Semisynthesis and biological activity of aminoacyl triesters of squamocin, an annonaceous acetogenin

Article abstract of DOI:10.1016/j.bmc.2005.03.029

A number of aminoacyl triesters of squamocin 1, a cytotoxic acetogenin isolated from the seeds of Annona reticulata, have been synthesized in two to three steps from protected (l)-aminoacids and squamocin 1 using standard coupling/deprotection procedures.

Full text of DOI:10.1016/j.bmc.2005.03.029

Bioorganic & Medicinal Chemistry 13 (2005) 3773–3781
Semisynthesis and biological activity of aminoacyl triesters of
squamocin, an annonaceous acetogenin
Romain A. Duval,a Philippe Duret,a Guy Lewin,a,* Eva Perisb and Reynald Hocquemillera
a
´
´
Laboratoire de Pharmacognosie (BioCIS, UMR 8076 CNRS), Faculte de Pharmacie, rue J.B. Clement,
ˆ
92296 Chatenay-Malabry Cedex, France
bLaboratorio de Farmacognosia, Facultad de Farmacia, Universitad de Valencia, Spain
Received 25 November 2004; accepted 11 March 2005
Available online 11 April 2005
Abstract—A number of aminoacyl triesters of squamocin 1, a cytotoxic acetogenin isolated from the seeds of Annona reticulata,
have been synthesized in two to three steps from protected (L)-aminoacids and squamocin 1 using standard coupling/deprotection
procedures. These semisynthetic analogs were tested on submitochondrial particles (SMP) for their complex I inhibitory activities,
and against KB 3-1 cells in vitro. All triesters derivatives exhibited a complete extinction of activity at the enzymatic level, correlated
to a reduced though modulated cytotoxicity in comparison with squamocin 1. This activity can apparently be considered as a func-
tion of the amphipathy of the analogs, the more amphiphilic ones being the more cytotoxic.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ity.14 This ambiguity led us to investigate whether acyl-
ation of these hydroxy groups by moieties containing
electron-rich functions (amino, imidazole, hydroxy-
methyl, phenol. . .) could maintain the native molecule
activities by acting even at some distance from the ali-
phatic backbone. a-Aminoacyl derivatives of a represen-
tative acetogenin correspond to ideal candidates for
such a study, since selective deprotection of the acyl res-
idues allows access to diverse protic analogs of the lead
inhibitor. Moreover, a very significant enhancement of
the hydrosolubility of such derivatives can be expected.
The chosen acetogenin to be modified was squamocin 1
(Fig. 1), a highly cytotoxic agent and potent respiratory
inhibitor among this class of natural products.9,14–17
Annonaceous acetogenins exhibit a broad range of bio-
logical activities (cytotoxic, antiparasitic, insecticide. . .)1–3
as a result of the inhibition of the mitochondrial
NADH-ubiquinone oxidoreductase (complex I),4,5 caus-
ing a decrease of ATP biosynthesis and cell death by
apoptotic mechanisms.6,7 In addition to their often spec-
tacular cytotoxicity against various sensitive and MDR
expressing cancer cell lines in vitro and in vivo,5,8–12
annonaceous acetogenins appear as potential antitumor
agents because of their selective inhibition of the plasmic
NADH oxidoreductase overexpressed and deregulated
in transformed cells.13 From a mechanistic point of
view, the precise interaction between these new type of
inhibitors and their huge mitochondrial target remains
unknown. Two observations were the basis of the pres-
ent study: (a) the transformation of the secondary alco-
hols of acetogenins into acetate or mesyl functions is
known to systematically lead to a disappearance of cyto-
toxicity;1,9 (b) such acylated or sulfonylated derivatives
can retain a high degree of enzymatic inhibitory activ-
2. Results and discussion
2.1. Semisynthesis
The Phe, Trp, and Pro a-aminoacids were selected to
evaluate the influence of the a-amino function on the
biological activity of triester squamocin derivatives.
Short chain polar a-aminoacids such as Ser and His
were selected to evaluate the influence of proximal protic
functions. On the other hand, considering that the
behavior of membrane partition of natural acetogenins
might be a key feature for their bioactivity,18,19 the crea-
tion of hydrophilic distal functions was targeted by
Keywords: Annonaceous acetogenins; Squamocin; Mitochondrial
inhibitors; Cytotoxicity.
*
Corresponding author. Tel.: +33 1 46 83 55 93; fax: +33 1 46 83 53
99; e-mail: guy.lewin@cep.u-psud.fr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.029

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R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
OH
OH
OH
O
34
O
O
1
28
24
15
5
O
Squamocin 1
37
Figure 1.
selecting the Glu, Tyr, and Lys a-aminoacids for the O-
acylation of squamocin 1. The desired a-aminoacyl tri-
esters of squamocin 1 were semisynthesized in two steps
using procedures from peptide chemistry. The native
acetogenin was first esterified with good yields in pres-
ence of an excess Na-BOC protected (L)-a-aminoacids,
using DCC and a catalytic amount of 4-DMAP
(Scheme 1).
ing triamino analog 2. This fact could reflect the prob-
able enzymatic interaction of the polyoxygenated domain
of bioactive acetogenins through its electron-donating
character, suggested by the strong inhibitory activities
of non-hydroxylated, acetylated or carbonylated
acetogenins.14
All fully or partially deprotected squamocin triesters
were tested against KB 3-1 cells in vitro (human epider-
moid nasopharyngeal carcinoma), and their cytotoxicity
evaluated in comparison with tri-O-TBDMS squamocin
1s tested as an inactive and metabolically inert deriva-
tive of the parent molecule25 (Table 2).
The a-amino groups of the Phe, Trp, and Lys and Pro
squamocin triesters 2p–5p were deprotected by use of
HF in pyridine (Scheme 2). In order to prevent epimeri-
zation of the sensitive lactonic C-36 stereocenter,
known to readily occur in polar protic media in the pres-
ence of a weak base,20,21 the corresponding ammonium
fluorides 2, 3, and 4a were purified without being turned
into their corresponding free bases. On the other hand,
the trifluoride salt of Pro squamocin triester 5 could
not be separated from residual pyridinium fluoride be-
cause of its complete solubility in aqueous phase, simi-
larly to the trifluoride salt of Trp squamocin triester 3.
Compound 5 was therefore obtained by treating 5p with
trifluoroacetic acid and purified by chromatography
over Sephadexꢁ LH-20.
In spite of similar or enhanced polarity in comparison
with the natural acetogenin, all compounds exhibited
strongly reduced cytotoxicities relatively to squamocin
1, but were 10–1000 times more active than its trisilyl
ether 1p in most cases. The most amphipathic triesters,
possessing free a-amino groups (as their ammonium)
appeared to be the more cytotoxic (Pro, Phe, Ne-(Z)-
Lys and Trp derivatives), followed by some Na-BOC
compounds with a deprotected lateral chain (Ser, His,
and Lys derivatives). The location of protic functions
was found to be an important factor for the activity:
apart from the amphiphilic character, it seemed that
the restoration of a polar environment close to the ali-
phatic chain of the acetogenin could account for the
residual cytotoxic activities of a-amino analogs. Thus,
the Ser triester 6 was 10–30 times less cytotoxic than
the last derivatives, despite the existence of three alcohol
functions similarly to squamocin 1. Identically, the Lys
triester 4b exhibited a twenty times lower activity than
its analog 4a. The presence of three hydrophilic carboxy-
lic acid (triester 8) or imidazole moieties (triester 9) did
not compensate for the peracylation of the hydroxyles
of squamocin 1, these analogues being weakly or not
cytotoxic. Similarly, the rigid triphenolic derivative 7,
bearing distant protic functions, was devoid of activity.
The lateral chain functions of several triesters were
deprotected using the following reagents, with accept-
able yields (Scheme 3): (a) Pd–C (10%)/1,4-cyclohexadi-
ene system in hot absolute ethanol,22,23 allowing a
smooth catalytic hydrogenolysis of the O-benzylether
groups of the Ser and Tyr triesters 6p and 7p, and of
the Ne-benzyloxycarbonyl and O-benzylester groups of
the Lys and Glu triesters 4p and 8p, respectively. In no
case, reduction of the terminal a,b-unsaturated lactone
was observed; (b) excess HOBT in THF for the detosyla-
tion of the imidazole nuclei24 of the Nim-Ts-His triester
9p.
2.2. Biological evaluation
A selection of squamocin analogs was tested against
complex I from beef heart SMP, and their inhibitory
activities evaluated (Table 1). Compounds 2p, 2, and 5
exhibited a complete absence of potency in comparison
with squamocin 1, its triacetylated derivative 1a and
rotenone, but were significantly more active than previ-
ously described tri-O-TBDMS squamocin 1s.25 More-
over, it has to be noticed the similar activities of the
tri-Na-BOC protected derivative 2p and its correspond-
3. Conclusion
A number of aminoacyl triesters of squamocin 1, a cyto-
toxic annonaceous acetogenin, have been semisynthe-
sized. Despite the promising hydrosolubilities of most
analogs, the derivatization of the secondary alcohols
of squamocin 1 resulted in a dramatic loss of activity,
although these triesters were cytotoxic relatively to the
inactive lipophilic triether 1p. As previously suggested,
our results show that amphipathy is a key factor for
the bioactivity of acetogenin-derived inhibitors, taking
into account that molecules possessing a terminal a,b-
Abbreviations: DCC: dicyclocarbodiimide; 4-DMAP: 4-dimethyl-
aminopyridine; HOBT: N-hydroxybenzotriazole; SMP: submitochon-
drial particles; THF: tetrahydrofurane.

R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
3775
Squamocin 1
(a)
ORp
ORp
ORp
O
34
O
O
1
28
24
15
5
O
O
2p-9p
37
R
Rp =
NHBOC
(R = protected lateral chain)
RpOH
Squamocin
triesters
Yield %
2p
3p
4p
5p
6p
7p
8p
9p
86
73
83
71
81
76
68
37
Nα-(BOC)-PheOH
Nα-BOC-TrpOH
Nα-(BOC)-Nε-(Z)-LysOH
Nα-(BOC)-ProOH
Nα-(BOC)-O-(Bn)-SerOH
Nα-(BOC)-O-(Bn)-TyrOH
Nα-(BOC)-O-(Bn)-GluOH
Nα-(BOC)-Nim-(Ts)-HisOH
Scheme 1. Semisynthesis of the protected 15,24,28-tri-Na-BOC-(aminoacyl)-squamocin derivatives 2p–9p. Reagents and conditions: (a) squamocin 1,
RPOH, DCC, cat. 4-DMAP, EtOAc or CH2Cl2, 0 ꢂC to rt.
BOC
NH
BOC
NH
BOC
NH
R
O
R
O
R
O
O
O
O
O
34
O
O
1
28
24
15
5
O
2p-5p
(a)
(a)
37
(b)
NH2.HF
O
TFA.HN
O
(a)
O
O
2
5
NH2.HF
H
N
O
NH2.HF
O
N
H
O
O
O
O
4a
3
Scheme 2. Deprotection of the Na-BOC groups of the triesters 2p–5p. Reagents and conditions: (a) 2p–4p, HF/Py, anisole, 0 ꢂC to rt, 2h (68% 2, 39%
3, 57% 4a); (b) 5p, TFA, CH2Cl2, 0 ꢂC, 2h (55%).
unsaturated lactone but devoid of amphiphily (c-meth-
ylbutenolides substituted by long aliphatic chains, some
type E acetogenins, squamocin triether 1p) are signifi-
cantly less active on mitochondrial complex I than most
of the obtained triesters.26,27 Beside, the restoration of a
protic environment as close as possible to the aliphatic
skeleton of the acetogenin seems to be responsible for
the superior cytotoxicity of certain squamocin triesters.
However, this activity appears to be poorly specific in
term of enzymatic inhibition, and the possibility of a

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R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
BOC
NH
BOC
NH
BOC
NH
R
O
R
O
R
O
O
O
O
O
34
O
O
1
28
24
15
5
O
4p, 6-9p
37
(a)
H
(b)
N
H2N
N
BOC
NH
(a)
H
N
O
O
O
(a)
(a)
BOC
4b
O
O
H
N
HO
BOC
9
H
O
HOOC
N
H
BOC
N
BOC
O
O
6
HO
O
O
8
7
Scheme 3. Deprotection of the lateral chains of triesters 4p and 6p–9p. Reagents and conditions: (a) 4p, 6–8p, Pd–C (10%), 1,4-cyclohexadiene,
EtOH, 40–65 ꢂC, 2–8 h (86% 4b, 52 %6, 60% 7, 32 %8); (b) 9p, HOBT, THF, rt, 5 h (42%).
Table 1. Complex I inhibitory activities (IC50 nM) of squamocin
triesters 2p, 2, and 5
Table 2. Cytotoxic activities (IC50 nM) of the squamocin triesters 2–9
Compound
KB 3-1a
Compound
NADH oxydasea
Squamocin 1
2.6 · 10ꢀ14
Squamocin 1
0.8 0.09
Tri-(ProNH.TFA)-squamocin 5
Tri-(PheNH2.HF)-squamocin 2
2.0 · 10ꢀ8
6.1 · 10ꢀ8
6.3 · 10ꢀ8
2.4 · 10ꢀ7
5.5 · 10ꢀ7
1.1 · 10ꢀ6
1.7 · 10ꢀ6
>10ꢀ5
Tri-(ProNH.TFA)-squamocin 5
Tri-(PheNH2.HF)-squamocin 2
Tri-(Na-BOC-Phe)-squamocin 2p
247.7 20.5
440.0 42.4
625.0 92.9
Tri-(Ne-Z-LysNH2.HF)-squamocin 4a
Tri-(TrpNH2.HF)-squamocin 3
Tri-(Na-BOC-SerOH)-squamocin 6
Tri-(Na-BOC-LysNH2)-squamocin 4b
Tri-(Na-BOC-HisNH)-squamocin 9
Tri-(Na-BOC-TyrOH)-squamocin 7
Tri-(Na-BOC-GluCOOH)-squamocin 8
Tri-(O-Ac)-squamocin 1ab
Tri-(O-TBDMS)-squamocin 1sb
Rotenoneb
5.0 1.3
>5000
5.4 0.6
a Tests performed on bovine heart submitochondrial particles (SMP).
b Reference compounds.
>10ꢀ5
Tri-(O-TBDMS)-squamocinb 1p
>10ꢀ5
NaFb
5.3 · 10ꢀ3
Paclitaxelc
2.1 · 10ꢀ11
a KB 3-1: human nasopharyngeal epidermo¨ıd carcinoma.
b Control compounds.
specific membrane perturbation by highly amphiphilic,
positively charged lipophilic compounds,28,29 at a cellu-
lar or mitochondrial level, cannot be excluded. Based on
partition studies, Shimada et al. proposed that the polar
domain of acetogenins locates at the water–lipids inter-
face of the inner mitochondrial membrane, allowing the
specific interaction of their terminal lactone with com-
plex I.18,19 Considering the increased hydrophilicity of
the central core of most semisynthesized triesters (i.e.,
their superior anchorage potential) but their dramatic
loss of activity, our results are not in favor of this mod-
eling and suggest a specific interaction between the poly-
oxygenated domain of annonaceous acetogenins and
mitochondrial complex I.
c Reference compound.
4. Experimental
NMR spectra were recorded on Bruker AC-200
(200 MHz) or Bruker AM-400 (400 MHz) spectrome-
ters. Mass spectra (MS or HRMS) were recorded on
Kratos MS-80 Rf. Optical rotations were measured
on a Schmidt–Haensch polarimeter E at 589 nm. Col-
umn chromatography was performed with silica gel 60
(9385 Merck), Florisilꢁ (Merck), Sephadexꢁ LH-20

R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
3777
(Pharmacia), or alumina 90 standard II–III (1097
Merck). TLC was performed on aluminum plates coated
with silica gel 60F254 (554 Merck) and revealed with sul-
furic vanillin reagent. Solvents used in this study were
simply redistilled before use.
4.92(m, 4H, H-15, NH 2), 5.04 (dq, 1H, H-36,
J = 1.5 Hz, J = 6.8 Hz), 7.17–7.29 (m, 16H, H-35, H-
arom.). ESIMS m/z 1064 [M+H]+, 1086 [M+Na]+.
4.2.3. 15,24,28-Tri-O-(Na-BOC-tryptophoyl)-squamocin
3p. To a solution of 104 mg (0.167 mmol) squamocin 1
in 2mL EtOAc were added 254 mg (0.836 mmol) Na-
BOC-tryptophane and catalytic 4-DMAP. The mixture
4.1. Extraction
Squamocin 1 was isolated in significant quantity from
the seeds of Annona reticulata, collected in Viet-Nam,
using a described procedure.30 Its chemical identity
was cooled to 0 ꢂC and
a solution of 172mg
(0.836 mmol) DCC in 1 mL EtOAc was added dropwise.
The reaction media was brought back to room temper-
ature and stirred for 15 h, then evaporated under re-
duced pressure. The residue was retaken in ether
(50 mL) and filtered, and the filtrate washed by water
(3 · 5 mL), dried (Na2SO4), and filtered. The filtrate
was evaporated in vacuo and the crude product chro-
matographed over a column of silica gel (EtOAc/cyclo-
hexane 50:50 v/v), furnishing 181 mg (73%) triester 3p
as an amorphous white solid. 1H NMR (CDCl3,
400 MHz) d 0.86 (t, 3H, H-34, J = 6.8 Hz), 1.20 (m,
2H, H-26), 1.35 (m, 2H, H-14), 1.40 (d, 3H, H-37,
1
was determined by extensive H and 13C NMR (includ-
ing HOHAHA, HMQC, and HMBC) experiments, and
comparison with an authentic sample previously iso-
lated from Annona cherimolia.9
4.2. Semisynthesis
4.2.1. 15,24,28-Tri-O-(Na-BOC-phenylalanyl)-squamocin
2p. To a solution of 120 mg (0.193 mmol) squamocin 1
in 2.5 mL EtOAc were added 256 mg (0.965 mmol)
Na-BOC-phenylalanine and catalytic 4-DMAP. The ob-
tained mixture was cooled to 0 ꢂC and a solution of
199 mg (0.965 mmol) DCC in 1 mL EtOAc was added
dropwise. The reaction medium was brought back to
room temperature and stirred 15 h, then evaporated
under reduced pressure. The residue was retaken in cyclo-
hexane (3 · 5 mL) and filtered on Whatman GF/A. The
crude product was chromatographed over a column of
silica gel (EtOAc/cyclohexane 30:70 v/v), furnishing
t
J = 6.8 Hz), 1.43 (s, 27H, Bu–O), 1.49 (m, 4H, H-27,
H-29), 1.54 (m, 4H, H-4, H-25), 1.61 (m, 4H, H-18, H-
21), 1.84 (m, 4H, H-17, H-22), 2.26 (t, 2H, H-3,
J = 7.6 Hz), 3.23 (m, 3H, Hb), 3.30 (m, 3H, Hb), 3.80
(m, 2H, H-19, H-20), 3.93 (m, 2H, H-16, H-23), 4.65
(m, 3H, Ha), 4.89 (m, 1H, H-24), 4.98 (m, 2H, H-15,
H-28), 5.0 (dq, 1H, H-36, J = 1,5 Hz, J = 6.8 Hz), 5.04
(m, 3H, NHBOC), 6.89–7.07 (m, 3H, H-20), 6.98 (d,
1H, H-35, J = 1.5 Hz), 7.0–7.59 (m, 13H, H-35, H-
arom.), 8.51–8.73 (m, 3H, NHind). ESIMS m/z 1505
[M+Na]+, 764 [M+2Na]2+. [a]D ꢀ10 (c 1, CHCl3).
1
226 mg (86%) triester 2p as a colorless resin. H NMR
(CDCl3, 400 MHz) d 0.87 (t, 3H, H-34, J = 6.8 Hz),
1.24 (m, 2H, H-26), 1.38 (s, 27H, Bu–O), 1.40 (d, 3H,
t
H-37, J = 6.8 Hz), 1.46 (m, 4H, H-27, H-29), 1.53 (m,
4H, H-4, H-14), 1.74 (m, 4H, H-18, H-21), 1.88 (m,
4H, H-17, H-22), 2.25 (t, 2H, H-3, J = 7.1 Hz), 2.99
(m, 3 H, Hb), 3.14 (m, 3H, Hb), 3.86 (m, 2H, H-19, H-
20), 3.96 (m, 2H, H-16, H-23), 4.55 (m, 3H, Ha), 4.82
(m, 1H, H-28), 4.85 (m, 1H, H-24), 4.97 (m, 4H, H-15,
NHBOC), 4.99 (dq, 1H, H-36, J = 1.3 Hz, J = 6.8 Hz),
6.98 (d, 1H, H-35, J = 1.3 Hz), 7.15–7.29 (m, 15H, H-
arom.). ESIMS m/z 1388 [M+Na]+. [aD] +2 8 c( 1,
CHCl3).
4.2.4. 15,24,28-Tri-O-(tryptophoyl)-squamocin tri-(ammo-
nium fluoride) 3. A solution of 40 mg (27.0 lmol) triester
3p in 0.4 mL anisole was cooled to 0 ꢂC and 0.4 mL of a
65% HF solution in pyridine was added dropwise. The
reaction medium was stirred at that temperature for
2h, then diluted by EtOAc (20 mL) and treated by
water (8 mL). The organic phase was decanted and
washed by water (3 · 5 mL), dried (Na2SO4), filtered,
and evaporated under reduced pressure. The crude
product was chromatographed over a column of Flori-
silꢁ (EtOAc/MeOH 80:20 v/v), furnishing 13 mg (39%)
triester 3 as a white powder. 1H NMR (MeOD,
400 MHz) d 0.93 (t, 3H, H-34, J = 6.8 Hz), 1.33 (d,
3H, H-37, J = 6.8 Hz), 1.73 (m, 4H, H-18, H-21), 1.81
(m, 4H, H-17, H-22), 2.26 (t, 2H, H-3, J = 7.8 Hz),
3.09 (m, 3H, Hb), 3.25 (m, 3H, Hb), 3.75 (m, 3H, Ha),
3.84 (m, 2H, H-19, H-20), 3.92 (m, 2H, H-16, H-23),
4.63–4.95 (m, 3H, H-15, H-24, H-28), 5.06 (dq, 1H, H-
36, J = 1.3 Hz, J = 6.8 Hz), 6.96–7.63 (m, 16H, H-35,
H-arom.). ESIMS m/z 1183 [M+H]+. HRESIMS m/z
1180.7180 (calcd for C70H96N6O10: 1180.7188).
4.2.2. 15,24,28-Tri-O-(phenylalanyl)-squamocin tri-(ammo-
nium fluoride) 2. A solution of 52mg (38.1 lmol) tri-
ester 2p in 0.5 mL anisole was cooled to 0 ꢂC and
0.5 mL of a 65% HF solution in pyridine was added
dropwise. The reaction medium was stirred at that tem-
perature for 2h, then diluted by EtOAc (10 mL) and
treated by water (5 mL). The organic phase was dec-
anted and washed by water (3 · 3 mL), dried (Na2SO4),
filtered, and evaporated under reduced pressure. The
crude product was chromatographed over a column of
Florisilꢁ (EtOAc/MeOH 90:10 v/v), furnishing 29 mg
1
(68%) triester 2 as a white powder. H NMR (MeOD,
4.2.5. 15,24,28-Tri-O-(Na-BOC-Ne-Z-lysyl)-squamocin
4p. To a solution of 83 mg (0.132mmol) squamocin 1
in 1.5 mL EtOAc were added 251 mg (0.66 mmol) Na-
BOC-Nx-Z-lysine and catalytic 4-DMAP. The mixture
400 MHz) d 0.88 (t, 3H, H-34, J = 6.8 Hz), 1.25 (m,
2H, H-26), 1.36 (d, 3H, H-37, J = 6.8 Hz), 1.48 (m,
2H, H-14), 1.53 (m, 2H, H-4), 1.67 (m, 4H, H-18, H-
21), 1.94 (m, 4H, H-17, H-22), 2.22 (t, 2H, H-3,
J = 7.3 Hz), 2.89 (m, 3H, Hb), 3.03 (m, 3H, Hb), 3.71
(m, 3H, Ha), 3.82 (m, 2H, H-19, H-20), 3.97 (m, 2H,
H-16, H-23), 4.83 (m, 1H, H-24), 4.86 (m, 1H, H-28),
was cooled to 0 ꢂC and
a solution of 136 mg
(0.660 mmol) DCC in 1 mL EtOAc was added dropwise.
The reaction media was brought back to room

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R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
temperature and stirred for 15 h, then evaporated under
reduced pressure. The residue was retaken in a toluene/
cyclohexane 50:50 v/v mixture (10 mL) and filtered over
Whatman GF/A. The filtrate was evaporated in vacuo
and the crude product chromatographed over a column
of silica gel (EtOAc/cyclohexane 50:50 v/v), furnishing
4.2.8. 15,24,28-Tri-O-(Na-BOC-prolyl)-squamocin 5p.
To a solution of 86 mg (0.138 mmol) squamocin 1 in
2mL EtOAc were added 149 mg (0.693 mmol) Na-
BOC proline and catalytic 4-DMAP. The mixture was
cooled to 0 ꢂC and a solution of 142mg (0.690 mmol)
DCC in 1 mL EtOAc was added dropwise. The reaction
media was brought back to room temperature and stir-
red for 20 h, then evaporated under reduced pressure.
The residue was retaken in a toluene/cyclohexane
70:30 v/v mixture (10 mL) and filtered over cotton.
The filtrate was evaporated in vacuo and the crude prod-
uct chromatographed over a column of silica gel
(EtOAc/cyclohexane 50:50 v/v), furnishing 120 mg
1
189 mg (83%) triester 4p as a colorless resin. H NMR
(CDCl3, 400 MHz) d 0.87 (t, 3H, H-34, J = 6.8 Hz),
1.26 (m, 2H, H-26), 1.37 (m, 6H, Hd), 1.39 (d, 3H, H-
37, J = 6.8 Hz), 1.42(s, 27H, tBu–O), 1.52(m, 7H, H-
4, H-14, Hb), 1.54 (m, 4H, H-27, H-29), 1.59 (m, 3H,
Hb), 1.64 (m, 4H, H-18, H-21), 1.79 (m, 6H, Hc), 1.88
(m, 4H, H-17, H-22), 2.25 (t, 2H, H-3, J = 7 Hz), 3.17
(m, 6H, He), 3.82 (m, 2H, H-19, H-20), 3.95 (m, 2H,
H-16, H-23), 4.22 (m, 3H, Ha), 4.86 (m, 2H, H-24, H-
28), 4.92 (m, 1H, H-15), 4.99 (dq, 1H, H-36,
J = 1.5 Hz, J = 6.8 Hz), 5.08 (s, 6H, HBn), 5.15 (m, 3H,
NHBOC), 6.97 (d, 1H, H-35, J = 1.5 Hz), 7.28–7.36 (m,
16H, H-arom.). ESIMS m/z 1733 [M+Na]+, 878
[M+2Na]2+. [a]D +6 (c 1, CHCl3).
1
(71%) triester 5p as a colorless resin. H NMR (CDCl3,
400 MHz) d 0.86 (t, 3H, H-34, J = 6.8 Hz), 1.30 (m, 2H,
H-26), 1.43 (s, 27H, tBu–O), 1.40 (d, 3H, H-37,
J = 6.8 Hz), 1.55 (m, 10H, H-4, H-14, H-25, H-27, H-
29), 1.69 (m, 4H, H-18, H-21), 1.89 (m, 6H, Hc), 1.93
(m, 4H, H-17, H-22), 1.94 (m, 3H, Hb), 2.19 (m, 3H,
Hb), 2.26 (t, 2H, H-3, J = 7.3 Hz), 3.48 (m, 3H, Hd),
3.52(m, 3H, H ), 3.85 (m, 2H, H-19, H-20), 3.98 (m,
d
2H, H-16, H-23), 4.29 (m, 3H, Ha), 4.85 (m, 1H, H-
28), 4.87 (m, 2H, H-15, H-24), 4.99 (dq, 1H, H-36,
J = 1.5 Hz, J = 6.8 Hz), 6.98 (d, 1H, H-35, J = 1.5 Hz).
ESIMS m/z 1238 [M+Na]+. [a]D ꢀ17 (c 1, CHCl3).
4.2.6. 15,24,28-Tri-O-(Nx-Z-lysyl)-squamocin tri-(ammo-
nium fluoride) 4a. A solution of 70 mg (40.9 lmol) tri-
ester 4p in 0.6 mL anisole was cooled to 0 ꢂC and
0.7 mL of a 65% HF solution in pyridine was added
dropwise. The reaction medium was stirred at that tem-
perature for 5 h then treated with MeOH (10 mL) and
evaporated to dryness. The residue was suspended in
water (8 mL), and the precipitate filtered, washed by
cyclohexane and air-dried. Thirty four milligrams
(57%) of triester 4a were obtained as an amorphous
4.2.9. 15,24,28-Tri-O-(prolyl)-squamocin tri-(ammonium
trifluororacetate) 5. A solution of 77 mg (63.4 lmol) tri-
ester 5p in 0.4 mL CH2Cl2 was cooled to 0 ꢂC and
0.8 mL TFA was added dropwise. The reaction medium
was stirred at that temperature for 2h then evaporated
under reduced pressure. The crude product was chro-
matographed over a column of Sephadexꢁ LH-20
(CH2Cl2 100%), furnishing 44 mg (55%) triester 5 as a
white resin. 1H NMR (CDCl3, 400 MHz) d 0.86 (t,
3H, H-34, J = 6.9 Hz), 1.39 (d, 3H, H-37, J = 6.8 Hz),
1.53 (m, 10H, H-4, H-14, H-25, H-27, H-29), 1.82 (m,
4H, H-18, H-21), 1.96 (m, 6H, Hc), 2.08 (m, 4H, H-17,
H-22), 2.10 (m, 3H, Hb), 2.41 (m, 3H, Hb), 2.24 (t,
2H, H-3, J = 7.7 Hz), 3.45 (m, 3H, Hd), 3.74 (m, 2 H,
H-19, H-20), 3.95 (m, 2H, H-16, H-23), 4.51 (m, 3H,
Ha), 4.89 (m, 3H, H-15, H-24, H-28), 4.98 (dq, 1H, H-
36, J = 1.3 Hz, J = 6.8 Hz), 6.96 (d, 1H, H-35,
J = 1.3 Hz). ESIMS m/z 916 [M+H]+. HRESIMS m/z
913.6396 (calcd for C52H87N3O10: 913.6391).
1
white powder. H NMR ((CD3)2CO), 400 MHz d 0.88
(t, 3H, H-34), 1.36 (d, 3H, H-37, J = 7 Hz), 1.55 (m,
2H, H-4), 1.73 (m, 4H, H-18, H-21), 1.91 (m, 4H, H-
17, H-22), 2.21 (t, 2H, H-3, J = 7 Hz), 3.12(m, 6H,
He), 3.84 (m, 2H, H-19, H-20), 3.96 (m, 2H, H-16, H-
23), 4.07 (m, 3H, Ha), 4.83 (m, 2H, H-24, H-28), 4.92
(m, 1H, H-15), 5.03 (m, 7H, H-36, HBn), 7.24–7.41 (m,
16H, H-35, H-arom.). ESIMS m/z 1431 [M+Na]+, 72 8
[M+2Na]2+
. HRESIMS m/z 1408.8754 (calcd for
C79H120N6O16: 1408.8761).
4.2.7. 15,24,28-Tri-O-(Na-BOC-lysyl)-squamocin 4b. To
a solution of 74 mg (43.3 lmol) triester 4p in 0.5 mL
absolute EtOH were added 222 mg (3 mass equiv) Pd–
C (10%). The mixture was Ar purged; then 150 lL
(1.60 mmol) 1,4-cyclohexadiene was added dropwise.
The reaction medium was heated to 40 ꢂC and stirred
for 2h at that temperature, then filtered over Whatman
GF/A. The filtrate was evaporated under reduced pres-
sure, the residue retaken in 3 mL MeOH and filtered
over Celiteꢁ-545, furnishing 46 mg (86%) triester 4b as
4.2.10. 15,24,28-Tri-O-(Na-BOC-O-Bn-seryl)-squamocin
6p. To a solution of 95 mg (0.153 mmol) squamocin 1 in
1 mL EtOAc were added 226 mg (0.765 mmol) Na-BOC-
O-Bn-serine and catalytic 4-DMAP. The mixture was
cooled to 0 ꢂC and a solution of 158 mg (0.767 mmol)
DCC in 1 mL EtOAc was added dropwise. The reaction
media was brought back to room temperature and stir-
red for 6 h, then evaporated under reduced pressure.
The residue was retaken in cyclohexane (3 · 3 mL) and
filtered over Whatman GF/A. The filtrate was evapo-
rated in vacuo and the crude product chromatographed
over a column of silica gel (EtOAc/cyclohexane 35:65
v/v), furnishing 180 mg (81%) triester 6p as a colorless
resin. 1H NMR (CDCl3, 400 MHz) d 0.85 (t, 3H,
H-34, J = 6.8 Hz), 1.22 (m, 2H, H-26), 1.40 (d, 3H,
1
a white resin. H NMR (CDCl3, 400 MHz) d 0.87 (t,
3H, H-34, J = 6.8 Hz), 1.35 (d, 3H, H-37, J = 6.8 Hz),
1.43 (m, 6H, Hd), 1.68 (m, 4H, H-18, H-21), 1.71 (m,
6H, Hb), 1.92(m, 10H, H , H-17, H-22), 2.23 (t, 2H,
c
H-3, J = 7.1 Hz), 2.68 (m, 6H, He), 3.81 (m, 2H, H-19,
H-20), 3.94 (m, 2H, H-16, H-23), 4.20 (m, 3H, Ha),
4.84 (m, 2H, H-24, H-28), 4.88 (m, 1H, H-15), 4.94
(dq, 1H, H-36, J = 1.5 Hz, J = 6.8 Hz), 5.19 (m, 3H,
NHBOC), 6.95 (d, 1H, H-35, J = 1.5 Hz). ESIMS m/z
1309 [M+H]+. [a]D ꢀ8 (c 1, EtOAc).
t
H-37, J = 6.8 Hz), 1.43 (s, 27H, Bu–O), 1.52(m, 2H,

R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
3779
H-25), 1.54 (m, 6H, H-4, H-27, H-29), 1.58 (m, 2H,
H-14), 1.63 (m, 4H, H-18, H-21), 1.88 (m, 4H, H-17,
H-22), 2.25 (t, 2H, H-3, J = 7.1 Hz), 3.68 (m, 3H, Hb),
3.77 (m, 2H, H-19, H-20), 3.85 (m, 4H, Hb, H-16/H-
23), 3.97 (m, 1H, H-16/H-23), 4.42 (m, 3H, Ha), 4.52
(s, 6H, HBn), 4.90 (m, 3H, H-15, H-24, H-28), 4.98
(dq, 1H, H-36, J = 1.5 Hz, J = 6.8 Hz), 5.41 (m, 3H,
NHBOC), 6.98 (d, 1H, H-35, J = 1.5 Hz), 7.25–7.34 (m,
15H, H-arom.). ESIMS m/z 1478 [M+Na]+, 750
[M+2Na]2+. [a]D +27 (c 1, CHCl3).
4.2.13. 15,24,28-Tri-O-(Na-BOC-tyrosyl)-squamocin 7.
To a solution of 66 mg (39.2 lmol) triester 7p in
0.6 mL absolute EtOH were added 198 mg (3 mass
equiv) Pd–C (10%). The mixture was Ar purged; then
200 lL (2.14 mmol) 1,4-cyclohexadiene was added drop-
wise. The reaction medium was heated to 65 ꢂC and stir-
red for 8 h at that temperature, then filtered over
Whatman GF/A. The filtrate was evaporated under re-
duced pressure and the residue chromatographed over
a small column of silica gel (EtOAc/cyclohexane 55:45
v/v), furnishing 33 mg (60%) triester 7 as a white resin.
1H NMR (CDCl3, 200 MHz) d 0.84 (t, 3H, H-34,
4.2.11. 15,24,28-Tri-O-(Na-BOC-seryl)-squamocin 6. To
a solution of 87 mg (59.8 lmol) triester 6p in 1 mL abso-
lute EtOH were added 261 mg (3 mass equiv) Pd–C
(10%). The mixture was Ar purged; then 175 lL
(1.86 mmol) 1,4-cyclohexadiene was added dropwise.
The reaction medium was heated to 45 ꢂC and stirred
for 5 h at that temperature, then filtered over Whatman
GF/A. The filtrate was evaporated under reduced pres-
sure and the residue chromatographed over a small col-
umn of silica gel (EtOAc/cyclohexane 70:30 v/v),
t
J = 6.8 Hz), 1.41 (s, 27H, Bu–O), 1.41 (d, 3H, H-37,
J = 6.8 Hz), 1.54 (m, 4H, H-4, H-14), 1.69 (m, 4H, H-
18, H-21), 1.90 (m, 4H, H-17, H-22), 2.22 (t, 2H, H-3,
J = 7 Hz), 2.98 (m, 6H, Hb), 3.83 (m, 3H, H-19, H-20,
H-16/H-23), 3.97 (m, 1H, H-16/H-23), 4.47 (m, 3H,
Ha), 4.71 (m, 1H, H-24/H-28), 4.83 (m, 1H, H-24/H-
28), 4.95 (m, 3H, NHBOC), 4.96 (dq, 1H, H-36,
J = 1.3 Hz, J = 6.8 Hz), 5.14 (m, 1H, H-15), 6.72(m,
6H, Hb), 6.97 (m, 7H, Ha, H-35). ESIMS m/z 1430
[M+NH4]+. HRESIMS m/z 1411.8289 (calcd for
C79H117N3O19: 1411.8281). [a]D +6 (c 1, EtOAc).
1
furnishing 37 mg (52%) triester 6 as a white resin. H
NMR (CD2Cl2, 400 MHz) d 0.86 (t, 3H, H-34,
J = 6.8 Hz), 1.27 (m, 2H, H-26), 1.37 (d, 3H, H-37,
J = 6.8 Hz), 1.44 (s, 27H, Bu–O), 1.51 (m, 2H, H-25),
4.2.14. 15,24,28-Tri-O-(Na-BOC-O-Bn glutamyl)-squamo-
cin 8p. To a solution of 112mg (0.180 mmol) squamocin
1 in 2mL EtOAc were added 303 mg (0.9 mmol) Na-
BOC-O-Bn-glutamate and catalytic 4-DMAP. The mix-
ture was cooled to 0 ꢂC and a solution of 185 mg
(0.90 mmol) DCC in 1 mL EtOAc was added dropwise.
The reaction media was brought back to room temper-
ature and stirred for 24 h, then evaporated under re-
duced pressure. The residue was retaken in a toluene
(3 · 5 mL) and filtered over Whatman GF/A. The fil-
trate was evaporated in vacuo and the crude product
chromatographed over a column of silica gel (EtOAc/
cyclohexane 35:65 v/v), furnishing 193 mg (68%) triester
t
1.49 (m, 2H, H-14), 1.54 (m, 6H, H-4, H-27, H-29),
1.54 (m, 4H, H-18, H-21), 1.78 (m, 4H, H-17, H-22),
2.22 (t, 2H, H-3, J = 7.3 Hz), 3.68 (m, 2H, H-19, H-
20), 3.75 (m, 3H, Hb), 3.93 (m, 3H, Hb), 3.96 (m, 1H,
H-23), 3.99 (m, 1H, H-16), 4.12 (m, 1H, Ha), 4.31 (m,
2H, Ha), 4.86 (m, 1H, H-24), 4.94 (m, 1H, H-28), 4.97
(dq, 1H, H-36, J = 1.5 Hz, J = 6.8 Hz), 5.09 (m, 1H,
H-15), 5.47–5.69 (m, 3H, NHBOC), 6.99 (d, 1H, H-35,
J = 1.5 Hz). ESIMS m/z 1202 [M+NH4]+. [a]D ꢀ6 (c
0.5, EtOAc).
1
4.2.12. 15,24,28-Tri-O-(Na-BOC-O-Bn-tyrosyl)-squamo-
cin 7p. To a solution of 101 mg (0.162mmol) squamocin
1 in 1.5 mL EtOAc were added 301 mg (0.812mmol)
Na-BOC-O-Bn-tyrosine and catalytic 4-DMAP. The
mixture was cooled to 0 ꢂC and a solution of 167 mg
(0.812mmol) DCC in 1 mL EtOAc was added dropwise.
The reaction media was brought back to room temper-
ature and stirred for 15 h, then evaporated under re-
duced pressure. The residue was retaken in a toluene/
cyclohexane 20:80 v/v (3 · 5 mL) and filtered over
Whatman GF/A. The filtrate was evaporated in vacuo
and the crude product chromatographed over a column
of silica gel (EtOAc/cyclohexane 30:70 v/v), furnishing
8p as a colorless resin. H NMR (CDCl3, 400 MHz) d
0.86 (t, 3H, H-34, J = 6.8 Hz), 1.27 (m, 2H, H-26),
t
1.40 (d, 3H, H-37, J = 6.8 Hz), 1.43 (s, 27H, Bu–O),
1.53 (m, 8H, H-4, H-14, H-27, H-29), 1.68 (m, 4H, H-
18, H-21), 1.91 (m, 4H, H-17, H-22), 1.93 (m, 3H,
Hb), 2.20 (m, 3H, Hb), 2.25 (t, 2H, H-3, J = 7.3 Hz),
2.46 (m, 6H, Hc), 3.79 (m, 2H, H-19, H-20), 3.93 (m,
2H, H-16, H-23), 4.32 (m, 3H, Ha), 4.86 (m, 2H, H-
24, H-28), 4.97 (m, 1H, H-15), 4.99 (dq, 1H, H-36,
J = 1.5 Hz, J = 6.8 Hz), 5.11 (s, 6H, HBn), 5.15 (m, 3H,
NHBOC), 6.97 (d, 1H, H-35, J = 1.5 Hz), 7.30–7.35 (m,
15H, H-arom.). ESIMS m/z 1604 [M+Na]+, 813
[M+2Na]2+. [a]D +5 (c 1, CHCl3).
1
208 mg (76%) triester 7p as a colorless resin. H NMR
(CDCl3, 400 MHz) d 0.87 (t, 3H, H-34, J = 6.8 Hz),
1.26 (m, 2H, H-26), 1.39 (s, 27H, Bu–O), 1.42(d, 3H,
4.2.15. 15,24,28-Tri-O-(Na-BOC-glutamyl)-squamocin 8.
To a solution of 90 mg (56.9 lmol) triester 8p in 1 mL
absolute EtOH were added 270 mg (3 mass equiv) Pd–
C (10%). The mixture was Ar purged; then 300 lL
(3.19 mmol) 1,4-cyclohexadiene was added dropwise.
The reaction medium was heated to 40 ꢂC and stirred
for 3 h at that temperature, then filtered over Whatman
GF/A. The filtrate was evaporated under reduced pres-
sure and the residue chromatographed over a column
of alumina (toluene/MeOH/AcOH 90:7:3 v/v/v), furnish-
t
H-37, J = 6.8 Hz), 1.50 (m, 4H, H-27, H-29), 1.54 (m,
6H, H-4, H-14, H-25), 1.72 (m, 4H, H-18, H-21), 1.89
(m, 4H, H-17, H-22), 2.25 (t, 2H, H-3, J = 7.1 Hz),
2.92 (m, 3H, Hb), 3.09 (m, 3H, Hb), 3.84 (m, 2H, H-
19, H-20), 3.99 (m, 2H, H-16, H-23), 4.49 (m, 3H,
Ha), 4.85 (m, 2H, H-24, H-28), 4.96 (dq, 1H, H-36),
4.97 (m, 3H, NHBOC), 5.01 (m, 1H, H-15), 5.02(s, 6H,
HBn), 6.88 (m, 6H, Hb), 6.96 (d, 1H, H-35), 7.08 (m,
6H, Ha), 7.25–7.43 (m, 15H, H-arom.). ESIMS m/z
1706 [M+Na]+. [a]D +22 (c 1, CHCl3).
1
ing 24 mg (32%) triester 8 as a colorless resin. H NMR
(CDCl3, 200 MHz) d 0.84 (t, 3H, H-34, J = 6,8 Hz), 1.35

3780
R. A. Duval et al. / Bioorg. Med. Chem. 13 (2005) 3773–3781
(d, 3H, H-37, J = 6.8 Hz), 1.92(m, H , 14H, H-17, H-
b
4.3.2. Cytotoxicities. Cytotoxicities were colorimetrically
evaluated through a 96 wells plate assay after 72h cell
exposure to the acetogenins. KB 3-1 cells were cultivated
in DullbeccoÕs MEM ÔglutamaxÕ (1 g/L glucose) enriched
with 10% FCS, in a moist atmosphere containing 5%
CO2. Each well of the test plate was inseminated at t0
with 100 lL of a 1.2 · 105 cell/mL cellular suspension.
Homogeneous dilutions were obtained from a freshly
prepared 10ꢀ3 M solution of the acetogenin in DMSO,
a 100 lL aliquote of this solution being diluted in
5.90 mL of the culture media. This obtained
2 · 10ꢀ5 M solution was further and extensively diluted
with 2% DMSO-precontaining culture media. After 24 h
cell growth, 100 lL of the dilutions were administrated
in triplicate into the wells. Final homogenization was en-
sured by a 3 min treatment on a microtitration plate.
After 72h contact, the test plates were emptied and di-
rectly treated with 200 lL of a 2.5 g/L methylene blue
solution in MeOH/water 50:50 v/v after the method of
Fleury et al.32 After 40 min contact, the plates were
emptied and the wells quickly rinsed under a very mild
flow of cold tap water (three times). The plates were
air-dried (50 ꢂC) and each well treated with 100 lL of
0.1 N HCl. Absorbances were recorded at 620 nm after
30 min incubation at 37 ꢂC and homogenization on a
microtitration plate, under a blank of 100 lL of 0.1 N
HCl. IC50 were expressed as the extrapolated concentra-
tions allowing 50% cell survival in comparison with
untreated controls.
18, H-21, H-22), 2.25 (t, 2H, H-3, J = 7 Hz), 2.42 (m,
6H, Hc), 3.83 (m, 2H, H-19, H-20), 4.02 (m, 2H, H-16,
H-23), 4.32 (m, 3H, Ha), 4.76 (m, 1H, H-24/H-28),
4.87 (m, 1H, H-24/H-28), 4.93 (m, 1H, H-15), 5.0 (dq,
1H, H-36, J = 1.3 Hz, J = 6.8 Hz), 5.33 (m, 3H,
NHBOC), 6.98 (d, 1H, H-35, J = 1.3 Hz). ESIMS m/z
1333 [M+Na]+.
4.2.16. 15,24,28-Tri-O-(Na-BOC Nim-Ts-histidyl)-squamo-
cin 9p. To a solution of 134 mg (0.215 mmol) squamocin
1 in 2mL EtOAc were added 441 mg (0.9 mmol) Na-
BOC-Nim-Ts-histidine and catalytic 4-DMAP. The mix-
ture was cooled to 0 ꢂC and a solution of 222 mg
(1.077 mmol) DCC in 1 mL EtOAc was added dropwise.
The reaction media was brought back to room tempera-
ture and stirred for 30 h, then evaporated under reduced
pressure. The residue was retaken in THF (3 · 7 mL) and
filtered over Whatman GF/A. The filtrate was evapo-
rated in vacuo and the crude product chromatographed
over a column of silica gel (EtOAc/cyclohexane 60:40
v/v), furnishing 143 mg (37%) triester 9p as a white resin.
1H NMR (CDCl3, 400 MHz) d 0.85 (t, 3H, H-34,
t
J = 6.9 Hz), 1.22 (m, 2H, H-26), 1.38 (s, 27H, Bu–O),
1.40 (d, 3H, H-37, J = 6.8 Hz), 1.49 (m, 2H, H-14),
1.51 (m, 4H, H-27, H-29), 1.53 (m, 4H, H-4, H-25),
1.67 (m, 4H, H-18, H-21), 1.89 (m, 4H, H-17, H-22),
2.25 (t, 2H, H-3, J = 7.4 Hz), 2.42 (s, 9H, CH3Ts), 2.98
(m, 6H, Hb), 3.83 (m, 2H, H-19, H-20), 3.93 (m, 2H,
H-16, H-23), 4.51 (m, 3H, Ha), 4.81 (m, 2H, H-24, H-
28), 4.84 (m, 1H, H-15), 4.97 (dq, 1H, H-36,
J = 1.6 Hz, J = 6.8 Hz), 5.64 (m, 3H, NHBOC), 6.97 (d,
1H, H-35, J = 1.6 Hz), 7.09 (s, 1H, H-40), 7.10 (s, 1H,
H-40), 7.15 (s, 1H, H-40), 7.33–7.79 (m, 12H, HTs), 7.88
(s, 1H, H-20), 7.89 (s, 2H, H-20). ESIMS m/z 921
[M+2Na]2+. [a]D +10 (c 1, CHCl3).
Acknowledgements
The authors wish to thank Prof. D. Cortes for the mea-
surement of the complex I inhibitory activities.
4.2.17. 15,24,28-Tri-O-(Na-BOC-histidyl)-squamocin 9.
To a solution of 180 mg (0.10 mmol) triester 9p in
1.5 mL anhydrous THF were added 162mg (1.2mmol)
HOBT, and the mixture was stirred at room tempera-
ture for 5 h. The reaction medium was filtered over
Whatman GF/A, and the filtrate evaporated under re-
duced pressure. The residue was filtered over alumina
(EtOAc/MeOH 90:10 v/v) and the crude product chro-
matographed in the same conditions, furnishing 56 mg
(42%) 9 as a colorless resin. 1H NMR (CDCl3,
References and notes
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´
`
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1065–1073.
t
200 MHz) d 0.84 (t, 3H, H-34), 1.35 (s, 27H, Bu–O),
1.39 (d, 3H, H-37, J = 6.8 Hz), 1.91 (m, 4H, H-17, H-
22), 2.22 (t, 2H, H-3, J = 7 Hz), 3.15 (m, 6H, Hb), 3.83
(m, 2H, H-19, H-20), 3.96 (m, 2H, H-16, H-23), 4.43
(m, 3H, Ha), 4.79 (m, 3H, H-15, H-24, H-28), 4.96
(dq, 1H, H-36, J = 1.3 Hz, J = 6.8 Hz), 5.50 (m, 3H,
NHBOC), 6.89 (s, 2H, H-40), 6.96 (d, 1H, H-35,
J = 1.3 Hz), 7.05 (s, 1H, H-40), 8.27 (s, 3H, H-20).
ESIMS 1335 [M+H]+. HRESIMS m/z 1333.8143 (calcd
for C70H111N9O16: 1333.8149).
4.3. Bioassays
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