Total synthesis and cytotoxicity of the marine natural product malevamide D and a photoreactive analog

Article abstract of DOI:10.3762/bjoc.10.29

The marine natural product malevamide D from the cyanobacterium Symploca hydnoides was synthesized for the first time. The final peptide coupling linked the dolaisoleuine and dolaproine subunits. The phenyl group of malevamide D was also functionalized wi

Full text of DOI:10.3762/bjoc.10.29

Total synthesis and cytotoxicity of the marine natural
product malevamide D and a photoreactive analog
Werner Telle1, Gerhard Kelter2, Heinz-Herbert Fiebig2, Peter G. Jones3
and Thomas Lindel*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 316–322.
1Institute of Organic Chemistry, TU Braunschweig, Hagenring 30,
38106 Braunschweig, Germany, 2Oncotest Institute for Experimental
Oncology GmbH, Am Flughafen 12–14, 79108 Freiburg, Germany
and 3Institute of Inorganic and Analytical Chemistry, TU
Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
doi:10.3762/bjoc.10.29
Received: 17 October 2013
Accepted: 03 January 2014
Published: 03 February 2014
Email:
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Thomas Lindel* - th.lindel@tu-braunschweig.de
* Corresponding author
Guest Editor: J. S. Dickschat
Keywords:
© 2014 Telle et al; licensee Beilstein-Institut.
License and terms: see end of document.
cytotoxicity; diazirines; dolastatin analogs; marine natural products;
peptides; total synthesis
Abstract
The marine natural product malevamide D from the cyanobacterium Symploca hydnoides was synthesized for the first time. The
final peptide coupling linked the dolaisoleuine and dolaproine subunits. The phenyl group of malevamide D was also functional-
ized with a photoreactive diazirine moiety, which was carried through seven reaction steps. Comprehensive assessment of the cyto-
toxicity in a panel of 42 human cancer cell lines revealed a geomean IC70 value of 1.5 nM (IC50 0.7 nM) for malevamide D,
whereas the photoreactive derivative proved to be less active by a factor of at least 200. COMPARE analysis indicated tubulin
interaction as likely mode of action of malevamide D.
Introduction
The depsipeptide malevamide D (1, Figure 1) belongs to the Although there has been no study regarding the mode of action,
dolastatin class of marine natural products and has been isolated it can be assumed that malevamide D (1) acts similarly to dolas-
from the cyanobacterium Symploca hydnoides by Scheuer and tatin 10 (3) by inhibiting the formation of microtubuli from
co-workers in 2002 (7.5 mg, 0.014% of the dry weight) [1]. tubulin and, thereby, cell division. Dolastatin-type natural prod-
Malevamide D (1) was reported to exhibit in vitro cytotoxicity ucts continue to be tested in clinical trials for cancer therapy [3].
in the subnanomolar range (IC50 values about 0.7 nM). Closely This includes an antibody conjugate of monomethylauristatin E
related to malevamide D (1) is isodolastatin H (2), which is also (MMAE), which has been approved for the treatment of
active in vivo, but slightly weaker than dolastatin 10 (3) [2]. Hodgkin lymphoma (Brentuximab vedotin) [4].
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Beilstein J. Org. Chem. 2014, 10, 316–322.
of the lithium enolate of t-BuOAc (Scheme 1). β-Hydroxyhexa-
noate 6 was obtained as a mixture of diastereomers (5:4), which
was separated by column chromatography (silica) on a multi-
gram scale. Treatment of (3R,4S)-6 with Meerwein's salt in
presence of proton sponge afforded Cbz-protected MMMAH
tert-butyl ester 7 in 87% yield. There are also other, enantiose-
lective syntheses of 7 [7] and Boc-protected versions of
MMMAH [8,9], but we intended to also have the (3S)-epimer of
7 at our disposal.
After hydrogenation of 7, the resulting secondary amine was
coupled with Cbz-valine (8) affording dipeptide 9 (DEPCl,
diethyl phosphorochloridate), 80% over two steps, Scheme 1).
N,N-Dimethylisoleucine (10), obtained by reductive dimethyla-
tion of isoleucine with aqueous formaldehyde and H2 on Pd/C
[10,11], was appended at the N-terminus (DEPC, diethyl phos-
phorocyanidate) affording tripeptide tert-butyl ester 11. Treat-
ment with TFA liberated the free acid 12, which was coupled
with the trifluoroacetate of dolaproine ester 13, liberated from
the N-Boc-protected analog [2].
Figure 1: Structures of the strongly cytotoxic marine natural products
malevamide D (1), isodolastatin H (2), and dolastatin 10 (3).
With the subsequent coupling step affording TBDPS-protected
malevamide D (14), we initially had difficulties. It proved to be
crucial to remove excess of TFA from both coupling partners 12
In this paper, we report the first total synthesis of malevamide D and 13 completely, by repeated evaporation of DCM solutions.
(1) and an assessment of its in vitro cytotoxicity in a panel of 42 Finally, a coupling yield of 68% was reached following the
cell lines. We also synthesized a diazirinylated analog of 1, DEPC protocol. Desilylation (TBAF in THF/H2O) afforded 1
which, by photocrosslinking, might contribute to the under- (HRESIMS found 755.49257, calcd. for C40H68NaN4O8
standing of the binding of 1 to tubulin.
755.49294) in 12% yield after 10 steps from valinol 4 (longest
linear sequence). The optical rotation of the synthesized ma-
terial ([α]D28 −37, c 0.09, MeOH) is a little smaller than
reported for the isolated natural product ([α]D26 −55, c 0.10,
Results and Discussion
Total synthesis of malevamide D
The natural products malevamide D (1), isodolastatin H (2), and MeOH) [1]. The 1H and 13C NMR spectra of 1 shows two sets
dolastatin 10 (3) share two β-methoxy-γ-amino acid building of signals in a ratio of ca. 1:1. Clearly separated in the
blocks. The closest structural analog of malevamide D (1) is 13C NMR spectrum are, for instance, the ester carbonyl signals
isodolastatin H (2) with an identical ester section composed of of the conformers (174.0 and 173.3 ppm), the carbonyl signals
dolaproine and (2S)-3-phenylpropan-1,2-diol. On the N-termi- of the pyrrolidine amide (170.5 and 170.6 ppm), and all phenyl
nal side, the isopropyl and two sec-butyl side chains of isodola- carbon signals. Of the eastern section, only three of the four
statin H (2) are replaced by one sec-butyl and two isopropyl pyrrolidine carbon signals are broad. Most of the carbon signals
side chains, respectively, in the case of malevamide D (1).
of the western tripeptide of malevamide D are broadened (see
Supporting Information File 1). The 1H NMR spectrum shows
The total synthesis of 1 adopts the strategy by Yamada and many broad and overlapping signals, the assignment of which
co-workers in their total synthesis of 2, which sets the first retro was only possible by careful interpretation of the 2D NMR data
cut at the tertiary amide bond between dolaisoleuine and set. The only isolated signals belonging to different signal sets
dolaproine [2]. The tert-butyl ester 7 of Cbz-protected are located at δH 3.84 (one of the diastereotopic OCH2 signals)
3-methoxy-5-methyl-4-(methylamino)hexanoic acid and 5.21 ppm (acylated carbinol). The two sets of signals are
(MMMAH) was obtained in multigram quantities within five probably caused by the presence of both conformers of the acyl
steps from Cbz-valine (8), adapting a convenient sequence pyrrolidine. An assignment of the data sets to either of the two
developed for dolaisoleuine by Pettit and co-workers [5]. Cbz- conformers was not possible. The occurrence of conformers has
protected N-methylvalinol (4) [6] was oxidized to the aldehyde been mentioned by Scheuer and co-workers and is also known
5 under Parikh–Doering conditions, followed by aldol addition for dolastatin 10 [12] and symplostatin 1 [13].
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Beilstein J. Org. Chem. 2014, 10, 316–322.
Scheme 1: Total synthesis of malevamide D (1). a) DMSO (16 equiv), NEt3 (5 equiv), pyridine·SO3 (5 equiv), 0 °C, 1 h, 91%. b) LDA (2.5 equiv),
t-BuOAc (1.3 equiv), THF, −78 °C, 1 h; separation of diastereomers, (3R,4S)-6: 47%. c) 1,8-bis(dimethylamino)naphthalene (2.5 equiv), Me3OBF4
(2.6 equiv), 4 Å MS, 1,2-DCE, 0 °C, 2 h, rt, 16 h, 87%. d) H2, Pd/C (5%), MeOH, rt, 1 h. e) 8 (3 equiv), NEt3 (4 equiv), DEPCl (2 equiv), DCM, 0 °C,
2 h, rt, 16 h, 80% from 7. f) H2, Pd/C (5%), MeOH, rt, 90 min. g) 10 (3 equiv), NEt3 (4 equiv), DEPC (1.5 equiv), DCM, 0 °C, 2 h, rt, 16 h, 83% from 9.
h) TFA, DCM, 0 °C, 90 min, quant. i) 13 (1 equiv), NEt3 (6 equiv), DEPC (1.3 equiv), DCM, 0 °C to rt, 16 h, 68%. j) TBAF·3H2O (5 equiv), THF/H2O
19:1, rt, 3 h, 71%. DEPCl: diethyl phosphorochloridate; DEPC: diethyl phosphorocyanidate.
As an alternative route to the N-terminal tripeptide 11, we also amine 16 appears to react too slowly to be able to compete with
investigated the coupling of the N-terminal dipeptide 15 with oxazolone formation.
MMMAH tert-butyl ester (16, Scheme 2). However, after treat-
ment of dipeptide 15 with secondary amine 16 (0.43 equiv) and Diazirinyl-substituted malevamide D
DEPC (17, 2.5 equiv), the only isolable product was Encouraged by the strong cytotoxicity of 1, we addressed the
oxazolylphosphate 18 (35%) without any amide formation synthesis of a photoactivatable analog. The question was
occurring. The structure elucidation of 18 required C–P whether such a derivative would still be cytotoxic. The phenyl
coupling constant analysis, which showed doublet 13C NMR ring of 1 was chosen as location of a trifluoromethyldiazirinyl
signals of oxazole C-5 (10 Hz) and C-4 (6.2 Hz). To the best of substituent. Pettit and co-workers have shown that introduction
our knowledge, this is only the second time that a peptide- of a phosphate moiety at the p-position of the phenyl ring of
derived oxazol-5-ylphosphate has been characterized. Earlier, auristatin PE did not lead to loss of cytotoxicity [3]. Recently,
Boyd and co-workers had converted an oxazolone to an we have synthesized a hemiasterlin derivative with a diazirinyl-
oxazolyl phosphate by treatment with excess diphenyl ated indole moiety by incorporation of a thermally stable
chlorophosphate [14]. In our case, a similar pathway is to be L-phototryptophan carrying a 1-azi-2,2,2-trifluoroethyl unit
assumed with the oxazolone being formed first. The secondary [15].
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Beilstein J. Org. Chem. 2014, 10, 316–322.
Scheme 2: Formation of oxazolylphosphate 18 on attempted DEPC-
mediated coupling of dipeptide 15.
Racemic photo phenylpropanediol 25 was obtained starting
from aryl(trifluoromethyl)ketone 19 (Scheme 3), which itself
was synthesized via Grignard monoallylation of 1,4-dibromo-
benzene, followed by dihydroxylation, dioxolane protection and
trifluoroacetylation of the remaining brominated position [16].
Refluxing of ketone 19 with NH2OH·HCl in pyridine led to
nearly quantitative hydrolysis of the dioxolane affording diol
oxime 20 as 95:5 mixture of E- and Z-isomers. However, if
solid NaOH was added to the reaction mixture and heated for
several hours, the dioxolane survived, affording a 1:1 mixture
of E/Z-oximes, which were tosylated after short work-up to (E)-
and (Z)-22.
Scheme 3: Synthesis of tosyloximes (Z)-22 and (E)-22, X-ray struc-
ture of (E)-22. a) NH2OH·HCl (1.5 equiv), pyridine, reflux, 2 h, 80%. b)
2,2-dimethoxypropane (12.4 equiv), p-TsOH·H2O (5 mol %), DMF, rt,
1 h, 95%. c) p-TsCl (1.05 equiv), NEt3 (1.3 equiv), DCM, 0 °C to rt,
18 h, 83%. d) (i) NH2OH·HCl, NaOH, EtOH, pyridine, reflux (for details
see Supporting Information File 1); (ii) p-TsCl (1.05 equiv), NEt3
(1.3 equiv), DCM, 0 °C to rt, 18 h, 74%.19F NMR chemical shifts (ppm)
in CDCl3.
Reprotection of the diol unit of 20 and tosylation of 21 afforded pyrrolidine, coupling with tripeptide 12 afforded O-silylated
tosyloxime (E)-22, which crystallized overnight from the neat diazirinyl-substituted malevamide D (29, 68%). Desilylation
mixture and was analyzed by X-ray crystallography [17]. Thus, (TBAF, THF/H2O 19:1) led to a product mixture, the sep-
it became possible to assign the 19F NMR chemical shifts of aration of which required semipreparative HPLC (RP-18,
both isomers ((E)-22: δF −66.9; (Z)-22: δF −61.9).
MeOH/H2O 9:1). Diazirinyl-substituted malevamide 30 was
obtained in 33% isolated yield as a mixture of two epimers
Conversion of (E)-22 to diaziridine 23 (colorless solid, δF differing at the carbinol center (HRESIMS found 863.48644,
−75.96, −75.93) was performed quantitatively (liquid NH3 in calcd. for C42H67NaF3N6O8, 863.48647). In the NMR spectra,
t-BuOMe, Scheme 4). As expected, the NMR spectra of the four sets of signals were observed because of additional cis/
colorless solid 23 showed two sets of signals. Oxidation of 23 trans isomerism of the acyl pyrrolidine moiety. As in the case
with I2/NEt3 to diazirine 24 (δF −65.7) and acidic deprotection of 1, the tripeptidic western half showed mostly broad signals,
afforded diazirine diol 25. Figure 2 shows the DSC (differential whereas the signals of the phenyl carbons are resolved better,
scanning calorimetry) profile of 25, which melts at 54 °C and even if not fully separated.
starts to decompose above 80 °C, indicating sufficient thermal
Cytotoxicity studies of malevamide D and
stability for biological studies. Monosilylation of the primary
hydroxy group of 25 afforded building block 26. Coupling with diazirinyl-substituted malevamide D
Boc-protected dolaproine (27, DCC/DMAP/CSA) yielded ester The synthesized 1 proved to be potently cytotoxic with a
28 as a mixture of two diastereomers. After deprotection of the geomean IC70 value of 1.5 nM (IC50 0.7 nM) in a panel of
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Beilstein J. Org. Chem. 2014, 10, 316–322.
42 human cancer cell lines (see Table 1 and Supporting Infor-
mation File 1). The cytotoxicity was about 10-fold higher than
that of paclitaxel. The breast cancer cell lines MAXF-401 (IC70
0.2 nM) and MCF-7 (0.5 nM), the colon cancer cell lines HT-29
(0.3 nM) and RKO (0.5 nM), the lung cancer cell line H460
(0.7 nM), the liver cancer cell line LIXF-575 (0.6 nM), and the
renal cancer cell line RXF-393 (0.4 nM) all proved to be very
sensitive. Against 36 of the 42 cell lines, IC70 values of below
10 nM were determined. The most resistant cell lines were
shown to be RXF-1781 (kidney), CXF-269 (colon), MEXF-276
Table 1: Cytotoxicities (IC70, IC50, nM) of malevamide D (1) against
selected human cancer cell lines.a
histotype
cell line
IC70
IC50
Colon
HT-29
0.3
5.0
0.7
0.4
0.2
0.6
1.0
4.0
0.8
0.4
0.7
0.5
0.2
0.6
0.4
0.3
0.2
0.4
0.7
1.0
0.6
0.3
0.5
0.4
Stomach
Lung, adeno
GXF-251
LXFA-629
Lung, large cell LXFL-529
Breast
MAXF-401
MEXF-462
OVXF-899
PAXF-1657
PRXF-22Rv1
Melanoma
Ovary
Pancreas
Prostate
Scheme 4: Synthesis of photo malevamide D 30. a) NH3(l), t-BuOMe,
−40 °C, 2 h, rt, 16 h, quant. b) I2 (1.2 equiv), NEt3 (2.2 equiv), Et2O,
0 °C, 15 min, rt, 1 h, 99%. c) THF, 2 N HCl, rt, 3 h, 97%. d) TBDPSCl
(1.1 equiv), imidazole (2.2 equiv), DMF, 0 °C, 1 h, 80%. e) 27
(0.97 equiv), DMAP (0.41 equiv), DCC (1 equiv), CSA (0.23 equiv),
0 °C to rt, 16 h, 69%. f) TFA, DCM, 0 °C, 90 min. g) 12 (1 equiv), NEt3
(6 equiv), DEPC (2.14 equiv), DCM, 0 °C, 150 min, 68%.
Mesothelioma PXF-1752
Kidney
Uterus
RXF-486L
UXF-1138L
aFor complete results in the panel of 42 human cancer cell lines and a
COMPARE analysis, see Supporting Information File 1.
h) TBAF·3H2O (5 equiv), THF/H2O 19:1, 0 °C, 2 h, 33%.
Figure 2: DSC curve of diazirine 25, heating rate 5 °C/min.
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(melanoma, IC70 about 50 nM, each), and in particular LXFA-
289 (lung, adenocarcinoma), PAXF-546L (pancreas), and PXF-
1118 (pleuramesothelioma, IC70 > 100 nM, IC50 about 50 nM,
each). Diazirinyl-substituted malevamide D 30 was about 200
times less cytotoxic than 1.
Supporting Information
Supporting Information File 1
Procedures of synthesis and biotest, X-ray data and 1H,
13C NMR spectra of selected compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-29-S1.pdf]
To obtain clues on the likely mode of action of 1 and 30, the in
vitro activity data were compared with those of 177 reference
compounds by COMPARE analysis. Individual IC50 values of a
test compound were correlated with those of 177 reference
compounds as determined for Oncotest's 42 cell line panel and
expressed quantitatively as Spearman correlation coefficients
(see Supporting Information File 1). Furthermore, 1 and 30
were correlated to each other. For compound 30, COMPARE
analysis revealed the highest similarities towards tubulin inter-
acting compounds like the vinca alkaloids vinfluine, vincristine,
vindesine and the taxoid compound docetaxel. Furthermore,
good correlations were detected towards PLK-1 inhibitors.
Similar results were obtained for 1. Determining a Spearman
coefficient of 0.742 between 1 and 30 suggested that these com-
pounds share the same mode of action, most probably antimi-
totic activity based on tubulin interaction.
Acknowledgements
We thank Merck KGaA (Darmstadt, Germany) for the generous
gift of chromatography materials. The BASF Group
(Ludwigshafen, Germany) and Honeywell Specialty Chemicals
Seelze GmbH (Seelze, Germany) are thanked for the donation
of solvents.
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