A macrolactonization approach to the stevastelins

Article abstract of DOI:10.1016/S0040-4039(02)00416-1

A synthesis of the stevastelins, a novel class of immunosuppressant agents, is reported based on a macrolactonization approach. This synthesis commenced with the stereoselective preparation of the stearic acid segment from tetradecanal using Evans asymmetric synthesis methodology and an aldol reaction with a thioester. After a high yielding coupling reaction between the fatty acid residue and the corresponding tripeptide, we proceeded with the macrolactonization key step. Thus, macrolactonizations of hydroxy acid 27 and dihydroxy acid 30, according to Yamaguchi conditions, afforded the corresponding 13-membered ring stevastelin derivatives 28 and 31 in 90 and 82% yields, respectively. In this latter case, the corresponding 15-membered lactone was not formed. Finally, depsipeptide derivative 31 was converted into stevastelin C3 (5).

Full text of DOI:10.1016/S0040-4039(02)00416-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2961–2965
A macrolactonization approach to the stevastelins
Francisco Sarabia,* Samy Chammaa and F. Jorge Lo´pez-Herrera
Departamento de Bioqu´ımica, Biolog´ıa Molecular y Qu´ımica Orga´nica, Facultad de Ciencias, Universidad de Ma´laga,
29071 Ma´laga, Spain
Received 12 February 2002; accepted 28 February 2002
Abstract—A synthesis of the stevastelins, a novel class of immunosuppressant agents, is reported based on a macrolactonization
approach. This synthesis commenced with the stereoselective preparation of the stearic acid segment from tetradecanal using
Evans asymmetric synthesis methodology and an aldol reaction with a thioester. After a high yielding coupling reaction between
the fatty acid residue and the corresponding tripeptide, we proceeded with the macrolactonization key step. Thus, macrolactoniza-
tions of hydroxy acid 27 and dihydroxy acid 30, according to Yamaguchi conditions, afforded the corresponding 13-membered
ring stevastelin derivatives 28 and 31 in 90 and 82% yields, respectively. In this latter case, the corresponding 15-membered lactone
was not formed. Finally, depsipeptide derivative 31 was converted into stevastelin C3 (5). © 2002 Elsevier Science Ltd. All rights
reserved.
Stevastelins A (1), B (2), A3 (3), B3 (4) and C3 (5) (Fig.
1), recently isolated from culture broths of Penicillium
sp. NK374186,1,2 represent a new class of natural cyclic
depsipeptides with intriguing biological properties as
immunosuppressive agents. The great level of interest
that immunosuppressants have elicited in the scientific
community is clearly indicated by the immunosuppres-
sive agents cyclosporin A3 or FK506,4 whose discover-
ies, biological evaluations and subsequent uses in
medicine have led to a substantial increase in the suc-
cess of organ and bone marrow transplantations. In
addition to these relevant and outstanding applications
in surgery, these compounds represent valuable bio-
chemical tools for the investigation of signal transduc-
tion pathways at the molecular level.5 Therefore,
immunosuppressants have occupied a prominent posi-
tion in the forefront of total synthesis, chemical biology
and medicine research and new natural products such
as rapamycin,6 sanglifehrin A,7 tamandarins8 or
pateamine A9 have also rapidly captured the attention
of the chemical and biomedical communities on
account of their broad range of immunosuppressant
actions. Initially, the stevastelins were discovered as
inhibitors of the IL-2 and IL-6 gene expressions. Thus,
like cyclosporin A or FK506,10 they exhibited growth-
inhibition activities against OKT3-stimulated human T
cell proliferation with an IC50 particularly striking for
stevastelin B (2) of 1.8 mg/mL and for B3 (4) of 0.42
O
O
O
OH
O
O
HN
O
HN
OH
O
N
H
NH HN
O
N
H
O
OAc
OR1
OR
OR2
O
Stevastelins A (1): R = SO3H
B (2): R = H
Stevastelins A3 (3): R1 = Ac; R2 = SO3H
B3 (4): R1 = Ac; R2 = H
C3 (5): R1 = H; R2 = H
Figure 1. Structures of Stevastelins A (1), B (2), A3 (3), B3 (4) and C3 (5).
Keywords: stevastelin; immunosuppressant; natural products; cyclic depsipeptide; stereoselective synthesis; macrolactonization.
* Corresponding author. Tel.: +34-952134258; fax: +34-952131941; e-mail: frsarabia@uma.es
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00416-1

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F. Sarabia et al. / Tetrahedron Letters 43 (2002) 2961–2965
mg/mL. However, further biological investigations11
revealed that these natural products, unlike the above
mentioned immunosuppressants, did not inhibit the
phosphatase activity of calcineurin. So it appears that
the novel mechanism of immunosuppressive action that
the stevastelins seem to be involved in remains unclear,
and its elucidation could lead to the disclosure of new
signal transduction pathways. These interesting and
unprecedented biological findings, coupled with the fact
that a total synthesis of the stevastelins has not been
reported to date, make the stevastelins very attractive
targets for chemists, biochemists and clinicians owing
to their potential utility in medicine. Structurally,12 the
stevastelins are comprised of a family of 13- and 15-
membered cyclic depsipeptides, characterized by two
main frameworks: a stearic acid unit and a peptidic
(Scheme 2). In this way, both key fragments, the acid
16 and the peptide 25, were ready for the coupling
reaction.
This coupling was performed again by using EDCI and
HOBt as coupling reagents, to obtain the amide 26 in
90% yield. The final step towards the corresponding
hydroxy acid, prior to the final macrolactonization, was
conducted with the cleavage of the allyl ester of 26
mediated by palladium(0) in the presence of
morpholine18 to obtain the hydroxy acid 27. This
hydroxy acid would give access to the 13-membered
stevastelins, and, in fact, macrolactonization of this
compound, under Yamaguchi conditions,19 afforded
the corresponding depsipeptide 2820 in a very good
yield (90%) (Scheme 3).
This methodology should provide a highly convergent
route to other members of the stevastelins if we are to
be able to accomplish the synthesis of the 15-membered
macrolactone derivatives of the stevastelins starting
from the same synthetic intermediates. Thus, we pre-
pared the diol 29 by reaction with camphorsulfonic acid
(CSA) in methanol in a moderate yield (60%) and, after
fragment that contains
L-serine, L-threonine and L-
valine which are connected by an ester and an amide
linkage. In addition, the stearic acid moiety contains a
tetrade fragment, whose absolute configuration has
been established only for stevastelin B (2)13 but no for
other members of the family.
Thus, we decided to attempt the synthesis of these
immunosuppressive agents, starting with the construc-
tion of the main structural units contained in the stev-
astelins: the stearic acid and the tripeptide derivative
fragments. Scheme 1 outlines the synthesis of the first
key important fragment. Accordingly, tetradecanal 6,
obtained by oxidation from the commercially available
tetradecanol, was reacted with the chiral boron enolate
of the oxazolidinone 7, following Evans’ asymmetric
methodology,14 to obtain in a 80% yield the syn aldol 8.
The conversion of this compound to the aldehyde 13
was achieved in five steps with excellent yields involving
selective protections and deprotections through com-
pounds 9, 10, 11 and 12 according to the synthetic
sequence depicted in Scheme 1. In order to complete
the synthesis of the stearic acid fragment, the anti aldol
was required from aldehyde 13. So, we proceeded with
an aldol reaction of 13 with the boron enolate of
tert-butyl thiopropionate 14,15 to obtain the desired
compound 15 in good yield (85%) as the only
diastereoisomer and containing the required relative
configuration of the tetrade. Finally, thioester 15 was
transformed into the acid 16 by reaction with hydrogen
peroxide in the presence of lithium hydroxide in 85%
yield. The correct absolute configuration of compound
16 was unequivocally confirmed by chemical methods.16
The synthesis of the peptide residue was achieved by
conventional methods starting from the corresponding
commercially available protected amino acids, which
were coupled by standard solution phase synthesis of
peptides,17 as outlined in Scheme 2. Thus, after prepa-
ration of the allyl ester of the protected serine deriva-
tive 17, the coupling with the threonine derivative 20
was carried out with N-ethyl-N%-[3-(dimethylamino)-
propyl]carbodiimide (EDCI) in the presence of 1-
hydroxybenzotriazole (HOBt) to obtain the dipeptide
21 in very good yield. Following the same synthetic
sequence, the tripeptide 25 was prepared in high yields
Scheme 1. Synthesis of the fatty acid residue of stevastelins,
the stearic acid derivative (16). Reagents and conditions: (a)
1.1 equiv. n-Bu2BOTf, 1.1 equiv. Et3N, CH2Cl2, 0°C, 0.5 h,
then 1.0 equiv. of 6, −78°C, 12 h, 80%. (b) 4.0 equiv. LiBH4,
THF, 0°C, 4 h, 95%. (c) 1.1 equiv. PivCl, CH2Cl2/pyr., 0°C,
2 h, 94%. (d) 1.2 equiv. TBSCl, 1.5 equiv. imidazole, DMF,
0°C, 12 h, 98%. (e) 2.1 equiv. DIBAL-H, CH2Cl2, −78°C, 0.5
h, 98%. (f) 2.0 equiv. (COCl)2, 2.5 equiv. DMSO, 4.0 equiv.
Et3N, CH2Cl2, −78°C, 0.5 h, 98%. (g) 1.5 equiv. of 14, 1.2
equiv. (Chx)2BCl, 1.2 equiv. Et3N, CH2Cl2, 0°C, 2 h, then 1.0
equiv. of 13, −78°C, 12 h, 85%. (h) 5.0 equiv. H2O2, 5.0 equiv.
LiOH, THF–H2O, 0°C, 1 h, 85%.

F. Sarabia et al. / Tetrahedron Letters 43 (2002) 2961–2965
2963
O
O
a
BnO
OH
BnO
O
NHBoc
NHR
18, R = Boc
19, R = H
17
b
O
c
BnO
OH
NHBoc
20
O
OBn
O
O
OH
NHBoc
23
BnO
N
H
NHR
O
21, R = Boc
22, R = H
c
b
OBn
O
O
H
N
RHN
N
24, R = Boc
25, R = H
H
O
O
b
OBn
Scheme 2. Synthesis of the peptide residue of stevastelins, the
tripeptide (25). Reagents and conditions: (a) 1.5 equiv. EDCI,
0.1 equiv. 4-DMAP, 1.5 equiv. allylic alcohol, CH2Cl2, 25°C,
8 h, 88%. (b) 2.5 equiv. TFA, CH2Cl2, 25°C, 0.5 h, 98% for
19, 98% for 22, 98% for 25. (c) 1.5 equiv. EDCI, 1.0 equiv.
HOBt, CH2Cl2, 0“25°C, 0.5 h, 85% for 21, 89% for 24.
Scheme 3. Coupling of stevastelin fragments (16) and (25)
and macrolactonization reaction. Reagents and conditions: (a)
1.5 equiv. EDCI, 1.0 equiv. HOBt, CH2Cl2, 0“25°C, 0.5 h,
90%. (b) 0.1 equiv. Pd[PPh3]4, 1.0 equiv. morpholine, THF,
0“25°C, 0.5 h, 98%. (c) 1.3 equiv. of 2,4,6-trichloroben-
zoylchloride, 2.2 equiv. Et3N, THF, 0°C, 1 h, then add to a
solution of 2.2 equiv. 4-DMAP in toluene (0.005 M based on
27), 75°C, 10 min, 90%.
cleavage of the allyl ester, the resulting dihydroxy acid
30 was subjected to a macrolactonization reaction
under Yamaguchi conditions. However, we obtained
the 13-membered macrolactone 3121 as a single product
(82%), not detecting any formation of the desired 15-
membered cyclic depsipeptide. To address this result,
we could include a protection step of the hydroxyl
group at C-3 of 26, followed by the cleavage of the silyl
ether of the hydroxyl group at C-5, to give an alter-
nated substrate for the macrolactonization reaction.
However, this strategy has already been attempted by
Chakraborty et al.,22 who has recently reported an
approach to the stevastelins wherein they describe no
success with the modified strategy. With the 13-mem-
bered macrolactone of the stevastelins in hand, we
undertook deprotection of the benzyl ethers of the
depsipeptide 31 by the action of boron trichloride to
obtain stevatelin C3 (5)23 (Scheme 4).
tones, and, in fact, we are currently exploring different
strategies to accomplish this goal and thereby give
access to a broad family of stevastelins and analogues
for further biological evaluations.
Acknowledgements
This work was financially supported by the Direccio´n
General de Investigacio´n y Cient´ıfica Te´cnica (Ref.
PB97-1091) and by the Direccio´n General de Universi-
dades e Investigacio´n, Consejer´ıa de Educacio´n y Cien-
cia, Junta de Andaluc´ıa (FQM 0158). We thank Dr. J.
I. Trujillo from The Scripps Research Institute (La
Jolla, CA) for assistance in the preparation of this
manuscript. We thank Unidad de Espectroscop´ıa de
Masas de la Universidad de Sevilla for exact mass
spectroscopic assistance.
In conclusion, we have designed an approach to the
stevastelins with the aim of delivering not only the
natural compounds but also analogues thereof. Initial
results have proven the convergence and efficiency of
our synthetic approach towards 13-membered ring stev-
astelins; however, an efficient synthetic approach for
the corresponding 15-membered rings has proved elu-
sive. Thus, the next challenge to address in this research
is to synthesize the corresponding 15-membered lac-

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F. Sarabia et al. / Tetrahedron Letters 43 (2002) 2961–2965
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stevastelin B (2) during structural elucidation studies.
Scheme 4. Macrolactonization of dihydroxy acid (30). Syn-
thesis of stevastelin C3 (5). Reagents and conditions: (a) 0.5
equiv. CSA, MeOH, 0“25°C, 3.0 h, 60%. (b) 0.1 equiv.
Pd[PPh3]4, 10.0 equiv. morpholine, THF, 0“25°C, 0.5 h,
98%. (c) 1.3 equiv. of 2,4,6-trichlorobenzoylchloride, 2.2
equiv. Et3N, THF, 0°C, 1 h, then add to a solution of 2.2
equiv. 4-DMAP in toluene (0.005 M based on 30), 75°C, 10
min, 82%. (d) 2.5 equiv. BCl3, CH2Cl2, −78°C, 0.5 h, 90%.
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20. Compound
28:
Rf=0.60
(silica
gel,
hex-
anes:AcOEt:MeOH, 15:4:1); 1H NMR (400 MHz,

F. Sarabia et al. / Tetrahedron Letters 43 (2002) 2961–2965
2965
CDCl3) l 7.30–7.09 (m, 11H, CH aromatics, NH), 6.66
(d, 1H, J=8.8 Hz, –NH), 6.60 (d, 1H, J=8.8 Hz, –NH),
5.15 (dd, 1H, J=8.8, 2.3 Hz, CHOCꢀO), 4.50–4.43 (m,
3H), 4.37–4.30 (m, 4H), 4.12 (dc, 1H, J=5.9, 2.3 Hz),
3.79–3.73 (m, 1H), 3.68 (dd, 1H, J=7.6, 2.9 Hz), 3.62–
3.51 (m, 1H), 2.34 (dc, 1H, J=7.6 Hz, 7.0 Hz, CH(CH3)),
2.12 (ds, 1H, J=7.0, 6.5 Hz, CH(CH3)), 1.70–1.57 (m,
1H, J=6.5, 2.3 Hz, CHOCꢀO), 4.51–4.43 (m, 3H), 4.38–
4.28 (m, 4H), 4.12 (dc, 1H, J=5.9, 2.3 Hz), 3.84 (dd, 1H,
J=5.9, 2.3 Hz), 3.83–3.71 (m, 2H), 2.41 (dc, 1H, J=7.6,
7.0 Hz, CH(CH3)), 2.20–2.11 (m, 1H), 1.65–1.55 (m, 1H),
1.50–1.31 (m, 1H), 1.28–1.11 (m, 24H, CH3(CH2)12−),
1.06 (d, 3H, J=7.0 Hz, CH(CH3)), 0.93 (d, 3H, J=7.0
Hz, CH(CH3)), 0.90 (d, 3H, J=6.5 Hz, CH(CH3)), 0.86–
0.75 (m, 9H, CH(CH3)2, CH3CH2); 13C NMR (50.3
MHz, CDCl3) l 175.9, 171.5, 160.0, 156.5, 137.8, 137.7,
137.6, 133.1, 129.4, 128.4, 128.3, 127.8, 127.7, 127.6,
121.3, 78.5, 74.7, 72.2, 71.4, 61.8, 58.6, 52.2, 44.3, 38.2,
35.2, 31.9, 31.1, 29.7, 29.6, 29.3, 26.1, 22.7, 19.3, 18.0,
16.4, 14.7, 14.1, 4.9; FAB HRMS (NBA) m/e 794.5287,
M+1 calcd for C46H71N3O8 794.5319.
1H), 1.50–1.32 (m, 1H), 1.25–1.07 (m, 27H, CH3(CH2)12−
,
CH(CH3)), 1.04 (d, 3H, J=7.0 Hz, CH(CH3)), 0.92 (d,
3H, J=7.0 Hz, CH(CH3)), 0.89 (d, 3H, J=7.0 Hz,
CH(CH3)), 0.83–0.75 (m, 18H, SiC(CH3)3, CH3(CH2)12−
,
CH(CH3)2), 0.01 (s, 3H, Si(CH3)2), 0.00 (s, 3H, Si(CH3)2);
13C NMR (50.3 MHz, CDCl3) l 175.7, 171.6, 160.0,
156.6, 146.1, 137.8, 137.7, 133.2, 129.5, 128.4, 128.3,
127.8, 127.7, 127.6, 121.3, 77.2, 76.5, 74.7, 72.2, 71.3,
61.8, 58.3, 52.1, 44.2, 37.2, 34.8, 31.9, 31.2, 30.3, 29.8,
29.7, 29.6, 29.5, 29.3, 25.8, 25.4, 22.7, 19.3, 18.0, 17.8,
16.4, 14.3, 14.1, 5.8, −3.6, −4.6; FAB HRMS (NBA) m/e
908.6175, M+1 calcd for C52H85N3O8Si 908.6184.
22. Chakraborty, T. K.; Ghosh, S.; Dutta, S. Tetrahedron
Lett. 2001, 42, 5085–5088.
23. All new compounds exhibited satisfactory spectroscopic
and analytical and/or accurate mass data. For the specific
case of stevastelin C3 (5), spectroscopic data of natural
stevastelin C3 were not reported by the Japanese group
(see Ref. 1), and this compound is pending to be com-
pared with an authentic sample of 5.
21. Compound
31:
Rf=0.60
(silica
gel,
hexanes:
AcOEt:MeOH, 12:7:1); 1H NMR (400 MHz, CDCl3) l
7.31–7.10 (m, 11H, aromatic CH, NH), 6.76 (d, 1H,
J=8.8 Hz, NH), 6.44 (d, 1H, J=8.2 Hz, NH), 5.15 (dd,