WO1996011683A1 - Nouveaux agents cytotoxiques - Google Patents

Nouveaux agents cytotoxiques Download PDF

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Publication number
WO1996011683A1
WO1996011683A1 PCT/US1995/013234 US9513234W WO9611683A1 WO 1996011683 A1 WO1996011683 A1 WO 1996011683A1 US 9513234 W US9513234 W US 9513234W WO 9611683 A1 WO9611683 A1 WO 9611683A1
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Prior art keywords
oxo
taxol
compounds
xylosyl
compound
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PCT/US1995/013234
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English (en)
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WO1996011683A9 (fr
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Hauser Chemical Research, Inc.
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Publication date
Application filed by Hauser Chemical Research, Inc. filed Critical Hauser Chemical Research, Inc.
Priority to AU40012/95A priority Critical patent/AU4001295A/en
Publication of WO1996011683A1 publication Critical patent/WO1996011683A1/fr
Publication of WO1996011683A9 publication Critical patent/WO1996011683A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel

Definitions

  • This invention relates to taxane derivatives. More particularly, this invention relates to derivatives of naturally occurring xylosyl substituted taxanes. These new compounds demonstrate surprising tubulin binding activity and cytotoxicity. "Although plant extracts have been used as anticancer agents for centuries, only a handful of plant-derived natural products have been found to show clinically useful activity, and taxol is clearly a member of this select group.” (Kingston, "The Chemistry of Taxol, Pharmac.
  • Taxol shown as composition 1 in Figure 1, is a compound that occurs in the bark of the Pacific yew tree as well as other members of the taxus species. Taxol has been identified as having significant tubulin binding activity (Schiff, P.B. et al.. “Promotion of Microtubule Assembly in vitro by Taxol," Nature, Vol. 277: 655-67 (Feb. 1979)), and when delivered to the cell, it has significant cytotoxicity. Taxol was recently approved for the treatment of refractory ovarian cancer by the United States Food and Drug Administration.
  • Taxol is unusual among cytotoxic agents in that its method of action is through stabilization of polymerized tubulin, i.e., "tubulin binding." Because this mechanism is different from conventional cytotoxic agents, it is a highly important addition to the arsenal of cancer therapy
  • Taxol is a complex molecule, and the specific attributes of its chemistry responsible for its tubulin binding activity have not been identified. Numerous taxol derivatives, having one or more substituted side groups, have been tested for tubulin binding activity with varying and unpredictable results. It is apparent from those tests that even minor changes in the taxol molecule may result in significantly different tubulin binding and cytotoxicity.
  • Taxotere i.e., compound 2 shown in Figure 1, which is a semisynthetic derivative of taxol with improved water solubility.
  • Taxotere is a registered trademark of Rhone-Poulenc Rorer. Taxotere has been compared with taxol in phase I clinical trials.
  • Taxotere 2 differences between taxol 1 and Taxotere 2 are minor (see Figure 1), enhanced in vitro tubulin binding activity is observed for Taxotere. Taxotere is slightly more active as a promoter of tubulin polymerization, 1.5 times more potent as an inhibitor of replication in mouse macrophage-like
  • J774.2 cells and in P388 murine leukemia cells at least five-fold more potent in taxol-resistant tumor cells.
  • Taxol itself is difficult to deliver to the target site in vivo due to its poor solubility in water and the need to use delivery media which themselves have certain deficiencies.
  • the synthetic taxol derivatives described herein have not previously been described, and the literature does not suggest that they would exhibit cytotoxicity and enhanced tubulin assembly. Indeed, changes to taxol at the C-7 site including acylation, attachment of polar groups and
  • the compounds of this invention have been tested for tubulin binding and cytotoxicity, using B-16 melanoma.
  • the compounds have been screened by the National Cancer Institute using a number of cancer cell lines with surprisingly good results. The National Cancer Institute has selected these compounds for further testing as
  • R represents Ac or H
  • R' represents: ;
  • xylopyranoside substituted taxanes are hemialdals.
  • the reduced form of the "oxo” materials are referred to as “oxo diols.”
  • oxo diols For example, the formation of "oxo-10-DAXT diol" from oxo-10-DAXT is shown in Figure 8. Unless otherwise indicated herein, "oxo" is intended to include both the hemialdals and the diols.
  • This invention relates to the treatment of certain 7-xylosyl substituted naturally occurring taxanes with an oxidizing agent sufficient to partially oxidize the xylosyl group and to generate in good yield the hemialdal
  • the hemialdal "oxo" compounds of the present invention are obtained by oxidation of naturally occurring xylosyl substituted taxanes.
  • the oxidation reactions of this invention are mild enough that their progress can be
  • HPLC chromatography
  • xylopyranoside are intended to mean the same thing and are used interchangeably.
  • introduction of an oxidizing agent to a xylopyranoside substituted taxane could cause oxidation at sites other than C-7 on the taxane ring, such as the 2'-hydroxy and the 10-position on 10-DAXT. In fact, oxidation does not appear to occur at these positions.
  • introduction of an oxidizing agent to mixtures containing glycoside substituted taxanes and other non-glycoside substituted taxane compounds does not result in oxidation of the other taxanes. Such mixtures occur in biomass or partial separations of extracts of such biomass. This selectivity enables the oxidative cleavage of the glycosides to be conducted at various stages during the isolation of taxol from Taxus brevifolia or other naturally-occurring materials.
  • the oxo compounds may be converted to taxol or taxol precursors or themselves may be used as cytotoxic agents.
  • the oxidation of the xylopyranoside side chain is accomplished by using an effective amount of an oxidizing agent.
  • Effective oxidizing agents include, but are not limited to, periodic acid and salts thereof, such as sodium or potassium periodate or metaperiodate, and lead
  • Additional oxidizing agents may include an effective amount of one or more oxidizing agents can be utilized. In particular, one or more of these oxidizing agents can be employed. The relative effectiveness of the various possible oxidizing agents depends upon the
  • the preferred oxidizing agent is periodate.
  • the oxidizing agent can be employed, but generally the oxidizing agent should be present in the range of 1-10 molar equivalents of oxidizing agent per mole of xylopyranoside taxane. Preferably, at least 2
  • the oxidation is preferably accomplished utilizing an effective dissolution amount of a taxane solvent which is compatible with the particular oxidizing agent or agents employed.
  • Typical solvents include tetrahydrofuran, water, acetone, dioxane, acetic acid, or mixtures thereof or other taxane solvents known to one of ordinary skill in the art.
  • Methanol and other alcohols are particularly unacceptable and should not be used, because they interact with the intermediate hemialdal compound.
  • oxidative conversion of the xylopyranoside functional group or mixtures thereof as the oxidizing agent is oxidized in two to twenty-four hours for solutions which are approximately 0.1 mg./ml. of taxane using 2-10 molar equivalents of the periodate reagent.
  • R represents Ac or H
  • R' represents:
  • R" represents:
  • the alcohol solvents e.g., methanol, used in the specific examples of the Rao patent will ensure that hemialdals are not formed.
  • the hemialdal side chain at the C-7 site in the "oxo" compounds of the present invention are in equilibrium with isomers of that side chain. These include hemialdals in open (i.e., non-cyclic) form. However, the equilibrium constant is such that the following hemialdal structure is greatly preferred.
  • the structure shown above is intended to include the isomers of the C-7 hemialdal side chain in equilibrium with it.
  • the hemialdal nature of the "oxo" compounds can be partially elucidated using spectroscopic analyses such as nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS).
  • NMR nuclear magnetic resonance spectroscopy
  • MS mass spectroscopy
  • the 7-xylopyranoside taxanes from the oxidation products, the "oxo" compounds.
  • several features of the NMR spectra are useful for determining the hemialdal structure of the "oxo" compounds as described in the preceding paragraph.
  • the high-field NMR spectra were acquired using dimethyl sulfoxide-d 6 as solvent with residual dimethyl sulfoxide as an internal standard.
  • 7-xylopyranoside taxane derivatives show well resolved resonances at 2.85, 3.05, 3.25, 3.60, 4.10, 4.25, 4.80, and 4.89 ppm. attributable to the xylopyranoside group.
  • the 1 H-NMR spectra of the "oxo" compounds generally show no resonances at the positions listed for the xylopyranoside group. However, the 1 H-NMR spectra of the "oxo" compounds consistently show new multiple resonances at 3.40, 4.15, 5.10, 6.45, 6.6 and 6.8 ppm. attributable to the hemialdal group.
  • the resonances in the 1 H-NMR spectra of the "oxo" compounds are generally not well resolved because the "oxo" compounds are an equilibrium mixture of the isomers at the C-7 site.
  • the 13 C-NMR spectra for the starting material, the 7-xylopyranoside taxanes, generally show a strong resonance in the region from 104 to 107 ppm for the
  • the mass spectral data for four compounds, "oxo-XT” (4), “oxo-10-DAXT” (6), “oxo-10-DAXTB” (10), and “oxo-10-DAXTC” (14), show molecular ion signals that correspond with a mass for the hemialdal structure plus the mass of sodium ion.
  • the additional mass of sodium ion is a commonly observed effect of the electrospray MS method. No strong ion signals were observed which would indicate the presence of the dialdehyde structure.
  • the hemialdal nature of the "oxo" compounds can be further elucidated by treating a solution of the "oxo" compound with a silylating agent, triethylsilyl chloride (see Figure 9).
  • a silylating agent triethylsilyl chloride
  • the product mixture from this reaction is stable to silica gel chromatography.
  • the purified silylated product shows three triethylsilyl groups when analyzed by NMR and MS (see Example 6).
  • the hemialdal-type structure has three reactive silylation sites available, the
  • dialdehyde-type structure has only one reactive silylation site. Therefore, the presence of three triethylsilyl groups in the product of the silylation is further evidence for the hemialdal-type structure as opposed to the dialdehyde-type structure.
  • Patent does not disclose or suggest that the dialdehydes demonstrate tubulin binding or cytotoxic activity.
  • Example 5 The process for converting "oxo" hemialdals to diols is illustrated in Example 5 and Figure 8.
  • the process involves treatment of a tetrahydrofuran/water/acetic acid solution of the "oxo" derivative with a reducing agent , such as sodiumcyanoborohydride) at room temperature for a period of approximately 24 hours.
  • a reducing agent such as sodiumcyanoborohydride
  • the reaction can be monitored by TLC or HPLC.
  • the diol product can be isolated by
  • the "oxo" compounds show good tubulin binding and cytotoxicity activity with in vitro testing.
  • the compounds of this invention have been successfully tested for tubulin binding and for their effect on B16 melanoma.
  • the compounds tested demonstrated improved tubulin binding and
  • cytotoxicity over the precursors from which they were derived.
  • oxo-XT (4), the tubulin binding and cytotoxicity data are comparable to results for taxol.
  • Example 7 illustrates tests performed to measure tubulin binding and the effect of various compounds on B16 melanoma.
  • Example 8 illustrates the results of "oxo" tests performed by the National Cancer Institute on the cytotoxic effects of various "oxo" compounds on a number of different cancer cell lines.
  • HPLC high-pressure liquid chromatography
  • a system consisting of a model L-6200 pump, Model AS-4000 or L-3000 UV/VIS/DAD detector (Hitachi Instruments, Inc.).
  • the system was equipped with an NEC 286 computer with 40M hard drive and Lab manager HPLC software (Hitachi Instruments, Inc.).
  • HPLC columns used included a 4.6 mm. ⁇ 250 mm. Phenyl column, packed with 5 ⁇ m diphenyl material (Supelco, Inc.); a 4.6 mm. ⁇ 250 mm., 5 ⁇ m, 60 angstrom Pentafluorophenyl (PFP) column (ES
  • Example 1 This example illustrates the conversion of XT, i.e., 7-xylosyl taxol, to "oxo-XT.”
  • Flash silica gel chromatography yielded the purified fully silylated material. A 20-30% EtOAc/hexane gradient elution was used, and 770 mg of the fully silylated compound was recovered. This corresponds to a yield of 65% for this step.
  • Example 2 This example illustrates the conversion of
  • 10-DAXT i.e., 10-deacetyl-7-xylosyl taxol
  • oxo-10-DAXT i.e., 10-deacetyl-7-xylosyl taxol
  • Example 3 This example illustrates the conversion of 10-DAXTB, i.e., 10-deacetyl- 7-xylosyl taxol B, to "oxo-10-DAXTB" .
  • Example 4 This example illustrates the conversion of
  • 10-DAXTC i.e., 10-deacetyl-7-xylosyl taxol C, to "oxo-10-DAXTC.”
  • Example 5 This example illustrates the reduction of "oxo-10-DAXT" to the 7-oxo-10-DAXT diol form.
  • Example 6 This example illustrates the silylation of "oxo-XT.”
  • Example 7 This example illustrates the results of
  • Tubulin free of microtubule-associated proteins was purified from bovine brain as described in Algaier, J.; Himes, R.H., "The Effect of Dimethyl Sulfoxide on the Kinetics of Tubulin Assembly” Biochim. Biophys. Acta, Vol 954, pp 235-243, 1998.
  • the assembly reaction was done at 37°C in PEM buffer (0.,1 M Pipes, pH 6.9, 1 mM EGTA, and 1 mM MgSo 4 ) at a protein concentration of 1 mg/ml (10 ⁇ K) in the presence of taxol or taxol analogs and 0.5 mM GTP.
  • the reaction was monitored by the increase in the apparent absorbance at 350 nm.
  • Taxol has been included in Table 1 for reference. In addition, each sample is compared to a control sample of taxol as reported in the columns: "ED 50 /ED 50 Taxol” (for Tubulin Assembly), and "ED 50 /ED 50 Taxol” (for B16
  • taxol shows a value of approximately 1 in these columns. A number less than 1 in these columns indicates greater activity than taxol. A number greater than 1 in these columns indicates lower activity than taxol.
  • test procedures employed by that organization for determining th cytotoxic effect of various materials.
  • the test procedures are generally described in Boyd, M.R. et al. "Data Display and Analysis Strategies for the NCI Disease-Oriented In Vitro Antitumor Drug Screen,” contained as Chapter 2 in
  • Tables 2 through 7 are summaries of data for different cytotoxic "oxo" compounds of this invention. In each table are listed the "panel" or type of human cancer cell line, the specific cell line (coded by the discoverer or the National Cancer Institute), and the log 10 values for: GI50 (for the oxo compound), GI50 TAX (for taxol), TGI (for the compound), TGI TAX (for taxol), LC50 (for the compound), an LC50 TAX (for taxol). The listed values are derived from dose response curves for each compound and for taxol.
  • GI50 As indicated in the Boyd article, the terms GI50, TGI and LC50 are defined as follows:
  • the GI50 value for a specific cell line is compared with the value listed for GI50 TAX.
  • the TGI value for a specific cell line is compared with the value listed for TGI TAX.
  • the LC50 value for a specific cell line is compared with the value listed for LC50 TAX. The lower number (more negative) of the two corresponds with a lower relative concentration needed to reach the GI50, TGI or LC50 parameter.
  • the compound is either more potent than taxol if it has a lower number, or less potent than taxol if it is a higher number.
  • the GI50 and GI50 TAX values for melanoma cell line M14 for oxo-10-DAXT and for taxol are -6.65 and -11.73, respectively.
  • taxol is more potent than oxo-10-DAXT.
  • the TGI and TGI TAX values for oxo-10-deacetyl-7-xylosyl taxol and taxol are -5.60 and -4.62, respectively.
  • Oxo-10-DAXT is more potent than taxol at this point in the dose response curve.
  • the other values in Tables 2 through 7 can be evaluated similarly.

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Abstract

Cette invention se rapporte à un procédé pour inhiber la croissance des cellules cancéreuses, ce système consistant à mettre ces cellules en contact avec une quantité efficace d'un composition comprenant le composé de la formule (I), où R représente Ac ou H; R' représente un composé de formule (II) ou (III) ou (IV) ou OH; et R' représente un composé de formule (V) ou (VI).
PCT/US1995/013234 1994-10-14 1995-10-16 Nouveaux agents cytotoxiques WO1996011683A1 (fr)

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AU40012/95A AU4001295A (en) 1994-10-14 1995-10-16 New cytotoxic agents

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US32327594A 1994-10-14 1994-10-14
US08/323,275 1994-10-14

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WO1996011683A9 WO1996011683A9 (fr) 1996-06-13

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5767297A (en) * 1997-02-05 1998-06-16 Ensuiko Sugar Refining Co., Ltd. Taxoid derivative and method of producing thereof
EP0882732A1 (fr) * 1997-06-03 1998-12-09 Ensuiko Sugar Refining Company, Limited Dérivés taxoides et procédé pour leur préparation
US5926992A (en) * 1994-09-06 1999-07-27 Daiwa Seiko, Inc. Intra-line fishing rod
EP1534674A2 (fr) * 2002-08-02 2005-06-01 Immunogen, Inc. Agents cytotoxiques renfermant des nouveaux taxanes puissants et utilisation therapeutique de ceux-ci
US7390898B2 (en) 2002-08-02 2008-06-24 Immunogen Inc. Cytotoxic agents containing novel potent taxanes and their therapeutic use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5356928A (en) * 1992-11-06 1994-10-18 Hauser Chemical Research, Inc. Cytotoxic agents

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5356928A (en) * 1992-11-06 1994-10-18 Hauser Chemical Research, Inc. Cytotoxic agents

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5926992A (en) * 1994-09-06 1999-07-27 Daiwa Seiko, Inc. Intra-line fishing rod
US6505430B1 (en) 1994-09-06 2003-01-14 Daiwa Seiko Inc. Intra-line fishing rod
US5767297A (en) * 1997-02-05 1998-06-16 Ensuiko Sugar Refining Co., Ltd. Taxoid derivative and method of producing thereof
EP0882732A1 (fr) * 1997-06-03 1998-12-09 Ensuiko Sugar Refining Company, Limited Dérivés taxoides et procédé pour leur préparation
EP1534674A2 (fr) * 2002-08-02 2005-06-01 Immunogen, Inc. Agents cytotoxiques renfermant des nouveaux taxanes puissants et utilisation therapeutique de ceux-ci
EP1534674A4 (fr) * 2002-08-02 2007-11-28 Immunogen Inc Agents cytotoxiques renfermant des nouveaux taxanes puissants et utilisation therapeutique de ceux-ci
US7390898B2 (en) 2002-08-02 2008-06-24 Immunogen Inc. Cytotoxic agents containing novel potent taxanes and their therapeutic use
US7414073B2 (en) 2002-08-02 2008-08-19 Immunogen Inc. Cytotoxic agents containing novel potent taxanes and their therapeutic use
US7495114B2 (en) 2002-08-02 2009-02-24 Immunogen Inc. Cytotoxic agents containing novel potent taxanes and their therapeutic use

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Publication number Publication date
AU4001295A (en) 1996-05-06

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