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The solution conformation of didemnin B, the most potent member of a family of depsipeptides that shows antitumour, antiviral, and immunosuppressive activity, has been studied in chloroform solution using n.m.r. spectroscopy. 1H and 13C spectra have been assigned from analysis of a number of two-dimensional homonuclear and heteronuclear chemical shift correlation experiments which confirm the recently corrected primary structure of the molecule. The conformation of the peptide has been deduced from measurements of the temperature dependence of the NH chemical shifts, analysis of coupling constant data and primarily through NOE effects observed in the rotating frame. Interproton distance bounds determined from a quantitative analysis of the ROE data provide 41 constraints from which a family of closely related structures were calculated using distance geometry algorithms. A type II beta-turn involving residues Thr6, Leu7, and Pro8 is well represented in the computed conformers as is a hydrogen bonding interaction between the NH of Leu3 and the carbonyl oxygen of Thr6. This latter interaction causes the linear portion of the structure to fold back over the depsipeptide ring, imparting to it a degree of structural stability as well as giving the molecule a somewhat globular character. Only one transannular hydrogen bond, between Ist1 NH and Leu3 carbonyl, stabilizes the conformation of the depsipeptide, which has an irregular non-planar configuration. The small temperature coefficients (less than 2.0 x 10(-3) ppm/degrees C) for the NHs of Ist1 and Leu3 are consistent with their involvement in these hydrogen bonding interactions. We find that many of the structural features observed in the crystalline form of didemnin B are conserved in solution. Analysis of the 13C spin-lattice relaxation rates of the protonated carbons reveals small variations in effective correlation times at specific sites in the molecule. The data suggests that the peptide segment encompassing residues Leu3 through to Thr6 is in a more motionally restricted part of the structure.

Didemnin B has undergone trials in cancer patients, and has antiviral and immunosuppressive properties. [(3)H]didemnin B was administered intraperitoneally (i.p.) to mice at 320 or 1280 microg/kg. Urine and feces were collected until 168 h, at which time the mice were killed and tissues collected. Additionally, [(3)H]didemnin B was given i.p. at 320 microg/kg, and mice were killed at 1-120 h post-dosing. Radiolabel increased rapidly in blood then rapidly declined. Most radiolabel in urine, feces and tissues represented parent compound. Concentrations of [(3)H]didemnin B were greatest in the liver > gallbladder > lower digestive tract congruent with pancreas > spleen > kidney congruent with adipose tissue congruent with urinary bladder with urine. The pancreas had the longest terminal half-life of the tissues and the highest radioactivity at 7 days. Intermediate concentrations were in the duodenum congruent with jejunum > lung > iliopsoas > stomach congruent with testes congruent with skin > heart. Low concentrations were in the humerus congruent with femur congruent with quadriceps congruent with triceps >> brain. Fecal excretion accounted for 45.9%-58.3% of the dose and declined after 24 h, followed by an increase, suggesting possible enterohepatic recycling or an impact of circadian rhythms. Urinary excretion accounted for 18.4%-25.2% of the dose, but was minimal after 24 h. The concentrations were highest in organs previously found to be sensitive in animals and humans. Didemnin B should be evaluated in animal models for treatment of pancreatic cancer.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel marine-derived depsipeptide, Aplidin", in human plasma. The method was validated to demonstrate the specificity, recovery, limit of quantitation (LOQ), accuracy, and precision of measurements. The calibration range for Aplidin R was established using Aplidin " standards from 0.05-50 ng/mL in blank human plasma. The multiple reaction monitoring, based on the transition m/z 1110.7 → 295.3, was specific for Aplidin R , and that based on the transition m/z 1112.6→297.3 was specific for didemnin B (the internal standard); no endogenous materials interfered with the analysis of Aplidin and didemnin B from blank human plasma. The assay was linear over the concentration range 0.05-50.0 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9979 to 0.9999. The mean intra- and interday accuracies for all calibration standards (n = 12) ranged from 97 to 106% (

Reductive amination, amide formation using BOP [(1H-1,2,3-benzotriazol-1-yloxy)tris(dimethylamino)-phosphonium hexafluorophosphate] and esterification via DCC (dicyclohexylcarbodiimide) were the key synthetic steps to generate an advanced intermediate in the preparation of a didemnin B analog based on a sugar scaffolding (2). This analog should provide insight into the bioactive conformation of didemnin B.

Of the six marine-derived compounds that have reached clinical trials as antitumor agents three-didemnin B, Aplidine, and ecteinascidin 743-are derived from tunicates. Di-demnin B (DB), a cyclic depsipeptide from the compound tunicate Trididemnum solidum, was the first marine-derived compound to enter Phases I and II clinical trials. The Phase II studies, sponsored by the U. S. National Cancer Institute, indicated complete or partial remissions with non-Hodgkins lymphoma, but cardiotoxicity caused didemnin B to be dropped from further study. The closely related dehydrodidemnin B (DDB, Aplidine) was isolated in 1988 from a second colonial tunicate, Aplidium albicans, and spectroscopic studies assigned a structural formula in which a pyruvyl group in DDB replaced the lactyl group in DB and syntheses of DDB have been achieved. Aplidine is more active than DB and lacks DB's cardiotoxicity. It was introduced by PharmaMar into Phase I clinical trials in January 1999. The second family of tunicate-derived antitumor agents are the ecteinascidins (ETs), from the mangrove tunicate Ecteinascidia turbinata. The antitumor extracts of E. turbinata were first described in 1969, but the small amount of ETs in E. turbinata prevented their isolation for over a decade. The structures of ETs have been assigned mainly by spectroscopy. Phase II clinical trials with ET 743 are underway. Future supplies of ET's should be available from aquaculture or synthesis.

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD- fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis. J. Cell. Biochem. 72:269-278, 1999. r 1999 Wiley-Liss, Inc.

(1S,2S,3R)-3-Hydroxy-2-methoxy-1-cyclohexanecarboxylic acid was synthesized for the construction of an amino acid to be used in a constrained ring didemnin B analog ( 2 ). The amine functionality was introduced into the cyclohexane ring by reductive amination using sodium triacetoxyborohydride or by oxime formation followed by reduction.

An X-ray crystal analysis of φ-lactone N-{1-(N-{4-{[3-hydroxy-5-methyl-1-oxo-4-(N-L-threonylamino)heptyl]oxy}-2,5-dimethyl-1,3-dioxohexyl}-L-leucyl}-L-prolyl}-N,O-dimethyl-L-tyrosine hydrobromide hydrate (1a), C 42 H 66 N 5 O 11 + .Br - .H 2 O, was obtained in order to determine the backbone folding of the macrocycle and to compare the results obtained with those reported previously for the natural product didemnin B (1b). Some differences were noted in the torsion angles of the two conformers of the hydrobromide salt, denoted (1a) and (1a'). The conformation of (1a') resembled the conformation of (1b) more closely than did that of (1a). Certain regions of both crystal backbones were more flexible than those in didemnin B ; however, the transannular hydrogen bonds in both (1a) and (1a') were somewhat stronger than in (1b).

[formula: see text] Tamandarin A (1), a newly isolated natural product similar in structure to didemnin B (2), was shown to be somewhat more active in vitro than 2 against pancreatic carcinoma with an ED50 value 1.5 to 2 ng/mL. We report here the first total synthesis of 1. The key steps include a practical stereoselective synthesis of the Hiv-isostatine unit, high-yielding linear precursor formation, a successful macrocyclization, and coupling of the macrocycle with the side chain to afford tamandarin A (1).

Of the six marine-derived compounds that have reached clinical trials as antitumor agents three-didemnin B, Aplidine, and ecteinascidin 743-are derived from tunicates. Di-demnin B (DB), a cyclic depsipeptide from the compound tunicate Trididemnum solidum, was the first marine-derived compound to enter Phases I and II clinical trials. The Phase II studies, sponsored by the U. S. National Cancer Institute, indicated complete or partial remissions with non-Hodgkins lymphoma, but cardiotoxicity caused didemnin B to be dropped from further study. The closely related dehydrodidemnin B (DDB, Aplidine) was isolated in 1988 from a second colonial tunicate, Aplidium albicans, and spectroscopic studies assigned a structural formula in which a pyruvyl group in DDB replaced the lactyl group in DB and syntheses of DDB have been achieved. Aplidine is more active than DB and lacks DB's cardiotoxicity. It was introduced by PharmaMar into Phase I clinical trials in January 1999. The second family of tunicate-derived antitumor agents are the ecteinascidins (ETs), from the mangrove tunicate Ecteinascidia turbinata. The antitumor extracts of E. turbinata were first described in 1969, but the small amount of ETs in E. turbinata prevented their isolation for over a decade. The structures of ETs have been assigned mainly by spectroscopy. Phase II clinical trials with ET 743 are underway. Future supplies of ET's should be available from aquaculture or synthesis.

Didemnin B is a depsipeptide extracted from the marine tunicate Trididemnin cyanophorum. This agent is a potent inhibitor of L1210 growth in vitro and has activity against murine B16 melanoma, P388 leukemia, and M5076 sarcoma in vivo. The results of preclinical toxicologic tests demonstrated abnormalities in clotting parameters thought to be secondary to drug-induced liver dysfunction. Thirty-five patients with advanced cancer received didemnin B according to a 5-day bolus schedule with dose levels ranging from 0.03 to 2.00 mg/m2/d. The dose-limiting toxicity was nausea and vomiting. Sporadic elevation of the hepatic enzyme level occurred but was not dose limiting. Two patients had anaphylactic symptoms possibly related to the 5% polyoxyethylated castor oil (Cremophor EL, BASF, Ludwigshafen, Germany) vehicle during the drug infusion. Clinical bleeding was not observed and myelosuppression was not significant. No partial or complete tumor responses were seen. The recommended Phase II dose for the 5-day schedule is 1.6 mg/m2/d.

Several homo- and heteronuclear two-dimensional NMR techniques were used to assign all H- and C-resonances of the two conformers A and B of [7-(N-methyl-L-leucine)]didemnin B. Didemnine is a biologically highly active cytostaticum and immunosuppressivum. The assignment of the aliphatic C-atoms were done by the inverse H,C-COSY with TOCSY transfer which connects complete proton spin systems and represents them on C-atoms. The structure of both conformers (A and B) in (D6)DMSO solution was derived from homo- and heteronuclear couplings (J), temperature dependencies of NH protons, and NOE effects. Distances determined from the latter were used for refinements by restrained MD calculations using the GROMOS program. The solution structure of [Me-L-Leu7]didemnin B (A and B) was compared to that of didemnin B. The backbone structure of the macrocyclic ring and of the linear side-chain moiety are very similar in conformer A and didemnin B, though the Ist1-Hip2 region of the ring is slightly ex tended in conformer A. This may be caused by the influence of the Me-L-Leu7 residue in A and may be responsible for its reduced biological activity in comparison to didemnin B. The more weakly populated conformer B exhibits a βVI turn in the linear side-chain moiety.

Several homo- and heteronuclear two-dimensional NMR techniques were used to assign all H- and C-resonances of the two conformers A and B of [7-(N-methyl-L-leucine)]didemnin B. Didemnine is a biologically highly active cytostaticum and immunosuppressivum. The assignment of the aliphatic C-atoms were done by the inverse H,C-COSY with TOCSY transfer which connects complete proton spin systems and represents them on C-atoms. The structure of both conformers (A and B) in (D6)DMSO solution was derived from homo- and heteronuclear couplings (J), temperature dependencies of NH protons, and NOE effects. Distances determined from the latter were used for refinements by restrained MD calculations using the GROMOS program. The solution structure of [Me-L-Leu7]didemnin B (A and B) was compared to that of didemnin B. The backbone structure of the macrocyclic ring and of the linear side-chain moiety are very similar in conformer A and didemnin B, though the Ist1-Hip2 region of the ring is slightly ex tended in conformer A. This may be caused by the influence of the Me-L-Leu7 residue in A and may be responsible for its reduced biological activity in comparison to didemnin B. The more weakly populated conformer B exhibits a βVI turn in the linear side-chain moiety.

We present the application of several homo- and heteronuclear 1D- and 2D-NMR techniques to assign the 1H-NMR chemical shifts of the dominant conformation of didemnin B (2; three different conformations in (D6)DMSO solution in the ratio 8:1:1) and its conformational analysis, as well as the solution conformation of didemnin A (1). The conformations were refined by restrained molecular-dynamics calculations using the GROMOS program and by MOMO, a novel personal-computer-based interactive molecular-graphics and molecular-mechanics package, using experimental distances (via a H…H pseudo potential function) as restraints. The solution structures of 1 and 2 obtained by GROMOS and MOMO calculations were compared with each other and related to the recently solved crystal structure of 2. Focusing on the main conformer, the two kinds of the distance-restrained conformational calculations for 2 yielded a ‘solution structure’ close to the crystal structure. Almost all of the 40 restrained H…H distances coincided (within the estimated standard deviations) with those observed in the crystal structure. One more hydrogen bond was detected in solution involving the lactoyl OH group (disordered in the crystal structure) and the dimethyltyrosine (Me2Tyr5) carbonyl O-atom. The macrocyclic ring system in the modeled solution structure of 1 exhibited a topology close to those of the solution and crystal structures of 2. The main difference between 1 and 2 could be traced back to a significant change in the Ψ angle of the N-methyl-D-leucine (MeLeu7) residue. In 1, the N-methyl moiety of MeLeu7 points inward within the macrocyclic ring toward the 1st and Hip region. We also tested the suitability of structures obtained from NMR data as ‘search fragments’ in the ‘Patterson search approach’ of crystal-structure analysis. It proved possible to resolve the crystal structure of 2 a posteriori with the Patterson search program PATSEE, in this way.