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

Summary 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 with a photoreactive diazirine moiety, which was carried through seven reaction steps. Comprehensive assessment of the cytotoxicity 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 dolastatin class of marine natural products and has been isolated from the cyanobacterium Symploca hydnoides by Scheuer and co-workers in 2002 (7.5 mg, 0.014% of the dry weight) [1].Malevamide D (1) was reported to exhibit in vitro cytotoxicity in the subnanomolar range (IC 50 values about 0.7 nM).Closely related to malevamide D (1) is isodolastatin H (2), which is also active in vivo, but slightly weaker than dolastatin 10 (3) [2].
Although there has been no study regarding the mode of action, it can be assumed that malevamide D (1) acts similarly to dolastatin 10 (3) by inhibiting the formation of microtubuli from tubulin and, thereby, cell division.Dolastatin-type natural products continue to be tested in clinical trials for cancer therapy [3].This includes an antibody conjugate of monomethylauristatin E (MMAE), which has been approved for the treatment of Hodgkin lymphoma (Brentuximab vedotin) [4].In this paper, we report the first total synthesis of malevamide D (1) and an assessment of its in vitro cytotoxicity in a panel of 42 cell lines.We also synthesized a diazirinylated analog of 1, which, by photocrosslinking, might contribute to the understanding of the binding of 1 to tubulin.

Results and Discussion
Total synthesis of malevamide D The natural products malevamide D (1), isodolastatin H (2), and dolastatin 10 (3) share two β-methoxy-γ-amino acid building blocks.The closest structural analog of malevamide D (1) is isodolastatin H (2) with an identical ester section composed of dolaproine and (2S)-3-phenylpropan-1,2-diol.On the N-terminal side, the isopropyl and two sec-butyl side chains of isodolastatin H (2) are replaced by one sec-butyl and two isopropyl side chains, respectively, in the case of malevamide D (1).
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 and 13 completely, by repeated evaporation of DCM solutions.An assignment of the data sets to either of the two conformers was not possible.The occurrence of conformers has been mentioned by Scheuer and co-workers and is also known for dolastatin 10 [12] and symplostatin 1 [13].As an alternative route to the N-terminal tripeptide 11, we also investigated the coupling of the N-terminal dipeptide 15 with MMMAH tert-butyl ester (16, Scheme 2).However, after treatment of dipeptide 15 with secondary amine 16 (0.43 equiv) and DEPC (17, 2.5 equiv), the only isolable product was oxazolylphosphate 18 (35%) without any amide formation occurring.The structure elucidation of 18 required C-P coupling constant analysis, which showed doublet 13 C NMR signals of oxazole C-5 (10 Hz) and C-4 (6.2 Hz).To the best of our knowledge, this is only the second time that a peptidederived oxazol-5-ylphosphate has been characterized.Earlier, Boyd and co-workers had converted an oxazolone to an oxazolyl phosphate by treatment with excess diphenyl chlorophosphate [14].In our case, a similar pathway is to be assumed with the oxazolone being formed first.The secondary amine 16 appears to react too slowly to be able to compete with oxazolone formation.

Diazirinyl-substituted malevamide D
Encouraged by the strong cytotoxicity of 1, we addressed the synthesis of a photoactivatable analog.The question was whether such a derivative would still be cytotoxic.The phenyl ring of 1 was chosen as location of a trifluoromethyldiazirinyl substituent.Pettit and co-workers have shown that introduction of a phosphate moiety at the p-position of the phenyl ring of auristatin PE did not lead to loss of cytotoxicity [3].Recently, we have synthesized a hemiasterlin derivative with a diazirinylated indole moiety by incorporation of a thermally stable L-phototryptophan carrying a 1-azi-2,2,2-trifluoroethyl unit [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-dibromobenzene, followed by dihydroxylation, dioxolane protection and trifluoroacetylation of the remaining brominated position [16].
Refluxing of ketone 19 with NH 2 OH•HCl in pyridine led to nearly quantitative hydrolysis of the dioxolane affording diol oxime 20 as 95:5 mixture of Eand 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.
Reprotection of the diol unit of 20 and tosylation of 21 afforded tosyloxime (E)-22, which crystallized overnight from the neat mixture and was analyzed by X-ray crystallography [17].Thus, it became possible to assign the 19

Cytotoxicity studies of malevamide D and diazirinyl-substituted malevamide D
The synthesized 1 proved to be potently cytotoxic with a geomean IC 70 value of 1.5 nM (IC 50 0.7 nM) in a panel of

Conclusion
Our cytotoxicity test results for 1 confirm those obtained by Scheuer and co-workers who determined IC 50 values of about 0.7 nM against four cell lines (mouse lymphoma P-388, human lung carcinoma A-549, human colon carcinoma HT-29, human melanoma MEL-28) [1].We had hoped that introduction of a Brunner-type diazirine unit in the para-position of the phenyl ring of 1 could lead to an analog that was still active, because it had been shown in several SAR studies that limited variation of the amine or alcohol component at the C-terminal amide or ester moieties of the dolastatin family members can be tolerated without much loss of cytotoxicity.This includes epimerization of the thiazole-carrying stereocenter of 3 [18], replacement of the phenyl by a methyl group, or removal of the thiazole ring [7].Auristatins, of which some undergo clinical trials, are characterized by lacking the thiazole ring of 3 [19].Pettit and co-workers prepared auristatin TP, which carries a phosphate unit in the para-position of the phenyl ring, and also two aminoquinoline derivatives that were as cytotoxic as dolastatin 10 [3].Antibody conjugates of monomethylauristatin E functionalized on the C-terminal side have also shown promising pharmacological profiles [20,21].However, compound 30 is at least 200 times less active than the natural product 1.Lower cytotoxicity may be associated with solubility problems.We will now search for more water-soluble, photoactivatable analogs of 1. Nunes and coworkers had synthesized a benzophenone-labelled derivative of the analog HTI-286, which competes with 3, and were able to identify α-tubulin as binding partner [22].
Finally, a coupling yield of 68% was reached following the DEPC protocol.Desilylation (TBAF in THF/H 2 O) afforded 1 (HRESIMS found 755.49257, calcd.for C 40 H 68 NaN 4 O 8 755.49294) in 12% yield after 10 steps from valinol 4 (longest linear sequence).The optical rotation of the synthesized material ([α] D 28 −37, c 0.09, MeOH) is a little smaller than reported for the isolated natural product ([α] D 26 −55, c 0.10, MeOH) [1].The 1 H and 13 C NMR spectra of 1 shows two sets of signals in a ratio of ca.1:1.Clearly separated in the 13 C NMR spectrum are, for instance, the ester carbonyl signals of the conformers (174.0 and 173.3 ppm), the carbonyl signals of the pyrrolidine amide (170.5 and 170.6 ppm), and all phenyl carbon signals.Of the eastern section, only three of the four pyrrolidine carbon signals are broad.Most of the carbon signals of the western tripeptide of malevamide D are broadened (see Supporting Information File 1).The 1 H NMR spectrum shows many broad and overlapping signals, the assignment of which was only possible by careful interpretation of the 2D NMR data set.The only isolated signals belonging to different signal sets are located at δ H 3.84 (one of the diastereotopic OCH 2 signals) and 5.21 ppm (acylated carbinol).The two sets of signals are probably caused by the presence of both conformers of the acyl pyrrolidine.

Table 1 :
Cytotoxicities (IC 70 , IC 50 , nM) of malevamide D (1) against selected human cancer cell lines.a For complete results in the panel of 42 human cancer cell lines and a COMPARE analysis, see Supporting Information File 1.
a Similar results were obtained for 1. Determining a Spearman coefficient of 0.742 between 1 and 30 suggested that these compounds share the same mode of action, most probably antimitotic activity based on tubulin interaction.