A comprehensive study of Sansalvamide A derivatives: The structure-activity relationships of 78 derivatives in two pancreatic cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2009.07.017

We report an extensive structure-activity relationship (SAR) of 78 compounds active against two pancreatic cancer cell lines. Our comprehensive evaluation of these compounds utilizes SAR that allow us to evaluate which features of potent compounds play a

Full text of DOI:10.1016/j.bmc.2009.07.017

Bioorganic & Medicinal Chemistry 17 (2009) 5806–5825
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A comprehensive study of Sansalvamide A derivatives: The structure–activity
relationships of 78 derivatives in two pancreatic cancer cell lines
Po-Shen Pan, Robert C. Vasko, Stephanie A. Lapera, Victoria A. Johnson, Robert P. Sellers, Chun-Chieh Lin,
*
Chung-Mao Pan, Melinda R. Davis, Veronica C. Ardi, 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:
We report an extensive structure–activity relationship (SAR) of 78 compounds active against two pancre-
atic cancer cell lines. Our comprehensive evaluation of these compounds utilizes SAR that allow us to
evaluate which features of potent compounds play a key role in their cytotoxicity. This is the first report
of 19 new second-generation structures, where these new compounds were designed from the first gen-
eration of 59 compounds. These 78 structures were tested for their cytotoxicity and this is the first report
of their activity against two pancreatic cancer cell lines. Our results show that out of 78 compounds, three
compounds are worth pursuing as leads, as they show potency of P55% in both cancer cell lines. These
Received 2 June 2009
Revised 9 July 2009
Accepted 10 July 2009
Available online 15 July 2009
Keywords:
Sansalvamide A
Pancreatic cancer
Macrocycle
Cyclic peptides
Natural products
Structure–activity relationship
Cytotoxicity
three compounds all have a common structural motif, two consecutive D-amino acids and an N-methyl
moiety. Further, of these three compounds, two are second-generation structures, indicating that we
can incorporate and utilize data from the first generation to design potency into the second generation.
Finally, one analog is in the mid nanomolar range, and has the lowest IC50 of any reported San A deriv-
ative. These analogs share no structural homology to current pancreatic cancer drugs, and are cytotoxic at
levels on par with existing drugs treating other cancers. Thus, we have established Sansalvamide A as an
excellent lead for killing multiple pancreatic cancer cell lines.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
supports further exploration of this class of compounds. All 11 of
the San A-amide derivates prepared by Silverman and co-workers
Natural products provide an excellent source of lead structures
for new drugs. These novel structures are critical for development
of new therapeutic small molecules that target unique biological
pathways. Sansalvamide A (San A) is one such natural product
(Fig. 1). San A, which was isolated from a marine fungus (Fusarium
ssp.), exhibits antitumor activity on multiple cancer cell lines.1–3
The natural product is a depsipeptide (Fig. 1) and, therefore, prone
to ring opening at the ester bond by esterases. Silverman and co-
workers found that the natural product peptide (where the
hydroxy acid is converted to an amino acid) was 10-fold more
active than the natural product depsipeptide.4,5 Thus, to avoid
deactivation via ring-opening all 78 derivatives reported by our
laboratory6,7 were synthesized as the San A peptide derivatives.
To date, the synthesis of 89 analogs have been reported, 78 by us
and 11 by Silverman et al.4 All 89 derivatives are referred to as
‘San A-amide’ derivatives. The cytotoxicity of San A-amide deriva-
tives against pancreatic,4,5,8 colon,3,6,9,10 breast, prostate, and mel-
anoma cancers4 clearly indicates San A-amide’s excellent potential
as a new therapeutic agent for the treatment of various cancers and
contain only L
-amino acids4,5,11,12 and these demonstrate reason-
able potency against the colon cancer cell line HCT-116. They
attribute potency against this cell line to the placement of multiple
N-methyl moieties on the macrocycle. However, our extensive
studies on HCT-11613,14 and our results reported here for pancre-
atic cancer cell lines PL45 and BxPC-3 indicate that placement of
multiple N-methyl moieties does not generate a potent molecule
Amino Acid III
Hydroxy Acid IV
Amino Acid IV
O
O
N
O
H2N
O
OH
H
O
HN
O
NH
HN
HN
O
NH
H3CO
O
HN
NHBoc
O
O
Amino Acid V
O
Amino
Acid II
Amino Acid I
Sansalvamide A
* Corresponding author.
E-mail address: mcalpine@sciences.sdsu.edu (S.R. McAlpine).
Figure 1. Retrosynthetic approach.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.017

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5807
against these two cell lines and that there are other factors that
contribute to the molecule’s potency.
commercially available chemical diversity (i.e., amino acids), are
efficiently synthesized, have defined 3D structures (which is typi-
cally required for good binding affinity for protein targets), are
effective at penetrating cell membranes, and are stable within
cells. To date, there are 720 clinically used peptide drugs or drug
candidates: 38% of these are in clinical trials, 56% are in advanced
preclinical phases, and 5% are on the market.17–19 These peptide
drugs are used as prostate and breast cancer antitumor agents,
HIV protease inhibitors, osteoporosis-treating drugs, and immuno-
suppressants.19–21 Thus, there is outstanding precedence for treat-
ing diseases with peptides.
Pancreatic cancer is the fifth most deadly cancer in the US. Only
10% of patients are eligible for surgery22 and less than 20% of pancre-
atic cancers respond to the current drugs of choice, one of which is
Gemcitabine (2,2-difluorodeoxycytidine).23 Although progress has
been made on improving survival rates, current therapy is far from
acceptable. Indeed, the five-year survival rate for patients with pan-
creatic cancers is still less than 5%.24 With such a poor rate of
response to current chemotherapeutic methods, there is an immedi-
ate need for new and effective pancreatic cancer treatments. This
work reports the SAR of 78 San A-amide derivatives against two
drug-resistant pancreatic cancer cell lines and establishes a struc-
tural template that can be used to design new compounds.
We report here an extensive structure–activity relationship
(SAR) of 78 compounds tested for their cytotoxicity against two
pancreatic cancer cell lines. This report includes the synthesis
and activity of 19 new compounds (second generation) that were
designed from the first generation of 59 compounds. These data
provide a global view of the San A series and their complex SAR
against PL45 and BxPC-3. Two of the three most potent com-
pounds’ structures are second-generation compounds and, thus,
their potency is reported here for the first time. These three struc-
tures clearly point to a common modality, where one N-methyl and
two consecutively placed D-amino acids are important for potency.
Further, one compound, 63, has cytotoxicity in the mid nanomolar
region, which is the lowest reported IC50 of any San A derivative.
Peptides are sometimes considered poor drugs for two reasons:
solubility and rapid degradation within cells. In order for linear
peptides to achieve 3D structures that will bind appropriately to
their protein targets, they are often composed of extended se-
quences of amino acids, making them insoluble. Cyclic peptides,
like San A, often perform better as drugs than linear peptides
because a small number of amino acids define a rigid 3D structure.
San A-amide analogs also have the advantage that they are lipo-
philic and therefore have rapid membrane absorption.15 Further,
cyclic peptides tend to have greater binding affinity for protein
targets than their linear counterparts or other small molecules
because they have restricted bond rotation and conformational
restraint, which may lock a molecule into an ideal binding mode.16
In addition, cyclic peptides degrade much slower than linear pep-
tides because proteases have difficulty cleaving amide bonds lo-
2. Results and discussion
2.1. Synthesis of all compounds
All 78 derivatives described here were constructed as the peptide
analogs (Fig. 1, where the hydroxy acid was exchanged for an amino
cated within
a
macrocycle.15 Cyclic peptides also have
O
R3
NRBoc
HO
O
O
HRN
O
OCH3
NHR
O
R2
R3
3
NRBoc
R
N
RN
RN
R1
1. (a)
2. (b)
1. (a)
2. (b)
HO
R2
H3CO
NRH
+
R1
R2
II
R1
I-II
O
O
MeO
O
I
O
R = H or Me
I-II-III (Fragment 1)
OH
NHBoc
O
O
R4
OH
R5
1. (a)
NR
O
5
R4
OMe
2. (c)
NRBoc
R5
NRH
IV
R3
IV-V (Fragment 2)
O
HRN
RN
R2
R3
O
MeO
RN
R1
O
R3
O
R4
O
R4
O
N
1. (d)
2. (b)
N
O
R
R
NR
RN
O
NR
RN
RN
R2
O
I-II-III (Fragment 1)
(a)
R2
O
O
Boc
3. (e)
R5
N
R
NR
O
RN
R1
O
R5
MeO
R4
R1
OH
O
NR
O
NRBoc
R5
Cyclized San A
Derivatives
Linear Pentapeptide
IV-V (Fragment 2)
Figure 2. Synthesis of macrocycles. Reagents and conditions: (a) coupling agent (TBTU (1.2 equiv), and/or HATU (0.75 equiv)), DIPEA (4 equiv), DCM (0.1 M); (b) TFA (20%),
anisole (2 equiv), DCM; (c) LiOH (4 equiv), MeOH; (d) LiOH (10 equiv), MeOH; (e) HATU (0.7 equiv), DEPBT (0.7 equiv), TBTU (0.7 equiv), DIPEA (6 equiv), DMF:DCM:CH3CN
(2:2:1) 0.01 M.

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P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
Figure 3. Amino acids used in the synthesis of 78 derivatives.
Figure 5. Compounds with alterations at positions II and/or I. Data is represented
Figure 4. Compounds with alterations at position I. Data is represented as % growth
as % growth inhibition relative to 1% DMSO control. Each data point is an average of
inhibition relative to 1% DMSO control. Each data point is an average of four wells
run in three assays. All assays were run for 72 h at 5 lM compound concentration.
four wells run in three assays. All assays were run for 72 h at 5
concentration.
lM compound

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5809
acidat position IV). Asuccinct syntheticprotocolhas beendeveloped
for the creation of these molecules.6,25 Our compounds have been
designedto explorehowtheplacementofN-methylmoieties, D-ami-
of theamine on residueII using TFAgave the freeamine, I–II(ꢀquan-
titative yields). Coupling of the dipeptide to monomer III (a–j) gave
the desired tripeptide (Fragment 1) in good yields (80–95%).28 The
synthesis of Fragment 2 was completed by coupling residue IV (a–
f) to residue V(a–g) to give dipeptide IV–V-Boc (90–95% yield).
The amine was deprotected on Fragment 1 using TFA and the acid
was revealed in Fragment 2 using lithium hydroxide. Fragment 1
andFragment2werecoupledusingmultiplecouplingagents,7,25,29,30
yielding 78 examples of linear pentapeptides (66–90% yield).28
In the case of the di- and tripeptide construction, acid/base
workup removed excess reagents and side products, and the
NMR indicated compounds did not usually require further purifica-
tion. Typically, it was only in the case of pentapeptides and macro-
cycles that we found it necessary to purify compounds via silica
chromatography, making the synthesis efficient. The purity of all
compounds was verified via NMR and/or LC–MS. The linear pep-
tides were then cyclized using similar conditions developed in
our laboratory.25 Upon cyclization, the final compounds were puri-
fied via flash chromatography and/or HPLC. When appropriate, the
side chains were deprotected (serines, lysines, and tyrosines). The
purity of all compounds was verified by NMR and LC–MS.25
no acids, aromatic, and hydrophilic amino acids impact the
cytotoxicity of the molecule. These derivatives have log P values
between 0.18 and 3.35 (see Supplementary data for a table), thus
meeting Lipinski’s rules for solubility and effective diffusion through
cellular membranes.26,27 Our synthetic approach utilized a conver-
gent solution-phase strategy in order toestablish a reliable and inex-
pensive route for the large-scaleproductionof compoundneeded for
additional biological studies (Fig. 2). Our outlined route provides
rapid and high-yielding synthesis of these compounds.
The synthesis of the San A-amide derivatives, were completed
using the amino acids shown in Figure 3 via the synthetic strategy
shown (Fig. 2). Using these amino acids, we were able to signifi-
cantly vary the San A-amide structures in order to explore the role
of stereochemistry, N-methyl moieties and aromatic or hydrophilic
amino acids in cytotoxicity.
The 78 structures and their cytotoxicity against the two pancre-
atic cancer cell lines are discussed in order of position. That is,
structures are arranged in order of amino acids that are altered
from the original San A peptide, where alterations to residues in
positions I–V are described in Figures 4–8, respectively.
2.2. Structure–activity relationships (SAR)
Using
2(1-H-benzotriazole-1-yl)-1,1,3-tetramethyl-uronium
tetrafluoroborate (TBTU), and diisopropylethylamine (DIPEA), acid
protected residue I (a–f) and N-Boc protected residue II (a–h) were
coupled to give the dipeptide I–II-Boc (80–94% yield). Deprotection
Although major efforts have been made, few truly novel classes
of compounds have shown activity against drug-resistant pancre-
atic cancer tumors.23,24 With this in mind, our work elucidates an
Figure 6. Compounds with alterations in positions III and/or I/II. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of four
wells run in three assays. All assays were run for 72 h at 5 M compound concentration.
l

5810
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
NH2
O
H2N
H2N
O
O
O
H2N
O
O
O
O
O
N
H
O
O
O
O
O
O
N
H
N
H
O
O
HN
NH
O
O
N
H
O
N
H
O
O
N
H
N
H
HN
HN
NH
N
HN
O
O
HN
HN
HN
NH
NH
O
HN
O
NH
NH
N
H
NH HN
O
O
O
O
O
HN
NH
NH HN
O
O
NH HN
O
NH HN
O
NH HN
O
NH HN
O
NH
O
O
NH HN
O
HO
Compound 35
Compound 36
Compound 37
Compound 38 Compound 39
Compound 40
Compound 41 Compound 42
CbzHN
CbzHN
CbzHN
H2N
CbzHN
O
O
O
O
O
O
N
O
O
O
O
O
O
O
N
O
O
N
N
H
N
O
N
H
O
O
N
H
N
NH
H
H
HN
N
HN
HN
NH
NH
NH
NH
H
O
HN
NH
N
NH
O
O
O
O
O
O
NH HN
O
NH HN
NH HN
NHHN
O
NH HN
O
O
NH HN
O
NHHN
O
O
O
O
O
Compound 43
Compound 44
Compound 45
Compound 46
Compound 47
Compound 48
Compound 49
Figure 7. Compounds with alterations at positions IV and/or I–III. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of four
wells run in three assays. All assays were run for 72 h at 5 M compound concentration.
l
understanding of the structure–activity relationship (SAR) of 78
San A derivatives against two drug-resistant pancreatic cancer cell
lines (PL45, BxPC-3).31 This SAR study describes an overview of the
cytotoxicity in cell-based assays for the San A series of molecules
and establishes a relationship between the structures and stereo-
chemistry of the active compounds’ amino acid side chains and
their growth inhibition against these two cell lines. We also high-
light three compounds, two of which are second generation, and
are potent (P55% cytotoxicity) in both cell lines. All three com-
pounds have similar structural motifs: an N-methyl incorporated
our compounds are consistently inhibiting drug-resistant pancre-
atic cancers and clearly establishes SAR. Potency exhibited by the
San A peptide 1 is shown so that comparisons can be made
between the natural product peptide and our synthetic analogs.
2.3. SAR position I
The histogram in Figure 4 shows the percent growth inhibition
produced by the addition of compounds (5 lM concentration) that
have changes to position I against two pancreatic cancer cell lines.
Compared to 1, two compounds show a moderate increased potency
against PL45 and BxPC-3: compound 2, which incorporated a
in two consecutive
D-amino acids, where one of these residues is
a
D-Phe. Given that there is an incomplete understanding of the
structure–activity relationship (SAR) of San A, we used a medicinal
chemistry approach to evaluate which structural features are key
to potency. This approach involves synthesizing analogs that have
both single and multiple changes relative to the San A natural
product peptide and testing them for potency. The core peptide
structure (Fig. 3) serves as a scaffold and compounds’ potency is
organized by position (I–V), highlighting amino acids altered from
the original San A-amide pentapeptide macrocycle (Figs. 4–8). Cell
proliferation was monitored by measuring how much 3H-thymi-
dine was incorporated into a cell’s DNA. This data can be analyzed
D
-phenylalanine, and 4 places a constrained aromatic moiety, an
L-tetrahydroisoquinoline amino acid (aa), at position I. However,
both compounds are only potent against one cell line, and thus they
are not leads. Compounds 5 and 6 show that a H-bonding element
(tyrosine and tryptophan, respectively) does not improve the cyto-
toxicity of the molecules over that of San A-amide. In summary, no
molecules with a change to position I are considered leads.
2.4. SAR position II
to determine
a
particular compound’s activity. 3H-thymidine
The histogram in Figure 5 shows the percent growth inhibi-
tion for compounds with changes made to position II, where
15 is a second-generation structure that also incorporates a
change at position I. Compound 7, which incorporates an
uptake assays were run using two distinguishable pancreatic can-
cer cell lines: PL45 and BxPC-3. Data shown gives percent growth
inhibition at 5 lM concentrations. Only growth inhibition values
greater than or equal to 55% are considered ‘potent’ and only those
compounds that surpass this threshold in both cell lines are
deemed lead structures. The use of two cell lines ensures that
N-methyl-
in growth inhibition over that of compound 1. Replacing the
-leucine with a -leucine at II (compound 8) slightly increased
L-leucine at position II, shows a significant decrease
L
D

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5811
O
O
O
O
O
O
O
O
O
N
O
O
O
O
O
O
N
H
N
H
H
O
O
N
O
O
O
O
N
N
H
HN
HN
NH
N
N
H
HN
HN
HN
H
NH
NH
HN
NH
O
HN
HN
HN
N
N
O
O
O
HN
NH
O
O
O
NH HN
O
N
NH HN
O
NH HN
O
NH
O
NH HN
O
O
O
Compound 50
Compound 51
Compound 52
Compound 53
Compound 54
Compound 55
Compound 56
ClCbzHN
O
O
O
O
N
O
O
O
O
N
O
N
HN
O
N
HN
O
O
O
N
O
O
N
O
N
H
H
H
HN
NH
HN
NH
HN
HN
NH
NH
NH
HN
NH
O
O
H
O
O
N
NH
O
O
O
NH HN
NH
NH HN
O
N
HN
O
NH HN
O
N
HN
NH HN
O
O
O
HO
O
O
O
O
O
O
Compound 63
Compound 61
Compound 62
Compound 57
Compound 58
Compound 59
Compound 60
CbzHN
H2N
H2N
O
O
O
O
N
O
O
O
N
H
O
O
N
N
H
O
O
O
N
O
O
O
N
HN
NH
N
H
O
NH
H
HN
HN
NH
HN
NH
O
O
H
NH
N
HN
NH
O
O
O
O
NH HN
O
BnO
NHHN
O
O
N
O
N
O
HN
NHHN
O
O
NH HN
O
O
O
Compound 64
Compound 65
Compound 66
Compound 67
Compound 68
Compound 69
Figure 8. Compounds with alterations at position V and/or I–IV. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of four
wells run in three assays. All assays were run for 72 h at 5 M compound concentration.
l
potency against PL45 but decreased potency against BxPC-3.
However, incorporation of an N-methyl- -leucine (compound 9)
show greater than 55% growth inhibition against one cell line.
In contrast to 1, which contains an isopropyl moiety at position
III, compounds containing a methyl 19 or an ethyl 20 moiety
were tested and showed significantly reduced potency against
both cell lines. Compound 21 contains an ethyl moiety at posi-
D
dramatically decreased the potency against both cell lines. Inclu-
sion of an aromatic moiety (compounds 10, 12, and 14), versus a
hydrophilic moiety (compounds 11 and 13) indicates that aro-
matic residues improve cytotoxicity relative to compounds with
polar residues at II (e.g., compare 10/12 to 13, and 11 to 14). A
new second-generation compound, 15, contains two aromatic
moieties. This molecule is cytotoxic against one cell line, show-
ing P55% cytotoxicity against one cell line, but it is poor against
a second cell line, as such it is not considered a valuable lead
structure.
tion III along with a
D-leucine at position II and shows lower
cytotoxicity than 8, which is its ‘parent’ compound. Interestingly,
placing either a hydrophilic element or an aromatic element at
position III, 22/24 or 23/25, respectively, does not lead to in-
creased potency against either cell line. Compounds 26 and 27
share an almost identical core structure, with the same residues
in positions I, II, IV, and V, but 26 has an
-ethyl glycine at III. Both compounds are below 55% against
both cell lines. Similarly 28 is also not potent. Compound 29,
which incorporates a -Phe at I, a -Leu at II, and a -valine at
D-valine and 27 has
D
2.5. SAR position III
D
D
D
III, shows some potency against both cell lines, but under the
55% required to make this molecule a lead. Compounds 30 and
31 involved alterations at both positions II and III, and both have
potency below 55% in each cell line. However, a new second-
generation compound, 32, exhibited improved cytotoxicity
against PL45 relative to both its starting molecules, 12 and 17,
but not against both cell lines. Comparing 32 to 33 and 34 show
The histogram in Figure 6 shows the percent inhibition of
growth by compounds with changes at position III (some of
which also contain changes at positions I and/or II), where com-
pounds 32–34 are all second-generation structures. Compound
16 incorporates an N-methyl-valine, compound 17 a
D-valine,
and compound 18, a N-methyl- -valine at position III. None
D

5812
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
O
O
O
O
O
O
O
O
O
O
O
O
N
H
N
H
O
O
O
O
N
H
N
H
O
O
N
N
H
N
HN
NH
NH
HN
HN
N
NH
NH
HN
NH
O
O
HN
NH
O
O
O
O
NH HN
O
NH HN
O
N
NH HN
O
NH HN
O
NH HN
O
O
Compound 1
Compound 7
Compound 16
Compound 50
Compound 70
Compound 71
O
O
O
O
O
O
O
O
O
O
O
O
N
HN
O
N
HN
NH
N
O
N
O
N
HN
O
N
H
N
N
H
N
NH
H
N
NH
NH
NH
NH
O
HN
NH
O
O
O
O
O
O
N
O
HN
N
HN
N
O
HN
N
HN
N
HN
N
O
HN
O
O
O
NH HN
O
O
O
O
O
O
Compound 72
Compound 73
Compound 74
Compound 75
Compound 76
Compound 77
Compound 78
Figure 9. Compounds with N-methyl moieties and their enantiomers. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of
four wells run in three assays. Error = 5%. All assays were run for 72 h at 5 M compound concentration.
l
that the incorporation of an N-methyl and two non-consecu-
tively placed -amino acids is not enough to produce compounds
2.7. SAR position V
D
with P55% cytotoxicity in PL45.
The histogram in Figure 8 shows the percent inhibition of
growth by compounds with changes at position V (where some
also include changes at position(s) I, II, III and/or IV), where com-
pounds 60–69 are all second-generation compounds. Compound
2.6. SAR position IV
50 incorporates an N-methy-
51 a -leucine, and compound 52 an N-methyl
and 52 exhibit good growth inhibition against both cell lines but
are not P55%. Interestingly 53, which contains an N-methyl- -leu-
cine at position IV and -leucine at position V, showed some prom-
ise in one cell line. Similar to previous observations, the inclusion
of a -Phe (at I) made 54 a promising lead structure. Incorporating
-leucine at positions IV and V gave non-toxic 55. Not surpris-
ingly compounds 56 and 57, which contain a -leucine at position
V and are structurally similar to non-potent compound 51, showed
poor growth inhibition against both cell lines. Interestingly,
compounds 57 and 58 have reduced potency compared to their
parent compounds 1, 17, 50, or 51. Compound 60 has a polar res-
idue, and as noted earlier, this property makes the molecule not
effective. 61 and 62 are both relatively non-toxic; yet 63 shows
reasonable potency in both cell lines, with cytotoxicity values of
L
-leucine at position 5, compound
The histogram in Figure 7 shows the percent inhibition of
growth by compounds with changes to position IV (where some
also include changes to position(s) I, II and/or III). Compounds
37, 42, 43, 47, and 49 are all second-generation structures. In
D
D
-leucine. Both 50
L
D
contrast to potent compound 17, which has a D-aa at III, 35 (D-aa
at position IV), shows 0% inhibition against both cell lines. Further,
the derivative with the N-methyl at III is non-potent, whereas com-
pound 36, which incorporates an N-methyl moiety at IV, showed
reasonable inhibition against both cell lines. Compound 37, which
D
a
D
D
has 2
D
-amino acids consecutively placed at III and IV, was not
-aa substitutions at positions
potent; neither was 38, which has
D
I, III, and IV. Compounds 39–43 all contain a lysine at position IV,
as well as other changes at various other positions. All five com-
pounds are uniformly poor growth inhibitors against both cell
lines. Not surprisingly, the hydrophobic compounds that are struc-
turally related to these polar compounds, 44–47, respectively, are
also relatively non-toxic and only show a low level of potency
against these cell lines. Further, the incorporation of an N-methyl
P55%. Similar to 54, compound 63, has two consecutive D-amino
acids and an N-methyl moiety. Consistent with previous observa-
tions, polar derivative 64 is not toxic, nor are derivatives 68 and
69. Comparison of compound 65 and 66 indicates that two consec-
utively placed D-amino acids play an important role in the potency
of 66, where 66 is also considered a lead. Compound 67 is based on
29 (Fig. 6), yet it does not perform as well as 29. In summary, three
D-valine at III, and a cyclohexyl moiety at position IV produced
compound 48, which showed improved cytotoxicity against both
cell lines relative to its parent (18). Finally, second-generation
compound 49, exhibited underwhelming cytotoxicity against both
cell lines.

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5813
Potent Compounds
100%
80%
60%
40%
20%
0%
2
36
53
54
63
12
17
48
66
25%
55%
23%
52%
25%
71%
59%
58%
65%
55%
51%
35%
45%
22%
30%
49%
70%
80%
PL45 5 M
BxPC-3 5 M
CbzHN
O
O
O
N
O
N
O
O
O
O
O
O
N
H
O
O
N
H
N
H
O
O
O
HN
NH
N
N
O
O
HN
HN
N
H
N
O
HN
O
O
HN
NH
O
NH
HN
O
O
NH HN
O
NH HN
O
NH HN
NH HN
NH HN
O
O
NH HN
O
O
O
Compound 2
Compound 36
Compound 53
Compound 54
Compound 63
Compound 54
O
O
O
O
O
N
H
N
H
O
O
N
O
N
NH
N
NH
HN
NH
O
O
O
HN
NH
O
O
H
N
NH HN
O
O
O
NH HN
O
O
NH HN
O
NHHN
O
O
Compound 12
Compound 17
Compound 48
Compound 66
Figure 10. Most potent compounds and the compounds from which they were designed. Data is represented as % growth inhibition relative to 1% DMSO control. Each data
point is an average of four wells run in three assays. All assays were run for 72 h at 5
lM compound concentration.
compounds, two being second generation, show greater than 55%
It is important to note that all three of these compounds have an
in each cell line: 54, 63, and 66.
N-methyl associated with two consecutive
D-amino acids, where
one of these is a -phenylalanine. In every case one can see that
D
2.8. SAR all
L
and
D
-amino amino acids
the final compound showed an average improved potency in both
cell lines over the compounds used to rationally design them. Com-
pound 54 was designed from the combination of several com-
pounds: 2, 36, and 53, and 63 was designed from compound 54.
Finally 66 was designed from 12, 17, and 48.
The histogram in Figure 9 shows the percent inhibition of growth
by compounds that contain all or all -amino acids. Compound 70 is
the enantiomer of 1, and its cytotoxicity is lower than that of 1.
Compound 71 is the enantiomer of 7 and, in addition to all -amino
L
D
D
Compound 54, which contains a N-methyl at IV and
acids at positions I and V, was designed from compounds 2, 36,
and 53, where 2 contained a -amino acid at I, 36 an N-methyl at
IV, and 53 had both a -amino acid at V and an N-methyl at IV.
D-amino
acids, it contains an N-methyl at position II. Although it is more
potent than 7, it is not as potent as 1. Compound 16 and 72 are enan-
tiomers with an N-methyl at position III, and both are non-potent.
Compound 50 with an N-methyl at V is potent and interestingly its
enantiomer 75 maintains relatively similar potency. Enantiomers
73 and 74, which contain 2-N-methyl moieties (positions II and V),
arealso notactive. Thesameis true forenantiomers76and77, which
also contain 2-N-methyl moieties (positions III and V). Finally com-
pound 78 with three N-methyls is not active. Thus, this series of mol-
ecules has no compounds with P55% cytotoxicity in any cell line.
D
D
Thus, although these three compounds, 2, 36, and 53 showed some
potential, 54 shows increased potency for both cell lines over that
shown by the three initial compounds from which they were
designed. Designed from 54 (this can be perceived by rotating 54
clockwise by one position), 63 maintained the motif of an
N-methyl followed by two consecutive D-amino acids. Its improved
cytotoxicity over that of 54 may result from a large hydrophobic
moiety placed at position IV or the addition of another aromatic
moiety at position 1. Compound 66 was designed from compounds
2.9. Design of potent compounds
12, 17, and 48 where it is comprised of an N-methyl
D-Phe at II
Shown in Figure 10 are the three most potent compounds: 54,
63, and 66 and the compounds from which they were designed.
Two of these three compounds are reported here for the first time.
(from 12), a -Val at III (from 17), a cyclohexyl moiety at IV (from
48), and the addition of a benzyl protected serine ether at V. Thus,
66 has three aromatic moieties placed around its macrocyclic back-
D

5814
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
bone and it is significantly more potent than its precursors. It is
interesting to note that several other derivatives, specifically 15
not display the highest cytotoxicity values against the two cell lines
at 5 M, it does show by far the best IC50 value, 500 nM in PL45. This
appears to result from the 5 M data point residing (consistently)
l
and 32, also contain two consecutive
D-amino acids and an
l
N-methyl moiety, yet these two compounds are only cytotoxic
against PL45, and in fact show very poor cytotoxicity against
BxPC-3. 15 has a tyrosine at I, and thus, this may impact its
cytotoxicity. 32 is structurally similar to 66, but it lacks the hydro-
phobic moieties at IV and V, which, as noted with 63, appear to
play an important role in the generating a cytotoxic molecule.
In summary, we report here for the first time three potent com-
pounds out of 78 derivatives, where potency was designed into our
compounds using known structural features that increase their
lower than the curve. Thus, overall we have found a single com-
pound (63) with a nanomolar IC50 value against these drug-resistant
cancer cell lines, as well as determined that 66 shows potential as a
lead. Both compounds are new derivatives, reported here for the first
time.
2.11. Summary of SAR results
The most important feature to emerge from this SAR study is
the observation that molecules that are potent contain two consec-
cytotoxicity, that is, N-methyls and D-amino acids. We successfully
generated compounds that show consistency across both pancre-
atic cancer cell lines, and have devised a general approach to
generating improved derivatives.
utive
acids. Indeed, all potent compounds contain two consecutive
-amino acids, one of which is a -phenylalanine, and an N-methyl
on or next to the -amino acids. It is easy to recognize this pattern
D-amino acids and an N-methyl located next to the D-amino
D
D
D
2.10. IC50 determination
by comparing the potency of 2, 36, and 53 to 54 and that of 12, 17,
and 48 to 66. Neither of the parent compounds are as cytotoxic as
54 and 66, respectively.
Inhibition constants were calculated for the three most potent
compounds by plotting five concentrations (50, 10, 0.5, and
Further, comparison of the potency of 65 to 66 clearly indicates
0.1
lM) and extracting data from the curves (Supplementary data).
that two
D-amino acids next to each other significantly improve the
All relationships are logarithmic in nature. We have identified two
compounds, 54 and 66, that show low micromolar cytotoxicity
and one compound, 63, that shows mid nanomolar cytotoxicity
against pancreatic cancer cell lines. The IC50 values for the most po-
tent compounds are shown in Figure 11, where these compounds
show up to ꢀ40-fold greater cytotoxicity than the parent natural
product peptide 1. It is interesting to note that although 63 does
compound’s cytotoxicity. Further validating our hypothesis is com-
parison of compounds 30 and 31, where 30 contains two
acids sequentially placed, and 31 contains two N-methyl
D
-amino
-amino
D
acids successively placed. Both have low cytotoxicity relative to
our lead compounds. Thus, it is not enough to have two sequential
D-amino acids, nor two N-methyl
D-amino acids to generate a lead
structure. Rather, as observed by others in this pentapeptide sys-
tem, D-amino acids play an important role in achieving a favorable
conformation.32–34
Finally, it is important to note that structurally similar com-
pound 56 contains 2 D-amino acids and an N-methyl but the D-ami-
no acids are not consecutive, and its potency is poor relative to that
of 54 or 66. Also note that 34 is not nearly as potent as other
compounds with consecutive
D-amino acids. Finally, more than
one N-methyl and/or more than two consecutively placed
acids are not potent (i.e., 61, 76, 77, and 78).
D-amino
In summary, all compounds found to exhibit cytotoxicity that
was equal to or greater than 55% in each pancreatic cancer cell line
contained a single N-methyl and two sequentially placed D-amino
acids. As such, we surmise that two factors are important for
potency: a single N-methyl in conjunction with two consecutively
placed D-amino acids, where one is a D-phenylalanine. This theory
is validated by several current examples described in the recent
literature, where cyclic peptides, specifically pentapeptides, with
O
O
O
O
O
N
N
H
H
HN
N
HN
NH
O
D
-amino acids lock the macrocycle into a specific conforma-
O
tion.32–34 Further, it is well established that these cyclic pentapep-
tides mimic beta and gamma turns and serve as templates for the
appropriate positioning of suitable binding motifs for proteins.35,36
NH HN
O
O
NH HN
O
Thus, we believe that the inclusion of two successive D-amino acids
coupled with a single N-methyl locks the macrocycle into a suit-
able position for binding to its biological target.
Compound 1
Compound 54
ClCbzHN
O
3. Conclusion
O
N
NH
O
N
HN
O
O
H
N
NH
O
We report here for the first time a comprehensive SAR evaluation
using two generations of compounds, where 19 new structures and
59 previously published structures were described and examined for
their cytotoxicity against two drug-resistant pancreatic cancer cell
lines. This global assessment included the determination that
O
NHHN
NH HN
O
O
O
O
placement of a single N-methyl and two consecutively placed D-amino
Compound 66
Compound 63
acids arerequired forcompounds to exhibitP55% cytotoxicity in both
cell lines, where one compound exhibited a 500 nM IC50. Two of the
three most potent compounds were second-generation compounds,
Figure 11. IC50s of potent compounds. Each data point is an average of four wells
run in three assays at 50, 10, 0.5, and 0.1 M.
l

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5815
and these two compounds had improved IC50s over the first-genera-
tion structure. Our three most potent small molecules target drug-
resistant pancreatic cancer cell lines, and share no homology with
other classes of chemotherapeutic agents and have reasonable C log P
values37 and molecular weights (600–738). Studies on third-genera-
tion compounds are on going and will be reported in due course.
lene chloride, and acetonitrile for pentapeptide couplings. The
amine (1.1 equiv) and acid (1 equiv) were weighed into a dry flask
along with 4–12 equiv of DIPEA and 1.1 equiv of TBTU. Anhydrous
methylene chloride was added to generate a 0.1 M solution. The
solution was stirred at room temperature and reactions were mon-
itored by TLC. Reactions were run for 1 h before checking via TLC. If
reaction was not complete additional 0.25 equiv were of HATU and
TBTU were added. If reaction was complete then workup was done
by washing with aqueous HCl (pH 1) and saturated sodium bicar-
bonate. (Note: if acetonitrile was used for the reaction, methylene
chloride was added to reaction upon workup and the resulting
solution was washed with the aqueous solvents.) After back
extraction of aqueous layers with methylene chloride, organic
layers were combined, dried over sodium sulfate, filtered and con-
centrated. Flash chromatography using a gradient of ethyl acetate–
hexane gave our desired peptide.
4. Experimental
4.1. General remarks
All coupling reactions were performed under argon atmosphere
with the exclusion of moisture. All reagents were used as received.
Anhydrous methylene chloride Dri Solv (EM), anhydrous tetrahy-
drofuran, anhydrous dimethylformamide, and anhydrous acetoni-
trile Dri Solv (EM) were obtained from VWR, and were packed
under nitrogen with a septum cap. Diisopropylethylamine (DIPEA)
was purchased from Aldrich, packaged under nitrogen in a sure
seal bottle. The coupling agent HATU came from: Applied Biosys-
tems at 850 Lincoln Center Dr. Foster City, CA 94404, USA. Tel.:
+1 800 327 3002 and the coupling agents TBTU NovaBiochem. DEP-
BT [3(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)] was
purchased from Aldrich (order number 49596-4). The 1H NMR
spectra were recorded on the Varian at 200 MHz or 400 MHz or
500 MHz. LC–MS was performed at San Diego State University
using HP1100 Finnigan LCQ. Flash column chromatography used
230–400 mesh 32–74 lm 60 Å silica gel from Bodman Industries.
4.3.2. General amine deprotection
Amines were deprotected using 20% TFA in methylene chloride
(0.1 M) with 2 equiv of anisole. The reactions were monitored by
TLC, where the TLC sample was first worked up in a mini-workup
using DI water and methylene chloride to remove TFA. Reactions
were allowed to run for 1–2 h and then concentrated in vacuo.
4.3.3. General acid deprotection
Acids were deprotected using ꢀ4 equiv of lithium hydroxide (or
until pH ꢀ11) in methanol (0.1 M). The peptide was placed in a
flask, along with lithium hydroxide and methanol and stirred over-
night. Within 12 h the acid was usually deprotected. Workup of
reactions involved the acidification of reaction solution using HCl
to pH 1. The aqueous solution was extracted three times with
methylene chloride, and the combined organic layer was dried,
filtered, and concentrated in vacuo.
4.2. Thymidine incorporation/growth inhibition assay
Proliferation of the PL-45 and BxPC-3 pancreatic cancer cells was
tested in the presence and absence of the compounds using 3H-thy-
midine uptake assays. Cells treated with the compounds were
compared to DMSO controls for their ability to proliferate as indi-
cated by the incorporation of 3H-thymidine into their DNA. Cells
were cultured in 96-well plates at a concentration of 2000–2500
4.3.4. Macrocyclization procedure (in situ)
All pentapeptides were acid and amine deprotected using con-
centrated HCl (eight drops per 0.3mmol of linear pentapeptide)
in THF (0.05 M). Anisole (2 equiv) was added to the reaction and
the reaction was stirred at room temperature. The reaction typi-
cally took 4 days, but TLC and LC–MS were used to monitor the
reaction every 12 h. LC–MS data typically indicated the reaction
was ꢀ50% complete after the first day. Addition of four drops of
concentrated HCl per 0.3 mmol of pentapeptide, stirring at RT
overnight and checking the reaction via LC–MS usually showed
ꢀ75% completion. On the fourth day verification of the presence
of the free amine and free acid and disappearance of the starting
linear protected pentapeptide permitted workup. The reaction
was concentrated in vacuo and the crude, dry, double deprotected
peptide (free acid and free amine) was dissolved in a minimum
solution of DMF: methylene chloride: acetonitrile (2:2:1 ratio).
Three coupling agents (DEPBT, HATU, and TBTU) were used at
ꢀ0.5–0.75 equiv each. These coupling agents were dissolved in a
calculated volume of dry 40% DMF, 40% methylene chloride, and
20% acetonitrile that would give a 0.01 M overall solution when in-
cluded in the volume used for the deprotected peptide. The cou-
pling agents were then added to the deprotected peptide
solution. DIPEA (6 equiv or more in order to neutralize the pH)
were then added to the reaction. The coupling agents are typically
not very soluble in acetonitrile, which is why a combination of
solvents is used.
cells/well in DMEM (Gibco) supplemented with L-glutamine, 10% fe-
tal bovine serum and 1% penicillin–streptomycin antibiotic. After
incubation for 6 h, the compounds were added. The compounds
were dissolved in DMSO at a final concentration of 1% and tested
at the concentrations indicated in the manuscript. The DMSO control
was also at 1%. After the cells had been incubated with the
compounds for 56 h, 1 mCi 3H-thymidine per well was added and
the cells were cultured for an additional 16 h (for the cells to have
a total of 72 h treatment), at which time the cells were harvested
usinga PHDcell harvester(CambridgeTechnologyInc.). The samples
were then counted (CPM) in a scintillation counter for 5 m.
Decreases in 3H-thymidine incorporation, as compared 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 deter-
mined using 0, 0.1, 0.5, 5, 10, and 50 lM of compound. All calcula-
tions including mean, SEM, and IC50 were performed on Excel.
4.3. Synthesis
For synthesis details of the first-generation compounds see Ref.
6. For second-generation compounds see Ref. 7. Potent compounds’
final characterization data are listed below.
After 1 h, TLC and LC–MS (where the LC–MS sample was worked
up prior to injection) indicated that a product spot was developing.
4.3.1. General peptide synthesis
All peptide coupling reactions were carried out under argon
with dry solvent, using methylene chloride for dipeptide and tri-
peptide couplings and a mixture of dimethylformamide, methy-
Some coupling reactions would not go to completion using only TBTU and
therefore ꢀ0.25 equiv of HATU, and/or DEPBT were used. In
a few cases up to
1.7 equiv of all three coupling reagents were used.

5816
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
The comparison Rf value in the product spot on TLC was the pro-
tected linear pentapeptide. The reactions were always complete
after 3 h, and monitoring the starting material deprotected penta-
peptide via LC–MS was the easiest method of determining comple-
tion. 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 methylene
chloride, the organic layers were combined, dried, filtered and con-
centrated. All macrocycles were purified by initially running a crude
plug of compound using an ethyl acetate/hexane gradient on silica
gel, then running a column on the isolated product. Finally, when
necessary reverse phase-HPLC was used for additional purification
using a gradient of acetonitrile and DI water with 0.1% TFA.
4.3.5.6. Dipeptide HO-Val-Leu-Leu-NHBoc. Dipeptide HO-Val-
Leu-Leu-NHBoc was synthesized following the ‘General acid depro-
tection’. This tripeptide was taken on to the next reaction without
further purification or characterization. (661 mg, 88% yield).
4.3.5.7. Pentapeptide MeO-
Pentapeptide MeO- -Tyr-N-Me-
thesized following the ‘General peptide synthesis’ procedure. Utilizing
503.4 mg (1.41 mmol, 1.0 equiv) of amine Meo- -Tyr- -MePhe-NH,
D-Tyr-N-Me-D-Phe-Val-Leu-Leu-NHBoc.
D
D-Phe-Val-Val-Leu-NHBoc was syn-
D
D
689 mg (1.55 mmol, 1.0 equiv) of acid HO-Val-Leu-Leu-NHBoc,
0.86 mL (3.5 equiv) of DIPEA, 91 mg (0.28 mmol, 0.2 equiv) of TBTU,
and 322 mg (0.85 mmol, 0.6 equiv) HATU,169 mg (0.56 mmol,
0.4 equiv) of DEPBT. The crude reaction was purified by column chroma-
tography (silica gel, EtOAc/Hex) to yield the pentapeptide (727 mg, 60%
yield).
4.3.5. Synthesis of compound 15
4.3.5.1. Dipeptide MeO-Val-Leu-NHBoc. Dipeptide MeO-Val-Leu-
NHBoc was synthesized following the ‘General peptide synthesis’
procedure. Utilizing 370 mg (2.2 mmol, 1.1 equiv) of amine OMe-
Val-NH2, 500 mg (2.0 mmol, 1.0 equiv) of acid HO-Leu-NHBoc,
1.4 mL (8 equiv) of DIPEA, 515.2 mg (1.6 mmol, 0.8 equiv) of TBTU,
305 mg (0.8 mmol, 0.4 equiv) The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the dipep-
tide (625 mg, 90% yield).
Rf: 0.5 (EtOAc:Hex 2:1).
1H NMR (400 MHz, CDCl3): d 0.6–0.8 (m, 6H), 0.8–1.0 (m, 12H),
1.5–1.8 (m, 5H), 2.6 (m, 2H), 2.8–3.4 (m, 4H), 3.7 (s, 3H), 4.2 (m,
a
H), 4.4–4.5 (m,
aH), 4.5–4.6 (m, aH), 4.6–4.7 (m, aH), 4.7–4.8
(m, H), 5.0 (d, 1H), 6.6–6.8 (br, 2H), 6.7–7.4 (m, 9H).
a
4.3.5.8. Macrocycle
D-Tyr-N-Me-D-Phe-Val-Leu-Leu. Macrocycle
D
-Tyr-N-Me- -Phe-Val-Leu-Leu was synthesized following the
D
Rf: 0.5 (EtOAc:Hex 1:2).
‘Macrocyclization procedure’. Utilizing 182.4 mg (0.27 mmol,
1.0 equiv) of linear pentapeptide, 0.19 mL (4 equiv) of DIPEA,
52.6 mg (0.16 mmol, 0.6 equiv) of TBTU, 41.5 mg (0.11 mmol,
0.4 equiv) HATU, and 16.3 mg (0.11 mmol, 0.4 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (18 mg, 10% yield).
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.4 (s, 9H), 1.6–
1.8 (m, 2H), 2.1–2.3 (m, 1H), 3.7 (s, 3H), 4.0–4.1 (m,
(m, H), 4.8–4.9 (br, 1H), 6.6 (d, 1H).
aH), 4.4–4.5
a
4.3.5.2. Dipeptide MeO-Val-Leu-NH2. Dipeptide MeO-Val-Leu-
NH2 was synthesized following the ‘General amine deprotection’.
This dipeptide was taken on to the next reaction without further
purification or characterization. (446 mg, 100% yield).
Rf: 0.5 (EtOAc:Hex 4:1).
1H NMR (400 MHz, CDCl3): d 0.6–1.0 (m, 18H), 1.6–1.8 (m, 4H),
1.8–2.0 (m, 1H), 2.9 (s, 3H), 2.6–3.2 (m, 4H), 4.1 (m,
H), 4.3–4.5 (m, H), 4.6–4.7 (m, H), 5.5 (m, H), 6.7–7.0 (m,
4H), 7.1–7.4 (m, 5H).
aH), 4.2 (m,
a
a
a
a
4.3.5.3. Tripeptide MeO-Val-Leu-Leu-NHBoc. Tripeptide MeO-
Val-Leu-Leu-NHBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 689 mg (2.82 mmol, 1.0 equiv)
of amine MeO-Val-Leu-NH2, 774 mg (3.11 mmol, 1.0 equiv) of acid
HO-Leu-NHBoc, 1.9 mL (4 equiv) of DIPEA, 544 mg (1.69 mmol,
0.6 equiv) of TBTU, 644 mg (1.69 mmol, 0.6 equiv) of HATU. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the tripeptide (887 mg, 60% yield).
Rf: 0.5 (EtOAc:Hex 1:1).
LC–MS: m/z calcd for C33H51N5O5 (M+1) = 598.39, found 598.3.
4.3.6. Synthesis of compound 32
4.3.6.1. Dipeptide MeO-Phe-N-Me-
D-Phe-NBoc. Dipeptide MeO-
Phe-N-Me- -Phe-NBoc was synthesized following the ‘General
D
peptide synthesis’ procedure. Utilizing 509 mg (1.51 mmol,
1.1 equiv) of amine MeO-Phe-NH2, 600 mg (1.37 mmol, 1.0 equiv)
of acid HO-N-Me-D-Phe-NBoc, 1.5 mL (4 equiv) of DIPEA, 828 mg
(2.58 mmol, 1.2 equiv) of TBTU. The crude reaction was purified
by column chromatography (silica gel, EtOAc/Hex) to yield the
dipeptide (520.1 mg, 86% yield).
1H NMR (200 MHz, CDCl3): d 0.8–1.1 (m, 12H), 1.4 (s, 9H), 2.8 (s,
3H), 3.7 (s, 3H), 4.0–4.2 (m,
6.7 (d, 2H).
aH), 4.4–4.6 (m, 2aH), 4.9 (d, 1H), 6.5–
Rf: 0.7 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H), 2.7 (s, 3H), 3.1–3.2 (m,
4.3.5.4. Dipeptide MeO-
-Tyr-N-Me- -Phe-NBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 318 mg (1.4 mmol, 1.1 equiv) of
amine MeO- -Tyr-NH2, 576 mg (1.3 mmol, 1.0 equiv) of acid HO-N-
Me- -Phe-NBoc, 1.4 mL (8 equiv) of DIPEA, 515.2 mg (1.6 mmol,
D-Tyr-N-Me-D-Phe-NBoc. Dipeptide MeO-
4H), 3.7 (s, 3H), 4.8–5.0 (m, 2aH), 7.1–7.3 (dd, 10H).
D
D
4.3.6.2. Dipeptide MeO-Phe-N-Me-D-Phe-NH. Dipeptide MeO-
D
Phe-N-Me- -Phe-NH was synthesized following the ‘General
D
D
amine deprotection’. This dipeptide was taken on to the next reac-
tion without further purification or characterization. (521 mg,
100% yield).
0.8 equiv) of TBTU, 380 mg (0.8 mmol, 0.6 equiv) of HATU. The crude
reaction was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the dipeptide (625 mg, 90% yield).
Rf: 0.5 (EtOAc:Hex 1:2).
4.3.6.3. Tripeptide MeO-Phe-N-Me-
tide MeO-Phe-N-Me- -Phe- -Val-NHBoc was synthesized follow-
ing the ‘General peptide synthesis’ procedure. Utilizing 521 mg
(1.5 mmol, 1.1 equiv) of amine MeO-Phe-N-Me- -Phe-NH,
302.3 mg (1.39 mmol, 1.0 equiv) of acid HO- -Val-NHBoc,
D-Phe-D-Val-NHBoc. Tripep-
1H NMR (200 MHz, CDCl3): d 1.4 (m, 9H), 2.8 (s, 3H), 2.9–3.4 (m,
D
D
4H), 3.7 (s, 3H), 4.7–4.9 (m, 2
(dd, 4H), 7.1–7.3 (m, 5H).
aH), 6.0 (br, 1H), 6.2 (br, 1H), 6.7–7.0
D
D
0.97 mL (4 equiv) of DIPEA, 268 mg (0.83 mmol, 0.6 equiv) of TBTU,
and 317 mg (0.83 mmol, 0.6 equiv) of HATU. The crude reaction
was purified by column chromatography (silica gel, EtOAc/Hex)
to yield the tripeptide (316 mg, 45% yield).
4.3.5.5. Dipeptide MeO-
D-Tyr-N-Me-D-Phe-NH. Dipeptide MeO-
D
-Tyr-N-Me- -Phe-NH was synthesized following the ‘General
D
amine deprotection’. This dipeptide was taken on to the next reac-
tion without further purification or characterization. (625 mg,
100% yield).
Rf: 0.3 (EtOAc:Hex 3:7).

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5817
1H NMR (200 MHz, CDCl3): d 0.5 (d, 6H), 1.4 (s, 9H), 2.9 (s, 3H),
3.1 (t, 4H), 3.7 (s, 3H), 4.1 (m, H), 4.8 (m, H), 5.0 (d, 1H), 5.5 (m,
H), 7.2-7.3 (dd, 10H).
Rf: 0.3 (EtOAc:Hex 3:7).
a
a
1H NMR (200 MHz, CDCl3): d 0.95 (d, 6H), 1.4 (s, 9H), 1.6–1.8 (m,
a
3H), 3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.1 (m,
6.6 (d, 1H), 7.1–7.4 (m, 5H).
aH), 4.8–5.0 (m, aH), 6.4–
4.3.6.4. Tripeptide MeO-Phe-D-MePhe-D-Val-NH2. Tripeptide MeO-
Phe-N-Me- -Phe- -Val-NH2 was synthesized following the ‘General
D
D
4.3.7.2. Dipeptide MeO-D-Phe-Leu-NH2. Dipeptide MeO-D-Phe-
amine deprotection’. This tripeptide was taken on to the next
reaction without further purification or characterization. (316 mg,
100% yield).
Leu-NH2 was synthesized following the ‘General amine deprotec-
tion’. This dipeptide was taken on to the next reaction without
further purification or characterization. (1029 mg, 100% yield).
4.3.6.5. Dipeptide MeO-Leu-Leu-NHBoc. Dipeptide MeO-Leu-Leu-
NHBoc was synthesized following the ‘General peptide synthesis’
procedure. Utilizing 401 mg (2.2 mmol, 1.1 equiv) of amine MeO-
Leu-NH2, 500 mg (2.0 mmol, 1.0 equiv) of acid HO-Leu-NHBoc,
1.4 mL (4 equiv) of DIPEA, 774 mg (1.2 mmol, 1.0 equiv) of TBTU.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the dipeptide (703 mg, 98% yield).
Rf: 0.35 (EtOAc:Hex 2.5:7.5).
4.3.7.3. Tripeptide MeO-
-Phe-Leu- -Val-NHBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 343 mg (1.17 mmol, 1.1 equiv) of
amine MeO- -Phe-Leu-NH2, 230.4 mg (1.06 mmol, 1.0 equiv) of acid
HO- -Val-NHBoc, 1.49 mL (8 equiv) of DIPEA, 408 mg (1.25 mmol,
1.2 equiv) of TBTU. The crude reaction was purified by column chro-
matography (silica gel, EtOAc/Hex) to yield the tripeptide
(443.8 mg, 85% yield).
D-Phe-Leu-D-Val-NHBoc. Tripeptide MeO-
D
D
D
D
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.4 (s, 9H), 1.6–
Rf: 0.7 (EtOAc:Hex 1:1).
1.8 (m, 6H), 3.7 (s, 3H), 4.0–4.1 (m,
(br, 1H), 6.4 (d, 1H).
aH), 4.5–4.7 (m,
aH), 4.8–4.9
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.5 (s, 12H), 1.6–
1.8 (m, 3H), 2.2 (m, 2H), 3.1 (m, 2H), 3.7 (s, 3H), 3.9–4.0 (dd, 1H),
4.4 (m,
aH), 4.9 (dd,
aH), 5.0 (m,
aH), 6.3 (d, 1H), 6.6 (m, 1H),
4.3.6.6. 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. (349 mg, 58% yield).
7.0–7.4 (m, 5H).
4.3.7.4. Tripeptide MeO-
D
-Phe-Leu-D-Val-NH2. Tripeptide MeO-
D-Phe-Leu-D-Val-NH2 was synthesized following the ‘General
amine deprotection’. This tripeptide was taken on to the next reac-
tion without further purification or characterization. (353.5 mg,
100% yield).
4.3.6.7. Pentapeptide MeO-Phe-N-Me-
D
-Phe-
D-Val-Leu-Leu-
NHBoc. Pentapeptide MeO-Phe-N-Me- -Phe-
D
D
-Val-Leu-Leu-NHBoc
was synthesized following the ‘General peptide synthesis’
procedure. Utilizing 316 mg (0.72 mmol, 1.1 equiv) of amine
MeO-Phe-N-Me-D-Phe-D-Val-NH2, 240 mg (0.65 mmol, 1.0 equiv)
of acid HO-Leu-Leu-NHBoc, 0.92 mL (8 equiv) of DIPEA, 126 mg
(0.39 mmol, 0.6 equiv) of TBTU, 174 mg (0.45 mmol, 0.7 equiv)
HATU, and 78 mg (0.26 mmol, 0.4 equiv) of DEPBT. The crude reac-
tion was purified by column chromatography (silica gel, EtOAc/
Hex) to yield the pentapeptide (172 mg, 34% yield).
4.3.7.5. Dipeptide MeO-Leu-Leu-NHBoc. Dipeptide MeO-Leu-
Leu-NHBoc was synthesized following the ‘General peptide syn-
thesis’ procedure. Utilizing 401 mg (2.2 mmol, 1.1 equiv) of amine
MeO-Leu-NH2, 500 mg (2.0 mmol, 1.0 equiv) of acid HO-Leu-
NHBoc, 1.4 mL (4 equiv) of DIPEA, 774 mg (1.2 mmol, 1.0 equiv)
of TBTU. The crude reaction was purified by column chromatogra-
phy (silica gel, EtOAc/Hex) to yield the dipeptide (627.4 mg, 87.2%
yield).
Rf: 0.3 (EtOAc:Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.7-0.9 (m, 18H), 1.4 (s, 9H), 1.4–
1.6 (m, 2H), 2.0–2.2 (m, 4H), 2.8 (m, 1H), 3.0 (m, 1H), 3.0–3.2 (m,
Rf: 0.35 (EtOAc:Hex 2.5:7.5).
1H NMR (400 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.4 (s, 9H), 1.6–
4H), 3.3 (s, 3H), 3.7 (s, 3H), 4.0–4.1 (m,
a
H), 4.3–4.4 (m,
a
H),
1.8 (m, 6H), 3.7 (s, 3H), 4.0–4.1 (br,
(br, 1H), 6.4 (d, 1H).
aH), 4.5–4.7 (m, aH), 4.8–4.9
4.6–4.7 (m,
aH), 5.1 (m,
aH), 5.3 (m,
aH), 7.1–7.3 (m, 10H).
4.3.6.8. Macrocycle Phe-N-Me-
D
-Phe-
D
-Val-Leu-Leu. Macrocycle
4.3.7.6. 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. (571.8 mg, 91% yield).
Phe-N-Me- -Phe-D-Val-Leu-Leu was synthesized following the
D
‘Macrocyclization procedure’. Utilizing 139 mg (0.21 mmol,
1.0 equiv) of linear pentapeptide, 0.3 mL (8 equiv) of DIPEA,
41 mg (0.13 mmol, 0.6 equiv) of TBTU, 65 mg (0.17 mmol,
0.8 equiv) HATU, and 13 mg (0.04 mmol, 0.2 equiv) of DEPBT. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) yield the macrocycle (2.3 mg, 0.07% yield).
Rf: 0.3 (EtOAc:Hex 1:1).
4.3.7.7. Pentapeptide MeO-
tapeptide MeO- -Phe-Leu- -Val-Leu-Leu-NHBoc was synthesized fol-
lowing the ‘General peptide synthesis’ procedure. Utilizing 443.8 mg
(1.1 mmol, 1.1 equiv) of amine MeO- -Phe-Leu- -Val-NH2, 354 mg
D-Phe-Leu-D-Val-Leu-Leu-NHBoc. Pen-
D
D
D
D
1H NMR (400 MHz, CD3OD): d0.8–1.0 (m, 18H), 1.3 (m, 2H), 4.4–
(1. mmol, 1.0 equiv) of acid HO-Leu-Leu-NHBoc, 1.44 mL (8 equiv) of
DIPEA, 198 mg (0.62 mmol, 0.6 equiv) of TBTU, and 235 mg
(0.62 mmol, 0.6 equiv) HATU. The crude reaction was purified by col-
umn chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide
(545 mg, 73% yield).
1.6 (m, 4H), 2.6 (m, 1H), 3.2–3.4 (m, 7H), 4.2 (m,
5.0 (m, 3 H), 7.2 (dd, 10H).
aH), 4.6 (m, aH),
a
LC–MS: m/z calcd for C36H51N5O5 (M+1) = 634.82, found 634.5.
4.3.7. Synthesis of compound 33
Rf: 0.6 (EtOAc:Hex 1:1).
4.3.7.1. Dipeptide MeO-D-Phe-Leu-NHBoc. Dipeptide MeO-
D
-
1H NMR (400 MHz, CD3OD): d 0.7–0.9 (m, 24H), 1.4 (s, 9H), 1.5–
1.7 (m, 6H), 2.2 (m, 2H), 3.0 (m, H), 3.1 (m, H), 3.7 (s, 3H), 4.0–4.2
Phe-Leu-NHBoc was synthesized following the ‘General peptide
synthesis’ procedure. Utilizing 951 mg (4.4 mmol, 1.1 equiv) of
(m, 2aH), 4.3–4.5 (m, 2aH), 4.6–4.7 (m, H), 7.0–7.4 (m, 5H).
amine MeO-D-Phe-NH2, 1000 mg (4.0 mmol, 1.0 equiv) of acid
HO-Leu-NHBoc, 2.8 mL (4 equiv) of DIPEA, 1545 mg (4.8 mmol,
1.2 equiv) of TBTU. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(1381 mg, 88% yield).
4.3.7.8. Macrocycle MeO-
D-Phe-Leu-D-Val-Leu-Leu-NHBoc. Mac-
rocycle -Phe-Leu- -Val-Leu-Leu was synthesized following the
D
D
‘Macrocyclization procedure’. Utilizing 226 mg (0.37 mmol,
1.0 equiv) of linear pentapeptide, 0.67 mL (10 equiv) of DIPEA,

5818
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
96.04 mg (0.3 mmol, 0.8 equiv) of TBTU, 113.7 mg (0.3 mmol,
0.8 equiv) HATU, and 44.8 mg (0.15 mmol, 0.4 equiv) of DEPBT.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) yield the macrocycle (1 mg, 0.5 % yield).
Rf: 0.3 (100% EtOAc).
4.3.8.7. Pentapeptide MeO-
D
-Phe-Leu-
D
-MeVal-Leu-Leu-NHBoc.
-Val-Leu-Leu-NHBoc was
Pentapeptide MeO- -Phe-Leu-N-Me-
D
D
synthesized following the ‘General peptide synthesis’ procedure.
Utilizing 278 mg (0.63 mmol, 1.1 equiv) of amine MeO-N-Me-
Val-Leu-Leu-NH2, 217 mg (0.57 mmol, 1.0 equiv) of acid HO-
D
-
-
D
1H NMR (500 MHz, CD3OD): d 0.7–0.9 (m, 24H), 1.2–1.7 (m, 9H),
Phe-Leu-NHBoc, 0.40 mL (5 equiv) of DIPEA, 172 mg (0.57 mmol,
1.0 equiv) of DEPBT, and 45 mg (0.11 mmol, 0.2 equiv) HATU. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the pentapeptide (229 mg, 55% yield).
Rf: 0.35 (EtOAc:Hex 1:1).
2.0–2.1 (m, H), 2.2 (m, 1H), 3.0 (m, H), 3.1 (m, H), 3.9 (d,
(dd, H), 4.4–4.6 (m, 2 H), 5.2 (t, H), 7.0–7.4 (m, 5H).
aH), 4.0
a
a
LC–MS: m/z calcd for C32H51N5O5 (M+1) = 586.78, found 586.6.
4.3.8. Synthesis of compound 34
1H NMR (400 MHz, CD3OD): d 0.7–0.9 (m, 24H), 1.4 (s, 9H), 1.5–
4.3.8.1. Dipeptide MeO-
Phe-Leu-NHBoc was synthesized following the ‘General peptide
synthesis’ procedure. Utilizing 480 mg (2.2 mmol, 1.1 equiv) of
D
-Phe-Leu-NHBoc. Dipeptide MeO-
D
-
1.7 (m, 6H), 3.1 (s, 3H), 3.2–3.4 (m, 2H), 3.5 (s, 3H), 4.1 (d,
(d, H), 4.4 (t, H), 4.5–4.6 (m, 2 H), 4.6–4.7 (m, 2H), 5.0 (d, 1H),
6.7 (d, 1H), 6.8 (m, 1H), 7.0 (d, 1H), 7.1–7.3 (m, 5H).
aH), 4.2
a
a
a
amine MeO-D-Phe-NH2, 505 mg (2.0 mmol, 1.0 equiv) of acid HO-
Leu-NHBoc, 1.4 mL (4 equiv) of DIPEA, 772 mg (2.4 mmol,
1.2 equiv) of TBTU. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(786 mg, 94% yield).
4.3.8.8. Macrocycle
D-Phe-Leu-N-Me-D-Val-Leu-Leu. Macrocycle
D
-Phe-Leu-N-Me- -Val-Leu-Leu was synthesized following the
D
‘Macrocyclization procedure’. Utilizing 193.2 mg (0.31 mmol,
1.0 equiv) of linear pentapeptide, 0.6 mL (11 equiv) of DIPEA,
50.3 mg (0.16 mmol, 0.5 equiv) of TBTU, 83.3 mg (0.22 mmol,
0.7 equiv) HATU, and 46.8 mg (0.16 mmol, 0.5 equiv) of DEPBT.
The crude reaction was purified by reverse phase-HPLC to yield
the macrocycle (15 mg, 8% yield).
Rf: 0.9 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m,
2H), 1.8–2.0 (m, 1H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br, 1H),
4.8 (br,
aH), 4.8–5.0 (q,
aH), 6.5 (m, 1H), 7.1–7.4 (m, 5H).
Rf: 0.25 (EtOAc:Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 24H), 1.3–1.8 (m, 9H),
4.3.8.2. Dipeptide HO-
D
-Phe-Leu-NHBoc. Dipeptide HO-D-Phe-
2.2 (m, 1H), 2.8 (s, 3H), 2.9–3.1 (m, 2H), 4.0 (m,
aH), 4.3 (m, aH),
Leu-NHBoc was synthesized following the ‘General acid deprotec-
tion’. This dipeptide was taken on to the next reaction without
further purification or characterization. (632 mg, 89% yield).
4.5 (m, 2 H), 4.8 (m, H), 7.0 (d, 1H), 7.2–7.3 (m, 5H), 7.6 (d,
a
a
1H), 8.3 (m, 1H), 8.7 (s, 1H).
LC–MS: m/z calcd for C33H53N5O5 (M+1) = 600.4, found 600.7.
4.3.8.3. Dipeptide MeO-Leu-Leu-NHBoc. Dipeptide MeO-Leu-
Leu-NHBoc was synthesized following the ‘General peptide syn-
thesis’ procedure. Utilizing 801 mg (4.4 mmol, 1.1 equiv) of amine
MeO-Leu-NH2, 1.0 g (4.0 mmol, 1.0 equiv) of acid HO-Leu-NHBoc,
2.8 mL (4 equiv) of DIPEA, 1.5 g (4.8 mmol, 1.2 equiv) of TBTU.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the dipeptide (1.4 g, 99% yield).
Rf: 0.9 (EtOAc:Hex 1:1).
4.3.9. Synthesis of compound 37
4.3.9.1. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-
Leu-NHBoc was synthesized following the ‘General peptide synthe-
sis’ procedure. Utilizing 476 mg (2.2 mmol, 1.1 equiv) of amine
MeO-Phe-NH2, 500 mg(2.0 mmol, 1.0 equiv)of acidHO-Leu-NHBoc,
1.4 mL(4 equiv)of DIPEA, 708 mg(2.2 mmol, 1.1 equiv)ofTBTU. The
crude reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the dipeptide (682 mg, 87% yield).
Rf: 0.9 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.4 (s, 9H), 1.6–
1.8 (t, 6H), 3.7 (s, 3H), 4.0–4.1 (dd,
(br, 1H), 6.4 (d, 1H).
aH), 4.5–4.7 (m, aH), 4.8–4.9
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m,
3H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br, 1H), 4.8 (br,
5.0 (q, H), 6.5 (m, 1H), 7.1–7.4 (m, 5H).
aH), 4.8–
a
4.3.8.4. 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. (1.2 g, 82% yield).
4.3.9.2. 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 purifica-
tion or characterization. (508 mg, 100% yield).
4.3.8.5. Tripeptide MeO-N-Me-
MeO-N-Me- -Val-Leu-Leu-NHBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 258 mg (2.3 mmol,
1.1 equiv) of amine MeO-N-Me- -Val-NH, 563 mg (1.6 mmol,
D-Val-Leu-Leu-NHBoc. Tripeptide
D
4.3.9.3. Tripeptide MeO-Phe-Leu-
D-Val-NHBoc. Tripeptide MeO-
Phe-Leu- -Val-NHBoc was synthesized following the ‘General pep-
D
D
tide synthesis’ procedure. Utilizing 960 mg (2.5 mmol, 1.1 equiv) of
amine MeO-Phe-Leu-NH2, 485 mg (2.2 mmol, 1.0 equiv) of acid
HO-D-Val-NHBoc, 1.6 mL (4 equiv) of DIPEA, 788 mg (2.5 mmol,
1.1 equiv) of TBTU. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the tripeptide
(951 mg, 87% yield).
1.0 equiv) of acid HO-Leu-Leu-NHBoc, 1.1 mL (4 equiv) of DIPEA,
620 mg (1.6 mmol, 1.0 equiv) of HATU. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (341 mg, 40% yield).
Rf: 0.5 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.9–1.1 (m, 18H), 1.5 (s, 9H), 1.6–
1.8 (m, 1H), 2.1 (m, 2H), 2.8 (s, 1H), 3.0 (d, 3H), 3.7 (s, 3H), 4.0–4.2
Rf: 0.7 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.9–1.1 (m, 12H), 1.5 (s, 9H), 1.6–
1.7 (m, 2H), 1.8 (s, 1H), 2.1 (m, 1H), 3.1 (m, 2H), 3.7 (s 3H), 3.9 (dd,
(br, 2H), 4.2 (m, aH), 5.0 (s, aH), 5.1 (s, aH), 5.2–5.4 (br, aH), 5.5 (m,
1H), 6.8 (d, 1H).
1H), 4.4 (br,
aH), 4.8 (dd, aH), 5.0 (d, aH), 6.3 (s, 1H), 6.6 (d, 1H),
7.1–7.3 (m, 5H).
4.3.8.6. Tripeptide
MeO-N-Me-D-Val-Leu-Leu-NH2. Tripeptide
MeO-N-Me-D-Val-Leu-Leu-NH2 was synthesized following the
4.3.9.4. Tripeptide MeO-Phe-Leu-D-Val-NH2. Tripeptide MeO-Phe-
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization.
(278 mg, 100% yield).
Leu- -Val-NH2 was synthesized following the ‘General amine depro-
tection’. This dipeptide was taken on to the next reaction without fur-
ther purification or characterization. (758 mg, 100% yield).
D

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5819
4.3.9.5. Dipeptide MeO-
Leu-NHBoc was synthesized following the ‘General peptide syn-
thesis’ procedure. Utilizing 801 mg (4.4 mmol, 1.1 equiv) of amine
D
-Leu-Leu-NHBoc. Dipeptide MeO-
D
-Leu-
4.3.10.3. Tripeptide MeO-Phe-N-Me-D-Phe-Val-NHBoc. Tripep-
tide MeO-Phe-N-Me- -Phe-Val-NHBoc was synthesized following
the ‘General peptide synthesis’ procedure. Utilizing 337 mg
(1.0 mmol, 1.1 equiv) of amine MeO-Phe-N-Me- -Phe-NH,
D
MeO-
D
-Leu-NH2, 1.0 g (4.0 mmol, 1.0 equiv) of acid HO-Leu-
D
NHBoc, 2.8 mL (4 equiv) of DIPEA, 1.5 g (4.8 mmol, 1.2 equiv) of
TBTU. The crude reaction was purified by column chromatography
(silica gel, EtOAc/Hex) to yield the dipeptide (1.4 g, 99% yield).
Rf: 0.9 (EtOAc:Hex 1:1).
196 mg (0.9 mmol, 1.0 equiv) of acid HO-Val-NHBoc, 0.7 mL
(4 equiv) of DIPEA, 116 mg (0.36 mmol, 0.4 equiv) of TBTU, and
240 mg (0.63mmol, 0.7 equiv) The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the tripep-
tide (324 mg, 63% yield).
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.4 (s, 9H), 1.6–
1.8 (t, 6H), 3.7 (s, 3H), 4.0–4.1 (dd,
(br, 1H), 6.4 (d, 1H).
aH), 4.5–4.7 (m,
aH), 4.8–4.9
Rf: 0.5 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 0.5–0.7 (m, 6H), 1.4 (s, 9H), 1.5 (m,
1H), 2.9 (s, 3H), 3.0–3.4 (m, 4H), 3.7 (s, 3H), 4.2 (m,
aH), 4.8 (m,
4.3.9.6. Dipeptide HO-
D
-Leu-Leu-NHBoc. Dipeptide HO-
D
-Leu-
a
H), 5.1 (d, 1H), 5.4 (m, H), 6.8 (d, 1H), 7.0–7.4 (m, 10H).
a
Leu-NHBoc was synthesized following the ‘General acid deprotec-
tion’. This dipeptide was taken on to the next reaction without fur-
ther purification or characterization. (1.2 g, 82% yield).
4.3.10.4. Tripeptide MeO-Phe-N-Me-
D-Phe-Val-NH2. Tripeptide
MeO-Phe-N-Me- -Phe-Val-NH2 was synthesized following the
D
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization.
(264 mg, 100% yield).
4.3.9.7. Pentapeptide MeO-Phe-Leu-
tapeptide MeO-Phe-Leu- -Val- -Leu-Leu-NHBoc was synthesized fol-
lowing the ‘General peptide synthesis’ procedure. Utilizing 758 mg
(1.9 mmol, 1.0 equiv) of amine MeO-Phe-Leu- -Val-NH2, 667 mg
(1.9 mmol, 1.0 equiv) of acid HO- -Leu-Leu-NHBoc, 3.6 mL (11 equiv)
D-Val-D-Leu-Leu-NHBoc. Pen-
D
D
D
4.3.10.5. Dipeptide MeO-Cha-Leu-NHBoc. Dipeptide MeO-Cha-
Leu-NHBoc was synthesized following the ‘General peptide syn-
thesis’ procedure. Utilizing 489.1 mg (2.2 mmol, 1.1 equiva.) of
amine MeO-Cha-NH2, 500 mg (2.0 mmol, 1.0 equiv) of acid HO-
Leu-NHBoc, 1.4 mL (8 equiv) of DIPEA, 708 mg (2.2 mmol,
1.0 equiv) of TBTU. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(710 mg, 88% yield).
D
of DIPEA, 373 mg (1.2 mmol, 0.6 equiv) of TBTU, 116 mg (0.38 mmol,
0.2 equiv) of DEPBT, and 441 mg (1.1 mmol, 0.6 equiv) HATU. The
crude reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the pentapeptide (379 mg, 27% yield).
Rf: 0.4 (EtOAc:Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 24H), 1.4 (s, 9H), 1.5–
1.7 (m, 6H), 2.8 (s, 3H), 3.0–3.2 (m, 1H), 3.3 (d, 1H), 3.6 (s, 3H), 4.1
Rf: 0.5 (EtOAc:Hex 1:2).
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.1–1.3 (m, 4H),
(d,
aH), 4.2 (d, aH), 4.4 (t, aH), 4.5–4.6 (m, 2aH), 4.6–4.7 (m, 2H),
6.1 (d, 1H), 7.1–7.3 (m, 5H), 7.4 (m, 2H), 7.6 (d, 1H).
1.4 (s, 9H), 1.6–1.8 (m, 9H), 3.7 (s, 3H), 4.0–4.2 (dd,
(m, H), 4.8–4.9 (br, 1H), 6.2 (d, 1H).
aH), 4.5–4.7
a
4.3.9.8. Macrocycle Phe-Leu-D-Val-D-Leu-Leu. Macrocycle Phe-
Leu- -Val- -Leu-Leu was synthesized following the ‘Macrocycliza-
D
D
4.3.10.6. Dipeptide HO-Cha-Leu-NHBoc. Dipeptide HO-Cha-Leu-
NHBoc was synthesized following the ‘General acid deprotection’.
This dipeptide was taken on to the next reaction without further
purification or characterization. (674 mg, 99% yield).
tion procedure’. Utilizing 257 mg (0.43 mmol, 1.0 equiv) of linear
pentapeptide, 0.9 mL (12 equiv) of DIPEA, 68.3 mg (0.21 mmol,
0.5 equiv) of TBTU, 113 mg (0.30 mmol, 0.7 equiv) HATU, and
63.7 mg (0.21 mmol, 0.5 equiv) of DEPBT. The crude reaction was
purified by reverse phase-HPLC to yield the macrocycle (1.4 mg,
1% yield).
4.3.10.7. Pentapeptide MeO-Phe-N-Me-D-Phe-Val-Cha-Leu-
NHBoc. Pentapeptide MeO-Phe-N-Me-D-Phe-Val-Cha-Leu-NHBoc
was synthesized following the ‘General peptide synthesis’ proce-
dure. Utilizing 264 mg (0.6 mmol, 1.1 equiv) of amine MeO-Phe-
N-Me-D-Phe-Val-NH2, 217.4 mg (0.54 mmol, 1.0 equiv) of acid
Rf: 0.25 (EtOAc:Hex 1:1).
1H NMR (400 MHz, CD3OD): d 0.7–1.0 (m, 24H), 1.3–1.8 (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),7.2 (d, 1H), 7.6 (d, 1H), 8.2
a
a
HO-Cha-Leu-NHBoc, 0.4 mL (4 equiv) of DIPEA, 70 mg (0.22 mmol,
0.4 equiv) of TBTU, and 165 mg (0.43 mmol, 0.8 equiv) HATU. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the pentapeptide (210 mg, 60% yield).
Rf: 0.5 (EtOAc:Hex 2:1).
(d, 1H), 8.6 (d, 1H), 8.7 (s, 1H).
LC–MS: m/z calcd for C32H51N5O5 (M+1) = 586.4, found 587.5.
4.3.10. Synthesis of compound 49
4.3.10.1. Dipeptide MeO-Phe-D-MePhe-NBoc. Dipeptide MeO-
1H NMR (400 MHz, CD3OD): d 0.4–0.6 (dd, 3H), 0.6–0.8 (dd, 3H),
0.8–1.0 (m, 6H), 1.0–1.2 (m, 4H), 1.4 (s, 9H), 1.6–1.8 (m, 9H), 3.0 (s,
Phe-N-Me- -Phe-NBoc was synthesized following the ‘General
D
peptide synthesis’ procedure. Utilizing 515.5 mg (2.4 mmol,
1.1 equiv) of amine OMe-Phe-NH2, 606 mg (2.2.0 mmol, 1.0 equiv)
of acid HO-N-Me-D-Phe-NBoc, 1.52 mL (4 equiv) of DIPEA, 767 mg
(2.4 mmol, 1.1 equiv) of TBTU. The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the dipep-
tide (436.7 mg, 55% yield).
3H), 2.8–3.1 (m, 2H), 3.1–3.4 (m, 2H), 3.7 (s, 3H), 4.1 (m,
4.4 (m, H), 4.4–4.6 (m, H), 4.7–4.9 (m, H), 5.0 (br, 1H), 5.4–5.5
(m, H), 6.4 (d, 1H), 6.6 (d, 1H), 6.8 (d, 1H), 7.0–7.4 (m, 10H).
aH), 4.3–
a
a
a
a
4.3.10.8. Macrocycle Phe-N-Me-D-Phe-Val-Cha-Leu. Macrocycle
Phe-N-Me- -Phe-Val-Cha-Leu was synthesized following the ‘Mac-
D
Rf: 0.5 (EtOAc:Hex 1:1).
1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H), 1.6 (m, 2H), 2.6–2.8 (d,
rocyclization procedure’. Utilizing 210 mg (0.25 mmol, 1.0 equiv)
of linear pentapeptide, 0.2 mL (4 equiv) of DIPEA, 57 mg
(0.18 mmol, 0.7 equiv) of TBTU, 67 mg (0.18 mmol, 0.7 equiv)
HATU, and 53 mg (0.18 mmol, 0.7 equiv) of DEPBT. The crude reac-
tion was purified by reverse phase-HPLC to yield the macrocycle
(28 mg, 14% yield).
3H), 3.2–3.2 (m, 4H), 3.7 (s, 3H), 4.6–5.0 (m, 2
7.2–7.4 (m, 10H), 8.1 (m, 1H).
aH), 6.9–7.1 (s, 1H),
4.3.10.2. Dipeptide MeO-Phe-N-Me-
D-Phe-NH. Dipeptide MeO-
Phe-N-Me- -Phe-NH was synthesized following the ‘General
D
Rf: 0.5 (EtOAc:Hex 3:1).
amine deprotection’. This dipeptide was taken on to the next reac-
tion without further purification or characterization. (337.1 mg,
100% yield).
1H NMR (400 MHz, CD3OD): d 0.4–0.5 (dd, 3H), 0.6–0.7 (dd, 3H),
0.8–1.0 (m, 6H), 1.0–1.2 (m, 4H), 1.6–1.8 (m, 9H), 3.0 (s, 3H), 2.8–

5820
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
3.1 (m, 4H), 4.1 (m,
(m, aH), 7.0–7.4 (m, 10H).
a
H), 4.2–4.3 (m,
a
H), 4.4–4.7 (m, 2
a
H), 5.1–5.2
chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide
(279 mg, 39% yield). Rf: 0.5 (EtOAc:Hex 1:1); 1H NMR (500 MHz,
CDCl3): d 1.1–1.2 (br, 6H), 1.4 (s, 20H), 2.0 (s, 3H), 3.0–3.1 (m,
2H), 3.1–3.2 (m, 2H), 3.4 (dd, 1H), 3.6 (m, 1H), 3.7 (s, 3H), 3.9
LC–MS: m/z calcd for C33H51N5O5 (M+1) = 598.39, found 598.3.
4.3.11. Synthesis of compound 60
4.3.11.1. Dipeptide MeO-Phe-N-Me-
(m, 2H), 4.3 (br,
(m, H), 4.7–4.8 (m,
7.2–7.4 (m, 15H).
a
H), 4.4 (m,
aH), 4.5 (m, aH), 4.5 (s, 2H), 4.6
D
-Phe-NBoc. Dipeptide MeO-
a
aH), 5.5 (m, 2H), 6.7 (m, 1H), 7.0 (d, 1H),
Phe-N-Me- -Phe-NBoc was synthesized following the ‘General
D
peptide synthesis’ procedure. Utilizing 258 mg (1.20 mmol,
1.1 equiv) of amine MeO-Phe-NH2, 500 mg (1.09 mmol, 1.0 equiv)
of acid HO-N-Me-D-Phe-NBoc, 0.95 mL (5 equiv) of DIPEA, and
4.3.11.8. Macrocycle Phe-
D-MePhe-Val-Cha-Ser(Bn). Macrocycle
Phe- -MePhe-Val-Cha-Ser(Bn) was synthesized following the
D
385 mg (1.20 mmol, 1.1 equiv) of TBTU. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the dipeptide (480 mg, 99% yield).
‘Macrocyclization procedure’. Utilizing 242 mg (0.32 mmol,
1.0 equiv) of linear pentapeptide, 0.22 mL (4 equiv) of DIPEA,
51 mg (0.16 mmol, 0.5 equiv) of TBTU, 61 mg (0.16 mmol,
0.5 equiv) HATU, and 48 mg (0.16 mmol, 0.5 equiv) of DEPBT. The
crude reaction was purified by reverse phase-HPLC to yield the
macrocycle (7.2 mg, 3.5% yield). Rf: 0.5 (EtOAc:Hex 9:1); 1H NMR
(400 MHz, CD3OD): 0.9–1.0 (br, 6H), 1.5–1.7 (m, 11H), 1.9 (m,
Rf: 0.5 (EtOAc:Hex 2:3); 1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H),
2.7 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 1H), 4.7 (m,
2aH), 7.1–7.3 (dd, 10H).
4.3.11.2. Dipeptide MeO-Phe-N-Me-D-Phe-NH. Dipeptide MeO-
2H), 3.0–3.1 (m, 4H), 3.6–3.7 (m, 2H), 4.1 (m,
aH), 4.2 (m, aH),
Phe-N-Me- -Phe-NH was synthesized following the ‘General
D
4.3 (m, 2 H), 4.4 (m, H), 4.5 (m, 1H), 5.1 (m, 1H), 5.3 (m, 1H),
a
a
amine deprotection’. This dipeptide was taken on to the next reac-
tion without further purification or characterization. (371 mg,
100% yield).
5.4 (m, 1H), 7.1–7.3 (dd, 10H); LC–MS: m/z calcd for C39H49N5O6
(M+1) = 648, found 650.2.
4.3.12. Synthesis of compound 61
4.3.11.3. Tripeptide MeO-Phe-N-Me-
MeO-Phe-N-Me- -Phe-Val-NHBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 371 mg (1.09 mmol,
1.1 equiv) of amine MeO-Phe-N-Me- -Phe-NH, 215 mg (0.99 mmol,
1.0 equiv) of acid HO-Val-NHBoc, 0.95 mL (5 equiv) of DIPEA,
349 mg (1.09 mmol, 1.1 equiv) of TBTU. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the tripeptide (491 mg, 92% yield). Rf: 0.5 (EtOAc:Hex 2:3); 1H
NMR (200 MHz, CDCl3): d 1.0 (d, 6H), 1.4 (s, 9H), 2.9 (s, 3H), 3.1
D-Phe-Val-NHBoc. Tripeptide
4.3.12.1. Macrocycle
D-Phe-Leu-N-Me-Val-
D-Leu-D-Phe. Macro-
D
cycle -Phe-Leu-N-Me-Val-
D
D
-Leu- -Phe was synthesized following
D
the ‘Macrocyclization procedure’. Utilizing 151 mg (0.23 mmol,
1.0 equiv) of linear pentapeptide, 0.61 mL (8 equiv) of DIPEA,
45.8 mg (0.14 mmol, 0.6 equiv) of TBTU, 52.5 mg (0.13 mmol,
0.6 equiv) HATU, and 41 mg (0.14 mmol, 0.6 equiv) of DEPBT. The
crude reaction was purified by reverse phase-HPLC to yield the
macrocycle (42 mg, 28% yield).
D
Rf: 0.5 (EtOAc:Hex 4:1).
(t, 4H), 3.7 (s, 3H), 4.1–4.3 (m, 2aH), 4.7–4.8 (m, 2aH), 5.0–5.1
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.3 (s, 1H), 1.5
(t, 4H), 1.7 (m, 1H), 2.8 (m, 1H), 3.0 (d, 2H), 3.2 (m, 2H), 3.2 (s, 3H),
(br, 1H), 5.5 (m, 1H), 7.2–7.3 (dd, 10H).
4.2 (m, 3
8.4 (d, 1H), 8.5 (s, 1H).
aH), 4.5 (m, 2aH), 7.1–7.3 (m, 10H), 7.7 (s, 1H), 7.9 (s, 1H),
4.3.11.4. Tripeptide MeO-Phe-N-Me-
D-Phe-Val-NH2. Tripeptide
MeO-Phe-N-Me- -Phe-Val-NH2 was synthesized following the
D
LC–MS: m/z calcd for C36H51N5O5 (M+1) = 633.82, found 634.7.
‘General amine deprotection’. This dipeptide was taken on to
the next reaction without further purification or characteriza-
tion. (400 mg, 100% yield).
4.3.13. Synthesis of compound 62
4.3.13.1. Macrocycle Phe-Leu-N-Me-Val-Leu-
D-Phe. Macrocycle
Phe-Leu-N-Me-Val-Leu- -Phe was synthesized following the ‘Mac-
D
4.3.11.5. Dipeptide MeO-Cha-Ser(Bn)-NHBoc. Dipeptide MeO-
Cha-Ser(Bn)-NHBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 412 mg (1.86 mmol,
1.1 equiv) of amine MeO-Cha-NH2, 500 mg (1.69 mmol,
1.0 equiv) of acid HO-Ser(Bn)-NHBoc, 1.4 mL (5 equiv) of DIPEA,
597 mg (1.86 mmol, 1.1 equiv) of TBTU. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to
yield the dipeptide (781 mg, 99% yield). Rf: 0.5 (EtOAc:Hex
2:3); 1H NMR (200 MHz, CDCl3): d 1.4 (s, 20H), 1.6–1.7 (m, 2H),
rocyclization procedure’. Utilizing 287 mg (0.44 mmol, 1.0 equiv)
of linear pentapeptide, 0.61 mL (8 equiv) of DIPEA, 84 mg
(0.26 mmol, 0.6 equiv) of TBTU, 100 mg (0.26 mmol, 0.6 equiv)
HATU, and 78 mg (0.26 mmol, 0.6 equiv) of DEPBT. The crude reac-
tion was purified by reverse phase-HPLC to yield the macrocycle
(75 mg, 26% yield).
Rf: 0.5 (EtOAc:Hex 4:1).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 18H), 1.3–1.8 (m, 6H),
2.7 (m, 1H), 2.9 (m, 2H), 3.1 (m, 2H), 3.2 (s, 3H), 4.4 (m, 3
H), 7.1–7.3 (m, 10H), 7.7 (s, 1H), 8.0 (s, 1H), 8.2 (d, 1H), 8.7 (s, 1H).
LC–MS: m/z calcd for C36H51N5O5 (M+1) = 633.82, found 635.1.
aH), 4.7 (m,
3.7 (s, 3H), 3.8 (d, 2H), 4.1 (m, 2H), 4.4 (s, 2H), 4.5 (d, 2
7.3–7.4 (dd, 5H).
aH),
2a
4.3.11.6. Dipeptide HO-Cha-Ser(Bn)-NHBoc. Dipeptide HO-Cha-
Ser(Bn)-NHBoc was synthesized following the ‘General acid depro-
tection’. This dipeptide was taken on to the next reaction without
further purification or characterization. (715 mg, 94% yield).
4.3.14. Synthesis of compound 63
4.3.14.1. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-
Leu-NHBoc was synthesized following the ‘General peptide synthe-
sis’ procedure. Utilizing 951 mg (4.4 mmol, 1.1 equiv) of amine
MeO-Phe-NH2, 1000 mg (4.0 mmol, 1.0 equiv) of acid HO-Leu-
NHBoc, 2.8 mL (4 equiv) of DIPEA, 1546 mg (4.8 mmol, 1.2 equiv)
ofTBTU. Thecrudereactionwaspurifiedbycolumnchromatography
(silica gel, EtOAc/Hex) to yield the dipeptide (1564 mg, 99% yield).
Rf: 0.8 (EtOAc:Hex 1:1).
4.3.11.7. Pentapeptide MeO-Phe-N-Me-
NHBoc. Pentapeptide MeO-Phe-N-Me-
D
-Phe-Val-Cha-Ser(Bn)-
-Phe-Val-Cha-Ser(Bn)-
D
NHBoc was synthesized following the ‘General peptide synthesis’
procedure. Utilizing 400 mg (0.91 mmol, 1.1 equiv) of amine
MeO-Phe-N-Me-D-Phe-Val-NH2, 370 mg (0.82 mmol, 1.0 equiv)
of acid HO-Cha-Ser(Bn)-NHBoc, 0.57 mL (4 equiv) of DIPEA,
132 mg (0.41 mmol, 0.5 equiv) of TBTU, and 156 mg (0.41 mmol,
0.5 equiv) HATU. The crude reaction was purified by column
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m,
2H), 3.0–3.1 (m, 2H), 3.7 (s, 3H), 4.8–5.1 (s, 2H), 4.1 (m,
H), 6.5 (d, 1H), 7.0–7.3 (m, 5H).
aH), 4,8 (m,
a

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5821
4.3.14.2. 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. (1165 mg, 100% yield).
62.3 mg (0.16 mmol, 0.6 equiv) HATU, and 49 mg (0.16 mmol,
0.6 equiv) of DEPBT. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) yield the macrocycle
(37.5 mg, 16.8% yield).
Rf: 0.5 (EtOAc:Hex 4:1).
4.3.14.3. Tripeptide MeO-Phe-Leu-N-Me-Val-NBoc. Tripeptide MeO-
Phe-Leu-N-Me-Val-NBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 510 mg (1.74 mmol, 1.1 equiv) of
amine MeO-Phe-Leu-NH2, 367 mg (1.58 mmol, 1.0 equiv) of acid HO-
N-Me-Val-NBoc, 1.1 mL (4 equiv) of DIPEA, 723 mg (1.9 mmol,
1.2 equiv) of HATU. The crude reaction was purified by column chroma-
tography (silica gel, EtOAc/Hex) to yield the tripeptide (585 mg, 73%
yield).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 12H), 1.2–1.6 (m, 8H),
1.7–1.9 (m, 1H), 2.1–2.3 (m, 2H), 2.9 (s, 3H), 2.7–3.1 (m, 4H), 4.1–
4.3 (m, 3
7.4 (m, 15H).
LC–MS: m/z calcd for C44H57ClN6O7 (M+1) = 818.41, found
818.2.
aH), 4.5 (m, aH), 4.7 (m, aH), 5.1 (d, 1H), 5.2 (s, 2H), 7.0–
4.3.15. Removal of carbobenzyloxy groups via acid to yield
compound 64
Rf: 0.6 (EtOAc:Hex 1:1).
1H NMR (400 MHz, CDCl3): d0.9–1.1 (m, 12H), 1.5 (s, 9H), 1.6–1.8
4.3.15.1. Macrocycle Phe-Leu-N-Me-
26 mg (0.03 mmol, 1.0 equiv) of compound 140 and 0.32 mL of
HBr to remove the protecting group on the Lysine at Position
D-Val-D-Lys-D-Phe. Utilizing
(m, 1H), 2.3 (m, 2H), 2.8 (s, 3H), 3.1 (m, 2H), 3.7 (s, 3H), 4.0 (d,
aH), 4.4
(m, H), 4.8 (m, H), 6.3 (d, 1H), 6.5 (d, 1H), 7.1–7.3 (m, 5H).
a
a
D-
Lys(2-Cl-Cbz). The crude reaction was taken on to the next reaction
without further purification or characterization (20.8 mg, 100%
yield).
4.3.14.4. Tripeptide MeO-Phe-Leu-N-Me-Val-NH. Tripeptide MeO-
Phe-Leu-N-Me-Val-NH was synthesized following the ‘General amine
deprotection’. This tripeptide was taken on to the next reaction without
further purification or characterization. (468 mg, 100% yield).
1H NMR (400 MHz, CD3OD): d 0.8–1.0 (m, 12H), 1.2–1.7 (m, 8H),
1.7–1.9 (m, 1H), 2.1–2.3 (m, 2H), 2.9 (s, 3H) 2.8–2.9 (m, 4H), 4.2 (m,
aH), 4.3 (m, 2aH), 4.5 (m, aH), 4.7 (m, aH), 5.0 (d, 1H), 7.0–7.3 (m,
4.3.14.5. Tetrapeptide MeO-Phe-Leu-N-Me-Val-D-Lys(2-Cl-Cbz)-
9H)
NHBoc. Tetrapeptide MeO-Phe-Leu-N-Me-Val-
D
-Lys(2-Cl-Cbz)-
LC–MS: m/z calcd for C36H52N6O5 (M+1) = 649.84, found 649.6
NHBoc was synthesized following the ‘General peptide synthesis’
procedure. Utilizing 468 mg (1.1 mmol, 1.1 equiv) of amine MeO-
Phe-Leu-N-Me-Val-NH, 436 mg (1.0 mmol, 1.0 equiv) of acid HO-
Lys(2-Cl-Cbz)-NHBoc, 1.27 mL (7 equiv) of DIPEA, 135 mg
(0.4 mmol, 0.4 equiv) of TBTU, and 320 mg (0.8 mmol, 0.8 equiv)
of HATU. The crude reaction was purified by column chromatog-
raphy (silica gel, EtOAc/Hex) to yield the dipeptide (675 mg, 80%
yield).
4.3.16. Synthesis of compound 65
4.3.16.1. Dipeptide MeO-Phe-N-Me-D-Phe-NBoc. Dipeptide MeO-
Phe-N-Me-
synthesis’ procedure. Utilizing 258 mg (1.20 mmol, 1.1 equiv) of amine
MeO-Phe-NH2, 500 mg (1.09 mmol, 1.0 equiv) of acid HO-N-Me-
D-Phe-NBoc was synthesized following the ‘General peptide
D-
Phe-NBoc, 0.95 mL (5 equiv) of DIPEA, and 385 mg (1.20 mmol,
1.1 equiv) of TBTU. The crude reaction was purified by column chroma-
tography (silica gel, EtOAc/Hex) to yield the dipeptide (480 mg, 99%
yield).
Rf: 0.35 (EtOAc:Hex 1:1).
1H NMR (400 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.4 (s, 9H), 1.4–
1.8 (m, 9H), 2.3 (m, 2H), 3.0 (s, 3H), 3.1–3.2 (m, 2H), 3.7 (s, 3H), 4.3
Rf 0.5 (EtOAc:Hex 2:3); 1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H),
2.7 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 1H), 4.7 (m,
(m,
aH), 4.4 (d, 1H), 4.6 (m, aH), 4.8 (m, aH), 4.9 (m, aH), 5.2 (s,
2H), 5.3 (d, 1H), 6.4 (d, 1H), 6.5 (d, 1H), 7.1–7.4 (m, 9H).
2aH), 7.1–7.3 (dd, 10H).
4.3.14.6. Tetrapeptide MeO-Phe-Leu-N-Me-Val-D-Lys(2-Cl-Cbz)-
4.3.16.2. Dipeptide MeO-Phe-N-Me-D-Phe-NH. Dipeptide MeO-
NH2. Tetrapeptide MeO-Phe-Leu-N-Me-Val- -Lys(2-Cl-Cbz)-NH2
D
Phe-N-Me- -Phe-NH was synthesized following the ‘General
D
was synthesized following the ‘General amine deprotection’. This
tetrapeptide was taken on to the next reaction without further
purification or characterization. (590 mg, 100% yield).
amine deprotection’. This dipeptide was taken on to the next reac-
tion without further purification or characterization. (371 mg,
100% yield).
4.3.14.7. Pentapeptide MeO-Phe-Leu-N-Me-Val-
D
D
-Lys(2-Cl-Cbz)-
-Phe-NHBoc. Pentapeptide MeO-Phe-Leu-N-Me-Val- -Lys(2-Cl-
-Phe-NHBoc was synthesized following the ‘General peptide
4.3.16.3. Tripeptide MeO-Phe-N-Me-
tide MeO-Phe-N-Me- -Phe-Val-NHBoc was synthesized following
the ‘General peptide synthesis’ procedure. Utilizing 371 mg
(1.09 mmol, 1.1 equiv) of amine MeO-Phe-N-Me- -Phe-NH,
215 mg (0.99 mmol, 1.0 equiv) of acid HO-Val-NHBoc, 0.95 mL
(5 equiv) of DIPEA, 349 mg (1.09 mmol, 1.1 equiv) of TBTU. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the tripeptide (491 mg, 92% yield). Rf:
0.5 (EtOAc:Hex 2:3); 1H NMR (200 MHz, CDCl3): d 1.0 (d, 6H), 1.4
D-Phe-Val-NHBoc. Tripep-
D
D
Cbz)-
D
synthesis’ procedure. Utilizing 570 mg (0.8 mmol, 1.1 equiv) of
amine MeO-Phe-Leu-N-Me-Val- -Lys(2-Cl-Cbz)-NH2, 195 mg
(0.7 mmol, 1.0 equiv) of acid HO- -Phe-NHBoc, 0.6 mL (5 equiv)
D
D
D
of DIPEA, 95 mg (0.3 mmol, 0.4 equiv) of TBTU, and 224 mg
(0.6 mmol, 0.8 equiv) HATU. The crude reaction was purified by
column chromatography (silica gel, EtOAc/Hex) to yield the penta-
peptide (549 mg, 79% yield).
(s, 9H), 2.9 (s, 3H), 3.1 (t, 4H), 3.7 (s, 3H), 4.1–4.3 (m, 2
aH), 4.7-
Rf: 0.3 (EtOAc:Hex 1:1).
4.8 (m, 2 H), 5.0–5.1 (br, 1H), 5.5 (m, 1H), 7.2–7.3 (dd, 10H).
a
1H NMR (400 MHz, CD3OD): d 0.7–0.9 (m, 12H), 1.4 (s, 9H), 1.3–
1.7 (m, 9H), 2.3 (m, 2H), 3.0 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), (d,
4.3.16.4. Tripeptide MeO-Phe-N-Me-
D-Phe-Val-NH2. Tripeptide
a
H), 4.3–4.4 (m, 2
a
H), 4.8–4.9 (m, 2aH), 5.0 (br,
aH), 5.2 (s, 2H),
MeO-Phe-N-Me- -Phe-Val-NH2 was synthesized following the
D
6.4–6.5 (dd, 2H), 6.9 (d, 1H), 7.1–7.5 (m, 14H).
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization.
(400 mg, 100% yield).
4.3.14.8. Macrocycle
Phe-Leu-N-Me-Val-
D
-Lys(2-Cl-Cbz)-
D-
Phe. Macrocycle Phe-Leu-N-Me-Val-
D
-Lys(2-Cl-Cbz)-
D
-Phe was
synthesized following the ‘Macrocyclization procedure’. Utilizing
228 mg (0.27 mmol, 1.0 equiv) of linear pentapeptide, 0.38 mL
(8 equiv) of DIPEA, 43.8 mg (0.14 mmol, 0.5 equiv) of TBTU,
4.3.16.5. Dipeptide MeO-Cha-Ser(Bn)-NHBoc. Dipeptide MeO-
Cha-Ser(Bn)-NHBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 412 mg (1.86 mmol, 1.1 equiv)

5822
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
of amine MeO-Cha-NH2, 500 mg (1.69 mmol, 1.0 equiv) of acid HO-
Ser(Bn)-NHBoc, 1.4 mL (5 equiv) of DIPEA, 597 mg (1.86 mmol,
1.1 equiv) of TBTU. The crude reaction was purified by column
chromatography (silica gel, EtOAc/Hex) to yield the dipeptide
(781 mg, 99% yield). Rf: 0.5 (EtOAc:Hex 2:3); 1H NMR (200 MHz,
CDCl3): d 1.4 (s, 20H), 1.6–1.7 (m, 2H), 3.7 (s, 3H), 3.8 (d, 2H), 4.1
crude reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the tripeptide (512 mg, 96% yield). Rf: 0.5 (EtOA-
c:Hex 2:3); 1H NMR (200 MHz, CDCl3): d 1.0–1.1 (m, 6H), 1.4 (s, 9H),
2.9 (m, 1H), 3.0 (s, 3H), 3.4–3.5 (br, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 2
aH),
4.8 (m, H), 5.0 (br, 1H), 5.4 (br, 1H), 7.2–7.3 (m, 10H).
a
(m, 2H), 4.4 (s, 2H), 4.5 (d, 2aH), 7.3–7.4 (dd, 5H).
4.3.17.4. Tripeptide MeO-Phe-N-Me-D-Phe-D-Val-NH2. Tripeptide
MeO-Phe-N-Me- -Phe- -Val-NH2 was synthesized following the
D
D
4.3.16.6. Dipeptide HO-Cha-Ser(Bn)-NHBoc. Dipeptide HO-Cha-
Ser(Bn)-NHBoc was synthesized following the ‘General acid depro-
tection’. This dipeptide was taken on to the next reaction without
further purification or characterization. (715 mg, 94% yield).
‘General amine deprotection’. This dipeptide was taken on to the
next reaction without further purification or characterization.
(417 mg, 100% yield).
4.3.17.5. Dipeptide MeO-Cha-Ser(Bn)-NHBoc. Dipeptide MeO-Cha-
Ser(Bn)-NHBoc was synthesized following the ‘General peptide syn-
thesis’ procedure. Utilizing 412 mg (1.86 mmol, 1.1 equiv) of amine
MeO-Cha-NH2, 500 mg (1.69 mmol, 1.0 equiv) of acid HO-Ser(Bn)-
NHBoc, 1.4 mL (5 equiv) of DIPEA, 597 mg (1.86 mmol, 1.1 equiv) of
TBTU. The crude reaction was purified by column chromatography (sil-
ica gel, EtOAc/Hex) to yield the dipeptide (780 mg, 99% yield). Rf: 0.5
(EtOAc:Hex 2:3); 1H NMR (200 MHz, CDCl3): d 1.4 (s, 20H), 1.5–1.6
4.3.16.7. Pentapeptide MeO-Phe-N-Me-
NHBoc. Pentapeptide MeO-Phe-N-Me-
NHBoc was synthesized following the ‘General peptide synthesis’
procedure. Utilizing 400 mg (0.91 mmol, 1.1 equiv) of amine
MeO-Phe-N-Me-D-Phe-NH, 370 mg (0.82 mmol, 1.0 equiv) of acid
HO-Cha-Ser(Bn)-NHBoc, 0.57 mL (4 equiv) of DIPEA, 132 mg
(0.41 mmol, 0.5 equiv) of TBTU, and 156 mg (0.41 mmol, 0.5 equiv)
HATU. The crude reaction was purified by column chromatography
(silica gel, EtOAc/Hex) to yield the pentapeptide (279 mg, 39%
yield). Rf: 0.5 (EtOAc:Hex 1:1); 1H NMR (500 MHz, CDCl3): d 1.1–
1.2 (br, 6H), 1.4 (s, 20H), 2.0 (s, 3H), 3.0–3.1 (m, 2H), 3.1–3.2 (m,
2H), 3.4 (dd, 1H), 3.6 (m, 1H), 3.7 (s, 3H), 3.9 (m, 2H), 4.3 (br,
D
-Phe-Val-Cha-Ser(Bn)-
-Phe-Val-Cha-Ser(Bn)-
D
(m, 2H), 3.7 (s, 3H), 3.9 (dd, 2H), 4.1 (br, 2H), 4.5 (s, 2H), 4.6 (m, 2
aH),
7.2–7.3 (s, 5H).
4.3.17.6. Dipeptide HO-Cha-Ser(Bn)-NHBoc. Dipeptide HO-Cha-
Ser(Bn)-NHBoc was synthesized following the ‘General acid depro-
tection’. This dipeptide was taken on to the next reaction without
further purification or characterization. (766 mg, 99% yield).
aH), 4.4 (m,
aH), 4.5 (m, aH), 4.5 (s, 2H), 4.6 (m, aH), 4.7–4.8 (m,
aH), 5.5 (m, 2H), 6.7 (m, 1H), 7.0 (d, 1H), 7.2–7.4 (m, 15H).
4.3.16.8. Macrocycle Phe-N-Me-D-Phe-Val-Cha-Ser(Bn). Macro-
4.3.17.7. Pentapeptide
MeO-Phe-N-Me-
D
-Phe- -Val-Cha-
D
cycle Phe-N-Me- -Phe-Val-Cha-Ser(Bn) was synthesized following
D
Ser(Bn)-NHBoc. Pentapeptide MeO-Phe-N-Me-
D-Phe-D-Val-Cha-
the ‘Macrocyclization procedure’. Utilizing 242 mg (0.32 mmol,
1.0 equiv) of linear pentapeptide, 0.22 mL (4 equiv) of DIPEA,
51 mg (0.16 mmol, 0.5 equiv) of TBTU, 61 mg (0.16 mmol,
0.5 equiv) HATU, and 48 mg (0.16 mmol, 0.5 equiv) of DEPBT. The
crude reaction was purified by reverse phase-HPLC to yield the
macrocycle (7.2 mg, 2.0% yield). Rf: 0.5 (EtOAc:Hex 9:1); 1H NMR
(400 MHz, CD3OD): d 0.9–1.0 (br, 6H), 1.4–1.6 (m, 11H), 1.6–1.7
(m, 2H), 2.2 (br, 1H), 3.0–3.1 (m, 4H), 3.6–3.8 (m, 2H), 4.4–4.5
Ser(Bn)-NHBoc was synthesized following the ‘General peptide
synthesis’ procedure. Utilizing 417 mg (0.94 mmol, 1.1 equiv) of
amine MeO-Phe-N-Me-D-Phe-D-Val-NH2, 386 mg (0.86 mmol,
1.0 equiv) of acid-Cha-Ser(Bn)-NHBoc, 0.60 mL (4 equiv) of DIPEA,
138 mg (0.43 mmol, 0.5 equiv) of TBTU, and 164 mg (0.43 mmol,
0.5 equiv) HATU. The crude reaction was purified by column chro-
matography (silica gel, EtOAc/Hex) to yield the pentapeptide
(210 mg, 28% yield). Rf: 0.5 (EtOAc:Hex 3:2); 1H NMR (500 MHz,
CDCl3): d 0.9–1.0 (m, 6H), 1.4 (s, 9H), 1.5–1.6 (s, 11H), 1.8 (m,
2H), 2.8 (s, 3H), 3.0 (m, 2H), 3.2 (m, 2H), 3.6 (s, 3H), 3.8 (m, 1H),
(m, 2aH), 4.5–4.6 (br, 2aH), 5.1 (m, 2H), 7.1 (m, 2H), 7.1–7.3 (dd,
15H)LC–MS: m/z calcd for C43H55N5O6 (M+1) = 738, found 738.36.
4.2 (m, 2aH), 4.4 (m, 2aH), 4.5 (m, 2H), 4.7 (m, aH), 5.0 (br, 1H),
4.3.17. Synthesis of compound 66
6.2 (d, 1H), 6.4 (d, 1H), 6.5 (d, 1H), 7.1–7.3 (dd, 15H).
4.3.17.1. Dipeptide MeO-Phe-N-Me-D-Phe-NBoc. Dipeptide MeO-
Phe-N-Me- -Phe-NBoc was synthesized following the ‘General
D
4.3.17.8. Macrocycle Phe-N -Me-D-Phe-D-Val-Cha-Ser(Bn). Mac-
peptide synthesis’ procedure. Utilizing 258 mg (1.20 mmol,
1.1 equiv) of amine MeO-Phe-NH2, 500 mg (1.09 mmol, 1.0 equiv)
of acid HO-N-Me-D-Phe-NBoc, 1.0 mL (5 equiv) of DIPEA, and
rocycle Phe-N-Me- -Phe- -Val-Cha-Ser(Bn) was synthesized fol-
D
D
lowing the ‘Macrocyclization procedure’. Utilizing 182 mg
(0.24 mmol, 1.0 equiv) of linear pentapeptide, 0.16 mL (4 equiv)
of DIPEA, 39 mg (0.12 mmol, 0.5 equiv) of TBTU, 46 mg (0.12 mmol,
0.5 equiv) HATU, and 36 mg (0.12 mmol, 0.5 equiv) of DEPBT. The
crude reaction was purified by reverse phase-HPLC to yield the
macrocycle (1.0 mg, 1.0% yield). Rf: 0.5 (EtOAc:Hex 3:1); 1H NMR
(400 MHz, CD3OD): d 0.9–1.0 (dd, 6H), 1.2–1.3 (m, 4H), 1.3–1.4
(m, 6H), 1.7–1.8 (m, 1H), 1.9 (br, 2H), 2.9 (s, 3H), 3.0–3.1 (m, 3H),
385 mg (1.20 mmol, 1.1 equiv) of TBTU. The crude reaction was
purified by column chromatography (silica gel, EtOAc/Hex) to yield
the dipeptide (480 mg, 99% yield).
Rf: 0.5 (EtOAc:Hex 2:3); 1H NMR (200 MHz, CDCl3): d 1.4 (s, 9H),
2.7–2.8 (s, 3H), 3.1–3.2 (s, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 1H), 4.9–5.0
(br, 2aH), 7.2–7.4 (dd, 10H).
3.1–3.2 (m, 3H), 3.7 (m, 2H), 3.8 (m, 2H), 3.9 (m,
aH), 4.2 (m,
4.3.17.2. Dipeptide MeO-Phe-N-Me-
D
-Phe-NH. Dipeptide MeO-
2a
H), 4.4 (m, 2 H, 4.5 (s, 2H), 7.1–7.3 (dd, 15H); LC–MS: m/z calcd
a
Phe-N-Me- -Phe-NH was synthesized following the ‘General
D
for C43H55N5O6 (M+1) = 738, found 738.5.
amine deprotection’. This dipeptide was taken on to the next reac-
tion without further purification or characterization. (371 mg,
100% yield).
4.3.18. Synthesis of compound 67
4.3.18.1. Dipeptide MeO-
Phe- -Val-NHBoc was synthesized following the ‘General peptide
synthesis’ procedure. Utilizing 476 mg (2.2 mmol, 1.1 equiv) of
amine MeO- -Phe-NH2, 500 mg (2.0 mmol, 1.0 equiv) of acid HO-
-Val-NHBoc, 1.4 mL (4 equiv) of DIPEA, and 773 mg (2.4 mmol,
D-Phe-D-Val-NHBoc. Dipeptide MeO-D-
D
4.3.17.3. Tripeptide MeO-Phe-N-Me-
tide MeO-Phe-N-Me- -Phe- -Val-NHBoc was synthesized following
the ‘General peptide synthesis’ procedure. Utilizing 371 mg
(1.09 mmol, 1.1 equiv) of amine MeO-Phe-N-Me- -Phe-NH,
215 mg (0.99 mmol, 1.0 equiv) of acid HO- -Val-NHBoc, 0.95 mL
(5 equiv) of DIPEA, 349 mg (1.09 mmol, 1.1 equiv) of TBTU. The
D-Phe-D-Val-NHBoc. Tripep-
D
D
D
D
D
1.2 equiv) of TBTU. The crude reaction was washed 2 Â 100 mL
pH 1 water and 10 Â 100 mL saturated NaHCO3 to yield the dipep-
tide (788 mg, 95% yield). Rf: 0.5 (EtOAc:Hex 35:65); 1H NMR
D

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5823
(200 MHz, CDCl3): d 0.95 (d, 6H), 1.5 (s, 9H), 1.6–1.8 (m, 3H), 3.2
4.3.18.9. Pentapeptide HO-
D
-Phe-
D
-Leu-
D
-Val-Leu-Ser(Bn)-NH2.
(m, 2H), 3.7 (s, 3H), 4.0–4.1 (m,
1H), 7.1–7.4 (m, 5H).
a
H), 4.8–5.0 (m,
a
H), 6.4–6.6 (d,
Pentapeptide HO- -Phe-
D
D
-Leu-D-Val-Leu-Ser(Bn)-NH2 was syn-
thesized following the ‘General amine deprotection’. This penta-
peptide was taken on to the next reaction without further
purification. (191 mg, 100% yield). LC–MS: m/z calcd for
C36H53N5O7 (M+1) = 668.8, found 669.0.
4.3.18.2. Dipeptide MeO-D-Phe-D-Val-NH2. Dipeptide MeO-D-
Phe- -Val-NH2 was synthesized following the ‘General amine
D
deprotection’. This dipeptide was taken on to the next reaction with-
out further purification or characterization. (596 mg, 100% yield).
4.3.18.10. Macrocycle
D-Phe-D-Leu-D-Val-Leu-Ser(Bn). Macrocy-
cle -Phe- -Leu- -Val-Leu-Ser(Bn) was synthesized following the
D
D
D
4.3.18.3. Tripeptide MeO-
MeO- -Phe- -Leu- -Val-NHBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 596 mg
(1.9 mmol, 1.1 equiv) of amine MeO- -Phe- -Leu-NH2, 379 mg
(1.7 mmol, 1.0 equiv) of acid HO- -Val-NHBoc, 1.2 mL (5 equiv)
of DIPEA, 672 mg (0.85 mmol, 2.0 equiv) of TBTU. The crude reac-
tion was washed 3 Â 100 mL saturated NaCl. It was then purified
by column chromatography (silica gel, EtOAc/Hex) to yield the
tripeptide (857 mg, 94% yield). Rf: 0.5 (EtOAc:Hex 1:1); 1H NMR
(200 MHz, CDCl3): d 0.9–1.0 (m, 12H), 1.5 (s, 12H), 1.6–1.8 (m,
3H), 2.1 (m, 1H), 3.1 (d, 2H), 3.7 (s, 3H), 3.9–4.0 (dd, 1H), 4.4
D
-Phe-
D
-Leu-
D
-Val-NHBoc. Tripeptide
‘Macrocyclization procedure’. Utilizing 220 mg (0.32 mmol,
1.0 equiv) of linear pentapeptide, 0.34 mL (8 equiv) of DIPEA,
53.0 mg (0.17 mmol, 0.5 equiv) of TBTU, 62.7 mg (0.17 mmol,
0.5 equiv) HATU, and 49.4 mg (0.17 mmol, 0.5 equiv) of DEPBT.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the macrocycle (36.5 mg, 17% yield). Rf:
0.5 (EtOAc:Hex 70:30); 1H NMR (500 MHz, CD3OD): d 0.8–1.1 (m,
18H), 1.4 (s, 3H), 1.6–1.8 (m, 7H), 2.1 (d, 1H), 2.2 (m, 1H), 3.2–3.4
D
D
D
D
D
D
(m, 1H), 3.8 (m, 2H), 4.2 (m, 3aH), 4.5 (m, aH), 4.7 (m, 2aH), 6.9 (d,
1H), 7.2–7.5 (m, 10H), 7.9 (d, 1H), 8.2 (d, 1H), 8.4 (d, 1H), 8.7 (d,
1H); LC–MS: m/z calcd for C36H51N5O6 (M+1) = 650.8, found 651.4.
(m,
aH), 4.9 (m, aH), 5.0 (d, aH), 6.3 (d, 1H), 6.5 (d, 1H), 7.0–
7.4 (m, 5H).
4.3.19. Synthesis of compound 68
4.3.19.1. Dipeptide MeO-Phe-Leu-NHBoc. Dipeptide MeO-Phe-
Leu-NHBoc was synthesized following the ‘General peptide syn-
thesis’ procedure. Utilizing 477 mg (2.2 mmol, 1.1 equiv) of amine
MeO-Phe-NH2, 500 mg (2.0 mmol, 1.0 equiv) of acid HO-Leu-
NHBoc, 1.4 mL (4 equiv) of DIPEA, 773 mg (2.4 mmol, 1.2 equiv)
of TBTU. The crude reaction was purified by column chromatogra-
phy (silica gel, EtOAc/Hex) to yield the dipeptide (785 mg, 99%
yield). 0.5 (EtOAc:Hex 1:4).
4.3.18.4. Tripeptide
MeO-D-Phe-D-Leu-D-Val-NH2. Tripeptide
MeO- -Phe- -Leu- -Val-NH2 was synthesized following the ‘Gen-
D
D
D
eral amine deprotection’. This dipeptide was taken on to the next
reaction without further purification or characterization. (502 mg,
100% yield).
4.3.18.5. Dipeptide MeO-Leu-Ser(Bn)-NHBoc. Dipeptide MeO-
Leu-Ser(Bn)-NHBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 339 mg (1.9 mmol, 1.1 equiv)
of amine MeO-Leu-NH2, 500 mg (1.7 mmol, 1.0 equiv) of acid
HO-Ser(Bn)-NHBoc, 1.2 mL (4 equiv) of DIPEA, 652 mg (2.0 mmol,
1.2 equiv) of TBTU. The crude reaction was washed 2 Â 100 mL
with pH 1 water and 10 Â 100 mL with saturated NaHCO3 to yield
the dipeptide (636 mg, 89% yield). Rf: 0.5 (EtOAc:Hex 35:65); 1H
NMR (200 MHz, CDCl3): d 0.9–1.0 (m, 6H), 1.4 (s, 9H), 1.6–1.8 (m,
1H NMR (200 MHz, CDCl3): d 0.9 (m, 6H), 1.4–1.5 (s, 9H), 1.6–1.7
(br, 3H), 3.1–3.2 (t, 2H), 3.7–3.8 (s, 3H), 4.0–4.2 (m,
aH), 4.7–4.9
(m, H, 1H), 6.5 (d, 1H), 7.0–7.2 (br, 2H), 7.3–7.4 (s, 3H).
a
4.3.19.2. 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. (584 mg, 100% yield).
5H), 3.5 (dd, 2H), 3.7 (s, 3H), 3.9–4.0 (dd, 1H), 4.2 (m,
2H), 4.6 (m, H), 5.3 (d, 1H), 6.9 (d, 1p), 7.3 (m, 5H).
aH), 4.5 (s,
a
4.3.19.3. Tripeptide MeO-Phe-Leu-Val-NHBoc. Tripeptide MeO-
Phe-Leu-Val-NHBoc was synthesized following the ‘General pep-
tide synthesis’ procedure. Utilizing 578 mg (1.98 mmol, 1.0 equiv)
of amine MeO-Phe-Leu-NH2, 433 mg (1.94 mmol, 1.02 equiv) of
acid HO-Val-NHBoc, 1.90 mL (6 equiv) of DIPEA, 466 mg
(1.44 mmol, 0.7 equiv) of TBTU, 275 mg (0.72 mmol, 0.4 equiv) of
HATU. The crude reaction was purified by column chromatography
(silica gel, EtOAc/Hex) to yield the tripeptide (886 mg, 91% yield).
Rf: 0.5 (EtOAc:Hex 2:3).
4.3.18.6. Dipeptide HO-Leu-Ser(Bn)-NHBoc. Dipeptide HO-Leu-
Ser(Bn)-NHBoc was synthesized following the ‘General acid depro-
tection’. This dipeptide was taken on to the next reaction without
further purification or characterization. (631 mg, 98% yield).
4.3.18.7. Pentapeptide MeO-
NHBoc. Pentapeptide MeO- -Phe-
was synthesized following the ‘General peptide synthesis’ proce-
dure. Utilizing 488 mg (1.2 mmol, 1.1 equiv) of amine MeO-
Phe- -Leu- -Val-NH2, 463 mg (1.1 mmol, 1.0 equiv) of acid HO-
D
-Phe-
D
-Leu-
D-Val-Leu-Ser(Bn)-
D
D
-Leu-D-Val-Leu-Ser(Bn)-NHBoc
1H NMR (200 MHz, CDCl3): d 0.8–1.0 (m, 12H), 1.4–1.5 (s, 9H),
1.6–1.7 (m, 3H), 2.0–2.2 (m, 1H), 3.1 (d, 2H), 3.7 (s, 3H), 3.8–3.9
D
-
D
D
(q, aH), 4.4–4.5 (m, aH), 4.8–4.9 (q, aH), 5.0 (d, 1H), 6.3 (d, 1H),
Leu-Ser(Bn)-NHBoc, 1.2 mL (6 equiv) of DIPEA, and 436 mg
(1.4 mmol, 1.2 equiv) of TBTU. The crude reaction was washed
2 Â 100 mL with pH 1 water and 10 Â 100 mL with saturated NaH-
CO3. It was then purified by column chromatography (silica gel,
EtOAc/Hex) to yield the pentapeptide (435 mg, 52% yield). Rf: 0.5
(EtOAc:Hex 65:35); 1H NMR (400 MHz, CD3OD): d 0.8–0.9 (m,
18H), 1.3 (s, 1H), 1.4 (s, 9H), 1.6–1.8 (m, 7H), 2.1 (m, 1H), 3.0–3.2
6.4–6.5 (d, 1H), 7.1 (m, 2H), 7.2–7.4 (m, 3H).
4.3.19.4. Tripeptide MeO-Phe-Leu-Val-NH2. Tripeptide MeO-Phe-
Leu-Val-NH2 was synthesized following the ‘General amine depro-
tection’. This dipeptide was taken on to the next reaction without
further purification or characterization. (705 mg, 100% yield).
(m, 2H), 3.6–3.8 (m, 4H), 4.2 (m,
4.5–4.7 (d, 3H), 7.1–7.3 (m, 10H), 7.8 (d, 1H), 8.0 (d, 1H), 8.1 (d, 1H).
a
H), 4.3 (m,
a
H), 4.4 (m, 2
a
H),
4.3.19.5. Dipeptide MeO-Lys(Cbz)-N-Me-Leu-NBoc. Dipeptide
MeO-Lys(Cbz)-N-Me-Leu-NBoc was synthesized following the
‘General peptide synthesis’ procedure. Utilizing 742 mg
4.3.18.8. Pentapeptide HO-
D
-Phe-
D
-Leu-D-Val-Leu-Ser(Bn)-NHBoc. (2.2 mmol, 1.1 equiv) of amine MeO-Lys(Cbz)-NH2, 500 mg
Pentapeptide HO- -Phe-
D
D
-Leu-
D
-Val-Leu-Ser(Bn)-NHBoc was
(2.0 mmol, 1.0 equiv) of acid HO-N-Me-Leu-NBoc, 1.4 mL
(4 equiv) of DIPEA, 458 mg (1.4 mmol, 0.7 equiv) of TBTU. The
crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the dipeptide (1.05 g, 91% yield).
synthesized following the ‘General acid deprotection’. This penta-
peptide was taken on to the next reaction without further purifica-
tion or characterization. (252 mg, 60% yield).

5824
P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
Rf: 0.5 (EtOAc:Hex 2:3).
for support of R.C.V. We thank NIH 1U54CA132379-01A1 for sup-
port of SRM and VA.
1H NMR (200 MHz, CDCl3): d 0.9–1.0 (t, 6H), 1.2–1.4 (m, 2H), 1.5
(s, 9H), 1.6–1.7 (m, 6H), 1.8–1.9 (m, 1H), 2.7–2.8 (s, 3H), 3.1–3.2 (q,
2H), 3.7 (s, 3H), 4.5–4.7 (br,
6.5 (br, 1H), 6.5 (br, 1H), 7.3 (s, 1H), 7.4 (s, 4H).
aH), 4.8–4.9 (br, aH), 5.1 (s, 2H), 6.4–
Supplementary data
Supplementary data (1H NMR data and LC–MS data for com-
pounds and their intermediates, C log P values, % inhibition, and a
list of compounds with their structural variation by position) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.07.017.
4.3.19.6. Dipeptide HO-Lys(Cbz)-N-Me-Leu-NBoc. Dipeptide HO-
Lys(Cbz)-N-Me-Leu-NBoc was synthesized following the ‘General
acid deprotection’. This dipeptide was taken on to the next reaction
without further purification or characterization. (947 mg, 93% yield).
4.3.19.7. Pentapeptide MeO-Phe-Leu-Val-Lys(Cbz)-N-Me-Leu-
NBoc. Pentapeptide MeO-Phe-Leu-Val-Lys(Cbz)-N-Me-Leu-NBoc
was synthesized following the ‘General peptide synthesis’ proce-
dure. Utilizing 705 mg (1.80 mmol, 1.1 equiv) of amine MeO-
Phe-Leu-Val-NH2, 940 mg (1.72 mmol, 1.0 equiv) of acid HO-
Lys(Cbz)-N-Me-Leu-NBoc, 2.4 mL (8 equiv) of DIPEA, 347 mg
(1.08 mmol, 0.6 equiv) of TBTU, 342 mg (0.90 mmol, 0.50 equiv)
HATU, and 216 mg (0.72 mmol, 0.4 equiv) DEPBT. The crude
reaction was purified by column chromatography (silica gel,
EtOAc/Hex) to yield the pentapeptide (573 mg, 37% yield).
Rf: 0.5 (EtOAc:Hex 7:3).
References and notes
1. Cueto, M.; Jensen, P. R.; Fenical, W. Phytochemistry 2000, 55, 223.
2. Hwang, Y.; Rowley, D.; Rhodes, D.; Gertsch, J.; Fenical, W.; Bushman, F. Mol.
Pharmacol. 1999, 55, 1049.
3. Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1999, 40, 2913.
4. Liu, S.; Gu, W. D. L.; Ding, X.-Z.; Ujiki, M.; Adrian, T. E.; Soff, G. A.; Silverman, R.
B. J. Med. Chem. 2005, 48, 3630.
5. Ujiki, M.; Milam, B.; Ding, X.-Z.; Roginsky, A. B.; Salabat, M. R.; Talamonti, M. S.;
Bell, R. H.; Gu, W.; Silverman, R. B.; Adrian, T. E. Biochem. Biophys. Res. Commun.
2006, 340, 1224.
6. Styers, T. J.; Kekec, A.; Rodriguez, R. A.; Brown, J. D.; Cajica, J.; Pan, P.-S.; Parry,
E.; Carroll, C. L.; Medina, I.; Corral, R.; Lapera, S.; Otrubova, K.; Pan, C.-M.;
Mcguire, K. L.; Mcalpine, S. R. Bioorg. Med. Chem. 2006, 14, 5625.
7. Rodriguez, R. A.; Pan, P.-S.; Pan, C.-M.; Ravula, S.; Lapera, S. A.; Singh, E. K.;
Styers, T. J.; Brown, J. D.; Cajica, J.; Parry, E.; Otrubova, K.; Mcalpine, S. R. J. Org.
Chem. 2007, 72, 1980.
8. Pan, P. S.; Curtis, F. A.; Carroll, C. L.; Medina, I.; Liotta, L. A.; Sharples, G.;
Mcalpine, S. R. Bioorg. Med. Chem. 2006, 14, 4731.
9. Otrubova, K.; Styers, T. J.; Pan, P.-S.; Rodriguez, R.; Mcguire, K. L.; Mcalpine, S. R.
Chem. Commun. 2006, 1033.
10. Carroll, C. L.; Johnston, J. V. C.; Kekec, A.; Brown, J. D.; Parry, E.; Cajica, J.; Medina,
I.; Cook, K. M.; Corral, R.; Pan, P.-S.; Mcalpine, S. R. Org. Lett. 2005, 7, 3481.
11. Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 3743.
12. Gu, W.; Liu, S.; Silverman, R. B. Org. Lett. 2002, 4, 4171.
13. Otrubova, K.; Lushington, G. H.; Vander Velde, D.; Mcguire, K. L.; Mcalpine, S. R.
J. Med. Chem. 2008, 51, 530.
14. Pan, P. S.; Mcguire, K.; Mcalpine, S. R. Bioorg. Med. Chem. Lett. 2007, 17, 5072.
15. Amidon, G. L.; Lee, H. J. Annu. Rev. Pharmacol. Toxicol. 1994, 34, 321.
16. Wenger, R. M. Prog. Allergy 1986, 38, 46.
17. Jarvis, L. M. Chem. Eng. News 2006.
18. Marx, V. Chem. Eng. News 2005, 83, 17. http://pubs.acs.org/cen/business/83/
i11/8311bus1.html.
19. The most recent peptide drugs include Byetta from Amylin: <http://
www.drugs.com/pro/byetta.html>http://www.drugs.com/pro/
byetta.html<http://www.drugs.com/pro/byetta.html><http://
www.drugs.com/pro/byetta.html>, and from Aileron comes stapled peptides:
http://pubs.acs.org/cen/coverstory/86/8622cover.html.
20. Singh, E. K.; Sellers, R. P.; Alexander, L. D.; Mcalpine, S. R. Curr. Opin. Drug Disc.
Dev. 2008, 11, 544.
21. Loffet, A. Eur. Peptide Soc. 2002, 8, 1.
1H NMR (400 MHz, CDCL3): d 0.8–1.0 (m, 18H), 1.3–1.4 (m, 2H),
1.5 (m, 12H), 1.6–1.7 (m, 5H), 1.7–1.8 (s, 1H), 1.8–1.9 (br, 1H), 2.2
(br, 1H), 3.7–3.8 (d, 3H), 3.0–3.2 (m, 4H), 3.7 (s, 3H), 4.2 (m,
aH),
4.3–4.4 (br, H), 4.5 (br, H), 4.6–4.7 (br, H), 4.8 (m, H), 4.9
a
a
a
a
(br, 1H), 5.1 (s, 2H), 6.6–6.8 (br, 2H), 6.9 (br, 1H) 7.1–7.2 (d, 2H),
7.2–7.3 (m, 5H), 7.3–7.4 (m, 4H).
4.3.19.8. Macrocycle Phe-Leu-Val-Lys(Cbz)-N-Me-Leu. Macrocy-
cle Phe-Leu-Val-Lys(Cbz)-N-Me-Leu was synthesized following
the ‘Macrocyclization procedure’. Utilizing 478 mg (0.61 mmol,
1.0 equiv) of linear pentapeptide, 0.63 mL (6 equiv) of DIPEA,
97.3 mg (0.30 mmol, 0.50 equiv) of TBTU, 115 mg (0.30 mmol,
0.50 equiv) HATU, and 90.7 mg (0.30 mmol, 0.50 equiv) of DEPBT.
The crude reaction was purified by column chromatography (silica
gel, EtOAc/Hex) to yield the macrocycle (47.6 mg, 10.5% yield).
Rf: 0.5 (EtOAc:Hex 4:1).
1H NMR (400 MHz, CDCL3): d 0.7–1.1 (m, 18H), 1.2 (m, 1H), 1.4–
1.6 (m, 6H), 1.6–1.8 (m, 3H), 1.9 (s, 2H), 1 2.7 (s, 3H), 2.9–3.0 (m,
1H), 3.1–3.3 (m, 3H) 3.9 (t,
(br, H), 5.0 (m, H), 5.1 (s, 2H, 1
7H), 7.3–7.4 (m, 6H), 7.7–7.8 (s, 1H).
a
H), 4.3 (m,
aH), 4.4 (br, aH), 4.4–4.6
a
a
a
H), 6.7 (br, 1H), 7.1–7.3 (m,
22. Murr, M. M.; Sarr, M. G.; Oishi, A. J.; Heerden, J. A. CA Cancer J. Clin. 1994, 44,
304.
LC–MS: m/z calcd for C41H60N6O7 (M+1) = 748.45, found 749.5.
23. Burris, H. A.; Moore, M. J.; Andersen, J.; Greem, M. R.; Rothenberg, M. I.;
Modiano, M. R.; Cripps, M. C.; Portenoy, R. K.; Sotorniolo, A. M.; Tarassaoff, P.;
Nelson, R.; Dorr, F. A.; Stephens, C. D.; Vonhoff, D. J. Clin. Oncol. 1997, 15, 2403.
24. Sener, S. F.; Fremgen, A.; Menck, H. R.; Winchester, D. P. J. Am. Coll. Surg. 1999,
189, 1.
4.3.20. Removal of carbobenzyloxy groups via acid to yield
compound 69
Utilizing 47.6 mg (0.06 mmol, 1 equiv) of pentapeptide Phe-
Leu-Val-Lys(Cbz)-N-Me-Leu, the carbobenzyloxy group was re-
moved by dissolving the compound in 33% Hydrogen bromine
(HBr) in glacial acetic acid (0.1 M). The crude reaction was concen-
trated down in vacuo and purified via reversed-phase HPLC.
(17.2 mg, 84% yield).
25. Styers, T. J.; Rodriguez, R. A.; Pan, P.-S.; Mcalpine, S. R. Tetrahedron Lett. 2006,
47, 515.
26. The C log P values were calculated using an algorithm. The log P value of a
compound, which is the logarithm of its partition coefficient between n-
octanol and water log(coctanol/cwater), is a well established measure of the
compound’s hydrophilicity. Low hydrophilicities and therefore high log P
values cause poor absorption or permeation. It has been shown for compounds
to have a reasonable probability of being well absorbt their log P value must
not be greater than 5.0. The distribution of calculated log P values of more than
3000 drugs on the market underlines this fact.
Rf: 0.5 (EtOAc:Hex 5:1).
1H NMR (400 MHz, CD3OD): d 0.7–1.1 (m, 18H), 1.2 (m, 1H),
1.4–1.6 (m, 6H), 1.6–1.8 (m, 3H), 1.9 (s, 2H), 2.7 (s, 3H), 2.9–3.0
27. Lipinskis rules state that the ideal drug contains no more than five H-doners,
no more than 10 H-acceptors, molecular weights of less than 500, and log P
values 65.0.
(m, 1H), 3.1–3.3 (m, 3H), 3.9 (t,
4.4–4.6 (br, H), 5.0 (m, H), 5.1 (s, 2H, 1
7.4 (m, 6H), 8.4 (s, 1H).
a
H), 4.3 (m,
aH), 4.4 (br, aH),
a
a
aH), 6.7 (br, 1H), 7.3–
28. Dipeptide and tripeptide structures were confirmed using 1H NMR. All linear
pentapeptides were confirmed using LC–MS and 1H NMR. (Note: 1H NMR were
taken for cyclized peptides, but due to their complexity, they were not seen as the
primaryconfirmationforcyclizedcompounds.)SeeSupplementarydataforspectra.
29. Geistlinger, T. R.; Mcreynolds, A. C.; Guy, R. K. Chem. Biol. 2004, 11, 273.
Multiple coupling agents reference.
LC–MS: m/z calcd for C33H54N6O5 (M+1) = 614.82, found 615.5.
Acknowledgments
30. Bolla, M. L.; Azevedo, E. V.; Smith, J. M.; Taylor, R. E.; Ranjit, D. K.; Segall, A. M.;
Mcalpine, S. R. Org. Lett. 2003, 5, 109.
31. Two cell lines were chosen to provide evidence that our compounds were
targeting a type of cancer, that is, pancreatic. PL-45 is a primary pancreatic
We thank San Diego State University, the Frasch foundation
(658-HF07), and CSUPERB for financial support. We thank the Ho-
well Foundation for support of R.C.V. We thank NIW T90DK07015

P.-S. Pan et al. / Bioorg. Med. Chem. 17 (2009) 5806–5825
5825
ductial adenocarcinoma and BxPC3 is the cell line of choice for xenograft
mouse model studies.
32. Chatterjee, J.; Mierke, D. F.; Kessler, H. J. Am. Chem. Soc. 2006, 128, 15164.
33. Heller, M.; Sukopp, M.; Tsomaia, N.; John, M.; Mierke, D. F.; Reif, B.; Kessler, H. J.
Am. Chem. Soc. 2006, 128, 13806.
34. Zhang, X.; Nikiforovich, G. V.; Marshall, G. R. J. Med. Chem. 2007, 50, 2921.
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Thornton, J. M. Eur. J. Biochem. 1996, 242, 352.
37. See Supplementary data for C log P values.