Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 4 to 5 amino acid residues in defined sequence
Reexamination Certificate
2000-03-21
2004-02-03
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
4 to 5 amino acid residues in defined sequence
C530S333000, C530S338000, C514S017400
Reexamination Certificate
active
06686445
ABSTRACT:
Dolastatin 15, a known and potent antineoplastic constituent of the Indian Ocean shell-less mollusk
Dolabella auricularia
, was utilized as the lead substance from which were developed a series of novel derivatives. The present invention relates to methods of synthetically producing these new agents and presents their in vitro evaluations against a variety of murine and human cancer cell lines, and against a selection of bacteria and fungi. The effect of these derivatives on the inhibition of tubulin polymerization is also disclosed. Surprisingly, all of the new compounds, in which the C-terminal (S)-dolapyrrolidinone unit (Dpy, 5) of Dolastatin 15 is replaced with a series of structurally diverse, more readily available and less expensive amides, show cancer cell growth inhibition activities which are quite comparable to those of Dolastatin 15 (see: U.S. Pat. No. 4,879,278, Pettit et al.) All of the new compounds were, however, less potent than Dolastatin 15 as inhibitors of tubulin polymerization. The structurally modified peptides also caused mitotic arrest in cultured cells and inhibited the growth of a Gram-negative bacterium.
Some of this work was funded by Outstanding Investigator Grant CA-44344-01-08 awarded by the Division of Cancer Treatment, National Cancer Institute, DHHS. The United States government may have certain rights to this invention.
Marine organisms are an exceptionally productive source of biologically active and medicinally important substances bearing unique structures (see: FAULKNER, D. J. 1994, Marine Natural Products,
Natural Products Reports
, 11, 355; KOBAYASHI, M., et al. 1994, Bioactive substances isolated from marine sponge, a miniature conglomerate of various organisms,
Pure and Applied Chemistry
, 819; and, KÖNIG, G. et al. 1994, Biological activities of selected marine natural products,
Planta Medica
, 60, 532-537). Illustrative are the Indian Ocean (see: PETTIT et al. 1993, The isolation of dolastatins 10-15 from the marine mollusk
Dolabella auricularia, Tetrahedron
, 49, 9151) and Japanese (see: NAKAMURA et al., 1995, Stereochemistry and total synthesis of Dolastatin E.
Tetrahedron Letters
, 36, 5059, and the references cited therein) varieties of the sea hare
Dolabella auricularia
, from which a large number of antineoplastic and/or cytostatic linear and cyclic peptides, designated the dolastatins, have been isolated. Most of these potentially important peptides contain unprecedented amino acid units. Among these, the linear peptides, dolastatin 15 (1) (see: PETTIT et al., 1989a, Isolation and structure of the cytostatic linear depsipeptide dolastatin 15
, Journal of Organic Chemistry
, 54, 6005) and dolastatin 10 (2) (see: PETTIT et al., 1987, The isolation and structure of a remarkable marine animal antineoplastic constituent: Dolastatin 10
, Journal of the American Chemical Society
, 109, 6883) have exhibited the most potent antineoplastic activity (see: U.S. Pat. Nos. 4,816,444; 4,879,278; 4,978,744 and 5,554,725; and Hu et al., 1993, Effects of dolastatins on human B-lymphocytic leukemia cell lines,
Leukemia Research
, 17, 333) and have been selected for clinical development. Indeed, Phase 1 clinical trials of dolastatin 10 (2) have been ongoing under the auspices of the U.S. National Cancer Institute since November, 1995.
Since 1984, considerable research efforts have been directed to exploring structural modifications of dolastatin 10 (2) (PETTIT et al., 1995, Antineoplastic Agents 337, Synthesis of Dolastatin 10 Structural Modifications,
Anticancer Drug Design
, 10, 529) and dolastatin 15 (1) for the purpose of developing new potential anticancer drugs. A number of structural modifications of these peptides have been investigated in order to alter the antineoplastic activity of the parent molecule and eliminate from each peptide where possible, the more synthetically challenging units, especially the phenylalanine-derived C-terminal segments. Preliminary structure/activity studies based on dolastatin 10 (2) suggested that the thiazole-containing C-terminal unit could be adequately replaced with &bgr;-phenethylamine without significant loss of activity, whereas certain other modifications led to moderate or more drastic loss of antiproliferative activity (see: PETTIT et al., 1995, Antineoplastic Agents 337, Synthesis of Dolastatin 10 Structural Modifications,
Anticancer Drug Design
, 10, 529) without significant change in inhibitory effects on tubulin assembly. In the case at hand, a series of structural modifications of dolastatin 15 were made in which the C-terminal dolapyrrolidinone unit (5) was replaced by various amides. In contrast to dolastatin 10, major structural changes in the C-terminal amide unit of dolastatin 15 had essentially no adverse affect upon the inhibitory effects of the depsipeptide against tumor cell growth, but did result in a moderate reduction in the inhibition of tubulin polymerization.
One major factor driving the present research arises from the inescapable fact that there is not enough
Dolabella auricularia
in the world to allow sufficient quantities of effective components to be isolated therefrom to meet the need of the cancer-afflicted population in a commercially feasible manner. Therefore, a commercially effective synthesis must be developed which is capable of replicating a molecule containing only those substituents which are effective to control or arrest or mitigate the spread of cancer cells through a human system inflicted therewith. It is toward that goal that the present invention is directed.
Materials and Methods
All amino acids (S-configurations) and derivatives which are discussed herein were used as obtained from Sigma-Aldrich Co. Other reagents (DEPC, DCC, EDC-HCl, HOBt, NMM, Et
3
N, 4-pyrrolidinopyridine, TFA, etc. {Abbreviations used: DEPC (diethylphosphorocyanidate), DCC (N,N′-dicyclo-hexylcarbodiimide), EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), WRK (Woodward's reagent K, 2-ethylphenylisoxazolium-3′-sulfonate), BroP (tris(dimethylamino) phosphonium bromide hexafluorophosphate), HOBt (1-hydroxybenzotriazole), NMM (4-methylmorpholine), Et
3
N (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran), EtOAc (ethyl acetate), AcOH (acetic acid), Z (benzyloxycarbonyl), Boc (tert-butyloxycarbonyl)}) described were also obtained from Sigma-Aldrich and used without further purification. Amines 6a-r were either redistilled or recrystallized. All solvents were redistilled, and solvent extracts of aqueous solutions were dried over anhydrous magnesium sulfate or sodium sulfate. THF was distilled from LiAlH
4
. Reactions were monitored by thin-layer chromatography using ANALTECH silica gel GF (0.25 mm) plates visualized by either UV irradiation or 3% ceric sulfate in 3 N H
2
SO
4
solution as appropriate. Crude products were purified by flash chromatography over silica gel (E. Merck, DARMSTADT, 70-230 mesh). The final peptide products (12a-r) were further purified by rapid gel permeation chromatography in methanol on a column of lipophilic SEPHADEX LH-20.
Melting points were measured with an ELECTROTHERMAL digital melting point apparatus, model IA9200, and are uncorrected. Optical rotation measurements were recorded on a PERKIN-ELMER 241 polarimeter in methanol (unless otherwise noted) at 25° C. IR spectra were obtained using a NICOLET FTIR Model MX-1 instrument. All
1
H-NMR spectra were observed on a VARIAN GEMINI 300 MHz instrument with CDCl
3
or DMSO-d
6
as solvent as noted. The
13
C-NMR spectra were obtained with a UNITY 500 MHz instrument in CDCl
3
. EIMS data were recorded with a MAT 312 mass spectrometer. Elemental analyses were determined by Galbraith Laboratories, Inc. located in Knoxville, Tenn.
All compounds synthesized were first evaluated for in vitro antitumor activity against murine P388 lymphocytic leukemia cells and against murine L1210 leukemia cells and human CA46 Burkitt lymphoma cells using techniques described by Hamel & Lin (see: HAMEL, E., and LIN, C. M., 1993, Interaction of combretastat
Flahive Erik J.
Pettit George R.
Arizona Board of Regents, acting for and on behalf of Arizona St
Low Christopher S. F.
Lukton David
Mybeck Richard R.
Rosenfield Susan Stone
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