Synthetic studies on stevastelins. 2. Synthesis of lipidic- and peptidic-modified analogues

Article abstract of DOI:10.1021/jo050628y

The synthesis of a series of stevastelin analogues with modification of the susbstituent at the C-2 position of the stearic acid chain (compounds 28 and 31), variation of the amino acids (compounds 41, 42, 73, and 78), or lacking the lipidic chain (compou

Full text of DOI:10.1021/jo050628y

Synthetic Studies on Stevastelins. 2.
Synthesis of Lipidic- and Peptidic-Modified Analogues
Francisco Sarabia,* Samy Chammaa, and Miguel Garc´ıa-Castro
Department of Biochemistry, Molecular Biology and Organic Chemistry, Faculty of Sciences,
University of Malaga, Campus de Teatinos s/n, 29071, Malaga, Spain
frsarabia@uma.es
Received March 31, 2005
The synthesis of a series of stevastelin analogues with modification of the susbstituent at the C-2
position of the stearic acid chain (compounds 28 and 31), variation of the amino acids (compounds
41, 42, 73, and 78), or lacking the lipidic chain (compound 91) is described. The replacement of
L-valine and L-threonine with other amino acids proceeded without difficulties for the synthesis of
analogues 41 and 42; however, the substitution of L-serine with simple amino acids, such as glycine
or L-alanine, proved to be elusive, which was adscribed to factors of conformational flexibility.
Finally, the substitution with L-valine or L-threonine proceeded without difficulties to provide the
analogues 73 and 78 respectively.
Introduction
search for new stevastelin-type immunosuppressive
agents, we decided to undertake the preparation of a
series of analogues. Taking into consideration previous
studies on the biological properties of the truncated
stevastelins,6 which lack the lipidic chain, and the data
revealing that this structural component was essential
for the immunosuppressive activity, we deemed it of
importance to ascertain the biological value of the sub-
stituents R1-R4 along the lipidic and peptidic fragments.
To explore these variations, a primary set of analogues
capable of elucidating the biological relevance of the 2-C-
alkyl substituent and the amino acids was designed
(Scheme 1). Accordingly, the synthetic strategy utilized
by Chida’s group4b proved to be the most appropriate
route for the preparation of the stevastelin analogues
through a convergent and flexible synthesis that permits
the facile modification of key structural elements by the
incorporation of the appropriate building blocks along a
common synthetic sequence, utilizing the epoxyalcohol
The preceding article in this issue,1 detailed synthetic
studies on the stevastelins, a new class of [13]- and [15]-
macrocyclic depsipeptides with intriguing immuno-
suppressive properties,2 that culminated with the total
synthesis of stevastelin B3,4 following the synthetic
strategy delineated by Chida’s group,4b which proved to
be the most effective, and stevastelin B35 via a trans-
lactonization reaction from the [15]-membered ring de-
rivative. In recapping our research studies involving the
* Corresponding author. Fax: 34-952 131941.
(1) See the preceding article on this issue: Sarabia, F.; Chammaa,
S. J. Org. Chem. 2005, 70, 7846-7857.
(2) For isolation of stevastelins, see: (a) Morino, T.; Masuda, A.;
Yamada, M.; Nishimoto, M.; Nishikiori, T.; Saito, S.; Shimada, N. J.
Antibiot. 1994, 47, 1341-1343. For structural determination, see: (b)
Morino, T.; Shimada, K.-I.; Masuda, A.; Nishimoto, M.; Saito, S. J.
Antibiot. 1996, 49, 1049-1051. For biological properties, see: (c)
Morino, T.; Shimada, K.-I.; Masuda, A.; Noriyuki, Y.; Nishimoto, M.;
Nishikiori, T.; Saito, S. J. Antibiot. 1996, 49, 564-568. (d) Shimada,
K.-I.; Morino, T.; Masuda, A.; Sato, M.; Kitagawa, M.; Saito, S. J.
Antibiot. 1996, 49, 569-574.
(5) For synthetic approaches of [13]-membered stevastelins, see: (a)
Sarabia, F.; Chammaa, S.; Lo´pez-Herrera, F. J. Tetrahedron Lett. 2002,
43, 2961-2965. For total synthesis of stevastelin B3, see: (b) Kuro-
sawa, K.; Matsuura, K.; Chida, N. Tetrahedron Lett. 2005, 46, 389-
392. (c) Chakraborty, T. K.; Ghosh, S.; Laxman, P.; Dutta, S.; Samanta,
R. Tetrahedron Lett. 2005, 46, 5447-5450.
(3) For synthetic approaches of [15]-membered stevastelins, see: (a)
Chakraborty, T. K.; Ghosh, S.; Dutta, S. Tetrahedron Lett. 2001, 42,
5085-5088. (b) Sarabia, F.; Chammaa, S.; Sa´nchez-Ruiz, A.; Lo´pez-
Herrera, F. J. Tetrahedron Lett. 2003, 44, 7671-7675.
(4) For total synthesis of stevastelin B, see: (a) Kohyama, N.;
Yamamoto, Y. Synlett 2001, 694-696. (b) Kurosawa, K.; Nagase, T.;
Chida, N. Chem. Commun. 2002, 1280-1281.
(6) Hamaguchi, T.; Masuda, A.; Morino, T.; Osada, H. Chem. Biol.
1997, 4, 279-286.
10.1021/jo050628y CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/30/2005
7858
J. Org. Chem. 2005, 70, 7858-7865

Synthesis of Modified Stevastelin Analogues
SCHEME 1. Structures of Stevastelins A (1) and B
(2) and Retrosynthetic Program for Stevastelin B
Analogues
SCHEME 2. Synthesis of the Acyclic Precursors
17 and 19 of Stevastelin Analogues 28 and 31a
a Reagents and Conditions: (a) i. 2.2 equiv of red-Al, THF, 0 f
25 °C, 18 h, 80% for 5; ii. 2.0 equiv of CuI, 6.0 equiv of 1.0 M
EtMgBr, -20 °C, 3 h, 97% for 6. (b) 3.0 equiv of Me2C(OMe)2, 0.05
equiv of CSA, DMF, 0 °C, 2 h, 57% for 7 plus 24% for the 3,5-
acetal derivative, 93% for 8. (c) 3.0 equiv of TBAF, THF, 25 °C, 4
days, 78% from 4. (d) 6.4 equiv of 10, 9.0 equiv of 2,4,6-
Cl3C6H2COCl, 9.0 equiv of Et3N, THF/toluene, 0 °C, 1.5 h, then
0.5 equiv of 4-DMAP, 0 °C, 2.5 h, 86% (6:1 epimeric mixture) for
11, 70% (6:1 epimeric mixture) for 13. (e) 0.1 equiv of 10% Pd/C-
ethylenediamine complex, H2, MeOH, 25 °C, 0.5 h. (f) 1.5 equiv of
15, 1.5 equiv of HOBt, 2.6 equiv of EDCI, DMF, 25 °C, 0.5 h, 80%
for 16 from 11, 80% for 18 from 13. (g) AcOH/H2O, THF, 25 °C,
18 h, 83% for 17, 81% for 19.
4 as the common intermediate for delivery of analogues,
as mentioned in the previous article.
Results and Discussion
Synthesis of 2-C-Alkyl Stevastelin Analogues. The
synthesis of the 2-C-alkyl-modified analogues commenced
with the treatment of epoxyalcohol 41 with various
nucleophiles, including red-Al7 and ethylmagnesium
bromide/CuI,8 to yield the oxirane ring-opening products
5 and 6, respectively, noting that for 5 the oxirane ring
opening was accompanied with TBS deprotection.9 Pro-
tection of the 1,3-diol as the acetal, followed by TBS
deprotection for 8, provided the corresponding alcohols
7 and 9 in good yields. The couplings of these alcohols
with the L-serine derivative 10 was accomplished by use
of the Yamaguchi protocol,10 to furnish the esters 11 and
13 in a 75% average yield but with 5-8% epimerization
at C-2 of the serine residue, as was observed with the
esterification of the corresponding esteric fragment con-
tained in the natural compound. The introduction of the
rest of the peptidic chain was undertaken under similar
conditions as described in the previous article, by cou-
pling of amines 12 and 14 with dipeptide 15, to obtain
acyclic depsipeptides 16 and 18. The macrocyclization
process was initiated with the acetal cleavage of 16 and
18 (Scheme 2) and selective oxidation of the primary
hydroxyl group by the sequential action of TEMPO/
NaClO11 and NaClO2 of the resulting diols 17 and 19 to
the acids 21 and 24 through aldehydes 20 and 23,
respectively (Scheme 3).
With the acyclic stevastelin precursors 21 and 24 in
hand, we proceeded with the macrocyclization reactions12
by treatment of the resulting seco amino acids 22 and
25, from the Boc deprotection of 21 and 24, with diethyl
cyanophosphonate (DECP)13 as previously described for
natural stevastelin B, to furnish the macrocyclic
depsipeptides 26 and 29 in 23% and 33% yields from diols
17 and 19, respectively. The preparation of the final 2-C-
alkyl analogues was accomplished in two steps, including
(11) Hansen, T. M.; Florence, G. J.; Lugo-Mas, P.; Chen, J.; Abrams,
J. N.; Forsyth, C. J. Tetrahedron Lett. 2003, 44, 57-59.
(12) (a) Hale, K. J.; Bhatia, G. S.; Frigerio, M. In The Chemical
Synthesis of Natural Products; Hale, K. J., Ed.; Sheffield Academic
Press: Sheffield, 2000; pp 349-415. (b) Sarabia, F.; Chammaa, S.;
Sa´nchez-Ruiz, A.; Mart´ın-Ortiz, L.; Lo´pez-Herrera, F. J. Curr. Med.
Chem. 2004, 11, 1309-1332.
(13) (a) Yamada, S.; Kasai, Y.; Shioiri, T. Tetrahedron Lett. 1973,
1595-1598. (b) Takuma, S.; Hamada, Y.; Shioiri, T. Chem. Pharm.
Bull. 1982, 30, 3147-3153.
(7) Ma, P.; Martin, V. S.; Masamune, S.; Sharpless, K. B.; Viti, S.
M. J. Org. Chem. 1982, 47, 1378-1380.
(8) Nicolaou, K. C.; Oshima, T.; Murphy, F.; Barluenga, S.; Xu, J.;
Winssinger, N. Chem. Commun. 1999, 809-810.
(9) Rajashekhar, B.; Kaiser, E. T. J. Org. Chem. 1986, 51, 1625-
1627.
(10) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M.
Bull. Chem. Soc. Jpn. 1979, 52, 1989-1993.
J. Org. Chem, Vol. 70, No. 20, 2005 7859

Sarabia et al.
SCHEME 3. Total Synthesis of 2-C-Alkyl
Stevastelin Analogues 28 and 31a
SCHEME 4. Synthesis of the Peptidic-Modified
Stevastelins 41 and 42a
a Reagents and Conditions: (a) 0.01 equiv of TEMPO, 0.1 equiv
of KBr, 15.0 equiv of NaHCO3, 2.4 equiv of NaClO, CH2Cl2/H2O,
0 °C, 1 h. (b) 5.0 equiv of NaClO2, 4.0 equiv of NaH2PO4, 87.0 equiv
of 2-methyl-2-butene, t-BuOH/H2O, 25 °C, 10 min. (c) TFA, CH2Cl2,
0 °C, 1 h. (d) 4.9 equiv of DEPC, 5.4 equiv of Et3N, DMF (1.0 mM
based on triols 17 and 19), 0 f 25 °C, 23% for 26 from 17, 33% for
29 from 19. (e) H2, 10% Pd/C, MeOH, 25 °C, 2 h. (f) 4.3 equiv of
Ac2O, pyridine, 0 °C, 2 h, 17% for 28 from 17, 33% for 31 from 19.
debenzylation and selective monoacetylation to yield the
2-demethyl and 2-ethyl stevastelin analogues 28 and 31
(Scheme 3) in 1.0% and 3.0% overall yields in 21 and 22
steps, respectively, from Evans’s oxazolidinone (see Scheme
1).14
a Reagents and Conditions: (a) 0.55 equiv of Boc-Leu-Thr-OH
or 0.55 equiv of Boc-Val-Val-OH, 1.07 equiv of HOBt, 1.6 equiv of
EDCI, DMF, 25 °C, 0.5 h, 99% for 33 and 83% for 35. (b) AcOH/
H2O, THF, 25 °C, 18 h. (c) 0.02 equiv of TEMPO, 0.1 equiv of KBr,
15.0 equiv of NaHCO3, 3.0 equiv of NaClO, CH2Cl2/H2O, 0 °C, 1.5
h. (d) 2.0 equiv of NaClO2, 2.8 equiv of NaH2PO4, 100.0 equiv of
2-methyl-2-butene, t-BuOH/H2O, 25 °C, 15 min. (e) 5.0 equiv of
TFA, CH2Cl2, 0 °C, 2 h. (f) 5.0 equiv of DEPC, 5.5 equiv of Et3N,
DMF (1.0 mM based on 33 and 35 respectively), 0 f 25 °C, 48 h.
(g) i. H2, 10% Pd/C, MeOH, 25 °C, 2 h for 41; ii. H2, 10% Pd(OH)2,
MeOH, 25 °C, 4 h for 42. (h) 4.3 equiv of Ac2O, pyridine, 0 °C,
2-3 h, 9% for 41 from 33, 18% for 42 from 35.
Synthesis of Peptidic-Modified Stevastelins. The
same route was projected for the preparation of the
peptidic-modified stevastelins, replacing the alkyl side
chains present in the natural stevastelins by groups that
may elucidate the biological role of the different R2, R3,
and R4 groups. In particular, the exchange of the iso-
propyl, hydroxyl, and hydroxymethyl groups correspond-
ing to the valine, threonine, and serine residues, respec-
tively, by isobutyl, methyl, and hydrogen groups was
considered as suitable changes to identify the effect of
these amino acid substituents on the biological activities.
In this context, the synthesis of the valine- and threonine-
modified analogues commenced from the advanced in-
termediate 32,1 which was coupled with the correspond-
ing dipeptides Boc-Leu-Thr(OH)-OH15 and Boc-Val-Val-
OH,16 by the action of EDCI/HOBt,17 to afford compounds
33 and 35 in 99% and 83% yields, respectively. Proceed-
ing in a similar manner as before for compounds 22 or
25, the acyclic acids 38 and 40 were efficiently synthe-
sized through diols 34 and 36 and aldehydes 37 and 39.
From these acids, the synthesis of the peptidic analogues
41 and 42 was achieved by implementation of the same
synthetic sequence as that described for stevasletin B (5),
with no significant difficulties along the synthetic route,
to give the stevastelin analogues 41 and 42 in 9% and
18% overall yields from 33 and 35, respectively (Scheme
4).
The replacement of the L-serine residue with another
amino acid was our next objective in this investigation
(15) Dipeptide Boc-Leu-Thr(OH)-OH was prepared from commer-
cially available Boc-Thr(OH)-OH and Boc-Leu-OH through the allyl
ester of L-threonine (H-Thr(OH)-Oallyl), which was coupled with Boc-
Leu-OH by treatment with EDCI/HOBt in a 72% overall yield from
Boc-Thr(OH)-OH. Finally, the dipeptide derivative Boc-Leu-Thr(OH)-
OAllyl was transformed into the acid by the action of Pd[PPh3]4/NMO.
(16) Uccello-Barretta, G.; Luliano, A.; Menicagli, R.; Peluso, P.;
Pieroni, E.; Salvadori, P. Chirality 1997, 9, 113-121.
(14) The overall yield of the total synthesis for all new stevastelin
analogues is referred to the starting oxazolidinone described in the
preceding article, according to Evans methodology: (a) Evans, D. A.
Aldrichimica Acta 1982, 15, 318-327. (b) Evans, D. A.; Bartroli, J.;
Shih, T. L. J. Am. Chem. Soc. 1981, 103, 2127-2129. (c) Evans, D. A.;
Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc. 1982, 104, 1737-1739.
(d) Evans, D. A.; Mathre, D. J.; Scott, W. L. J. Org. Chem. 1985, 50,
1830-1835. (e) Evans, D. A.; Scott, J. M.; Ennis, M. D. J. Org. Chem.
1993, 58, 471-485.
(17) Gao, X.-M.; Ye, Y.-H.; Bernd, M.; Kutscher, B. J. Pept. Sci. 2002,
8, 418-430.
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Synthesis of Modified Stevastelin Analogues
SCHEME 5. Toward Serine-Modified Stevastelin
Analoguesa
to complete the first set of representative stevastelin
analogues for structure-activity relationship (SAR) stud-
ies. As indicated before, we deemed of interest the
removal or the replacement of the acetyloxymethylene
group of L-serine by other groups, leading us to consider
the amino acids glycine or L-alanine as the most ap-
propriate candidates. Thus, coupling of alcohol 43 with
amino acid derivatives 44 and 45 was undertaken ac-
cording to the Keck procedure18 to afford the correspond-
ing esters 46 and 48 in 85% and 70% yields, respectively.
The cleavage of the N-Cbz group of these products by
hydrogen in the presence of Pd/C-ethylendiamine com-
plex catalyst19 was followed by the linkage to the dipep-
tide 15, delivering the tripeptide derivatives 50 and 52,
respectively. The preparation of these compounds for the
macrocyclization process was conducted using the same
synthetic sequence as that for the previously described
analogues, with no difficulties encountered during the
acetal hydrolysis, to provide triols 51 and 53, followed
by selective oxidation and Boc cleavage, yielding the
acyclic depsipeptide precursors 54 and 55. The macro-
cyclization reactions were performed under similar condi-
tions as previously described, but unfortunately, these
macrolactamization reactions did not proceed as desired,
resulting in a complex mixture of products, without the
formation of the cyclized products 56 and 57. Similarly,
unsuccessful results were encountered when we at-
tempted all the possible macrocyclizations for the glycine-
modified stevastelin analogue, from the other three
possible acyclic precursors 58, 59, and 60 (Scheme 5).
Presumably, the large degree of conformational flexibility
that these precursors possess, as a consequence of the
replacement of the bulky benzyloxymethyl group of the
L-serine amino acid residue by the more flexible glycine
or L-alanine, could explain these disappointing results
in the macrocyclization reactions.20
In light of these discouraging although interesting
results, we opted to introduce L-valine and L-threonine
moeities instead of a L-serine residue, with the goal of
demonstrating the importance of the conformational
flexibility for the macrocyclization reaction, as well as
preparing analogues to be considered for biological evalu-
ations. Thus, esters 61 and 63, prepared by esterification
of alcohol 43 with the commercially available amino acids
derivatives Z-Val-OH and Z-Thr(Bn)-OH via the Yamagu-
chi and Keck procedures, respectively, were subjected to
chemical modifications, in a similar way as before,
through derivatives 62, 65, 66, and 69 for the L-valine-
modified analogue and 64, 67, 68, and 71 for the
L-threonine derivative, to yield the corresponding acyclic
precursors 70 and 72. To our delight, Boc deprotections
of 70 and 72 followed by macrocyclizations of their
resulting ammonium trifluoroacetate salts under the
action of DEPC, furnished the cyclic depsipeptides 73 and
74 in reasonably good yields (30% overall yield from 65
a Reagents and Conditions: (a) 2.0 equiv of 44 or 45, 2.0 equiv
of DCC, 1.0 equiv of 4-DMAP, 0.5 equiv of CSA, CH2Cl2, 25 °C,
0.5 h, 85% for 46, 70% for 48. (b) 0.1 equiv of 10% Pd/C-
ethylenediamine complex, H2, MeOH, 25 °C, 15 min. (c) 1.5 equiv
of 15, 1.7 equiv of HOBt, 2.8 equiv of EDCI, DMF, 25 °C, 0.5 h,
72% for 50 from 46, 62% for 52 from 48. (d) AcOH/H2O, 25 °C, 14
h, 91% for 51, 99% for 53. (e) Exact conditions as steps a, b, and
c in Scheme 3, 75% for 54, 83% for 55. (f) 5.0 equiv of DEPC, 5.5
equiv of Et3N, DMF, 25 °C, 20-72 h, decomposition in both cases.
for 73 and 25% overall yield from 67 for 74) as compared
to previous macrolactamizations. While the stevastelin
analogue 73 was already accomplished, the preparation
of the stevastelin analogue 78 required additional steps
for completion, which were carried out without difficulty
through a sequence of selective protection-deprotection
reactions, including silylation of 74, debenzylation of the
resultant silyl ether 75, acetylation, and final desilylation
of the acetate 77, completing the synthesis in 24 steps
for the longest linear sequence and 2.0% overall yield
from the Evans’s oxazolidinone (see Schemes 1 and 6).
Synthesis of Truncated Stevastelins. Finally, we
decided to examine the influence of the lipidic chain in
the biological properties of stevastelins. Despite the fact
that truncated stevastelins proved to be inactive, dem-
onstrating that the lipidic chain seemed to be essential
for their biological activities,6 it was not demonstrated,
however, if the reason for this lack of activity was caused
exclusively by the absence of the lipidic chain or by the
combination of the lack of either the lipidic chain as the
(18) (a) Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394-
2395. (b) Kiho, T.; Nakayama, M.; Kogen, H. Tetrahedron 2003, 59,
1685-1697.
(19) Sajiki, H.; Hattori, K.; Hirota, K. J. Org. Chem. 1998, 63, 7990-
7992.
(20) Glycine residues contained in linear peptidic chains are known
to enhance cyclization processes, see: (a) Kessler, H.; Hass, B. Int. J.
Pept. Protein Res. 1993, 39, 36. Despite this, we found in the literature
examples of glycine-containing peptides which did not cyclize via a
macrolactamization reaction. See: (b) Jeremic, T.; Linden, A.; Heim-
gartner, H. Helv. Chim. Acta 2004, 87, 3056-3079.
J. Org. Chem, Vol. 70, No. 20, 2005 7861

Sarabia et al.
SCHEME 6. Synthesis of the Peptidic-Modified
Stevastelins 73 and 78a
SCHEME 7. Synthesis of the Truncated
Stevastelin 91a
a Reagents and Conditions: (a) 0.1 equiv of 10% Pd/C-eth-
ylenediamine complex, H2, MeOH, 25 °C, 15 min, 90% for 62, 97%
for 64. (b) 1.2 equiv of 15, 1.2 equiv of HOBt, 1.8 equiv of EDCI,
DMF, 25 °C, 0.5 h, 51% from 61 for 65, 83% from 63 for 67. (c)
AcOH/H2O, THF, 25 °C, 14 h, 99% for 66 and 68. (d) 0.02 equiv of
TEMPO, 0.1 equiv of KBr, 20.0 equiv of NaHCO3, 3.0 equiv of
NaClO, CH2Cl2/H2O, 0 °C, 0.5 h, 88% for 69 and 71. (e) 2.5 equiv
of NaClO2, 2.0 equiv of NaH2PO4, 96.0 equiv of 2-methyl-2-butene,
t-BuOH/H2O, 25 °C, 15 min, 96% for 70, 99% for 72. (f) 5.0 equiv
of TFA, CH2Cl2, 0 °C, 1 h, 99%. (g) 5.0 equiv of DEPC, 5.5 equiv
of Et3N, DMF (1.0 mM based on 65 and 67), 0 f 25 °C, 20 h, 30%
for 73 from 65, 25% for 74 overall yield from 67. (h) 1.3 equiv of
TBSOTf, 2.0 equiv of 2,6-lutidine, CH2Cl2, 0 °C, 15 min. (i) H2,
10% Pd(OH)2, MeOH, 25 °C, 0.5 h. (j) 4.3 equiv of Ac2O, 4-DMAP
cat., pyridine, 0 °C, 1 h. (k) HF‚pyridine (excess), THF, 0 °C, 0.5
h, 36% overall yield from 74.
a Reagents and Conditions: (a) 1.5 equiv of 10, 1.5 equiv of
EDCI, 0.1 equiv of 4-DMAP, CH2Cl2, 0 °C, 1 h, 80%. (b) 0.1 equiv
of 10% Pd/C-ethylenediamine complex, H2, MeOH, 25 °C, 15 min,
99%. (c) 1.1 equiv of 15, 1.1 equiv of HOBt, 1.6 equiv of EDCI,
DMF, 25 °C, 0.5 h, 72%. (d) AcOH/H2O, THF, 25 °C, 18 h. (e) 0.02
equiv of TEMPO, 0.1 equiv of KBr, 15.0 equiv of NaHCO3, 3.0
equiv of NaClO, CH2Cl2/H2O, 0 °C, 1.5 h, 97%. (f) 2.0 equiv of
NaClO2, 2.8 equiv of NaH2PO4, 100.0 equiv of 2-methyl-2-butene,
t-BuOH/H2O, 25 °C, 15 min, 90%. (g) 5.0 equiv of TFA, CH2Cl2, 0
°C, 2 h. (h) 5.0 equiv of DEPC, 5.5 equiv of Et3N, DMF (1.0 mM
based on 82), 0 f 25 °C, 48 h, 30% overall yield from 82. (i) 1.3
equiv of TBSOTf, 2.0 equiv of 2,6-lutidine, CH2Cl2, 0 °C, 5 min. (j)
H2, 10% Pd(OH)2, MeOH, 25 °C, 0.5 h. (k) 10.0 equiv of Ac2O, 0.02
equiv of 4-DMAP, pyridine, 0 °C, 3 h. (k) HF‚pyridine (excess),
THF, 0 °C, 0.5 h, 56% overall yield from 88.
tetrad system contained at C-2/C-4 positions. At this
juncture, we chose analogue 91 in order to clarify such
uncertainty. The synthesis, outlined in Scheme 7, com-
menced with the advanced precursor 7921 and proceeded
in a synthetic course identical to that for previous
analogues that afforded in an efficient way the coveted
analogue 91, through compounds 80-90 without major
difficulties along its synthesis route (Scheme 7).
represent an interesting set of compounds capable of
providing essential information about the structure-
activity relationships for the stevastelin family. Chemi-
cally, we have demonstrated that the route designed by
the Chida group proved to be the most adequate to reach
the stevastelin group of cyclic depsipeptides. However,
the conformational flexibility of the acyclic precursors
seems to be essential for a successful macrocyclization
reaction, thus concluding that this conformational re-
striction represents a significant hurdle for the imple-
mentation of the synthetic strategy for the synthesis of
a library of amino acid-modified stevastelins. From the
biological point of view, preliminary inhibition studies
against different phosphatases (VHR, CD45, and PP2B)
revealed a notable decrease of activity for all these
analogues compared with stevastelin B (IC50 ) 19.8 µM
against VHR), providing interesting information about
the highly specific binding to the VHR receptor. Further
biological investigations, including inhibition studies of
T-cell proliferation, of these and other related compounds
Conclusions
In conclusion, we have described the synthesis of a
series of stevastelin analogues, in which either the 2-C-
methyl group was replaced by another alkyl group, the
amino acids contained in the structure of the natural
substance were substituted by other residues, or the
lipidic chain was removed. The resultant series of
stevastelin analogues 28, 31, 41, 42, 73, 78, and 91
(21) (a) Ziegler, F. E.; Kneisley, A.; Thottathil, J. K.; Wester, R. T.
J. Am. Chem. Soc. 1988, 110, 5434-5442. (b) Horita, K.; Oikawa, Y.;
Yonemitsu, O. Chem. Pharm. Bull. 1989, 37, 1698-1704.
7862 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis of Modified Stevastelin Analogues
tography (silica gel, 4% MeOH in CHCl3) to obtain cyclic
are in progress, which may represent high interest for
the discovery and design of new potential therapeutic
agents.22
depsipeptide 28 (7 mg, 44%) as a white solid: Rf ) 0.28 (silica
gel, 8% MeOH in CHCl3). [R]25 ) -60.6° (c ) 0.35, DMSO).
D
1H NMR (400 MHz, DMSO-d6): δ ) 0.85 (t, J ) 7.0 Hz, 3 H,
CH3CH2), 0.87-0.92 (m, 9 H, CH(CH3), CH(CH3)2), 0.99 (d, J
) 5.9 Hz, 3 H, (CH3)CH(OH)), 1.17-1.27 (m, 22 H, 11 × CH2),
1.37-1.49 (m, 1 H, CH2CHOC(dO)), 1.53-1.64 (m, 2 H, CH2-
CHOC(dO), CH(CH3)), 1.89-1.99 (m, 1 H, CH(CH3)2), 1.98 (s,
3 H, OCOCH3), 2.13 (dd, J ) 12.9, 5.9 Hz, 1 H, CH2CONH),
2.28 (dd, J ) 12.9, 7.5 Hz, 1 H, CH2CONH), 3.77 (sext, J )
5.9 Hz, 1 H, (CH3)CH(OH)), 3.83 (dd, J ) 9.1, 8.6 Hz, 1 H,
CHNH(Val)), 3.91-3.99 (m, 1 H, CH(OH)), 4.15-4.24 (m, 2
H, CHNH(Ser), CHNH(Thr)), 4.29 (dd, J ) 11.1, 7.5 Hz, 1 H,
CH2OAc), 4.34 (dd, J ) 11.1, 5.9 Hz, 1 H, CH2OAc), 4.59 (d, J
) 4.8 Hz, 1 H, CH(OH)), 4.69-4.75 (m, 1 H, CHOC(dO)), 4.85
(d, J ) 5.4 Hz, 1 H, (CH3)CH(OH)), 6.97 (d, J ) 8.6 Hz, 1 H,
NH(Ser)), 8.08 (d, J ) 8.6 Hz, 1 H, NH(Val)), 8.75 (d, J ) 7.0
Hz, 1 H, NH(Thr)).13C NMR (100 MHz, DMSO-d6): δ ) 10.5,
14.2, 19.6, 19.7, 20.0, 20.8, 22.3, 25.6, 28.9, 29.0, 29.20, 29.26,
29.29, 29.31, 29.6, 30.2, 31.5, 41.4, 42.5, 51.6, 58.1, 61.5, 61.9,
67.2, 67.5, 79.0, 168.9, 170.3, 170.4, 171.3, 171.5. FAB HRMS
(NBA): m/e 664.4144, M + Na+; calcd for C33H59N3O9 664.4149.
Cyclic Depsipeptide 29. The resulting ammonium tri-
fluoroacetate 25 (obtained from 76 mg of triol 19, ∼0.092
mmol) was subjected to the macrolactamization reaction in a
manner similar to that for the preparation of 26 to afford cyclic
depsipeptide 29 (22 mg, 33% over four steps from triol 19) as
a colorless oil: Rf ) 0.38 (silica gel, 55% EtOAc and 5% MeOH
in hexanes). 1H NMR (400 MHz, CDCl3): δ ) 0.85 (t, J ) 7.0
Hz, 3 H, CH3CH2), 0.90-0.98 (m, 9 H, CH(CH3)2, CH-
(CH2CH3)), 1.01 (d, J ) 7.0 Hz, 3 H, CH(CH3)), 1.12 (d, J )
6.4 Hz, 3 H, (CH3)CH(OH)), 1.17-1.29 (m, 22 H, 11 × CH2),
1.39-1.50 (m, 1 H, CH2CHOC(dO)), 1.66-1.76 (m, 4 H, CH2-
CHOC(dO), CH(CH2CH3), CH(CH3)), 1.98-2.10 (m, 1 H,
CH(CH3)2), 2.24-2.32 (m, 1 H, CH(CH2CH3)), 3.66 (bs, 1 H,
CH(OH)), 3.90 (dd, J ) 9.6, 5.3 Hz, 1 H, CH2OBn), 3.92-4.00
(m, 1 H, CH2OBn), 4.11-4.20 (m, 1 H, CHNH(Val)), 4.44 (dq,
J ) 6.4, 2.1 Hz, 1 H, (CH3)CH(OH)), 4.43-4.50 (m, 2 H,
CHNH(Ser), CHNH(Thr)), 4.48 (d, J ) 11.3 Hz, 1 H, CH2Ph),
4.53 (d, J ) 11.3 Hz, 1 H, CH2Ph), 4.87 (dt, J ) 7.5, 6.4 Hz, 1
H, CHOC(dO)), 6.70 (bs, 1 H, NH), 7.21-7.35 (m, 6 H, Ph,
NH), 7.61 (d, J ) 8.1 Hz, 1 H, NH). FAB HRMS (NBA): m/e
740.4827, M + Na+; calcd for C40H67N3O8 740.4826.
Experimental Section
Acyclic Depsipeptides 22 and 25. General Procedure.
To a solution of the crude dihydroxyacid (∼0.069 mmol) in
anhydrous CH2Cl2 (7.0 mL) was added TFA (1.3 mL) at 0 °C.
The reaction mixture was stirred for 1 h at that temperature,
and after that time, the solvents were evaporated under
reduced pressure, and the crude product was diluted with
toluene (5.0 mL) and concentrated again, repeating this
operation twice. The resulting ammonium trifluoroacetate salt
(22 and 25), obtained in quantitative yield, was used for the
next step not requiring further purification.
Trihydroxy Cyclodepsipeptide 27. The resulting am-
monium trifluoroacetate 22 (obtained from 152 mg of triol 17,
∼0.191 mmol) was dissolved in anhydrous DMF (190 mL, 1
mM based on the triol 17), and the solution was cooled to 0
°C. DEPC (156 µL, 0.936 mmol, 4.9 equiv) and anhydrous TEA
(143 µL, 1.03 mmol, 5.4 equiv) were sequentially added at 0
°C, and the reaction mixture was stirred for 18 h at ambient
temperature. After this time, the crude mixture was diluted
with diethyl ether and washed with a saturated aqueous NH4-
Cl solution. After separation of both phases, the aqueous layer
was extracted with more diethyl ether, and the final combined
organic solution was sequentially washed with saturated
aqueous NH4Cl solution (2 × 10 mL), H2O (1 × 10 mL), and
brine (1 × 10 mL), dried (MgSO4), filtered, and concentrated
in vacuo. The crude product was purified by flash column
chromatography (silica gel, 35% EtOAc and 5% MeOH in
hexanes) to afford impure cyclic depsipeptide 26 (30 mg, 23%
overall yield from 17). (FAB HRMS (NBA): m/e 712.4512, M
+ Na+; calcd for C38H63N3O8 712.4513.) This crude was
dissolved in MeOH (3 mL), and 10% Pd(OH)2/C (40 mg) was
added. The reaction was allowed to proceed under an atmo-
sphere of H2 at ambient temperature, and after 1 h, the
suspension was filtered through a silica gel pad. The solid was
washed with MeOH and CH2Cl2, and the combined clear
organic solution was concentrated under reduced pressure to
obtain crude product 27 which was purified by flash column
chromatography (silica gel, 40% EtOAc and 10% MeOH in
hexanes) to afford cyclodepsipeptide 27 (19 mg, 17% over 5
steps from triol 17) as a colorless oil: Rf ) 0.31 (silica gel,
50% EtOAc and 10% MeOH in hexanes). 1H NMR (400 MHz,
DMSO-d6): δ ) 0.82-0.92 (m, 12 H, CH3CH2, CH(CH3), CH-
(CH3)2), 1.02 (d, J ) 5.9 Hz, 3 H, (CH3)CH(OH)), 1.17-1.25
(m, 22 H, 11 × CH2), 1.46-1.56 (m, 3 H, CH2CHOC(dO),
CH(CH3)), 1.86-1.96 (m, 1 H, CH(CH3)2), 2.11 (dd, J ) 13.4,
6.4 Hz, 1 H, CH2CONH), 2.28 (dd, J ) 13.4, 5.9 Hz, 1 H, CH2-
CONH), 3.58-3.70 (m, 2 H, CH2OH), 3.84-3.92 (m, 3 H,
CHNH(Val), CH(OH), (CH3)CH(OH)), 4.03 (dt, J ) 7.5, 6.4 Hz,
1 H, CHNH(Ser)), 4.12-4.20 (m, 1 H, CHNH(Thr)), 4.58 (d, J
) 4.8 Hz, 1 H, OH), 4.68-4.75 (m, 1 H, CHOC(dO)), 4.83 (dd,
J ) 5.9, 5.3 Hz, 1 H, CH2OH), 4.96 (d, J ) 5.3 Hz, 1 H, OH),
7.22 (d, J ) 7.0 Hz, 1 H, NH(Thr)), 8.00 (d, J ) 9.1 Hz, 1 H,
NH(Val)), 8.32 (d, J ) 7.5 Hz, 1 H, NH(Ser)). 13C NMR (100
MHz, DMSO-d6): δ ) 10.1, 14.0, 19.2, 19.3, 20.2, 22.1, 25.5,
28.7, 28.8, 28.95, 29.0, 29.03, 29.5, 29.7, 31.3, 41.1, 42.4, 54.9,
58.5, 59.8, 61.2, 66.7, 67.4, 77.9, 169.7, 170.1, 171.1, 171.5. FAB
HRMS (NBA): m/e 622.4044, M + Na+; calcd for C31H57N3O8
622.4043.
Cyclic Depsipeptide 31. Hydrogenolysis of 29 (18 mg,
0.025 mmol) and subsequent selective monoacetylation of the
resulting trihydroxy cyclodepsipeptide 30 (15 mg) were per-
formed exactly as described above for the preparation of 28 to
furnish, after purification by flash column chromatography
(silica gel, 4% MeOH in CHCl3), cyclic depsipeptide 31 (7 mg,
44% overall two steps), as a white solid, together with the
diacetylated compound (5 mg, 28%). [31]: Rf ) 0.34 (silica gel,
50% EtOAc and 10% MeOH in hexanes). [R]25D ) -29.7° (c )
1
0.3, CHCl3). H NMR (400 MHz, DMSO-d6): δ ) 0.72 (d, J )
7.0 Hz, 3 H, CH(CH3)), 0.79-0.90 (m, 12 H, CH3CH2, CH-
(CH3)2, CH(CH2CH3)), 0.99 (d, J ) 6.4 Hz, 3 H, (CH3)CH(OH)),
1.16-1.29 (m, 22 H, 11 × CH2), 1.29-1.40 (m, 1 H, CH2CHOC-
(dO)), 1.46-1.57 (m, 3 H, CH2CHOC(dO), CH(CH2CH3)),
1.60-1.70 (m, 1 H, CH(CH3)), 1.93-2.00 (m, 1 H, CH(CH2-
CH3)), 1.97 (s, 3 H, CH3CO2), 2.04-2.13 (m, 1 H, CH(CH3)2),
3.68-3.72 (m, 1 H, CH(OH)), 3.89 (dd, J ) 10.7, 7.0 Hz, 1 H,
CH2OAc), 4.05 (dd, J ) 10.7 Hz, 1 H, CHNH(Val)), 4.14-4.24
(m, 1 H, (CH3)CH(OH)), 4.28 (dd, J ) 9.7, 2.1 Hz, 1 H, CHNH-
(Thr)), 4.40 (dd, J ) 10.7, 6.4 Hz, 1 H, CH2OAc), 4.74-4.81
(m, 1 H, CHNH(Ser)), 4.89 (d, J ) 4.8 Hz, 1 H, (CH3)CH(OH)),
4.94 (dt, J ) 9.1, 4.8 Hz, 1 H, CHOC(dO)), 5.60 (d, J ) 5.4
Hz, 1 H, CH(OH)), 7.75 (d, J ) 8.6 Hz, 1 H, NH(Ser)), 7.95 (d,
J ) 10.2 Hz, 1 H, NH(Val)), 8.42 (d, J ) 9.7 Hz, 1 H, NH(Thr)).
13C NMR (100 MHz, DMSO-d6): δ ) 6.6, 12.2, 14.2, 19.3, 19.7,
20.79, 20.83, 22.3, 24.8, 25.5, 28.9, 29.0, 29.1, 29.19, 29.23,
29.25, 29.29, 30.2, 30.6, 31.5, 32.3, 40.8, 50.0, 54.7, 57.9, 61.3,
62.7, 67.0, 75.3, 79.2, 169.8, 170.5, 170.6, 171.8, 174.1. FAB
Cyclic Depsipeptide 28. To a solution of triol 27 (15 mg,
0.025 mmol, 1.0 equiv) in pyridine (3.0 mL) was added acetic
anhydride (23 µL, 0.25 mmol, 10.0 equiv) at 0 °C. After stirring
for 2 h at this temperature, MeOH was added, and after 5
min, the resulting solution was concentrated in vacuo. The
obtained crude product was purified by flash column chroma-
(22) Bialy, L.; Waldmann, H. Angew. Chem., Int. Ed. 2005, 44,
3814-3839.
J. Org. Chem, Vol. 70, No. 20, 2005 7863

Sarabia et al.
HRMS (NBA): m/e 692.4464, M + Na+; calcd for C35H63N3O9
and CH2Cl2. The resulting organic solution was concentrated
in vacuo, and the obtained crude product was dissolved in
pyridine (2.0 mL) and subjected to the action of acetic
anhydride (29 µL) at 0 °C. After stirring for 3 h at this
temperature, the reaction mixture was quenched by addition
of MeOH and concentrated. The crude product was purified
by flash column chromatography (silica gel, 10% AcOEt in
hexanes) to obtain stevastelin analogue 42 (4 mg, 18% overall
yield from 36) as a white solid: Rf ) 0.52 (silica gel, 100%
EtOAc). [R]25D ) -43.3° (c ) 0.30, CHCl3).1H NMR (400 MHz,
DMSO-d6): δ ) 0.73 (d, J ) 7.0 Hz, 3 H, CH(CH3)), 0.80 (d, J
) 7.0 Hz, 3 H, CH(CH3)2), 0.82-0.87 (m, 9 H, CH3CH2, CH-
(CH3)2), 0.89 (d, J ) 6.4 Hz, 3 H, CH(CH3)2), 1.14 (d, J ) 7.5
Hz, 3 H, CH(CH3)), 1.16-1.30 (m, 22 H, 11 × CH2), 1.37-1.47
(m,1 H, CH2CHOC(dO)), 1.49-1.59 (m, 1 H, CH2CHOC(dO)),
1.64-1.72 (m, 1 H, CH(CH3)), 1.96 (s, 3 H, OC(dO)CH3), 2.00-
2.17 (m, 2 H, 2 × CH(CH3)2), 2.21 (dq, J ) 7.5, 3.2 Hz, 1 H,
CH(CH3)), 3.60-3.64 (m, 1 H, CH(OH)), 3.96 (dd, J ) 10.7 Hz,
1 H, CHNH(Val)), 4.03 (dd, J ) 10.7 Hz, 1 H, CH2OAc), 4.29
(dd, J ) 9.7, 4.8 Hz, 1 H, CHNH(Val)), 4.48 (dd, J ) 11.3, 4.8
Hz, 1 H, CH2OAc), 4.66-4.73 (m, 1 H, CHNH(Ser)), 4.84-4.90
(m, 1 H, CHOC(dO)), 5.43 (d, J ) 3.2 Hz, 1 H, CH(OH)), 7.89
(d, J ) 10.2 Hz, 1 H, NH(Val)), 7.98 (d, J ) 9.7 Hz, 1 H,
NH(Ser)), 8.02 (d, J ) 8.1 Hz, 1H, NH(Val)). 13C NMR (100
MHz, DMSO-d6): δ ) 7.2, 14.1, 16.1, 17.4, 19.2, 19.4, 19.5,
20.7, 22.2, 25.4, 28.8, 28.9, 29.0, 29.1, 29.2, 29.8, 31.2, 31.4,
31.7, 46.2, 49.9, 57.1, 61.4, 62.7, 74.8, 78.9, 169.3, 170.3, 170.6,
171.1, 175.2. FAB HRMS (NBA): m/e 676.4518, M + Na+;
calcd for C35H63N3O8 676.4513.
Cyclic Depsipeptides 73 and 74. The transformation of
compounds 65 (93 mg, 0.121 mmol) and 67 (77 mg, 0.085
mmol) to cyclic depsipeptide 73 and 74, respectively, followed
the same synthetic sequence as that described above for cyclic
depsipeptide 26, through triols 66 (88 mg, 99%) and 68 (75
mg, 99%), aldehydes 69 (77 mg, 88%) and 71 (66 mg, 88%),
acids 70 (76 mg, 96%) and 72 (66 mg, 99%), and their
corresponding ammonium trifluoroacetates, to obtain, after
purifications by flash column chromatography (silica gel, 35%
EtOAc, 5% MeOH in hexanes and 33% acetone in toluene,
respectively) cyclic depsipeptides 73 (23 mg, 30% yield over 5
steps) and 74 (15 mg, 25% yield over 5 steps) as colorless oils.
[73]: Rf ) 0.33 (silica gel, 50% EtOAc, 5% MeOH in hexanes).
1H NMR (400 MHz, DMSO-d6): δ ) 0.72 (d, J ) 7 Hz, 3 H,
CH(CH3)), 0.84 (d, J ) 7 Hz, 9 H), 0.91 (d, J ) 7 Hz, 12 H, 4
× CH3CH), 1.02 (d, J ) 6.4 Hz, 3 H, (CH3)CH(OH)), 1.12 (d, J
) 7.5 Hz, 3 H,), 1.17-1.37 (m, 37 H), 1.36-1.46 (m, 1 H,
CH(CH3)), 1.47-1.59 (m, 1 H, CH(CH3)), 1.74-1.84 (m, 1 H,
CH(CH3)), 2.04-2.24 (m, 1 H, CH(CH3)), 2.31-2.34 (s, 1 H,
CH(CH3)2), 3.57-3.63 (m, 3 H,), 3.96 (t, J ) 8.6 Hz, 1 H,
CHO-), 4.06-4.18 (m, 6 H), 4.22 (dd, J ) 9.1, 2.1 Hz, 2 H,),
4.30 (t, J ) 7 Hz, 2 H, (CHNH), 4.81-4.87 (m, 1 H), 4.98-
5.06 (m, 1 H), 7.49 (d, J ) 8.1 Hz, 1 H, CHNH(Thr(OH)), 7.92
(d, J ) 10.2 Hz, 1 H, NH), 8.25 (d, J ) 8.6 Hz, 1 H, NH). FAB
HRMS (NBA): m/e 648.8705, M + Na+; calcd for C34H63N3O7
648.8697. [74]: Rf ) 0.25 (silica gel, 33% acetone in toluene).
1H NMR (400 MHz, DMSO-d6): δ ) 0.77 (d, J ) 7.3 Hz, 3 H,
CH(CH3)), 0.81-0.88 (m, 6 H, CH3CH2, CH(CH3)), 0.91 (d, J
) 7.3 Hz, 3 H, CH(CH3)), 0.98 (d, J ) 6.1 Hz, 3 H, CH(CH3)),
1.01 (d, J ) 6.1 Hz, 3 H, CH(CH3)), 1.16 (d, J ) 6.7 Hz, 3 H,
CH(CH3)), 1.09-1.27 (m, 22 H, 11 × CH2), 1.40-1.64 (m, 2 H,
CH2CHOC(dO)), 1.72-1.87 (m, 1 H, CH(CH3)), 1.94-2.09 (m,
1 H, CH(CH3)2), 2.13-2.26 (m, 1 H, CH(CH3)), 3.64-3.74 (m,
1 H, CH(OH)), 3.96 (dd, J ) 11.0, 10.4 Hz, 1 H, CHNH(Val)),
4.03-4.16 (m, 2 H, (CH3)CH(OBn), (CH3)CH(OH)), 4.26 (dd,
J ) 9.8, 1.2 Hz, 1 H, CHNH(Thr)), 4.36 (d, J ) 6.1 Hz, 1 H,
CH(OH)), 4.45 (d, J ) 11.6 Hz, 1 H, CH2Ph), 4.59 (d, J ) 11.6
Hz, 1 H, CH2Ph), 4.70 (dd, J ) 9.2, 3.0 Hz, 1 H, CHNH(Thr)),
4.81-4.93 (m, 1 H, CHOC(dO)), 5.08 (d, J ) 4.9 Hz, 1 H,
CH(OH)), 7.31 (s, 5 H, Ph), 7.64 (d, J ) 9.2 Hz, 1 H, NH), 7.69
(d, J ) 9.8 Hz, 1 H, NH), 8.31 (d, J ) 9.8 Hz, 1 H, NH).
692.4462.
Cyclic Depsipeptide 41. To a solution of the crude acid
38 (∼0.064 mmol) in anhydrous CH2Cl2 (2.0 mL) was added
TFA (0.5 mL) at 0 °C. The reaction mixture was stirred for 2
h at that temperature, and after that time, the solvents were
evaporated under reduced pressure to obtain the corresponding
ammonium trifluoroacetate salt, which was used for the next
step without further purification. The resulting ammonium
trifluoroacetate (∼0.064 mmol) was dissolved in anhydrous
DMF (64 mL, 1 mM based on the triol 34), and the solution
was cooled to 0 °C. DEPC (53 µL, 0.32 mmol, 5.0 equiv) and
TEA (49 µL, 0.35 mmol, 5.5 equiv) were sequentially added at
0 °C, and the reaction mixture was stirred for 48 h at 25 °C.
After this time, the reaction mixture was concentrated under
high vacuum (0.5 mm of Hg) at 50 °C, and the resultant crude
product was purified by flash column chromatography (silica
gel, 40% toluene, 56% EtOAc and 4% MeOH) to obtain the
corresponding cyclic depsipeptide (12 mg, partially impurified).
To a solution of this macrocycle (12 mg) in MeOH (2 mL)
was added 10% Pd/C (20 mg), and the reaction was allowed to
proceed under an atmosphere of H2 at 25 °C. After 2 h, the
suspension was filtered, and the solid was washed with MeOH
and CH2Cl2. The resulting organic solution was concentrated
in vacuo, and the obtained crude product was dissolved in
pyridine (1.5 mL) and subjected to the action of acetic
anhydride (14 µL) at 0 °C. After stirring for 2 h at this
temperature, the reaction mixture was quenched by addition
of MeOH and concentrated. The crude product was purified
by flash column chromatography (silica gel, 3% MeOH in
CHCl3) to obtain stevastelin analogue 41 (4 mg, 9% overall
yield from 34) as a white solid: Rf ) 0.30 (silica gel, 5% MeOH
in CHCl3). [R]25 ) -26.0° (c ) 0.20, CHCl3). 1H NMR (400
D
MHz, DMSO-d6): δ ) 0.75 (d, J ) 7.0 Hz, 3 H, CH(CH3)),
0.83-0.88 (m, 6 H, CH3CH2, CH(CH3)2), 0.90 (d, J ) 6.4 Hz, 3
H, CH(CH3)2), 0.97 (d, J ) 5.9 Hz, 3 H, (CH3)CH(OH)), 1.10
(d, J ) 7.5 Hz, 3 H, CH(CH3)), 1.17-1.30 (m, 22 H, 11 × CH2),
1.43-1.59 (m, 4 H, CH2CHO(dO), CH2CH(CH3)2), 1.67-1.80
(m, 2 H, CH(CH3), CH2CH(CH3)2), 1.98 (s, 3H, OC(dO)CH3),
2.21 (dq, J ) 7.5, 3.2 Hz, 1 H, CH(CH3)), 3.61-3.64 (m, 1 H,
CH(OH)), 3.97 (dd, J ) 10.7, 6.4 Hz, 1 H, CH2OAc), 4.14-
4.19 (m, 1 H, (CH3)CH(OH)), 4.22 (dd, J ) 9.7, 2.7 Hz, 1 H,
CHNH(Thr)), 4.37 (dd, J ) 10.7, 7.0 Hz, 1 H, CH2OAc), 4.38-
4.45 (m,1 H, CHNH(Leu)), 4.67 (ddd, J ) 7.5 Hz, 1 H, CHNH-
(Ser)), 4.84-4.89 (m, 1 H, CHOC(dO)), 4.92 (d, J ) 4.3 Hz, 1
H, (CH3)CH(OH)), 5.35 (d, J ) 5.4 Hz, 1 H, CH(OH)), 7.92 (d,
J ) 8.6 Hz, 1 H, NH(Ser)), 7.95 (d, J ) 10.2 Hz, 1 H, NH(Leu)),
8.19 (d, J ) 9.1 Hz, 1 H, NH(Thr)). 13C NMR (100 MHz, DMSO-
d6): δ ) 7.1, 14.1, 16.0, 20.5, 20.7, 22.0, 22.3, 22.9, 24.7, 25.6,
28.87, 28.91, 29.07, 29.13, 29.17, 29.21, 31.5, 46.6, 50.2, 53.0,
57.9, 62.5, 66.8, 74.8, 79.3, 169.5, 170.3, 172.3, 175.0, 175.5.
Cyclic Depsipeptide 42. To a solution of the crude acid
40 (∼0.052 mmol) in anhydrous CH2Cl2 (2.0 mL) was added
TFA (0.5 mL) at 0 °C. The reaction mixture was stirred for
1.5 h at that temperature, and after that time, the solvents
were evaporated under reduced pressure to obtain the corre-
sponding ammonium trifluoroacetate salt, which was used for
the next step without further purification. The resulting
ammonium trifluoroacetate (∼0.052 mmol) was dissolved in
anhydrous DMF (52 mL, 1 mM based on the diol 36), and the
solution was cooled to 0 °C. DEPC (43 µL, 0.26 mmol, 5.0 equiv)
and TEA (41 µL, 0.29 mmol, 5.5 equiv) were sequentially added
at 0 °C, and the reaction mixture was stirred for 48 h at 25
°C. After this time, the reaction mixture was concentrated
under high vacuum (0.5 mm of Hg) at 50 °C, and the resultant
crude product was purified by flash column chromatography
(silica gel, 70% EtOAc in hexanes) to obtain the corresponding
cyclic depsipeptide (11 mg).
To a solution of this macrocycle (11 mg) in MeOH (2 mL)
was added 20% Pd(OH)2 (20 mg), and the reaction was allowed
to proceed under an atmosphere of H2 at 25 °C. After 4 h, the
suspension was filtered, and the solid was washed with MeOH
Silyl Ether 75. A solution of 74 (15 mg, 0.0208 mmol, 1.0
equiv) in CH2Cl2 (1.0 mL) was treated at 0 °C with 2,6-lutidine
7864 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis of Modified Stevastelin Analogues
(5 µL, 0.0416 mmol, 2.0 equiv) and tert-butyldimethylsilyl
trifluoromethanesulfonate (6.2 µL, 0.027 mmol, 1.3 equiv).
After stirring for 15 min at 0 °C, MeOH was added, and the
crude mixture was diluted with diethyl ether. The organic
solution was washed with a saturated aqueous NH4Cl solution,
and the resulting biphasic mixture was separated, the aqueous
layer extracted with diethyl ether, and the combined organic
phase was washed with water and brine, dried (MgSO4), and
concentrated under reduced pressure to give silyl ether 75 (18
mg), which was used in the next step without purification.
Diol 76. A solution of compound 75 (18 mg, 0.0208 mmol)
in MeOH (2.5 mL) was treated with 10% Pd(OH)2-C/25 mg),
and the reaction was allowed to proceed under an H2 atmo-
sphere at ambient temperature. After 30 min, the reaction was
complete, as judged by TLC, and the suspension was filtered,
the solids washed with MeOH and CH2Cl2, and the resulting
organic solution concentrated under vacuum, to obtain diol 76
(14 mg), which was used in the next step without purification.
Cyclic Depsipeptide 78. To a solution of cyclic depsipep-
tide 76 (14 mg, 0.019 mmol) in pyridine (2.0 mL) was added
Ac2O (28 µL) and 4-DMAP (0.4 mg) at room temperature. After
stirring for 1 h at this temperature, the reaction was quenched
by addition of MeOH, and the resulting crude mixture was
concentrated under reduced pressure to obtain crude product
77. After solving the resulting product 77 in THF (2.0 mL),
HF‚pyridine (70% HF, 2.0 mL) was added at 0 °C. After
stirring at this temperature for 40 min, a saturated aqueous
NaHCO3 solution was added, followed by addition of CH2Cl2.
After separation of the phases, the organic layer was washed
with water and brine, filtered, dried (MgSO4), and concen-
trated under reduced pressure. The resulting crude product
was subjected to purification by flash column chromatography
(silica gel, 4% MeOH in CHCl3) to obtain cyclic depsipeptide
78 (5 mg, 36% overall yield from 67) as a white solid: Rf )
H, (CH3)CH(OH)), 5.20 (dq, J ) 6.4, 5.9 Hz, 1 H, (CH3)-
CH(OAc)), 7.70 (d, J ) 9.1 Hz, 1 H, NH(Thr(OAc))), 7.88 (d, J
) 10.2 Hz, 1 H, NH(Val)), 8.25 (d, J ) 9.7 Hz, 1 H, NH(Thr-
(OH))). 13C NMR (100 MHz, DMSO-d6): δ ) 7.3, 14.0, 15.8,
16.6, 19.0, 19.5, 20.8, 21.1, 22.1, 25.5, 28.7, 28.97, 29.04, 29.9,
31.3, 46.2, 54.1, 58.6, 61.1, 66.8, 68.6, 74.2, 78.0, 168.5, 169.7,
170.5, 171.2, 175.1. FAB HRMS (NBA): m/e 692.4461, M +
Na+; calcd for C35H63N3O9 692.4462.
Cyclic Depsipeptide 91. Desilylation of compound 90 was
carried out exactly in the same manner as that described
before for cyclic depsipeptide 78, by treatment with HF‚
pyridine (70% HF, 2.0 mL) to obtain, after purification by flash
column chromatography (silica gel, 75% EtOAc and 5% MeOH
in hexanes), cyclic depsipeptide 91 (10 mg, 56% from 88 over
3 steps) as a colorless oil: Rf ) 0.24 (silica gel, 85% EtOAc,
10% hexanes, 5% MeOH). [R]25 ) -110° (c ) 0.11, CHCl3).
D
1H NMR (400 MHz, DMSO-d6): δ )0.73 (d, J ) 6.4 Hz, 3H,
CH3CH), 0.90 (d, J ) 6.4 Hz, 3H, CH3CH), 0.92 (d, J ) 6.4
Hz, 3H, CH3CH), 1.01 (d, J ) 6.4 Hz, 3H, CH3CHOH), 1.10
(d, J ) 7.5 Hz, 3H, CH3CHCONH), 1.92-2.07 (m, 2H, CH3CH,
CH(CH3)2), 2.00 (s, 3H, CH3CO2), 2.20-2.30 (m, 1H, CH3CH-
CONH), 3.64-3.70 (m, 1H, CHOH), 3.86 (dd, J ) 10.7 Hz, 1H,
CH2OCO), 3.94-4.01 (m, 2H, CH2OCO, CHCH(CH3)2), 4.03-
4.10 (m, 1H, CH3CHOH), 4.18-4.25 (m, 2H, CH2OAc), 4.26
(dd, J ) 9.1, 2.1 Hz, 1H, CHCHOH), 4.58 (d, J ) 6.4 Hz, 1H,
CHOH), 4.60-4.66 (m, 1H, CHCH2OAc), 5.13 (d, J ) 4.8 Hz,
CH3CHOH), 7.55 (d, J ) 9.7 Hz, 1H, NH(Val)), 7.76 (d, J )
8.1 Hz, 1H, NH(Ser)), 7.83 (d, J ) 9.7 Hz, 1H, NH(Thr)).13C
NMR (100 MHz, DMSO-d6): δ ) 9.1, 13.7, 19.4, 19.8, 20.8,
30.8, 33.3, 46.3, 51.9, 58.6, 61.4, 62.9, 66.5, 67.0, 71.3, 168.2,
170.4, 171.1, 171.5, 175.5.
Acknowledgment. This work was financially sup-
ported by Fundacio´n Ramo´n Areces and the Direccio´n
General de Investigacio´n (BQU2001-1576) and fellow-
ships from Fundacio´n Ramo´n Areces (S.C.). We thank
Dr. J. I. Trujillo from Pfizer (St. Louis, MO) for as-
sistance in the preparation of this manuscript. We thank
Unidad de Espectroscop´ıa de Masas of Seville and
Granada Universities for exact mass spectroscopic as-
sistance.
0.34 (silica gel, 10% MeOH in CHCl3). [R]25 ) -34.4° (c )
D
0.25, CHCl3). 1H NMR (400 MHz, DMSO-d6): δ ) 0.72 (d, J )
7.0 Hz, 3 H, CH(CH3)), 0.82-0.87 (m, 6 H, CH3CH2, CH(CH3)),
0.90 (d, J ) 6.4 Hz, 3 H, CH(CH3)), 1.03 (d, J ) 6.4 Hz, 3 H,
CH(CH3)), 1.11 (d, J ) 7.5 Hz, 3 H, CH(CH3)), 1.19 (d, J ) 6.4
Hz, 3 H, CH(CH3)), 1.17-1.27 (m, 22 H, 11 × CH2), 1.35-1.46
(m, 1 H, CH2CHOC(dO)), 1.48-1.59 (m, 1 H, CH2CHOC(d
O)), 1.73-1.82 (m, 1 H, CH(CH3)), 2.00 (s, 3 H, OC(dO)CH3),
2.04-2.13 (m, 1 H, CH(CH3)2), 2.20 (dq, J ) 7.5, 4.3 Hz, 1 H,
CH(CH3)), 3.64 (bs, 1 H, CH(OH)), 3.97 (dd, J ) 10.7, 10.2
Hz, 1 H, CHNH(Val)), 4.04-4.11 (m, 1 H, (CH3)CH(OH)), 4.25
(dd, J ) 9.7, 2.7 Hz, 1 H, CHNH(Thr(OH))), 4.68 (dd, J ) 9.1,
5.9 Hz, 1 H, CHNH(Thr(OAc))), 4.82-4.87 (m, 1 H, CHO(Cd
O)), 4.90 (d, J ) 5.9 Hz, 1 H, CH(OH)), 5.00 (d, J ) 4.8 Hz, 1
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for all other compounds, and
1H and 13C NMR spectra for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO050628Y
J. Org. Chem, Vol. 70, No. 20, 2005 7865