Synthesis of cyclo-β-tripeptides and their biological in vitro evaluation as antiproliferatives against the growth of human cancer cell lines

Article abstract of DOI:10.1002/1522-2675(20011017)84:10<2924::AID-HLCA2924>3.0.CO;2-E

A number of cyclo-β-tripeptides and their linear precursors were subjected to primary biological evaluation for cancer-cell growth inhibition (one-dose, three-cell essay), and the five most active ones were then tested in the anti-tumor screen of the National Cancer Institute (Bethesda, USA) with 60 human cancer cell lines. Growth inhibition values GI₅₀ in the one-digit micromolar, and in one case in the nanomolar range were obtained. The effects show selectivities for certain types of cancer cells and for certain cell lines within these types; the screen includes leukemia, non-small-cell lung, colon, and central-nervous-system (CNS) cancer, melanoma, ovarian, renal, prostate, and breast cancer cell lines. The synthesis and full characterization of two new cyclo-β-peptides, (β³-HSer(OBn))₃ (11) and (β³-HMet)₃ (12) are described. Other cyclo-β-peptides included in this investigation are (βAsp(Bn))₃ (13), (β-HGlu(Bn))₃ (14), and (β-HAla)₃ (16), compounds which had been previously prepared by us. Strongest activities were measured with the cyclo-β-peptides bearing benzyl-ester or benzyl-ether groups in the side chains. The cytotoxic activity of the compounds included in this investigation is much lower (LC₅₀ > 100 μM) than their antiproliferative activity (GI₅₀).

Full text of DOI:10.1002/1522-2675(20011017)84:10<2924::AID-HLCA2924>3.0.CO;2-E

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Helvetica Chimica Acta ± Vol. 84 (2001)
Synthesis of Cyclo-b-tripeptides and Their Biological in vitro Evaluation as
Antiproliferatives against the Growth of Human Cancer Cell Lines
by Karl Gademann1) and Dieter Seebach*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule Zürich,
Hönggerberg, CH-8093 Zürich
Dedicated to the memory of Professor Luigi M. Venanzi
A number of cyclo-b-tripeptides and their linear precursors were subjected to primary biological evaluation
for cancer-cell growth inhibition (one-dose, three-cell essay), and the five most active ones were then tested in
the anti-tumor screen of the National Cancer Institute (Bethesda, USA) with 60 human cancer cell lines. Growth
inhibition values GI50 in the one-digit micromolar, and in one case in the nanomolar range were obtained.
The effects show selectivities for certain types of cancer cells and for certain cell lines within these types;
the screen includes leukemia, non-small-cell lung, colon, and central-nervous-system (CNS) cancer,
melanoma, ovarian, renal, prostate, and breast cancer cell lines. The synthesis and full characteriza-
tion of two new cyclo-b-peptides, (b3-HSer(OBn))3 (11) and (b3-HMet)3 (12) are described. Other cyclo-
b-peptides included in this investigation are (b-Asp(Bn))3 (13), (b-HGlu(Bn))3 (14), and (b-HAla)3
(16), compounds which had been previously prepared by us. Strongest activities were measured with the
cyclo-b-peptides bearing benzyl-ester or benzyl-ether groups in the side chains. The cytotoxic activity of
the compounds included in this investigation is much lower (LC50 > 100 mm) than their antiproliferative
activity (GI50).
Introduction. ± Oligomers of b-amino acids have recently emerged as a new class of
peptidomimetics with a strong folding preference for various secondary structures
simply based on different substitution patterns of the b-amino acid building blocks (for
reviews, see [1 ± 6]). Over the last five years, all common protein-structural elements
such as three different helices [7 ± 14], turns [13 ± 15], pleated sheet, and hairpin
structures [10][13][14][16 ± 18] have been identified. More possible structures wait to
be elucidated [15][19][20]. b-Peptides are resistant to the degradation by proteinase
enzymes [10][21][22] and their building blocks, the b-amino acids are non mutagenic
according to Amesꢀ tests [21]. Hence, these oligomers may serve as peptidomimetics for
the use in medicinal chemistry.
We were the first to demonstrate that b-peptides can possess biological activity by
mimicking the peptide hormone somatostatin with a cyclo-b-tetrapeptide [23][24].
Rational design then led to a linear b-tetrapeptide with a 100-fold more potent,
nanomolar, selective affinity for the human somatostatin receptor 4 [25]. In addition
three other biologically active b-peptides have been designed as cholesterol uptake
inhibitors [26] and antibiotics [27 ± 29].
1
)
Part of the dissertation No. 13556 of K. G., ETH-Zürich.

Helvetica Chimica Acta ± Vol. 84 (2001)
2925
Cyclic b-peptides, however, have received less attention, so far. While cyclo-b-
tripeptides2) of type A (which resemble the bacterial Fe chelator enterobactin (B))
have been prepared as long as thirty years ago (from achiral or racemic b-amino acids
[33 ± 37]; anthranilic acid [38] and enantiomerically pure b-amino acids [10][39 ± 42]),
their structures in the solid state (achiral cyclo(b-HGly) [43]) and in solution [42] have
been investigated only recently. Thus, cyclo-b-tripeptides appear to stack to indefinite
tube-like hollow structures, so called nanotubes in the solid state. These structures are
characterized by a polar hollow center with apolar side chains. Moreover, due to
the unidirectional alignment of the CˆO groups, a large dipole moment results. In
addition, the proper arrangement of the side chains could introduce molecular
function such as catalysis or binding of metal ions (cf. B). Hence, we wondered
whether those unique properties of cyclo-b-tripeptides could be accompanied by
biological activities, i.e., interaction with living cells. We envisioned, for example,
inclusion of these compounds in cell membranes or walls, which should result in
cytotoxic or antibiotic properties. Hence, a series of cyclo-b-tripeptides was pre-
pared and their antiproliferative properties against cancer cell lines in vitro was
evaluated.
Synthesis. ± There are many methods for the EPC (enantiomerically pure
compound) synthesis of b-amino acids. These approaches have been summarized in
several review articles [44 ± 47]. The large variety of available a-amino acids [48]
renders them ideal starting materials for the synthesis of b3-amino acids. Hence, the
Arndt-Eistert homologation of a-amino acids via diazo ketones [49] was used as a
simple route, on the laboratory scale, with N-phtaloyl [50][51] and N-carbamate
protection [52] on the amino groups. The diazo ketones are decomposed in the Wolff
rearrangement by using light, heat, or Ag salts to give the ketene intermediates that can
be trapped by various nucleophiles [53 ± 57]. When the nucleophile is H2O, the
corresponding b3-amino acids are isolated, and, with alcohols, the b3-amino acid esters
are formed. An orthogonal protecting-group strategy was selected for b-peptide
2
)
It is interesting to note that the larger cyclo-b-tetrapeptides have been found to be somatostatin analogues
[23][24], molecular scaffolds [30][31] and artificial transmembrane ion channels [32].

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Helvetica Chimica Acta ± Vol. 84 (2001)
synthesis in solution: Boc3) for the N-terminus, methylester for the C-terminus, and Bn
groups for the side chains.
The homologation of the suitably protected a-amino acids Met and Ser was
effected, according to well-established routes [10][53]. In a one-pot procedure, the
i
carboxylic acid functionalities were activated in THF at 208 (ClCO2Et or ClCO2 Bu,
4
Et3N), and subsequently CH2N2 ) was acylated by the resulting mixed anhydrides to
give the diazo esters 1 and 2 (Scheme 1). Purification by flash chromatography (for 1)
or by recrystallization and flash chromatography (for 2) afforded these compounds in
yields between 70 and 80%. It is interesting to note that no methylation by the strong
alkylating reagent CH2N2 of the S-atom of Met was observed under the reaction condi-
tions. This reaction can be conveniently carried out on a multigram scale. Transforma-
tion of 0.2 mol of Boc-Met gave 38.8 g of the diazo ketone 1 after flash chromatography.
Scheme 1. Preparation of b-Amino Acid Derivatives 3 ± 6
a) ClCO2Et, Et3N, THF then CH2N2, Et2O. b) For R ˆ H: CF3CO2Ag (cat.), Et3N, THF, H2O; for R ˆ MeO:
CF3CO2Ag (cat.), Et3N, MeOH.
‡
The Wolff rearrangement, catalyzed by Ag /Et3N5) in a THF/H2O mixture, gave
the b3-amino acids Boc-b3-HMet-OH (3) and Boc-b3-HSer(OBn)-OH (5) in excellent
yields (80 ± 95%). The other two building blocks required, b-amino esters 4 and 6, were
obtained in nearly quantitative yield via decomposition of the corresponding diazo
esters 1 and 2 in the presence of MeOH as the nucleophile. The cheaper PhCOOAg was
used instead of the CF3COOAg. However, this exchange is only possible, when the
products are purified by flash chromatography6).
3
)
)
)
)
For abbreviations, see Exper. Part.
Caution: The generation and handling of CH2N2 requires special precautions.
The CF3COO counterion is assumed to be exchanged to give the catalytically active Ag /Et3N species.
The purification by crystallization is hampered by the high crystallinity of the by-product, benzoic acid.
4
5
6
‡

Helvetica Chimica Acta ± Vol. 84 (2001)
2927
The synthesis of the b-peptides 7 ± 10 was accomplished according to traditional
protocols7). The Boc group of the b-amino esters 4 and 6 were removed by CF3COOH
in CH2Cl2 and subsequent coupling with 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride (EDC)/1-hydroxy-1H-benzotriazole (HOBt) furnished the b-
dipeptides 7 and 9 in high yields. Another cycle of chain elongation resulted in the b-
tripeptides 8 and 10. Both b-peptides have high melting points and low solubility. The
higher yield of b-peptide 8 (84%) as compared to 10 (39%) is the result of modified
workup procedures8). All b-peptides were fully characterized (1H- and 13C-NMR, IR
spectroscopy, [a]D, MS spectrometry, and elemental analyses).
Having the fully protected b-tripeptide precursors 8 and 10 at hand, it was of
importance to choose a clean and efficient cyclization method. There are numerous
approaches for the cyclization of a-peptides, which often cyclize in miserable yields.
The route developed by Schmidt et al. via pentafluorophenyl active esters [58 ± 60] was
the method of choice9), as it has been previously employed for b-peptides [10]. The
very insoluble b-tripeptide 10 could only be dissolved in millimolar concentrations in
CF3CH2OH, and precipitated partially upon addition of aqueous base. In addition, 50
equiv. of NaOH had to be used in order to cleave the C-terminal methyl ester. After
prolonged reaction times, the b-tripeptide acid could be isolated in nearly quantitative
yield. Subsequent coupling with pentafluorophenol in DMF using EDC gave the b-
peptide active ester, which was terminally N-deprotected by CF3COOH in CH2Cl2 to
give the trifluoracetate salt. This compound was dissolved in MeCN and added via
syringe pump to a dilute (3.3 mm) solution of EtN(i-Pr)210) in MeCN at elevated
temperatures (708) over several hours (Scheme 2). The cyclic b-tripeptide 11
7
)
)
)
For the solution phase synthesis of other b-peptides carrying functionalized side chaines (Boc/Bn strategy),
see, e.g., [41].
8
9
Washing the crude b-peptide with a variety of solvents instead of the usual liquid-liquid extraction, and
aqueous workup.
This method was originally developed for Z-protected peptides. After transformation to the active ester,
the Z group was cleaved hydrogenolytically, and the peptide was subsequently cyclized in situ. It may be
speculated that the metal surface on which the cyclization occurs has a positive effect on the rate and yield
of the cyclization [58 ± 60].
10
)
Hünigꢁs base.

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Helvetica Chimica Acta ± Vol. 84 (2001)
precipitated out of the hot and dilute reaction mixture and could be easily isolated by
simple filtration. This b-peptide 11 is insoluble in essentially all common organic
solvents, except for TFA. NMR Spectra could only be measured in a mixture of CDCl3
and TFA. In addition, a very high melting point (>2158) is observed. The analogous
cyclo-b-tripeptide 12 (containing three protected b-HSer residues) was prepared by the
same method, albeit in somewhat lower yield, it also has a poor solubility and a high
melting point.
Scheme 2. Deprotection, Activation, and Cyclization to Give the Cyclo-b-tripeptides 11 and 12
a) NaOH/H2O/CF3CH2OH. b) C6F5OH, EDC, DMF, 08. c) TFA, CH2Cl2. d) Slow addition to a dilute soln. of
EtN(i-Pr)2 in MeCN, 708.
Biological Evaluation. ± To study the influence of the side chains on the
antiproliferative activity, as well as the effect of cyclization, several derivatives
previously prepared by us were also submitted to the National Cancer Institute (NCI)
for testing. Beside the ether- and thioether containing cyclo-b-tripeptides 11 and 12,
prepared as described above, we submitted the Asp and homologated Glu derived
cyclo-b-tripeptides 13 [41] and 14 [41], the protected triolide derived from malic acid 15
[61], as well as (b-HAla)3 (16) [40], bearing three Me groups as side chains. Finally, the
linear precursors 7 ± 10 were included in the tests.
All compounds were tested in a first one-dose, three-cell-line assay by the NCI
(Table 1). The linear di- and tri-b-peptides 7, 8, and 10 displayed antiproliferative
activity. Also the cyclo-(b-HSer)3 was considered active, whereas the thio derivative
cyclo-(b-HMet)3 failed to show significant activity under the test conditions. It is
interesting to note that the antiproliferative activity is lost when cyclizing the linear,
fully protected b-HMet precursor 10 to the cyclo-b-tripeptide 12. The cyclo-b-peptide

Helvetica Chimica Acta ± Vol. 84 (2001)
2929
Table 1. Primary Biological Evaluation of the Growth Inhibition by Compounds 7 ± 16 in a One-Dose, Three-
Cell Assay. Compounds showing significant growth inhibition with one cell line at 100 mm concentration were
considered active.
Compound
Activity
Compound
Activity
Boc-(b-HSer)2-OMe (7)
Boc-(b-HSer)3-OMe (8)
Boc-(b-HMet)2-OMe (9)
Boc-(b-HMet)3-OMe (10)
Cyclo(b-HSer)3 (11)
active
active
inactive
active
active
Cyclo(b-HMet)3 (12)
Cyclo(b-Asp)3 (13)
Cyclo(b-HGlu)3 (14)
Triolide 15
inactive
active
active
inactive
inactive
Cyclo(b-HAla)3 (16)
13, derived from the only proteinogenic b-amino acid H2N-Asp-OH, was shown to
inhibit the growth of the cancer cells, and also compound 14 with doubly homologated
side chains (an amino adipic acid or b-HGlu derivative). Apparently, amide
functionalities in the backbone are required for antiproliferative activity, since
replacement of the N- by O-atoms (13 ! 15) results in complete loss of biological
activity. Also the cyclo-b-peptide with methyl side chains was found to be inactive,
indicating that a certain functionalization of the side-chains and/or the presence of
aromatic Bn groups is necessary for biological activity.
From the results of the preliminary tests, the compounds 7, 8, 10, 11, 13, and 14 were
chosen for evaluation in vitro against 60 human cancer cell lines derived from nine
cancer-cell types (leukemia, non-small-cell lung cancer, colon cancer, CNS cancer,
melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer). For each

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compound and for each cell line, dose-response curves were obtained with five different
concentrations (10-fold dilutions), and the concentration (in mm) causing 50% cell
Table 2. Growth-Inhibition (GI50) Values (in mm) of Compounds 7, 8, 10, 11, 13, and 14 Tested in the Anti-Tumor
Screen of the NCI Consisting of 60 Human Cancer Cell Lines (NSCLC ˆ non-small-cell lung cancer). All values
are the concentrations needed to achieve 50% growth inhibition of the given human cancer cell line. The
compounds are tested at a minimum of five concentrations (with 10-fold dilution each). A 48-h continuous drug
exposure protocol is used, and a sulforhodamine B protein assay is used to estimate growth relative to a control.
The values for b-peptide 13 are the mean values of two independent experiments (except for the CCRF-CEM
cell line). The mean values of each cancer cell line were calculated only of the active cell lines (GI50 < 100 mm),
and only if at least three cell lines were thus considered active (n.a. ˆ not available).
Cell line
11
13
14
7
8
10
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
> 100
93
0.07
0.66
4
4
39
> 100
n.d.
> 100
> 100
> 100
> 100
> 100
> 100
40
> 100
68
1
> 100
> 100
> 100
40
> 100
> 100
> 100
> 100
35
4
6
56
40
39
> 100
42
4
Mean
36
9
> 100
44
n.a.
36
NSCLC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
> 100
18
12
> 100
55
> 100
> 100
> 100
> 100
34
21
> 100
47
> 100
> 100
36
> 100
16
19
21
23
26
17
25
35
40
16
42
47
30
69
35
4
> 100
52
> 100
> 100
> 100
> 100
35
26
> 100
21
> 100
> 100
22
n.d.
78
Mean NSCLC
28
35
24
24
33
39
Colon cancer
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
> 100
32
> 100
> 100
> 100
> 100
91
71
> 100
> 100
> 100
> 100
> 100
26
> 100
> 100
> 100
> 100
> 100
28
n.d.
> 100
37
> 100
> 100
> 100
> 100
14
> 100
72
> 100
> 100
31
30
46
56
Mean Colon
n.a.
n.a.
n.a.
38
43
n.a.
CNS Cancer
Melanoma
SF-268
SF-295
SF-539
SNB-75
U251
41
19
15
3
15
20
19
1
n.d.
9
20
34
35
25
28
42
89
96
13
93
20
22
17
13
20
6
22
5
13
12
Mean CNS
18
13
10
28
67
19
LOX IMVI
M14
SK-MEL2
SK-MEL28
SK-MEL-5
UACC-257
> 100
> 100
28
> 100
> 100
> 100
> 100
87
> 100
> 100
17
> 100
> 100
> 100
53
33
37
59
55
> 100
72
> 100
> 100
> 100
58
24
> 100
56
13
> 100
> 100
> 100
> 100
46
> 100
69
Mean
n.a.
n.a.
n.a.
42
62
52

Helvetica Chimica Acta ± Vol. 84 (2001)
Table 2 (cont.)
2931
Cell line
11
13
14
7
8
10
Ovarian
IGROVI
> 100
81
> 100
> 100
> 100
20
98
50
98
> 100
> 100
> 100
6
34
28
19
34
25
23
100
100
4
100
24
56
22
20
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
> 100
> 100
> 100
> 100
21
> 100
29
60
20
Mean ovarian
50
n.a.
51
27
65
30
Renal cancer
768-0
A498
17
12
17
21
28
23
8
8
17
30
27
39
30
30
18
24
81
34
21
37
ACHN
CAKI-1
RXF 393
TK-10
UO-31
> 100
11
18
> 100
> 100
13
20
> 100
> 100
2
> 100
21
n.d.
> 100
> 100
16
19
> 100
6
> 100
32
> 100
Mean renal
15
21
8
31
36
25
Prostate
PC-3
DU-145
> 100
> 100
> 100
> 100
> 100
> 100
27
22
13
23
> 100
87
Mean prostate
> 100
> 100
> 100
25
18
n.a.
Breast cancer
MCF 7
> 100
> 100
27
> 100
> 100
14
> 100
> 100
14
20
22
37
26
34
52
34
12
> 100
81
35
33
> 100
25
38
> 100
39
NCI/ADR-RES
MDA-MB-231
HS 578T
MDA-MB-435
MDA-N
40
13
5
30
40
69
63
> 100
> 100
62
> 100
> 100
> 100
> 100
> 100
45
T-47D
Mean breast
43
n.a.
21
32
37
47
growth inhibition (GI50) relative to the control was calculated. The results are collected
in Table 2.
The cyclo(b-HSer)3 (11) displays moderate antiproliferative activity against the
leukemia cell lines K-562 (4 mm) and MOLT-4 (6 mm), against several CNS cancer cell
lines (3 mm for SNB-75, mean 18 mm), and against renal cancer (mean 15 mm), whereas
only weak or no activity was measured for most of the other cell lines.
However, its derivative cyclo(Asp(OBn))3 (13) possesses strong growth inhibiting
properties against several leukemia cell lines. Submicromolar values have been
measured for CCRF-CEM (0.07 mm) and for HL-60 TB (0.66 mm). In addition, this
compound shows moderate-to-good antiproliferative activities in vitro against CNS
cancer (SNB-75 cell line with 1 mm, mean 13 mm) and renal cancer cell lines. Again, only
weak to no activity has been determined with 13 for in vitro activity against colon
cancer, melanoma, ovarian, and several breast cancer cell lines, indicating a certain
selectivity in this assay.
The doubly homologated Glu derived cyclo-b-peptide 14, sharing the BnOCO
groups with 13, displays no antiproliferative activity against leukemia cell lines.
However, the cyclotripeptide 14 exhibits activity against CNS (mean 10 mm) and renal
(mean 8 mm) cancer cell lines, which is stronger than that observed with 13.

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Helvetica Chimica Acta ± Vol. 84 (2001)
Furthermore, some good values were recorded for some non-small-cell lungs cancer
(HOP-92, 4 mm), ovarian (SK-OV-3, 6 mm), and breast (HS 578T, 5 mm) cancer cell
lines, whereas only weak or no activity was found in the prostate, melanoma, and colon
cancer tests.
To our surprise, the linear precursors tested did show a broad, yet weak-to-
moderate activity against many of the cell lines tested. Thus, the dipeptide 7 has the
broadest, i.e., least selective activity (it was considered active for 52 of 60 cell lines
evaluated), with no high values having been found. The longer-chain linear tripeptides
8 and 10 appear to be more selective (compare the values obtained for 7 and 8 for
leukemia, and of 7 and 10 for colon cancer cell lines). In addition, some good activities
have been detected for the linear peptides 8 (OVCAR-4, 4 mm) and for 10 (HL-60 TB,
1 mm).
The b-peptides included in this investigation have also been tested for cytotoxic
activity; ca. 90% of the LC50 values were > 100 mm, i.e., outside of the measuring range
of the test. This means that the recorded antiproliferative activities are selective against
human cancer cells!
Conclusion. ± Several b-peptides have been evaluated in vitro as antiproliferatives
against the growth of human cancer cell lines. It is interesting to see that several cyclic
tri-b-peptides, as well as two b-dipeptides and a b-tripeptide, display antiproliferative
activity by inhibiting the growth of several cancer cell lines. The b-peptide cyclo(b-
Asp)3 13 is the most potent compound in this series displaying submicromolar activity
(GI50 values down to 70 nm) against two leukemia cell lines. This compound appears to
inhibit preferentially the growth of leukemia, CNS, and renal cell lines, and only little or
no activity has been found for the other cell lines evaluated.
The presence of an amide backbone in the cyclo-b-tripeptides as well as of Bn
groups appear to be necessary for biological activity. In addition, it looks like
cyclization is enhancing the growth inhibition effect.
The antiproliferative properties of some of the evaluated b-peptides are an
additional demonstration of a biological activity of b-peptides. Given the remarkable,
complete stability of b-peptides against microbial, fungal, and mammalian peptidases
[22], the spectrum of their biological activity is now broadened from mimicking a
natural a-peptidic hormone [23 ± 25], inhibiting a protein receptor [26], and having
antibacterial activity [27 ± 29], to inhibiting the growth of human cancer cells.
We thank the National Cancer Institute, Bethesda, MD, for the biological evaluation of our compounds in
their standard 60 cell lines assay, as part of the Developmental Therapeutics Program. We are indebted to Mr. C.
Czekelius and Ms. N. Becker for their skillful technical assistance as part of the requirements for the laboratory
course in advanced organic chemistry. We thank Dr. C. M. Krell for providing a sample of the triolide 15 and Dr.
J. L. Matthews for a sample of 16. Novartis AG is gratefully acknowledged for continuing financial support,
Degussa AG for providing methionine and SOLVAY AG for donating CF3COOH.
Experimental Part
1. General. Abbreviations: Boc: (tert-butoxy)carbonyl, DCC: 1,3-dicyclohexylcarbodiimide, EDC: 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, FAB: fast atom bombardment, FC: flash chro-
matography, HOBt: 1-hydroxy-1H-benzotriazole, h.v.: high vacuum, 0.01 ± 0.1 Torr, b-HXxx: b-homoamino
acid, NMM: N-methylmorpholine, TFA: CF3COOH, TFE: 2,2,2-trifluoroethanol. THF was freshly distilled

Helvetica Chimica Acta ± Vol. 84 (2001)
2933
over K under Ar before use. Et3N was distilled over CaH2 and stored over 4-Š molecular sieves. Solvents for
chromatography and workup procedures were distilled from Sikkon (anh. CaSO4; Fluka). Boc-Ser(OBn)-OH
was purchased from Senn Chemicals. Boc-Met-OH was prepared from Met [62]. Compounds 13, 14 [41], 15 [61],
and 16 [40] were prepared according to the literature procedures. All other reagents were used as received from
Fluka. TLC: Merck silica gel 60 F254 plates; detection with UV or dipping into a soln. of anisaldehyde (9.2 ml),
AcOH (3.75 ml), conc. H2SO4 (12.5 ml), and EtOH (338 ml), followed by heating. FC: Fluka silica gel 60 (40 ±
63 mm); at ca. 0.3 bar. Anal. HPLC: Knauer HPLC system (pump type 64, EuroChrom 2000 integration
package, degaser, UV detector (variable-wavelength monitor)), Macherey-Nagel C8 column (Nucleosil 100-5 C8
(250 Â 4 mm)). Prep. HPLC: Knauer HPLC system (pump type 64, programmer 50, UV detector (variable-
wavelength monitor)), Macherey-Nagel C8 column (Nucleosil 100-7 C8 (250 Â 21 mm)). M.p.: Büchi 510
apparatus; uncorrected. Optical rotations: Perkin-Elmer 241 polarimeter (10 cm, 1 ml cell) at r.t. IR Spectra:
Perkin-Elmer 782 spectrophotometer. NMR Spectra: Bruker AMX-500 (1H: 500 MHz, 13C: 125 MHz), AMX-
400 (1H: 400 MHz, 13C: 100 MHz), ARX-300 (1H: 300 MHz), Varian Gemini-300 (1H: 300 MHz, 13C: 75 MHz),
or Gemini-200 (1H: 200 MHz, 13C 50 MHz); chemical shifts (d) in ppm downfield from Me4Si (ˆ0 ppm); J
values in Hz. MS: VG Tribrid (EI) or Hitachi Perkin-Elmer RHU-6M (FAB in a 3-nitrobenzyl-alcohol matrix)
spectrometer; in m/z (% of basis peak). Elemental analyses were performed by the Microanalytical Laboratory
of the Laboratorium für Organische Chemie der ETH-Zürich.
2. Preparation of Diazo Ketones. General Procedure 1 (GP 1). Caution: The generation and handling of
CH2N2 requires special precautions [63][64]. The N-Boc-protected a-amino acid was dissolved in THF (0.2m)
under Ar. The soln. was cooled to 158, and Et3N (1 equiv.) and ClC(O)OEt or ClC(O)OiBu (1 equiv.) were
added. After 15 min, the suspension was allowed to warm to 08, and a soln. of CH2N2 in Et2O was added until a
strong yellow color persisted. The suspension was allowed to warm to r.t. and stirred for 3 h. The excess
of CH2N2 was destroyed by the addition of few drops of AcOH. The mixture was diluted with H2O, and the
bulk of THF was evaporated. After addition of Et2O, the mixture was washed with sat. NaHCO3, NH4Cl, and
NaCl solns. The org. layer was dried (MgSO4) and evaporated. FC or recrystallization gave the pure diazo
ketones.
3. Homologation of the Diazo Ketones to the Corresponding b-Amino Acids. General Procedure 2 (GP 2).
The diazo ketone was dissolved in THF/H2O 9 :1 (v/v) (0.25m). Under the exclusion of light, the soln. was
cooled to ca. 258, and a soln. of CF3CO2Ag (0.11 equiv.) in Et3N (2.3 equiv.) was added. The mixture was
allowed to warm to r.t. and stirred for 3 h in the dark. THF was evaporated, and the residue was dissolved in
Et2O. The b-amino acid was extracted with sat. NaHCO3 soln., and the H2O layer was carefully acidified at 08 to
pH 2 ± 3 and extracted with Et2O. The combined org. layers were dried (MgSO4) and evaporated. The resulting
b-amino acids were used without further purification.
4. Homologation of the Diazo Ketones to the Corresponding b-Amino Acid Methyl Esters. General
Procedure 3 (GP 3). The diazo ketone was dissolved in MeOH (0.25m). Under the exclusion of light, the soln.
was cooled to ca. 258, and a soln. of CF3CO2Ag or PhCO2Ag (0.11 equiv.) in Et3N (2.3 equiv.) was added. The
mixture was allowed to warm to r.t. and stirred for 3 h in the dark. The solvent was evaporated and the residue
was dissolved in Et2O. The org. layer was washed with sat. NaHCO3, NH4Cl and NaCl solns., dried (MgSO4),
and evaporated.
5. Boc-Deprotection with TFA. General Procedure 4 (GP 4). The Boc-protected compound was dissolved in
CH2Cl2 (0.25m) and cooled to 08. The same amount (v/v) of TFA was added, and the mixture was stirred for 1 h
at 08 and for 1 h at r.t. The solvent was evaporated, the residue was twice taken up in CHCl3 and evaporated. The
resulting trifluoroacetate was dried under h.v. and used without further purification.
6. Peptide Coupling with EDC and HOBt. General Procedure 5 (GP 5). The trifluoroacetate of the amino
fragment was dissolved in CHCl3 (0.5m) and cooled to 08. Et3N (4 equiv.), HOBt (1.2 equiv.), a soln. of the Boc-
protected fragment (1 equiv.) in CHCl3 (0.25m), and EDC (1.2 equiv.) were added, and the resulting mixture
was stirred for 30 min at 08. The mixture was then allowed to warm to r.t. and stirred for additional 12 h (TLC
control). The mixture was diluted with CHCl3 and was washed with 1m HCl, sat. NaHCO3, and sat. NaCl solns.
The org. layer was dried (MgSO4) and evaporated, and the resulting peptides were either purified or used
without further purification.
tert-Butyl N-{(1S)-3-Diazo-1-[2-(methylsulfanyl)ethyl]-2-oxopropyl}carbamate (1). Boc-Met-OH (49.9 g,
0.2 mol) was transformed according to GP 1. FC (Et2O/pentane 1:1) gave compound 1 (38.8 g, 71%). Yellow
solid. M.p. 62.5 ± 63.58. Rf (Et2O/pentane 1:1) 0.20. [a]D
ˆ
16.7 (c ˆ 1.03). IR (CHCl3): 858w, 1020w, 1045w,
1163s, 1369s, 1443w, 1496s, 1641s, 1708s, 2111s, 2920w, 2981m, 3008m, 3432w. 1H-NMR (300 MHz, CDCl3): 1.43
(s, t-Bu); 1.79 ± 2.13 (m, MeSCH2CH2); 2.10 (s, Me); 2.54 (t, J ˆ 7.3, MeSCH2CH2); 4.32 (br., CH); 5.23 (br. d,
J ˆ 7.5, NH); 5.50 (br. s, CHN2). 13C-NMR (75 MHz, CDCl3): 15.47; 28.32; 30.10; 32.00; 54.20; 56.47; 80.17;

2934
Helvetica Chimica Acta ± Vol. 84 (2001)
‡
‡
155.36; 193.35. EI-MS: 199 (35, [M t-BuOH] ), 189 (22), 172 (26, [M Boc] ), 161 (31), 157 (22), 146 (53),
143 (50), 115 (20), 104 (43), 61 (41), 57 (100). Anal. calc. for C11H19N3O3S (273.11): C 48.33, H 7.01, N 15.37;
found: C 48.42, H 7.02, N 15.26.
tert-Butyl N-{(1S)-[(Benzyloxy)methyl]-3-diazo-2-oxopropyl}carbamate (2). Boc-Ser(OBn)-OH (33.5 g;
113 mmol) was transformed according to GP 1. Recrystallization (AcOEt/(i-Pr)2O/pentane) and FC (Et2O/
pentane 1 :2 to 1:1) gave compound 2 (28.6 g, 79%). Yellow needles. M.p. 81 ± 838 ([41]: 87 ± 888). [a]D
(c ˆ 1.00, CHCl3). ([41]: ˆ 24.2 (c ˆ 1.13, CHCl3)). NMR Data in agreement with those in [41].
ˆ
23.6
(S)-3-{[(tert-Butoxy)carbonyl]amino}-5-(methylsulfanyl)pentanoic Acid (3). Diazo ester
1
(2.0 g,
7.31 mmol) was transformed according to GP 2. Recrystallization (AcOEt) gave compound 3 (1.6 g, 83%).
Off-white crystals. M.p. 82.5 ± 83.58. [a]D
ˆ
19.4 (c ˆ 1.03). IR (CHCl3): 850w, 928w, 1050w, 1166s, 1368m,
1
1393w, 1503s, 1708s, 2930w, 2981m, 3008m, 3435w. H-NMR (300 MHz, CDCl3): 1.44 (s, t-Bu); 1.76 ± 1.95 (m,
MeSCH2CH2); 2.10 (s, Me); 2.17 ± 2.61 (m, MeSCH2CH2, CH2CO); 4.01 (br., CH); 5.05 (br. d, J ˆ 8.1, NH);
5.97 (br., OH). 13C-NMR (75 MHz, CDCl3): 15.56; 28.35; 30.75; 33.88; 38.88; 46.74; 79.75; 155.49; 176.55. MS:
‡
‡
‡
547 (30, [2 M ‡ Na] ), 525 (44, [2 M ‡ H] ), 262 (100, [M H] ). Anal. calc. for C11H21NO4S (263.35): C 50.17,
H 8.04, N 5.32; found: C 50.24, H 7.83, N 5.32.
Methyl (S)-3-{[(tert-Butoxy)carbonyl]amino}-5-(methylsulfanyl)pentanoate (4). Diazo ester 1 (10.0 g;
36.59 mmol) was transformed according to GP 3. FC (Et2O/pentane 2 :3), subsequent thorough drying under
h.v., and storing at 208 gave compound 4 (9.6 g, 95%). Colorless needles. Rf (Et2O/pentane 2 :3) 0.28. M.p.
36 ± 378. [a]D
ˆ
22.1 (c ˆ 1.12). IR (CHCl3): 1054w, 1166s, 1289w, 1368m, 1392w, 1438m, 1501s, 1707s, 2981m,
3008m, 3435w. 1H-NMR (300 MHz, CDCl3): 1.41 (s, t-Bu); 1.75 ± 1.84 (m, MeSCH2CH2); 2.08 (s, MeS); 2.43 ±
2.58 (m, MeSCH2CH2, CH2CO); 3.66 (s, MeO); 3.96 ± 4.04 (m, CH); 5.02 (d, J ˆ 8.4, NH). 13C-NMR (75 MHz,
CDCl3): 15.54; 28.35; 30.76; 34.01; 38.90; 46.91; 51.71; 79.41; 155.29; 171.92. EI-MS: 221 (24), 157 ( 100), 129
(47), 128 (51), 102 (28), 61 (28), 59 (26). Anal. calc. for C12H23NO4S (277.38): C 51.96, H 8.36, N 5.05; found: C
51.83, H 8.51, N 5.06.
(R)-4-(Benzyloxy)-3-{[(tert-butoxy)carbonyl]amino}butanoic Acid (5). Diazo ester 2 (12.72 g; 40 mmol)
was transformed according to GP 2. Drying under h.v. gave compound 5 (9.6 g; 78 %). White crystals. M.p.
1
104 ± 1068. ([41]: 73 ± 748). H-NMR (200 MHz, CDCl3): in agreement with those in [41].
Methyl (R)-4-(Benzyloxy)-3-[(tert-butoxy)carbonyl]amino}butanoate (6). Diazo ester
2 (24.4 g,
76.4 mmol), 1.92 g (8.4 mmol) of PhCO2Ag, and 30.8 ml (220 mmol) of Et3N were transformed according to
GP 3. FC (Et2O/pentane 1:2) gave 6 (22.63 g; 92%). Colorless solid. Rf (Et2O/pentane 2 :3) 0.47. M.p. 34 ± 358.
[a]D
ˆ
12.7 (c ˆ 2.90, CHCl3). 1H-NMR (300 MHz, CDCl3): 1.43 (s, t-Bu); 2.62 (d, J ˆ 6.5, CH2CO2Me); 3.48 ±
3.63 (m, CH2O, MeO); 4.16 (br., CHN); 4.50 (s, PhCH2); 5.18 (br., NHBoc); 7.24 ± 7.36 (m, 5 arom. H).
13C-NMR (300 MHz, CDCl3): 28.37; 36.15; 47.28; 51.63; 71.03; 73.19; 79.45; 127.63; 127.73; 128.39; 137.92;
155.92; 171.94. IR (CHCl3): 3675w, 3613w, 3487w, 3446w, 3026w, 2974w, 2865w, 1278s, 1706s, 1513m, 1503s,
‡
1436m, 1366s, 1247s, 1222s, 1166s, 1068m, 932m, 802s, 772s, 704s, 694m. FAB-MS: 647.5 (11.0, [2 M ‡ H] ), 324.2
(97.4, [M ‡ H] ), 268.2 (72.4, [M tBu ‡ 2 H] ), 224.2 (100.0, [M Boc ‡ 2 H] ). Anal. calc. for C17H25NO5
‡
‡
‡
(323.39): C 63.14, H 7.79, N 4.33; found: C 62.97, H 7.56, N 4.42.
Boc-b3-HSer(OBn)-b3-HSer(OBn)-OMe (7). The ester 6 (5.50 g, 17 mmol) was deprotected according to
GP 4 and reacted with 5 according to GP 5. FC (Et2O/pentane) gave 7 (6.28 g, 72%). Colorless powder. M.p.
1
104 ± 1078. Rf 0.2 (Et2O/pentane). H-NMR (500 MHz, CDCl3): 1.42 (s, t-Bu); 2.43 ± 2.53 (m, CH2CO); 2.55 ±
2.63 (m, CH2CO); 3.45 ± 3.49 (m, CH2O); 3.53 ± 3.61 (m, CH2O); 3.62 (s, MeO); 4.07 (br., NCH); 4.41 ± 4.47 (m,
NCH); 4.49 (s, PhCH2); 5.44 (br., NH); 6.36 (d, J ˆ 7.9, NH); 7.27 ± 7.36 (m, 10 arom. H). 13C-NMR (125 MHz,
CDCl3): 28.61; 35.61; 37.93; 45.88; 47.70; 51.70; 70.50; 71.11; 73.23; 73.25; 127.69; 127.73; 127.76; 127.84; 128.44;
‡
‡
128.46; 137.77; 138.00; 155.46; 170.26; 171.83. FAB-MS: 1029.6 (9, [2 M ‡ H] ); 515.3 (92, [M ‡ H] ); 415.3
‡
(100, [M Boc] ). Anal. calc. for C28H38N2O7 (514.61): C 65.35, H 7.44, N 5.44; found: C 65.40, H 7.34, N 5.49.
Boc-b3-HSer(OBn)-b3-HSer(OBn)-b3-HSer(OBn)-OMe (8). b-Peptide 7 (5.66 g, 11 mmol) was depro-
tected according to GP 4 and reacted with 5 according to GP 5. Recrystallization from AcOEt gave 8 (6.5 g,
1
84%). Colorless crystals. M.p. 140 ± 1428. H-NMR (300 MHz, CDCl3/CD3OD 2 :1): 1.38 (s, t-Bu); 2.37 ± 2.39
(m, CH2CO); 2.45 ± 2.62 (m, 2 CH2CO); 3.34 ± 3.51 (m, 3 CH2O); 3.57 (s, MeO); 4.00 ± 4.40 (br., NCH); 4.26 ±
4.29 (m, NCH); 4.37 ± 4.41 (m, NCH); 4.45 (s, PhCH2); 4.49 (s, 2 PhCH2); 7.21 ± 7.32 (m, 15 arom. H). 13C- NMR
(75 MHz, CDCl3/CD3OD 2 :1): 32.00; 39.69 (2 C); 41.99; 49.96; 50.74; 51.61; 55.50; 56.21; 63.96; 82.71; 83.47;
96.5; 131.61; 131.66; 132.26; 141.61; 141.66; 141.75; 159.90; 174.80; 175.19; 175.95. FAB-MS: 728.5 (34, [M ‡
‡
‡
‡
Na] ); 706.5 (60, [M ‡ H] ); 606.4 (100, [M Boc] ). Anal. calc. for C39H51N3O9 (705.84): C 66.36, H 7.28, N
5.95; found: C 66.46, H 7.28, N 5.94.
Boc-b3-HMet-b3-HMet-OMe (9). Compound 4 (1.51 g, 5.44 mmol) was deprotected according to GP 4 and
reacted with 3 according to GP 5. Recrystallization (AcOEt) gave 9 (1.75 g, 76%). Yellowish powder. M.p. 135 ±

Helvetica Chimica Acta ± Vol. 84 (2001)
2935
1378. [a]D
ˆ
22.7 (c ˆ 1.01). IR (CHCl3): 812w, 928w, 1052w, 1168s, 1367m, 1438m, 1497s, 1514s, 1702s, 2919w,
3008m, 3428w. 1H-NMR (300 MHz, CDCl3): 1.42 (s, t-Bu); 1.73 ± 1.92 (m, 4 MeSCH2CH2); 2.09 (s, MeS); 2.09 (s,
MeS); 2.31 ± 2.59 (m, 2 MeSCH2, CH2CO); 3.69 (s, MeO); 3.90 ± 3.97 (m, CH); 4.31 ± 4.39 (m, CH); 5.37 (br. d,
J ˆ 5.9, NH); 6.36 (d, J ˆ 8.7, NH). 13C-NMR (75 MHz, CDCl3): 15.57; 28.40; 30.79; 30.86; 33.32; 33.98; 38.24;
‡
40.84; 45.45; 47.63; 51.87; 79.41; 155.60; 170.35; 171.97. FAB-MS: 423 (62, [M ‡ H] ), 323 (100, [M ‡ H
‡
Boc] ). Anal. calc. for C18H34N2O5S2 (422.61): C 51.16, H 8.11, N 6.63; found: C 51.08, H 7.96, N 6.56.
Boc-b3-HMet-b3-HMet-b3-HMet-OMe (10). b-Peptide 9 (1.25 g; 2.97 mmol) was deprotected according to
GP 4 and reacted with 3 according to GP 5. FC (CH2Cl2/MeOH 20 :1) gave 10 (658 mg, 39%). Colorless solid.
M.p. 179 ± 1828. Rf (CH2Cl2/MeOH 20 :1) 0.21. 1H-NMR (500 MHz, CDCl3): 1.44 (s, t-Bu); 1.74 ± 1.98 (m,
3 MeSCH2CH2); 2.09 (s, MeS); 2.10 (s, MeS); 2.10 (s, MeS); 2.32 ± 2.58 (m, 3 MeSCH2, 3 CH2CO); 3.70 (s,
MeO); 3.97 ± 4.03 (br., NCH); 4.22 (br., NCH); 4.35 (m, NCH); 5.33 (br., NH); 6.52 (br., NH); 6.86 (br., NH).
13C-NMR (125 MHz, CDCl3): 15.59; 15.61; 28.43; 30.81; 30.87; 30.98; 33.24; 33.30; 34.36; 38.34; 40.16; 41.07;
‡
45.72; 45.91; 46.70; 47.65; 51.94; 155.75; 170.47; 170.62; 172.15. FAB-MS: 1135.5 (5, [2 M ‡ H] ); 568.2 (100,
‡
‡
[M ‡ H] ); 482.2 (62, [M Boc] ). Anal. calc. for C24H45N3O6S3 (567.83): C 50.77, H 7.99, N 7.40; found: C 50.95,
H 7.82, N 7.46.
Cyclo(b3-HSer(OBn)-b3-HSer(OBn)-b3-HSer(OBn)) (11). b-Peptide 8 (4.94 g, 7.0 mmol) was dissolved in
TFE (60 ml). NaOH (8.4 g, 210 mmol) in H2O (42 ml) was added, and the resulting mixture was stirred for 1 d at
r.t. and for 6 h at 408. The pH value of the resulting soln. was adjusted to 2 ± 3 with 1m HCl soln., and the soln. was
extracted with AcOEt (3Â). The org. layer was dried (MgSO4) and evaporated. The resulting Boc-b-peptide
acid (4.0 g; 5.8 mmol) was dissolved in DMF (300 ml) and cooled under Ar to 08. Pentafluorophenol (1.29 g,
7 mmol) and EDC (1.34 g, 7.0 mmol) were added, and the resulting soln. was stirred at r.t. overnight. The solvent
was evaporated, and the residue was taken up in CHCl3 and washed with 1m HCl and sat. NaCl solns. The solvent
was removed under reduced pressure and dried under h.v. The resulting b-peptide ester was deprotected
according to GP 4, dissolved in 280 ml of MeCN (0.025m) and added by syringe pump over 7 h to a soln. of
EtN(i-Pr)2 (1.36 g, 10.5 mmol) in MeCN (1700 ml, 3.3 mm) at 708. The suspension was stirred at r.t. overnight.
The resulting precipitate was filtered and purified by carefully washing with 0.5m HCl, 0.5m NaOH, sat. NaCl
solns., and MeCN to give 11 (810 mg, 20% based on 8). Colorless powder. M.p. > 2158. IR (KBr): 3286s, 3088w,
3031w, 2858m, 2794w, 1652s, 1559s, 1452m, 1364m, 1314w, 1271w, 1198m, 1130s, 1099s, 1027m, 734m, 696s, 668m,
1
609w, 527w, 466w. H-NMR (400 MHz, CDCl3/10 drops CF3CO2D): 2.54 ± 2.64 (m, 3 CH2CO); 3.53 ± 3.64 (m,
3 BnOCH2); 4.42 ± 4.46 (br., 3 NCH); 7.25 ± 7.38 (m, 15 arom. H). 13C-NMR: 32.55; 37.78; 48.44; 69.96; 73.58;
‡
‡
109.21; 128.22; 128.62; 128.77; 136.07; 173.26. FAB-MS: 1185.6 (15, [2 M ‡ K] ), 1167.6 (6, [2 M ‡ Na] ), 1147.7
‡
‡
‡
‡
(7, [2 M ‡ H] ), 612.3 (6, [M ‡ K] ), 596.3 (57, [M ‡ Na] ), 574.3 (100, [M ‡ H] ).
Cyclo(b3-HMet-b3-HMet-b3-HMet) (12). Compound 10 (503 mg, 0.89 mmol) was dissolved in TFE (7.1 ml,
0.03m soln.). NaOH (1.77 g, 44.3 mmol) in H2O (8.9 ml) were added, and the resulting mixture was stirred for
2 d. The pH value of the resulting soln. was adjusted to 2 ± 3 with 1m HCl soln. and extracted with AcOEt. The
org. layer was dried (MgSO4) and evaporated. The resulting Boc-b-peptide acid (475 mg, 0.86 mmol) was
dissolved in DMF (43 ml; 0.02m) and cooled under Ar to 08. Pentafluorophenol (171 mg, 0.93 mmol) and EDC
(172 mg, 0.90 mmol) were added, and the resulting soln. was stirred at r.t. for 8 h. The solvent was evaporated,
the residue was taken up in CHCl3 and washed with 1m HCl and sat. NaCl solns. The solvent was removed under
reduced pressure and dried under h.v. The resulting b-peptide ester (555 mg, 0.77 mmol) was deprotected
according to GP 4 and dissolved in MeCN (31 ml, 0.025m). This soln. was added by syringe pump over 4 h to a
soln. of EtN(i-Pr)2 (0.24 ml, 1.39 mmol) in MeCN (420 ml, 3.3mm) at 708. The resulting precipitate was filtered
and purified by washing with 0.5m HCl, 0.5m NaOH, and sat. NaCl solns. to give 12 (125 mg, 32% based on 10).
Off-white solid. M.p. > 2158. IR (KBr): 3281s, 3079w, 2913m, 2850w, 1653s, 1559s, 1438m, 1368w, 1294w, 1207w,
1
1145w, 951w, 695w, 632w. H-NMR (300 MHz, CDCl3/10 drops TFA): 1.84 ± 1.92 (m, 3 MeSCH2CH2); 2.11 (s,
3 MeS); 2.46 ± 2.61 (m, 3 CH2CO); 2.81 (m, 3 MeSCH2); 4.48 (br., 3 CH); 7.54 (br., 3 NH). 13C-NMR (75 MHz,
‡
CDCl3/10 drops TFA): 14.91; 29.97; 32.85; 40.74; 48.51; 173.91. FAB-MS: 909.3 (9, [2 M ‡ K] ), 871.4 (6,
‡
‡
‡
[2M ‡ H] ), 498.1 (17.3), 458.2 (17 [M ‡ Na] ), 436.2 (100, [M ‡ H] ).
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Received May 17, 2001