Synthesis of Thelepamide via Catalyst-Controlled 1,4-Addition of Cysteine Derivatives and Structure Revision of Thelepamide

Article abstract of DOI:10.1021/acs.orglett.7b03706

The first enantioselective total synthesis and structural reassignment of (-)-thelepamide, a cytotoxic tetraketide-amino acid from the marine worm Thelepus crispus, is reported. A convergent approach provides access to all thelepamide diastereomers in six steps from four simple building blocks. Key features of the synthesis include the application of Melchiorre's organocatalytic thia-Michael reaction and a sonication-assisted assembly of an unprecedented N,O-acetal-hemiacetal moiety. The corrected structure was confirmed by NMR-DFT analysis.

Full text of DOI:10.1021/acs.orglett.7b03706

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Thelepamide via Catalyst-Controlled 1,4-Addition of
Cysteine Derivatives and Structure Revision of Thelepamide
Tobias Seitz,† Ramon E. Millan,‡ Dieter Lentz,† Carlos Jimenez,‡ Jaime Rodríguez,*,‡
́
́
́
and Mathias Christmann*,†
†Freie Universitat Berlin, Institute of Chemistry and Biochemistry, Takustraße 3, 14195 Berlin, Germany
̈
‡
́
́
Centro de Investigaciones Cientıficas Avanzadas (CICA), Departamento de Quımica, Facultade de Ciencias, Universidade da
Coruna, 15071 A Coruna, Spain
̃
̃
S
* Supporting Information
ABSTRACT: The first enantioselective total synthesis and structural
reassignment of (−)-thelepamide, a cytotoxic tetraketide−amino acid from
the marine worm Thelepus crispus, is reported. A convergent approach provides
access to all thelepamide diastereomers in six steps from four simple building
blocks. Key features of the synthesis include the application of Melchiorre’s
organocatalytic thia-Michael reaction and a sonication-assisted assembly of an
unprecedented N,O-acetal−hemiacetal moiety. The corrected structure was
confirmed by NMR−DFT analysis.
arine sponges and tunicates are an important source of
between the tetraketide part of the molecule and a suitable
M
structurally diverse bioactive secondary metabolites.1 In
cysteine derivative. In synthetic direction, two approaches to
form this bond in a stereoselective fashion were considered. First,
an SN2 substitution of a tosylate leaving group under inversion of
configuration was investigated. As a second option, we envisaged
an asymmetric catalyst-controlled thia-Michael addition6 of
cysteine derivatives to (E)-oct-5-en-4-one and a subsequent
diastereoselective reduction of the keto group.
2
́
2013, the Rodrıguez group reported the isolation of
(−)-thelepamide from the tidal zone-derived marine worm
Thelepus crispus. Thelepamide (1) possesses an unprecedented
N,O-acetal−hemiacetal motif that caught our interest as novel
Val-Cys peptide mimetic.3 While thelepamide’s carbon backbone
is identical to that of spongiacysteine (2), isolated by Kigoshi and
co-workers4 from a marine sponge, both molecules differ in the
oxidation pattern at C2, C2″ and the N-methyl group (Figure 1).
The first synthetic route commenced with a Ru-catalyzed
reduction7 of β-ketoester 3 followed by Weinreb amide
formation8 to afford β-hydroxyamide 4 in 70% yield over two
steps and 99% ee (Scheme 1). This material was subjected to
ethylmagnesium bromide in Et2O at 0 °C to give hydroxy ketone
5 in 64% yield.9 The C3 stereogenic center was generated using
an Evans−Tishchenko reduction.10 The expected anti-config-
uration in 6 was confirmed using Rychnovsky’s acetonide
method.11 After the free hydroxyl group had been converted to
the corresponding tosylate 7,12 we carried out SN2 reactions with
D- and L-N-Boc-Cys-OMe. Due to partial epimerization at C2′
during the SN2-reaction and problems occurring in the
subsequent steps toward thelepamide, we decided to abandon
this route in favor of the diastereoselective thia-Michael
approach. Nevertheless, thioethers 9 and 10 turned out to be
useful as reference standards for the following transformations.
In our second-generation approach, the synthetic sequence
commenced with a stereoselective 1,4-addition of L-8 to known
enone 1113 (Table 1). Enantioselective thia-Michael additions
have been studied by Wynberg since the late 1970s.14 For our
substrate, 20 mol % of unmodified cinchona alkaloids 12a−d
gave only moderate selectivities using dichloromethane as the
solvent (entries 1−4). With Takemoto’s thiourea catalyst 12e15
Figure 1. Structures of (−)-thelepamide (1: proposed) and
spongiacysteine (2).
The relative configurations of thelepamide’s four stereogenic
centers were established on the basis of ROESY, J-based
configurational analysis, and by comparison of calculated and
experimental NMR shifts using Goodman’s DP4 method.5
Motivated by the unique 1,3-oxazolidin-2-one, the selective yet
moderate biological activity against the leukemia cell line CCRF-
CEM (IC50 = 13.2 μM), we decided to pursue its chemical
synthesis.
In order to validate the structural assignment and probe
structure−activity by synthetic analogues, a flexible synthetic
access to thelepamide was sought. We identified the carbon−
sulfur bond at C3 in thelepamide as the key disconnection
Received: November 29, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03706
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 1. First-Generation Route to Thelepamide
Table 1. Optimization of the Thia-Michael Addition
under the same conditions, the selectivity increased to 6.6:1 in
favor of 13 (entry 5).16 Surprisingly, using the enantiomeric
Takemoto catalyst 12f, no selectivity toward 14 could be induced
(entry 6). In 2008, Melchiorre17 reported that 1:2 adducts of
cinchona alkaloid amines18 and N-Boc-phenylglycine (Phg)
derivatives19 provide superior selectivities in thia-Michael
reactions. Using their conditions at 0 °C provided 13 with
12.9:1 selectivity (entry 7). Lowering the temperature to −28 °C
increased the selectivity to 24.5:1 but also raised the reaction
time to 168 h (entry 8). Changing the amino acid to its
enantiomer lowered the selectivity to 16.7:1, demonstrating its
minor yet relevant influence (entry 9). Additionally, and in order
to gain access to 14, we screened the catalysts based on the
pseudoenantiomeric cinchona alkaloids. Catalyst 12h provided
14 in 9.3:1 ratio at 0 °C which increased to 12.9:1 at −28 °C
(entries 10 and 11). The cinchonine-based catalyst 12i gave 14 in
12:1 selectivity at 0 °C and 17.4:1 at −28 °C (entries 12 and 13).
Again, the mismatching alkaloid−Phg combination lowered the
selectivity to a 5:1 ratio (entry 14). On a preparative scale, 13 and
14 were obtained in 98% and 99% yield, respectively. It should be
noted that this approach might also be useful in the
stereoselective preparation of cysteine−S conjugates, which
play an important role as precursor of plant aromas as well as in
the formation of natural scents of cats and humans.20
b
c
entry
cat.
solvent
temp (°C)
time (h)
dr (13:14)
1
12a
12b
12c
12d
12e
12f
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
PhMe
23
23
18
18
1.9:1.0
2.3:1.0
2
3
23
18
1.0:1.2
4
23
18
1.0:1.7
5
23
30
6.6:1.0
6
23
30
1.0:1.1
7
12g
12g
0
35
12.9:1.0
24.5:1.0
16.7:1.0
1.0:9.3
8
PhMe
PhMe
−28
−28
0
168
168
35
d
9
12g
12h
12h
12i
10
11
12
13
14
PhMe
PhMe
−28
0
168
35
1.0:12.9
1.0:12.0
1.0:17.4
1.0:5.0
PhMe
12i
PhMe
PhMe
−28
−28
168
168
e
12i
a
Reaction conditions (entries 1−6): 1.10 equiv of 11, 1.00 equiv of L-
8, cat. (20 mol %), CH2Cl2 (0.10 M), Reaction conditions (entries 7−
14): 2.00 equiv of 11, 1.00 equiv L-8, cat. (20 mol %), PhMe (0.24 M).
b
c
Reaction was run until starting material L-8 was fully consumed. The
diastereomeric ratio was determined via GC analysis. Reaction was
carried out with L-N-Boc-phenylglycine. Reaction was carried out with
d
e
D-N-Boc-phenylglycine.
Having developed a catalyst-controlled access to both
diastereomers 13 and 14, we then turned our attention to the
diastereoselective reduction of the keto group and the
completion of the synthesis of the proposed stereoisomer of
thelepamide. As shown in Scheme 2, diastereoselective reduction
of β-substituted ketone 13 with zinc borohydride21 yielded a
separable 3.8:1 mixture (89%). The relative and absolute
configuration of the minor diastereomer 15a was confirmed by
X-ray-crystallography. The major diastereomer 15b was acylated
with propionic acid using the Steglich protocol22 to give ent-10,
the enantiomer of 10 obtained in the initial route (Scheme 1).
The Boc group of 15b was removed with TFA in CH2Cl2 (15b →
16b), followed by acylation with 2-ketoisovaleric acid to give α-
ketoamide 17b. Subsequently, methyl ester cleavage with lithium
hydroxide afforded carboxylic acid 18b in 75% yield. The
reaction was accompanied by epimerization at C2′. Formation of
the 5-hydroxyoxazolidin-4-one was achieved via N,O-acetal
formation with formaldehyde by sonicating the reaction under
B
DOI: 10.1021/acs.orglett.7b03706
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
ratios. From a previous project,24 we were alerted that NMR
Scheme 2. Second-Generation Route to Thelepamide
spectra can be strongly affected by deprotonation of a carboxylic
1
acid moiety. Gratifyingly, the H NMR spectrum of 3,5-epi-1
sodium carboxylate was superimposable with the original
thelepamide spectrum.
In the 13C NMR spectrum, the 44−71 ppm region was most
indicative for comparing the thelepamide diastereomers (Figure
3). The sodium carboxylate of 3,5-epi-1 and the reported
Figure 3. Comparison of the 44−71 ppm 13C NMR spectral region of
three synthetic thelepamide diastereomers with that of isolated material.
spectrum were nearly identical with an overall median 13C NMR
shift deviation of <0.1 ppm. These results were supported by
HPLC comparison with an authentic thelepamide sample.25
While the previous DFT-NMR analysis2 was carried out
assuming that 1 was present as free carboxylic acid, we repeated
the computational analysis with the corresponding carboxylates.
The sodium carboxylates of four possible thelepamide
diastereomers (1a−d, see Figure 4) were initially submitted to
a conformational search with the Macromodel program using the
protocol of Hoye et al.26 Thus, 85 conformers for 1a, 67 for 1b,
90 for 1c, and 51 for 1d were found within a 3.0 kcal/mol
window. All conformers were classified by energy and
frequencies using the B3LYP27/6-31+G(d,p) functional. Finally,
basic conditions.23 At this step it was necessary to maintain the
reaction between 24 and 26 °C in order to suppress the
formation of side products. Thus, the proposed structure of
thelepamide (1) was obtained in 19% yield over six steps as 1:1
diastereomeric mixture at C2″.
In an analogous fashion to the route described above, 3,5-epi-
thelepamide (3,5-epi-1) obtained from 14 and 5-epi-thelepamide
(5-epi-1) obtained from 15a were synthesized in six steps with
23% and 9% overall yield, respectively (Figure 2). Surprisingly, a
comparison of the original thelepamide spectra with those of all
obtained diastereomers showed a clear mismatch in both the 1H
and 13C NMR spectra. The differences manifested in the
magnitude of the chemical shifts as well as the diastereomeric
Figure 4. Proposed thelepamide (1) and four diastereomeric
carboxylates (1a−d) used in the DFT calculations.
Figure 2. Structures of thelepamide diastereomers 3,5-epi-1 and 5-epi-1.
C
DOI: 10.1021/acs.orglett.7b03706
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
the combination MPW1PW9128/6-311+G(2d,p)-polarizable
continuum model was used for proton and carbon chemical
shift calculations.29 The sets of 1H and 13C chemical shifts were
compared by mean absolute error (MAE), R2 of δcalcd/δexpt, by
the linear regression of calculated (δscaled) and by the statistical
DP4+ parameter developed by Sarotti and co-workers.30 A 100%
probability DP4+ value for both carbon and proton chemical
shifts in favor to 1d was in agreement with the synthetic 3,5-epi-1
configuration.
Comparison of the optical rotation of (−)-thelepamide and
3,5-epi-1 showed them to be equal in sign and magnitude further
confirming the configuration to be 3S,5R,2′R. From the NMR
data, we could not make a direct assignment of the C2″-
configuration of the major thelepamide diastereomer. However,
DFT calculations suggest the configuration to be 2″R (1d).
In order to probe the reported biological activity for
thelepamide, the three synthetic diastereomers were tested
against the CCRF-CEM cell line. Unfortunately, no cytotoxicity
for concentrations up to 100 μM could be detected.
Unit, FMP, Berlin, Germany, for preparing the cytotoxicity
assays.
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synthesis of (−)-thelepamide. Key steps include a catalyst
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b03706.
Procedures and spectroscopic data (PDF)
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2008, 23, 369.
Accession Codes
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CCDC 1587470 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: jaime.rodriguez@udc.es.
*E-mail: m.christmann@fu-berlin.de.
ORCID
Jaime Rodríguez: 0000-0001-5348-6970
Mathias Christmann: 0000-0001-9313-2392
Notes
(31) Saunders, C. M.; Tantillo, D. J. Mar. Drugs 2017, 15, 171.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The present research was financed in part by Grants from
́
Ministerio de Economia y Competitividad of Spain (AGL2015-
63740-C2-2-R and INMUNOTOP-RTC-2016-4611-1). J.R. and
R.E.M. acknowledge Xunta de Galicia and CESGA for the
computational facilities. We thank Silke Radetzki, Screening
D
DOI: 10.1021/acs.orglett.7b03706
Org. Lett. XXXX, XXX, XXX−XXX