Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
  • C07D487/18—Bridged systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
  • C07D471/14—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
  • C07D519/04—Dimeric indole alkaloids, e.g. vincaleucoblastine

Definitions

• Vinca alkaloids originally isolated from the leaves of the periwinkle plant [Vinca rosea Linn., now Cantharanthus roseus (L.) G. Don] [Noble et al., Ann. N. Y. Acad. Sci. 1958 76:882-894; Noble, Lloydia 1964 27:280-281; Svoboda et al., J. Am.
• Vinblastine and vincristine are the most widely recognized members of the vinca alkaloids as a result of their clinical use as antitumor drugs, and their discovery represent one of the earliest important contributions that plant-derived natural products have made to cancer chemotherapy.
• Ness et al . In The Alkaloids; Brossi et al . Eds.; Academic: San Diego, 1990 37:229-240; Pearce, In The Alkaloids; Brossi et al. Eds.; Academic: San Diego, 1990 37:145- 204; Kuehne, In The Alkaloids; Brossi et al . Eds.; Academic: San Diego, 1990 37:77-132.
• vincristine are important clinical agents and are used in combination therapies for treatment of
• Hodgkin's disease 80% cure rate
• ovarian cancer breast cancer
• head and neck cancer 80% cure rate
• non-Hodgkin ' s lymphoma vinblastine
• vincristine (vincristine) .
• the semi-synthetic vinca alkaloid vindesine ( la) a derivative of vinblastine, is used to treat lung cancer and acute leukemia and less often for melanoma, and breast cancer.
• vindesine a derivative of vinblastine
• Vinblastine (1) CH 3 C-OCH 3 —O-C-CH 3
• Vindesine (1a) —CH 3 —C-NH 2 —OH use is the instances of treatment relapse with the emergence of tumor resistance derived from
• Pgp P-glycoprotein
• Vinblastine and vincristine were among the first small molecules shown to bind tubulin and to inhibit microtubule formation and mitosis.
• Ness et al . In The Alkaloids; Brossi et al . Eds.; Academic: San Diego, 1990 37:229-240; Pearce, In The Alkaloids; Brossi et al . Eds.; Academic: San Diego, 1990 37:145- 204; Kuehne, In The Alkaloids; Brossi et al . Eds.; Academic: San Diego, 1990 37:77-132; Fahy, Curr.
• vinca alkaloids share a common binding site on tubulin.
• the relative overall affinities for beta-tubulin binding are vincristine>vinblastine> vinorelbine>vinflunine, but there is no significant difference in the affinity of all four drugs for tubulin heterodimers .
• Vinflunine is not very potent in vitro yet is active in vivo, and this has been attributed to its superior cellular uptake.
• Vincristine has found wide use in the treatment of hematologic malignancies including leukemias and lymphomas . It is also widely used in pediatric solid tumors and, in the past, in small cell lung cancer. Vinblastine is an important component of the combination regimen that is curative for testicular cancer. Vindesine is used in the treatment of leukemia, lymphoma, melanoma, breast cancer, and lung cancer. Vinorelbine is quite different and has found use mainly in breast cancer and non-small cell lung cancer. Structural formulas for vinblastine, vincristine and vindesine are shown below .
• the present inventor and co-workers reported the total synthesis of vinblastine [Ishikawa et al., J. Am. Chem. Soc. 2008 130:420-421; Ishikawa et al., J. Am. Chem. Soc. 2009 131:4904-4916] and its extension to the total synthesis of related natural products including vincristine and key analogues that utilizes a one-pot, two-step, biomimetic Fe(III)- promoted single electron oxidative coupling of catharanthine and vindoline and a subsequent
• the present invention contemplates a vinca alkaloid compound such as vinblastine, vincristine or vindesine that is substituted at the 20' -position with a urea or a thiourea group whose proximal nitrogen atom that is directly bonded to the 20'- position carbon atom is secondary and whose distal nitrogen is mono- or more preferably disubstituted; i.e., contains one or more preferably two
• proximal and distal nitrogens are illustrated below in the partial structure of a contemplated compound showing atoms near the 20'- position and an urea derivative bonded to the 20'- carbon atom via the proximal urea nitrogen, with the distal urea nitrogen bonded to substituents R ⁇ and R5.
• one of the distal nitrogen R ⁇ or substituents can be hydrido.
• R ⁇ and R > are independently a) a straight or branched chain hydrocarbyl group that has 1 to about 6 carbon atoms and is free of tertiary or quaternary carbon atoms, b) an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are
• a ring structure of b) or c) or d) above can be optionally substituted with 1, 2 or 3 substituents selected from the group consisting of C ⁇ -Cg- hydrocarbyl, C;j_-Cg-hydrocarbyloxy, phenyl, halogen, perfluoro-C]_-C5-hydrocarbyl, perfluoro-C ] _-Cg- hydrocarbyloxy, nitro and mixtures thereof.
• An above contemplated 20 ' -substituted vinca alkaloid can also be further substituted with a fluoro group at the 10' -position of the molecule.
• a pharmaceutically acceptable salt of such a contemplated compound is also contemplated.
• a preferred vinca alkaloid is a 20' -urea- substituted or thiourea-substituted vinblastine, vincristine or vindesine that can optionally also be substituted at the 10' position with a fluoro group rather than a hydrido as are illustrated by the structural Formula I shown below. These three compounds are structurally very similar and each
• Y is 0 or S, preferably 0, and and R. are independently hydrido or a substituent as discussed below, except that R 4 and R- 3 are both hydrido only when Z is F.
• R ⁇ and R ⁇ is a straight or branched chain hydrocarbyl group other than a tertiary or quaternary hydrocarbyl group that has 1 to about 6 carbon atoms.
• Z is H
• at least one of R ⁇ and R ⁇ is other than hydrido, and more preferably both of R ⁇ and R ⁇ are other than hydrido.
• R ⁇ and R ⁇ > can also be an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur.
• R ⁇ and R ⁇ can be an aralkyl or heteroaralkyl group containing 5- or 6-membered ring atoms and 1-3 carbons in the alkyl portion in a third aspect.
• the two substituents on the distal nitrogen atom (R ⁇ and R ⁇ ) together with that nitrogen atom can form a single 5- or 6-membered ring or a fused ring system containing two rings, each of which can contain a 5- or 6-members and can also contain one or two
• hetero atoms that can independently be nitrogen, oxygen or sulfur in a fourth aspect of the invention .
• Each of the before described rings or ring systems is optionally substituted with 1, 2 or 3 substituents selected from the group consisting of Ci -Cg-hydrocarbyl , Ci -Cg-hydrocarbyloxy, phenyl, halogen, perfluoro-Ci -Cg-hydrocarbyl, perfluoro-C ] _-
• a pharmaceutical composition containing a cancer cell proliferation-inhibiting amount of a compound of a contemplated 20 ' -urea-substituted or thiourea-substituted vinca alkaloid such as
• a method of treating a diagnosed cancer is also contemplated. That treatment can be carried out in vitro or in vivo as within a mammalian subject, and comprises contacting the cancerous cells as by administering a therapeutically effective amount, e.g., a cancer cell proliferation-inhibiting amount, of a before-defined contemplated 20' -urea- or thiourea-substituted vinca alkaloid compound to a cancerous cell culture or to a mammalian subject, and preferably repeating that contacting (administration) over time to at least inhibit the growth
• a therapeutically effective amount e.g., a cancer cell proliferation-inhibiting amount
• the cancer cells so treated include those usually treated by an unsubstituted, parental vinca alkaloid compound such as vinblastine, vincristine or vindesine, and include hematologic cancer cells such as leukemia or lymphoma cells, as well as cells of carcinomas, sarcomas, melanomas, neuromas and the like.
• the present invention has several benefits and advantages.
• a preferred 20' -urea- or thiourea-substituted vinca alkaloid compound is about equal to about ten times more potent as a cytotoxic agent against a colorectal carcinoma cancer cell line than is a parental, unsubstituted vinca alkaloid such as vinblastine.
• One advantage of the invention is that a preferred 20' -urea- or thiourea-substituted vinca alkaloid compound is about equal to about eighty times more potent against multiple drug resistant colorectal carcinoma cancer cell lines than is a parental, unsubstituted vinca alkaloid compound such as vinblastine.
• a contemplated 20' -urea- or thiourea-substituted vinca alkaloid compound is about equal to about ten times more potent as a cytotoxic agent against a leukemia cell line than is the parental, unsubstituted vinca alkaloid .
• Another advantage of the invention is that a contemplated 20' -urea- or thiourea-substituted vinca alkaloid compound is relatively easy to synthesize .
• an element means one element or more than one element.
• hydrocarbyl is used herein as a short hand term for a non-aromatic group that includes straight and branched chain aliphatic as well as alicyclic groups or radicals that contain only carbon and hydrogen.
• alkyl, alkenyl and alkynyl groups are contemplated, whereas aromatic hydrocarbons such as phenyl and naphthyl groups, which strictly speaking are also hydrocarbyl groups, are referred to herein as aryl groups or radicals, as discussed hereinafter.
• hydrocarbyl groups contain a chain of 1 to about 6 carbon atoms, and preferably 1 to about 4 carbon atoms .
• hydrocarbyl group is an alkyl group.
• a generalized, but more preferred substituent can be recited by replacing the descriptor "hydrocarbyl” with “alkyl” in any of the substituent groups enumerated herein.
• alkyl radicals examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like.
• suitable alkenyl radicals include ethenyl (vinyl), 2-propenyl, 3-propenyl, 1,4- pentadienyl, 1, -butadienyl, 1-butenyl, 2-butenyl, 3-butenyl, hexenyl, hexadienyl and the like.
• alkynyl radicals examples include ethynyl,
• hydrocarbyl ether is referred to as a
• hydrocarbyloxy rather than a “hydrocarboxy” group as may possibly be more proper when following the usual rules of chemical nomenclature.
• Illustrative hydrocarbyloxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, allyloxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, cyclohexenyloxy groups and the like .
• Carbocyclic alone or in combination, means a hydrocarbyl radical that contains 3 to about 8 carbon atoms, preferably about 3 to about 6 carbon atoms, and is cyclic.
• Examples of such cyclohydrocarbyl radicals include cyclopropyl, cyclobutyl,
• aryl alone or in combination, means a phenyl or naphthyl or other radical as recited hereinafter that optionally carries one two or three independently substituents selected from a C ] _-Cg-hydrocarbyl group, C ⁇ -Cg-hydrocarbyloxy group, a phenyl group, a perfluoro-C ] _-Cg-hydrocarbyl group, a perfluoro-Ci -Cg-hydrocarbyloxy group, a halogen
• heterocyclyl (heterocyclo ) is a 5- or 6-membered ring that contains 1 to 4 hetero atoms
• non-carbons in the ring that independently are nitrogen, oxygen or sulfur atoms in a saturated or partially unsaturated ring that is optionally substituted on one or more ring carbon atoms by a halogen, hydrocarbyl, hydrocarbyloxy, oxo group, and the like, and/or on a secondary ring nitrogen atom
• hydrocarbyl group (i.e., -NH-) by a hydrocarbyl group, wherein a hydrocarbyl or hydrocarbyloxy group contains 1 to about 6 carbon atoms, and preferably 1 to about 4 carbon atoms.
• heterocyclyl groups are pyrrolidinyl , piperidinyl, piperazinyl, di- and tetrahydropyridyl, 4- (C]_-Cg-hydrocarbyl ) -piperidinyl,
• heteroaryl group is an aromatic heterocyclic ring that preferably contains one, or two, or three or four atoms in the ring other than carbon. Those heteroatoms can independently be nitrogen, sulfur or oxygen.
• a heteroaryl group can contain a single 5- or 6-membered ring or a fused ring system having two 6-membered rings or a 5- and a 6-membered ring.
• heteroaryl groups include 6-membered ring substituents such as pyridyl, pyrazyl, pyrimidinyl, and pyridazinyl; 5-membered ring substituents such as 1,3,5-, 1,2,4- or 1,2,3- triazinyl, imidazyl, furanyl, thiophenyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, 1,2,3-, 1,2,4-, 1,2,5-, or 1 , 3 , 4-oxadiazolyl and xsothiazolyl groups; 6-/5-membered fused ring substituents such as benzothiofuranyl, isobenzothiofuranyl,
• tetrahydroisoquinolinyl tetrahydroisoindolinyl, and anthranilyl groups
• 6-/ 6-membered fused rings such as 1,2-, 1,4-, 2,3- and 2 , 1-benzopyronyl , quinolinyl, isoquinolinyl , cinnolinyl, quinazolinyl, and 1 , 4-benzoxazinyl groups.
• halogen means fluorine, chlorine or bromine.
• halohydrocarbyl means a hydrocarbyl radical having the significance as defined above wherein one or more hydrogens are replaced with a halogen. Examples of such
• halohydrocarbyl radicals include chloromethyl
• perfluorohydrocarbyl means a hydrocarbyl group wherein each hydrogen has been replaced by a fluorine atom.
• perfluorohydrocarbyl groups in addition to trifluoromethyl above, are perfluorobutyl, perfluoroxsopropyl, perfluorododecyl and perfluorodecyl .
• a halohydrocarbyloxy substituent is a halogenated ether such as a trifluoromethoxy group and the like.
• the present invention contemplates a 20'- urea- or thiourea-substituted vinca alkaloid compound or pharmaceutically acceptable salt of such a compound. More particularly, a contemplated vinca alkaloid compound that is substituted at the 20'- position with a urea or thiourea group whose proximal nitrogen atom that is directly bonded to the
• 20' carbon atom is secondary (has one hydrogen atom bonded to it) and whose distal nitrogen is contains one, and more preferably contains two non-hydrido substituents (the and groups in the formula below) when the vinca alkaloid comopound does not contain a 10'-fluoro group.
• a preferred vinca alkaloid compound is a 20' -substituted vinblastine, vincristine or vindesine that can optionally be further substituted at the 10' -position with a fluoro group and corresponds in structure to Formula I below:
• Vinblastine (1) CH 3 — C-OCH 3 — 0-C-CH 3
• Vincristine (2) CH — C-OCH 3 — 0-C-CH 3
• hydrido independently selected from the group consisting of hydrido, a) a straight or branched chain or cyclic hydrocarbyl group that has 1-6 carbon atoms, and preferably 2-6 carbon atoms, that is free of tertiary or quaternary carbon atoms, b) an aromatic or
• aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are
• a third substituent, c) is an aralkyl or heteroaralkyl group containing 5 or 6 ring atoms of which up to three ring atoms can independently be nitrogen, oxygen or sulfur and contains 1-3 carbons in the alkyl portion.
• substituents of a)-d) above is other than hydrido, and more preferably, both R ⁇ and R- 3 substituents are other that hydrido when Z is H.
• Z is F
• R 4 and R ⁇ can be H, but it is preferred that only one be H and more preferred that both of R 4 and R- 3 be other than H.
• the single or fused ring system of d) can contain one or two additional hetero atoms that can independently be nitrogen, oxygen or sulfur.
• Each of the rings or ring systems of b) , c) and d) above can optionally be substituted with 1, 2 or 3 substxtuents selected from the group consisting of C ] _-Cg- hydrocarbyl, C ⁇ -Cg-hydrocarbyloxy, phenyl, halogen, perfluoro-C ] _-C -hydrocarbyl , perfluoro-C ] _-Cg- hydrocarbyloxy, nitro and mixtures thereof.
• both of R ⁇ and R ⁇ can be hydrido only when Z is F, but preferably, at least one of R ⁇ and R ⁇ is other than hydrido, and more preferably both of R ⁇ and R ⁇ are other than hydrido.
• One preferred R ⁇ and/or R ⁇ substxtuent is a straight or branched chain hydrocarbyl group that has 1-6 carbon atoms, and preferably 2-6 carbon atoms, that is free of tertiary or quaternary carbon atoms.
• hydrocarbyl substxtuent groups have been generally discussed previously. This hydrocarbyl group excludes those substxtuents that contain a tertiary carbon atom as in a t-butyl group [(0113)30-] or a quaternary carbon as is present in a neo-pentyl group [ (CH3) 3CH2-] .
• a second group of distal nitrogen substxtuents is an aromatic or aliphatic carbocyclic or heterocyclic ring structure that contains one or two rings, up to twelve ring atoms, and up to four ring atoms that are independently nitrogen, oxygen or sulfur. That ring structure is optionally substituted with 1, 2 or 3 substituents (ring substituents ) that are themselves selected from the group consisting of C]_-Cg- hydrocarbyl, Ci -Cg-hydrocarbyloxy, phenyl, halogen, perfluoro-C ] _-Cg-hydrocarbyl, perfluoro-C]_-Cg- hydrocarbyloxy, nitro and mixtures thereof.
• Illustrative ring structures include phenyl,
• a contemplated aromatic or aliphatic carbocyclxc or heterocyclic ring structure and/or substituent can contain 1-3 of its own
• substituents but preferably contains only one such as a halogen like chloro or fluoro, a C]_-Cg-hydrocarbyl group such as methyl, a Ci -Cg-hydrocarbyloxy group such as methoxy, a perfluoro-C ] _-Cg-hydrocarbyl group such as trifluoromethyl or a perfluoro-C]_-Cg- hydrocarbyloxy group such as pentafluoroethoxy .
• a halogen like chloro or fluoro a C]_-Cg-hydrocarbyl group such as methyl
• Ci -Cg-hydrocarbyloxy group such as methoxy
• a perfluoro-C ] _-Cg-hydrocarbyl group such as trifluoromethyl or a perfluoro-C]_-Cg- hydrocarbyloxy group such as pentafluoroethoxy .
• One preferred nitrogen substituent contains one ring that contains 3-6 carbon atoms in the ring.
• Another preferred substituent is an aromatic ring structure that contains one or two rings.
• One preferred aromatic ring contains one substituent.
• Another contemplated group of distal nitrogen atom and/or R ⁇ substituents is an aralkyl or heteroaralkyl group containing 5 or 6 ring atoms of which up to three ring atoms can independently be nitrogen, oxygen or sulfur and 1-3 carbons in the alkyl portion.
• the aromatic ring portion of a contemplated ring structure is optionally substituted with 1, 2 or 3 substituents that are selected from the group consisting of C]_-Cg-hydrocarbyl, C]_-Cg- hydrocarbyloxy, phenyl, halogen, perfluoro-C]_-Cg- hydrocarbyl, perfluoro-C ] _-Cg-hydrocarbyloxy, nitro and mixtures thereof, as are discussed above.
• Zero or one ring substituent other than hydrogen is preferred .
• alkyl portion of an aralkyl or heteroaralkyl group can contain 1 to 3 carbon atoms .
• Illustrative non-ring-substituted aralkyl groups include benzyl and phenethyl groups, whereas
• heteroaralkyl groups include
• a distal urea nitrogen atom can also form a ring structure together with the two and
• That nitrogen-containing ring structure can contain a single 5- or 6- membered ring or contain a fused two ring structure in which the rings are both 5-membered, or both 6-membered, or in which one ring is 5-membered and the other is 6- membered.
• a contemplated ring structure can also contain one or two additional hetero atoms (non- carbon atoms) in the ring(s) that can be
• This nitrogen-containing ring is preferably a 6-membered ring or a 5-/ 6-membered fused ring system.
• 20'-Urea- or thiourea-substituted vinblastine compounds are illustrated hereinafter that contain distal nitrogen atoms that are present in 5-, 6-, 6/6- and 5/6- membered rings .
• nitrogen-containing rings can be aromatic as in the case of an 1-imidazyl
• 2- (1, 3, 2-dioxazolyl) group but are more usually aliphatic such as 1-piperidinyl , 1-pyrrolidinyl , 1-piperazinyl , 1-morpholinyl, 1-thiomorpholinyl , isoquinolinyl, tetrahydroisoindolinyl and the like.
• a -NR ⁇ R5 cyclic substituent that contains a free hydrogen atom such as that of a secondary nitrogen (-NH-) present on the 4-position nitrogen of piperazine ring are blocked as with a t-Boc, F-moc, acetyl, C]_-Cg-hydrocarbyl such as methyl, or other group during synthesis and
• a urea nitrogen atom-containing ring structure is optionally substituted with 1, 2 or 3 substituents that are selected from the group consisting of C]_-Cg-hydrocarbyl, C]_-Cg- hydrocarbyloxy, phenyl, halogen, perfluoro-C ] _-Cg- hydrocarbyl, perfluoro-C]_-Cg-hydrocarbyloxy, nitro and mixtures thereof.
• a phenyl substituent bonded to a piperidinyl group or the aromatic portion of a ring structure formed by a -NR ⁇ RS cyclic structure can contain 1-3 substituents independently selected from the group consisting Ci-Cg-hydrocarbyl, C ] _-Cg- hydrocarbyloxy, perfluoro-C ⁇ -Cg-hydrocarbyl ,
• Another aspect of the invention is a doubly substituted vinca alkaloid compound.
• This compound contains a 20' -urea or thiourea group as discussed above, and also contains an added fluoro (F)
• a contemplated 20' -urea- or thiourea- substituted vinca alkaloid compound can also be used in the manufacture of a medicament (pharmaceutical composition) that is useful at least for inhibiting the proliferation (growth) of hematologic cancer cells such as leukemia or lymphoma cells, as well as cells of carcinomas, sarcomas, melanomas, neuromas and the like.
• a contemplated compound, medicament or pharmaceutical composition containing the same inhibits that growth by contacting those cancerous cells in vitro, or in vivo as in a subject in need thereof, as is a parent compound.
• pharmaceutically acceptable salts, buffers and the like are present that collectively are referred to as pharmaceutically acceptable diluents as compared to those that can be present in a composition that is not intended for pharmaceutical use, as in an in vitro assay.
• a compound of the invention can be provided for use by itself, or as a pharmaceutically
• the contemplated compounds are amines.
• Parental vinblastine has reported pKa values of 5.4 and 7.4, whereas vincristine has reported pKa values of 6.04 and 7.67.
• Both compounds are sold commercially as their sulfate salts.
• Vindesine is reported to have pka values of 6.04 and 7.67 [The Merck Index, 12 th ed., Merck and Co., Whitehouse Station, NJ, 1996, page 1704].
• Vindesine is also commercially available as the sulfate salt.
• Exemplary salts useful for a contemplated compound include but are not limited to the
• ethanesulfonate glucoheptanoate , glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide , hydroiodide, 2-hydroxy- ethanesulfonate , lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,
• palmoate palmoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate.
• the salts can also be used as an aid in the isolation, purification or
• the acid used and the salt prepared need not be pharmaceutically acceptable.
• a contemplated compound is active in in vitro assay studies at nanomolar to micromolar amounts .
• a contemplated compound is present in the composition in an amount that is sufficient to provide a
• a contemplated pharmaceutical composition contains a cancerous cell proliferation-inhibiting amount of a contemplated 20' -urea- or thiourea- substituted vinca alkaloid compound or a
• compositions can be administered to mammalian cells in vitro as in a cell culture to contact those cells, or the cells can be contacted in vivo as in a living, host mammal in need.
• anti-neoplastic drugs such as a 20 ' -substituted vinca alkaloid contemplated here are administered parenterally in vivo in a weight amount per square meter of the recipient's body surface area (bsa) .
• this amount is typically about 1 to about 20 mg/m ⁇ bsa, and about one-half those amounts for children, with an amount being chosen so that the maximal amount does not cause leukopenia.
• Children weighing about 10 kg or less are typically dosed at about 0.05 mg/kg.
• vinblastine sulfate is typically administered to adults at 3.7 mg/m 2 bsa for the first dose, 5.5 mg/m 2 bsa for the second weekly dose, 7.4 mg/m 2 bsa for the third weekly dose, 9.25 mg/m 2 bsa for the fourth weekly dose and 11.1 mg/m 2 bsa for the fifth weekly dose.
• Dosages typically do not exceed 18.5 mg/m 2 bsa, and should not be increased if the white-cell count falls to approximately 3000 cells/mm 3 .
• Usual dosages for adults are about 5.5 to 7.4 mg/m 2 bsa.
• Dosages of a contemplated 20' -position urea- or thiourea-substituted vinca alkaloid compound or its pharmaceutically acceptable salt typically do not exceed those of the parent compound and can be less .
• a contemplated composition is typically administered in vivo to a subject in need thereof a plurality of times within one month, such as weekly, and can be administered over a period of several months to several years . More usually, a
• contemplated composition is administered a plurality of times over a course of treatment.
• a contemplated 20'-urea- or thiourea-substituted vinca alkaloid compound is administered to treat the same disease state in the same amount and at the same intervals as is a
• a contemplated 20' -urea- or thiourea-substituted vinca alkaloid can be utilized as a first course of treatment, and is preferably administered if there is relapse after a first or later course of treatment, particularly where multiple drug resistance is shown or suspected (indicated) .
• a contemplated pharmaceutical composition can be administered orally (perorally) or
• parenterally which is preferred, in a formulation containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
• parenteral as used herein includes subcutaneous injections, intravenous (which is most preferred), intramuscular, intrasternal injection, or infusion techniques .
• Formulation of drugs is discussed in, for example, Hoover, John E.,
• Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules .
• the amount of a contemplated compound in a solid dosage form is as discussed previously, an amount sufficient to provide a concentration of about 0.5 nM to about 1000 nM, preferably about 1 nM to about 50 nM, in the serum or blood plasma.
• a solid dosage form can also be administered a plurality of times during a one week time period.
• a compound of this invention is ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the
• capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.
• the dosage forms can also comprise buffering agents such as sodium citrate, magnesium or calcium carbonate or
• Tablets and pills can additionally be prepared with enteric coatings .
• a contemplated pharmaceutical composition is preferably adapted for parenteral administration.
• a pharmaceutical composition is preferably in liquid form when administered, and most preferably, the liquid is an aqueous liquid, although other liquids are contemplated as discussed below, and a presently most preferred composition is an injectable preparation .
• injectable preparations for example, sterile injectable aqueous or oleaginous solutions or suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• suitable dispersing or wetting agents and suspending agents for example, sterile injectable aqueous or oleaginous solutions or suspensions.
• preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 , 3-butanediol .
• a nontoxic parenterally acceptable diluent or solvent for example, as a solution in 1 , 3-butanediol .
• acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline.
• liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration.
• Sterile water solutions of a 20' -urea- or thiourea-substituted vinca alkaloid active component or sterile solution of the active component in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
• a contemplated 20' -urea- or thiourea- substituted vinca alkaloid is provided as a dry powder that is to be dissolved in an appropriate liquid medium such as sodium chloride for injection prior to use.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides .
• fatty acids such as oleic acid find use in the preparation of an injectable composition.
• Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.
• Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions .
• a mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a contemplated compound is administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.
• a sample to be assayed such as cells and tissue can be used.
• These in vitro compositions typically contain the water, sodium or potassium chloride, and one or more buffer salts such as and acetate and phosphate salts, Hepes or the like, a metal ion chelator such as EDTA that are buffered to a desired pH value such as pH 4.0 -8.5, preferably about pH 7.2-7.4,
• the pharmaceutical composition is in unit dosage form.
• the pharmaceutical composition is in unit dosage form.
• the pharmaceutical composition is in unit dosage form.
• composition is divided into unit doses containing appropriate quantities of the active compound.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, in vials or ampules.
• a contemplated 20' -urea- or thiourea-substituted vinca alkaloid is administered with one or more other antineoplastic compounds.
• Such joint therapy is well known in the art, with other drugs such as cisplatin, 5-fluorouracil and the like being co-administered. That co-administration is usually physically separate administrations of each compound that are timed so that the two or more active agents can act in concert .
• Chemis ry The targeted vinblastine C20'-urea and -thiourea analogues were prepared by two methods (Scheme 1) .
• Method 2 was used. This method entailed treating 20'- aminovinblastine ( 7 ) with p-nitrophenyl-chloroformate to provide the activated carbamate 13 , which was then treated with a series of amines to provide the additional C20'-urea analogues.
• the vinblastine C20 ' -thiourea analogues were prepared also using two complementary methods (Scheme 2). Thus, treatment of 20 ' -aminovinblastine ( 7 ) with a small series of commercially available
• isothiocyanates (Method 3) versus isocyanates provided the corresponding C20 ' -thiourea analogues.
• treatment of 20 ' -isothiocyano- vinblastine ( 5 ) [Leggans et al . , Org. Lett. 2012
• N, IV-disubstituted C20'-urea and -thiourea vinblastine analogues were also prepared using Methods 2 and 4 and commercially available N, N-disubstituted amines. Additionally, the
• carbamate analogue 45 was synthesized for direct comparison with what proved to be the potent C20 ! - urea and -thiourea derivatives.
• vinblastine (1) was treated with the N, I ⁇ f-dimethylcarbamyl chloride to provide the C20 ' -carbamate 45 (below) .
• the C20 ' -substituted vinblastine analogues were examined for cell growth inhibitory activity against the HCT116 (human colon cancer) .
• HCT116/VM46 resistant human colon cancer tumor cell lines, the latter of which exhibits resistance (100- fold) to vinblastine through overexpression of Pgp.
• R CH 2 (2-pyridyl) (33) 24 5.6 670
• Terminal IV-alkyl substituents were not only well tolerated, but provided significant enhancements in activity, improving on the potency of 11 and providing derivatives that substantially surpass that of vinblastine itself. Most notable of these are the urea derivatives 14-18, bearing small IV-alkyl substituents. Some of those compounds exhibited IC 5 o values of 700-800 pM against HCT116, improving activity against HCT116 about 10-fold relative to 11, and substantially surpassing the potency of
• heteroaromatic ring (furan or pyridine) provided 33 and 34 , which displayed comparable activity to the parent 31 , matching or slightly exceeding the potency of vinblastine.
• N,N- disubstituted ureas and thioureas were also examined in order to establish whether the terminal urea nitrogen could be fully substituted or whether the derivatives require or benefit from the presence of an N-H H-bond donor, below.
• all the N,N- disubstituted ureas exhibited potent cell growth inhibitory activity matching or surpassing the activity of vinblastine and indicating that a terminal H-bond donor site is not important to their functional activity.
• H-donor capabilities of the former may be important.
• a L1210 murine leukemia cell line
• HCT116 human colon cancer cell line
• HCT116/VM46 resistant human colon cancer cell line, Pgp over-expression
• ND Not Done.
• the C20' -position within vinblastine represents a key site amenable to functionalization capable of simultaneously enhancing potency and presumably decreasing relative Pgp transport central to clinical resistance.
• R 4 H
• R 5 CH 3 ( 45 ) 60 8500
• R 4 and R 5 CH 3 ( 46 ) 980 >10000
• the 20 '- (methylamino) vinblastine ( 45 ) was enlisted to establish whether the active urea derivatives require or benefit from the H-bond donor site of the derivatized C20' -amine.
• treatment of 45 with ethyl isocyanate provided the W-methyl urea 47 for comparison with 15 .
• HCT116 700-fold decrease in activity between 15 and 47
• 35 contains the large biphenyl urea substituent yet matches the functional activity of vinblastine, whereas 39 bears the much smaller N, Ai-dimethylurea whose functional activity slightly exceeds that of vinblastine ( ⁇ 3-fold) .
• these binding studies confirmed that 39 binds tubulin with a slightly better affinity than vinblastine and further established that even 35 , bearing the large biphenyl substituent, remarkably binds with an affinity matching or even slightly exceeding that of vinblastine .
• the C20' urea derivative Compound 58 was examined in a tubulin binding assay conducted by measuring the competitive displacement of 3 H- vinblastine from porcine tubulin Illustrated below [Owellen et al., Biochem. Pharmacol. 1977 26:1213- 1219] .
• the binding studies established that Compound 58 binds tubulin with a higher affinity than
• vinblastine establishing that its enhanced potency in the cell growth functional assays correlates directly with its target tubulin binding affinity and suggests that the improved intrinsic activity is a direct result of the inhibition of microtubule formation.
• vinblastine ( 1 ) and Compounds 55 , 57 and 58 were examined offsite at an independent laboratory in a more comprehensive human tumor 15-cell line panel including cell lines of clinical interest from breast, lung, colon, prostate and ovary tissue
• PA-1 4. 6 0. 11 0. 19 0. 11
• the average IC 50 value for vinblastine in this human tumor cell line panel was 6.1 nM, excluding the two cell lines for which it was inactive, and the comparative average IC Q values were 310 pM, 200 pM, and 200 pM for Compounds 55, 57 and 58, respectively, representing average
• the C20'-site can also be a superb site for modulating the physical and chemical properties of the drug that impact additional features including Pgp efflux, in vivo drug distribution, selective cellular uptake, and metabolism.
• Compound 57 was found to bind tubulin with a higher affinity than vinblastine, confirming that its enhanced potency observed in the cell growth functional assays correlates with its target tubulin binding affinity.
• Examination of Compounds 55, 57 and 58 in a human tumor 15-cell line panel revealed that these C20' urea analogs are on average 20- to 30-fold more potent than vinblastine across a broad spectrum of clinically relevant human cancer cell lines (range of 10-200-fold more potent), displaying low pM IC50 values (40-450 pM for 58) .
• the C20' position within vinblastine represents a key site amenable to functionalization capable of simultaneously improving tubulin binding affinity, substantially enhancing biological potency, and presumably decreasing relative Pgp transport central to clinical resistance.
• HCT116/VM46 Cold, vinblastine resistant
• Isocyanates used include: ethyl isocyanate, n-butyl isocyanate, t-butyl isocyanate, cyclohexyl isocyanate, phenyl isocyanate, benzyl isocyanate, phenethyl isocyanate, o-methoxyphenyl isocyanate, ra-methoxyphenyl
• isocyanate isocyanate, p-methoxyphenyl isocyanate, p-fluoro- phenyl isocyanate, p-chlorophenyl isocyanate, p-tolyl isocyanate, p-trifluoromethylphenyl isocyanate, furfuryl isocyanate and p-biphenyl isocyanate.
• the amines used include: methylamine, propylamine, isopropylamine , cyclopropylamine , dimethylamine , diethylamine , 2- (aminomethyl) pyridine, morpholine, piperidine, ethanolamine , pyrrolidine, N-methylpiperazine , tetrahydropyridine ,
• the amines used include: cyclohexylamine , 4-fluorophenethylamine and dimethylamine .
• a solution 20 ' -aminovinblastine (8.8 mg, 0.011 mmol) in THF (3 mL) was treated with a 37% formaldehyde in water solution (4 ⁇ , 0.05 mmol).
• the reaction mixture was stirred for 4 h at 25 °C and then was treated with sodium cyanoborohydride (12 mg, 0.20 mmol).
• the reaction mixture was stirred for 1 h at 25 °C and then was quenched with distilled 3 ⁇ 40 (3 mL) .
• the mixture was extracted with 10% MeOH in
• Iron (III) chloride hexahydrate 55 mg, 0.21 mmol was added to a solution of ( - ) -vindoline (19 mg, 0.041 mmol) and 10 1 -fluorocatharanthine [Gotoh et al., ACS Med. Chem. Lett. 2011, 2:948-952] (15 mg, 0.041 mmol) in CF 3 CH 2 OH (0.1 mL) , aqueous 0.1 N HC1
• iron (III) oxalate hexahydrate (198 mg, 0.41 mmol) in degassed 3 ⁇ 40 (40 mL) was cooled to 0 °C and placed under Ar.
• CS 3 (215 mg, 1.23 mmol) was added to the mixture at 0 °C, followed by the vindoline coupling solution and NaBH 4 (31 mg, 0.81 mmol) in H 2 0 (1 mL) .
• the resulting mixture was stirred for 30 minutes before being quenched by addition of 28-30% aqueous NH 4 OH (4 mL) .
• a solution 51 (1.6 mg, 0.0019 mmol) in THF (3 mL) was treated with ethyl isocyanate (2 ⁇ 0.025 mmol) .
• the reaction mixture was stirred for 4 hours at 25 °C and then was concentrated under reduced pressure.
• L1210 ATCC #CCL-219, mouse lymphocytic leukemia, see Supporting Information
• HCT116 ATCC #CCL-247, human colorectal carcinoma
• HCT116/VM46 a vxnblastine-resistant strain of HCT116 cells in culture.
• a population of cells (> 1 x 10 6 cells/mL as determined with a hemocytometer ) was diluted with an appropriate amount of Dulbecco- modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) to a final
• phosphatase assay was used to establish the IC5 0 values as follows: the media in each cell was removed and 100 ⁇ of phosphatase solution (100 mg phosphatase substrate in 30 mL 0.1 M NaOAc, pH 5.5, 0.1% Triton X-100 buffer) was added to each well.

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