WO2004106291A1 - Composes d'uree haloethyle et leur utilisation en vue d'attenuer, d'inhiber ou de prevenir la migration de cellules cancereuses - Google Patents

Composes d'uree haloethyle et leur utilisation en vue d'attenuer, d'inhiber ou de prevenir la migration de cellules cancereuses Download PDF

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WO2004106291A1
WO2004106291A1 PCT/CA2004/000771 CA2004000771W WO2004106291A1 WO 2004106291 A1 WO2004106291 A1 WO 2004106291A1 CA 2004000771 W CA2004000771 W CA 2004000771W WO 2004106291 A1 WO2004106291 A1 WO 2004106291A1
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carcinoma
substituted
aryl
alkyl
nrr
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PCT/CA2004/000771
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Rene C. Gaudreault
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Imotep Inc.
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Priority to CA002568607A priority Critical patent/CA2568607A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/08—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D263/16—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D263/28—Nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/30—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by halogen atoms, or by nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/32—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/40—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/42—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/45—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups at least one of the singly-bound nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfonamides
    • C07C311/47—Y being a hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the present invention pertains to the field of therapeutics for diseases or disorders related to cancer cell migration, in particular to therapeutically active haloethyl urea derivatives and their use to inhibit cancer cell migration.
  • a cancer is a malignant tumour of potentially unlimited growth. It is primarily the pathogenic replication (a loss of normal regulatory control) of various given types of cells found in the human body. By select mutation resulting from a primary lesion, the DNA of a cancer cell evolves and converts the cell into an autonomous system. Invasion and metastasis are the most insidious and life-threatening aspects of cancer.
  • Metastasis of the primary tumour produces secondary tumours and disseminated cancer.
  • Therapy for metastasis currently relies on a combination of early diagnosis and aggressive treatment, which may include radiotherapy, chemotherapy or hormone therapy.
  • the high mortality rate for many cancers indicates that improvements are needed in the prevention and treatment of metastasis.
  • WO 97/00956 describes the use of an antibody raised against an adhesion protein on endothelial and muscle cells for inhibiting tumour metastasis.
  • U.S. Patent No. 6,015,893 describes oligonucleoside compounds useful in inhibiting the expression of focal adhesion kinase protein.
  • U.S. Patent No. 5,700,830 describes a method for inhibiting the adherence between cancerous cells and noncancerous structures in a mammal, comprising the administration to the mammal of a nitric oxide-releasing compound.
  • Urea-based compounds have been described for diverse indications, including as herbicides (U.S. Patent No. 3,885,954), as prophylactics against gastrointestinal and cardiovascular disorders (U.S. Patent No. 4,707,478), as anti-parasitic agents (U.S. Patent No. 4,707,478), as anti-athersclerotic agents (U.S. Patent No. 4,623,662), as treatments for gastrointestinal, spasmolytic and ulcerogenic disorders (U.S. Patent No. 4,304,786) and as anti-cancer agents (for example, U.S. Patent Nos. 3,968,249; 4,973,675 and 4,803,223).
  • a class of l-aryl-3-(2- chloroethyl)urea derivatives have been described as anti-cancer agents (U.S. Patent Nos. 5,530,026 and 5,750,547, and International Patent Application WO 00/61546) and as ⁇ -tubulin inhibitors (International Patent Application WO 01/447504).
  • An object of the present invention is to provide novel halo urea compounds for the inhibition of cancer cell motility/migration.
  • compound of formula I there is provided compound of formula I:
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (Ci- ⁇ ) alkyl, (Ca-Ci ⁇ ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(C ⁇ -C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O-aryl, -O-CH 2 -aryl, -S-(C ⁇ -C 16 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C ⁇ 6 ) alkynyl, -S- aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 ) cycl
  • a method of attenuating, inhibiting or preventing cancer cell migration in a mammal comprising administering an effective amount of a compound of formula I.
  • Figure 1 presents a graph illustrating the ability of the compound 1 (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) and cDDP (D) to inhibit tumour cell growth in a dose and time dependent manner.
  • Figure 2 depicts the microtubule depolymerization and cytoskeleton disruption induced by compounds of the invention.
  • Figure 3 presents a Western Blot illustrating the generation of an alkylated form of ⁇ -tubulin by compound 1 and compound 5.
  • Figure 4 presents a Western Blot illustrating the generation of an alkylated form of ⁇ -tubulin by compounds of the invention.
  • Figure 5 presents graphs illustrating the ability of compounds of the invention to inhibit MDA- MB-231 cancer cell migration in the wound assay.
  • Figure 6 presents graphs illustrating the ability of the compound I (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) to inhibit HT1080 cell migration.
  • Figure 7 presents graphs illustrating the ability of the compound 1 (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) and cDDP to impede the growth of two unrelated tumour cell lines in the chick chorioallantoic membrane (CAM) assay.
  • Figure 8 presents graphs illustrating the ability of compound 1, 5, 2 and 30 to impede the growth of CS1 cell line in the chick chorioallantoic membrane (CAM) assay
  • Figure 9 presents a graph illustrating the inhibition of carcinoma cell growth in Balb/c mice.
  • Figure 10 presents a graph illustrating that extracellular matrices and serum do not protect tumour cells against CEU toxiity in clonogenic assay.
  • the invention provides for the use of haloethyl urea derivatives of Formula I for attenuating, inhibiting or preventing cancer cell motility /migration.
  • cancer cell migration is a key feature of cancer cell invasion and metastasis.
  • the invention provides the use of the compounds of the invention to inhibit cancer progression and/or in the prevention of cancer metastasis.
  • halogen refers to fluorine, bromine, chlorine, and iodine atoms.
  • hydroxyl refers to the group -OH.
  • thiol or “mercapto” refers to the group -SH, and -S(O) 0- 2.
  • lower alkyl refers to a straight chain or branched, or cyclic, alkyl group of 1 to 16 carbon atoms. This term is further exemplified by such groups as methyl, ethyl, 72-propyl, i- propyl, 72-butyl, t-butyl, 1-butyl (or 2-methylpropyl), cyclopropylmethyl, z ' -amyl, n-amyl, hexyl and the like.
  • substituted lower alkyl refers to lower alkyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom of the lower alkyl moiety.
  • lower alkenyl refers to a straight chain or branched hydrocarbon of 2 to 16 carbon atoms having at least one carbon to carbon double bond.
  • substituted lower alkenyl refers to lower alkenyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl, alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom to produce a stable compound.
  • lower alkynyl refers to a straight chain or branched hydrocarbon of 2 to 16 carbon atoms having at least one carbon to carbon triple bond.
  • substituted lower alkynyl refers to lower alkynyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl, alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom to produce a stable compound.
  • alkyl alkenyl refers to a group -R-CR-CR'"R"", where R is lower alkyl, or substituted lower alkyl, R', R", R"" are each independently selected from hydrogen, halogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.
  • alkyl alkynyl refers to a group -R-C ⁇ CR where R is lower alkyl or substituted lower alkyl, R is hydrogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.
  • alkoxy refers to the group -OR, where R is lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or substituted cycloheteroalkyl as defined below.
  • acyl refers to groups -C(O)R, where R is hydrogen, lower alkyl substituted lower alkyl, aryl, substituted aryl.
  • aryloxy refers to groups -OAr, where Ar is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl group as defined below.
  • amino refers to the group NRR', where R and R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, cycloalkyl, or substituted hetaryl as defined below or acyl.
  • amido or “amide” refers to the group -C(O)NRR', where R and R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl as defined below.
  • Carboxyl refers to the group -C(O)OR, where R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl and the like as defined.
  • aryl refers to an aromatic carbocyclic group having at least one aromatic ring (e.g., phenyl or biphenyl) or multiple condensed rings in which at least one ring is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl, 9-fluorenyl etc.).
  • substituted aryl refers to aryl optionally substituted with one or more functional groups, e.g., halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano.
  • functional groups e.g., halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alky
  • heterocycle refers to a saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl, quinolinyl, indolizinyl, indanyl or benzo[b]thienyl) and having at least one hetero atom, such as N, O or S, within the ring.
  • a single ring e.g., morpholino, pyridyl or furyl
  • multiple condensed rings e.g., naphthpyridyl, quinoxalyl, quinolinyl, indolizinyl, indanyl or benzo[b]thienyl
  • hetero atom such as N, O or S
  • substituted heterocycle refers to heterocycle optionally substituted with, halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
  • heteroaryl or “hetar” refer to a heterocycle in which at least one heterocyclic ring is aromatic.
  • substituted heteroaryl refers to a heterocycle optionally mono or poly substituted with one or more functional groups, e.g., halogen, hydroxyl, thiol, lower allcyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
  • functional groups e.g., halogen, hydroxyl, thiol, lower allcyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, al
  • aralkyl refers to the group -R-Ar where Ar is an aryl group and R is lower alkyl or substituted lower alkyl group.
  • Aryl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, alkoxy, alkyl thio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • heteroalkyl refers to the group -R-Het where Het is a heterocycle group and R is a lower alkyl group.
  • Heteroalkyl groups can optionally be unsubstituted or substituted with e.g., halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • heteroarylalkyl refers to the group -R-HetAr where HetAr is an heteroaryl group and R lower alkyl or substituted loweralkyl.
  • Heteroarylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • cycloalkyl refers to a cyclic or polycyclic alkyl group containing 3 to 15 carbon.
  • these may be multiple condensed rings in which one of the distal rings may be aromatic (e.g. tetrahydronaphthalene, etc.).
  • substituted cycloalkyl refers to a cycloalkyl group comprising one or more substituents with, e.g halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
  • cycloheteroalkyl refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced with a heteroatom (e.g., N, O, S or P).
  • substituted cycloheteroalkyl refers to a cycloheteroalkyl group as herein defined which contains one or more substituents, such as halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • substituents such as halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • alkyl cycloalkyl refers to the group -R-cycloalkyl where cycloalkyl is a cycloalkyl group and R is a lower alkyl or substituted lower alkyl.
  • Cycloalkyl groups can optionally be unsubstituted or substituted with e.g. halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • treatment refers to improvement in the recipient's status as well as prophylaxis.
  • the improvement can be subjective or objective and related to features such as symptoms or signs of the disease or condition being treated. Prevention of deterioration of the recipient's status is also encompassed by the term.
  • ameliorate or “amelioration” includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease being treated, both temporary and long-term.
  • the compounds according to the instant invention include compounds of the following general formula:
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (Ci-Ci ⁇ ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(C ⁇ -C ⁇ 6 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O- aryl, -O-CH 2 -aryl, -S-(C ⁇ -C ⁇ 6 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8
  • the compound of the invention is one in which RI and R2 are each independently selected from H, (C ⁇ -C 6 ) alkyl and ( -C 6 ) alkoxy.
  • B is phenyl, substituted with one or more substituents indepnednetly selected from the list as shown above.
  • B is phenyl substituted with halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (C C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl , wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR*, -OC(O)R, -C(O)NR'R', -OR', wherein R and R' are as defined above.
  • X is F, CI, Br or I
  • RI and R2 are as defined above, and
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from, (C;[-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C2-C 16 ) alkynyl, -O ⁇ d-C ⁇ ) alkyl, -O-(C2-C 16 ) alkenyl, -O-(C2-C 16 ) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH 2 -aryl, -S-(C !
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R', -OC(S)R, -C(O)R,
  • substituents of the compounds of formula (I) are as follows: X is F, CI, Br or I; RI and R2 are as defined above, and
  • B is phenyl substituted with at least one substituent selected from, (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -d 6 ) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH 2 -aryl, -S-(d-C 16 ) alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R*, -OC(S)R', -C(O)R,
  • X is F, CI, Br or I
  • RI and R2 are as defined above, and
  • B is phenyl substituted with at least one group selected from, (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl,
  • alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR, -O-aryl, -OC(O)R, -C(O)R, -
  • R and R are as defined above.
  • X is F CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from (d-C 16 ) alkyl, (C 2 -C 1 ) alkenyl, (C2-C 16 ) alkynyl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH 2 -aryl, -S-(d-C 16 ) alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R',
  • the compounds of formula (I) include the compounds of formula (II):
  • X is F CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O-aryl, -O-CH 2 -aryl, -S-(C C 16 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S- aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 ) cycloalky
  • X is CI or Br.
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine;
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R",
  • X is F, CI, Br or I
  • B is phenyl substituted with at least one substituent selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH 2 -aryl, -S-(d-C 16 ) alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R',
  • substituents of the of the compounds of formula (I) are as follows: X is F, CI, Br or I;
  • B is phenyl substituted with at least one group selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl,
  • alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR, -O-aryl, -OC(O)R, -C(O)R, -
  • R and R are as defined above.
  • X is F, CI, Br or I
  • B is substituted with at least one group selected from aryl, -O-aryl, -O-CH 2 -aryl and halo.
  • X is F, CL Br or l
  • B is substituted with at least one substituent selected from the group of (d-C 16 ) alkyl, (d-C 16 ) alkynyl or -O-alkyl; wherein: said alkyl is substituted with at least one substituent selected from the group of-CN, -O-alkyl, -
  • alkyny and -O-alkyl are substituted with at least one substituent selected from -CN, -
  • R is as defined above.
  • X is F, CL Br or l
  • B is substituted with at least one group selected from -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O) 2 OR, -S(O)NRR, -S(O)ONRR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR; wherein R is as defined above.
  • the compounds of formula (I) include the compounds of formula (III):
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C ⁇ 6 ) alkynyl, aryl, -O-(d-C 16 ) allcyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O- aryl, -O-CH 2 -aryl, -S-(d-d 6 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 )
  • substitutents of formula (I) are as follows:
  • X is F CI, Br or I
  • RI and R2 are each independently selected from the group of H, (d-C 6 ) alkyl, (C ⁇ -C 6 ) hydroxy allcyl, (d-C 6 ) alkoxy, (C 3 -C 7 ) cycloalkyl, halo substituted (d-C 6 ) alkyl, halo di- substituted (d-C 6 ) alkyl, halo tri-substituted (d-C 6 ) alkyl and halo;
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O- aryl, -O-CH 2 -aryl, -S-(d-C 16 )alkyl, -S-(C 2 -C ⁇ 6 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 ) cycl
  • substituents of the compounds of formula (I) are as follows: X is F, CI, Br or I;
  • RI and R2 are each independently selected from the group of H, (C ⁇ -C 6 ) alkyl, (d-C 6 ) hydroxy alkyl, (d-C 6 ) alkoxy, (C 3 -C 7 ) cycloalkyl, halo substituted (d-C 6 ) alkyl, halo di-substituted (d- C 6 ) alkyl, halo tri-substituted (C ⁇ -C 6 ) alkyl and halo;
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, -O-(d-d 6 ) alkyl, -O-(C 2 -C 16 ) alkenyl,
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R', -C(O)R,
  • X is F, C Br or l
  • RI and R2 are each independently selected from the group of H, (C ⁇ -C 6 ) alkyl, (d-C 6 ) hydroxy alkyl, (C ⁇ -C 6 ) alkoxy, (C -C 7 ) cycloalkyl, halo substituted (d-C 6 ) alkyl, halo di-substituted (Ci-
  • B is phenyl substituted with at least one group selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl,
  • alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR', -O-aryl, -OC(O)R', -C(O)R', -
  • R and R' are as defined above.
  • the compounds of formula (I) include the compounds of formula (IV):
  • R3 is selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR, -NO 2 , -CN, -C(O)R, -C(S)R,-OC(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -C(S)NR(SR), -CH(CN) 2 , -CH[C(O)R] 2 , -CH[C(S)R] 2 , -CH[C(O)OR] 2 , -CH[C(O)OR] 2 , -CH
  • R3 is selected from halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (d-C 16 ) alkyl, (C 2 -C 16 ) allcenyl, (C -C 16 ) alkynyl, wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR, -OC(O)R, -C(O)NR'R', -OR', wherein R and R are as defined above.
  • the compounds of formula (I) include the compounds of formula (V):
  • R3 is selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR, -NO 2 , -CN, -C(O)R, -C(S)R,-OC(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -C(S)NR(SR), -CH(CN) 2 , -CH[C(O)R] 2 , -CH[C(S)R] 2 , -CH[C(O)OR] 2 , -CH[C(O)OR] 2 , -CH
  • R3 is selected from halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (C C ⁇ 6 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR,
  • the compounds according to formula (I) include those listed below: l-(2-Chloro-ethyl)-3-772-tolyl-urea; l-(2-Chloro-ethyl)-3-(3-ethyl-phenyl)-urea; l-(2-Chloro-ethyl)-3-(3-methoxy-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(4-hydroxy-butyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[4-(3-hydroxy-propyl)- ⁇ henyl]-urea; l-(2-Chloro-ethyl)-3-(3-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(5-hydroxy-pentyl)-phenyl]-urea;
  • Acetic acid 4- ⁇ 3-[3-(2-chloro-ethyl)-ureido]-phenyl ⁇ -butyl ester;
  • Compounds of Formula I wherein X is Br or I may undergo rearrangement to provide a rearrangement product. Such rearrangement products are considered to be within the scope of the present invention.
  • the present invention contemplates the compounds of Fonnula I wherein X is Br or I as a form of pro-drugs, for which both the pro-drug form and the rearrangement product may have activity in inhibiting proliferation of cancer cells.
  • this invention includes the pharmaceutically acceptable salts of the compounds defined by Formula I, II, and III.
  • a compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of organic and inorganic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to salts of the compounds of the above formula which are substantially non-toxic to living organisms.
  • Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts.
  • Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as -toluenesulfonic acid, methanesulfonic acid, oxalic acid, 7-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like
  • organic acids such as -toluenesulfonic acid, methanesulfonic acid, oxalic acid, 7-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • salts examples include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenyl
  • the pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
  • Salts of amine groups may also comprise quarternary ammonium salts wherein the amino nitrogen carries a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety.
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
  • bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like, hi one embodiment, the base addition salt is a potassium or sodium salt.
  • any salt of this invention is usually not of a critical nature, as long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
  • This invention further encompasses the pharmaceutically acceptable solvates of the compounds of Formula I, II or III. Many of these compounds can combine with solvents such as water, methanol, ethanol and acetonitrile to form pharmaceutically acceptable solvates such as the corresponding hydrate, methanolate, ethanolate and acetonitrilate.
  • the compounds of the present invention have multiple asymmetric (chiral) centers. As a consequence of these chiral centers, the compounds of the present invention occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All asymmetric forms, individual isomers and combinations thereof, are within the scope of the present invention.
  • R and S are used herein as commonly used in organic chemistry to denote the absolute configuration of a chiral center, according to the Cahn-higold-Prelog system.
  • the stereochemical descriptor R rectus
  • S sinister
  • R rectus
  • S sinister
  • the priority of groups is decided using sequence rules as described by Cahn et al, Angew. Chem., 78, 413-447, 1966 and Prelog, V. and Helmchen, G.; Angew. Chem. Int. Ed. Eng, 21, 567-583, 1982).
  • the older D-L system is also used in this document to denote relative configuration, especially with reference to amino acids and amino acid derivatives.
  • a Fischer projection of the compound is oriented so that carbon-1 of the parent chain is at the top.
  • D is used to represent the relative configuration of the isomer in which the functional (determining) group is on the right side of the carbon atom at the chiral center and "L", that of the isomer in which it is on the left.
  • the stereochemistry of the Formula I, II and III compounds may be critical to their potency as agonists or antagonists.
  • the relative stereochemistry is preferably established early during synthesis, which avoids stereoisomer separation problems later in the process. Subsequent synthetic steps then employ stereospecific procedures so as to maintain the preferred configuration.
  • Non-toxic metabolically labile esters and amides of compounds of Formula I, II or III are ester or amide derivatives that are hydrolyzed in vivo to afford said compounds of Formula I, II or III and a pharmaceutically acceptable alcohol or amine.
  • Examples of metabolically labile esters include esters formed with (C ⁇ -C 6 ) alkanols in which the alkanol moiety may be optionally substituted by a (d-C 16 ) alkoxy group, for example, methanol, ethanol, propanol and methoxyethanol.
  • Examples of metabolically labile amides include amides formed with amines such as methylamine.
  • the compounds of Formula I can be used for attenuating, inhibiting or preventing cancer cell migration in a mammal in need of such therapy.
  • the compounds of the invention are used to attenuate, inhibit or prevent cancer cell migration.
  • the compounds can be used alone or they can be used as part of a multi-drug regimen in combination with known therapeutics.
  • cancers include, but are not limited to leukaemia, carcinomas, adenocarcinomas, melanomas and sarcomas.
  • Carcinomas, adenocarcinomas and sarcomas are also frequently referred to as "solid tumours," examples of commonly occurring solid tumours include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, ovary, pancreas, prostate, stomach and uterus, non- small cell lung cancer and colorectal cancer.
  • leukaemia refers broadly to progressive, malignant diseases of the blood-forming organs. Leukaemia is typically characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow but can also refer to malignant diseases of other blood cells such as erythroleukaemia, which affects immature red blood cells. Leukaemia is generally clinically classified on the basis of (1) the duration and character of the disease - acute or chronic; (2) the type of cell involved - myeloid (myelogenous), lymphoid (lymphogenous) or monocytic, and (3) the increase or non-increase in the number of abnormal cells in the blood - leukaemic or aleukaemic (subleukaemic).
  • Leukaemia includes, for example, acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leuk
  • tumour generally refers to a tumour which originates in connective tissue, such as muscle, bone, cartilage or fat, and is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumour sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemorrhagic
  • melanoma is taken to mean a tumour arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex
  • carcinomas that originate in cells that make organs which have glandular (secretory) properties or that originate in cells that line hollow viscera, such as the gastrointestinal tract or bronchial epithelia. Examples include, but are not limited to, adenocarcinomas of the breast, lung, pancreas and prostate.
  • Additional cancers encompassed by the present invention include, for example, Hodgkin's Disease, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumours, primary brain tumours, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, gliomas, testicular cancer, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, mesothelioma and medulloblastoma.
  • the compounds of Formula I can be used for attenuating, inhibiting or preventing cancer cell proliferation in a mammal in need of such therapy.
  • the novel compounds described by Formula I of the invention are used to attenuate, inhibit or prevent cancer cell proliferation.
  • the compounds can be used alone or they can be used as part of a multi-drug regimen in combination with known therapeutics.
  • the therapeutic compounds of Formula I are capable of attenuating, inhibiting, or preventing cancer cell migration in vivo.
  • compounds within Formula I will demonstrate different activities in their ability to attenuate, inhibit, or prevent cancer cell migration and to treat the diseases associated with such migration.
  • the ability of the compounds to attenuate, inhibit, or prevent cancer cell migration can be initially determined in vitro if desired.
  • the present invention thus contemplates a preliminary in vitro screening step to further characterize compounds suitable for incorporation into the therapeutic compositions.
  • a number of standard tests to determine the ability of a compound to attenuate, inhibit, or prevent cancer cell migration are known in the art and can be employed to test the compounds of Formula I. Exemplary procedures are described herein.
  • Candidate compounds of Fonnula I can be tested in vitro and in vivo to determine their activity in inhibiting cancer cell migration and metastasis formation.
  • the ability of a compound to inhibit migration of neopalstic cells can be assessed in vitro using standard cell migration assays.
  • assays are conducted in multi-well plates, the wells of the plate being separated by a suitable membrane into top and bottom sections.
  • the membrane can be coated with an appropriate compound, the selection of which is dependent on the type of cell being assessed and can be readily determined by one skilled in the art. Examples include collagen, gelatine or Matrigel.
  • chemo-attractant such as soluble fibronectin, EGM-2, IL-8, ⁇ -FGF, ⁇ -FGF and the like
  • chemo-attractant such as soluble fibronectin, EGM-2, IL-8, ⁇ -FGF, ⁇ -FGF and the like
  • An aliquot of the test cells together with the test compound is added to the upper chamber, typically various dilutions of the test compound are tested. After a suitable incubation time, the membrane is rinsed, fixed and stained. The cells on the upper side of the membrane are wiped off, and then randomly selected fields on the bottom side are counted.
  • Neoplastic cell migration can also be assessed in vitro using the wound healing assay described by Alper et al. (J Natl Cancer hist. 2001 93(18):1375-84) and Tamura et al. (Science 1998;280:1614-7). Briefly, a wound is created in a cell monolayer by the gentle removal of the attached cells. The migration of the cells into the wound is then observed at different time points and in the absence or presence of a test compound to determine the effect of the test compound on cell migration.
  • compositions may be desirable to determine the ability of the compositions to inhibit cell migration of certain specific cancer cell lines, for example drug- resistant or highly metastatic cell lines and that appropriate cell lines can be selected accordingly.
  • Suitable neoplastic cell lines are available from the American Type Culture Collection (ATCC), which currently provides 950 cancer cell lines, and other commercial sources, hi one embodiment of the present invention, in vitro testing of the candidate compounds is conducted in a human cancer cell-line.
  • ATCC American Type Culture Collection
  • suitable human cancer cell-lines for in vitro testing of the compounds of the present invention include, but are not limited to, breast cancer cell-lines MCF- 7, t47D and MDA-MB-23, colon cancer cell-lines CaCo and LoVo, ovarian cancer cell-line SKOV3, prostate cancer cell-line DU-145, chronic myeloid leukaemia cell-line K562 and bladder cancer cell-line T24.
  • Toxicity of the candidate compounds can also be initially assessed in vitro using standard techniques.
  • human primary fibroblasts can be treated in vitro with a compound of Formula I.
  • Cells are then tested at different time points following treatment for their viability using a standard viability assay, such as the assays described above or the trypan-blue exclusion assay.
  • Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS).
  • FACS fluorocytometer cell sorter
  • the ability of the candidate compounds to inhibit tumour growth or metastasis in vivo can be detennined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, hie, New York, NY).
  • xenograft models in which a human tumour has been implanted into an animal.
  • xenograft models of human cancer include, but are not limited to, human solid tumour xenografts in mice, implanted by sub-cutaneous injection and used in tumour growth assays; human solid tumour isografts in mice, implanted by fat pad injection and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.
  • xenograft models of human cancer include, but are not limited to, human solid tumour xenografts in mice, implanted by sub-cutaneous injection and used in tumour growth assays; human solid tumour isografts in mice, implanted by fat pad injection and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.
  • the candidate compounds can be tested in vivo on solid tumours using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumour fragment, or implanted with an appropriate number of cancer cells, on day 0.
  • Subcutaneous xenografts metastasize infrequently and seldom invade adjacent tissue, therefore, rate of tumour growth or delay of significant tumour growth are the endpoints used in this model.
  • the animals bearing tumours are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumours, tumours are allowed to develop to the desired size, animals having insufficiently developed tumours being eliminated.
  • the selected animals are distributed at random to undergo the treatments and controls. Suitable controls will be dependent on the actual compound being tested and may include untreated animals.
  • Chemotherapy generally begins from 1 to 22 days after grafting, depending on the type of tumour, and the animals are observed every day.
  • the compounds can be administered prior to tumour implantation, for example, about 7 days prior.
  • the compounds of the present invention can be administered to the ammals, for example, orally, by i.p. injection or bolus infusion.
  • the different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial.
  • tumours are measured about 2 or 3 times a week until the tumour reaches a pre-determined size and / or weight, or until the animal dies if this occurs before the tumour reaches the predetermined size / weight.
  • the animals are then sacrificed and the tissue histology, size and / or proliferation of the tumour assessed.
  • Orthotopic xenograft models are an alternative to subcutaneous models and may more accurately reflect the cancer development process.
  • tumour cells are implanted at the site of the organ of origin and develop internally. Daily evaluation of the size of the tumours is thus more difficult than in a subcutaneous model.
  • GFP green fluorescent protein
  • a recently developed technique using green fluorescent protein (GFP) expressing tumours in non-invasive whole-body imaging can help to address this issue (Yang and al, Proc. Nat. Aca. Sci, (2000), pp 1206-1211). This technique utilises human or murine tumours that stably express very high levels of the Aqueora vitoria green fluorescent protein.
  • the GFP expressing tumours can be visualised by means of externally placed video detectors, allowing for monitoring of details of tumour growth, angiogenesis and metastatic spread.
  • the use of this model thus allows for simultaneous monitoring od several features associated with tumour progression and has high preclinical and clinical relevance.
  • the ability of the candidate compound to inhibit formation of a solid tumour can also be assessed in the chick chorioallantoic membrane (CAM) assay using published protocols (Brooks et al, in Methods in Molecular Biology, Vol. 129, pp. 257-269 (2000), ed. A.R. Howlett, Humana Press Inc., Totowa, NJ).
  • CAM chick chorioallantoic membrane
  • the animals are grafted with a particular number of cells, and the anti-tumour activity is detennined by the increase in the survival time of the treated mice relative to the controls.
  • compositions of the present invention can be employed.
  • the animals are treated before or after injection or implantation of the neoplastic cells into the animal.
  • the spread of the neoplastic cells from the site of injection for example spread to the lungs and/or lymphoid nodes, is then monitored over a suitable period of time by standard techniques.
  • LLCl Lewis lung
  • the cells are administered intravenously to normal non- immune-compromised mice thus allowing for immediate dissemination of cancerous cells. Treatment can be initiated several days before injection of the LLCl cells in order to observe a preventive effect or immediately after injection of the cells in order to observe an attenuating effect. After about 14 days, the mice are sacrificed, the lungs removed and fixed and the number and size of lung tumours determined. The intravenous route of administration for the LLCl cells in this model allows for rapid evaluation of treatments.
  • LLCl cells are injected subcutaneously to allow the growth of a primary tumour, which is then surgically removed once a certain size is obtained. Following removal of the primary tumour, treatment is initiated for about 14 days, after which the animals are sacrificed and tumours counted as in the intravenous model. The primary tumour is removed in this model is recommended as it can be metastasis-suppressing.
  • In vivo toxic effects of the compounds of Formula I can be evaluated by standard techniques, for example, by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.
  • Table 1 Examples of xenograft models of human cancer
  • the compounds of the invention can be submitted to other standard tests, such as cytotoxicity tests, stability tests, bioavailability tests and the like.
  • the compounds of the invention will need to meet certain criteria in order to be suitable for human use and to meet regulatory requirements.
  • standard in vitro and in vivo tests can be conducted to determine information about the metabolism and pharmacokinetic (PK) of the compound which can be used to design human clinical trials.
  • PK pharmacokinetic
  • the safety profile of the compounds can be determined in at least two non-human species and then the compounds will progress into Clinical Trials in order to ftirther evaluate their efficacy in attenuating the metastasis of tumours and to obtain regulatory approval for therapeutic use.
  • Clinical Trials progress through phases of testing, which are identified as Phases I, II, III, and IV.
  • Phases I, II, III, and IV In vitro and in vivo information about the metabolism and pharmacokinetic (PK) of the compounds determined from pre-clinical studies facilitates the design of initial Phase I and Phase II clinical studies.
  • Phase I clinical trials are normally performed in healthy human volunteers or in advanced cancer patients.These studies are conducted to investigate the safety, tolerability and PK of the compositions and to help design Phase II studies, for example, in terms of appropriate doses, routes of administration, administration protocols. Phase I studies could incorporate pharmacodynamic assays to evaluate proof of principle in inhibition of target inhumans. An adequate pharmacodynamic endpoint would be to determine the inhibitory activity measured from the plasma of healthy volunteers.
  • Phase I studies allow the selection of safe dose levels for Phase II studies.
  • An important factor in the protocol design of the Phase II studies is the adequate recruitment of the patient population to be studied based on stringent selection criteria defining the demographics (age, race and sex) of the study, the previous medical history of the patient, the type of cancer and stage of its development as well as any previous cancer treatment history. The latter factor can be important when the composition is intended as an adjuvant to first line therapy rather than a treatment to refractory disease.
  • a protocol for Phase II studies typically specifies baseline data that can be used to characterise the population, to evaluate the success of randomization in achieving balance of important prognostic factors, and to allow for consideration of adjusted analyses. Staging of the cancers of interest
  • Staging of the cancer being investigated can be important and, when possible, patients should be recruited such that the cancer stage is as homogeneous as possible across the population to facilitate statistical analysis and interpretation of the data.
  • methods and criteria for staging of a cancer vary depending on the particular cancer being investigated.
  • Clinical biomarkers can be defined as follows (Atkinson A et al: Clin. Pharmacol. Ther. 69, 89-95 (2001):
  • Biomarker a characteristic that is objectively measured and evaluated as an indicator of normal biological process, pathogenic process, or pharmacological response to a therapeutic intervention.
  • Clinical endpoint a characteristic or variable that reflects how a patient feels or functions, or how long a patient survives.
  • Surrogate endpoint biomarker intended to substitute for a clinical endpoint.
  • a clinical investigator uses epidemiological, therapeutic, pathophysiological, or other scientific evidence to select a surrogate endpoint that is expected to predict benefit, harm or the lack of benefit or harm.
  • the FDA defines a surrogate endpoint, or marker, as a laboratory measurement or physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint that is a direct measure of how a patient feels, functions or survive and is expected to predict the effect of the therapy.
  • Phase III trials focus on determining how the compound compares to the standard, or most widely accepted, treatment.
  • patients are randomly assigned to one of two or more "arms".
  • one arm will receive the standard treatment (control group) and the other arm will be treated with the compound (investigational group).
  • Phase IV trials can be used to further evaluate the long-term safety and effectiveness of the compound. Phase IV trials are less common than Phase I, II and III trials and would take place after the compound has been approved for standard use.
  • urea moiety is achieved using the 4-dimethylaminopyridine-catalyzed reaction of the relevant 4-alkylaniline (VI) with di-tert-butyldicarbonate in dichloromethane followed by the trapping of the in situ generated isocyanate with the appropriate (R)- or ( ⁇ S)-2-aminoalcohol (see Knolker, et al, Synlett (1996) 502-504).
  • This procedure ensures a racemization-free synthesis of urea under mild conditions and circumvents side reactions such as the formation of symmetrical disubstituted urea (see Knolker, et al, Synlett (1997) 925-928).
  • the present invention provides methods of treating diseases characterized by neoplastic cell migration in a mammal comprising administering an effective amount of one or more compounds of Formula I, or non-toxic metabolically-labile esters or amides thereof, or pharmaceutically acceptable salts thereof.
  • the compounds of the present invention are typically formulated prior to administration.
  • the present invention thus provides pharmaceutical compositions comprising one or more compounds of Formula I and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients.
  • Pharmaceutical compositions comprising one or more compounds of Formula I in combination with one or more known cancer chemotherapeutics are also contemplated by the present invention.
  • Compounds of the general Formula I or pharmaceutical compositions comprising the compounds may be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles, hi the usual course of therapy, the active compound is incorporated into an acceptable vehicle to form a composition for topical administration to the affected area, such as hydropohobic or hydrophilic creams or lotions, or into a form suitable for oral, rectal or parenteral administration, such as syrups, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositiories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectables, or solutions.
  • parenteral as used herein includes subcutaneous injections, intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal injection or infusion techniques.
  • compositions intended for oral use may be prepared in either solid or fluid unit dosage forms.
  • Fluid unit dosage form can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • An elixir is prepared by using a hydroalcoholic (e.g., ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent.
  • Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.
  • Solid fonnulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
  • Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gxun tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbit
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or fl-propyl- /j-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or fl-propyl- /j-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol,
  • compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution, hi addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.
  • the compound(s) of the general Formula I may be administered, together or separately, in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter and polyethylene glycols.
  • the compounds can be formulated into solutions, suspensions, and ointments appropriate for use in the eye (see, for example, Mitra (ed.), (1993) Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y.; Havener, (1983) Ocular Pharmacology, CN. Mosby Co., St. Louis).
  • compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000).
  • therapeutic compounds are administered systemically to patients, for example, by bolus injection or continuous infusion into a patient's bloodstream.
  • one or more compounds of Formula I are administered systemically to a patient in need of therapy.
  • the compounds can be administered prior to, or after, administration of the chemotherapeutic agents, or they can be administered concomitantly.
  • the one or more chemotherapeutic may be administered systemically, for example, by bolus injection or continuous infusion, or it may be administered orally.
  • the dosage to be administered is not subject to defined limits, but it will usually be an effective amount. It will usually be the equivalent, on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects.
  • the compositions may be formulated in a unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Examples of ranges for the compound(s) in each dosage unit are from about 0.05 to about 100 mg, or more usually, from about 1.0 to about 30 mg.
  • Daily dosages of the compounds of the present invention will typically fall within the range of about 0.01 to about 100 mg/kg of body weight, in single or divided dose.
  • the actual amount of the compound(s) to be administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • the above dosage range is given by way of example only and is not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing harmful side effects, for example, by first dividing the larger dose into several smaller doses for administration throughout the day.
  • kits containing the compounds of the invention for use in the inhibition of cancer cell migration in a mammal in need of such therapy.
  • Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects , approval by the agency of manufacture, use or sale for human administration.
  • the liquid solution can be an aqueous solution, for example a sterile aqueous solution.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to a patient or applied to and mixed with the other components of the kit.
  • kits of the invention may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components.
  • the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient.
  • Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • EtOAc ethyl acetate
  • THF tetrahydrofuran
  • EtOH ethanol
  • TLC thin layer chromatography
  • GC gas chromatography
  • HPLC high pressure liquid chromatography
  • m-CPBA m-chloroperbenzoic acid
  • Et 2 O diethyl ether
  • DMSO dimethyl sulfoxide
  • DBU l,8-diazabicyclo-[5.4.0]undec-7-ene
  • MTBE methyl t-butyl ether
  • FDMS field desorption mass spectrometry.
  • This compound was prepared according to the process of Example 1, except that 4-cyclohexyl aniline was used instead of -t-butyl aniline.
  • the final product was recrystallized from THF/hexane to obtain 82% yield.
  • This compound was prepared according to the process of Example 1, except that 4-heptylaniline was used instead of ⁇ -t-butyl aniline.
  • the final product was recrystallized from THF/hexane to obtain 93% Yield.
  • This compound was prepared according to the process of Example 1, except that 4-iodoaniline was used instead of -t-butyl aniline.
  • the final product was recrystallized from THF/hexane. Yield 60%
  • This compound was prepared according to the process of Example 1, except that 4- benzyloxyaniline was used instead of ⁇ -t-butyl aniline.
  • This compound was prepared according to the process of Example 1, except that 4- hydroxyaniline was used instead of ⁇ -t-butyl aniline. The final product was recrystallized from
  • This compound was prepared according to the process of Example 1, except that 3'- aminoacetanilide was used instead of ⁇ -t-butyl aniline.
  • the final product was recrystallized from ethyl acetate/methanol/hexane. Yield 46%.
  • This compound was prepared according to the process of Example 1, except that 3-amino-N- butylbenzenesulfonmide was used instead of ⁇ -t-butyl aniline.
  • the final product was recrystallized from from ethanol/water. Yield 50%.
  • This compound was prepared according to the process of Example 1, except that 3-(l- hydroxyethyl) aniline was used instead of ⁇ -t-butyl aniline.
  • Acetic acid 5-(3-tert-butoxycarbonylamino-phenyl)-pentyl ester (88 mg, 0.27 mmol) was dissolved in a mixture of trifluoroacetic acid (4.5 mL) and water (0.5 mL) and the solution was stirred for 10 min at room temperature. The solvents were evaporated and the residue was taken up in ethyl acetate. The organic phase was washed with a saturated solution of NaHCO (twice), brine, dried over Na 2 SO and concentrated in vacuo to give acetic acid 5-(3-aminophenyl)pentyl ester (36 mg, 60%).
  • 6-(3-aminophenoxy)hexanoic acid ethyl ester was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 19. Purified by flash chromatography (hexane/ethyl acetate 3/2). Yield 85%.
  • 2-(hept-l-ynyl)aniline (628 mg, 3.35 mmol) was dissolved in ethanol (20 mL) and placed in a hydrogenation bottle with 10% Pd/C (88 mg). The bottle was filled with 40 psi of hydrogen and shaken for 4 h. The product was filtered through Celite, and the solvent was removed in vacuo to obtain 2-heptanylaniline. The aniline was used without any further purification (602 mg, %)
  • the purification technique was flash chromatography on silica gel : Hexane / AcOEt (70 / 30).
  • 5-(3-nitrophenyl)pent-4-yn-l-ol (1.0 Eq, 0.970 mmol, 0.200 g) in Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H 2 (38 PSI), ETOH (10 mL) was hydrogenated as described in Example 32 to yield 5-(3-aminophenyl)pentan-l-ol.
  • the product was purified by flash chromatography on silica gel : Hexane / AcOEt (70 / 30).
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 5-Hexyn-l-ol (2.58 Eq, 20.670 mmol, 2.030 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO 3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32.
  • 6-(3-nitrophenyl)hex-5-yn-l-ol (1.0 Eq, 0.958 mmol, 0.210 g) was hydrogenated with Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H 2 (38 PSI), ETOH (10 mL) as described in Example 32.
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 3-Butyn-l-ol (2.58 Eq, 20.670 mmol, 1.449 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO 3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32.
  • l-(2-chloroethyl)-3-(3-hydroxyphenyl)urea (1MO-365) was obtained as described in example 39.
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 4-pentyn-l-ol (2.67 Eq, 21.400 mmol, 1.800 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32. The solvent was removed and the residue was purified by flash chromatography on silica gel: hexane / AcOEt (70 / 30).
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 3-Butyn-l-ol (2.58 Eq, 20.670 mmol, 1.449 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32. The solvent was removed and the residue was purified by flash chromatography on silica gel: Hexane / AcOEt (65 / 35).
  • 3-nitrobenzylalcohol (1.0 Eq, 1.959 mmol, 0.300 g) was mixed with triethylamine (3.0 Eq, 5.880 mmol, 0.595 g), acetic anhydride (3.0 Eq, 5.880 mmol, 0.600 g) and 4-py ⁇ olidinopyridine (0.02 Eq, 0.039 mmol, 0.006 g) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (75 / 25).
  • 6-(3-nitro ⁇ henyl)hex-5-yn-l-ol (1.0 Eq, 1.140 mmol, 0.255 g) was mixed with triethylamine (3.0 Eq, 3.420 mmol, 0.346 g), acetic anhydride (3.0 Eq, 3.420 mmol, 0.349 g) and 4- pyrrolidmopyridine (0.02 Eq, 0.023 mmol, 0.003 g) ) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: Hexane / AcOEt (75 / 25).
  • 6-(3-aminophenyl)hex-5-ynyl acetate (1.0 Eq, 0.643 mmol, 0.168 g) in Pd / C 10% (0.07 Eq, 0.047 mmol, 0.050 g), H 2 (38 PSI), and ETOH (10 mL) was hydrogenated and purified as described in Example 32. The solvent was removed and the product was purified by flash cliromatography on silica gel: dichloromethane / MeOH (99 / 1).
  • Example 61 l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pent-l-ynyl)-phenyl]-urea (58);
  • Example 62 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid ethyl ester (59);
  • Example 63 l-(2-Chloro-ethyl)-3-[3-(5-methoxy-pentyl)-phenyl]-urea (60);
  • Example 64 ⁇ 3-[3-(2-Chloro-ethyl)-ureido]-phenyl ⁇ -acetic acid (61);
  • Example 66 l-(2-Chloro-ethyl)-3-(3-heptyl-phenyl)-urea (63);
  • Example 68 Pentanedioic acid mono- ⁇ 3-[3-(2-chloro-ethyl)-ureido]-phenyl ⁇ ester (65);
  • Example 70 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid (67);
  • Example 72 l-(2-Chloro-ethyl)-3-(3-hexyl-phenyl)-urea (69);
  • Example 74 6- ⁇ 3-[3-(2-Chloro-ethyl)-ureido]-phenyl ⁇ -hexanoic acid ethyl ester (71);
  • Example 78 l-(2-Chloro-ethyl)-3-[3-(4-hydroxy-but-l-ynyl)-phenyl]-urea (75);
  • Example 80 3-[3-(2-Chloro-ethyl)-ureido]-phenyl ⁇ -acetic acid ethyl ester (77);
  • Example 81 Acetic acid 3- ⁇ 3-[3-(2-chloro-ethyl)-ureido]-phenyl ⁇ -propyl ester (78).
  • the compounds can be assayed initially for their ability to inhibit cell growth (i.e. their cytotoxicity) in vitro using standard techniques, hi general, cells of a specific test cell line are grown to an appropriate density (e.g. approximately 1 x 10 4 ) and the candidate compound is added. After an appropriate incubation time (for example 48 to 74 hours), cell survival is assessed, for example, by using the resazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 11:48-50; O'Brien et al, (2000) Eur. J. Biochem. 267:5421-5426 and U.S. Patent No.
  • the candidate compounds can also be tested in vitro for their ability to inhibit anchorage- independent growth of tumour cells.
  • Anchorage-independent growth is known in the art to be a good indicator of tumourigenicity. In general, anchorage-independent growth is assessed by plating cells from an appropriate cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with the compound can then be compared with that of cells treated with an appropriate control (as described above).
  • Cells were grown in an appropriate medium. Cell growth inhibition was assessed using a modified Alamar Blue assay as described by Lancaster et al. (U.S. Patent No. 5,501,959). For the proliferating state, cells were seeded in 96-well plates and preincubated for 24h (or 72 hours for the quiescent state). After addition fresh medium containing increasing concentrations of the candidate compound, cells were incubated at 37°C for 48 hours. The culture medium was removed, cells were washed and contacted with a resazurin solution. Cell survival was calculated from fluorescence (excitation, 485 mn; emission, 590 nm) measured with a FL 600 Reader (BioTek Instruments). Cell growth inhibition was expressed as the dose of drug required to inhibit cell growth by 50%) (GI 50 ). Values are the means of at least three independent detenninations.
  • GI5 0 is the dose required to inhibit cell growth by 50%. Different values obtained from different synthetic batches of compound.
  • EXAMPLE 84 MDA-MB-231 Cell Growth Inhibition by Compounds of the Invention
  • the human tumour cell line MDA-MB-231 was grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. Cells were inoculated into 96 well microtiter plates in 100 ⁇ L at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of the cell line. After cell inoculation, the microtiter plates was incubated at 37° C, 5 % CO 2 , and 100 % relative humidity for 24 h prior to addition of experimental drugs.
  • TCA trichloro acetic acid
  • the plates were incubated for an additional 48 h at 37°C, 5 % CO2, and 100 %> relative humidity.
  • the assay was terminated by the addition of cold TCA.
  • Cells were fixed in situ by the gentle addition of 50 ⁇ l of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water.
  • Sulforhodamine B (SRB) solution 100 ⁇ L) at 0,1% (w/v) in 1 % acetic acid (v/v) was added to each well, and plates were incubated for 10 minutes at room temperature.
  • GI 50 is the dose required to inhibit cell growth by 50%.
  • EXAMPLE 85 Inhibition of tumour cell growth by the compounds of the instant invention in a dose- and time-dependent manner.
  • M21 and HT1080 tumour cell lines were inoculated into 96 well tissue culture plates in 100 ⁇ L containing 2 X 10 3 cells and were incubated at 37 °C. After 24 h, freshly solubilized drugs in DMSO were diluted in fresh medium. Aliquots of 100 ⁇ l containing escalating concentration of drugs (0.3 ⁇ M to 100 ⁇ M; A) compound 1, B) compound 2, C) tBEU and D) cDDP) were added to the appropriate microtiter wells already containing 100 ⁇ l of culture medium. The cells were incubated for different period of time ranging from 3 h to 48 h.
  • Bound stain was solubilized with 10 mM trizma base, and the absorbance was read using a ⁇ Quant Universal Microplate Specfrophotometer (Biotek, Winooski, VT) at 585 nm. The results were compared with those of a control reference plate fixed on the freatment day and the growth inhibition percentage was calculated for each drug contact period. The growth inhibition percentage is expressed as the mean of triplicates for each drug contact period, compared with those of a control reference plate fixed on the day of the treatment.
  • the cytotoxicity of the test compounds was compared with that of a classical and strong alkylating agent, namely cisplatin (cDDP).
  • cDDP cisplatin
  • the antimicrotubule agents colchicine, vinblastin and paclitaxel were also tested in this assay but was found not cytotoxic until they reach 48 h of exposure (data not shown). When they were in contact for less than 6 h with either cell lines, virtually none of the tested compounds showed inhibition of tumour cell growth and proliferation. However, as the time of contact between the test compounds and tumour cells was increased from 6 to 48 h, the comparable GI 50 of compound 1 and 5 markedly shifted to the left hand-side of the graph ( Figure 1 A and B) and this was shown in the low micromolar range for all tumour cell lines tested.
  • M21 cells were seeded at lxlO 5 cells in in 35-mm Petri dishes and incubated for 16 h at 37 °C.
  • Cells were treated for 24h with either 100 ⁇ M of compounds 1 and 5 or tBEU or classical antimicrotubule agents (50 ⁇ M of cisplatin (cDDP), 25 ⁇ M of colchicine (COL), 5 ⁇ M of vinblastine (VINB) or 50 ⁇ M of paclitaxel (TAX)).
  • cDDP cisplatin
  • COL colchicine
  • VINB vinblastine
  • TAX paclitaxel
  • the cells were penneabilized and blocked with 0.1 % saponin and 3 % (w/v) BSA in PBS, during 1 h at 37 °C.
  • the cells were then further incubated during 1 h at 37 °C with anti-tubulin (clone TUB2.1, that is specific to ⁇ -tubulin and does not cross-react with other-tubulin isoforms; Sigma-Aldrich; St-Louis, MO) (1: 200) in 0.1 % saponin and 3 % BSA in PBS.
  • anti-tubulin clone TUB2.1, that is specific to ⁇ -tubulin and does not cross-react with other-tubulin isoforms; Sigma-Aldrich; St-Louis, MO
  • the cells were washed three times with PBS containing 0.05 % of Tween 20 and incubated 1 h at 37 °C in blocking buffer containing anti-mouse IgG Alexa-488 (1 : 1000), DAPI (2.5 ⁇ g/ml in PBS) to stain nuclei and Rhodamine-labeled phalloidin (1 : 600) to stain the actin cytoskeleton.
  • the observations were made using a Nikon Eclipse E800 microscope (Tokyo, Japan) equipped with a 40X objective. Images were captured as 16 bit TIFF files with a Hamamatsu ORCA ER cooled (-20°C) digital camera (Photonics Management Management Corp., Bridgewater, N.J.) driven by SimplePCI AIC software (Compix Inc. C Imaging systems, Pennsylvania). Representative fields are shown from three separate experiments.
  • Paclitaxel stabilizes the microtubules, thus inhibiting their depolymerization, whereas the others rather blocking their polymerization, inducing therefore a depolymerization phenotype ( Figure 2).
  • the compound 1 effect on ⁇ -tubulin was indeed drastically different from what was observed after paclitaxel treatment, not as severe as vinblastine, but rather similar to that observed after colchicine cell exposure.
  • the bioisosteric derivative compound 5 showed a similar microtubule dissolution activity as compound 1.
  • tBEU did not exhibit any effect on the microtubule network nor did affected the filamentous structure of actin.
  • EXAMPLE 87 Generation of an alkylated form of ⁇ -tubulin by compound 1 and compound 5.
  • M21 cells or MDA-MB-231 cells were washed in ice-cold PBS, pooled and then solubilized in buffer containing 62.5 mM Tris, pH 6.8, 2 % SDS, 6 M urea, 10 % glycerol, 0.00125 % bromophenol blue, and 720 mM ⁇ - mercaptoethanol.
  • the cell extracts were boiled for 5 min, separated on 10 % SDS-PAGE electrophoresis gel and transfe ⁇ ed onto nitrocellulose membrane.
  • the membranes were blocked for 1 h at 37 °C with 5 % (w/v) milk in Tris buffered saline (TBS) containing 0.1 % Tween 20 (TBST) and then incubated, 1 h at 37 °C, with the appropriate antibody diluted in 5 % milk in TBST.
  • TBS Tris buffered saline
  • TBST Tween 20
  • the apparition of an additional immunoreactive band of ⁇ -tubulin was evaluated with the monoclonal anti-tubulin antibody (1: 500).
  • Membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1: 2500) (Amersham Canada, Oakville, Canada) diluted in 5 % milk in TBST, 1 h at room temperature, followed by chemiluminescent detection, using an enhanced chemoluminescence (ECL) detection kit (Amersham Pharmacia Biotech).
  • a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1: 2500) (Amersham Canada, Oakville, Canada) diluted in 5 % milk in TBST, 1 h at room temperature, followed by chemiluminescent detection, using an enhanced chemoluminescence (ECL) detection kit (Amersham Pharmacia Biotech).
  • ECL enhanced chemoluminescence
  • EXAMPLE 88 Generation of an alkylated form of ⁇ -tubulin by exemplary compounds 1, (R)24; S(24); 28; 5; (R)23; 81, 82, 83 and 84 of the invention
  • Human breast carcinoma cell line, MDA-MB-231 was grown in RPMI 1640 medium supplemented with 10% bovine calf serum iron supplemented (Hyclone, Road Logan, Utah) in a humidified atmosphere at 37°C in 5% CO 2 . After trypsinization with 1 mL trypsin- EDTA, cells were seeded in 12 well-plates. Each well received around 220 000 cells. After an incubation of 24 hours, the culture medium was removed and drugs, diluted in fresh medium, added.
  • Each of the ten drugs was dissblved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 30 ⁇ M. Eight incubation times for each drug was performed, i.e. 0, 1, 2, 4, 8, 12, 24 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 ⁇ L of cold PBS. Both cell aliquots are mixed.
  • Cell suspension 100 ⁇ L from the cell suspension was pipetted and used to determine cell concentration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 60 000 cells/35 ⁇ L in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated. SDS-PAGE Analysis and Immunoblotting of /3-tubulin. Samples (60 000 cells) were analyzed by 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane.
  • Membranes were then incubated with PBSMT [PBS (pH 7.4), 5% fat-free dry milk, and 0.1% Tween-20]. After Ponceau staining, the membrane is immersed with a solution of 1:500 monoclonal anti-/3-tubulin (clone TUB 2.1, Sigma) for 1 h at room temperature. Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse imnrunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
  • EXAMPLE 89 Generation of an alkylated form of ⁇ -tubulin by exemplary compounds 1; (R)24; (S)24; (R) 85; (S) 85, 5; (R)23; (S)23; (R) 86, (S) 86 of the invention (II)
  • Cell suspension 100 ⁇ L from the cell suspension was pipetted and used to determine cell concenfration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 60 000 cells/35 ⁇ L in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated.
  • Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse immunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
  • EXAMPLE 90 Generation of an alkylated form of ⁇ -tubulin by exemplary compounds 53; 57; 30; 63, 31, 28, 66, 47, 49, 68, 69 and 87 of the invention
  • Each of the ten drugs was dissolved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 5 ⁇ M. Eight incubation times for each drug was performed, i.e. 2, 7, 17, 25 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 ⁇ L of cold PBS. Both cell aliquots are mixed.
  • Cell suspension 100 ⁇ L from the cell suspension was pipetted and used to determine cell concentration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 100 000 cells/25 ⁇ L in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated.
  • MDA-MB-231 cells were grown in DMEM plus 5% FBS (Hyclone, Road Logan, Utah) and L-glutamine without antibiotics. Cells were seeded at 3xl0 5 cells in 6 well plate and grown to confluence. 16 hours prior the experiments, the different drugs were diluted in fresh medium and added to the wells. For a series of measurements, the concentrations selected were 0,5, 3 and 5 ⁇ M. For the other series of measurements, the concentrations selected were 0,25, 0,5 and 1 ⁇ M. The following day, the tip of a cell scraper was used to scratch the bottom of each well, producing a "wound" of roughly 1000 ⁇ m.
  • the medium was washed to remove the detached cells, and two pictures per well were taken at 0, 3, 6, 8 and 24 hours. By superimposing a hemacytometer grid over the picture, the distance between the two fronts was measured at numerous positions. Migration velocity was determined by a graph of the "wound" front distance over time.
  • the assay was repeated at least twice for each drug, at each concentration. For IMO- 236, the concentration at 3 and 5 ⁇ M were too toxic for the cells.
  • EXAMPLE 92 Inhibition of HT1080 tumour cell migration by compounds 1 and 5.
  • the chemotaxis motility of HT1080 was assessed using Boyden chambers. Briefly, the underside of TranswellTM migration chamber membranes (8.0- mm pore size) were coated with collagen IV as described previously (filardo et al., J. Cell Biol. 130:441-450 (1995); Kle ke et al. J. Cell Biol. 127:859-866 (1994) and as modified by Petitclerc et al. (Petitclerc et al. J. Biol. Chem. 275:8051-8061 (2000)).
  • the cells were pre-incubated or not for 16 h with escalating concentrations of different compounds of the invention, then they were added to the top of a collagen IN coated TranswellTM membrane (8.0 mm pore size), separating the lower and upper part of a Boyden chamber in the presence of same drugs. Soluble fibronectin (25 ⁇ g/ml) was added to the lower chamber to induce chemotaxis.
  • the cells were allowed to migrate for 4-6 h at 37 °C, fixed and stained for quatification. The number of migrating cells per well were counted. The results expressed the mean ⁇ s.e. of triplicates.
  • EXAMPLE 93 The compounds of the instant invention impede the growth of two unrelated tumour cell lines in the chick chorioallantoic membrane (CAM) assay.
  • CAM chick chorioallantoic membrane
  • Human HT1080 fibrosarcoma and hamster CS1 melanoma cell lines were used to assess the antitumoural activity of CEUs in the chick chorioallantoic assay (Petitclerc et al. J. Biol. Chem. 275:8051-9061 (2000); Kim et al. Cell 94:353-362 (1998); Lyu et al. Int. J. Cancer 77:257-263 (1998)).
  • day-0 fertilized chicken eggs were purchased from Couvoirs Victoriaville (Victoriaville, QC, Canada).
  • the eggs were incubated for 10 days in a Pro-FI egg incubator fitted with an automatic egg turner, before being transferred to a RoU-X static incubator for the rest of the incubation time (incubators were purchased from Lyon Electric, Chula Vista, San Diego).
  • the eggs were kept at 37 °C in a 60 % humidity atmosphere for the whole incubation period.
  • a hobby drill (Dremel; Racine, WI)
  • a hole was drilled on the side of the egg, and a negative pressure was applied to create a new air sac.
  • a window was opened on this new air sac and was covered with transparent adhesive tape to prevent contamination.
  • HT1080 3.5 x 10 5 cells/egg
  • CS1 5 x 10 6 cells/egg
  • the tested drugs were injected intravenously in 10-12 eggs in a small volume (100 ⁇ l).
  • the eggs were incubated until day, at which time the embryos were euthanized at 4 °C, followed by decapitation. Tumours were collected, pictures were taken to illustrate the different groups and the tumour-wet weights were recorded. In all experiments, the number of dead embryos from the different groups was monitored for any sign of toxicity.
  • Panel A and B of Figure 7 show that incubation of CS1 -derived tumours on the CAM with compound 1 or compound 5 resulted in a significant dose-dependent reduction of the tumours size, as observed also with cDDP (Figure 7D). Moreover, both test compounds also inhibited the formation of HT1080 tumour mass in the same concentration range (data not shown). It is noteworthy that the non-alkylating homologue tBEU failed to influence the growth of CS1 tumours ( Figure 7C), supporting the assumption that the antitumoural effect of compound 1 and compound 5 was dependent on their alkylating activity. In the same experimental settings, only 10 ⁇ g/egg of cDDP was sufficient to inhibit tumour cell growth.
  • EXAMPLE 94 Compounds 1, 2, 5 and 30 impede the growth of CS1 tumour cell line in the chick chorioallantoic membrane (CAM) assay.
  • EXAMPLE 95 Clonogenic survival of M21 cells on extracellular matrices (ECMs).
  • tumour microenvironment could modulate the tumour cells' ability to resist to chemotoxic agents such as cDDP, triggering a pro-survival signal through integrins (Hazlehurst et al. Oncogene 19:4319-4327 (2000); Rintoul et al. Clin. Sci. (Lond) 102:417-424 (2002): Damiano et al. Blood 93:1658-1667 (1999).
  • M21 skin melanoma cells were plated (1 X 10 cells in 100-n ⁇ m Petri dishes) on different ECMs and challenged with test compounds of the instant invention or the strong alkylating agent cDDP, as previously described (Deschesnes et al. Mol. Biol. Cell. 12:1569-1582 (2001)). Briefly, native and heat-denatured type IN collagen (50 ⁇ g/ml), fibronectin (25 ⁇ g/ml) and fibrin (50 ⁇ g/ml) were used to coat non-tissue culture plates ( ⁇ unc). After washes with serum-free DMEM, cells were plated in serum-containing or serum-free media on the different matrices for 16 h.
  • native and heat-denatured type IN collagen 50 ⁇ g/ml
  • fibronectin 25 ⁇ g/ml
  • fibrin 50 ⁇ g/ml

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Abstract

L'invention concerne des composes d'urée haloéthyle tels que décrits dans la formule (I) et leur utilisation en tant qu'agent thérapeutique dans l'atténuation, l'inhibition, ou la prévention de migration et de prolifération de cellules cancéreuses.
PCT/CA2004/000771 2003-05-28 2004-05-28 Composes d'uree haloethyle et leur utilisation en vue d'attenuer, d'inhiber ou de prevenir la migration de cellules cancereuses WO2004106291A1 (fr)

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