Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
  • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
  • C07D407/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

• This invention relates to the fields of pharmaceutical and organic chemistry and provides novel cryptophycin compounds useful as anti-microtubule agents.
• Neoplastic diseases characterized by the proliferation of cells not subject to the normal control of cell growth, are a major cause of death in humans and other mammals. Clinical experience in cancer chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases.
• microtubule system of eucaryotic cells is a major component of the cytoskeleton and is a dynamic assembly and disassembly; this is heterodimers of tubulin are polymerized and form microtubule.
• Microtubules play a key role in the regulation of cell architecture, metabolism, and division. The dynamic state of microtubules is critical to their normal function. With respect to cell division, tubulin is polymerized into microtubles that form the itotic spindle. The microtubules are then depolymerized when the mitotic spindle's use has been fulfilled. Accordingly, agents which disrupt the polymerization or depolymerization of microtubules, and thereby inhibit mitosis, comprise some of the most effective cancer chemotherapeutic agents in clinical use.
• the compounds claimed herein possess fungicidal properties as well. Further, such agents having the ability to disrupt the microtubule system can be useful for research purposes.
• cryptophycin compounds are known in the literature; however, cryptophycin compounds having even greater solubility and stability are desired. Further, a broader library of cryptophycin compounds could provide additional treatment options for the patient suffering from cancer. Applicants have now discovered novel cryptophycin compounds which can be prepared using total synthetic methods and are therefore well suited for development as pharmaceutically useful agents. Conveniently, commercially available amino acids are utilized to complete the cryptophycin ring system.
• the presently claimed invention provides novel cryptophycin compounds of Formula I
• Ar is selected from the group consisting of phenyl or any simple unsubstituted, substituted aromatic, heteroaromatic group, C 1 -C 12 alkyl, C 2 -C 12 alkene, C 2 -C 12 alkyne, NR 51 R 52 , OR 53 , and Formula Ar'
• R 51 is selected from the group consisting of hydrogen and C 1 -C 3 alkyl
• R 52 is selected from the group consisting of hydrogen and C 1 -C 3 alkyl
• R 53 is selected from the group consisting of C1-C 12 alkyl
• R 54 is selected from the group consisting of hydrogen, Ci-C ⁇ alkyl, simple aromatic, phenyl, COOR 57 , PO 3 H, SO 3 H, SO 2 R 58 , NR 59 R 60 , NHOR 61 , NHCHR 61 ', CN, N0 2 , halogen, OR 62 , and SR 63
• R 55 is selected from the group consisting of hydrogen, C_-C 6 alkyl, simple aromatic, phenyl, COOR 57 , PO 3 H, SO 3 H, SO 2 R 58 , NR 59 R 60 , NHOR 61 , NHCHR 61 ', CN, N0 2 , halogen, OR 62 , and SR 63
• R 56 is selected from the group consisting of hydrogen, C 1 -C 6
• R 58 is selected from the group consisting of hydrogen and C 1 -C 12 alkyl
• R 59 is selected from the group consisting of hydrogen, (C ⁇ Ce ) alkyl and fluorenylmethoxycarbonyl (F OC) ;
• R 60 is selected from the group consisting of hydrogen and (Ci-Ce) alkyl
• R 61 is selected from the group consisting of hydrogen, OR 64 , CH 2 NHR 65 , NHR 65 and fluorenylmethoxycarbonyl (FMOC);
• R 61 ' is selected from the group consisting of hydrogen, OR 64 ,
• R 62 is selected from hydrogen and Ci-C ⁇ alkyl
• R 63 is selected from hydrogen and C 1 -C 6 alkyl; R 64 is selected from the group consisting of hydrogen, (Ci-
• R 65 is selected from the group consisting of hydrogen and C ⁇ -C6 alkyl, NH 2 , and fluorenylmethoxycarbonyl (FMOC) ;
• R 66 is selected from the group consisting of hydrogen and i-C ⁇ alkyl and fluorenylmethoxycarbonyl (FMOC) ;
• R 67 is selected from the group consisting of hydrogen and Ci-C ⁇ alkyl;
• R 1 and R 2 are each independently selected from the group consisting of halogen, OH, SH, ammo, mono (Cj-Ce) alkylammo, di (C ⁇ -C 6 ) alkylamino, tri (Ci-Ce) alkylammonium, (C ⁇
• R 1 and R 2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring or monoalkylphosphate ring; or R 1 and R 2 may be taken together with Ci ⁇ and C 1 7 to _ orm a second bond between Ci ⁇ and C 17 ; R 3 is a lower alkyl group;
• R 7 is selected from the group consisting of H, a lower alkyl group, and the side chains of all D- and L- ammo acids;
• R 31 is selected from the group consisting of P, S, (C 1 -C 12 ) alkyl, B, R 32 ' and Si;
• R 32 is selected from the group consisting of ammo acid, carbohydrate, amino sugar, (saccharide) q , C (O) C -Ce ) alkylR 38 , and
• R 34 is (C 1 -C 4 ) alkyl
• R 35 is hydrogen or (C 1 -C 3 ) alkyl
• R 36 is OH, halo, (C 1 -C 3 ) alkyl, OR 34 , N0 2 , NH 2 and heteromatic;
• R 38 is COOR 39 , , NH 2 , and amino acid;
• R 39 is H or (C ⁇ -C 6 ) alkyl;
• R 40 , R 41 , and R 42 are each independently selected from the group consisting of hydrogen, OR 43 , halo, NH 2 , NO 2 , OP0 3 (R 6 ) 2 , -0(C ⁇ -C 6 )alkyl ⁇ henyl, and R 45 ;
• R 43 is C 1 -C6 alkyl;
• R 45 is selected from the group consisting of an aromatic group and a substituted aromatic group;
• R 46 is selected from the group consisting of H, Na, and -
• the present invention provides pharmaceutical formulations, a method for disrupting a microtubule system using an effective amount of a compound of Formula I, a method for inhibiting the proliferation of mammalian cells comprising administering an effective amount of a compound of Formula I, and a method for treating neoplasia in a mammal comprising administering an effective amount of a compound of Formula I. Also, provided is a method for controlling a mycotic infection comprising administering to an animal infected with or susceptible to infection with a fungi, an antifungally effective amount of a compound of Formula I.
• the term "simple alkyl” shall refer to C 1 -C 7 alkyl wherein the alkyl may be saturated, unsaturated, branched, or straight chain. Examples include, but are in no way limited to, methyl, ethyl, n-propyl, ISO- propyl, n-butyl, propenyl, ethenyl, sec-butyl, n-pentyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert pentyl, sec-pentyl, methylated pentyl groups and the like.
• alkenyl refers to an alkyl group, as defined above, having from one to three double bonds.
• alkynyl refers to an alkyl group, as defined above, having at least one triple bond. It is especially preferred that alkynyl has only one triple bond.
• Ci-C n ' alkyl; wherein n' is an integer from 1 to 12 means an alkyl group having from one to the indicated number of carbon atoms.
• the Ci-C n - alkyl can be straight or branched chain.
• D- and L- ammo acid refers to and includes both the D- and L- forms of the following ammo acids: alanme, leucine, isoleucme, valme, serine, glutamate, glutamme, aspartate, tryptophan, lysme, argimne, tyrosme, histid e, methionme, phenylalanine, asparagme, cyste e, glyc e, proline, and threon e.
• side chains of all D- and L- ammo acids means the group attached to the carbon that is attached to both the organic acid and the ammo group. For example, but not limited to -CH ⁇ for alanme, CH ? CH (CH-,) - for leucine, and so on.
• ammo acid means an organic acid containing an am no group.
• the term includes both naturally occurring and synthetic ammo acids, therefore, the ammo group can be, but is not required to be, attached to the carbon next to the acid.
• the ammo acid group is attached to the parent molecule via the acid functionality.
• the term shall refer to, but is in no way limited to (CH 2 ) 2 NH C00H, CH 2 CH (NH 2 ) CH 2 COOH, CH 3 CH ? (NH )CH 2 COOH, CH3SCH2CH2 (NH 2 ) CHCOOH and the like.
• the term “carbohydrate” refers to a class of substituents made up of carbon, hydrogen, and oxygen wherein hydrogen and oxygen are in the same proportions as in water or nearly the proportions as water.
• the term “carbohydrate” further refers to an aldehyde or ketone alcohol or a compound which on hydrolysis produces and aldehyde or ketone.
• the term “carbohydrate” is as commonly understood by the skilled artisan. For example, the term refers to, but is in no way limited to, C 12 H 22 O 11 and C 6 H 10 O 5 .
• amino sugar refers to a carbohydrate group containing from one to three amino substituents at any available position on the carbohydrate molecule .
• saccharide refers to carbohydrate subunits to form disaccharides or polysaccharides .
• the term means for example, but in no way limited to, lactose, maltose, sucrose, fructose, starch, and the like.
• substituted phenyl shall refer to a phenyl group with from one to three non- hydrocarbon substituents which may be independently selected from the group consisting of simple alkyl, Cl, Br, F, and I.
• substituted benzyl shall refer to a benzyl group with from one to three non- hydrocarbon substitutents which may be independently selected from the group consisting of simple alkyl, Cl, Br, F, and I wherein such substituents may be attached at any available carbon atom.
• cycloalkyl refers to a saturated C 3 -C ⁇ cycloalkyl group wherein such group may include from zero to three substituents selected from the group consisting of C 1 -C 3 alkyl, halo, and OR 22 wherein R 22 is selected from hydrogen and C 1 -C 3 alkyl. Such substituents may be attached at any available carbon atom. It is especially preferred that cycloalkyl refers to substituted or unsubstituted cyclohexyl .
• Lower alkoxyl group means any alkyl group of one to five carbon atoms bonded to an oxygen atom.
• lower alkyl group means an alkyl group of one to six carbons and includes linear and non- linear hydrocarbon chains, including for example, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert pentyl, sec-pentyl, and methylated pentyl groups.
• allylically substituted alkene means any alkene having from one to seven carbon atoms which contains an allyl substitution on it.
• unsaturated lower alkyl means a lower alkyl group as defined supra . , except that Ci is obviously not envisioned in this instance, wherein from one to two double bonds are present in the unsaturated lower alkyl substituent.
• the term “lower alkyl-C 3 -C 5 cycloalkyl” refers to C ⁇ -C h alkyl substituted with a C 3 -C 5 cycloalkyl group.
• a preferred lower alkyl-C 3 -C 5 cycloalkyl group is -C__ 2 -cyclopropyl; wherein the group is attached to the cryptophycin core structure at R 9 via the CH 2 .
• epoxide ring means a three- membered ring whose backbone consists of two carbons and an oxygen atom.
• aziridine ring means a three-membered ring whose backbone consists of two carbon atoms and a nitrogen atom.
• sulfide ring means a three-membered ring whose backbone consists of two carbon atoms and a sulfur atom.
• episulfide ring means a three-membered ring whose backbone consists of two carbon atoms and a sulfur atom.
• sulfate group means a five membered ring consisting of a carbon- carbon-oxygen-sulfur-oxygen backbone with two additional oxygen atoms connected to the sulfur atom.
• cyclopropyl ring means a three member ring whose backbone consists of three carbon atoms.
• inonoalkylphosphate ring means a five membered ring consisting of a carbon-carbon-oxygen-phosphorous-oxygen backbone with two additional oxygen atoms, one of which bears a lower alkyl group, connected to the phosphorous atom.
• simple unsubstituted aromatic group refers to common aromatic rings having 4n+2 electrons in a monocyclic conjugated system, for example, but not limited to: phenyl, furyl, pyrrolyl, thienyl, pyridyl and the like, or a bicyclic conjugated system, for example but not limited to indolyl or naphthyl.
• simple substituted aromatic group refers to a phenyl group substituted with a single group selected from the group consisting of halogen and lower alkyl group.
• heteromatic group refers to aromatic rings which contain one or more non-carbon substituent selected from the group consisting of oxygen, nitrogen, and sulfur. It is most preferred, but not limited to, an aromatic ring having from three to eight members wherein at least one member of the ring system is a heteroatom and the remaining members of the ring system are carbon.
• halogen or “halo” refers to those members of the group on the periodic table historically known as halogens. Methods of halogenation include, but are not limited to, the addition of hydrogen halides, substitution at high temperature, photohalogenation, etc., and such methods are known to the skilled artisan.
• the term “mammal” shall refer to the Mammalia class of higher vertebrates.
• the term “animal” shall include, but is not limited to, mammals, reptiles, amphibians, and fish.
• the term “mammal” includes, but is not limited to, a human.
• the term “treating” as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established.
• the cryptophycin compounds claimed herein can be useful for veterinary health purposes as well as for the treatment of a human patient.
• Suitable inert organic solvents include those known to the skilled artisan, for example, but not limited to, tetrahydrofuran (THF) and dimethylformamide (DMF) . DMF is especially preferred.
• Aqueous based solvents may be appropriate for some of the processes utilized herein. The pH of such aqueous solvents may be adjusted as desired to facilitate the process.
• R 6 is chloro methoxy phenyl
• R 7 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, or isopentyl;
• R 7 is isopropyl
• R 3 is methyl
• Ar' is phenyl with substituent selected from the group consisting of NR 59 R 60 , NHOR 61 , and NHCHR 61 ';
• R 1 and R 2 form an epoxide ring;
• R 7 is methyl
• R 2 is a glycinate; I) R 2 is an acylate;
• L) a compound of Formula I is used for the treatment of cancer in a mammal; M) a compound of Formula I is used as an antifungal agent;
• Ar is phenyl substituted with one or two from the group consisting of OH, OCH 3 , halo, and methyl;
• Q) R 2 is selected from the group consisting of halogen, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate, OR 31 , and SR 31 ; ;
• R) a compound of Formula I is used for the treatment of a fungal infection;
• S) Ar' is para ethyl substituted phenyl; and T) Ar' is para methyl substituted phenyl.
• the present invention provides a method of alleviating a pathological condition caused by hyperproliferating mammalian cells comprising administering to a subject an effective amount of a pharmaceutical or veterinary composition disclosed herein to inhibit proliferation of the cells.
• the method further comprises administering to the subject at least one additional therapy directed to alleviating the pathological condition.
• the pathological condition is characterized by the formation of neoplasms.
• the neoplasms are selected from the group consisting of mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, pancreatic adenocarcmoma, central nervous system (CNS) , ovarian, prostate, sarcoma of soft tissue or bone, head and neck, gastric which includes pancreatic and esophageal, stomach, myeloma, bladder, renal, neuroendocrme which includes thyroid and non-Hodgkin's disease and Hodgkm's disease neoplasms.
• CNS central nervous system
• neoplastic refers to a neoplasm, which is an abnormal growth, such growth occurring because of a proliferation of cells not subject to the usual limitations of growth.
• anti-neoplastic agent is any compound, composition, admixture, co-mixture, or blend which inhibits, eliminates, retards, or reverses the neoplastic phenotype of a cell.
• Anti-mitotic agents may be classified into three groups on the basis of their molecular mechanism of action. The first group consists of agents, including colchicme and colcemid, which inhibit the formation of microtubules by sequestering tubulin.
• the second group consists of agents, including vmblastme and vmcristme, which induce the formation of paracrystallme aggregates of tubulin.
• Vmblastme and vmcristme are well known anticancer drugs: their action of disrupting mitotic spindle microtubules preferentially inhibits hyperproliferative cells.
• the third group consists of agents, including taxol, which promote the polymerization of tubulin and thus stabilizes microtubules.
• Such compositions can also be provided together with physiologically tolerable liquid, gel, or solid carriers, diluents, adjuvants and excipients .
• physiologically tolerable liquid, gel, or solid carriers diluents, adjuvants and excipients .
• Such carriers, adjuvants, and excipients may be found in the U.S . Pharmacopeia, Vol.
• the present invention further provides a pharmaceutical composition used to treat neoplastic disease containing at least one compound of Formula I and at least one additional anti-neoplastic agent.
• Anti-neoplastic agents which may be utilized in combination with Formula I compounds include those provided in the Merck Index 11, pp 16-17, Merck & Co., Inc. (1989). The Merck Index is widely recognized and readily available to the skilled artisan.
• ant eoplastic agents may be antimetabolites which may include but are in no way limited to those selected from the group consisting of methotrexate, 5-fluorouracil, 6- mercaptopurme, cytosme, arabinoside, hydroxyurea, and 2- chlorodeoxyadenosine .
• the anti-neoplastic agents contemplated are alkylating agents which may include but are no way limited to those selected from the group consisting of cyclophosphamide, mephalan, busulfan, paraplatm, chlorambucil, and nitrogen mustard.
• the anti-neoplastic agents are plant alkaloids which may include but are in no way limited to those selected from the group consisting of vmcristme, vmblastme, taxol, and etoposide.
• the anti-neoplastic agents contemplated are antibiotics which may include, but are m no way limited to those selected from the group consisting of doxorubicm, daunorubicm, mitomyc C, and bleomycm.
• the anti-neoplastic agents contemplated are hormones which may include, but are in no way limited to those selected from the group consisting of calusterone, diomostavolone, propionate, epitiostanol, mepitiostane, testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate, flutamide, nilutamide, and trilotane.
• the anti-neoplastic agents contemplated include enzymes which may include, but are in no way limited to those selected from the group consisting of L-Asparginase and aminoacridine derivatives such as, but not limited to, amsacrine.
• Additional anti- neoplastic agents include those provided by Skeel, Roland T., "Antineoplastic Drugs and Biologic Response Modifier: Classification, Use and Toxicity of Clinically Useful Agents" Handbook of Cancer Chemotherapy (3rd ed.), Little Brown & Co. (1991) .
• compositions can be administered to mammals for veterinary use.
• domestic animals can be treated in much the same way as a human clinical patient.
• the dosage required for therapeutic effect will vary according to the type of use, mode of administration, as well as the particularized requirements of the individual hosts. Typically, dosages will range from about 0.001 to 1000 mg/kg, and more usually 0.01 to 10 mg/kg of the host body weight. Or, alternatively, dosages within these ranges can be administered as a bolus or IV injection, until the desired therapeutic benefits are obtained.
• drug dosage as well as route of administration, must be selected on the basis of relative effectiveness, relative toxicity, growth characteristics of tumor and effect of Formula I compound on cell cycle, drug pharmacokinetics, age, sex, physical condition of the patient and prior treatment, which can be determined by the skilled artisan.
• the compound of Formula I may be formulated into therapeutic compositions as natural or salt forms.
• Pharmaceutically acceptable non-toxic salts include base addition salts which may be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
• Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as for example, hydrochloric or phosphoric acids or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Additional excipients which further the invention are provided to the skilled artisan for example in the U.S. Pharmacopeia .
• anti- neoplastic compositions may be formulated for oral administration. Such compositions are typically prepared as liquid solution or suspensions or in solid forms. Oral formulation usually include such additives as binders, fillers, carriers, preservatives, stabilizing agents, e ulsifiers, buffers, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained relsease formulations, or powders, and typically contain 1% to 95% of active ingedient. More preferably, the composition contains from about 2 % to about 70% active ingredient.
• compositions of the present invention may be prepared as injectables, either as liquid solutions, suspensions, or emulsions; solid forms suitable for solution in or suspension in liquid prior to injection.
• injectables may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intrathecally, or intrapleurally.
• the active ingredient or ingredients are often mixed with diluents, carriers, or excipients which are physiologically tolerable and compatible with the active ingredient (s) .
• Suitable diluents and excipients are for example, water, saline, dextrose, glycerol, or the like and combinations thereof.
• the compositions may contain minor amounts of auxilary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
• the invention further provides methods for using Formula I compounds to inhibit the proliferation of mammalian cells by contacting these cells with a Formula I compound in an amount sufficient to inhibit the proliferation of the mammalian cell.
• a preferred embodiment is a method to inhibit the proliferation of hyperproliferative mammalian cells.
• hyperproliferative mammalian cells are mammalian cells which are not subject to the characteristic limitations of growth (programmed cell death for example) .
• a further preferred embodiment is when the mammalian cell is human.
• the invention further provides contacting the mammalian cell with at least one Formula I compound and at least one anti-neoplastic agent. The types of anti- neoplastic agents contemplated are discussed supra .
• the invention further provides methods for using a compound of Formula I to inhibit the proliferation of hyperproliferative cells with drug-resistant phenotypes, including those with multiple drug-resistant phenotypes, by contacting said cell with a compound of Formula I in an amount sufficient to inhibit the proliferation of a hyperproliferative mammalian cell.
• a preferred embodiment is when the mammalian cell is human.
• the invention further provides contacting a Formula I compound and at least one additional anti-neoplastic agent, discussed supra .
• the invention provides a method for alleviating pathological conditions caused by hyperproliferating mammalian cells for example, neoplasia, by administering to a subject an effective amount of a pharmaceutical composition containing Formula I compound to inhibit the proliferation of the hyperproliferating cells.
• pathological condition refers to any pathology arising from the proliferation of mammalian cells that are not subject to the normal limitations of growth. Such proliferation of cells may be due to neoplasms as discussed supra .
• the neoplastic cells are human.
• the present invention provides methods of alleviating such pathological conditions utilizing a compound of Formula I in combination with other therapies, as well as other anti-neoplastic agents.
• the effectiveness of the claimed compounds can be assessed using standard methods known to the skilled artisan. Examples of such methods are as follows:
• the minimum inhibitory concentration against the panel of 84 Cryptococcus neoformans isolates is determined to illustrate the desired antifungal activity.
• the compounds are screened for minimum inhibitory concentrations against KB, a human nasopharyngeal carcinoma cell line, LoVo, a human colorectal adenocarcinoma cell line using The Corbett assay, see Corbett, T.H. et al . Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development, pp 35-87, Kluwer Academic Publishers: Norwell, 1992. see also, Valeriote, et al. Discovery and Development of Anticancer Agents; Kluwer Academic Publishers, Norwell, 1993 is used for the evaluation of compounds.
• the most active compounds are further evaluated for cytotoxicity against four different cell types, for example a murine leukemia, a murine solid tumor, a human solid tumor, and a low malignancy fibroblast using the Corbett assay.
• the compounds are further evaluated against a broad spectrum of murine and human tumors implanted in mice, including drug resistant tumors.
• Tumor burden (mean tumor burden in treated animals versus mean tumor burden in untreated animals) are used as a further assessment. T/C values that are less than 42% are considered to be active by National Cancer Institute Standards; T/C values less than 10% are considered to have excellent activity and potential clinical activity by National Cancer Institute standards.
• Vinblastine Vinblastine, cytochalasin B, tetramethylrhodamine isothiocyanate (TRITC) -phalloidin, sulforhodamine B (SRB) and antibodies against ⁇ -tubulin and vimentin are commercially available from recognized commercial vendors.
• BME Basal Medium Eagle containing Earle's salts
• FBS Fetal Bovine Serum
• the Jurkat T cell leukemia line and A-10 rat aortic smooth muscle cells are obtained from the American Type Culture Collection and are cultured in BME containing 10% FBS and 50 ⁇ g/mL gentamycin sulfate.
• Human ovarian carcinoma cells (SKOV3) and a sub-line which has been selected fro resistance to vinblastine (SKVLBl) were a generous gift from Dr. Victor Ling of the Ontario Cancer Institute. Both cell lines are maintained in BME containing 10% FBS and 50 ⁇ g/mL gentamycin sulfate.
• Vinblastine is added to a final concentration of l ⁇ g/mL to SKVLBl cells 24 hours after passage to maintain selection pressure for P-glycoprotein- overexpressing cells.
• Cell proliferation assays are performed as described by Skehan et al .
• cultures are treated with the indicated drugs as described in Skehan and total cell numbers are determined by counting the cells in a hemacytometer .
• the percentage of cells in mitosis are determined by staining with 0.4% Giemsa in PBS followed by rapid washes with PBS.
• At least 1000 cells per treatment are scored for the presence of mitotic figures and the mitotic index is calculated as the ration of the cells with mitotic figures to the total number of cells counted.
• A-10 cells are grown to near-confluency on glass coverslips in BME/10% FBS. Compounds in PBS are added to the indicated final concentrations and cells are incubated for an additional 24 hours. For the staining of microtubules and intermediate filaments, the cells are fixed with cold methanol and incubated with PBS containing 10% calf serum to block nonspecific binding sites. Cells are then incubated at 37_C for 60 mm. with either monoclonal anti- ⁇ -tubulm or with monoclonal anti-vimentm at dilutions recommended by the manufacturer. Bound primary antibodies are subsequently visualized by a 45-mmute incubation with fluoresce -con ugated rabbit antimouse IgG.
• the coverslips are mounted on microscope slides and the fluorescence patterns are examined and photographed using a Zeiss Photomicroscope 111 equipped with epifluorescence optics for fluorescem.
• a Zeiss Photomicroscope 111 equipped with epifluorescence optics for fluorescem.
• cells are fixed with 3% paraformaldehyde, permeabilized with 0.2%
• Triton X-100 and chemically reduced with sodium borohydride (lmg/ML) .
• PBS containing lOOnM TRITC-phalloidin is then added and the mixture is allowed to incubate for 45 mm. at 37_C.
• the cells are washed rapidly with PBS before the coverslips are mounted and immediately photographed as described above.
• Aortic smooth muscle (A-10) cells are grown on glass coverslips and treated with PBS, 2 ⁇ M cytochalasm B, lOOnM v blastme or lOnM cryptophycin compounds . After 24 hours, microtubules and vimentm intermediate filaments are visualized by indirect immunofluorescence and microfilaments are stained using TRITC - phalloidin. The morphological effects of each drug is examined. Untreated cells displayed extensive microtubule networks complete with perinuclear microtubule organizing centers. Vimentm intermediate filaments were also evenly distributed throughout the cytoplasm, while bundles of microfilaments were concentrated along the major axis of the cell.
• Cytochalasin B caused complete depolymerization of microfilaments along with the accumulation of paracrystalline remnants. This compound did not affect the distribution of either microtubules or intermediate filaments. The cryptophycin treated microtubules and vimentin intermediates are observed for depletion of microtubules, and collapse of rimentin intermediate filaments.
• A-10 cells are treated for 3 hours with 0 or lO ⁇ M taxol before the addition of PBS, lOOnM vinblastine or lOr ⁇ M cryptophycin compound. After 24 hours, microtubule organization is examined by immunofluorescence as described above. Compared with those in control cells, microtubules in taxol-treated cells were extensively bundled, especially in the cell polar regions. As before, vinblastine caused complete depolymerization of microtubules non-pretreated cells. However, pretreatment with taxol prevented microtubule depolymerization in response to vinblastine. Similarly, microtubules pretreated with taxol are observed with cryptophycin treatment.
• A-10 cells are treated with either lOOnM vinblastine or lOnM cryptophycins for 24 hr . , resulting in complete microtubule depolymerization.
• the cells are then washed and incubated in drug-free medium for periods of 1 hour or 24 hours.
• Microtubules repolymerized rapidly after the removal of vinblastine, showing significant levels of microtubules after 1 hour and complete morphological recovery by 24 hour.
• Cells are visualized for microtubule state after treatment with a cryptophycin compound of this invention at either 1 hour or 24 hours after removal of the cryptophycin compounds .
• SKOV3 cells are treated with combinations of cryptophycins and vinblastine for 48 hours. The percentages of surviving cells are then determined and the IC 50 S for each combination is calculated.
• SKVLBl cells are resistant to natural product anticancer drugs because of their over expression of P- glycoprotein.
• Taxol caused dose-dependent inhibition of the proliferation of both cell lines with IC 50 S for SKOV3 and SKVLBl cells of 1 and 8000nM, respectively.
• Vinblastine also inhibited the growth of both cell lines, with IC 50 s of 0.35 and 4200nM for SKOV3 and SKVLBl cells, respectively.
• Cryptophycins compounds of this invention demonstrate activity with an IC 50 S of from about 1 to about 1000pm for SKOV3 and SKVLBl cells.
• the present invention provides novel cryptophycin compounds which are potent inhibitors of cell proliferation, acting by disruption of the microtubule network and inhibition of mitosis.
• Cryptophycin compounds disrupt microtubule organization and thus normal cellular functions, including those of mitosis.
• Classic anti-microtubule agents such as colchicme and Vmca alkaloids, arrest cell division at mitosis. It seems appropriate to compare the effect of one of these agents on cell proliferation with the cryptophycin compounds.
• the Vmca alkaloid v blastme was selected as representative of the classic anti-microtubule agents.
• the effect of cryptophycin compounds and v blastme on the proliferation and cell cycle progression of the Jurkat T-cell leukemia cell line is compared. Since antimitotic effects are commonly mediated by disruption of microtubules in the mitotic spindles, the effects of cryptophycin compounds on cytoskeletal structures are characterized by fluorescence microscopy. Immunofluorescence staining of cells treated with either a cryptophycin compound or vmblastme demonstrate that both compounds cause the complete loss of microtubules. Similar studies with SKOV3 cells can show that the anti-microtubule effects of cryptophycin compounds are not unique to the smooth muscle cell line.
• a dilution of 1:20 dimethylsulfoxide solution m PBS was prepared such that the final concentration was 10 ⁇ g/ml.
• Serial 1:3 dilutions using PBS (.5ml previous sample of 1ml PBS) were prepared.
• Falcon 2054 tubes were used for the assay.
• a lOul sample of each dilution of test compound was added in triplicate to wells of GC3 plates. The plates were incubated for 72 hours at about 37 C.
• a 10 ⁇ l sample of stock 3- [4, 5-dimethyl-2-yl] -2, 5-diphenyltetrazolium bromide salt ("MTT" 5 mg/ml in PBS) was added to each well. The plates were incubated for about an hour at 37 C.
• the preparation of the compounds of this invention can be completed using several protocols involving an activated ester followed by chromatography and acid induced deblocking where necessary.
• Preparation of any ester wherein R 1 or R 2 is derived from a carboxylic acid includes a variety of technologies employing acid chlorides, anhydrides, and common activating reagents (eg., carbodiimides) . " Any solvent other than participating alcohols can be used. Any mild bases and/or catalysts (amines, carbonates) can be used to aid in esterification.
• carba ates to the corresponding salts could be effected with any strong acid, namely, mineral acids comprised of hydrogen halides, hydrogen sulfates, hydrogen phosphates, hydrogen nitrates, hydrogen perchlorates, or strong organic acids such as tri luoroacetic, p-toluenesulfonic, and methanesulfonic.
• strong acid namely, mineral acids comprised of hydrogen halides, hydrogen sulfates, hydrogen phosphates, hydrogen nitrates, hydrogen perchlorates, or strong organic acids such as tri luoroacetic, p-toluenesulfonic, and methanesulfonic.
• strong organic acids such as tri luoroacetic, p-toluenesulfonic, and methanesulfonic.
• the same acids could be used to produce new salts from the corresponding free base.
• ester starting material can be prepared, for example, as follows:
• R 6 has the meaning defined supra .
• the necessary reaction time is related to the starting materials and operating temperature.
• the optimum reaction time for a given process is, as always, a compromise which is determined by considering the competing goals of throughput, which is favored by short reaction times, and maximum yield, which is favored by long reaction times .
• Step 1 Methyl 5-Phenylpent-2 (E) -enoate.
• a solution of trimethyl phosphonoacetate (376 g, 417 mL, 2.07 mol) in THF (750 L) was stirred at 0 °C in a 3L 3-neck round bottom flask equipped with a mechanical stirrer and 2 inlet.
• To the chilled solution neat tetramethyl guanidme (239 g, 260 L, 2.07 mol) was added dropwise via an addition funnel. The chilled clear pale yellow solution was stirred for 25 minutes at 0 °C.
• the reaction temperature was allowed to raise to -15°C.
• the reaction was quenched slowly withlN HCl (150 mL) .
• a spatula was employed to breakup the the semi-solid and IN HCl (200 mL) was added making the mixture more fluid.
• Concentrated HCl (625 mL) was charged to form a two phase system.
• the layers were separated and the product extracted with t- BuOMe .
• the organic layer was dried over MgS04 and concentrated in vacuo to yield a clear pale yellow oil, 247.8g.
• the crude product was distilled at 145 °C/0.25mm Hg yielding 209.7g, 86.2%.
• EIMS m/z 162 (1:M+) 144 (16), 129 (7), 117 (9) 108 (6), 92 (17), 91 (100), 75 (5), 65 (12), HREIMS m/z 162, 1049 (C11H14O, D -0.4 mmu); UV lmax (e) 206 (9900), 260 (360); IR nmax 3356, 2924, 1603, 1496, 1454, 970, 746, 700 cm -1 ; 1 E
• the resulting mixture was cooled to - 20 °C and treated with Ti (O-i-Pr) 4 (9.2 mL, 0.031 mol), followed by the addition of t-butylhydroperoxide (4.0 M in CH 2 CI 2 182 mL, 0.78 mol) at a rate to maintain the temperature 2 -20 °C.
• t-butylhydroperoxide 4.0 M in CH 2 CI 2 182 mL, 0.78 mol
• the reaction mixture was stirred for another 30 mm, and then treated with a solution of the allylic alcohol (50 g, 0.31 mol) in CH 2 CI 2 (30 mL) at a rate to maintain the temperature 2 -20 °C.
• the reaction was stirred at the same temperature for 5 h, then filtered into a solution of ferrous sulfate heptahydrate (132 g) and tarta ⁇ c acid (40 g) m water (400 mL) at 0 °C.
• the mixture was stirred for 20 mm, then transferred to a separatory funnel and extracted with t- BuOMe (2x200 mL) .
• the combined organic phase was stirred with 30% NaOH solution containing NaCl, for 1 h at 0 °C.
• the layers were again separated, and the aqueous phase extracted with t-BuOMe.
• the combined organic phase was washed with brine, dried over MgS0 4 and concentrated to yield 52.8 g as an amber oil.
• Step 4 ⁇ 2R, 3R) -2-hydroxy-3-methyl-5-phenylpentan-l-ol .
• Step 6 (21?, 3K)-2-[ ( ert-Butyldimethylsilyl) oxy] -S-methyl- B-phenylpent-l-yl Tosylate.
• a solution of the tosylate (100 g, 0.29 mol) and triethylamine (81.0 mL, 0.58 mol) in CH 2 CI 2 (1200 mL) was treated with neat TBS-OTf (99 mL, 0.43 mol) dropwise with continued stirring for another 20 min. The reaction was washed twice with brine, dried over MgS ⁇ 4 and concentrated to dryness.
• Step 8 (2R, 31?) -2- [ ( ert-Butyldimethylsilyl) oxy] -3-methyl- 5-phen ⁇ lpent- (£) -en-l-yl Tosylate.
• a solution of the bromide 100 g, 0.186 mol
• acetonitrile 700 mL
• DBU 83.6 mL, 0.557 mol
• the tosylate (50 g, 0.11 mol) was dissolved in DMSO (1 L) and treated with KCN (14.2 g, 0.22 mol) and water (25 mL) , and the resulting mixture was stirred at 60 °C under nitrogen for 18 h. After cooling to room temperature, the reaction mixture was partitioned between EtOAc (1 L) and water (1 L) . The aqueous phase was extracted with EtOAc (500 mL) , and the combined organic phase was washed with brine and dried over Na 2 S ⁇ . Flash chromatography over silica with CH 2 CI 2 afforded the desired nitrile in 92% yield.
• Step 10 Methyl (5S, 61?) -5- [ (tert-Butyldimethylsilyl) oxy] - 6-methyl- ⁇ -phenylocta-2 (E) ,7 (E) -dienoate.
• the nitrile 14.67 g, 46.5 mmol
• a 1.5M solution of DIBAL in toluene (37.2 mL, 55.8 mmol) was added dropwise with vigorous stirring. Upon complete addition, the cooling bath was removed and the reaction was stirred at room temperature for 1 h. The reaction mixture was carefully poured into IN HCl and the mixture stirred at room temperature for 30 min.
• the layers were separated, and the organic phase was washed with a saturated aqueous solution of sodium potassium tartrate (2x) , brine and dried over Na 2 S0 4 .
• the volatiles were removed in vacuo, and the crude pale yellow oil was used directly in the subsequent condensation.
• the crude aldehyde from above was dissolved in THF (90 mL) and treated with trimethyl phosphonoacetate (9.03 L, 55.8 mmol) and tetramethylguanidine (7.0 mL, 55.8 mmol) at room temperature under nitrogen.
• the reaction mixture was stirred for 16 h, then partitioned between EtOAc (200 mL) and water (100 L) .
• the reaction was diluted with diethyl ether (200 ml) then extracted with IN HCl followed by 5% NaHC0 3 .
• the combined 5% NaHC0 3 aqueous layers were then acidified with IN HCl to pH 2 and extracted with diethyl ether (600 ml).
• the combined ether extracts were then washed with brine and dried over NaS0 .
• the ether was removed in vacuo yielding a yellow solid (3a) weight 714 mg (98%) .
• epoxide (8a): beta-iso er, retention time (2X3.9mmX150rnm Novapak C 18 columns, 80% CH 3 CN/H 2 0, 1.0 ml/min, 1 220 nm) 7.51 min.
• epoxide (9a): alpha-isomer, retention time (2X3.9mmX150mm Novapak C 18 columns, 80% CH 3 CN/H 2 0, 1.0 ml/mm, 1 220 nm) 8.33 min.
• Epoxides (8b) and (9b) were prepared according to the procedure for (8a) and (9a) except a different column was used for the purification.
• epoxide (8b): beta-isomer, retention time (2X3.9mmX150mm Novapak C 18 columns, 70% CH 3 CN/H 2 0, 1.0 ml/min, 1 220 nm) 14.6 min.
• epoxide (9b): alpha-isomer, retention time (2X3.9mmX150mm Novapak C 18 columns, 70% CH 3 CN/H ? 0, 1.0 ml/min, 1 220 nm) 16.6 min.

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  • 1997-08-28 EP EP97939664A patent/EP0923564A4/de not_active Withdrawn
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