys(Boc)-Leu-O-
Resin
Pentapeptide H-Val-Leu-Phe-N-Me-Lys(Boc)-Leu-O-Resin was
synthesized following the Section 11.10 procedure. A positive nin-
hydrin test served to verify Fmoc deprotection. The resin was then
dried in vacuo for 24 h in a vacuum desiccator.
Tripeptide Fmoc-Phe-Lys(Boc)-Leu-O-Resin was synthesized
following the Section 11.8 procedure. Using the NH2-Lys(Boc)-
Leu-O-Resin prepared above, the Phe residue was incorporated
using 941.48 mg (2.43 mmol, 3 equiv) of Fmoc-Phe-OH, 371.8 mg
(2.43 mmol, 3 equiv) of HOBt, and 0.756 mL of DIC (6 equiv).
Completion of the coupling reaction was verified by a negative

6850
R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
ninhydrin test. The reaction mixture was then drained to leave the
amine-protected resin-bound tripeptide.
12.19.11. Macrocycle
Macrocycle -Val-Leu-Phe-Lys-Leu was synthesized following
the ‘General amine deprotection’. Utilizing 80.2 mg (0.11 mmols,
1.0 equiv) of Macrocycle -Val-Leu-Phe-Lys(Boc)-Leu, 1.83 mL of
D-Val-Leu-Phe-Lys-Leu
D
D
12.19.4. Tripeptide NH2-Phe-Lys(Boc)-Leu-O-Resin
methylene chloride, 0.45 mL of TFA (20% TFA, 0.05 M) to remove
the BOC protecting group on the Lysine. The crude reaction was ta-
ken on to the next reaction without further purification or charac-
terization (68.7 mg, quantitative yield).
Tripeptide NH2-Phe-Lys(Boc)-Leu-O-Resin was synthesized fol-
lowing the ‘General solid phase amine deprotection’ procedure. A
positive ninhydrin test served to verify Fmoc removal.
1H NMR (400 MHz, CD3OD): d 0.8–1.1 (m, 20H), 1.2 (m, 2H), 1.4
(m, 2H), 1.5–1.7 (m, 5H), 1.8 (m, 1H), 2.0 (m, 2H), 2.8 (m, 3H), 3.1
12.19.5. Tetrapeptide Fmoc-Leu-Phe-Lys(Boc)-Leu-O-Resin
Tetrapeptide Fmoc-Leu-Phe-Lys(Boc)-Leu-O-Resin was synthe-
sized following the Section 11.8 procedure. Using the NH2-
Phe-Lys(Boc)-Leu-O-Resin prepared above, the Leu residue was
incorporated using 858.8 mg (2.43 mmol, 3 equiv) of Fmoc-Leu-
OH, 371.8 mg (2.43 mmol, 3 equiv) of HOBt, and 0.756 mL of DIC
(6 equiv). 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 tetrapeptide.
(m, 1H), 3.2 (m, 1H), 3.7 (m,
aH), 3.8 (m, aH), 4.2 (m, aH), 4.5 (m,
a
H), 4.7 (m, H), 7.1–7.3 (m, 5H), 7.6 (d, 1H), 8.0 (d, 1H), 8.6 (d,
a
1H), 8.7 (d, 1H).
LCMS: m/z calcd for C32H52N6O5 (M+1) = 601.79, found 601.7.
12.20. Synthesis of compound 39
12.20.1. Dipeptide Fmoc-N-Me-Val-Leu-O-Resin
Dipeptide Fmoc-N-Me-Val-Leu-O-Resin was synthesized fol-
lowing the Section 11.8 procedure. Using 1610.3 mg (1.3 mmol,
1 equiv) of H-Leu-O-Resin, the N-Me-Val residue was incorporated
using 1378 mg of Fmoc-N-Me-Val-OH (3.9 mmol, 3 equiv), 597 mg
(3.9 mmol, 3 equiv) of HOBt, and 1.21 mL (6 equiv) of DIC. Comple-
tion 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.
12.19.6. Tetrapeptide NH2-Leu-Phe-Lys(Boc)-Leu-O-Resin
Tetrapeptide NH2-Leu-Phe-Lys(Boc)-Leu-O-Resin was synthe-
sized following the ‘General solid phase amine deprotection’ proce-
dure. A positive ninhydrin test served to verify Fmoc removal.
12.19.7. Pentapeptide Fmoc-D-Val-Leu-Phe-Lys(Boc)-Leu-O-
Resin
Pentapeptide Fmoc-D-Val-Leu-Phe-Lys(Boc)-Leu-O-Resin was
synthesized following the Section 11.8 procedure. Using the NH2-
Leu-Phe-Lys(Boc)-Leu-O-Resin prepared above, the Val residue
was incorporated using 824.5 mg (2.43 mmol, 3 equiv) of Fmoc-
12.20.2. Dipeptide H-N-Me-Val-Leu-O-Resin
Dipeptide H-N-Me-Val-Leu-O-Resin was synthesized following
the ‘General solid phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal.
D-Val-OH, 371.8 mg (2.43 mmol, 3 equiv) of HOBt, 0.756 mL of
DIC (6 equiv). 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.
12.20.3. Tripeptide Fmoc-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin
Tripeptide Fmoc-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin was syn-
thesized following the Section 11.8 procedure. Using the H-N-
Me-Val-Leu-O-Resin prepared above, the Lys(2-Cl-Z) residue was
incorporated using 1050 mg (1.95 mmol, 3 equiv) of Fmoc-Lys(2-
Cl-Z)-OH, 265 mg (1.95 mmol, 3 equiv) of HOAt, and 0.604 mL of
DIC (6 equiv). 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 tripeptide.
12.19.8. Pentapeptide NH2-D-Val-Leu-Phe-Lys(Boc)-Leu-O-Resin
Pentapeptide NH2- -Val-Leu-Phe-Lys(Boc)-Leu-O-Resin was
D
synthesized following the Section 11.10 procedure. A positive nin-
hydrin test served to verify Fmoc removal. The resin was then dried
in vacuo for 24 h in a vacuum desiccator.
12.19.9. Double deprotected pentapeptide NH2-D-Val-Leu-Phe-
Lys(Boc)-Leu-OH
12.20.4. Tripeptide NH2-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin
Tripeptide NH2-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin was synthe-
sized following the ‘General solid phase amine deprotection’ proce-
dure. A positive ninhydrin test served to verify Fmoc removal.
Double deprotected pentapeptide NH2-
Leu-OH was synthesized following the Section 11.11 procedure.
Utilizing the 1391.3 mg of dried NH2- -Val-Leu-Phe-Lys(Boc)-
Leu-O-Resin, 7 mL of 2,2,2-trifluoroethanol and 7 mL of CH2Cl2.
The resin slurry was stirred for 24 h, after which it was filtered,
washed with additional CH2Cl2, and dried in vacuo for 24 h
(386 mg, 66% yield).
D-Val-Leu-Phe-Lys(Boc)-
D
12.20.5. Tetrapeptide Fmoc-
Resin
D-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-
Tetrapeptide Fmoc-
synthesized following the Section 11.8 procedure. Using the
NH2-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin prepared above, the -Phe
residue was incorporated using 755 mg (1.95 mmol, 3 equiv) of
Fmoc- -Phe-OH, 298 mg (1.95 mmol, 3 equiv) of HOBt, and
0.604 mL of DIC (6 equiv). 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 tetrapeptide.
D-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin was
12.19.10. Macrocycle
D-Val-Leu-Phe-Lys(Boc)-Leu
D
Macrocycle -Val-Leu-Phe-Lys(Boc)-Leu was synthesized follow-
D
ing the ‘Macrocyclization procedure’. Utilizing 232 mg (0.32 mmols,
1.0 equiv) of linear pentapeptide, 0.33 mL (6 equiv) of DIPEA,
72.5 mg (0.22 mmols, 0.7 equiv) of TBTU, 85.8 mg (0.22 mmols,
0.7 equiv) HATU, and 67.5 mg (0.22 mmols, 0.7 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (100.8 mg, 44.5% yield).
D
Rf: 0.8 (100% EtOAc).
12.20.6. Tetrapeptide NH2-
Resin
D
-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-
1H NMR (400 MHz, CD3OD): d 0.8–1.2 (m, 20H), 1.3–1.7 (m,
18H), 1.8 (m, 1H), 2.0 (m, 2H), 2.9 (m, 3H), 3.1 (m, 1H), 3.6 (m,
Tetrapeptide
NH2-D-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin
a
H), 3.8 (m,
(m, 5H).
LCMS: m/z calcd for C37H60N6O7 (M+1) = 701.91, found 702.1.
a
H), 4.2 (m,
a
H), 4.5 (m,
a
H), 4.6 (m,
a
H), 7.1–7.3
was synthesized following the ‘General solid phase amine depro-
tection’ procedure. A positive ninhydrin test served to verify Fmoc
removal.

R. P. Sellers et al. / Bioorg. Med. Chem. 18 (2010) 6822–6856
6851
12.20.7. Pentapeptide Fmoc-Phe-
Leu-O-Resin
D
-Phe-Lys(2-Cl-Z)-N-Me-Val-
(1.95 mmol, 3 equiv) of HOAt, and 0.604 mL of DIC (6 equiv). Com-
pletion of the coupling reaction was verified by a negative ninhydrin
test. The reaction mixture was then drained to leave the amine-pro-
tected resin-bound tripeptide.
Pentapeptide
Resin was synthesized following the Section 11.8 procedure. Using
the NH2- -Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin prepared above,
Fmoc-Phe-D-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-
D
the Phe residue was incorporated using 755 mg (1.95 mmol,
3 equiv) of Fmoc-Phe-OH, 298 mg (1.95 mmol, 3 equiv) of HOBt,
0.604 mL of DIC (6 equiv). 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.
12.21.4. Tripeptide NH2-
D-Lys(Z)-N-Me-Val-Leu-O-Resin
Tripeptide NH2- -Lys(Z)-N-Me-Val-Leu-O-Resin was synthe-
D
sized following the ‘General solid phase amine deprotection’ proce-
dure. A positive ninhydrin test served to verify Fmoc removal.
12.21.5. Tetrapeptide Fmoc-
D-Phe-D-Lys(Z)-N-Me-Val-Leu-
O-Resin
12.20.8. Pentapeptide NH2-Phe-
Leu-O-Resin
D
-Phe-Lys(2-Cl-Z)-N-Me-Val-
Tetrapeptide Fmoc-
synthesized following the Section 11.8 procedure. Using the NH2-
-Lys(Z)-N-Me-Val-Leu-O-Resin prepared above, the -Phe residue
was incorporated using 755 mg (1.95 mmol, 3 equiv) of Fmoc-
-Phe-OH, 298 mg (1.95 mmol, 3 equiv) of HOBt, and 0.604 mL of
DIC (6 equiv). 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 tetrapeptide.
D-Phe-D-Lys(Z)-N-Me-Val-Leu-O-Resin was
Pentapeptide NH2-Phe-
D
-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Re-
D
D
sin was synthesized following the Section 11.10 procedure. A posi-
tive ninhydrin test served to verify Fmoc removal. The resin was
then dried in vacuo for 24 h in a vacuum desiccator.
D
12.20.9. Double deprotected pentapeptide NH2-Phe-D-Phe-
Lys(2-Cl-Z)-N-Me-Val-Leu-OH
Double deprotected pentapeptide NH2-Phe-
N-Me-Val-Leu-OH was synthesized following the Section 11.11
procedure. Utilizing the 2078.6 mg of dried NH2-Phe- -Phe-
D
-Phe-Lys(2-Cl-Z)-
12.21.6. Tetrapeptide NH2-
Resin
D
-Phe-
D-Lys(Z)-N-Me-Val-Leu-O-
D
Tetrapeptide NH2-D-Phe-D-Lys(Z)-N-Me-Val-Leu-O-Resin was
Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin, 10.39 mL of 2,2,2-trifluoroeth-
anol and 10.39 mL of CH2Cl2. The resin slurry was stirred for
24 h, after which it was filtered, washed with additional CH2Cl2,
and dried in vacuo for 24 h (323 mg, 59.5% yield).
synthesized following the ‘General solid phase amine deprotection’
procedure. A positive ninhydrin test served to verify Fmoc removal.
12.21.7. Pentapeptide Fmoc-Phe-D-Phe-D-Lys(Z)-N-Me-Val-Leu-
O-Resin
12.20.10. Macrocycle Phe-
D
-Phe-Lys(2-Cl-Z)-N-Me-Val-Leu
Pentapeptide Fmoc-Phe-
was synthesized following the Section 11.8 procedure. Using the
NH2- -Phe-Lys(2-Cl-Z)-N-Me-Val-Leu-O-Resin prepared above,
the Phe residue was incorporated using 755 mg (1.95 mmol,
3 equiv) of Fmoc-Phe-OH, 298 mg (1.95 mmol, 3 equiv) of HOBt,
0.604 mL of DIC (6 equiv). 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.
D-Phe-D-Lys(Z)-N-Me-Val-Leu-O-Resin
Macrocycle Phe- -Phe-Lys(2-Cl-Z)-N-Me-Val-Leu was synthe-
D
sized following the ‘Macrocyclization procedure’. Utilizing
160 mg (0.19 mmols, 1.0 equiv) of linear pentapeptide, 0.335 mL
(10 equiv) of DIPEA, 58.3 mg (0.153 mmols, 0.8 equiv) of TBTU,
49.3 mg (0.153 mmols, 0.8 equiv) HATU, and 46 mg (0.153 mmols,
0.8 equiv) of DEPBT. The crude reaction was purified by column
purification to yield the macrocycle (15.4 mg, 9.79% yield).
Rf: 0.25 (EtOAc/Hex 3:1).
D
1H NMR (400 MHz, CD3OD): d0.7–1.0 (m, 12H), 1.3–1.4 (m, 2H),
1.5–1.6 (m, 4H), 1.7–1.8 (m, 2H), 2.7 (m, 1H), 2.9 (m, 2H), 3.1 (t,
12.21.8. Pentapeptide NH2-Phe-
D
-Phe-D-Lys(Z)-N-Me-Val-Leu-
4H), 3.2 (s, 1H), 4.2 (t,
aH), 4.4 (t, aH), 4.7 (t, aH), 5.1 (s, 1H),7.0–
O-Resin
7.2 (m, 13H), 7.4 (d, 1H).
Pentapeptide NH2-Phe-D-Phe-D-Lys(Z)-N-Me-Val-Leu-O-Resin
LCMS: m/z calcd for C44H57ClN6O7 (M+1) = 817, found 818.5.
was synthesized following the Section 11.10 procedure. A positive
ninhydrin test served to verify Fmoc removal. The resin was then
dried in vacuo for 24 h in a vacuum desiccator.
12.21. Synthesis of compound 40
12.21.1. Dipeptide Fmoc-N-Me-Val-Leu-O-Resin
12.21.9. Double deprotected pentapeptide NH2-Phe-D-Phe-D-
Dipeptide Fmoc-N-Me-Val-Leu-O-Resin was synthesized fol-
lowing the Section 11.8 procedure. Using 1610.3 mg (1.3 mmol,
1 equiv) of H-Leu-O-Resin, the N-Me-Val residue was incorporated
using 1378 mg of Fmoc-N-Me-Val-OH (3.9 mmol, 3 equiv), 597 mg
(3.9 mmol, 3 equiv) of HOBt, and 1.21 mL (6 equiv) of DIC. Comple-
tion 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.
Lys(Z)-N-Me-Val-Leu-OH
Double deprotected pentapeptide NH2-Phe-
Me-Val-Leu-OH was synthesized following the Section 11.11 pro-
cedure. Utilizing the 2094 mg of dried NH2-Phe- -Phe- -Lys(Z)-
N-Me-Val-Leu-O-Resin, 10.47 mL of 2,2,2-trifluoroethanol and
10.47 mL of CH2Cl2. The resin slurry was stirred for 24 h, after
which it was filtered, washed with additional CH2Cl2, and dried
in vacuo for 24 h (301 mg, 57.9% yield).
D-Phe-D-Lys(Z)-N-
D
D
12.21.2. Dipeptide H-N-Me-Val-Leu-O-Resin
12.21.10. Macrocycle Phe- -Phe-
D
D-Lys(Z)-N-Me-Val-Leu
Dipeptide H-N-Me-Val-Leu-O-Resin was synthesized following
the ‘General solid phase amine deprotection’ procedure. A positive
ninhydrin test served to verify Fmoc removal.
Macrocycle Phe- -Phe-D-Lys(Z)-N-Me-Val-Leu was synthesized
D
following the ‘Macrocyclization procedure’. Utilizing 150 mg
(0.187 mmols, 1.0 equiv) of linear pentapeptide, 0.294 mL (9 equiv)
of DIPEA, 42.1 mg (0.131 mmols, 0.7 equiv) of HATU, 49.9 mg
(0.131 mmols, 0.7 equiv) TBTU, and 39.3 mg (0.131 mmols,
0.7 equiv) of DEPBT. The crude reaction was purified by reverse
phase-HPLC purification to yield the macrocycle (8.1 mg, 5.52%
yield).
12.21.3. Tripeptide Fmoc-
Tripeptide Fmoc- -Lys(Z)-N-Me-Val-Leu-O-Resin was synthe-
sized following the Section 11.8 procedure. Using the H-N-Me-Val-
Leu-O-Resin prepared above, the -Lys(Z) residue was incorporated
using 980 mg (1.95 mmol, 3 equiv) of Fmoc- -Lys(Z)-OH, 265 mg
D-Lys(Z)-N-Me-Val-Leu-O-Resin
D
D
D
Rf: 0.3 (EtOAc/Hex 1:1).