Constrained Derivatives of Stylostatin 1. 1. Synthesis and Biological Evaluation as Potential Anticancer Agents

Article abstract of DOI:10.1021/jm030943h

Hydroxyaminolactams have been used as constrained surrogates of the Ser-Leu dipeptide in the synthesis of analogues of the cycloheptapeptide stylostatin 1 (2). The rate of cyclization through formation of the Ile-Pro amide bond allowed us to prove that th

Full text of DOI:10.1021/jm030943h

J . Med. Chem. 2003, 46, 5825-5833
5825
Con str a in ed Der iva tives of Stylosta tin 1. 1. Syn th esis a n d Biologica l Eva lu a tion
a s P oten tia l An tica n cer Agen ts
Pilar Forns,† J ordi Piro´,† Carmen Cuevas,‡ Mo`nica Garc´ıa,†,§ Mario Rubiralta,†,§ Ernest Giralt,§,| and
Anna Diez†,§,
*
Laboratori de Qu´ımica Orga`nica, Facultat de Farma`cia, Universitat de Barcelona, 08028-Barcelona, Spain,
Pharma Mar, S.A., Av de los Reyes, 1, Polı´gono Industrial La Mina Norte, 28770-Colmenar Viejo, Madrid, Spain,
IRBB-Parc Cientı´fic de Barcelona, c/ J osep Samitier, 1-5, 08028-Barcelona, Spain, and Departament de Qu´ımica Orga`nica,
Facultat de Qu´ımica, Universitat de Barcelona, 08028-Barcelona, Spain
Received J uly 1, 2003
Hydroxyaminolactams have been used as constrained surrogates of the Ser-Leu dipeptide in
the synthesis of analogues of the cycloheptapeptide stylostatin 1 (2). The rate of cyclization
through formation of the Ile-Pro amide bond allowed us to prove that the valerolactams used
induced a turn in the linear precursor. Ring closure at the Pro-Phe amide bond was much
quicker and provided access to larger amounts of the target structures, with high purity. The
conformation of ψ-stylostatin 4 was compared to that of native stylostatin 1 using NMR analysis.
The ability of three ψ-stylostatins and the native stylostatin 1 to inhibit growth of cancer cell
lines was tested. None of the compounds showed activity below 1 µM. A possible relationship
between the decrease in activity and the presence of the piperidone Ser-Leu surrogate is
considered.
In tr od u ction
Cyclic peptides are of biological and chemico-phar-
maceutical interest.1 Structurally their mobility is more
restricted than that of their linear counterparts so that
they adopt fewer conformations and, as a result, show
higher specificity for the target receptors.2 In addition
they are often more stable in vivo. They therefore
represent promising drug candidates.
Cyclic L-D peptides usually show a strong tendency
to self-assemble in tubular arrays which are held
together by parallel or antiparallel â-sheet interactions.3
This property, related to their diverse biological activi-
ties, depends on the ring size of the peptide. In this
regard, the mode of action of small cyclopeptides re-
mains to be properly explained.
The characteristic of small cyclic peptides that inter-
ests us is the tendency of their linear precursors to
exhibit preorganization, which facilitates their cycliza-
tion to give specific compounds.4 We intended to test
the properties of 3-aminohydroxylactams type 15 as
constrained surrogates of the Ser-Leu dipeptide (Figure
1). In general lactams of type 1 induce a â-turn6 in linear
F igu r e 1. Chemical structures of {Ser-Leu} pseudodipeptide,
native stylostatin 1, and proposed Ψ-stylostatins.
peptides. Therefore, the construction of a cyclic peptide
from a linear precursor that contains the aminopiperi-
at the Ser-Leu and Ile-Pro residues. Both its biological
properties and its structure were appropriate for our
studies.
We planned to compare the synthesis of native
stylostatin 1 and ψ-stylostatins 3 and 4 in order to
evaluate the cyclization rate. We also intended to study
possible conformational variations and the differences
in anticancer activity.
done should be faster than that of the native cyclopep-
tide. The cycloheptapeptide stylostatin 1 (Figure 1) was
isolated from the marine sponge Stylotella sp.7 It showed
a cell growth inhibition ED50 of 0.8 µg/mL against P388
lymphocytic leukemia cells, and the 3D-structure, elu-
cidated by the authors,7 showed two â-turns centered
* Corresponding author. Tel.: +34-934037105; fax: +34-934037225;
e-mail: adiez@pcb.ub.es.
† Laboratori de Qu´ımica Orga`nica, Universitat de Barcelona.
‡ Pharma Mar, S.A.
Resu lts a n d Discu ssion
Syn t h esis via Ile-P r o Am id e Bon d F or m a t ion .
We planned to obtain the cyclopeptides by solid-phase
§ IRBB-Parc Cient´ıfic de Barcelona.
| Departament de Qu´ımica Orga`nica, Universitat de Barcelona.
10.1021/jm030943h CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/21/2003

5826 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Forns et al.
Sch em e 1
Ta ble 1. Conditions Assayed for the Cyclization of Compounds 6 and 8
entry
1
reagents
solvent
concentration, M
10-3
temperature, °C
time, h
2
2:9a
3:10a
BOP (3 equiv)
DIPEA (10 equiv)
BOP (3 equiv)
DMF
DMF
DMF
DMF
-10
1:0 reported (67%)
- -
1:1.25
- -
1:4
1:9
2
3
4
5
6
10-3
-10
-10
rt
24
21
3
DIPEA (10 equiv)
PyBOP (3 equiv)
DIPEA (10 equiv)
PyBOP (3 equiv)
DIPEA (10 equiv)
P yBOP (3 equiv)
DIP EA (10 equiv)
PyBOP (3 equiv)
DIPEA (10 equiv)
10-3
1:3
1:1.6
1:1
_
1.5:1
1:1
10-3
DCM/DMF (97:3)
7.8 × 10-4
7.7 × 10-4
r t
5
3.5:1
1.3:1
ACN
rt
6.5
a
Ratio obtained from analytical HPLC.
Sch em e 2
5). In this case an equimolar mixture of 2 and 9 was
obtained.
After isolation by semipreparative HPLC, compounds
2 and 9 showed the same molecular peak by ES-MS (743
Da). Amino acid analysis showed the presence of Ile in
the minor isomer, which was thus identified as native
stylostatin 1 (2), whereas compound 9 contained D-allo-
Ile instead (see Supporting Information). Therefore,
epimerization on the R-carbon of Ile had taken place
during the cyclization.11
synthesis of the linear precursors followed by cyclization
in solution. In this way we would be able to follow the
kinetics of the cyclization step by HPLC analysis.
Bourne et al.8 have reported syntheses of stylostatin 1
through formation of either the Ile-Pro or the Phe-Asn
amide bond and found that the former was more
efficient, leading to stylostatin 1 as a single compound
in 67% yield. Following this first report, we prepared
the linear heptapeptides 6 and 8,9 starting from com-
mercial Fmoc-Ile Wang resin in DMF and using a
mixture of HBTU, HOBt, and DIPEA as the activating
agent (Scheme 1).10 Final deprotection and cleavage
were performed simultaneously by treatment of resins
5 and 7 with 95% TFA in water. The linear peptides 6
and 8 (>85% pure by HPLC) were identified by ES-MS
and by amino acid analysis.
Cyclization of compound 8 was also optimized. As
above, we obtained the best results using DCM:DMF
(97:3) as the solvent, but here the nonepimerized
product 3 was the major product (Table 1, entry 5), as
shown by amino acid analysis following HPLC purifica-
tion.
Syn th esis via P r o-P h e Am id e Bon d F or m a tion .
To avoid epimerization of Ile and to try to improve the
synthesis of stylostatin 112,13 and its analogues, we
performed the head to tail cyclization through the Pro-
Phe amide bond. The required linear peptides 11 and
12 were synthesized in solid phase, using Fmoc-Pro
anchored to commercial Cl-TrtCl resin14 (Scheme 3) and
the HBTU/HOBt/DIPEA system in DMF. Peptides 11
and 12 were obtained >85% pure (HPLC) in 55 and 52%
chemical yield, respectively, and were identified by ES-
MS and amino acid analysis.
We then tested the cyclization conditions (Scheme 2),
using 5 mg samples of compound 6. Treatment of 6 with
BOP/DIPEA in DMF at -10 °C8 gave a mixture of two
cyclic compounds in a 1:4 ratio (Table 1, entry 1).
Prolonging reaction times, increasing the temperature,
or changing the activating reagent to PyBOP did not
modify the results. The best results were obtained when
DCM:DMF (97:3) was used as the solvent (Table 1, entry
The cyclization of compounds 11 and 12 was per-
formed both in DMF and in DCM:DMF, using PyBOP
as the activating agent15 in the presence of DIPEA
(Scheme 4). In all cases the cyclization was complete in
5 min. The expected cyclopeptides, stylostatin 2 and

Constrained Derivatives of Stylostatin 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5827
Sch em e 3
Ta ble 2. Comparative Cyclization of Compounds 6 and 8
conformation of the Ile-Pro amide bond. This is in
agreement with the reported X-ray diffraction struc-
ture,7 thus validating the method for determining the
conformation of pseudostylostatins. Comparison of the
∆δâγ values of the two isomers of pseudostylostatins 3
and 4 made it clear that they correspond to the expected
cis and trans isomers (Table 3). In both cases the cis
isomer is the major one.
time, h
6:2:9
8:3:10
2
28
6.4:1.2:2.4
0:3.6:6.4
0:7.8:2.2
- -
Sch em e 4
Interestingly, epi-stylostatin 9 and epi-ψ-stylostatins
10 and 13 showed only the trans Ile-Pro conformation
in DMSO-d6 (∆δâγ < 6 ppm).
Solven t Ad d ition Exp er im en t (%DMSO). We per-
formed additional NMR experiments in order to probe
further the conformational differences between stylo-
statin 1 (2) and Ψ-stylostatin 4. We first wanted to know
which amide protons were involved in hydrogen bonding
or were not accessible to the solvent, since this would
indicate orientation toward the inside of the macrocycle.
To ensure that the hydrogen bonds were intramolecular,
we first ran the spectrum at various concentrations of
the compounds in DMSO (0.25-16 mM) and in CDCl3
+ 0.5% DMSO (0.25-1 mM).17 No chemical shift dif-
ferences were observed by changing concentration,
which confirmed that the results would not be influ-
enced by intermolecular interactions.
We then compared the chemical shifts of the amide
NH protons in a gradient of DMSO in CDCl3. Protons
that are non-hydrogen-bonded, and totally accessible to
the solvent, should quickly establish a hydrogen bond
with DMSO so that their signal shifts downfield,
whereas protons that do not shift significantly may be
either hydrogen-bonded or not accessible to the solvent.
The spectra of stylostatin 1 (2) showed that the side
chain Asn amide protons and Ile-NH are totally acces-
sible to the solvent, but that most of the other NH
protons appear to be involved in hydrogen bonds (Figure
2a). The Ser-NH signal shift started at 8% DMSO,
suggesting limited access to solvent or weak hydrogen
bonding.
ψ-stylostatin 3, were obtained very pure (>95% by
HPLC) as the only reaction products, and in good
yields. Neither epimerization nor dimerization was
observed.
Once the cyclization had been optimized, we hydro-
genolyzed compounds 3 and 10 using Pd/C as the
catalyst to obtain ψ-stylostatins 4 and 13, which were
identified by MS and amino acid analysis (Scheme 5).
Kin etic Stu d ies. To study the influence of the
constrained Ser-Leu surrogate 1a on the cyclization
process, we followed the kinetics of the transformation
of compounds 6 and 8 by analytical HPLC (Scheme 2,
Table 2).16 The reaction was carried out on 40 mg
samples in DCM/DMF using PyBOP/DIPEA. The reac-
tion of compound 8 was complete after 2 h, whereas 6
required 28 h to be fully transformed. Thus, the
introduction of the pseudopeptide {Ser(Bn)-Leu} 1a
increased the cyclization rate 14-fold, suggesting that
the restricted pseudopeptides may induce a bent con-
formation of the linear precursor.
Str u ctu r a l NMR Stu d ies. As expected, the NMR
data of compound 2 in DMSO-d6 were in agreement
with those reported for native stylostatin 1.7 However,
1
the H NMR spectra of Ψ-cyclopeptides 3 and 4 showed
two sets of signals (Table 3), indicating the presence of
two conformers in DMSO-d6. We considered that this
slow interconversion could be due to Ile-Pro amide bond
rotamers. A characteristic feature of cis X-Pro dipep-
tides, compared to the corresponding trans X-Pro iso-
mers, is the chemical shift difference between the â and
γ carbon atoms of Pro: ∆δâγ g 8 ppm for the cis isomers,
whereas ∆δâγ < 6 ppm for the trans.1b For native
stylostatin 1, ∆δâγ ) 9 ppm, which indicates a cis
It was interesting that the spectrum of ψ-stylostatin
4 in CDCl3 showed very broad signals, especially for the
amide protons. On addition of DMSO the signals gained
resolution, reaching a maximum at 8% DMSO. In this
range the chemical shifts do not vary, and only a cis

5828 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Forns et al.
Sch em e 5
Ta ble 3. Proline Relevant 13C NMR Chemical Shifts in
DMSO-d6
This experiment showed that the replacement of the
Ser-Leu dipeptide by the piperidone surrogate affects
the conformational response of the cyclopeptide to
solvent variation. The lack of the Leu NH may contrib-
ute to this behavior, and the fact that the Ile-Pro trans
conformation is visible for ψ-stylostatin 4 indicates an
increased structural flexibility with respect to compound
2. In addition, the trans isomer becomes significant in
the presence of DMSO. This suggests that in the trans
isomer the amide protons are oriented toward the
external side of the cycle, and that hydrogen bonding
with the solvent may stabilize the conformation.
2
3
4
9
10
13
δCâ
29.7 29.0/27.9 29.2/28.0 28.8
20.7 21.0/24.9 21.0/25.1 24.3
28.4
24.5
3.9
28.5
24.4
4.1
δCγ
Ddbg
9
8/3
8.2 /2.9
4.5
Ile-Pro
conformation
cis
cis:trans cis:trans trans trans trans
) 1.6:1 ) 1.8:1
Tem p er a tu r e Coefficien ts (∆δ/δT). Our second
experiment consisted of calculating the temperature
coefficients of the amide protons in DMSO solution in
order to confirm their hydrogen-bonding state.18 In
general, absolute values above 4 ppb/K indicate that the
proton is non-hydrogen-bonded and fully accessible to
the solvent, whereas coefficients below 3 ppb/K indicate
a hydrogen-bonded state. Intermediate values would
indicate that the proton is in equilibrium between a
hydrogen-bonded and a non-hydrogen-bonded state.
The spectrum of stylostatin 1 (2) in DMSO showed
that only the Ser and Ile-NH amide protons are free
(Table 4). This corresponds to the conformation that
Pettit et al. observed by X-ray diffraction,7 in which the
hydrogen bonds of the two â-turns involve the Phe-NH
and the Ala-NH. The authors also indicate a third
hydrogen bond between the Leu-NH and the carbonyl
of the side chain of Asn. The fourth hydrogen bond is
not apparent from the X-ray diffraction analysis, but
the orientation of the Asn-NH toward the inner part of
the cycle is clear.
F igu r e 2. (a) 1H NMR chemical shifts of NH protons of
stylostatin 1 (2) at different proportions of DMSO-d6 in CDCl3
(3 mM, 25 °C). (b) H NMR chemical shifts of NH protons of
ψ-stylostatin 1 (cis-4) at different proportions of DMSO-d6 in
CDCl3 (3 mM, 25 °C).
For ψ-stylostatin 4 (Table 4), the first observation was
that increasing temperature led to a predominance of
the cis Ile-Pro conformer, and a dramatic loss in
resolution of signals corresponding to the Ile-Pro trans
1
Ile-Pro conformation is observed (Pro ∆δâγ ) 9 ppm, in
8% DMSO). With continued addition of DMSO, a second
set of signals, corresponding to the trans Ile-Pro con-
former (Pro ∆δâγ ) 2.9 ppm, in DMSO), appears, which
increases with the proportion of DMSO up to a 1.9:1
cis:trans proportion in DMSO.
The chemical shifts of the amide protons in the
solvent gradient show no major variations (Figure 2b),
and the fact that they are deshielded (δ > 7.5) suggests
that they are mostly accessible to the solvent or hydro-
gen bonded.
Ta ble 4. Temperature Coefficients (|∆δ/∆Τ| in ppb/K) in
DMSO
stylostatin 1 (2)
ψ-stylostatin (cis-4)
Phe
Asn
Ser
Leu
Ala
Ile
Asn-NHA
Asn-NHB
2.3
0.3
5.4
2.5
0.9
6.5
2.9
3.9
3.5
2
4.8
- -
1
4.7
3.2
- -a
a
The signals were masked in the 7.05-7.20 region.

Constrained Derivatives of Stylostatin 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5829
Ta ble 5. Growth Inhibition Effect (GI50 in µΜ) Found for the
that the presence of the Ser-Leu piperidone surrogate
slightly decreases the activity of the compounds. The
conformational alteration is possibly responsible for this
difference, although this explanation would not account
for the observation that the epi analogue 9 (only trans)
is more active than the others.
Compounds Tested
2
4
9
13
prostate tumors DU-145
8.44 10.6
3.55
9.82
3.13
4.03
3.48
5.02
4.06
5.55
4.32
4.94
7.22
LN-caP
SKOV-3
IGROV
2.44
3.77
3.35
4.29
4.16
4.64
4.28
4.97
7.13
3.49 2.60
4.99 3.04
3.33 2.58
4.97 3.30
3.86 4.39
6.69 3.06
5.20 3.46
5.10 5.03
9.31 6.85
ovarian tumors
IGROV-ET
Con clu sion
SK-BR-3
MEL-28
A-549
melanoma
NSCL
We have obtained five derivatives of the cyclohepta-
peptide stylostatin 1 (2), and we have established a new
and improved synthetic route for the synthesis of
stylostatin 1 (2). We have shown that the piperidone
surrogate {Ser-Leu} induces a turn in the linear peptide
which facilitates the cyclization.
pancreas
PANC-1
HT-29
colon
LOVO
LOVO-DOX
K-652
11.5
8.52
3.42
8.83 4.55 12.7
8.84 6.77 12.4
leukemia
3.59 1.94
4.32
The NMR data showed that the effect of introducing
the piperidone ring in the structure was much more
dramatic than expected. The hydrogen bonding pattern
of the molecule 4 is completely altered. As a result, the
molecule is more flexible and establishes a slow equi-
librium between cis and trans Ile-Pro amide bond
conformations, which is favored by the hydrogen bond-
ing solvent DMSO. We have evaluated the anticancer
activity of compounds 2, 4, 9, and 13. None of them is
active (GI50) below 1 µM, but the differences observed
in activity suggest that the piperidone ring can cause a
loss of activity.
conformer, to the point that only the data for the cis
conformer were reliable.
Of the four hydrogen bonds that stabilize the Ile-Pro
cis conformation of the native stylostatin 1 (2), ψ-stylo-
statin cis-4 lacks that of the Leu-NH, the Phe-NH
appears to be in equilibrium between a hydrogen-
bonded and a non hydrogen-bonded state, the Asn-NH
hydrogen bond becomes weaker, and only the Ala-NH
hydrogen bond is maintained. These data clearly indi-
cate that the cis conformer of ψ-stylostatin 4 is less
stable than that of the native peptide 2. This lower
degree of structuring implies a greater flexibility for the
molecule. In turn, this would allow the slow equilibrium
with the Ile-Pro trans conformer to take place.
From the NMR experiments, we can conclude that on
introducing the piperidone surrogate the turn centered
on the Ser-Leu dipeptide is maintained but that the
other stabilizing hydrogen bonds are significantly modi-
fied, with the result that the molecule becomes more
flexible, allowing for the onset Ile-Pro trans-conforma-
tion in DMSO.
Upon heating of the sample,19 the proportion of the
Ile-Pro trans isomer of 4 diminishes with respect to cis-
4. However, we did not observe a coalescence of the
signals to an intermediate shift, as it is usual in simple
amide rotamers. The fact that the two conformers
showed two independent sets of signals indicates that
cis-4 and trans-4 are in a very slow equilibrium.
It is noteworthy that this X-Pro trans conformation
is the only one observed in the three “epi” analogues 9,
10, and 13. This is understandable, since the Ile-NH
amide proton stabilizes the second â-turn of the native
stylostatin 1 (2) structure. The different orientation of
the substituents about the R-carbon of D-allo-Ile prob-
ably exerts a steric effect that favors the D-allo-Ile-Pro
trans conformer.
Exp er im en ta l Section
Abbr evia tion s. Abbreviations used for amino acids and
peptides follow the rules of the IUPAC-IUB Commission of
Biochemical Nomenclature.20 The following additional ab-
breviations are used: BOP, benzotriazol-1-yloxytris(dimethyl-
amino)phosphonium ; Cl-TrtCl-resin, 2-chlorotrityl chloride
resin; DCM, dichlorometane; DHB, 2,5-dihydroxybenzoic acid;
DIPEA, N,N-diisopropylethylamine; DIPCDI, N,N-diisopropyl-
carbodiimide; DMF, N,N-dimethylformamide; DMSO, di-
methyl sulfoxide; ES-MS, electrospray mass spectrometry;
EtOAc, ethyl acetate; Fmoc-OSu, 9-fluorenylmethoxycarbonyl-
N-hydroxysuccinimide; HPLC, high performance liquid chro-
matography; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium ; HOBt, 1-hydroxybenzotriazole; MALDI-TOF,
matrix-assisted laser desorption ionization-time-of-flight; MeOH,
methanol; NMR, nuclear magnetic resonance; PyBOP, benzo-
triazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium ; SPS, solid-
phase synthesis; SRB, sulforhodamine B; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; UV, ultraviolet. Amino acid
symbols denote the L-configuration unless stated otherwise.
Gen er a l. Solid-phase syntheses were carried out at 25 °C
in polypropylene syringes (5/10 mL) fitted with a polyethylene
porous disk. Solvents and soluble reagents were removed by
suction. Washings were performed with DMF (5 × 1 min) and
CH2Cl2 (5 × 1 min) using 10 mL of solvent per gram of resin
for each wash. Flash chromatography was eluted through SiO2
(silica gel 60 A CC, 230-240 mesh ASTM, SDS). TLC analysis
was done on SiO2 plates (F254, Macherey-Nagel) and devel-
oped with the solvent described in each case for flash chro-
matography. The spots were located by UV light and K2MnO4
reagent. Purification of reagents and solvents was effected
according to standard methods.21 Optical rotations were
measured on a Perkin-Elmer 241 MC polarimeter. MALDI-
TOF and ES-MS analyses of peptide samples were performed
in a Perspective Biosystems Voyager DE RP, using DHB
matrixes, and in a Micromass VG-quattro spectrometer.
Bioa ssa y Da ta . Since stylostatin 1 (2) had been
reported as cytostatic against P388 lymphocyte leuke-
mia cells (ED50 of 0.8 µg/mL),7 we tested the ability of
compounds 2, 4, and their epi analogues 9 and 13 to
inhibit the growth of a series of cancer cell lines (see
Experimental Section and Supporting Information). The
colorimetric tests showed that the most active compound
is epi-stylostatin 9, which shows a GI50 of 1.94 µM
against the K-652 leukemia cell line. However, none of
these compounds is active below 1 µM (GI50 shown in
Table 5).
1H and 13C NMR spectra of compounds 1a and 1b were
recorded on a Varian Gemini-200, in CDCl3. NMR experiments
of the peptides were carried out using a Bruker 600-MHz NMR
spectrometer. The samples were approximately 3 mM in
DMSO-d6. Chemical shifts were reported in δ (ppm) relative
to the undeuterated fraction of DMSO-d6 and to TMS. Amino
Despite the lack of growth inhibitory activity, a
comparison of the GI50 of 2 vs 4 and of 9 vs 13, shows

5830 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Forns et al.
acid analyses were performed on a Beckman system Gold 6300
at the Serveis Cient´ıfico-Te`cnics (UB).
were suspended in tert-butyl methyl ether and precipitated
by centrifugation. The peptide was then purified and charac-
terized according to the following general protocol.
(3R,4S,5R)-5-Ben zyloxy-N-[(1S)-1-ter t-bu toxyca r bon yl-
3-m eth ylbu tyl]-3-flu or en yloxycar bon ylam in o-4-h ydr oxy-
p ip er id in -2-on e (F m oc-{Ser (Bn )-Leu }-OtBu ). To a solution
of amine 1a 5b (282 mg, 0.69 mmol) in acetone (3 mL) were
added Fmoc-OSu (299 mg, 0.88 mmol) and NaHCO3 (185 mg,
2.21 mmol). The mixture was stirred at room temperature for
8 h. The solvent was evaporated, and the residue was
partitioned in brine and CH2Cl2. The aqueous phase was
extracted with CH2Cl2, and the combined organic layers, dried
and evaporated, gave an oil. Purification by flash chromatog-
raphy (hexane:EtOAc, 3:1) yielded the Fmoc-protected amino-
piperidone (412 mg, 98%) as an oil: [R]20D -21° (c 1.0, CHCl3);
1H NMR (CDCl3) 0.88 (d, J ) 6.3 Hz, 3H, CH(CH3)2), 0.92 (d,
J ) 6.3 Hz, 3H, CH(CH3)2′), 1.44 (s, 9H, C(CH3)3), 1.5-1.7 (m,
3H, CH(CH3)2, CH2), 3.34 (dd, J ) 13.2 and 3.0 Hz, 1H, H-6),
3.42 (dd, J ) 13.1 and 2.4 Hz, 1H, H-6′), 4.0-4.2 (m, 2H, H-4,
H-5), 4.24 (t, J ) 6.9 Hz, 1H, CH-Fmoc), 4.40 (d, J ) 6.7 Hz,
2H, CH2-Fmoc), 4.60 (dd, J ) 9.0 and 4.8 Hz, 1H, H-3), 4.70
(d, J ) 12.3 Hz, 1H, CH2Ph), 4.86 (d, J ) 12.3 Hz, 1H, CH2Ph),
5.16 (t, J ) 7.8 Hz, 1H, NCH), 6.00 (d, J ) 4.0 Hz, 1H, NH),
7.25-7.50 (m, 9H, Ph, H2 and H3-Fmoc), 7.61 (d, J ) 11.2
Hz, 2H, H1-Fmoc), 7.78 (d, J ) 8.2 Hz, 2H, H4-Fmoc); 13C NMR
(CDCl3) 21.0 (CH(CH3)2), 23.2 (CH(CH3)2′), 24.3 (CH(CH3)2),
27.9 (C(CH3)3), 36.6 (CH2), 44.1 (C6), 46.9 (CH-Fmoc), 54.7
(NCH), 55.6 (C3), 67.6 (CH2-Fmoc), 72.8 (C5), 72.9 (CH2Ph),
73.5 (C4), 81.9 (C(CH3)3), 120.3 (C4-Fmoc), 126.9 (C1-Fmoc),
128.0-128.5 (Ph-C2, Ph-C3, Ph-C4, C2 and C3-Fmoc), 137.7
(Ph-C1), 141.1 (C5-Fmoc), 143.5 (C6-Fmoc), 158.7 (CO car-
bamate), 167.8 (CO), 170.1 (CO); MS m/z 628 (M+, 2%). Anal.
Calcd for C37H44N2O7: C, 70.68; H, 7.05; N, 4.46. Found: C,
70.65; H, 7.08; N, 4.43.
Gen er a l Meth od for P u r ifica tion a n d Ch a r a cter iza -
tion of th e P ep tid es. The compounds were purified on a
Waters HPLC system using a SymmetryPrep C18 reversed-
phase preparative column (7.8 × 300 mm) with 7 µm packing
material. The crude peptides (50-100 mg) were dissolved in
water/acetonitrile; 1/2 (1 mL), filtered and injected through a
Rheodyne injector with 1 mL sample loop. The mobile phases
used were A: 0.05% aqueous TFA and B: 0.05% TFA in
acetonitrile, with a linear gradient from 10%B to 45%B over
40 min (3 mL/min). Fractions were manually collected at 1
min intervals. Elution of the peptide was determined simul-
taneously from the absorbances at 220 and 254 nm (Waters
2487). Fractions were reinjected to assess purity on analytical
reversed-phase HPLC (Waters NovaPak C18, 3.9 × 150 mm, 4
µm). In this case, we used a linear gradient from 0%B to
100%B over 30 min for peptides characterization. Pure frac-
tions were combined and lyophilized. Each peptide showed
[M + H+] and/or [M + Na+] peaks in its mass spectrum, and
each was >90% pure. The structure was confirmed by amino
acid analysis and NMR. The NMR signal assignments required
2D-HSQC, 2D-TOCSY, COSY (H,H), and 2D-NOESY experi-
ments. The samples for NMR were prepared approximately 3
mM in DMSO-d6.
Stylosta tin 1 P r ecu r sor (6). Operating as in the general
method, using Fmoc-Ile Wang resin (600 mg, 0.57 mmol/g),
we obtained compound 6 (H-Pro-Phe-Asn-Ser-Leu-Ala-Ile-OH).
The required Fmoc-amino acids were Fmoc-Ala-OH, Fmoc-
Leu-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-
OH, and Fmoc-Pro-OH. Peptide 6 (195 mg, 75%) was obtained
as a white solid with a purity of >85% after HPLC purification
(tR 14.4 min, characterization conditions). ES-MS calcd for
2(S)-2-[(3R, 4S,5R)-5-Ben zyloxy-3-(flu or en yloxycar bon -
ylam in o)-4-h ydr oxy-2-oxo-1-piper idin yl]isoh exan oic Acid
(F m oc-{Ser (Bn )-Leu }-OH). A mixture of Fmoc-{Ser(Bn)-
Leu}OtBu (500 mg, 0.82 mmol), CH2Cl2 (3 mL), and TFA (3
mL) was stirred at room temperature for 2.5 h. After evapora-
tion of the solvent, the solid residue was triturated several
times with cold Et2O to provide Fmoc-{Ser(Bn)-Leu}-OH (380
C
36H56N8O10 760.9, found m/z 761.1 [M + H+].
Stylosta tin 1 P r ecu r sor (11). Atta ch m en t of th e F ir st
Am in o Acid . A solution of Fmoc-Pro-OH (81 mg, 0.24 mmol,
0.5 equiv) and DIPEA (84 µL, 0.48 mmol, 2 equiv) in DCM (1
mL) was added to the Cl-TrtCl-resin (300 mg, 1.6 mmol/g),
and the reaction mixture was gently stirred for 1 h. MeOH
(0.8 mL) was then added, and after stirring for 10 min, the
Fmoc-Pro-O-TrtCl resin (0.8 mmol/g) was sequentially washed
with DCM and DMF. Operating as in the general method
for synthesis and purification, from the Fmoc-Pro-O-TrtCl
resin, and Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-
Ser(OtBu)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Phe-OH, peptide
11 (100 mg, 55%) was obtained as a white solid with a purity
of >85% as determined by HPLC (tR 14.1 min, characterization
conditions). ES-MS calcd for C36H56N8O10 760.9, found m/z
761.3 [M + H+].
ψ-Stylosta tin P r ecu r sor (8). Operating as in the general
method for the synthesis and purification, from Fmoc-Ile Wang
resin, and Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH,
Fmoc-Pro-OH, Fmoc-Ile-OH, and Fmoc-{Ser-Leu}-OH, we
obtained compound 8 (H-Pro-Phe-Asn-{Ser-Leu}-Ala-Ile-OH).
The linear ψ-peptide 8 (231 mg, 76%) was obtained as a white
solid and >85% pure, as determined by HPLC (tR 18.2 min,
characterization conditions). ES-MS calcd for C45H64N8O11
892.4, found m/z 893.2 [M + H+].
ψ-Stylosta tin P r ecu r sor (12). Operating as in the general
method for the synthesis and purification, from Fmoc-Pro-O-
TrtCl resin (0.8 mmol/g, obtained as for the preparation of 11),
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-{Ser(Bn)Leu}-OH, Fmoc-
Asn(Trt)-OH, and Fmoc-Phe-OH, we obtained ψ-peptide 12 (H-
Phe-Asn-{Ser(Bn)-Leu}-Ala-Ile-Pro-OH). The linear γ-peptide
12 (94.5 mg, 52%) was obtained as a white solid >85% pure,
as determined by HPLC (tR 17.6 min, characterization condi-
tions). ES-MS calcd for C45H64N8O11 892.4, found m/z 892.9
[M + H+].
Gen er a l P r oced u r e for Cycliza tion . To a solution of the
linear heptapeptides in DMF at room temperature were added
BOP or PyBOP and DIPEA. The cyclization was monitored
by analytical reversed-phase HPLC. When cyclization was
completed, the solvent was removed under reduced pressure
mg, 80%) as a white solid. Mp 197 °C (Et2O). [R]20 -41° (c
D
0.5, MeOH); 1H NMR (CD3OD) 0.94 (d, J ) 6.6 Hz, 6H,
CH(CH3)2), 1.6-2.0 (m, 3H, CH(CH3)2, CH2), 3.5-3.6 (m, 2H,
H-6), 4.1-4.2 (m, 2H, H-4, H-5), 4.34 (t, J ) 6.6 Hz, 1H, CH-
Fmoc), 4.42 (d, J ) 6.6 Hz, 2H, CH2-Fmoc), 4.50 (d, J ) 9.6
Hz, 1H, H-3), 4.79 (d, J ) 12 Hz, 1H, CH2Ph), 4.86 (d, J ) 12
Hz, 1H, CH2Ph), 5.31 (dd, J ) 10.8 and 3.9 Hz, 1H, NCH),
7.25-7.30 (m, 5H, Ph), 7.40 (t, J ) 6 Hz, 2H, H2-Fmoc), 7.47
(t, J ) 7.2 Hz, 2H, H3-Fmoc), 7.79 (d, J ) 7.2 Hz, 2H, H1-
Fmoc), 7.89 (d, J ) 7.2 Hz, 2H, H4-Fmoc); 13C NMR (CD3OD)
21.4 (CH(CH3)2), 23.7 (CH(CH3)2′), 25.2 (CH(CH3)2), 37.8 (CH2),
44.6 (C6), 48.1 (CH-Fmoc), 56.8 (C3, NCH), 68.3 (CH2-Fmoc),
71.1 (C5), 73.3 (CH2Ph), 75.7 (C4), 120.8 (C4-Fmoc), 126. 4 (C1-
Fmoc), 128.5-129.5 (Ph-C2, Ph-C3, Ph-C4, C2 and C3-Fmoc),
139.6 (Ph-C1), 142.5 (C5-Fmoc), 145.3 (C6-Fmoc), 159.3 (CO
carbamate), 171.2 (CO); MS m/z 572 (M+, 2%).
Gen er a l Meth od for th e Solid -P h a se P ep tid e Syn th e-
sis. The synthesis of linear peptides was accomplished manu-
ally by the Fmoc methodology using either the Fmoc-Ile Wang
resin or the Cl-Trt-Cl resin as solid supports. The N-Fmoc-
amino acids were activated using HBTU (2 equiv), HOBt (2
equiv), and DIPEA (1.5 equiv) in DMF for 10 min, before their
addition to the resin. The coupling reactions were maintained
for 4 h, and their completion was verified by the ninhydrin
(Kaiser) test. The coupling process was repeated under the
same conditions in case of positive ninhydrin test. Removal of
the Fmoc group was performed by incubation in 20% piperidine
in DMF (v/v) (2 × 1 min and 2 × 10 min). After the final
deprotection step, the obtained peptide resin was dried in
vacuum and treated with TFA/H2O (95/5, v/v, 15 mL/g resin)
for 4 h at room temperature to cleave the peptide from the
resin and remove the side chain protecting groups. The solid
support was filtered off and washed with TFA several times.
The solvent was evaporated and the obtained oily products

Constrained Derivatives of Stylostatin 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5831
to yield a light yellow oil, which was purified by semiprepara-
tive RP-HPLC. After lyophilization of the pure cyclopeptides
they were white solids.
as in the general method to yield stylostatin 1 (2, 9.6 mg, 50%)
as a white solid.
Cycliza tion of P ep tid e 8 To Give Ben zyl-ψ-stylosta tin
(3) a n d epi-Ben zyl-ψ-stylosta tin (10). Following the general
procedure, from linear peptide 8 (20 mg, 0.022 mmol), PyBOP
(14 mg, 0.027 mmol), and DIPEA (9.2 µL, 0.053 mmol),
cyclization was completed after 2 h as determined by analytical
reversed-phase HPLC. The crude product was purified by
semipreparative RP-HPLC and lyophilized to yield ψ-stylo-
statin 3 (6.2 mg, 32%) and its Ile epimer 10 (3.7 mg, 19%) as
white solids. Ben zyl-ψ-stylosta tin (3): Analytical HPLC (tR
19.5 min, characterization conditions); ES-MS calcd for
Cycliza tion of 6 To Give Stylosta tin 1 (2) a n d epi-
Stylosta tin (9). Following the general procedure for cycliza-
tion, from compound 6 (20 mg, 0.026 mmol), PyBOP (16.2 mg,
0.031 mmol), and DIPEA (11 µL, 0.063 mmol), the cyclization
was completed after 28 h, as determined by analytical reversed-
phase HPLC. The crude product was purified by semiprepara-
tive RP-HPLC and lyophilized to yield stylostatin 1 (2) (4.5
mg, 23%) and its Ile epimer 9 (7.7 mg, 40%) as white solids.
Stylosta tin 1 (2): Analytical HPLC (tR 15.6 min, character-
ization conditions); ES-MS calcd for C36H54N8O9 742.4, found
C
45H62N8O10 874.4, found m/z 875.5 [M + H+]. Tr a n s-3
1
1
m/z 743.4 [M + H+]; NMR (DMSO-d6) ser in e unit, H 3.67-
r ota m er : NMR (DMSO-d6) {ser in e} unit, H 3.00-3.05 (m,
1H, H-5), 3.32-3.42 (m, 1H, H-5), 3.65 (br t, J ) 7.3 Hz, 1H,
H-2), 4.03-4.06 (m, 1H, H-4), 4.12 (br d, J ) 8.4 Hz, 1H, H-3),
4.61 (d, J ) 12 Hz, 1H, CHAPh), 4.67 (d, J ) 12 Hz, 1H,
CHBPh), 5.10-5.20 (br s, 1H, OH), 7.10-7.34 (m, 5H, H-Ph),
9.07 (br s, 1H, NH); 13C 41.6 (C-5), 55.3 (C-2), 67.7 (C-3), 70.7
(OCH2Ph), 73.3 (C-4), 125.9-128.6 (C-Ph); a la n in e unit, 1H
1.30 (d, J ) 7 Hz, 3H, H-3), 4.25-4.34 (m, 1H, H-2), 7.71 (br
s, 1H, NH); 13C 18.6 (C-3), 50.2 (C-2); isoleu cin e unit, 1H 0.92
(d, J ) 6.7 Hz, 3H, H-5), 0.79-0.86 (m, 3H, C(3)-CH3), 1.36-
1.45 (m, 2H, H-4), 1.92-1.95 (m, 1H, H-3), 4.50-4.56 (m, 1H,
H-2), 7.16-7.20 (m, 1H, NH); 13C 11.2 (C-5), 16.0 (C(3)-CH3),
3.69 (m, 2H, H-3), 3.85 (dd, J ) 9.2 and 4 Hz, 1H, H-2), 8.46
(d, J ) 3.6 Hz, 1H, NH); 13C 58.8 (C-2), 60.4 (C-3); a la n in e
unit, 1H 1.13 (d, J ) 6.5 Hz, 3H, H-3), 4.44-4.46 (m, 1H, H-2),
7.32 (d, J ) 7.2 Hz, 1H, NH); 13C 15.9 (C-3), 47.4 (C-2);
isoleu cin e unit, 1H 0.77-0.82 (m, 9H, C(3)-CH3, H-5), 1.22-
1.30 (m, 1H, H-4), 1.50-1.55 (m, 1H, H-4), 1.71 (m, 1H, H-3),
4.02 (dd, J ) 8.7 and 5 Hz, 1H, H-2), 8.65 (d, J ) 4.8 Hz, 1H,
NH); 13C 10.5 (C-5), 14.4 (C(3)-CH3), 24.4 (C-4), 35.2 (C-3),
56.6 (C-2); p r olin e unit, 1H 0.68-0.77 (m, 1H, H-4), 1.49-
1.60 (m, 2H, H-4 and H-3), 2.15 (dd, J ) 12.4 and 7.1 Hz, 1H,
H-3), 2.6 (t, J ) 10.6 Hz, 1H, H-5), 3.23 (dt, J ) 10.6 and 7.9
Hz, 1H, H-5), 4.42 (d, J ) 7.1 Hz, 1H, H-2); 13C 20.7 (C-4),
29.7 (C-3), 45.1 (C-5), 60.1 (C-2); p h en yla la n in e unit, 1H 3.02
(under the H2O signal, 1H, H-3), 3.14 (dd, J ) 14 and 4 Hz,
1H, H-3), 4.15 (ddd, J ) 12, 7.8 and 4.4 Hz, 1H, H-2), 7.12 (d,
J ) 7.1 Hz, 2H, H-oPh), 7.20 (t, J ) 7.3 Hz, 1H, H-pPh), 7.27
1
22.9 (C-4), 37.2 (C-3), 54.4 (C-2); p r olin e unit, H 1.60-1.65
(m, 1H, H-4), 1.65-1.70 (m, 1H, H-3), 1.77-1.83 (m, 1H, H-3),
1.90-2.00 (m, 1H, H-4), 3.33-3.50 (m, 1H, H-5), 3.70-3.75
(m, 1H, H-5), 3.90-3.96 (m, 1H, H-2); 13C 24.9 (C-4), 27.9 (C-
1
3), 46.8 (C-5), 61.4 (C-2); p h en yla la n in e unit, H 3.26-3.32
(m, 1H, H-3), 3.75-3.80 (m, 1H, H-3), 3.75-3.80 (m, 1H, H-2),
(d, J ) 7.3 Hz, 2H, H-mPh), 8.58 (d, J ) 7.8 Hz, 1H, NH); 13
C
7.10-7.34 (m, 5H, H-Ph), 8.43 (br s, 1H, NH); 13C 33.6 (C-3),
36.8 (C-3), 57.5 (C-2), 126.3 (C-pPh), 128.2 (C-mPh), 128.4 (C-
oPh); leu cin e unit, 1H 0.77-0.82 (m, 3H, H-5), 0.83 (d, J )
6.2 Hz, 3H, H-5), 1.48-1.52 (m, 1H, H-4), 1.53-1.63 (m, 2H,
H-3), 4.27-4.31 (m, 1H, H-2), 8.03 (d, J ) 9.6 Hz, 1H, NH);
13C 21.0 and 22.9 (C-5), 24.2 (C-4), 39.9 (C-3), 51.3 (C-2);
1
56.4 (C-2), 125.9-128.9 (C-Ph); leu cin e unit, H 0.74 (d, J )
6.2 Hz, 3H, H-5), 0.79-0.86 (m, 3H, H-5), 1.36-1.42 (m, 1H,
H-4), 1.55-1.60 (m, 2H, H-3), 5.03-5.08 (m, 1H, H-2); 13C 20.3
and 23.0 (C-5), 23.0 (C-4), 34.1 (C-3), 52.8 (C-2); a sp a r a gin e
1
unit, H 2.50-2.60 (m, 1H, H-3), 2.27 (dd, J ) 15 and 5.2 Hz,
1
a sp a r a gin e unit, H 3.04-3.08 (m, 2H, H-3), 4.27-4.31 (m,
1H, H-3), 4.70-4.76 (m, 1H, H-2), 6.92 (br s, 1H, CONH2),
1H, H-2), 7.26-7.30 (m, 1H, CONH2), 7.74 (d, J ) 5.4 Hz, 1H,
NH), 7.78 (sa, 1H, CONH2); 13C 35.7 (C-3), 49.5 (C-2). epi-
Stylosta tin (9): Analytical HPLC (tR 16.9 min, characteriza-
tion conditions); ES-MS calcd for C36H54N8O9 742.9, found m/z
7.16-7.20 (m, 1H, CONH2), 8.15 (d, J ) 8.8 Hz, 1H, NH); 13
C
37.5 (C-3), 50.0 (C-2). Cis-3 r ota m er : NMR (DMSO-d6)
{ser in e} unit, 1H 3.05 (br d, J ) 10.5 Hz, 1H, H-5), 3.32-
3.42 (m, 1H, H-5), 3.65 (br t, J ) 7.3 Hz, 1H, H-2), 3.96-3.99
(m, 1H, H-4), 4.03-4.06 (m, 1H, H-3), 4.59 (d, J ) 12 Hz, 1H,
CHAPh), 4.66 (d, J ) 12 Hz, 1H, CHBPh), 5.10-5.20 (br s, 1H,
OH), 7.10-7.34 (m, 5H, H-Ph), 8.96 (d, J ) 6.1 Hz, 1H, NH);
13C 42.3 (C-5), 56.2 (C-2), 68.5 (C-3), 70.7 (OCH2Ph), 73.5 (C-
4), 125.9-128.6 (C-Ph); a la n in e unit, 1H 1.30 (d, J ) 7 Hz,
3H, H-3), 4.50-5.56 (m, 1H, H-2), 7.94 (d, J ) 8.8 Hz, 1H,
1
743.9 [M + H]+; NMR (DMSO-d6) ser in e unit, H 3.72 (ddd,
J ) 11.6, 4.8 and 3 Hz, 1H, H-3), 3.96-4.03 (m, 1H, H-3), 4.17-
4.25 (m, 1H, H-2), 5.13 (m, 1H, OH), 7.22-7.28 (m, 1H, NH);
13C 55.5 (C-2), 62.2 (C-3); a la n in e unit, 1H 1.23 (d, J ) 7.3
Hz, 3H, H-3), 4.25-4.27 (m, 1H, H-2), 7.22-7.27 (m, 1H, NH);
1
13C 18.1 (C-3), 48.7 (C-2); D-a llo-isoleu cin e unit, H 0.73 (d,
J ) 7 Hz, 3H, C(3)-CH3), 0.81-0.86 (m, 3H, H-5), 0.91-0.97
(m, 1H, H-4), 1.27-1.33 (m, 1H, H-4), 1.84-1.90 (m, 1H, H-3),
4.37 (t, J ) 9 Hz, 1H, H-2), 7.46 (br s, 1H, NH); 13C 11.2 (C-5),
14.0 (C(3)-CH3), 26.0 (C-4), 34.8 (C-3), 53.6 (C-2); p r olin e
unit, 1H 1.54-1.64 (m, 1H, H-3), 1.74-1.80 (m, 2H, H-4), 1.8-
1.91 (m, 1H, H-3), 3.3 (under the H2O signal, 1H, H-5), 3.48
(dt, J ) 10.2 and 5.8 Hz, 1H, H-5), 4.08 (t, J ) 7.2 Hz, 1H,
H-2); 13C 24.3 (C-4), 28.8 (C-3), 46.8 (C-5), 59.6 (C-2); p h en yl-
1
NH); 13C 16.2 (C-3), 47.2 (C-2); isoleu cin e unit, H 0.75 (d, J
) 6.4 Hz, 3H, H-5), 0.79-0.86 (m, 3H, C(3)-CH3), 1.10-1.17
(m, 1H, H-4), 1.48-1.53 (m, 1H, H-4), 1.60-1.65 (m, 1H, H-3),
4.08 (dd, J ) 8.4 and 6 Hz, 1H, H-2), 7.99 (d, J ) 6 Hz, 1H,
NH); 13C 10.8 (C-5), 14.2 (C(3)-CH3), 24.2 (C-4), 36.2 (C-3),
55.2 (C-2); p r olin e unit, 1H 1.07-1.16 (m, 1H, H-4), 1.54-
1.60 (m, 1H, H-4), 1.65-1.70 (m, 1H, H-3), 2.05 (dd, J ) 12
and 6.5 Hz, 1H, H-3), 2.78 (br t, J ) 10.3 Hz, 1H, H-5), 3.24-
3.30 (m, 1H, H-5), 4.56-4.62 (m, 1H, H-2); 13C 21.0 (C-4), 29.0
(C-3), 44.9 (C-5), 59.9 (C-2); p h en yla la n in e unit, 1H 2.90 (dd,
J ) 13.8 and 10.7 Hz, 1H, H-3), 3.17 (dd, J ) 13.8 and 4.5 Hz,
1H, H-3), 4.36 (ddd, J ) 10.8, 7.5 and 4.5 Hz, 1H, H-2), 7.10-
7.34 (m, 5H, H-Ph), 8.52 (d, J ) 7.5 Hz, 1H, NH); 13C 37.0
(C-3), 55.2 (C-2), 125.9-128.6 (C-Ph); leu cin e unit, 1H 0.76
(d, J ) 6.2 Hz, 3H, H-5), 0.79-0.86 (m, 3H, H-5), 1.42-1.47
(m, 1H, H-4), 1.60-1.65 (m, 2H, H-3), 5.10 (dd, J ) 10.7 and
5.5 Hz, 1H, H-2); 13C 20.4 and 23.0 (C-5), 22.9 (C-4), 34.2 (C-
3), 53.0 (C-2); a sp a r a gin e unit, 1H 2.50-2.60 (m, 1H, H-3),
4.45 (dd, J ) 13 and 6 Hz, 1H, H-2), 6.98 (br s, 1H, CONH2),
7.44 (s, 1H, CONH2), 7.56 (d, J ) 7.3 Hz, 1H, NH); 13C 37.2
(C-3), 49.6 (C-2).
1
a la n in e unit, H 3.09 (br t, J ) 14 Hz, 1H, H-3), 3.16 (dd, J
) 14 and 4.4 Hz, 1H, H-3), 3.92-4.00 (m, 1H, H-2), 7.16 (d, J
) 7.1 Hz, 2H, H-oPh), 7.20 (t, J ) 7.3 Hz, 1H, H-pPh), 7.28 (t,
J ) 7.5 Hz, 2H, H-mPh), 8.69 (br s, 1H, NH); 13C 34.3 (C-3),
56.3 (C-2), 126.3 (C-pPh), 128.2 (C-mPh), 128.4 (C-oPh);
1
leu cin e unit, H 0.81-0.86 (m, 3H, H-5), 0.89 (d, J ) 6.8 Hz,
3H, H-5), 1.45 (ddd, J ) 14, 9.2 and 4.6 Hz, 1H, H-3), 1.54-
1.64 (m, 1H, H-3), 1.65-1.73 (m, 1H, H-4), 3.92-4.00 (m, 1H,
H-2), 8.34 (br s, 1H, NH); 13C 21.0 and 22.7 (C-5), 24.0 (C-4),
1
39.3 (C-3), 52.0 (C-2); a sp a r a gin e unit, H 2.66 (dd, J ) 15.4
and 7 Hz, 1H, H-3), 2.75 (br d, J ) 15.4 Hz, 1H, H-3), 4.23-
4.31 (m, 1H, H-2), 6.91 (br s, 1H, CONH2), 7.46 (br s, 1H,
CONH2), 8.10 (br s, 1H, NH); 13C 35.5 (C-3), 50.5 (C-2).
Cycliza t ion of P ep t id e 11 To Give St ylost a t in 1 (2).
Operating as in the general method, from peptide 11 (20 mg,
0.026 mmol), PyBOP (16.2 mg, 0.031 mmol), and DIPEA (11
µL, 0.063 mmol), the cyclization was completed after 5 min.
The crude product was purified by semipreparative RP-HPLC
epi-Ben zyl-ψ-stylosta tin 10: Analytical HPLC (tR 20.5
min, characterization conditions); ES-MS calcd for C45H62N8O10
874.4, found m/z 875.6 [M + H+]; NMR (DMSO-d6) {ser in e}
unit, 1H 3.05 (dd, J ) 12.5 and 2.3 Hz, 1H, H-5), 3.36 (dd, J )
12.5 and 5.6 Hz, 1H, H-5), 3.55 (t, J ) 6 Hz, 1H, H-3), 3.99-

5832 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Forns et al.
1
4.02 (m, 1H, H-4), 4.00-4.04 (m, 1H, H-3), 4.60 (d, J ) 12 Hz,
1H, CHAPh), 4.69 (dd, J ) 12 Hz, 1H, CHBPh), 5.41 (br s, 1H,
(C-5), 54.9 (C-2), 65.0 (C-4), 67.8 (C-3); a la n in e unit, H 1.31
(d, J ) 6.8 Hz, 3H, H-3), 4.49-4.56 (m, 1H, H-2), 7.85 (d, J )
8.7 Hz, 1H, NH); 13C 16.3 (C-3), 47.2 (C-2); isoleu cin e unit,
1H 0.75-0.80 (m, 9H, H-5 and C(3)-CH3), 1.06-1.15 (m, 1H,
H-4), 1.50-1.54 (m, 1H, H-4), 1.54-1.65 (m, 1H, H-3), 4.05-
4.10 (m, 1H, H-2); 13C 10.9 (C-5), 14.3 (C(3)-CH3), 24.2 (C-4),
36.2 (C-3), 55.3 (C-2); p r olin e unit, 1H 1.05-1.15 (m, 1H, H-4),
1.52.1.58 (m, 1H, H-4), 1.65-1.70 (m, 1H, H-3), 2.05 (dd, J )
12 and 6 Hz, 1H, H-4), 2.78 (br t, J ) 10 Hz, 1H, H-5), 3.24-
3.30 (m, 1H, H-5), 4.58 (d, J ) 7.3 Hz, 1H, H-2); 13C 24.2 (C-
OH), 7.25-7.35 (m, 5H, H-Ph), 9.2 (d, J ) 6 Hz, 1H, NH); 13
C
42.1 (C-5), 56.6 (C-2), 69.2 (C-4), 71.2 (OCH2Ph), 73.4 (C-3),
1
127.1, 127.2, 127.9 (C-Ph); a la n in e unit, H 1.35 (d, J ) 7.6
Hz, 3H, H-3), 4.23 (m, 1H, H-2), 8.395 (d, J ) 8.5 Hz, 1H, NH);
1
13C 16.0 (C-3), 49.0 (C-2); D-a llo-isoleu cin e unit, H 0.65 (d,
J ) 6.7 Hz, 3H, C(3)-CH3), 0.88 (t, J ) 7.1 Hz, 3H, H-5), 0.97-
1.05 (m, 1H, H-4), 1.19-1.25 (m, 1H, H-4), 1.59-1.65 (m, 1H,
H-3), 4.67-4.72 (m, 1H, H-2), 6.77 (d, J ) 8.5 Hz, 1H, NH);
13C 11.5 (C-5), 13.0 (C(3)-CH3), 26.5 (C-4), 37.5 (C-3), 52.0 (C-
2); p r olin e unit, 1H 1.45-1.54 (m, 1H, H-3), 1.77-1.84 (m,
2H, H-4), 1.85-1.89 (m, 1H, H-3), 3.45 (dt, J ) 9.6 and 6.4
Hz, 1H, H-5), 3.58 (dt, J ) 9.6 and 7 Hz, 1H, H-5), 3.90 (dd, J
) 8 and 5 Hz, 1H, H-2); 13C 24.5 (C-4), 28.4 (C-3), 46.8 (C-5),
1
4), 29.2 (C-3), 45.1 (C-5), 60.0 (C-2); p h en yla la n in e unit, H
2.89 (dd, J ) 13.8 and 10.8 Hz, 1H, H-3), 3.12-3.20 (m, 1H,
1
H-3), H 4.36 (ddd, J ) 12.3, 7.5 and 4.6 Hz, 1H, H-2), 7.10-
7.30 (m, 5H, H-Ph), 8.51 (d, J ) 7.74 Hz, 1H, NH); 13C 36.5
(C-3), 55.3 (C-2), 125.8 (C-pPh), 127.9 (C-mPh), 128.6 (C-oPh);
1
1
60.4 (C-2); p h en yla la n in e unit, H 3.02-3.08 (m, 1H, H-3),
leu cin e unit, H 0.76-0.90 (m, 9H, H-5), 1.55-1.65 (m, 2H,
3.28-3.34 (under water, 1H, H-3), 3.94 (br s, 1H, H-2), 7.13
(d, J ) 7 Hz, 2H, H-oPh), 7.19 (t, J ) 7 Hz, 1H, H-pPh), 7.27
(d, J ) 7 Hz, 2H, H-mPh), 8.44 (br s, 1H, NH); 13C 34.5 (C-3),
55.6 (C-2), 126.0 (C-pPh), 128.0 (C-mPh), 128.8 (C-oPh);
leu cin e unit, 1H 0.71 (d, J ) 6.6 Hz, 3H, H-5), 0.83 (d, J )
6.7 Hz, 3H, H-5), 1.45-1.54 (m, 1H, H-4), 1.59-1.64 (m, 2H,
H-3), 1.73 (ddd, J ) 15, 11 and 4 Hz, 1H, H-3), 5.08 (dd, J )
12 and 4 Hz, 1H, H-2); 13C 20.5 and 23 (C-5), 23.0 (C-4), 34
(C-3), 53.5 (C-2); a sp a r a gin e unit, 1H 2.14 (br d, J ) 15.6
Hz, 1H, H-3), 2.45 (dd, J ) 15.6 and 9.5 Hz, 1H, H-3), 4.77
(m, 1H, H-2), 6.72 (br s, 1H, CONH2), 7.26-7.30 (br s, 1H,
CONH2), 7.60 (d, J ) 6 Hz, 1H, NH); 13C 36.5 (C-3), 49.3 (C-
2).
H-4), 1.60-1.70 (m, 1H, H-3), 5.08 (dd, J ) 11 and 4.6 Hz,
1H, H-2); 13C 20.8 and 23.2 (C-5), 22.8 (C-4), 34.3 (C-3), 53.0
1
(C-2); a sp a r a gin e unit, H 2.51 (dd, J ) 15 and 5.5 Hz, 1H,
H-3), 2.57 (dd, J ) 15 and 7 Hz, 1H, H-3), 4.45 (dd, J ) 13
and 6.5 Hz, 1H, H-2), 6.95 (br s, 1H, CONH2), 7.42 (br s, 1H,
CONH2), 7.60 (d, J ) 7.3 Hz, 1H, NH); 13C 37.1 (C-3), 49.5
(C-2).
Hyd r ogen a tion of epi-Ben zyl-ψ-stylosta tin (10) To
Yield epi-ψ-Stylosta tin (13). Operating as above, from
compound 10 (9.4 mg, 0.015 mmol) we obtained ψ-stylostatin
13 (6 mg, 73%). Analytical HPLC (tR 16.9 min, characterization
conditions); ES-MS calcd for C38H56N8O10 784.4, found m/z
785.4 [M + H+]; NMR (DMSO-d6) {ser in e} unit, 1H 3.03-
3.10 (m, 1H, H-5), 3.17 (dd, J ) 12 and 5 Hz, 1H, H-5), 3.50-
3.52 (m, 1H, H-2), 3.85 (br s, 1H, H-3), 4.02 (br s, 1H, H-4),
5.16 (d, J ) 4 Hz, 1H, C(5)-OH), 5.20 (br s, 1H, C(4)-OH),
9.15 (br s, 1H, NH); 13C 44.5 (C-5), 56.2 (C-2), 65.0 (C-4), 69.8
Cycliza tion of Lin ea r P ep tid e 12 To Give Ben zyl-ψ-
stylosta tin (3). From linear peptide 12 (20 mg, 0.022 mmol),
PyBOP (14 mg, 0.027 mmol), and DIPEA (9.2 µL, 0.053 mmol),
the cyclization was completed after 5 min. Purification and
lyophilization of the reaction product yielded benzyl-ψ-stylo-
statin (3, 11.7 mg, 61%) as a white solid.
1
(C-3); a la n in e unit, H 1.37 (d, J ) 7.5 Hz, 3H, H-3), 4.24 (q,
J ) 7.5 Hz, 1H, H-2), 8.32 (d, J ) 8.6 Hz, 1H, NH); 13C 16.2
1
(C-3), 49.0 (C-2); D-a llo-isoleu cin e unit, H 0.65 (d, J ) 6.7
Hyd r ogen a tion of Ben zyl-ψ-stylosta tin (3) To Give
ψ-Stylosta tin (4). A solution of the cyclic pseudopeptide 3
(15.4 mg, 0.018 mmol) in MeOH (5 mL), was hydrogenated at
room temperature and Pat in the presence of Pd-C (10%, 5
mg). The reaction, monitored by analytical HPLC of filtered
samples, was completed after 5 h. The catalyst was filtered
off, the solvent was evaporated, and the crude was purified
by semipreparative RP-HPLC (purification conditions) to yield
ψ-stylostatin 4 (10.5 mg, 81%). Analytical HPLC (tR 15.9 min,
characterization conditions); ES-MS calcd for C38H56N8O10
784.4, found m/z 785.4 [M + H+]. Tr a n s-4 r ota m er : NMR
(DMSO-d6) {ser in e} unit, 1H 3.02-3.10 (m, 2H, H-5), 3.61 (t,
J ) 6 Hz, 1H, H-2), 3.87 (ddd, J ) 7.3, 5.2 and 2 Hz, 1H, H-3),
3.95-4.00 (m, 1H, H-4), 5.05 (d, J ) 5.2 Hz, 1H, C(4)-OH),
5.13 (d, J ) 4 Hz, 1H, C(3)-OH), 9.05 (br s, 1H, NH); 13C 45.9
Hz, 3H, C(3)-CH3), 0.88 (d, J ) 7.3 Hz, 3H, H-5), 1.00 (q, J )
7 Hz, 1H, H-4), 1.20 (br s, 1H, H-4), 1.59-1.64 (m, 1H, H-3),
4.69 (d, J ) 7 Hz, 1H, H-2); 13C 11.5 (C-5), 13.2 (C(3)-CH3),
1
22.8 (C-4), 37.4 (C-3), 51.8 (C-2); p r olin e unit, H 1.53 (br s,
1H, H-3), 1.78-1.83 (m, 1H, H-4), 1.82-1.90 (m, 2H, H-3 and
H-4), 3.42-3.50 (m, 1H, H-5), 3.55-3.62 (m, 1H, H-5), 3.89-
3.92 (m, 1H, H-2); 13C 24.4 (C-4), 28.5 (C-3), 46.8 (C-5), 60.2
(C-2); p h en yla la n in e unit, 1H 3.03-3.10 (m, 1H, H-3), 3.25-
3.40 (under the H2O signal, 1H, H-3), 3.87-3.92 (m, 1H, H-2),
7.13 (d, J ) 7.1 Hz, 2H, H-oPh), 7.19 (t, J ) 7.5 Hz, 1H,
H-pPh), 7.27 (t, J ) 7.5 Hz, 2H, H-mPh), 8.48 (br s, 1H, NH);
13C 34.7 (C-3), 55.8 (C-2), 125.8 (C-pPh), 127.9 (C-mPh), 128.7
1
(C-oPh); leu cin e unit, H 0.74 (d, J ) 6.3 Hz, 3H, H-5), 0.87
(d, J ) 7.5 Hz, 3H, H-5), 1.60-1.70 (m, 2H, H-4 and H-3), 1.75
(ddd, J ) 15, 11.6 and 3.5 Hz, 1H, H-4), 5.05 (dd, J ) 11.8
and 3.8 Hz, 1H, H-2); 13C 20.3 and 23.2 (C-5), 22.8 (C-4), 34.3
(C-3), 53.6 (C-2); a sp a r a gin e unit, 1H 2.06-2.12 (m, 1H, H-3),
2.42 (dd, J ) 15.6 and 9.5 Hz, 1H, H-3), 4.77 (br s, 1H, H-2),
6.67 (br s, 1H, CONH2), 7.23 (br s, 1H, CONH2), 7.65 (d, J )
6 Hz, 1H, NH); 13C 36.5 (C-3), 49.2 (C-2).
1
(C-5), 55.9 (C-2), 65.3 (C-4), 69.0 (C-3); a la n in e unit, H 1.32
(d, J ) 6.8 Hz, 3H, H-3), 4.25-4.33 (m, 1H, H-2), 7.68 (br s,
1H, NH); 13C 18.6 (C-3), 50.3 (C-2); isoleu cin e unit, 1H 0.80-
0.90 (m, 3H, C(3)-CH3), 0.92 (d, J ) 6.4 Hz, 3H, H-5), 1.40-
1.46 (m, 2H, H-4), 1.91-1.96 (m, 1H, H-3), 4.50-4.55 (m, 1H,
H-2); 13C 11.3 (C-5), 16.1 (C(3)-CH3), 21 (C-4), 37.3 (C-3), 54.5
(C-2); p r olin e unit, 1H 1.65-1.70 (m, 1H, H-3), 1.71-1.78 (m,
1H, H-4), 1.78-1.84 (m, 1H, H-3), 1.95-2.00 (m, 2H, H-4),
3.42-3.46 (m, 1H, H-5), 3.70-3.75 (m, 1H, H-5), 3.91-3.95
(m, 1H, H-2); 13C 25.1 (C-4), 28.0 (C-3), 46.8 (C-5), 61.5 (C-2);
p h en yla la n in e unit, 1H 3.30-3.40 (under DMSO signal, 2H,
H-3), 3.74-3.80 (m, 1H, H-2), 7.10-7.30 (m, 5H, H-Ph), 8.43
(br s, 1H, NH); 13C 33.7 (C-3), 56.5 (C-2), 125.8 (C-pPh), 127.9
In h ibition of Cell Gr ow th by Color im etr ic Assa y.22
A
colorimetric type of assay, using the SRB reaction, has been
adapted for a quantitative measurement of cell growth and
viability, following the technique described by Skehan et al.:
23 cells are seeded in 96-well microtiter plates, at 5 × 103 cells
per well in aliquots of 195 µL of RPMI medium, and they are
allowed to attach to the plate surface by growing in drug free
medium for 18 h. Afterward, samples are added in aliquots of
5 µL (dissolved in DMSO/H2O 3:7). After 48-72 h exposure,
the antitumor effect is measured by the SRB methodology:
cells are fixed by adding 50 µL of cold 50% (wt/vol) trichloro-
acetic acid and incubating for 60 min at 4 °C. Plates are
washed with deionized water and dried; 100 µL of SRB solution
(0.4wt %/vol in 1% acetic acid) is added to each microtiter well
and incubated for 10 min at room temperature. Unbound SRB
is removed by washing with 1% AcOH. Plates are air-dried,
and bound stain is solubilized with Tris buffer. Optical
densities are read on an automated spectrophotometer plate
1
(C-mPh), 128.6 (C-oPh); leu cin e unit, H 0.76-0.90 (m, 9H,
H-5), 1.40-1.46 (m, 2H, H-4), 1.50-1.70 (m, 1H, H-3), 5.00-
5.04 (m, 1H, H-2); 13C 20.8 and 23.2 (C-5), 23 (C-4), 34.3 (C-3),
52.8 (C-2); a sp a r a gin e unit, 1H 2.26 (d, J ) 5.2 Hz, 1H, H-3),
2.28 (d, J ) 5.2 Hz, 1H, H-3), 4.70-4.78 (m, 1H, H-2), 6.93 (br
s, 1H, CONH2), 7.16-7.22 (br s, 1H, CONH2), 8.15 (d, J ) 9
Hz, 1H, NH); 13C 37.5 (C-3), 50.0 (C-2). Cis-4 r ota m er : NMR
1
(DMSO-d6) {ser in e} unit, H 3.02-3.10 (m, 1H, H-5), 3.12-
3.20 (m, 1H, H-5), 3.63 (m, 1H, H-2), 3.95-4.00 (m, 1H, H-3),
4.05-4.10 (m, 1H, H-4), 5.01 (br s, 1H, C(4)-OH), 5.26 (d, J
) 4 Hz, 1H, C(3)-OH), 8.82 (d, J ) 6 Hz, 1H, NH); 13C 44.4

Constrained Derivatives of Stylostatin 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5833
(7) Pettit, G. R.; Srirangam, J . K.; Herald, D. L.; Erickson, K. L.;
Doubeck, D. L.; Schmidt, J . M.; Tackett, L. P.; Bakus, G. J .
Antineoplastic agents. 251. Isolation and structure of stylostatin
1 from the Papua New Guinea marine sponge Stylotella auran-
tium. J . Org. Chem., 1992, 57, 7217-7220, and the “Additions
and Corrections” appeared in: Ibid. J . Org. Chem. 1993, 58,
3222.
(8) Bourne, G. T.; Meutermans, W. D. F.; Alewood, P. F.; McGeary,
R. P.; Scanlon, M.; Watson, A. A.; Smythe, M. L. A backbone
linker for Boc-based peptide synthesis and on-Resin cycliza-
tion: synthesis of stylostatin 1. J . Org. Chem. 1999, 64, 3095-
3101.
reader at a single wavelength of 490 nm. Data analyses are
generated automatically by LIMS implementation at Pharma
Mar and some parameters for cellular responses are calculated.
Using control OD values (C), test OD values (T) and time
zero OD values (T0), calculations are made for the following
parameters: GI50 ) concentration that causes 50% Growth
Inhibition, means the growth inhibition effect, calculated
from: 100 x [(T - T0)/C - T0)] ) 50; TGI ) total growth
inhibition, signifies a cytostatic effect: 100 x [(T - T0)/C -
T0)] ) 0; LC50 ) concentration that causes 50% cell killing,
means the cytotoxic effect calculated from: 100 × [(T - T0)/
T0)] ) -50.
(9) For the synthesis of 8, compound 1a (ref 5b) was converted
to Fmoc-{Ser(Bn)-Leu}-OH derivative (see Experimental Sec-
tion).
Ack n ow led gm en t. Support for this research was
provided by the CIRIT (Generalitat de Catalunya)
through grants 1999SGR-00077 and 2001SGR-00082,
and by the MCYT (Ministerio de Ciencia y Tecnolog´ıa,
Spain) through grants PB97-0976, 2FD97-0293, and
BQU2001-3228.
(10) Pseudodipeptide Fmoc-{Ser(Bn)-Leu}-OH showed no coupling/
deprotection peculiarities.
(11) In contrast to what had previously been reported,8 no dimer-
ization products were detected.
(12) Cyclization through the Phe-Asn8 and through the Leu-Ala13
amide bonds have been reported as less efficient than by
formation of Ile-Pro.8
(13) Rosenbaum, C.; Waldmann, H. Solid-phase synthesis of cyclic
peptides by oxidative cyclative cleavage of an aryl hydrazide
linker-synthesis of stylostatin 1. Tetrahedron Lett. 2001, 42,
5677-5680.
(14) The bulk of this resin is known to prevent the formation of 2,5-
dioxopiperazine when preparing C-terminal proline peptides:
Barlos, K.; Gatos, D.; Scha¨fer, W, Angew. Chem., Int. Ed. Engl.
1991, 30, 590-593.
(15) We also assayed DIPCDI/HOBt as the coupling reagent. Com-
pound 11 was satisfactorily converted into native stylostatin 1,
both in DMF (7 days) and in DCM:DMF (24 h). However,
compound 12 gave a complex mixture of products within which
the target cyclopeptide 3 was not detected.
Su p p or tin g In for m a tion Ava ila ble: Amino acid analysis
of stylostatin 1 (2) and epistylostatin 9. HPLC monitorization
of the comparative cyclization of linear peptides 6 and 8. NMR
spectra of ψ-stylostatin 4: (a) full set in DMSO (cis and trans
conformers); (b) full set in CDCl3:DMSO (92:8) (only cis
conformer); (c) comparative figure of variable temperature
NMR spectra (6.6-9.2 ppm). Full bioassay results. This
information is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(16) The fact that this reaction was slower made it more appropriate
for this study, despite the epimerization.
(17) Stylostatin 1 (2) is insoluble in CDCl3.
(1) Lambert, J . N.; Mitchell, J . P.; Roberts, K. D. The synthesis of
cyclic peptides. J . Chem. Soc., Perkin Trans. 1 2001, 471-484.
(b) Kessler, H. Peptide conformations. Part 19. Conformation
and biological effects of cyclic peptides. Angew. Chem., Int. Ed.
Engl. 1982, 21, 512-523.
(2) For an example, see: J iang, L.; Moehle, K.; Dhanapal, B.;
Obrecht, D.; Robinson, J . A. Combinatorial biomimetic chemis-
try: parallel synthesis of a small library of â-hairpin mimetics
based on loop III from human platelet-derived growth factor B.
Helv. Chim. Acta 2000, 83, 3097-3112.
(3) Vollmer, M. S.; Clark, T. D.; Steinem, C.; Ghadiri, M. R.
Photoswitchable hydrogen-bonding in self-organized cylindrical
peptide systems. Angew. Chem., Int. Ed Engl. 1999, 38, 1598-
1601. (b) Clark, T. D.; Buehler L. K.; Ghadiri, M. R. Self-
assembling cyclic â3-peptide nanotubes as artificial trans-
membrane ion channels. J . Am. Chem. Soc. 1998, 120, 651-
656. c. Clark, T. D.; Buriak, J . M.; Kobayashi, K.; Isler, M. P.;
McRee, D. E.; Ghadiri, M. R. Cylindrical â-sheet peptide as-
semblies. J . Am. Chem. Soc. 1998, 120, 8949-8962.
(4) Kubik, S.; Goddard, R. A new cyclic pseudopeptide composed of
(L)-proline and 3-aminobenzoic acid subunits as a ditopic receptor
for the simultaneous complexation of cations and anions. J . Org.
Chem. 1999, 64, 9475-9486. (b) Kubik, S. Large increase in
cation binding affinity of artificial cyclopeptide receptors by an
allosteric effect. J . Am. Chem. Soc. 1999, 121, 5846-5855. (c)
Endo, K.; Takahashi, H. Unnatural benzoic amino acids and
their derivatives. IV. Synthesis of novel peptidomimetics, cyclic
hexamers of unnatural amino acids, 2,5-disubstituted 3-amino-
benzoic acids. Heterocycles 1999, 51, 337-344.
(18) Ferna´ndez, M. M.; Diez, A.; Rubiralta, M.; Montenegro, E.;
Casamitjana, N.; Kogan, M. J .; Giralt, E. Spirolactams as
conformationally restricted pseudopeptides: synthesis and con-
formational analysis. J . Org. Chem. 2002, 67, 7587-7599. (b)
Estiarte, M. A.; Rubiralta, M.; Diez, A.; Thormann, M.; Giralt,
E. Oxazolopiperidin-2-ones as type II′ â-turn mimetics: synthesis
and conformational analysis. J . Org. Chem. 2000, 65, 6992-
6999. (c) Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.;
Scolastico, C. Conformational preferences of peptides containing
reverse-turn mimetic bicyclic lactams. Inverse γ-turns versus
type-II′ â-turns. Insights into â-hairpin stability. Eur. J . Org.
Chem. 1999, 389-400.
(19) We recorded the spectra in the range between 0 and 65 °C.
(20) IUPAC-IUB Commission on Biochemical Nomenclature. Symbols
for amino acid derivatives and peptides. Recommendations 1971.
J . Biol. Chem. 1972, 247, 977-983.
(21) Armarego, W. L. F.; Perrin, D. D. Purification of Laboratory
Chemicals, 4th ed.; Butterworth-Heinemann: Oxford, 2000.
(22) Faircloth, G. T.; Stewart, D.; Clement, J . J . A simple procedure
for the quantitative measurement of cytotoxicity assay. J . Tiss.
Cult. Methods 1988, 11, 201-205. (b) Monks, A.; Scudiero, D.;
Skehan, Ph.; Shoemaker, R.; Paull, K.; Vistica, D., Hose, C.;
Langley, J .; Cronise, P.; Vaigro-Wolf, A.; Gray-Goodrich, M.;
Campbell, H.; Mayo, J .; Boyd, M. Feasibility of a high-flux
anticancer drug screen using a diverse panel of cultured human
tumor cell lines. J . Natl. Cancer Inst. 1991, 83, 757-766. (c)
Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: Application to proliferation and cytotoxicity assay. J
Immunol. Methods 1983, 65, 55-63.
(5) Piro´, J .; Rubiralta, M.; Giralt, E.; Diez, A. Solid phase synthesis
of enantiomerically pure polyhydroxyvalerolactams. Tetrahedron
Lett. 2001, 42, 871-873. (b) Piro´, J .; Rubiralta, M.; Giralt, E.;
Diez, A. 3-Amino-2-piperidones as constrained pseudopeptides:
preparation of a new Ser-Leu surrogate. Tetrahedron Lett. 1999,
40, 4865-4868.
(6) For the first application of valerolactams to induce turns, see:
Freidinger, R. M.; Perlow, D. S.; Veber, D. F. Protected lactam-
bridged dipeptides for use as conformational constraints in
peptides. J . Org. Chem. 1982, 47, 104-109.
(23) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screening
J . Natl. Cancer Inst. 1990, 82, 1107-1112.
J M030943H