Structure - Activity relationship studies of targeting ligands against breast cancer cells

Article abstract of DOI:10.1021/jm9012032

A series of LXY3 (1) analogues were designed and synthesized. Their binding affinity was demonstrated using MDA-MB-231 breast cancer cells adherence inhibition assay. Further structure-activity relationship was obtained. Analogue 29 was discovered to have 3.5-fold increase of the binding affinity. Fluorescent microscopy and in vivo and ex vivo imaging studies demonstrated that 29 is an efficient in vivo targeting agent against α3 integrin of MDA-MB-231 breast tumor xenograft implant. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9012032

6744 J. Med. Chem. 2009, 52, 6744–6751
DOI: 10.1021/jm9012032
Structure -Activity Relationship Studies of Targeting Ligands against Breast Cancer Cells
Nianhuan Yao, Wenwu Xiao, Leah Meza, Harry Tseng, Mathida Chuck, and Kit S. Lam*
Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center,
University of California Davis, 4501 X Street, Sacramento, California 95817
Received August 12, 2009
A series of LXY3 (1) analogues were designed and synthesized. Their binding affinity was demonstrated
using MDA-MB-231 breast cancer cells adherence inhibition assay. Further structure-activity relation-
ship was obtained. Analogue 29 was discovered to have 3.5-fold increase of the binding affinity.
Fluorescent microscopy and in vivo and ex vivo imaging studies demonstrated that 29 is an efficient in
vivo targeting agent against R3 integrin of MDA-MB-231 breast tumor xenograft implant.
Introduction
radiolabeling and drug targeting.18-20 “The one-bead-one-
compound” (OBOC) combinatorial library method, devel-
oped in our lab,21,22 was widely used to identify peptide
and peptidomimetic ligands against various interesting re-
ceptors.23-25 Using this approach, we recently identified a
Integrins are heterodimeric cell surface proteins consisting
of R and β subunits that mediate extracellular matrix and
cell-cell interactions and regulate cellular activities such as
adhesion, migration, proliferation, and differentiation.1-3
Many integrins have been found to play an important role in
cancer invasion and metastasis, tumor angiogenesis, immune
functions, and tissue repair.4,5 Integrin R3β1 is a promiscuous
receptor reported to bind laminin, collagen, fibronectin, and
entactin.6-8 It participates in the phagocytosis of extracellular
matrix molecules by human breast cancer cells.9 Integrin R3β1
is also involved in the initial anchoring of MDA-MB-231
human breast cancer cells to cortical bone10 and the adhesion
and spreading of metastatic breast carcinoma cells to the
lymph node stroma.11 Increasing evidence that integrin R3β1
is significantly up-regulated on MDA-MB-231 cells9,12,13
suggests that R3β1 would be of great interest as a promising
receptor to develop targeting agents for delivering radionu-
clides, toxins, or cytotoxic agents to the breast tumor site.
Immunotargeting using monoclonal antibodies is a promis-
ing approach for early detection and treatment of cancer.14-16
Solid tumors, however, have proven less responsive in part
because of difficulties in the tumor selective delivery of anti-
bodies and potential cytolytic effectors.17 To circumvent these
problems, peptides and peptidomimetics have been inten-
sively studied for tumor imaging and targeting therapy be-
cause of their rapid clearance and high tumor penetration.
The best-studied is RGD containing ligands developed for
novel cyclic octapeptide
1
(cdGTyr(3-NO2)GHypNc,a
LXY3) (Figure 1) to specifically bind to R3 integrin on
MDA-MB-231 breast cancer cells. Further in vivo and ex
vivo imaging studies revealed that 1 effectively targeted R3
integrin on MDA-MB-231 breast tumor xenograft model.26
Here, with the aim to understand structure-activity rela-
tionship (SAR) and develop novel 1 analogues with higher
binding affinity, we performed a series of structural modifica-
tions on the peptide portion of 1. Their binding affinities to
MDA-MB-231 cell lines were evaluated using high efficiency
adherence inhibition assay. Modifications of Tyr(3-NO2) on
position 4 successfully yielded several derivatives with higher
affinities than 1.
Results and Discussions
In our search of novel ligands for breast cancer cells
through screening various random OBOC libraries, cyclic
octapeptides were found to bind to MDA-MB-231 cell lines.
Further “alanine scanning” on the positive-cyclic octapeptide
clearly revealed that residues D-Cys-1, Gly-3, Gly-5, and D-
Cys-8 are necessary for MDA-MB-231 cell binding. Subse-
quently, D-Asp-2, L-Hyp-6, and small polar amino acids in
position 7 were found to be essential for binding against
MDA-MB-231 cells in screenings of highly focused OBOC
cyclic octapeptide libraries.26
*To whom correspondence should be addressed. Phone: (916) 734-
8012. Fax: (916) 734-7946. E-mail:Kit.lam@ucdmc.ucdavis.edu.
aAbbreviations: Dab, R,γ-diaminobutyric acid; Tyr(3-NO2), 3-nitro-
tyrosine; HoSer, homoserine; Hyp, hydroxyproline; Thz, L-thiaproline;
Homotyr, homotyrosine; Fmoc, fluorenylmethoxycarbonyl; ESI, elec-
trospray ionization; NMR, nuclear magnetic resonance; HOBt,
N-hydroxybenzotriazole; DIC, N,N0-diisopropylcarbodiimide; PyBrop,
bromotripyrrolidinophosphonium hexafluorophosphate; DIEPA, N,N-
diisopropylethylamine; NMM, N-methylmorpholine; RP-HPLC, re-
verse-phase high performance liquid chromatography; TFA, trifluor-
oacetic acid; DMF, N,N-dimethylformamide; DCM, dichloromethane.
Abbreviations used for amino acids and designation of peptides follow
the rules of the IUPAC-IUB Commission of Biochemical Nomenclature
in J. Biol. Chem. 1972, 247, 977-983.
On the basis of above preliminary structure-activity rela-
tionship (SAR) information, we determined whether changes
to certain structural aspects of the peptide segment of 1
influenced binding affinity by replacing the disulfide bridge
and systematically modifying amino acid residues from the C
to N termini of the sequence. Derivatives are grouped on
amino acid substitutions or modifications, including five
separate segments of 1: (i) replacement of the disulfide bridge;
(ii) substitution of Asn; (iii) substitution or modifications of
Hyp; (iv) substitution of Tyr(3-NO2); (v) derivatization of the
N-terminus (Figure 1).
r
pubs.acs.org/jmc
Published on Web 10/16/2009
2009 American Chemical Society

Article
LXY1 was previously characterized as a specific ligand
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6745
Replacement of the Cystine Bridge. Disulfide bonds are
metabolically and chemically unstable structural features in
many naturally occurring and synthetic peptides.27 Replace-
ment of the S-S motif of a cystine bridge with more stable
alternatives, such as carbon-carbon bond, carbon-carbon
double bond, and amide bond,28-33 has long been regarded
as an attractive strategy to alleviate these problems. Syn-
thesis of the dicarba analogues of 1 (2) was achieved by ring-
close metathesis31-35 (Scheme 1). The linear precursor pep-
tide was prepared on Rink amide resin by Fmoc chemistry.36
D-Allyglycine was incorporated in place of D-Cys to facilitate
cyclization and retain original ring size as well. Ring-closing
metathesis of resin bound linear peptide was carried out in
1,2-dichloroethane at 60 °C for 30 h by using 25% Hoveyda-
Grubbs second-generation ruthenium catalyst. The resulting
cyclic olefinic peptides were cleaved from the resin by TFA
treatment in the usual manner, followed by Fmoc deprotec-
tion. The configuration of the double bond was established
by measurement of the J coupling constants between the
olefinic protons. The high J values observed (22.5 Hz) is
indicative of trans stereochemistry for the newly formed
double bond.
against R3 integrin of MDA-MB-231 breast cancer cells with
binding affinity Kd = 0.4 ( 0.07 μM via flow cytometry.26
This method iscomplicated, tedious, and time-consuming and
consequently not suitable for high throughput screening. To
circumvent these problems, a simple adherence inhibition
assay23 was developed to determine the half-maximal inhibi-
tory concentration (IC50) of the analogues for their ability to
inhibit binding of MDA-MB-231 cells to immobilized LXY1.
The relative binding affinity of these novel derivatives are
shown in Table 1.
Cyclization with an amide bond is the general strategy
to synthesize a cyclic peptide. To maintain the same ring
size as 1, Fmoc-D-Asp(OAll) and Fmoc-D-Dap(Alloc) were
selected to couple to the C and N termini, respectively,
Figure 1. Structure of 1.
Table 1. Analogues of 1 and Their IC50
compd
N-terminus
D-Cys 1
Tyr(3-NO2) 4
Hyp 6
Asn 7
D-Cys 8
IC50 (μM)
1
7.4
2
D-allyglycine
D-Dap
D-allyglycine
D-Asp
no
>25
>25
>25
>25
17.1
13.6
16.9
15.3
18.5
10.5
>25
21.4
24.1
>25
19.8
>25
>25
>25
>25
>25
11.9
>25
11.3
23.7
11.9
>25
6.3
3
4
D-Lys
Asp
5
D-Pen
D-pen
6
Asp
Dab
7
8
Pro
9
3, 4-dehydro-Pro
trans-4-fluoro-Pro
Thz
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36-48
49-58
(2s,4s)-4-phenyl pyrrolidine-2-carboxylic acid
azetidine-2-carboxylic acid
pipecolic acid
HomoTyr
Tyr(3-Cl)
Tyr(3-I)
Tyr(3-NH2)
Tyr(3-OCH3)
Tyr(3,5-Br)
Tyr(3,5-I)
Tyr(3,5-2NO2)
Phe(2-F)
Phe(3-F)
Phe(4-F)
Phe(3-CF3)
Phe(4-CF3)
Phe(3,4-2F)
Phe(3,5-2F)
Phe(3,4,5-3F)
Phe(2,3,4,5,6-5F)
Phe(2-NO2)
Phe(3-NO2)
Phe(2-Cl)
2.1
5.9
11.7
19.6
3.1
19
Phe(3-CN)
>25
>25
>25
aldehyde
isocyanate

6746 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Scheme 1. Synthesis Route of 2
Yao et al.
instead of D-Cys. Following assembly of linear peptide on
the Rink resin, OAll and Alloc group were removed simul-
taneously with tetrakis(triphenylphosphine)palladium (0.24
equiv) and phenylsilane (20 equiv) in DCM. The subsequent
cyclization was carried out in solid phase with PyBOP/
DIEPA as coupling reagents instead of DIC to afford
3 (Figure 2). In the synthesis of cyclic peptide 4, Fmoc-D-
Lys(Alloc), which possesses a long space side chain,
was used to simulate the approximate length of the disulfide
bridge. Due to the truncation of the C-terminus (D-Cys),
Fmoc-L-Asp-OAll was first coupled to the Rink resin
instead of Fmoc-L-Asn. L-Asp residue was automati-
cally transformed to L-Asn, followed by cyclic peptide
4 cleavage from the resin. Introduction of bulky penicilla-
mine can often induce resistance to enzymatic clea-
vage. Cyclic peptide 5 was obtained by substituting D-Cys
with D-Pen.
In comparison with 1, cystine bridge replacement with a
carbon-carbon double bond, amide bond, or penicillamine
bridge resulted in drastic loss of binding affinity. These
results indicate that cysteine bridge disulfide bonds are
necessary for the MDA-MB-231 cells binding.
Substitution of Asn with Asp and Dab. In our previous
OBOC libraries,26 various natural and unnatural amino
acids, including polar, hydrophobic, and charged amino
acids, were introduced to position 7. Small polar amino
acids, such as Asn, Thr, and homoserine, were preferred at
position 7. To further investigate the importance of small
polar amino acids, small charged amino acids (Asp and Dab)
instead of Asn were introduced to position 7 to give 6 and 7,
respectively. When substituting with negatively charged
amino acid (Asp), binding affinity to MDA-MB-231 breast
cancer cells was reduced 2.3-fold. Additionally, when sub-
stituting with positive-charged amino acid (Dab), binding
affinity to MDA-MB-231 breast cancer cells was reduced
1.8-fold. These observations suggest that a hydrophilic
residue is required at position 7, and an electrostatic inter-
action between ligand 1 and R3 intergrin may not exist
around residue Asn.
Substitution of Hyp with Analogues of Pro and Modifica-
tions to Hyp. Our initial OBOC library screenings estab-
lished that hydroxyproline at position 6 is very important
for binding affinity.26 This amino acid limits the rotation
about the NR-CR bond and can induce β-turn conforma-
tion.37,38 To further probe this structure effects, cyclic
amino acids were substituted for Hyp (Figure 3). Proline
(8), 3, 4-dehydroproline (9), and trans-4-fluoro-L-proline
(10) reduced the binding affinity 2- to 3-fold. Thioproline
(11) resulted in a slight decrease in binding affinity. (2s,4s)-
4-Phenylpyrrolidine-2-carboxylic acid (12) and the homo-
logous cyclic amino acids, four-membered azetidine-2-car-
boxylic acid (13) and six-membered pipecolic acid (14),
resulted in a significant loss of binding affinity. Taken
together, these findings indicate that polar hydroxyproline

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6747
may be important for cell binding. Encouraged by these
results, we therefore hypothesized that further modification
of hydroxyproline may increase binding affinity. To test
this assumption, we replaced hydroxyproline with (2S,4R)-
4-amino-1-pyrrolidine-2-carboxylic acid, which can pro-
vide the extra amino group for easily reacting with different
carboxylic acids. As shown in Supporting Information
(Scheme S1), commercially available (2S,4R)-Fmoc-4-ami-
no-1-Boc-pyrrolidine-2-carboxylic acid (Fmoc(2S,4R)-
Abpc) was first converted to (2S,4R)-Alloc-4-amino-1-
Fmoc-pyrrolidine-2-carboxylic acid. Linear peptide was
assembled on 20% down-substituted TentaGel S NH2
(2 g, loading 0.27 mmol/g). After Alloc deprotection,
38 various carboxylic acids (Supporting Information Table
S1) were coupled to amino group of (2S,4R)-Abpc.
On-bead cyclization was carried out in the mixture solvent
including water, acetic acid, and DMSO (75:5:20) for 48 h.
The synthetic route is shown in Supporting Information
(Scheme S2). To rapidly evaluate and compare binding
affinity to 1, on-bead cell binding assay was utilized. No
cell binding was observed with these analogues within 5 h
compared to 1 (Figure S1), indicating that modifications of
Hyp led to a decrease in binding affinity.
Substitution of Tyr(3-NO2) with Analogues of Phe and Tyr.
Various natural amino acids, unnatural amino acids, lysine
derivatives, and phenylalanine derivatives were introduced
to position 4 during the initial synthesis of OBOC
libraries.26 Tyr(3-NO2) at position 4 was determined to
show strong binding affinity. To further investigate the
importance of Tyr(3-NO2), a series of analogues of tyrosine
and phenylalanine instead of Tyr(3-NO2) were introduced
to position 4 to afford compounds 15-35 (Figure 4).
Replacement of the nitro group of Tyr(3-NO2) with differ-
ent halogens (chloro(16) and iodo(17)), amino group
(18), and methoxyl (19) resulted in a significant dec-
rease in binding affinity. Introduction of extra halogens
(bromo(20) and iodo(21)) to the aromatic ring dramatically
reduced the binding affinity, while introduction of an extra
nitro group (22) to the aromatic ring slightly reduced
binding affinity, which indicated that a polar group (NO2)
that can generate hydrogen bonds is favorable to cell
binding.
When Tyr(3-NO2) was substituted with phenylalanine
analogues, compounds with a nitro group at the 3 position
(33) or fluorine at the 3 and 5 (29) positions of the aromatic
ring had 2- to 4-fold higher binding affinity than 1. 28
[Phe(3,4-2F)] and 30 [Phe(3,4,5-3F)] were as active as 1. In
contrast, compounds with fluorine, trifluoromethyl, and
nitro group at the 2 or 4 position of the aromatic ring
(23, 25, 27, 32, 34) or cyano group at the 3 position of
aromatic ring (35) showed lower affinity. Compounds
with single fluorine and trifluoromethyl at position 3 of
the aromatic ring (24, 26) and the analogue with five
fluorines (31) had slightly less affinity. Taking all of these
findings into account, the position of a nitro group and
fluorine appeared to be relevant to binding affinity; more-
over, the hydroxyl group of Tyr(3-NO2) was not required for
cell binding.
Figure 2. Strutures of 1 analogues with various bridges.
Figure 3. Building blocks used for the substitution of hydroxyproline.

6748 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Yao et al.
Figure 4. Unnatural amino acids used for the substitution of Tyr(3-NO2).
Modifications to the N-Terminus. The previous molecular
interaction between peptide ligand 1 and R3 integrin de-
monstrated that the D158A mutation resulted in a remark-
able decrease in binding affinity to 1,26 indicating that
electrostatic interaction between Asp (R3 integrin) and
N-terminus of 1 may facilitate cell binding. The reductive
alkylation of the N-terminal amino group could render the
N-terminus more basic and introduce diverse groups.39
A series of analogues of 1 were therefore designed and
synthesized with the N-terminal amino group alkylated
with various aliphatic and aromatic substituents. Linear
peptide 1 was first assembled on Rink resin. Unhindered
amino group of the N-terminus then underwent reductive
alkylation with excess aldehydes (Figure 6) and sodium
cyanoborohydride (NaBH3CN) in DMF to give products
36-48 (Figure 5). It is important to note that aromatic
aldehydes commonly afforded monoalkylated products.
Aliphatic aldehydes with less steric hindrance generated
monoalkylated and dialkylated products (38, 40). Urea, an
attractive pharmacophore, can generate hydrogen bonds
Figure 5. Structure of 1 analogues with N terminal modifications.
primary amino group was treated with various isocyanates
(Figure 6) and NMM in DMF to provide products 49-58
with a urea group.39 Compared to 1, all N-terminal deriva-
tives showed a remarkable decrease in binding affinity,
suggesting that the free N-terminal amino group is crucial
for cell binding.
and potentially increase binding affinity. The N-terminus

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6749
Figure 6. Building blocks used for modifications to N terminus.
Conclusions
(0.1% TFA) to 100% CH3CN (0.1% TFA) at a flow rate of
1.0 mL/min. Preparative HPLC was performed on a System
Gold 126NMP solvent module (Beckman) with a C18 column
(Vydac, 5 μm, 2.5 cm i.d. ꢀ 25 cm). A gradient elution of 0-60%
B over 25 min, followed by 60-100% B over 25 min, followed
by 100% B for 5 min, was used at a flow rate of 7 mL/min
(solvent A is H2O/0.1% TFA; solvent B is acetonitrile/
0.1% TFA). Anti R3 integrin antibodies were purchased from
Chemicon (CHEMICON International, Inc.). MDA-MB-231
cells were obtained from American Type Culture Collection
(Manassas,VA).
Cyclic peptide 1 has been systematically modified from
C- to N-terminus to provide 58 analogues. Their binding affi-
nities to R3β1 integrin in MDA-MB-231 breast cancer cells
were evaluated by using an adherence inhibition assay. One of
the ligands (29) demonstrated a 3.5-fold increase of binding
affinity compared to 1. Fluorescence microscopy(Figure S2)
and in vivo and ex vivo imaging studies (Figure S3) further
demonstrated that 29 efficiently targeted R3 integrin on
MDA-MB-231 breast tumor implant in a xenograft model.
WepreviouslydiscoveredthatD-Asp-2, Gly-3, and Gly-5were
critical for MDA-MB-231 cell binding. The absence of a side
chain in Gly possibly provided the conformational flexibility
of the cyclic peptide backbone and facilitated the appropriate
spatial orientation of pharmacophores, resulting in binding
affinity enhancement. For the optimal binding affinity, this
report further demonstrates that the peptide portion of the
targeting ligands of MDA-MB-231 breast cancer cells must
contain (1) the disulfide linkage of D-cystine, (2) short polar
amino acid (Asn) at position 7, (3) β-turn inducible hydro-
xyproline at position 6, (4) phenylalanine analogues with very
specific arrangement of polar group (F of NO2) on the
aromatic ring, and (5) a free N-terminal amino group. These
features are required for MDA-MB-231 breast cancer cell
binding.
Synthesis of 1 Analogue with Various Bridges. Rink amide
resin (0.25 g, loading 0.6 mmol/g) was swollen in DMF for 2 h.
After Fmoc deprotection with 20% piperidine in DMF twice
(5 min, 15 min), the beads were washed sequentially with DMF,
MeOH, DCM, MeOH, and DMF, three times each. The beads
were then subjected to stepwise assembly of Fmoc-D-allylgly-
cine, Fmoc-Asn(Trt)-OH, Fmoc-Hyp(OtBu)-OH, Fmoc-Gly-
OH, Fmoc-Tyr(3-NO2)-OH, Fmoc-Gly-OH, Fmoc-D-Asp-
(OtBu)-OH, and Fmoc-D-allylglycine using Fmoc solid-phase
chemistry.36 Finally, the beads were sequentially washed with
DMF, MeOH, DCM and dried under vacuum. The beads were
swelled in 3 mL of anhydrous 1,2-dichloroethane (DCE) in a
dried flask under a nitrogen atmosphere for 30 min. The
Hoveryda-Grubbs catalyst (23 mg, 0.15 mmol) in 2 mL of
DCE was added to the flask, and the mixture was stirred at 60 °C
for 40 h. The beads were washed with DCM, MeOH, and DMF.
Following Fmoc deprotection, the beads were dried under
vacuum and treated with a TFA cocktail containing 82.5%
TFA/5% phenol/5% thioanisole/5% H2O/2.5% triisopropylsi-
lane (TIS) at room temperature for 3 h. The cleavage solution
was collected, concentrated, and precipitated in cold diethyl
ether. The crude product was purified by preparative reverse-
phase high performance liquid chromatography (RP-HPLC) to
give 2. 1H NMR (DMSO-d6, 500 MHz): δ ppm 12.40 (1H, brs),
10.80 (1H, brs), 8.74 (1H, d, J = 10 Hz), 8.69 (1H, d, J = 5 Hz),
8.31 (1H, d, J = 5 Hz), 8.18 (1H, d, J = 5 Hz), 8.05 (1H, brs),
7.95 (1H, d, J = 5 Hz), 7.79 (1H, S), 7.48 (1H, brs), 7.41 (1H, d,
J = 10 Hz), 7.39 (1H, d, J = 10 Hz), 7.04 (1H, d, J = 5 Hz), 7.01
(1H, d, J = 10 Hz), 6.91 (1H, brs), 6.86 (1H, brs), 5.52 (1H, m),
5.45 (1H, m), 5.22 (1H, brs), 4.53 (2H, m), 4.38 (2H, m),
Experimental Section
Materials. TentaGel S NH2 resin (90 μm, 0.26 mmol/g) was
purchased from Rapp Polymere GmbH (Tubingen, Germany).
€
Rink amide MBHA resin (0.5 mmol/g), amino acid derivatives,
HOBt, and DIC were purchased from GL Biochem (Shanghai,
China). All solvents and other chemical reagents were purchased
from Aldrich (Milwaukee, WI) and were analytical grade.
ESIMS was performed with Finnigan LCQ. Analytical HPLC
was performed on a Waters 2996 HPLC system equipped with
a 4.6 mm ꢀ 150 mm Waters Xterra MS C18 5.0 μm column
and employed a 20 min gradient from 100% aqueous H2O

6750 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Yao et al.
4.27 (2H, m), 4.00 (2H, m), 3.96 (2H, m), 3.65 (2H, m), 3.46
(2H, m), 3.05 (2H, m), 2.1-2.8 (overlap, m), 2.02 (1H, m), 1.87
(1H, m). ESI-MS m/z: calcd for C34H45N11O15 [M þ 1]þ, 848.3;
found 848.6. Purity 99%
was recorded with a 96-well microscope reader at 570 nm. IC50
was calculated on the basis of the inhibition curves resulting
from the concentration-dependent inhibition.
As described as above, Fmoc-D-Asp (OAll), Fmoc-Asn-
(Trt)-OH, Fmoc-Hyp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr-
(3-NO2)-OH, Fmoc-Gly-OH, Fmoc-D-Asp(OtBu)-OH, and
Fmoc-D-Dap(Alloc) were sequentially assembled on Rink
amide resin (0.25 g, loading 0.6 mmol/g) using Fmoc solid-phase
chemistry.36 The beads were treated with Pd(Ph3)4 (0.24 equiv)
and PhSiH3 (20 equiv) in DCM for 30 min, twice. After washing,
PyBrop (5equiv) and DIEPA (10 equiv) in DMF were added to
the beads. The reaction proceeded at room temperature until a
Kaiser test was negative. Following Fmoc deprotection, the
beads were subjected to TFA cocktail cleavage to afford 3. ESI-
MS m/z: calcd for C33H44N12O16 [M þ 1]þ, 865.3; found 865.6.
Purity 98%.
Acknowledgment. This work was supported by U.S.
Department of Defense Breast Cancer Research Program’s
Multidisciplinary Postdoctoral Training Award Contract No.
W81XWH-06-1-0447 (N.Y.) and National Institutes of
Health Grants NIH R01CA115483 and U19CA113298. We
thank David Olivos for editorial assistance.
Supporting Information Available: Synthetic procedures,
MS data, and biological data. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
As described for the synthesis of 3, Fmoc-L-Asp-OAll,
Fmoc-Hyp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr(3-NO2)-
OH, Fmoc-Gly-OH, Fmoc-D-Asp(OtBu)-OH, and Fmoc-D-
Lys(Alloc) were sequentially assembled to Rink amide resin to
give 4. ESI-MS m/z: calcd for C32H44N10O14 [M þ 1]þ, 793.3;
found 793.6. Purity 99%.
After the assembly of Fmoc-D-Pen(Trt), Fmoc-Asn(Trt)-OH,
Fmoc-Hyp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr(3-NO2)-
OH, Fmoc-Gly-OH, Fmoc-D-Asp(OtBu)-OH, and Fmoc-D-
Pen(Trt) to Rink amide resin (0.25 g, loading 0.6 mmol/g), the
beads were dried and treated with a TFA cocktail. Following
precipitation in cold diethyl ether, the crude peptides were
dissolved in 100 mL of 50 mM NH4HCO3 buffer. Activated
charcoal (100 mg) was added to the buffer. The solution was
stirred at room temperature until the Ellman test was negative.
The reaction solution was filtered, collected, and lyophilized.
The crude product was purified by preparative reverse-phase
high performance liquid chromatography (RP-HPLC) to give 5.
ESI-MS m/z: calcd for C36H51N11O15S2 [M þ 1]þ, 942.3; found
942.6. Purity 98%.
Synthesis of Cyclic Peptide 1 Derivatives with Substitution of
Asp, Hyp, and Tyr(3-NO2). Compounds 6-35 were synthesized
as 5 using Fmoc chemistry.36 Peptide cyclization was carried out
in the 50 mM NH4HCO3 buffer with activated charcoal. The
crude product was purified by preparative reverse-phase high
performance liquid chromatography (RP-HPLC). The final
products were characterized by ESI-MS (Supporting In-
formation). The purity of the final products was determined
by analytical HPLC (Supporting Information).
Synthesis of Cyclic Peptide 1 Derivatives with N-Terminal
Modification. Linear peptide cdGTyr(3-NO2)GHypNc was first
assembled to Rink amide resin (6 g) using Fmoc chemistry.36
After Fmoc deprotection, the beads were split into 21 portions.
Eleven different aldehydes (5 equiv) (Figure 6) and NaCNBH3
(16 equiv) in DMF were added to each portion of beads.
Additionally, 10 various isocyonates (16 equiv) (Figure 6) and
NMM (16 equiv) were added to each portion of beads. Each
mixture was shaken overnight at room temperature. The beads
were then completely washed and dried under vacuum. After
cleavage from the beads, the crude products were cyclized and
purified to give 36-58 (Supporting Information).
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