PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PA TENT COOPERATION TREA TI' (PCT)
`WO 98/QJ174
`
`WORLD INTI'J..LEcnJAL PROPERTY ORGANIZATION
`lnlemational Bureau
`
`(51) International Patent ·c1assUlc:atlon 6 :
`A6IK 3V445
`
`(11) International Publication Number:
`
`Al
`
`(43) International Publication Date:
`
`29 January 1998 (29.01.98)
`
`(21) International Application Number:
`
`PCT/US97/ l 2 I 58
`
`(22) International Filing Date:
`
`14 July 1997 ( 14.07.97)
`
`(30) Priority Data:
`60/022,863
`
`24 July 1996 (24.07.96)
`
`us
`
`BRISTOL-MYERS SQUIBB COMPANY
`(71) Applicant:
`ruSJUS]; P.O. Box 4000, Princeton, NJ 08543-4000 (US).
`
`(81) Designated States: AL, AM; AT, AU, AZ,·BB, BG, BR, RY,
`CA,. CH, CN. C'Z. DE, DK, EE, ES, Fl, GB, GE, HU,
`IL, IS, JP, KE, KG, KP, KR, KZ, LK •. LR,:LS, LT, LU,
`LV. MD. MG, MK, MN. MW. MX.'NO; Nz; PL, PT, RO,
`RU, SD, SE, SG, SI, .SK;· TJ, TM, TR, TT, UA, UG, UZ,
`VN, ARIPO patent (GH, KE, LS, MW, SD, SZ. UG,.ZW),
`t:urasian patent (AM, AZ, BY, KG,.KZ. MD, RU, TJ.:TM),
`&!ropean paient. (AT, BE, CH, DE, DK, ES; Fl. FR; GB.
`GR, m. IT, LU, MC, NL, PT, SE>.: OAPi patent csi;:. RJ,
`Cf, CG, Cl, CM, GA, GN, ML,. MR, NE, SN, TD, TG).
`
`(72) Inventor: FIRESTONE, Raymond, A.; 59 Barnes Road,
`Stamford, CT 06902 (US).
`
`Published
`With internaJional search report ..
`
`(74) Agents: RODNEY, Burton ct al.; Bristol-Myers Squibb Com(cid:173)
`pany, P.O. Box 4000, Princeton, NJ 08543-4000 (US).
`
`(54) Title: METHOD FOR TREATING TUMORS HAVING HIGH LDL REQUIREMENTS EMPLOYING~ INHIBITORS
`
`(57) Abstract
`
`A method is provided for treating hematologic iumors and solid tumors, including ccnain types of leukemias and mc~talic tumors,
`having high LDL r«juirements employing a dclipidating agent such as an MTP inhibitor to substantially reduce LDL blood levels. In
`addition, a method is provided for treating tumon of the above types having high LDL requirements, especially hematologic tumors such
`as ccnain leukemias, employing a dclipidating compound to substantially remove native LDL, and then administering. a cytotoxic agent
`carried in reconstituted LDL.
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`Codes used to identify S1a1es pany to the PCf on the fronr pages of pamphlets publishing international applications under rhe PCT.
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`Portugal
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`RU
`Russian Fcde'81ion
`SD
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`METHOD FO~ TREATING TUMORS HAVING
`. HIGH LDL·REQUIREMENTS EMPLQYING MTP INfiIBITQRS
`
`Field of the Invention
`The present invention relates to a met~od
`for treating cancers having high LDL requireme~ts
`employing a delipidating agent, which preferably is
`an MTP inhibitor, alone or in combination with a
`cytotoxic agent.
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`Background of. the Invention
`It is known that cancer cells need
`cholesterol to make new cell membrane. The
`cholesterol is supplied by either de novo synthesis
`or from low-density lipoprotein (LDL) , or both,
`Firestone, R.A. et al, "Selective Delivery of.
`Cytotoxic Compounds to Cells by the LDL Pathway, J.
`Med. Chem., 1984, 27, 1037-1043. Firestone et al
`describe a series of cytotoxic compounds that are
`compatible with reconstituted LDL and may be
`delivered with reconstituted LDL against cancers
`that copiously internalize LDL.
`Firestone, R.A., "Low-Density Lipoprot~l.n
`as a Vehicle for Targeting Antitumor Compounds to
`25 Cancer Cells", Bioconjugate Chemistry, 1994, 5, pp
`105-113, at page 105, in the "Introduction",
`discusses problems associated with cancer treatment
`as follows:
`"It is difficult to eradicate cancer cells
`in vivo because they share with normal cells, for
`the most part, the same biochemical machinery._
`There is no cytotoxic substance that is completely
`selective for malignant cells, and all those
`presently in use cause dose-limiting toxic side
`effects. For this reason there is a growing
`emphasis on targeting, i.e., selective delivery of
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`drugs to tumors in ways that bypass normal.body
`tissues.
`"Among the vehicles that can be useq fqr
`this purpose is low-density lipoprotein (LDL) .. a
`normal blood constituent that is the body's
`principal means for delivery of cholesterol to
`tissues. Cholesterol, a major constituent of
`mammalian cell membranes, is obtained by cells·
`either by making it themselves or by picking it up.
`from LDL or both. Cancer cells, like all dividing
`ones, need large amounts of cholesterol becaus.e.
`they are making new membrane. There is ample
`evidence that many types of cancer cells indeed
`have unusually great LDL requirements. The
`evidence is 2..;.fold: measurements of LDL uptake by
`tumor cells and depletion of LDL in the blood of
`cancer patients res\llting from ·high uptake by _the
`tumor (vide infra). Thus. if LDL could be made to
`carry antitumor drugs, it would serve as a
`targeting vehicle. This concept was proposed in
`1981-2 (1,2) and has been reviewed several times
`since then (3-7) ."
`( 1) Gal, D. , Ohashi, J. . MacDonald, p ~ c. ,
`Buchsbaum, H.J., and Simpson, E.R.
`(1981) Low-
`25 _density lipoprotein as a potential vehicle for
`chemotherapeutic agents and radionucleotides in the
`management of gynecologic neoplasms. Am. J.
`Obstet. Gynecol. 139, 877.
`(2) Counsel!, R.E., and Pohland, R.C.
`(1982) Lipoproteins as potential site-specific
`delivery systems for diagnostic and therapeutic
`J. Med. Chem. 25, 1115.
`agents.
`(3)
`van Berkel, T.J.C. (1993) Drug
`targeting: application of endogenous carriers for
`site-specific delivery of drugs. J. Control-led
`Release 24, 145.
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`(4) Vitols, S. (1991)° Uptake of low(cid:173)
`density lipoprotein by malignant cell--possible
`therapeutic applications. Cancer Cells 3, 488.
`(5)
`deSmidt, P.C., and Van Berkel, T.J.C:
`(1990) LDL-mediated drug targeting. Crit. Revs.
`Thera. Drug Carrier Syst. 7~ 99.
`Peterson, C., Masquelier, M., Rudling,
`(6)
`M., Soderberg, K., and Vitols, s. (1991)
`Lipoproteins, malignancy and anticancer agents.
`(U.S.} 5, 175.
`Targeted Diagn. Ther.
`(1987) Transport of
`(7)
`Catapano, A.L.
`cytotoxic compounds to cells via the LDL receptor
`pathway. Med. Sci. Res. 15, 411.
`At page 105 under the topic "LDL
`15 Uptake ... ", Firestone, supra, lists numerous tumor
`types that have especially high LDL requirements
`including acute myeloid leukemia (AML), human
`monocytic (FAB-M5) and myelomonocytic (FAB-M4)
`leukemias, chronic myeloid leukemia in blast.
`20 crisis, solid tumors such as epidermoid cervical
`cancer EC-50, endometrial adenocarcinoma AC-258,
`gastric carcinoma and parotid adenoma, G2 heptorna,
`squamous lung cancer, choriocarcinoma, brain tumors
`such as medulloblastoma, oligodendroglioma, gliorna
`25 V-251MG, and malignant menigioma, as well as tumor
`cells that are exceptionally metastatic
`(Schroeder, F., Kier, A.B. Olson, C.D., and
`Dempsey, N.E.
`(1984) Correlation of tumore
`metastasis with sterol carrier protein and plasma
`30 membrane sterol levels. Biochem. Biophys. Res.
`Commun. 124, 283, and
`Cambien, F., Ducimetiere, P., and Richard,
`J. (1980) Total serum cholesterol and cancer
`mortaiity in a middle-aged male population. Am. J.
`35 Epidemiol. 112, 388),
`tumor cells that are exceptionally
`aggressive
`(Rudling, M.J., Stahle, L., Peterson,
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`C.O., and Skoog, L. (1986) Content of low density
`lipoprotein receptors in breast cancer tissue
`related to survival of patients. Brit. Med. J. ·
`292, 580;
`Peterson, c., Vitols, S., Rudling, M. '·
`Blomgren, H., Edsmyr, F., and Skoog, L.
`(1985)
`Hypocholesterolemia in cancer patinets may .be
`caused by elevated LDL receptor activities in
`malignant cells. Med. Oncol. Tumor Pharmacother.
`2, 143;
`Muller, C. P. , Wagner, A. U. , Maucher, C. ', .
`and Steinke, B.
`(1989) Hypocholesterolemia, an
`unfavorable feature of prognostic value in chronic
`myeloid leukemia. Eur. J. Hematol. 43, 235),
`and tumor cells that are exceptionally
`undifferentiated
`( Ponec, M. , Havekes, L . , Kempenaar, J. , .
`Lavrijsen, S., Wijsman, M., Boonstra, J., and
`Vermeer, B.J. (1985) Calcium-mediated regulation of
`the low density lipoprotein receptor and
`intracellular cholesterol synthesis in human
`J: Cell Physiol. 125 9.8;
`epide:anal keratinocytes.
`Zyada, L.E., Hassan, H.T., Rees, J.K.H.,
`and Ragab, M. H.
`(1990) The relation between
`hypocholesterolernia and degree of maturation in
`acute rnyeloid leukemia. Hematol. Oncol. 8, 65;
`Ponec, M. , . Havekes, L. , Kempenaar; J. ,
`Lavrisen, S., and Vermeer, B.J. (1984) Defective
`low-density lipoprotein metabolism in cultured,
`normal transfo:aned and malignant keratinocytes: J.
`Invest. Dermatol. 83, 436).
`Firestone, supra, on page 107 under the
`topic "Reconstitution of LDL With Cytotoxic Drugs"
`states as follows,
`"In order to kill tumors with drugs that
`are targeted in LDL, the drugs must somehow be
`bound to the LDL in such a way that (1) they cannot
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`escape from it while traveling in the blood enroute(cid:173)
`to the tumor,
`(2) their cytotoxicity is chem~cally
`or physically masked while LDL-bound, and then ·
`restored after entering the target cells, (3) in
`5 quantity X killing power there is enough drug t·o
`kill cancer cells contained in the reconstituted
`LDL (r-LDL), whose uptake is limited by the number
`of LDL receptors on the tumor cells and their rate
`of internalization, and (4) the presence of Apo B
`and its binding power to LDL receptors are
`retained. The ability of the drug, once rele'a~ed:
`from its carrier, to escape from lysosomes must.
`also be taken in account (76) ."
`((76) Burton, R., et al (1975) The
`permeability properties of rat liver lysosomes to
`nucleotides. Biochem. Soc. Trans. 3, 1251).
`On page 109, under the topic "Removal of
`LDL From the Patient Before Treatment", Firestone,
`supra, states as follows,
`"During treatment, drug-bearing r-LDL must
`compete with native LDL for access to LDL receptors
`on the tumor cells, requiring elevated doses ,of r(cid:173)
`LDL. This can be countered by removing LDL from
`the patients' blood Cdelipidation} prior to
`treatment ( 139-141) . Although restoration of
`normal LDL levels takes days (141), it might be
`best to delipidate immediately prior to treatment
`because it induces upregulation of LDL receptors
`throughout the body (142), and it is unknown
`30 whether upregulation in this way would be greater
`for tumor or normal cells."
`( (139) Franceschini, G., Busnach, G.,
`Calabresi, L., Chiesa, G., Gianfranceschi, G.,
`Zoppl, F., Minetti, L., and Sirtori, C.R. (1991)
`35 Predictability of low-density lipoprotein levels
`during apheretic treatment of hypercholesterolemia.
`Eur. J. Clin. Invest. 21, 209.
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`(140) Saal, S.D., Parker, T.S., Gordon,
`B.R., Studebaker, J., Hudgins, L., Ahrens, E.H.,,
`(1986) Removal _of low-deri.sity
`Jr., and Rubin, A.L.
`lipoproteins in patients by extracorporeal
`immunoadsorption. Am. J. Med. 80, 583.
`(141) Parker, T.S., Gordon, B.R., Saal,
`S .D., Rubin, A. L., and Ahrens, E .H., Jr. (1986)
`Plasma high density lipoprotein is increased in: man
`when low density lipoprotein (LDL) is lowered by
`10 LDL-pheresis. Proc. Nat. Acad. Sci. U.S.A. 83,.
`777.
`
`15
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`25
`
`(142) Goldstein, J .L., _and Brown M.S.
`(1977) The low-density lipoprotein pathway and its
`relation to atherosclerosis. Annu. Rev. Biochem.
`46, 897).
`The microsomal triglyceride transfer ·
`protein (MTP) catalyzes the transport of
`triglyceride (TG), cholesteryl ester (CE), and
`phosphatidylcholine (PC) between small unilamell~r
`20 vesicles (SUV). Wetterau & Zilversmit, Chem. Phys.
`Lipids .J..a, 205-22 (1985). When transfer rates are
`expressed as the percent of the donor lipid
`transferred per time, MTP expresses a dis tin.ct
`preference for neutral lipid transport (TG and CE),
`relative to phospholipid transport. The protein
`from bovine liver has been isolated and
`characterized. Wetterau & Zilversmit, Chem Phys.
`Lipids .la, 205-22 (1985). Polyacrylamide gel
`electrophoresis (PAGE) analysis of the purified_
`30 protein suggests that the transfer protein is a·
`complex of two subunits of apparent molecular
`weights 58,000 and 88,000, since a single band was
`present when purified MTP was electrophoresed under
`nondenaturing condition, while two bands of
`apparent molecular weights 58,000 and 88,000 were
`identified when electrophoresis was performed in
`the presence of sodium dodecyl sulfate (SDS).
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`These two polypeptides are hereinafter referred to
`as 58 kDa and 88 kDa, respectively, or the 59, kDa
`and the 88 kDa component of MTP, respectively, or
`the low molecular weight subunit and the high
`5 molecular weight subunit of MTP, respectively.
`Characterization of the 58,000 molecular
`weight component of bovine MTP indicates that. it is
`the previously characterized multifunctional
`protein, protein disulfide isomerase (POI).
`JO Wetterau et al., J Biol. Chern. 265, 9800-7 · (1990).
`The presence of PD! in the transfer protein is
`supported by evidence showing that (1) the amino
`terminal 25 amino acids of the bovine 58,000 kDa
`component of MTP is identical to that of bovine
`.
`POI, and (2) disulfide isomerase activity was. .
`expressed by bovine MTP following the dissociation
`of the 58 k.Da - 88 k.Da protein complex.
`In
`addition, antibodies raised against bovine PDI, a
`protein which by itself has no TG transfer
`activity, were able to immunoprecipitate bovine TG
`transfer activity from a solution containing
`purified bovine MTP.·
`PDI normally plays a role in the folding
`and assembly of newly synthesized disulfide bonded
`proteins within the lumen of the endoplasmic
`reticulum. Bulleid & Freedman, Nature l.J..2., 649-51
`(1988). It catalyzes the proper pairing of
`cysteine residues into disulfide bonds, thus
`catalyzing the proper folding of disulfide bonded
`30 proteins.
`In addition, PDI has been reported to be
`identical to the beta subunit of human prolyl 4-
`hydroxylase. Koivu et al., J. Biol. Chem. ~.
`6447-9 (1987). The role of PDI in the bovine
`transfer protein is not clear. It does appea~ to
`be an essential component of the transfer protein
`as dissociation of PDI from the BB kDa component of
`bovine MTP by either low concentrations of a
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`denaturant (guanidine HCl), a chaotropic agent
`(sodium perchlorate), or a nondenaturing detergent
`(octyl glucoside) results in a loss of transfer
`activity.
`.Wetterau et al , Biochemistry J..Q, 9728-
`3 5 (1991) .
`Isolated bovine POI has no apparent
`lipid transfer activity, suggesting that either the
`88 kDa polypeptide is the transfer protein or that ·
`it confers transfer activity to the protein
`complex.
`The tissue and subcellular distribution of
`MTP activity in rats has been investigated.
`Wetterau & Zilversmit, Biochero Biophys. Acta .!l22,
`610-7 (1986). Lipid transfer activity was found in
`liver and intestine. Little or no transfer
`15 · activity was f"ound in plasma, brain, heart, or
`kidney. Within the liver, MTP was a soluble
`protein located within the lumen of the microsomal
`fraction. Approximately equal concentrations were
`found in the smooth and rough microsomes.
`Abetalipoproteinemia is an autosomal
`recessive disease characterized by a virtual
`absence of plasma lipoproteins which contain
`apolipoprotein B (apoB) . Kane & Havel in The
`Metabolic Basis of Inherited Disease, Sixth
`edition, 1139-64 (1989). Plasma TG levels may be
`as low as a few mg/dL, and they fail to rise after
`fat ingestion . . Plasma cholesterol levels are often
`only 20-45 mg/dL. These abnormalities are the
`result of a genetic defect in the assembly and/or
`secretion of very low density lipoproteins (VLDL)
`in the liver and chylomicrons in the intestine.
`The molecular basis for this defect has not been
`previously determined.
`In subjects examined,
`triglyceride, phospholipid, and cholesterol
`synthesis appear normal. At autopsy, subjects are
`free of atherosclerosis. Schaefer et_al., Clin
`Chem . .JA, B9-12 (1988). A link between the apoB
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`gene and abetalipoproteinemia has been excluded in
`several families. Talmud et al., J. Clin. Inyest .
`.a2., 1803-6 (1988) and Huang et al., Am. J. Huin
`Genet
`.A§., 1141-8 (1990).
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`Subjects with abetalipoproteinemia are
`afflicted with numerous maladies. Kane & Havel,
`supra. Subjects have fat rnalabsorption and·TG
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`accumulation in their enterocytes and hepatocytes.
`Due to the absence of TG-rich plasma lipoproteins;
`there is a defect in the transport of fat-soluble•
`vitamins such as vitamin ·E. This results in
`acanthocytosis of erythrocytes, spinocerePellar·
`ataxia with degeneration of the fasciculus cuneatus
`and gracilis, peripheral neuropathy, degenerative
`pigmentary retinopathy, and ceroid myopathy.
`Treatment of abetalipoproteinemic subjects inciudes
`dietary restriction of fat intake and dietary
`supplementation with vitamins A, E and K.
`In yitro, MTP catalyzes the transport of
`lipid molecules between phospholipid membranes.
`Presumably, it plays a similar role in viyo, and
`thus plays some role in lipid metabolism. The.
`subcellular (lumen of the microsomal fraction) and
`tissue distribution (liver and intestine) of MTP
`have led to speculation that it plays a role in the
`assembly of plasma lipoproteins, as these are the
`sites of plasma lipoprotein assembly. Wetterau &
`Zilversmit, Biochem. Biophys. Acta .B.1.2, 610-7
`(1986). The ability of MTP to catalyze the
`transport of TG between membranes is consistent
`with this hypothesis, and suggests that MTP may
`catalyze the transport of TG from its site of
`synthesis in the endoplasmic reticulum (ER)
`membrane to nascent lipoprotein particles within
`the lumen of the ER.
`Olof sson and colleagues have studied
`lipoprotein assembly in HepG2 cells. Bostrom~
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`21...:_, J. Biol. Chern. 2..QJ., 4434-42 (1988). Their
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`results suggest small precursor lipoproteinsbecome
`larger with time. This would be consistent with·
`the addition or transfer of lipid molecules to
`nascent lipoproteins as they are assembled. MTP:
`may play a role in this process.
`In support of
`this hypothesis, Howell and Palade, J. CelL BiOl.
`.22,, 833-45 (1982), isolated nascent lipoproteiris'
`from the hepatic Golgi fraction of rat liver.
`10 There was a spectrwn of sizes of particles present
`. with varying lipid and protein compositions.
`Particles of high density lipoprotein (HDL} .
`density, yet containing .apoB, were found. Higgins
`and Hutson, J Lipid Res 25, 1295-1305 (1984),
`reported lipoproteins isolated from Golgi were
`consistently larger than those· from the endoplasmic
`reticulum, again suggesting the assembly of
`lipoproteins is a progressive event. However,
`there is no direct evidence in .the prior art
`demonstrating that MTP plays a role in lipid
`metabolism or the assembly of.plasma lipoprotein.
`Recent reports (Science, Vol. 258, page
`999, 1992; D. Sharp et al, Nature, Vol. 365, page
`65, 1993) demonstrate that the defect causing
`abetalipoproteinemia is in the MTP gene, and as a
`result, the MTP protein.
`Individuals with
`abetalipoproteinemia have no MTP activity, as a
`result of mutations in the MTP gene, some of which
`have been characterized. These results indicate
`that MTP is required for the synthesis of apoB
`containing lipoproteins, such as VLDL,
`the
`precursor to LDL.
`It therefore follows that
`inhibitors of MTP would inhibit the synthesis of
`VLDL arid LDL, thereby lowering VLDL levels, LDL
`levels, cholesterol levels, and triglyceride levels
`in animals and man.
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`Canadian Patent Application No. 2, 0.91, 102
`published March 2, 1994 (corresponding to U.S;
`application Serial No. 117,362, filed September;:' 3,
`1993 (file DC2lb)) which is incorporated herein by
`reference), reports MTP inhibitors which also:block
`the
`ipoproteins in a human hepatic cell lirie
`(HepG2 cells). This provides further support.for
`the proposal that an MTP inhibitor would lower apoB
`containing lipoprotein and lipid levels in y;i.yQ.
`10 This Canadian patent application discloses a m~thod
`for identifying the MTP inhibitors
`
`which has the name 2-(1-(3, 3-diphenylpropyl)~4-
`piperidinyl)-2, 3-dihydro-3-oxo-llj-isoindole
`hydrochloride and
`
`I 5
`
`F
`which has the name l-[3-(6-fluoro-1-tetralanyl)(cid:173)
`methyl]-4-0-methoxyphenyl piperazine.
`
`20
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`25
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`
`Pescription of the Inyention
`In accordance with the present invention, a
`method is provided for treating tumors having high
`LDL requirements which method includes the step of
`administeri_ng to a manunalian species in need of
`treatment a therapeutically effective amount of a
`delipidating agent to substantially reduce LDL
`blood levels.
`In the above method, the delipidating agent
`may be optionally administered in combination with
`a cytotoxic agent.
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`
`In addition, in accordance with the present
`invention, a method is provided for treating tumors
`having high LDL requirements, especially
`hematologic tumors, which method includes the steps
`5 of administering to a mammalian species in need of
`treatment a therapeutically effective amount of a
`delipidating agent to substantially remove LDL
`(that is, native LDL), and administering a
`cytotoxic agent carried in reconstituted LDL (rLDL-
`drug conjugate) .
`The delipidating compound to be employed in
`the methods of the invention may be an LDL lowering
`compound which lowers LDL down to less than 20% of
`normal (that is less than 20% of 150 mg/dl that is
`30 mg/dl), preferably down to less than.10% of
`normal (that is less than 15 mg/dl) and most
`preferably to substantially zero LDL. Examples of
`delipidating agents which may be employed herein
`include MTP inhibitors, statins, fibrates and
`resins or combinations thereof, with MTP inhibitors
`being preferred.
`The reconstituted LDL (employed as a
`carrier for the cytotoxic agent in the above
`method) may be prepared according to the procedures
`described in the review article Firestone, R.A.,
`Low-Density Lipoprotein as a Vehicle for Targeting
`Antitumor Compounds to Cancer Cells, Bioconjugate
`Chemistry, 1994, 5, 105-113, such as disclosed in.
`the following references cited by Firestone, supra:
`(78) Krieger, M., Brown, M.S., Faust, J.R.,
`and Goldstein, J.L. (1978) Replacement of
`endogenous cholesteryl esters of low density
`lipoprotein with exogenous cholesteryl linoleate,
`J. Biol. Chem. 253, 4093.
`(79) Krieger, M., McPhaul, J.J., Goldstein,
`J.L., and Brown, M.S. (1979) Replacement of neutral
`lipids of low density lipoprotein with esters of
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`long chain unsaturated fatty acids, J. Biol. Chem.
`254, 3845.
`(1987) Preparation of
`(104) Lundberg, B.
`drug-low density lipoprotein complexes for de~ivery
`S of antitumoral drugs via the low density
`lipoprotein pathway, Cancer Res. 47, 4105, and
`Gene M. Dubowchik and Raymond A. Firestone,
`Tet. Lett. 35, 4523, 1994.
`The cytotoxic agent may be incorporated· in:
`the reconstituted LDL to form an LDL-drug conjugate
`following the procedure described in the Fire~tone
`review article, supra, especially as described in
`cited reference (104) Lundberg, supra.
`MTP 1nhibitors to be employed in the
`15 methods of the invention include MTP inhibitors
`disclosed in Canadian Patent Application No.·
`2,091,102 described hereinbefore (correspondingto
`U.S. Application Serial No. 117,362), U.S.
`Application Serial No. 472,067, filed June 6, i995
`(file DC2le), U.S. Application Serial No. 548,811
`(file DC2lh), U.S. provisional application No ..
`60/017,224, (file HX79a*), U.S. provisional
`application No. 60/017,253, (file HX82*) and U.S.
`provisional application No. 60/017,254, (file
`25 HX84*).
`All of the above U.S. applications are
`incorporated herein by reference.
`The MTP inhibitors disclosed in U.S.
`Application Serial No. 472,067, filed June 6, 1995
`(file DC2le) are piperidine compounds of the
`structure
`
`R2
`
`0
`
`R'tc.N-cN-R'
`
`R4
`
`or
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`5
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`10
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`15
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`25
`
`or
`
`or
`
`or
`
`0
`. where a is -c- .or
`II
`
`0
`II
`-S-
`11
`0
`
`X Is: CHR8, - C- ·CH- CH·
`"
`• I
`I
`0
`Rs
`R10
`
`or
`
`-C= C·;
`I
`I
`Rs R10
`
`Ra, R9 and RlO are independently hydrogen, alkyl,
`alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,
`heteroarylalkyl, cycloalkyl, or cycloalkylalkyl;
`
`Y is -(CR:z>m- or -~-
`0
`wherein m is 2 or 3;
`Rl is alkyl~ alkenyl, alkynyl, aryl,
`heteroaryl, arylalkyl wherein alkyl has at least 2
`carbons, diarylalkyl, arylalkenyl, diarylalkenyl,
`arylalkynyl, diarylalkynyl, diarylalkylaryl,
`heteroarylalkyl wherein alkyl has at least 2
`carbons, cycloalkyl, or cycloalkylalkyl wherein
`alkyl has at least 2 carbons, all optionally
`substituted throu~h available carbon atoms with l,
`2, 3 or 4 groups selected from halo, haloalkyl,
`alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl,
`alkylmercapto, arylmercapto, cycloalkyl, cyclo-
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`alkylalkyl, heteroaryl, fluorenyl, heteroarylalkyl,
`hydroxy or oxo;
`or Rl is a fluorenyl-type group of the
`structure
`
`5
`
`-R11_z1
`
`or
`
`_ R11_ z1
`
`or
`
`~
`R1i<' ~ R14
`
`lt.
`
`R12_ z2
`
`I/ ~
`R13~ ~ R14
`a
`
`k
`
`or
`
`10
`
`J2
`Rl is an indenyl-type group of the structure
`
`or
`
`or
`
`or
`
`E
`
`(a= 2,3 or 4)
`
`f
`
`or
`
`_ R11_ z1
`
`R16a
`
`R15a
`
`15
`
`zl and z2 are the same or different and are
`independently a bond, 0, S,
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`
`s II
`0
`
`,
`
`t1
`
`, -N--C- ,. -C-
`I
`II
`U
`alkyl O
`O
`
`H
`or · -9-:
`OH
`
`-NH-C-
`o
`with the proviso that with respect to ~' at: leas·t
`one of zl and z2 will be other than a bond; R11 is
`a bond, alkylene, alkenylene or alkynylene of up to·
`10 carbon atoms; arylene or mixed arylene-alkylene;
`Rl2 is hydrogen, alkyl, alkenyl, aryl, haloalkyl,
`trihaloalkyl, trihaloalkylalkyl, heteroaryl,
`heteroarylalkyl, arylalkyl, arylalkenyl, cy~lo-
`alkyl, aryloxy, alkoxy, arylalkoxy or cycloalkyl(cid:173)
`alkyl, with the provisos that
`(1) when Rl2 is H, aryloxy, alkoxy or
`-NH-C-
`, -N -C -
`u
`t
`U
`o
`alkyl O
`
`-C-
`II
`o
`
`arylalkoxy, then z2 is
`or a bond and
`(2) when z2 i.s a bond, Rl/. cannot be
`heteroaryl or heteroarylalkyl;
`Z is bond, 0, S, N-alkyl, N-aryl, or
`alkylene or alkenylene from 1 to 5 carbon atoms;
`Rl3, Rl4, RlS, and Rl6 are independently hydrogen,
`alkyl, halo, haloalkyl, aryl, cycloalkyl,
`cycloheteroalkyl, alkenyl, alkynyl, hydroxy;
`alkoxy, nitro, amino, thio, alkylsulfonyl,
`arylsulfonyl, alkylthio, arylthio, aminocarbonyl,
`alkylcarbonyloxy, arylcarbonylamino,
`alkylcarbonylamino, arylalkyl, heteroaryl,
`heteroarylalkyl or aryloxy;
`RlSa and Rl6a are independently hydrogen;
`alkyl, halo, haloalkyl, aryl, cycloalkyl, cyclo(cid:173)
`heteroalkyl, alkenyl, alkynyl, alkoxy, alkyl-
`sulfonyl, arylsulfonyl, alkylthio, arylthio, amino(cid:173)
`carbonyl, alkylcarbonyloxy, arylcarbonylamino,
`alkylcarbonylamino, arylalkyl, heteroaryl,
`heteroarylalkyl, or aryloxy;
`or Rl is a group of the structure
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`R17
`-(CH2)p~
`R18
`
`wherein p is 1 to 8 and R17 and R18 are each
`independently H, alkyl, alkenyl, aryl, arylalkyl,
`heteroaryl, heteroarylalkyl, cycloalkyl or
`cycloalkylalkyl at least one of R17 and RlB being
`other than H;
`or Rl is a group of the structure
`
`5
`
`15
`
`20
`
`ff20
`-R1s--<
`n2'
`10 wherein ·R19 is aryl or heteroaryl;
`R20 is aryl or heteroaryl;
`R21 is H, alkyl, aryl, alkylaryl, arylalkyl,
`aryloxy, arylalkoxy, heteroaryl, heteroarylalkyl,
`heteroarylalkoxy, cycloalkyl, cycloalkylalkyl or
`cycloalkylalkoxy;
`independently hydrogen,
`R2, R3, R4 are
`halo, alkyl, alkenyl, alkoxy, aryloxy, aryl,
`arylalkyl, alkylmercapto, arylmercapto, cycloalkyl,
`cycloalkylalkyl, heteroaryl, heteroarylalkyl,
`hydroxy or haloalkyl;
`Rs is independently alkyl, alkenyl, alkyrlyl,
`aryl, alkoxy, aryloxy, arylalkoxy, heteroaryl,
`arylalkyl, heteroarylalkyl, cycloalkyl, cycloalkyl(cid:173)
`alkyl, polycycloalkyl, polycycloalkylalkyl,
`cycloalkenyl, cycloheteroalkyl, heteroaryloxy,
`cycloalkenylalkyl, polycycloalkenyl, polycyclo(cid:173)
`alkenylalkyl, heteroarylcarbonyl, amino,
`alkylamino, arylamino, heteroarylamino,
`cycloalkyloxy, eycloalkylamino, all optionally
`substituted through available carbon atoms.with l,
`2, 3 .or 4 groups selected from hydrogen, halo,
`alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl,.
`alkynyl, cycloalkyl, cycloalkylalkyl,
`cycloheteroalkyl, cycloheteroalkylalkyl, aryl,
`heteroaryl, arylalkyl, arylcyclo-alkyl,
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`
`arylalkenyl, arylalkynyl, aryloxy, aryloxyalkyl,
`arylalkoxy, arylazo, heteroaryloxo .• hetero(cid:173)
`arylalkyl, heteroarylalkenyl, heteroaryloxy,
`hydroxy, nitre, cyano, amino, substituted amino,
`thiol, alkylthio, arylthio, heteroarylthio,
`arylthio.alkyl, alkylcarbonyl, arylcarbonyl,
`arylaminocarbonyl, alkoxycarbonyl, aminocarbonyl,
`alkynylaminocarbonyl, alkylaminocarbonyl,
`alkenylaminocarbonyl, alkylcarbonyloxy,
`arylcarbonyloxy, alkylcarbonylamino,
`arylcarbonylamino, arylsulfinyl, arylsulfinylalk}'l, ·
`arylsulfonyl, alkylsulfonyl, arylsulfonylamino,
`heteroarylcarbonylamino, heteroarylsulfinyl,
`heteroarylthio, heteroarylsulfonyl, alkyisulfinyl;
`R6 is hydrogen or C1-C4 alkyl or C1-C4
`alkenyl; all optionally substituted with 1, 2, J"or
`4 groups which may independently be any of the
`substituents listed in the definition of Rs set out
`above;
`
`20
`
`R7 is alkyl, aryl or arylalkyl wherein alkyl
`by itself or as pa.rt of arylalkyl is optionally
`
`substituted with oxo ( W ) ;
`
`and
`
`25
`
`are the same or different and are independently
`selected from heteroaryl containing 5- or 6-ring
`members; and
`
`30
`
`N-oxides
`thereof; and
`pharmaceutically acceptable salts thereof;
`with the provisos that where in the first
`formula X is CH2, and R2, R3 and R4 are each H,
`then Rl will be other than 3,3-diphe