Total syntheses and re-assignment of configurations of the cyclopeptides lissoclinamide 4 and lissoclinamide 5 from Lissoclinum patella

Article abstract of DOI:10.1039/a909360e

The total synthesis of lissoclinamide 4 and lissoclinamide 5, which are novel oxazoline/thiazole/thiazoline-based cyclopeptides isolated from the ascidian ("sea squirt") Lissoclinum patella, are described. The synthesis of the bis-thiazole based lissoclin

Full text of DOI:10.1039/a909360e

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Total syntheses and re-assignment of configurations of the
cyclopeptides lissoclinamide 4 and lissoclinamide 5 from
Lissoclinum patella
1
Christopher D. J. Boden and Gerald Pattenden*
School of Chemistry, Nottingham University, Nottingham, UK NG7 2RD
Received (in Cambridge, UK) 26th November 1999, Accepted 21st December 1999
Published on the Web 22nd February 2000
The total synthesis of lissoclinamide 4 and lissoclinamide 5, which are novel oxazoline/thiazole/thiazoline-based
cyclopeptides isolated from the ascidian (“sea squirt”) Lissoclinum patella, are described. The synthesis of the
bis-thiazole based lissoclinamide 5 necessitated the development of practical solutions to the elaboration of
enantiomerically pure valine and phenylalanine substituted thiazoles. To overcome the problems associated with
the configurational lability of enantiopure amino acid substituted thiazolines, the thiazoline-based cyclopeptide
lissoclinamide 4 was synthesised using a novel strategy whereby both the oxazoline and the thiazoline rings in the
natural product were formed simultaneously in a “one-pot” double cyclodehydration sequence from an appropriate
thioamide/amide cyclopeptide precursor. Based on our synthetic work the stereochemistries 2 and 1 published for
natural lissoclinamide 4 and lissoclinamide 5 were re-assigned to 16 and 15 respectively.
In recent years the marine environment has revealed itself as a
rich source of novel and unusual secondary metabolites, many
of which have shown considerable promise as lead compounds
for development of therapeutic agents.1 A particularly fascinat-
ing family of marine metabolites is the heterocycle-containing
cyclopeptides known as lissoclinamides isolated from the
ascidian (“sea squirt”) Lissoclinum patella.2 These metabolites
show structures which feature an oxazoline ring together with
one or more thiazoles and/or thiazolines, derived from unusual
amino acid residues, incorporated in a cyclic heptapeptide
e.g. lissoclinamide 5, 1, lissoclinamide 4, 2, and lissoclinamide 7,
3.3–5 Structurally related compounds which have also been
isolated from other ascidians include cyclodidemnamide 4,6
mollamide 5,7 and ascidiacyclamide 6.8 All of these cyclo-
peptides show a range of biological properties, particularly
immunoregulatory, antibiotic and antitumoral, but including
enzyme-inhibitory activity. Cyclopeptides can, of course,
assume a range of conformations, each of which can have
significantly different biological properties. Many marine
organisms are well known to accumulate inorganic salts in con-
siderable quantities and we,9 and others,10–13 have intimated that
lissoclinamide–metal conjugates, involving the various hetero-
atom ligands associated with their macrocyclic cavities, could
play a significant role in the biological properties of these
metabolites. With the aim of investigating the capacity of
lissoclinamides and related cyclopeptides to bind metals,
and correlating these data with the biological profiles of the
metabolites and their metal conjugates, we have examined
synthetic routes to the 31-Val and 21-Phe cycloheptapeptides
lissoclinamide 4 214 and lissoclinamide 5 1.15
In the light of the potential for cyclopeptides as therapeutic
agents, it is not surprising that synthetic investigations within
this family of natural products have been intensive.16 Spectro-
scopic methods, together with X-ray studies in the cases of
mollamide 5 and ascidiacyclamide 6 have played a crucial role
in the assignment of the structures of marine cyclopeptides, but
in several other instances these structures have needed signifi-
cant revision following synthetic investigations.16 The correct
assignment of the chiral centres adjacent to the thiazole, and
especially the thiazoline, rings in structures 1→6 is particularly
problematic since these centres are prone to undergo epimeris-
ation during the usual methods of cyclopeptide hydrolytic
degradation and during their synthesis.17 The design we
adopted for the synthesis of the structure 1 proposed for the
bisthiazole-based cyclopeptide lissoclinamide 5 is shown in
DOI: 10.1039/a909360e
J. Chem. Soc., Perkin Trans. 1, 2000, 875–882
This journal is © The Royal Society of Chemistry 2000
875

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Scheme 1
Scheme 1. Thus, we decided to prepare the thiazole-containing
compounds were not identical! With knowledge of the ease
tri- and tetra-peptides 9 and 10 respectively, then to condense
these two fragments at the Pro–Thr amide bond, leading to 8,
in readiness for macrocyclisation via the Thz–Phe amide bond
(Thz = thiazole) producing “pre-lissoclinamide 5” 7. Cyclo-
dehydration of 7 using the precedent established by Shioiri and
co-workers18 and by Schmidt and Gleich19 in their pioneering
work on the syntheses of the patellamides A–D, would then be
expected to lead to 1.
with which thiazole (and thiazoline) based cyclopeptides are
known to undergo epimerisation during hydrolytic degrad-
ation, alongside further analysis of the NMR data recorded for
our synthetic 1 and for natural lissoclinamide 5, we re-assigned
the stereochemistry of the natural product to (21R,31S) i.e. 15.
In model studies we compared and contrasted a range of
methods for the synthesis of enantiopure amino acid based thi-
azoles, including the (R)-Val and (S)-Phe derived thiazoles 12
and 14.20,21 Both of these compounds were easily prepared, with
Furthermore we carried out a total synthesis of this stereo-
structure, starting from (S)-valine and from (R)-phenylalanine,
using the strategy described previously. This synthesis gave
1
lissoclinamide 5 15, which displayed H NMR and 13C NMR
spectroscopic data which were identical to those found for the
natural product isolated from L. patella.
Although the thiazoline/thiazole-based cyclopeptide lisso-
clinamide 4 2 had earlier been assigned the (21S,31R) stereo-
structure, in light of our synthetic studies with lissoclinamide 5
(i.e. 15 versus 1), we re-assigned its stereochemistry to that
shown in structure 16, i.e. (21R,31S) before embarking on its
total synthesis. Hitherto there had been no reported total syn-
thesis of any thiazoline-based cyclopeptide.22 Our synthetic
plan for the synthesis of 16 envisaged the simultaneous form-
ation of the thiazoline and oxazoline rings in 16 from a suitable
dihydroxy precursor 17, via a “one-pot” double cyclodehydra-
tion sequence in the final step. The cyclothiopeptide 17, in turn,
was to be derived from macrocyclisation of the heptapeptide
thioamide produced from the fragments 18 and 19 (Scheme 3).
Thus, the Boc-protected (R)-thiazole 20 was first deblocked
using TFA in CH2Cl2 and the resulting amine was then coupled
with Boc-(S)-Ser-OH using DCC–HOBt leading to the tripep-
tide 21 in 71% yield. The tripeptide 21 was next deblocked
with TFA, and the free amine generated was thioacylated with
the (S)-valine derived thioacylating reagent 22 introduced by
Zacharie et al.23 producing the tetrapeptide 23 with no
detectable racemisation of the centre at C-31 (Scheme 4).
Boc-deprotection of 23, by the usual method, and coupling of
the resulting amine with Boc-allothreonine, finally gave the key
intermediate pentapeptide 18 in 76% yield. Further deprotec-
tion of 18 and coupling to the Boc-(S)-Phe-Pro-OH 19 using
DCC–HOBt in DMF next gave the heptapeptide 24. Saponifi-
cation of 24 to the carboxylic acid 25, followed by Boc depro-
tection and cyclisation using DPPA–NMM–DMF at room
temperature for 48 h, then gave the cyclopeptide 17, whose
Scheme 2 Reagents and conditions: i) Cys-OMe hydrochloride, EtOH,
25 ЊC; ii) MnO2, CH2Cl2, 25 ЊC; iii) CF3CO2H, CH2Cl2, 25 ЊC; iv)
Boc-Pro-aThr-OH, DCC, HOBt, NMM, CH2Cl2, 0–5 ЊC; v) LiOH,
THF–H2O; vi) Phe-Pro-OMe TFA salt, FDPP, NMM, MeCN, 25 ЊC.
>98% ee, by straightforward condensation reactions between
cysteine ester HCl and the N-Boc protected imino ethers 11 and
13 derived from (R)-valine and from (S)-phenylalanine respec-
tively. Deprotection of the Boc residue in 12 and coupling of the
free amine to Boc-Pro-aThr-OH† produced the tetrapeptide 9
in 84% yield, and saponification of 14 and coupling of the result-
ing carboxylic acid to (S)-Phe-Pro-OMe led to the correspond-
ing tripeptide 10 (Scheme 2). Saponification of the ester 10 and
condensation with the amine produced after Boc-removal from
9, using DCC–HOBt conditions, next led to the heptapeptide 8
in a gratifying 70% yield based on 10. Hydrolysis of the thiazole
ester in 8 with lithium hydroxide, followed by removal of the
Boc group from the crude carboxylic acid and treatment of the
resulting amino acid TFA salt with diphenylphosphorazide
(DPPA) and N-methylmorpholine (NMM) in DMF (2 days)
then afforded the cyclopeptide 7 in 35–40% yield from 9.
Dehydration of the allothreonine residue in 7 by treatment
with excess thionyl chloride at 0–4 ЊC for 36 h then gave the
“lissoclinamide 5” 1 in 73% yield, as a foam. Comparison of
the 1H NMR and 13C NMR spectroscopic data of the synthetic
and natural lissoclinamide showed, however, that the two
† aThr = -allothreonine.
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NMR spectroscopic data showed that it existed as a mixture
of several rotamers (Scheme 5). Finally, treatment of 17 with
Burgess’ reagent17 in refluxing THF produced the lissoclin-
amide 16 in 71% yield, which showed 1H NMR and 13C NMR
spectroscopic data, together with chiroptical data, which
matched corresponding data reported for natural lissoclin-
amide 4.
In summary, we have shown by total synthesis that the struc-
tures assigned to lissoclinamide 5 and lissoclinamide 4 have the
(21R,31S) stereochemistries, 15 and 16 respectively, and not the
corresponding stereochemistries, 1 and 2, published previously.
Furthermore, by establishing practical solutions to the syn-
thesis of enantiopure amino acid substituted thiazoles and to
the elaboration of chiral thiazoline cyclopeptides, it should be
possible to effect the synthesis of any stereodefined thiazole/
thiazoline based cyclopeptide, whether it be naturally occurring
or not, in the future.
In previous publications we have expressed the view that
metal cation complexation by cyclopeptides in vivo may facili-
tate the transport of such cations across hydrophobic mem-
branes. If this hypothesis is correct, then we would expect
any natural cyclopeptide to be formed from amino acids with
stereochemistries which lead to epimers of the cyclopeptide
which: a) adopt an “open” conformation in aqueous media, so
as to allow the various O- and N-ligands in the macrocycle
to attract a solvated metal cation which can then enter the
cavity without undue steric hindrance, and b) are capable of
forming a hydrophobic “shield” around the complexed cation
(metal–ligand conjugate) so as to allow transport through cell
membranes.
We decided to examine the compliance of the predicted
and revised structures for lissoclinamide 4, viz 2 and 16, with
these criteria by carrying out conformational searches for the
uncomplexed natural products in water using the MMFF94s
forcefield of Halgren24 and the GB/SA solvation model as
implemented in MacroModel 5.5.25 As can be seen from Fig.
1, a comparison of the preferred global minima for structures
2 and 16 in the aqueous phase clearly shows that both struc-
tures have an “open” lower face which would facilitate metal
entry and bindings. However, calculations on hypothetical
Zn2ϩ complexes, carried out using the same forcefield in the
vacuum phase show a remarkable difference in the “shield-
ing” abilities of the two structures (Fig. 2). Thus, although
the upper face of the macrocyclic ring can be covered by a
combination of the 31-Val isopropyl group and the 11-Phe
aromatic ring in both 2 and 16, the 21-(R)-Phe structure 16 is
able to cap the lower face of the molecule as well with the
aryl ring of the 21-(R)-Phe residue, whereas 2 is prevented
from doing so by the “equatorial” orientation of the 21-(S)-
Phe side chain. It is notable that the 21-(R)-Phe configuration
is also present in the even more cytotoxic bis-thiazoline
lissoclinamide 7, 26.26
Scheme 3
In contemporaneous synthetic studies we have developed
efficient syntheses of the related cyclopeptides cyclodidemn-
amide 427 and mollamide 5,28 carried out metal binding studies
(particularly to Cu2ϩ and Zn2ϩ) of selected cyclopeptides,29 and
examined the cyclooligomerisation of thiazole and oxazole
Scheme 4 Reagents and conditions: i, 50% TFA–CH2Cl2, 25 ЊC; ii,
Boc-Ser-OH (2.5 eq), DCC ϩ HOBt (1.25 eq), Et3N (1 eq), CH2Cl2,
0 ЊC; iii, NaHCO3, CH2Cl2–H2O; iv, 22, DMF, 0–5 ЊC; v, Boc-aThr-OH,
DCC-HOBt, iPr2NEt, DMF, 0–25 ЊC.
Scheme 5 Reagents and conditions: i, TFA, CH2Cl2; ii, 19, DCC–HOBt, iPr2NEt, DMF, 25 ЊC; iii, NaOH, MeOH–H2O; iv, NMM, DPPA, DMF,
25 ЊC, 48 h; v, Burgess reagent, THF, 65 ЊC, 30 min.
J. Chem. Soc., Perkin Trans. 1, 2000, 875–882
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Fig. 1 Global minima for i) 2 and ii) 16 in aqueous phase, calculated
with the MMFFs forcefield and GB/SA solvation model.
Fig. 2 Complexes of i) 2 and ii) 16 with Zn2ϩ, as calculated using the
MMFFs forcefield in vacuum phase.
and 13C NMR spectra were confirmed by two dimensional
homonuclear (1H) and/or heteronuclear (1H/13C) correlation
spectroscopy. Matrix dimensions for two dimensional spectra
1
were either 1024 points × 256 columns (homonuclear H) or
1
2048 points × 128 columns (heteronuclear H/13C), and were
recorded on a Bruker DRX500 instrument. Mass spectra
were recorded on an AE1 MS-902, MM-70E or VG Autospec
spectrometer using electron ionisation (EI) or fast atom
bombardment (FAB) techniques. Flash chromatography was
performed on Merck silica gel 60 as the stationary phase and
the solvents employed were either of analytical grade or were
distilled before use. Solvents were removed on a Büchi rotary
evaporator using water aspirator pressure.
based amino acids leading to novel synthetic heterocyclic-
based cyclopeptides.30 These studies are presented in other
publications and, in the case of cyclodidemnamide, in the
accompanying paper.
Experimental
General details
Molecular mechanics calculations were carried out on a
Silicon Graphics Indy R4600PC workstation using the
MacroModel program, version 5.5. The global minima for the
various structures were located by a 1000 conformer Monte-
Carlo search using the MMFF94s (planar amide nitrogen
atoms) forcefield; aqueous phase calculations utilised the GB/
SA solvation model.31
Optical rotations were measured on a JASCO DIPA-370 polar-
imeter; [α]D values were determined at 25 ЊC and are recorded in
units of 10Ϫ1 deg cm2 gϪ1. Infrared spectra were obtained using
a Perkin-Elmer 1600 series FT-IR instrument as dilute solu-
tions in spectroscopic grade chloroform. Proton NMR spectra
were recorded on either a Bruker DRX500 (500 MHz), a
Bruker AM400 (400 MHz) or a Bruker DPX360 (360 MHz)
spectrometer as dilute solutions in deuterochloroform or d6-
dimethyl sulfoxide. Chemical shifts are recorded relative to a
solvent standard and the multiplicity of a signal is designated
by one of the following abbreviations: s = singlet; d = doublet;
t = triplet; q = quartet; br = broad; m = multiplet; app =
apparent. All coupling constants, J, are reported in hertz.
Carbon-13 NMR spectra were obtained using the instruments
indicated for 1H above, at frequencies of 125 MHz, 100.6 MHz
and 90 MHz respectively. The spectra were recorded as dilute
solutions in deuterochloroform or d6-dimethyl sulfoxide with
chemical shifts reported relative to a solvent standard on a
broad band decoupled mode and the multiplicities obtained
Boc-(R)-Val-Thz-OMe, 12
The thiazole was prepared from cysteine ester HCl and the (R)-
imino ether 11 according to the procedure described previ-
ously,20 and showed: δH(500 MHz, DMSO, 90 ЊC) 8.36 (1H, s),
7.17 (1H, d, J 7.0 Hz), 4.59 (1H, dd, J 8.4, 6.9 Hz), 3.80 (3H, s),
2.25 (1H, septet, J 6.8 Hz), 1.40 (9H, s), 0.95 (3H, d, J 6.75 Hz),
0.92 (3H, d, J 6.75 Hz). δC(125 MHz, DMSO, 90 ЊC) 173.9 (s),
160.9 (s), 154.8 (s), 145.3 (s), 127.8 (d), 78.3 (s), 58.6 (d), 51.3
(q), 31.8 (d), 27.9 (q), 18.8 (q), 17.8 (q). m/z (ESMS) 337.1195
(Mϩ ϩ Na); C14H22N2O4NaS requires 337.1198. The enan-
tiomer of 12 was prepared in a similar manner and showed
identical spectroscopic data.
1
using a DEPT sequence. Where required, assignment for H
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Boc-(S)-Phe-Thz-OMe, 14
The crude trifluoroacetic acid salt from Boc-(S)-Phe-Pro-
OMe (2.259 g, 6.00 mmol) was prepared by acidolysis with 1:3
TFA–dichloromethane followed by concentration in vacuo, and
then redissolved in dichloromethane (20 ml). After cooling to
0–4 ЊC Hünig’s base (2.55 ml, 15.0 mmol) was added, followed
10 minutes later by the crude carboxylic acid and finally
pentafluorophenyl diphenylphosphinate (FDPP) (2.323 g, 6.05
mmol). The mixture was stirred at 0–4 ЊC for 30 minutes and
then at 25 ЊC for 3 hours. The solvent was removed under
reduced pressure and the residue was purified by flash chrom-
atography on silica gel (EtOAc–light petroleum, 1:1) to give the
tetrapeptide (2.40 g, 3.95 mmol, 71%) as a pale yellow foam:
δH(500 MHz, DMSO, 100 ЊC) 8.10 (1H, s), 7.87 (1H, br s), 7.29
(8H, m), 7.23 (2H, m), 5.08 (1H, ddd, J 10.6, 6.8, 5.3 Hz), 5.04
(1H, br s), 4.44 (1H, br s), 3.76 (1H, br s), 3.69 (3H, br s), 3.47
(1H, ddd, J 10.2, 6.7, 6.5 Hz), 3.37 (1H, dd, J 13.9, 5.3 Hz), 3.16
(2H, 4 line m), 3.04 (1H, dd, J 13.7, 6.8 Hz), 2.21 (1H, br s), 1.95
(4H, br s), 1.37 (9H, s). δC(125 MHz, DMSO, 100 ЊC) 173.7 (s),
171.5 (s), 169.0 (s), 154.5 (s), 148.5 (s), 137.3 (s), 136.4 (s), 129.0
(d), 128.7 (d), 127.6 (d), 125.9 (d), 123.2 (s), 78.3 (s), 58.4 (d),
54.1 (d), 51.1 (q), 46.2 (t), 39.7 (t), 37.2 (t), 28.1 (t), 27.8 (q),
24.05 (t). m/z (ESMS) 629.2456 (M ϩ Na); C32H38N4O6NaS
requires 629.2410. The 21-(R)-Phe epimer of 10 showed δH(360
MHz, DMSO, 90 ЊC) 8.23 (1H, d, J 8.1 Hz), 8.16 (1H, s), 7.83
(1H, d, J 8.4 Hz), 7.34–7.28 (8H, m), 7.23 (2H, m), 4.99 (2H,
m), 4.38 (1H, dd, J 8.55, 4.9 Hz), 3.76 (1H, m), 3.66 (3H, s),
3.49 (1H, m), 3.39 (1H, m), 3.18–3.09 (2H, 5 line m), 3.04 (1H,
3 line m), 2.19 (1H, 6 line m), 1.94 (2H, m), 1.85 (2H, m), 1.33
(9H, s). δC(90 MHz, DMSO, 90 ЊC) 173.9 (s), 171.7 (s), 169.1
(s), 154.65 (s), 148.6 (s), 137.5 (s), 136.6 (s), 129.2 (d), 128.9 (d),
127.8 (d), 126.2 (d), 126.05 (d), 123.6 (s), 78.4 (s), 58.6 (d), 54.1
(d), 51.5 (q), 46.4 (t), 39.7 (t), 37.2 (t), 28.3 (t), 27.9 (q), 24.3 (t).
The thiazole was prepared from cysteine ester HCl and the (S)-
imino ether 13 according to the procedure described previ-
ously,20 and showed: δH(500 MHz, DMSO, 90 ЊC) 8.37 (1H, s),
7.29 (5H, m), 7.23 (1H, m), 5.08 (1H, ddd, J 9.85, 5.0, 4.2 Hz),
3.88 (3H, s), 3.37 (1H, dd, J 14.0, 5.0 Hz), 3.14 (1H, dd, J 14.0,
9.85 Hz), 1.35 (9H, s). δC(125 MHz, DMSO, 90 ЊC) 174.1 (s),
160.9 (s), 154.5 (s), 145.4 (s), 137.4 (s), 128.7 (d), 128.1 (d), 127.7
(d), 125.9 (d), 78.3 (s), 54.2 (d), 51.3 (q), 39.7 (t), 27.8 (q).
m/z (ESMS) 385.1200 (M ϩ Na); C18H22N2O4NaS requires
385.1198. The enantiomer 20 was prepared in the same manner
and, as expected, showed identical spectroscopic data.
Boc-aThr-(R)-Val-Thz-OMe, 9
Trifluoroacetic acid (2.5 ml) was added in one portion to a
solution of the thiazole 12 (472 mg, 1.50 mmol) in dichloro-
methane (10 ml). The solution was stirred at 25 ЊC for 2 hours,
then diluted with toluene (4 ml) and concentrated in vacuo. A
further 4 ml of toluene was added, and the resulting mixture
was then reconcentrated in vacuo to leave the crude amine TFA
salt as a gum. Hünig’s base (865 µl, 5.0 mmol) was added to
a solution of the salt in dichloromethane (15 ml), and the
solution was then cooled to 4 ЊC and stirred for 15 min. Boc-
allothreonine (ca. 1.20 mmol) was added, followed by 1-hydroxy-
benzotriazole (162 mg, 1.20 mmol) and finally DCC (268 mg,
1.30 mmol). The mixture was stirred at 0–5 ЊC for 2 hours and
then at room temperature for 5 hours. It was then filtered (to
remove precipitated DCU) and washed sequentially with 10%
citric acid solution (10 ml), 10% NaHCO3 (10 ml), water (10 ml)
and finally brine (10 ml). The solution was dried (MgSO4) and
concentrated in vacuo to leave the crude tripeptide. Purification
by flash chromatography on silica gel (1:1 EtOAc–light petrol-
eum eluant) gave the tripeptide (350 mg, 842 µmol, 70%) as a
white foam: [α]D25 ϩ7 (c = 2.54, CHCl3). νmax (CHCl3)/cmϪ1 3429,
1716, 1369. δH (360 MHz, CDCl3) 0.86 (3H, d, J 6.8 Hz,
(CH3)2CH), 0.90 (3H, d, J 6.8 Hz, (CH3)2CH), 1.21 (3H, d,
J 6.4 Hz, CH3CHOH), 1.32 (9H, s, (CH3)3), 2.33–2.38 (1H,
m, (CH3)2CH), 3.86 (3H, s, OCH3), 4.00–4.07 (1H, m, CH3-
CHOH), 4.15 (1H, br s, CHNHCOO), 5.16 (1H, dd, J 6.1, 8.9
Hz, CHCH(CH3)2), 5.84 (1H, d, J 6.5 Hz, NHCOO), 7.64 (1H,
d, J 8.1 Hz, NHCOCH), 8.04 (1H, s, SCH). δC (90.5 MHz,
CHCl3) 17.1 (q), 19.1 (q), 27.8 (q), 32.7 (d), 52.1 (q), 56.3 (d),
59.1 (d), 68.3 (d), 79.8 (s), 127.0 (d), 146.0 (s), 155.7 (s), 161.4
(s), 171.0 (s), 172.1 (s). m/z (FAB) 416.1862 (MHϩ, C18H30-
N3O6S requires 416.1862), 416 (29), 360 (20%).
Boc-(S)-Phe-Thz-(S)-Phe-Pro-aThr-(R)-Val-Thz-OMe, 8
Aqueous sodium hydroxide (1 M, 1.05 ml, 1.05 mmol) was
added dropwise over ca. 5 min to a stirred solution of the
tetrapeptide 10 (607 mg, 1.00 mmol) in THF–water (10 ml, 4:1)
at 0–4 ЊC. The solution was stirred at 0–4 ЊC for 30 min and
then at room temperature for 1 h. Ether (15 ml) and water
(5 ml) were added, and the heterogeneous mixture was then
acidified to pH 1 using 2 M HCl. The aqueous layer was separ-
ated and extracted with ethyl acetate (2 × 15 ml). The combined
organic layers were washed with water (15 ml) and brine (10
ml), then dried (Na2SO4) and concentrated in vacuo to leave the
crude carboxylic acid.
The tripeptide 9 (312 mg, 750 µmol) was acidolysed by
stirring it in a 20% TFA–dichloromethane solution at 0–4 ЊC
for 4 hours. Work up, as previously described, gave the crude
TFA salt as a gum. The salt was redissolved in DMF (5 ml),
then Hünig’s base (425 µl, 2.50 mmol) was added at 0–4 ЊC and
the solution was stirred for 10 min. The crude carboxylic acid
was added, followed by 1-hydroxybenzotriazole (135 mg, 1.00
mmol) and finally DCC (206 mg, 1.00 mmol). The mixture was
stirred at 0 ЊC for 18 hours and then at room temperature for
6 hours, before being worked up as described for the tetrapep-
tide. The crude product was purified by flash chromatography
on silica gel (methanol–ethyl acetate 1:39 eluant) to give the
heptapeptide 8 (475 mg, 534 µmol, 71% based on the tripeptide)
as a pale yellow foam: δH(500 MHz, DMSO, 100 ЊC) 8.32 (1H,
s), 8.08 (1H, s), 8.02 (1H, d, J 8.1 Hz), 7.87 (1H, d, J 8.1 Hz),
7.67 (1H, br s), 7.28 (8H, m), 7.23 (2H, m), 5.09 (3H, m), 4.49
(2H, br s), 4.34 (3H, m), 4.00 (1H, app t, J 6.1 Hz), 3.74 (1H,
br s), 3.60 (1H, app t, J 4.1 Hz), 3.50 (2H, m), 3.37 (1H, dd,
J 14.4, 5.0 Hz), 3.17 (2H, m), 3.04 (1H, dd, J 12.2, 7.2 Hz), 2.36
(2H, m), 2.10–1.85 (4H, m), 1.36 (9H, s), 1.18 (3H, d, J 6.3
Hz), 1.00 (3H, d, J 6.4 Hz), 0.96 (3H, d, J 4.5 Hz); δC(125 MHz,
DMSO, 90 ЊC) 173.8 (s), 172.2 (s), 170.8 (s), 169.7 (s), 169.3 (s),
160.3 (s), 159.4 (s), 154.5 (s), 148.6 (s), 145.7 (s), 137.3 (s), 129.9
(d), 129.7 (d), 127.6 (d), 125.9 (d), 123.2 (d), 78.3 (s), 66.7 (d),
The (S)-Val derived epimer of 9 showed: δH(500 MHz,
DMSO, 100 ЊC) 8.35 (1H, s), 7.92 (1H, d, J 7.75 Hz), 6.28 (1H,
d, J 6.75 Hz), 5.06 (1H, dd, J 8.4, 6.6 Hz), 4.43 (1H, br s), 4.03
(1H, dd, J 8.4, 6.1 Hz), 3.94 (1H, br s), 3.87 (3H, s), 2.37 (1H,
septet, J 6.75 Hz), 1.43 (9H, s), 1.14 (3H, d, J 6.3 Hz), 0.99 (3H,
d, J 6.8 Hz), 0.95 (3H, d, J 6.7 Hz). δC(125 MHz, DMSO,
100 ЊC) 172.2 (s), 170.2 (s), 160.8 (s), 154.8 (s), 145.4 (s), 127.7
(d), 78.1 (s), 66.5 (d), 60.2 (d), 56.2 (d), 51.2 (q), 31.8 (d),
27.8 (q), 19.2 (q), 18.7 (q), 17.5 (q). m/z (ESMS) 438.1687
(Mϩ ϩ Na); C18H29N3O6NaS requires 438.167.
Boc-(S)-Phe-Thz-Phe-Pro-OMe, 10
Aqueous sodium hydroxide (1 M, 7.0 ml, 7.0 mmol) was added
over 5 min to a stirred solution of the thiazole 14 (2.005 g, 5.54
mmol) in THF–MeOH–water (2:2:1). The solution was stirred
at 25 ЊC for 90 min and then concentrated in vacuo to remove
the THF and methanol; the residual aqueous solution was
washed with ether (2 × 10 ml) and acidified to pH 1 using 2 M
HCl. The mixture was extracted with ethyl acetate (3 × 30 ml)
and the extract was then washed successively with water (5 ml)
and brine (20 ml), then dried (MgSO4) and concentrated in
vacuo to leave the crude acid as an oil, which was used without
further purification.
J. Chem. Soc., Perkin Trans. 1, 2000, 875–882
879

View Article Online
65.7 (d), 60.1 (t), 59.5 (d), 58.5 (d), 54.7 (t), 54.1 (d), 51.5 (d),
46.5 (d), 39.7 (q), 37.3 (t), 31.85 (d), 27.75 (q), 23.9 (t), 19.4 (q),
18.7 (q), 17.6 (q), 13.6 (q). m/z (ESMS) 912.3433 (M ϩ Na);
C44H55N7O9NaS2 requires 912.3400.
(1H, t, J 6.7 Hz), 3.05 (1H, dd, J 13.1, 4.8 Hz), 2.85 (1H, dd,
J 13.1, 9.6 Hz), 2.75 (1H, m), 2.69 (1H, m), 2.53 (1H, dd, J 13.8,
11.3 Hz), 2.10 (1H, m), 2.04 (1H, m), 1.93 (1H, m), 1.78 (1H,
m), 1.63 (1H, m), 1.34 (3H, d, J 6.3 Hz), 1.17 (3H, d, J 6.6 Hz),
0.71 (3H, d, J 6.3 Hz). δC(100 MHz, CDCl3, 25 ЊC) 170.0 (s),
168.5 (s), 167.8 (s), 165.4 (s), 158.5 (s), 157.0 (s), 147.9 (s), 146.1
(s), 134.3 (s), 134.1 (s), 128.1 (d), 128.1 (d), 127.1 (d), 127.0 (d),
125.8 (d), 125.4 (d), 121.9 (d), 120.8 (d), 65.5 (d), 60.6 (d), 54.2
(d), 51.7 (d), 51.2 (d), 45.0 (t), 40.6 (t), 39.0 (t), 29.7 (d), 27.5 (t),
23.2 (t), 18.7 (q), 18.6 (q), 18.5 (q). m/z (FAB) 758.2815 (MHϩ);
C38H44N7O6S2 requires 758.2795.
The (21R,31S) diastereomer of 8 showed: δH(360 MHz,
DMSO, 100 ЊC) 8.37 (1H, s), 8.18 (1H, d, J 8.15 Hz), 8.09 (1H,
s), 7.91 (1H, d, J 8.1 Hz), 7.68 (1H, br s), 7.29 (8H, m), 7.24
(2H, m), 5.07 (3H, m), 4.56 (1H, br s), 4.52 (1H, m), 4.39 (1H,
m), 3.96 (1H, m), 3.86 (3H, s), 3.73 (1H, br s), 3.51 (1H, m),
3.37 (1H, dd, J 13.9, 5.1 Hz), 3.21 (2H, m), 3.13 (1H, dd,
J 13.9, 9.6 Hz), 2.34 (2H, m), 2.10–1.83 (4H, m), 1.35 (9H, s),
1.18 (3H, d, J 6.3 Hz), 0.98 (3H, d, J 6.3 Hz), 0.96 (3H, d, J 6.3
Hz). δC(90 MHz, DMSO, 90 ЊC) 174.5 (s), 173.3 (s), 171.6 (s),
170.6 (s), 161.6 (s), 155.3 (s), 149.3 (s), 146.1 (s), 137.4 (s), 129.8
(d), 129.7 (d), 128.7 (d), 128.5 (d), 126.8 (d), 126.7 (d), 124.1 (d),
79.1 (s), 67.7 (d), 60.2 (t), 59.1 (d), 57.1 (q), 54.8 (t), 52.3 (d),
52.1 (d), 47.4 (d), 38.0 (t), 32.5 (d), 31.5 (d), 29.2 (t), 28.5 (q),
24.9 (t), 20.3 (q), 19.6 (q), 18.4 (q).
Lissoclinamide 5, 1, and revised structure 15
Thionyl chloride (500 µl) was added dropwise to a stirred solu-
tion of the macrocyclic heptapeptide 7 (34 mg, 45 µmol) in
dichloromethane (5 ml) at 0–4 ЊC, and the mixture was stirred
at 0–4 ЊC for 30 minutes and then left to stand at 3–5 ЊC for
36 h. The mixture was carefully added to a rapidly stirred
potassium carbonate solution (10%), and then extracted
with ethyl acetate (3 × 15 ml). The combined extracts were
washed with water (10 ml) and brine (10 ml), dried (Na2SO4)
and then concentrated in vacuo to leave the crude product.
Purification by flash chromatography on silica gel (methanol–
ethyl acetate 1:99 eluant) gave the “lissoclinamide 5” 1 (23.5
mg, 31.8 µmol, 71%) as a white foam. [α]2D5 ϩ64 (c = 0.54,
CHCl3). νmax (CHCl3)/cmϪ1 3380, 2927, 1667. δH (360 MHz,
CDCl3) 0.94 (3H, d, J 6.7 Hz, (CH3)2), 1.09 (3H, d, J 6.6 Hz,
(CH3)2), 1.48 (3H, d, J 6.3 Hz, CH3CHO), 1.61–2.07 (3H, m,
CHHЈCH2N, CH2N), 2.14–2.22 (1H, m, CH2CHN), 2.72–2.82
(1H, m, (CH3)2CH), 2.83–2.90 (1H, m, CH2N), 3.00 (1H, dd,
J 3.1, 13.6 Hz, CH2Ph), 3.04 (1H, dd, J 4.6, 13.5 Hz, CH2Ph),
3.27 (1H, dd, J 7.9, 13.5 Hz, CH2Ph), 3.54–3.58 (1H, m,
CH2N), 3.73 (1H, dd, J 7.0, 13.5 Hz, CH2Ph), 4.19 (1H, d,
J 5.8 Hz, CHCHNC), 4.45 (1H, app t, J 7.6 Hz, CH2CHN),
4.79–4.86 (1H, m, OCH), 4.90 (1H, app t, J 8.4 Hz, (CH3)2CH-
CH), 4.99–5.05 (1H, m, OCCHCH2Ph), 5.33–5.39 (1H, app q,
J 7.4 Hz, SCCHCH2Ph), 7.13–7.35 (10H, m, ArH), 7.43 (1H,
d, J 7.8 Hz, (CH3)2CHCHNH), 7.92 (1H, s, SCH), 8.04 (1H,
s, SCH), 8.14 (1H, d, J 8.8 Hz, OCCH(CH2Ph)NH), 8.47
(1H, d, J 7.5 Hz, SCCH(CH2Ph)NH). δC (90.5 MHz, CDCl3)
19.4 (q), 19.9 (q), 21.6 (q), 25.3 (t), 29.1 (t), 31.4 (d), 39.5 (t),
41.7 (t), 47.2 (t), 51.9 (d), 54.6 (d), 56.3 (d), 58.5 (d), 75.3 (d),
80.6 (d), 123.4 (d), 124.4 (d), 127.1 (d), 127.1 (d), 128.3 (d),
128.6 (d), 129.6 (d), 129.8 (d), 135.6 (s), 136.6 (s), 148.2 (s),
148.8 (s), 159.8 (s), 161.5 (s), 169.0 (s), 169.2 (s), 170.0 (s), 171.2
(s). m/z (FAB) 740.2690 (MHϩ); C38H42N7O5S2 requires
740.2689.
“Pre-lissoclinamide” 5, 7
Aqueous sodium hydroxide solution (1 M, 400 µl, 400 µmol)
was added in one portion to the heptapeptide 8 (140 mg, 158
µmol) dissolved in THF–methanol (5 ml, 1:1) and the mix-
ture was stirred for 90 min. It was then concentrated to a
small volume (removing the THF and methanol), and the
residue was diluted with water (5 ml), acidified to pH 1 using
2 M HCl and extracted with ethyl acetate (3 × 10 ml). The
combined extracts were washed with water (10 ml) and brine
(8 ml), dried (MgSO4) and concentrated in vacuo to leave the
crude carboxylic acid (131 mg, 95% mass recovery) as a yel-
low foam. The acid was dissolved in a 50% solution of TFA
in dichloromethane (4 ml) and stirred at 25 ЊC for 2 h. The
solvents were removed in vacuo using the toluene addition
procedure described for 8 to leave a gummy residue, which
was dissolved in DMF (30 ml). N-Methylmorpholine (70 µl,
500 µmol) was added, followed by DPPA (44 µl, 200 µmol)
and the resulting solution was stirred at 0–4 ЊC for 2 h, and
then allowed to stand at room temperature for 2 days. Work
up as for the heptapeptide, followed by flash chromatography
on silica gel (methanol–ethyl acetate 1:39 eluant) gave the
macrolactam (42 mg, 55.5 µmol, 35%) as a white foam: [α]D
ϩ26 (c = 0.16, CHCl3). νmax (CHCl3)/cmϪ1 3382, 2927, 1675,
1631, 1546, 1493, 1463. δH (360 MHz, CDCl3) 0.86 (3H, d,
J 6.7 Hz, (CH3)2), 1.05 (3H, d, J 6.7 Hz, (CH3)2), 1.25 (3H,
d, J 6.8 Hz, CH3CHOH), 1.78–1.98 (1H, m, CH2CH2N),
1.99–2.13 (2H, m, CH2CH2N, CH2CH), 2.18–2.31 (1H, m,
CH2CH), 2.45–2.51 (1H, m, (CH3)2CH), 3.06–3.14 (1H, m,
CH2N), 3.16–3.23 (3H, m, CHHЈPhЈ, CH2Ph), 3.46–3.51 (1H,
m, CHHЈPhЈ), 3.63–3.69 (1H, m, CH2N), 4.06–4.14 (3H, m,
CHOH, OCHCH, CH2CH), 4.41 (1H, br s, OH), 4.92 (1H,
app t, J 8.0 Hz, (CH3)2CHCH), 5.14 (1H, ddd, J 6.2, 6.2, 9.5
Hz, CHCH2Ph), 5.62–5.68 (1H, m, CHCH2Ph), 6.68 (1H, d,
J 8.0 Hz, OCHCHNH), 7.16–7.37 (10H, m, ArH), 7.91 (1H,
s, SCH), 8.06 (1H, s, SCH), 8.17 (1H, d, J 7.6 Hz, (CH3)2-
CHCHNH), 8.24 (1H, d, J 9.5 Hz, NCOCH(CH2)NH), 8.59
(1H, d, J 8.3 Hz, SCCHNH). δC (90.5 MHz, CDCl3) 19.1 (q),
19.5 (q), 19.6 (q), 25.5 (t), 28.7 (t), 33.3 (d), 39.1 (t), 42.0 (t), 47.9
(t), 52.2 (d), 52.6 (d), 58.8 (d), 60.6 (d), 62.8 (d), 67.1 (d), 123.6
(d), 124.3 (d), 127.1 (d), 127.3 (d), 128.5 (d), 128.8 (d), 129.6 (d),
129.7 (d), 135.3 (s), 136.2 (s), 148.2 (s), 148.5 (s), 160.4 (s), 160.5
(s), 168.5 (s), 169.5 (s), 169.6 (s), 171.4 (s); m/z (FAB) 758.2798
(MHϩ); C38H44N7O6S requires 758.2795.
Data for revised stereostructure 15: [α]D25 Ϫ35 (c = 0.19,
CHCl3). νmax (CHCl3)/cmϪ1 3697, 2977, 2929, 2872, 1682, 1636,
1602, 1110. δH (500 MHz, CDCl3) 0.80 (3H, d, J 6.6 Hz,
(CH3)2), 1.09 (3H, d, J 6.6 Hz, (CH3)2), 1.47 (3H, d, J 6.3 Hz,
CH3CHO), 1.73–1.77 (2H, m, CH2CH2N), 1.86–1.92 (1H, m,
CHHЈCHN), 2.08–2.15 (2H, m, CHHЈN, CHHЈCHN), 2.77
(1H, dd, J 10.0, 13.2 Hz, CH2Ph), 2.77–2.83 (1H, m, (CH3)2-
CH), 2.91 (1H, dd, J 10.2, 12.5 Hz, CH2Ph), 3.27 (1H, dd, J 4.5,
12.4 Hz, CH2Ph), 3.27 (1H, obs. m, CHHЈN), 3.90 (1H, dd,
J 4.1, 13.2 Hz, CH2Ph), 4.31 (1H, d, J 3.9 Hz, OCHCH), 4.58
(1H, app t, J 7.8 Hz, CH2CHN), 4.86–4.91 (2H, m, CH3CHO,
OCCHCH2Ph), 5.20 (1H, app t, J 10.3 Hz, (CH3)CHCH),
5.43–5.47 (1H, m, SCCHCH2Ph), 7.23–7.35 (10H, m, ArH),
7.91 (1H, s, SCH), 7.93 (1H, d, J 9.9 Hz, (CH3)CHCHNH),
8.08 (1H, s, SCH), 8.72 (1H, d, J 7.2 Hz, OCCH(CH2Ph)NH),
9.24 (1H, d, J 5.8 Hz, SCCH(CH2Ph)NH). δC (90.5 MHz,
CDCl3) 19.9 (q), 20.3 (q), 25.1 (t), 28.8 (t), 32.9 (d), 40.8 (t), 42.8
(t), 47.1 (t), 53.9 (d), 54.5 (d), 55.1 (d), 56.6 (d), 75.2 (d), 82.6
(d), 123.0 (d), 123.1 (d), 127.1 (d), 127.3 (d), 128.6 (d), 128.6 (d),
129.7 (d), 129.9 (d), 136.0 (d), 136.2 (d), 167.6 (s), 168.6 (s),
171.0 (s), 171.4 (s). m/z (FAB) 740.2690 (MHϩ); C38H42N7O5S2
requires 740.2689.
The (21R,31S) diastereomer of 7 showed: δH(400 MHz,
CDCl3, 25 ЊC) 9.44 (1H, d, J 5.6 Hz), 8.61 (1H, d, J 7.6 Hz),
9.16 (1H, d, J 9.1 Hz), 7.82 (1H, s), 7.65 (1H, s), 7.345 (4H,
app dt, J 16.45, 7.4 Hz), 7.24 (4H, m), 6.82 (2H, br s), 5.57
(1H, br s), 5.48 (1H, ddd, J 5.6, 4.7, 4.2 Hz), 5.065 (1H, dd,
J 11.2, 9.1 Hz), 4.80 (1H, t, J 6.5 Hz), 4.51 (1H, ddd, J 9.6, 7.6, 4.8
Hz), 4.17 (1H, dd, J 13.8, 4.15 Hz), 4.08 (1H, t, J 7.8 Hz), 3.75
880
J. Chem. Soc., Perkin Trans. 1, 2000, 875–882

View Article Online
Boc-(S)-Ser-(R)-Phe-Thz-OMe, 21
Boc-aThr-(S)-Val-ꢀ(CS-NH)-(S)-Ser-(R)-Phe-Thz-OMe, 18
Trifluoroacetic acid (5 ml) was added in one portion to a solu-
tion of the thiazole 20 (1.8 g, 5.0 mmol) in dichloromethane (20
ml). The resulting solution was stirred at 25 ЊC for 2 h, then
diluted with toluene (5 ml) and concentrated to low volume. A
further 5 ml of toluene was added, and the solution was then
re-concentrated to leave the crude amine hydrotrifluoroacetate
as a gum. The gum was redissolved in dichloromethane (30 ml)
and the solution was then cooled to 4 ЊC and Hünig’s base
(2.55 ml, 15.0 mmol) was added in one portion. After 15 min
Boc-(S)-serine (1.2 g, 6.00 mmol) was added, followed by
1-hydroxybenzotriazole (811 mg, 6.00 mmol) and finally DCC
(1.341 g, 6.50 mmol). The mixture was stirred at 0–5 ЊC for
2 h, then at room temperature for 1 h and filtered (to remove
precipitated DCU). The filtrate was washed with 10% citric
acid solution (10 ml), 10% NaHCO3 (10 ml), water (10 ml)
and finally brine (10 ml), then dried (MgSO4) and concen-
trated to leave the crude tripeptide. Purification by flash
chromatography on silica gel (1:1 ethyl acetate–light petrol-
eum eluant) gave the tripeptide (1.6 g, 3.55 mmol, 71%) as a
white foam: δH(500 MHz, DMSO, 90 ЊC) 8.37 (1H, s), 8.33
(1H, d, J 8.1 Hz), 7.25 (4H, m), 7.21 (1H, m), 6.16 (1H, br s),
5.41 (1H, ddd, J 8.95, 8.1, 5.5 Hz), 4.43 (1H, br s), 4.04 (1H,
m), 3.88 (3H, s), 3.52 (2H, m), 3.39 (1H, dd, J 14.05, 5.5 Hz),
3.24 (1H, dd, J 14.05, 8.95 Hz), 1.42 (9H, s). δC(125 MHz,
DMSO, 90 ЊC) 172.5 (s), 170.1 (s), 160.9 (s), 154.7 (s), 145.3
(s), 137.0 (s), 128.8 (d), 128.3 (d), 127.8 (d), 126.0 (d),
78.1 (s), 61.5 (t), 56.8 (d), 52.1 (d), 51.4 (q), 39.7 (t), 27.8 (q).
m/z (ESMS) 472.1490 (M ϩ Na); C21H27N3O6NaS requires
472.1518.
Trifluoroacetic acid (2 ml) was added in one portion to a solu-
tion of the tetrapeptide 23 (565 mg, 1.00 mmol) in dichloro-
methane (8 ml). The resulting solution was stirred at 4 ЊC for
3 h then diluted with toluene (5 ml) and concentrated in vacuo
to leave the crude TFA salt as a gum. The gum was dissolved in
DMF (6 ml) and the solution was then cooled to 4 ЊC. Hünig’s
base (510 µmol) was added, followed by Boc-allothreonine (ca.
1.0 mmol of crude material from Boc-protection step), HOBt
(142 mg, 1.05 mmol) and finally DCC (227 mg, 1.10 mmol).
The mixture was stirred at 0–4 ЊC for 90 min and then at 25 ЊC
for 24 h, after which the solution was diluted with ethyl acetate
(40 ml) and water (20 ml). The separated aqueous layer was
extracted with more ethyl acetate (20 ml), and the combined
organic layers were washed with water (4 × 30 ml) and brine (10
ml), then dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel (methanol–
dichloromethane 1:19 eluant) to give the pentapeptide (507 mg,
761 µmol, 76%) as an off-white foam. δH(500 MHz, DMSO,
90 ЊC) 9.55 (1H, d, J 6.9 Hz), 8.37 (1H, d, J 8.1 Hz), 8.35 (1H,
s), 7.58 (1H, d, J 8.4 Hz), 7.26 (4H, m), 7.21 (1H, m), 6.31 (1H,
br s), 5.41 (1H, ddd, J 9.0, 8.1, 5.6 Hz), 5.05 (1H, dd, J 6.3, 6.3
Hz), 4.63 (1H, dd, J 8.4, 6.3 Hz), 3.97 (1H, dd, J 8.4, 6.3 Hz),
3.91 (1H, dq, J 6.3, 6.3 Hz), 3.88 (3H, s), 3.67 (1H, dd, J 11.0,
6.4 Hz), 3.64 (1H, dd, J 11.0, 5.2 Hz), 3.41 (1H, dd, J 14.1, 5.6
Hz), 3.23 (1H, dd, J 14.1, 9.0 Hz), 2.20 (1H, dqq, J 6.3, 6.8, 6.8
Hz), 1.43 (9H, s), 1.13 (3H, d, J 6.3 Hz), 0.93 (3H, d, J 6.8 Hz),
0.90 (3H, d, J 6.8 Hz). δC(125 MHz, DMSO, 90 ЊC) 203.4 (s),
172.2 (s), 170.0 (s), 167.9 (s), 160.8 (s), 154.9 (s), 145.3 (s),
137.0 (s), 128.8 (d), 128.3 (d), 127.7 (d), 126.0 (d), 78.2 (s),
66.6 (d), 63.3 (d), 60.7 (d), 60.5 (t), 60.2 (d), 52.3 (d), 51.3
(q), 39.7 (t), 32.6 (d), 27.9 (q), 19.2 (q), 19.0 (q), 17.1 (q).
m/z (ESMS) 688.2474 (Mϩ ϩ Na); C30H43N5O8NaS2 requires
688.2451.
Boc-(S)-Val-ꢀ(CS-NH)-(S)-Ser-(R)-Phe-Thz-OMe, 23
Trifluoroacetic acid (5 ml) was added in one portion to a solu-
tion of the tripeptide 21 (1.35 g, 3.00 mmol) in dichloromethane
(20 ml). The resulting solution was stirred at 4 ЊC for 3 h then
diluted with toluene (5 ml) and concentrated, leaving the crude
TFA salt as a gum. The gum was redissolved in ethyl acetate (30
ml) and 10% NaHCO3 (20 ml) and the two layers were then
separated. The aqueous phase was extracted with more ethyl
acetate (3 × 20 ml) and the combined organic phases were
then washed with half-saturated brine. The combined aqueous
phases were extracted continuously with 200 ml dichloro-
methane for 12 h, and the extract was combined with the ethyl
acetate layers, then dried (Na2SO4) and concentrated in vacuo to
leave the crude amino alcohol. The residue was redissolved in
anhydrous DMF (10 ml) and the solution was cooled to 0–4 ЊC.
A solution of the (S)-thioamidating reagent 22 (1.05 g, 3.00
mmol)23 in DMF (10 ml) was introduced by careful dropwise
addition to the stirred solution over 5–10 minutes. The resulting
mixture was stirred at 4 ЊC for 1 h and then at 25 ЊC for 4 h,
and finally diluted with ethyl acetate (100 ml). The organic
extract was washed with water (4 × 20 ml) and brine (20 ml),
then dried (MgSO4) and concentrated in vacuo to leave the
crude product. Purification by flash chromatography on silica
gel (ethyl acetate–methanol, 98:2) gave the tetrapeptide (1.24 g,
2.2 mmol, 73%) as a white foam: δH(500 MHz, DMSO, 90 ЊC)
9.46 (1H, d, J 7.2 Hz), 8.53 (1H, d, J 8.1 Hz), 8.36 (1H, s), 7.26
(4H, m), 7.12 (1H, m), 6.27 (1H, d, J 8.4 Hz), 5.42 (1H, ddd,
J 9.05, 8.1, 5.55 Hz), 5.075 (1H, dd, J 12.9, 5.7 Hz), 4.285
(1H, dd, J 8.8, 6.5 Hz), 3.88 (3H, s), 3.68 (1H, dd, J 10.9, 6.0
Hz), 3.63 (1H, dd, J 10.9, 5.2 Hz), 3.43 (1H, dd, J 14.1, 5.55
Hz), 3.22 (1H, dd, J 14.1, 9.05 Hz), 2.15 (1H, dqq, J 5.7, 6.8,
6.8 Hz), 1.405 (9H, s), 0.91 (3H, d, J 6.8 Hz), 0.87 (3H, d,
J 6.8 Hz). δC(125 MHz, DMSO, 90 ЊC) 203.9 (s), 172.2 (s),
168.05 (s), 160.8 (s), 154.7 (s), 145.3 (s), 137.0 (s), 128.8 (d),
128.3 (d), 127.7 (d), 126.0 (d), 78.2 (s), 65.8 (d), 60.6 (t), 60.4,
52.3 (d), 51.3 (q), 39.7 (t), 32.4 (d), 27.8 (q), 19.0 (q), 17.1 (q).
m/z (ESMS) 587.1960 (Mϩ ϩ Na); C26H36N4O6NaS2 requires
587.1974.
Boc-Phe-Pro-aThr-(S)-Val-ꢀ(CS-NH)-(S)-Ser-(R)-Phe-Thz-
OMe, 24
The pentapeptide 18 (708 mg, 1.06 mmol) was converted into
the corresponding TFA salt by treatment with 20% TFA in
dichloromethane at 4 ЊC for 3 h, followed by dilution with
toluene and concentration as described for previous steps.
The crude TFA salt was dissolved in DMF (5 ml) at 4 ЊC and
Hünig’s base (510 µl, 3.00 mmol) was added, followed by Boc-
(S)-Phe-Pro-OH (435 mg, 1.20 mmol), HOBt (169 mg, 1.25
mmol) and finally DCC (268 mg, 1.30 mmol). The mixture was
stirred at 0–4 ЊC for 90 min and then 25 ЊC for 24 h. It was then
diluted with ethyl acetate (40 ml) and water (20 ml), and the
separated aqueous layer was extracted with more ethyl acetate
(20 ml). The combined organic layers were washed with water
(4 × 30 ml) and brine (10 ml), then dried (MgSO4) and concen-
trated in vacuo. Purification by flash chromatography on silica
gel (methanol–dichloromethane 1:14 eluant) gave the hepta-
peptide (643 mg, 719 µmol, 68%) as a pale yellow foam: δH(500
MHz, DMSO, 90 ЊC) 9.52 (1H, d, J 7.0 Hz), 8.37 (1H, d, J 7.8
Hz), 8.35 (1H, s), 7.68 (1H, br s), 7.56 (1H, d, J 8.2 Hz), 7.28
(4H, m), 7.22 (1H, m), 5.40 (1H, ddd, J 9.0, 8.1, 5.5 Hz), 5.05
(1H, app q, J 6.65 Hz), 4.63 (1H, dd, J 8.3, 6.3 Hz), 4.49 (2H, br
s), 4.29 (1H, app t, J 7.1 Hz), 3.97 (1H, br s), 3.88 (3H, s), 3.66
(3H, m), 3.48 (1H, m), 3.43 (1 H, dd, J 14.1, 5.5 Hz), 3.23 (1H,
dd, J 14.1, 9.0 Hz), 3.00 (1H, br s), 2.84 (1H, dd, J 13.9, 8.4 Hz),
2.24 (1H, m), 2.05–1.83 (4H, br m), 1.35 (9H, s), 1.16 (3H, d,
J 6.3 Hz), 0.94 (3H, d, J 6.8 Hz), 0.91 (3H, d, J 6.8 Hz). δC(125
MHz, DMSO, 90 ЊC) 203.3 (s), 172.3 (s), 171.2 (s), 169.6 (s),
167.9 (s), 160.8 (s), 154.4 (s), 145.3 (s), 137.2 (s), 137.0 (s), 128.9
(d), 128.8 (d), 128.3 (d), 127.7 (d), 127.6 (d), 126.0 (d), 125.8 (d),
78.0 (s), 66.8 (d), 63.6 (d), 60.8 (d), 60.6 (t), 59.3 (d), 58.6 (d),
53.4 (d), 52.3 (d), 51.3 (q), 46.4 (t), 39.7 (t), 37.0 (t), 32.4 (d),
28.2 (t), 27.8 (q), 24.1, 19.4 (q), 19.9 (q), 17.1 (q). m/z (ESMS)
932.3682 (Mϩ ϩ Na); C44H59N7O10NaS2 requires 932.3663.
J. Chem. Soc., Perkin Trans. 1, 2000, 875–882
881

View Article Online
2 For a review of L. patella and other Ascidian metabolites see:
B. S. Davidson, Chem. Rev., 1993, 93, 1771.
Pre-lissoclinamide 4, 17
Aqueous sodium hydroxide solution (1 M, 400 µl, 400 µmol)
was added in one portion to a solution of the heptapeptide 24
(132 mg, 147 µmol) in THF–methanol (5 ml, 1:1) and the mix-
ture was stirred at 25 ЊC for 90 min and then concentrated to a
small volume (removing the THF and methanol). The residue
was diluted with water (5 ml), then acidified to pH 1 using 2 M
HCl and extracted with ethyl acetate (3 × 10 ml). The combined
extracts were washed with water (10 ml) and brine (10 ml), dried
(MgSO4) and concentrated in vacuo to leave the crude carb-
oxylic acid 25 (129 mg, ~100% mass recovery) as a yellow foam.
The acid was dissolved in a solution of TFA in dichloro-
methane (4 ml 20%) and then stirred at 0–4 ЊC for 4 h. The
solvents were removed in vacuo to leave a gummy residue.
The residue was dissolved in DMF (35 ml), and N-methyl-
morpholine (70 µl, 500 µmol) was then added followed by
DPPA (44 µl, 200 µmol). The resulting mixture was stirred at
4 ЊC for 2 h, and then allowed to stand at room temperature for
2 days. Work up as described for the intermediate heptapeptide,
followed by purification by flash chromatography on silica gel
(methanol–dichloromethane 1:9 eluant) gave the cyclopeptide
diol (34 mg, 44 µmol, 30%) as a white foam. NMR spectroscopy
revealed a complex mixture of at least three rotamers, which
could not be resolved to any useful extent. m/z (ESMS)
800.2854 (Mϩ ϩ Na); C38H47N7O7NaS2 requires 800.2876.
3 D. F. Sesin, S. J. Gaskell and C. M. Ireland, Bull. Soc. Chim. Belg.,
1986, 95, 853.
4 For the first isolation of lissoclinamides 4 and 5 see: B. M. Degnan,
C. J. Hawkins, M. F. Lavin, E. J. McCaffrey, D. L. Parry, A. L. van
den Brenk and D. J. Watters, J. Med. Chem., 1989, 32, 1349.
5 For the second reported isolation of lissoclinamides see: F. J.
Schmitz, M. B. Ksetbati, J. S. Chang, J. L. Wang, M. B. Hossain,
D. van der Helm, M. H. Engel, A. Serban and J. A. Silfer, J. Org.
Chem., 1989, 54, 3463.
6 S. G. Toske and W. Fenical, Tetrahedron Lett., 1995, 36, 8355.
7 A. R. Carroll, B. F. Bowden, J. C. Coll, D. C. R. Hockless, B. W.
Skelton and A. H. White, Aust. J. Chem., 1994, 47, 61.
8 T. Ishida, M. Tanaka, M. Nabae, M. Inoue, S. Kato, Y. Hamada
and T. Shioiri, J. Org. Chem., 1988, 53, 107 and references therein.
9 For a review see: J. P. Michael and G. Pattenden, Angew. Chem., Int.
Ed. Engl., 1993, 32, 112.
10 A. L. Van den Brenk, D. P. Fairlie, G. R. Hanson, L. R. Gahan,
C. J. Hawkins and A. Jones, Inorg. Chem., 1994, 33, 2280.
11 A. L. Van den Brenk, K. A. Byriel, D. P. Fairlie, L. R. Gahan,
G. R. Hanson, C. J. Hawkins, A. Jones, C. H. L. Kennard,
B. Moubaraki and K. S. Murray, Inorg. Chem., 1994, 33, 3549.
12 P. Wipf, S. Venkatraman, C. P. Miller and S. J. Geib, Angew. Chem.,
Int. Ed. Engl., 1994, 33, 1516.
13 P. Comba, R. Cusack, D. P. Fairlie, L. R. Gahan, G. R. Hanson,
U. Kazmaier and A. Ramlow, Inorg. Chem., 1998, 37, 6721.
14 For preliminary publication see: C. D. J. Boden and G. Pattenden,
Tetrahedron Lett., 1994, 35, 8271.
15 For preliminary publication see: C. D. J. Boden and G. Pattenden,
Tetrahedron Lett., 1995, 36, 6153.
Lissoclinamide 4, 16
16 See for example: (a) Y. Hamada, M. Shibata and T. Shioiri,
Tetrahedron Lett., 1985, 26, 6501; (b) Y. Hamada, M. Shibata and
T. Shioiri, Tetrahedron Lett., 1985, 26, 5159; (c) Y. Hamada,
M. Shibata and T. Shioiri, Tetrahedron Lett., 1985, 26, 5155.
17 P. Wipf and P. C. Fritch, Tetrahedron Lett., 1994, 35, 5397.
18 Y. Hamada, M. Shibata and T. Shioiri, Tetrahedron Lett., 1985, 26,
5155.
The cyclopeptide diol 17 (21.0 mg, 27 µmol) was dissolved in
THF (4 ml) and Burgess’ reagent (15.0 mg, 63 µmol) was added
in one portion. The resulting solution was heated under reflux
in a nitrogen atmosphere for 20 minutes, then cooled to room
temperature and diluted with ethyl acetate (10 ml) and water
(6 ml). The separated aqueous layer was extracted with more
ethyl acetate (2 × 8 ml) and the combined organic extracts were
then washed with water (7 ml) and brine (10 ml), dried (MgSO4)
and concentrated to leave the crude product. Purification by
flash chromatography on silica gel (methanol–ethyl acetate
1:39) gave lissoclinamide 4 (14.2 mg, 19.2 µmol, 71%) as a
white foam: δH(400 MHz, CDCl3, 25 ЊC); in agreement with
published values. δC(100 MHz, CDCl3, 25 ЊC) 173.6 (s), 171.5
(s), 170.65 (s), 170.5 (s), 169.6 (s), 168.0 (s), 159.8 (s), 148.4 (s),
136.1 (s), 136.0 (s), 130.0 (d), 128.7 (d), 127.4 (d), 127.3 (d),
123.3 (d), 82.5 (d), 80.1 (d), 77.5 (d), 75.2 (d), 56.8 (d), 55.6 (d),
54.4 (d), 54.3 (d), 47.1 (t), 42.5 (t), 40.5 (t), 34.0 (t), 33.4 (d), 28.6
(t), 25.4 (t), 21.95 (q), 20.2 (q), 19.5 (q). m/z (ESMS) 764.2641
(Mϩ ϩ Na); C38H43N7O5NaS2 requires 764.2665.
19 U. Schmidt and P. Gleich, Angew. Chem., Int. Ed. Engl., 1985, 24,
569.
20 M. North and G. Pattenden, Tetrahedron, 1990, 46, 8267; cf.
Y. Hirotsu, T. Shiba and T. Kaneko, Bull. Chem. Soc. Jpn., 1967, 40,
2945.
21 C. D. J. Boden, G. Pattenden and T. Ye, Synlett, 1995, 417.
22 For a synthesis of the bis-thiazoline based lissoclinamide 7, reported
contemporaneously, see: P. Wipf and P. C. Fritch, J. Am. Chem. Soc.,
1996, 118, 12358.
23 B. Zacharie, G. Sauvé and C. Penney, Tetrahedron, 1993, 49, 10489.
24 T. A. Halgren, J. Comp. Chem., 1996, 17, 490.
25 F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp,
M. Lipton, C. Caufield, G. Chang, T. Hendrickson and W. C. Still,
J. Comp. Chem., 1990, 11, 440; see also http://www.schrodinger.com/
macromodel.html.
26 For an SAR study of Lissoclinamide
7 and some epimeric
analogues, see: P. Wipf, P. C. Fritch, S. J. Geib and A. M. Sefler,
J. Am. Chem. Soc., 1998, 120, 4105.
27 C. D. J. Boden, M. C. Norley and G. Pattenden, Tetrahedron Lett.,
1996, 37, 9111; M. C. Norley and G. Pattenden, Tetrahedron Lett.,
1998, 39, 3087.
28 B. McKeever and G. Pattenden, Tetrahedron Lett., 1999, 40, 9317.
29 D. J. Freeman, G. Pattenden, A. F. Drake and G. Siligardi, J. Chem.
Soc., Perkin Trans. 2, 1998, 129.
30 A. Bertram, J. S. Hannam, K. A. Jolliffe, F. González-López de
Turiso and G. Pattenden, Synlett, 1999, 1723.
31 W. C. Still, A. Tempczyk, R. C. Hawley and T. Hendrickson,
J. Am. Chem. Soc., 1990, 112, 6127.
Acknowledgements
We thank the EPSRC for support (Fellowship to C. D. J. B.)
and Zeneca for support via their Strategic Fund Initiative. We
also thank Dr J. M. Clough (Zeneca Agrochemicals) for his
interest in this work and Professor F. J. Shmitz for providing us
1
with a copy of the H NMR spectrum of naturally occurring
lissoclinamide 4.
References
1 For extensive reviews and bibliography see: D. J. Faulkner, “Marine
Natural Products”, in Nat. Prod. Rep., 1998, 15, 113; this is an
ongoing annual series, beginning in 1986.
Paper a909360e
882
J. Chem. Soc., Perkin Trans. 1, 2000, 875–882