Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
  • C07D471/14—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
  • C07F7/0803—Compounds with Si-C or Si-Si linkages
  • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
  • C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
  • C07F7/0803—Compounds with Si-C or Si-Si linkages
  • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
  • C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
  • C07F7/0814—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring said ring is substituted at a C ring atom by Si

Definitions

• the present invention relates to novel compounds and methods of preparation thereof and, particularly, to firing expanded camptothecin derivatives or analogs and to methods of preparation of such camptothecin analogs.
• Camptothecins are DNA topoisomerase I inhibitors now being used as anticancer drugs.
• Topotecan (tpt) and CPT-11 are the first two members in the camptothecin family to gain Food and Drug Administration full approval status (topotecan in 1996 as second-line therapy for advanced epithelial ovarian cancer, topotecan again in 1998 for the treatment of small cell lung cancer, CPT-11 in 1998 as first- line therapy for colon cancer) .
• Several other analogs of the camptothecin family such as GI-147211C, DX8951f, 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin are in various stages of pre-clinical and clinical evaluation.
• campothecins in clinical use undergoes relatively rapid hydrolysis in the bloodsteam resulting in a marked loss of anticancer activity. It is the key -hydroxylactone pharmacophore within clinically relevant camptothecins that hydrolyzes at physiological pH to yield a biologically- inactive and potentially toxic hydroxy carboxylate form. Fassberg, J. and Stella, V.J., " A Kinetic and Mechanistic Study of the Hydrolysis of Camptothecin and Some Analogues", J. Pharm. Sci .
• campto >thecin The structures of campto >thecin and some of its important analogs are shown below:
• camptothecin and 9-AC both open rapidly and essentially completely to almost negligible 0.2 % lactone levels at equilibrium. While the presence of HSA promotes lactone ring opening for camptothecin and 9-AC, red blood cells and lipid bilayers in general preferentially bind the electroneutral lactone forms of camptothecins over their respective negatively-charged carboxylate lactone forms. Burke, T.G., Staubus, A.E., Mishra, A.K. , and Malak, H. , "Liposomal Stabilization of Camptothecin' s Lactone Ring", J. Am. Chem. Soc.
• camptothecin family of drugs Although substantial strides have been made in the development of the camptothecin family of drugs, it remains very desirable to develop improved compounds in this family of drugs and to develop improved synthetic routes for producing such drugs.
• the present invention provides generally for a compound having the following formula (1) :
• R 1 and R 2 are independently the same or different and are hydrogen, -C(0)R f wherein R f is an alkyl group, an alkoxy group, an amino group or a hydroxy group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryloxy group, an acyloxy group, -OC(0)OR d , wherein R d is an alkyl group, -OC(0)NR a R b wherein R a and R b are independently the same or different, H, -C(0)R f , an alkyl group or an aryl group, a halogen, a hydroxy group, a nitro group, a cyano group, an azido group, a formyl group, a hydrazino group, an amino group, -SR, wherein R c is hydrogen, -C(0)R f wherein R f is an alkyl group, an alkoxy group, an amino group or
• R 3 is H, a halogen atom, a nitro group, an amino group, a hydroxy group, or a cyano group; or R 2 and R 3 together form a chain of three or four members selected from the group of CH, CH 2 , 0, S, NH, or NR 1S , wherein R 15 is an d-C 6 alkyl group ;
• R 4 is H, F, an amino group, a C1-3 alkyl group, a C2-3 alkenyl group, a C2-3 alkynyl group, a trialkylsilyl group or a C1-3 alkoxy group; ,
• R 5 is a Ci-10 alkyl group, an alkenyl group, an alkynyl group, or a benzyl group;
• R 6 is -Si(R ⁇ R 9 R 10 ) or - (R 7 ) Si (R 8 R 9 R 10 ) , wherein R 7 is an alkylene group, an alkenylene group, or an alkynylene group; and R 8 , R 9 and R 10 are independently a Ci-io alkyl group, a C2-10 alkenyl group, a C2-10 alkynyl group, an aryl group or a -(CH 2 ) N R 1:L group, wherein N is an integer within the range of 1 through 10 and R 11 is a hydroxy group, an alkoxy group, an amino group, an alkylamino group, a dialkylamino group, a halogen atom, a cyano group, -SR C or a nitro group;
• R 1S is -C(0)R f or H
• R 1 and R 2 together may, for example, form a group of the formula -0(CH 2 ) n O- wherein n represents the integer 1 or 2.
• R 2 and R 3 together may, for example, form a group of the formula -0(CH 2 ) n O- wherein n represents the integer 1 or 2.
• R 5 is preferably an ethyl group, an allyl group, a benzyl group or a propargyl group. Most preferably, R 5 is an ethyl group. Preferably, R 4 is H.
• R a and R 9 are methyl groups
• R 10 is a tert-butyl group or a methyl group
• R 1 is H and R 3 is H.
• R 2 may, for example, be H, NH 2 or OH.
• R 13 is preferably H.
• R 16 is preferably H or an alkyl group. Most preferably, R 16 is H or -C(0)R f , wherein R f is an alkyl group. Most preferably, R 1S is H.
• the present invention also provides a method of synthesizing a compound having the formula
• X is a radical precursor.
• X is Cl , Br or I.
• X is Br or I .
• the present invention also provides a compound having the formula
• R 12 is preferably H or -C(0)R f , -C(0)OR d or -C(0)NR a R b ;
• the present invention further provides compounds having the formulas
• the present invention also provides a compound having the formula
• the present invention also provides a compound having the formula
• R 15 is a d-Cg alkyl group.
• the present invention further provides a compound having the formula
• R 14 is SiMe 3 , I, or Br.
• the present invention provides a method of synthesizing a compound having the following formula:
• Y is chlorine, bromine or iodine
• step (b) treating the compound formed in step (a) with an organometallic reagent having the structure:
• M is Li, Na, K, MgY, or ZnY under suitable conditions to form a compound having the structure:
• step (c) treating the compound formed in step (b) under suitable conditions with acid to form a compound having the structure :
• step (d) treating the compound formed in step (c) under suitable conditions of halogenative desilylation to form a compound having the structure :
• step (e) treating the compound in step (d) with acid or iodotrimethylsilane under suitable conditions for demethylation to provide a compound of the following structure :
• step (f) treating the compound in step (e) with a lithium base or a sodium base in the presence of an inorganic lithium salt to deprotonate the nitrogen atom,
• step (g) reacting of the resulting deprotonated species of step (f) with a compound of the following structure:
• Z is I, Br, Cl , a mesylate group or a tosylate group, and under suitable conditions to cause the formation of the compound of the following structure:
• all compounds of the present invention including the ⁇ -hydroxylactone group can exist in racemic form, enantiomerically enriched form, or enantiomerically pure form.
• the formulas of such compounds as set forth herein cover and/or include each such form.
• radical precursor (s) refers generally to those functional groups that cleave to generate radicals under standard conditions of chain or non-chain radical reactions. Common radical precursors are the halogens
• alkyl refer generally to both unsubstituted and substituted groups unless specified to the contrary. Unless otherwise specified, alkyl groups are hydrocarbon groups and are preferably C ⁇ -C ⁇ s (that is, having 1 to 15 carbon atoms) alkyl groups, and more preferably C ⁇ -C ] _o alkyl groups, and can be branched or unbranched, acyclic or cyclic. The above definition of an alkyl group and other definitions apply also when the group is a substituent on another group (for example, an alkyl group as a substituent of an alkylamino group or a dialkylamino group) .
• aryl refers to phenyl or naphthyl .
• alkoxy refers to -OR d , wherein R d is an alkyl group.
• aryloxy refers to -OR e , wherein R e is an aryl group.
• acyl refers to -C(0)R f .
• alkynyl refers to a straight or branched chain hydrocarbon group with at least one triple bond, preferably with 2-15 carbon atoms, and more preferably with 2-10 carbon atoms
• alkylene (for example, -C ⁇ CR h or -CH 2 -C ⁇ CR h ) .
• alkenylene and alkynylene refer to bivalent forms of alkyl, alkenyl and alkynyl groups, respectively.
• alkyl groups can be substituted with a wide variety of substituents to synthesize homocamptothecin analogs retaining activity.
• alkyl groups may preferably be substituted with a group or groups including, but not limited to, a benzyl group, a phenyl grou , an alkoxy group , a hydroxy group , an ammo group (including, for example, free ammo groups, alkylamino, dialkylamino groups and arylam o groups) , an alkenyl group, an alkynyl group and an acyloxy group.
• R a and R b are preferably independently hydrogen, an acyl group, an alkyl group, or an aryl group.
• Acyl groups may preferably be substituted with
• R f is an alkyl group, a haloalkyl group (for example, a perfluoroalkyl group), an alkoxy group, an ammo group and a hydroxy group.
• Alkynyl groups and alkenyl groups may preferably be substituted with (that is, R 9 and R h are preferably) a group or groups including, but not limited to, an alkyl group, an alkoxyalkyl group, an ammo alkyl group and a benzyl group .
• acyloxy refers to the group -OC(0)R d .
• alkoxycarbonyloxy refers to the group -OC(0)OR d .
• Amino and hydroxy groups may include protective groups as known in the art.
• Preferred protective groups for amino groups include te t-butyloxycarbonyl , formyl , acetyl, benzyl, p-methoxybenzyloxycarbonyl , trityl .
• Other suitable protecting groups as known to those skilled in the art are disclosed in Greene, T. , Wuts, P.G.M., Protective Groups in Organic Synthesis, Wiley (1991) , the disclosure of which is incorporated herein by reference.
• R 1 , R 2 , R 3 , R 6 , R 7 and R 8 are preferably not excessively bulky to maintain activity of the resultant camptothecin analog.
• R 1 , R 2 , R 3 , R 6 , R 7 and R 8 are preferably not excessively bulky to maintain activity of the resultant camptothecin analog.
• R 1 , R 2 , R 3 , R 6 , R 7 and R 8 are preferably not excessively bulky to maintain activity of the resultant camptothecin analog.
• R 2 , R3 , R s , R 7 and R 8 independently have a molecular weight less than approximately 250. More preferably R 1 , R 2 , R 3 , R 6 , R 7 and R 8 independently have a molecular weight less than approximately 200.
• camptothecin analogs of the present invention can be prepared for pharmaceutical use as salts with inorganic acids such as, but not limited to, hydrochloride, hydrobromide , sulfate, phosphate, and nitrate.
• the camptothecin analogs can also be prepared as salts with organic acids such as, but not limited to, acetate, tartrate, fumarate, succinate, citrate, methanesulfonate, p-toluenesulfonate, and stearate.
• Other acids can be used as intermediates in the preparation of the compounds of the present invention and their pharmaceutically acceptable salts.
• the E-ring (the lactone ring) may be opened with alkali metal such as, but not limited to, sodium hydroxide or calcium hydroxide, to form opened E-ring analogs of compounds of formula (1) as set forth in the compounds of formula (2) .
• alkali metal such as, but not limited to, sodium hydroxide or calcium hydroxide
• the intermediates thus obtained are more soluble in water and may be purified to produce, after treatment with an acid, a purified form of the camptothecin analogs of the present invention.
• the E-ring may also be modified to produce analogs of compounds of formula (1) with different solubility profiles in water or other solvents.
• Methods to achieve this goal include, but are not limited to, opening the E-ring with hydroxide or a water-soluble amino group or functionalizing the hydroxy group at position 20 of the E-ring with a water- soluble group such as a polyethylene glycol group or an acyl group .
• a water- soluble group such as a polyethylene glycol group or an acyl group .
• Such groups can be introduced either on the homocamptothecin derivative or at an earlier stage in the synthesis.
• the analogs thus prepared act as pro-drugs. In other words, these analogs regenerate the compounds of formula (1) (with the closed E-ring structure) when administered to a living organism. See , Greenwald, R.B. et al . , J. Med .
• Alkyl esters resulting from acylation at C20 will result in more Hpophilic pro-drugs that may not hydrolyze until the alkyl group is enzymatically cleaved.
• the present invention also provides a method of treating a patient, which comprises administering a pharmaceutically effective amount of a compound of formulas (1) and/or (2) or a pharmaceutically acceptable salt thereof.
• the compound may, for example, be administered to a patient afflicted with cancer and/or leukemia.
• the compounds of the present invention may also act as antiviral (for example, anti-HIV) agents and antiparasitic agents.
• the compounds of formulas (1) and/or (2) may be administered by any conventional route of administration, including, but not limited to, intravenously, intramuscularly, orally, subcutaneously, intratumorally, intradermally, and parenterally.
• the pharmaceutically effective amount or dosage is preferably between 0.01 to 60 mg of one of the compounds of formulas (1) and (2) per kg of body weight.
• the pharmaceutically effective amount or dosage is preferably between 0.1 to 40 mg of one of the compounds of formulas (1) and (2) per kg of body weight.
• a pharmaceutically effective amount or dosage contains an amount of one of the compounds of formulas (1) and/or (2) effective to display antileukemic, antitumor (anticancer), antiviral and/or antiparisitic behavior.
• Pharmaceutical compositions containing as an active ingredient one of the compounds of formulas (1) and/or (2) or a pharmaceutically acceptable salt thereof m association with a pharmaceutically acceptable carrier or diluent are also within the scope of the present invention.
• the present invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising any of the compounds of formulas (1) and (2) and a pharmaceutically acceptable carrier.
• the composition may, for example, contain between 0.1 mg and 3 g, and preferably between approximately 0.1 mg and 500 mg of the compounds of formulas (1) and/or (2), and may be constituted into any form suitable for the mode of administration.
• the A,B,E- or B,E-rmg modified camptothecins of the present invention 1) display enhanced stability m the presence of HSA through elimination or minimization of the highly preferential binding by HSA of carboxylate over lactone forms; 2) display high levels of lipophilicity which promote reversible associations of the lactone forms of the drugs with red blood cells, thereby slowing and restricting the extent of drug hydrolysis; and 3) display improved stability m aqueous solution.
• novel blood-stable silyl-substituted homocamptothecin (referred to herein as ⁇ -hydroxylactone silatecans or homosilatecans (hST) ) derivatives of the present invention can be prepared by significant modification of a total synthesis approaches set forth in U.S. Patent Application No. 09/212,178, entitled CAMPTOTHECIN ANALOGS AND METHODS OF PREPARATION THEREOF and filed December 15, 1998 and U.S. Patent Application No. 09/007,872, entitled NOVEL INTERMEDIATES IN THE SYNTHESIS OF CAMPTOTHECIN AND RELATED COMPOUNDS AND SYNTHESIS THEREOF and filed January 15, 1998 the disclosure of which are incorporated herein by reference. Novel intermediates were synthesized to carry out the cascade radical annulation of the present invention.
• the novel homocamptothecins of the present invention contain A,B- or B-ring modifications which decrease the preferential carboxylate over lactone binding by human albumin. These modifications in the A,B-rings also markedly enhance lipophilicity and promote lactone associations with lipid bilayers present in blood.
• the new compounds also contain an expanded ⁇ -hydroxylactone E-ring which improved the overall stability of the agents without loss of potency.
• the E-ring expanded ⁇ -hydroxylactone silatecans of the present invention display IC S0 values in the range of 2 to 115 nM.
• the compounds of the present invention (several of which are described in the formula above) , as a result of their novel structural substitutions, have superior human plasma and human blood stabilities than the agents described by Lavergne et al .
• E-ring modified camptothecins of the present invention has led to the identification of the most blood-stable camptothecins displaying intrinsic potency yet to be identified.
• An additional benefit of these new agents is that they do not display any significant interspecies variations in blood stabilities such as those of 9-AC and camptothecin described in Mi, Z. and Burke, T.G. "Marked Interspecies Variations Concerning the Interactions of Camptothecin with Serum Albumins : A Frequency-Domain Fluorescence Spectroscopic Study", Biochemistry, 33 : 12540- 12545 (1994) .
• This very attractive feature should greatly facilitate the drug development process and the translation of experimental observations and dosing schedules developed in animal models to the clinic.
• Figure 1 illustrates synthesis of precursors for the cascade radical reactions.
• Figure 2a and 2b illustrate synthesis of new AB-ring modified homocamptothecin/homosilatecan derivatives.
• Figure 3 illustrates a typical fluorescence fluorescence emission spectra for a homosilatecan (1 ⁇ M 7-trimethylsilyl-10-aminohomocamptothecin (DB-38)) in the presence and absence of lipid bilayer membranes.
• FIG. 4 illustrates a comparison of the equilibrium binding of four novel homosilatecans to SUVs composed of electroneutral dimyristoylphosphatidylcholine (DMPC) in PBS with data acquired for camptothecin (CPT) and topotecan (TPT) as well.
• DMPC electroneutral dimyristoylphosphatidylcholine
• Figure 5 illustrates the marked dependence of the the fluorescence emission spectra of 1 ⁇ M 7-t- butyldimethylsilyl-10-hydroxyhomocamptothecin (DB-91) on the presence of water.
• Figure 6 illustrates the fluorescence emission spectra of 1 ⁇ M 7-t-butyldimethylsilyl-10-hydroxy- homocamptothecin (DB-91) in solutions of phosphate-buffered saline (PBS) at pH 7.4 and in PBS at pH 7.4 containing albumin-free red blood cells at a concentration of (10 ⁇ 1) x 10 6 cell/ ⁇ L.
• PBS phosphate-buffered saline
• Figure 7 illustrates the fluorescence emission spectra of prior art compound 7-ethyl-10-hydroxycamptothecin (SN-38) in solutions of phosphate-buffered saline (PBS) at pH 7.4 and in PBS at pH 7.4 containing albumin-free red blood cells at a concentration of (10 ⁇ 1) x 10 6 cell/ ⁇ L.
• PBS phosphate-buffered saline
• Figure 8 illustrates the pH dependence of the stability of 1 ⁇ M 7-trimethylsilyl-10-aminohomocamptothecin (DB38) in solutions of phosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0 as determined using HPLC methods .
• PBS phosphate-buffered saline
• Figure 9 illustrates the pH dependence of the stability of 1 ⁇ M 7-t-butyldimethylsilylhomocamptothecin (DB81) in solutions of phosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0 as determined using HPLC methods .
• PBS phosphate-buffered saline
• Figure 10 illustrates the pH dependence of the stability of 1 ⁇ M 7-t-butyldimethylsilyl-10-aminohomo- camptothecin (DB90) in solutions of phosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0 as determined using HPLC methods.
• PBS phosphate-buffered saline
• Figure 11 illustrates the pH dependence of the stability of 1 ⁇ M of 7-t-butyldimethylsilyl-10-hydroxyhomo- camptothecin (DB91) in solutions of phosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0 as determined using HPLC methods.
• PBS phosphate-buffered saline
• Figure 12 illustrates the improved stabilities of four novel homosilatecans of the current invention in PBS solution as determined using HPLC methods.
• Figure 13 depicts the improved stabilities of four novel homosilatecans of the current invention in PBS/HSA as determined by HPLC methods .
• Figure 14 depicts the human plasma stabilities of four novel homosilatecans of the current invention as determined using HPLC methods .
• Figure 15 depicts the stabilities of four novel homosilatecans of the current invention in PBS suspensions containing physiologically-relevant concentrations [ (5 ⁇ 1) x 10 6 cell/ ⁇ L] of albumin-free red blood cells.
• Figure 16 illustrates the improved human blood stabilities of four novel homosilatecans of the current ⁇ invention as determined using HPLC methods.
• Figure 17 illustrates the improved human blood stabilities of four novel homosilatecans of this invention relative to clinically-relevant agents of the prior art which include 9-aminocamptothecin (9AC) , camptothecin (CPT) , topotecan (TPT) and SN-38.
• 9AC 9-aminocamptothecin
• CPT camptothecin
• TPT topotecan
• FIG. 1 shows the synthesis of a key iodopyridone 9, which can be used to make the compounds of formula 1.
• the synthesis of 9 starts from enol ether 3, an intermediate in the synthesis of camptothecin and analogs. See U.S. Patent Application No. 09/007,872, the disclosure of which is incorporated herein by reference. Dihydroxylation followed by oxidative cleavage provides the keto formate 5, which is then extended by a Reformatsky reaction to give 6.
• the formyl group is cleaved in this reaction, and acid promoted cleavage of the t-butyl ester directly results in ⁇ -hydroxylactone 7.
• This compound is then converted to the iodopyridone 9 by a sequence of 1) iodinative desilylation, and 2) demethylation.
• Figure 2a shows the conversion of iodopyridone 9 to several model AB modified homocamptothecin derivatives.
• N-Propargylation of 9 under optimized conditions provides radical precursors 10a,b.
• the cascade radical annulations of these precursors with the indicated isonitriles give products la,c,e,g.h.
• Products la,c,e were then deprotected by standard means to provide the target drug candidates lb,d,f in the indicated overall yields.
• Compounds lg and lh do not require deprotection.
• the synthesis of additional novel compounds of the present invention are described in the Examples section of the present application.
• Lavergne and coworkers disclose two ways to make homosilatecan derivatives, but both have serious limitations.
• the first involves the conversion of a standard camptothecin derivative to a homocamptothecin derivative by a series of steps involving disassembly of the normal lactone and reassembly of the homologated lactone.
• the second route involves a total synthesis using a palladium catalyzed cyclization to form ring C.
• This route is limited by the availability of A-ring precursors and by the ability of the substituents thereon to survive the many subsequent steps of the synthesis. Furthermore, the synthesis does not appear to offer the possibility to introducing many B ring substituents, including the substituents described herein.
• radical cascade synthetic schemes of the present invention are much more tolerant and flexible and can be used to make homocamptothecin derivatives with many A- , B-, or A/B substitution patterns, as shown, for example, in Figure 2b.
• various reagents can be used in the radical cascade including, but not limited to, hexamethylditin, hexamethyldisilane, or tetrakis (trimethylsilyl) silane .
• the source of energy for this reaction can be a sun lamp or an ultraviolet lamp.
• temperature is preferably set between approximately 25 and 150°C. More preferably, the temperature is set at approximately 70°C.
• solvents include benzene, toluene, benzotrifluoride, acetonitrile, THF and tert- butanol.
• substituents on the alkyne (R 6 ) and the isonitrile (R ⁇ R) in the synthetic schemes of the present invention because of the mildness of the reaction conditions.
• allyl derivatives can be substituted instead and the same final products are formed, albeit in lower yield.
• the substituent on C20 (R s ) can also be widely varied since it derives from readily available allyl alcohols.
• Substituted esters can also be used in the Reformatsky reaction to provide compounds with subsitutents on C20a (R 13 ) .
• the compounds of this invention might be used in racemic form for chemotherapy, it is more preferable to use samples that are exclusively or predominately the biologically active enantiomer at C20. Because of a change in priorities in the Cahn-Ingold-Prelogs Rules for assignment of absolute configuration, the C20 S enantiomer of a standard camptothecin generally has the same relative configuration as the C20 R enantiomer of the corresponding homocamptothecin.
• Racemic or enantiomerically enriched samples of homocamptothecin derivatives can be separated into their individual components by standard methods of liquid chromatography using commercially available chiral columns. See Lavergne, O.; et al . , "Homocamptothecins : Synthesis and Antitumor Activity of Novel E-Ring Modified Camptothecin Analogs," J. Med. Chem. , 41 , 5410-5419 (1998) .
• Biochemistry 33 12540-12545; and Mi, Z., Malak, H. , and
• camptothecin lactone opens rapidly and completely to the carboxylate form with a t 1/2 value of 11 mm and an almost negligible percentage of lactone at equilibrium value of 0.2%.
• camptothecin displayed enhanced stability (t 1/2 value of 22 mm and a percentage of lactone at equilibrium value of 5.3 %) .
• the enhanced stability of camptothecin lactone in human blood was found to be a result of drug associations with the lipid bilayers of red blood cells. Camptothecin binds erythrocyte membranes, the drug localizes within the acyl chain region, and accordingly remains protected from hydrolysis .
• topotecan In contrast to the low levels of lactone remaining at equilibrium in whole human blood for camptothecin and 9- aminocamptothecin ( ⁇ 0.5 % and 5.3 %, respectively), topotecan (11.9 %) , CPT-11 (21.0 %) , and SN-38 (19.5 %) all display improved blood stabilities. While lactone levels at equilibrium for topotecan are 20-fold greater than for 9- aminocamptothecin, the corresponding levels of lactone for
• CPT-11 and SN-38 are approximately 40-fold greater than in the case of 9-aminocamptothecin.
• the significant gains in the relative stabilities of topotecan, CPT-11, and SN-38 can be correlated to their favorable interactions with HSA. It is believed that structural substituents at the 7- and 9- positions hinder and prevent the preferential binding of the carboxylate drug forms by HSA.
• the technique of time- resolved fluorescence anisotropy has recently been used to demonstrate that, under experimental conditions where camptothecin carboxylate associates with HSA and tumbles in solution closely associated with the protein, the carboxylate forms of topotecan and CPT-11 do not associate with HSA.
• direct spectroscopic evidence has been obtained which indicates that HSA preferentially binds the lactone form of this agent, thereby shifting the lactone -carboxylate equilibrium to the lactone.
• HSA plays an important role in determining the relative human blood stabilities of the camptothecins.
• camptothecin and 9-aminocamptothecin the protein acts as a sink for the carboxylate drug form, binding the opened ring species and thereby shifting the lactone-carboxylate equilibria to the right.
• topotecan, CPT-11, and SN-38 no such preferential binding of the carboxylate drug form by HSA is observed.
• HSA preferentially binds the lactone form of SN-38 which thereby promotes higher circulatory levels of this biologically active species.
• camptothecins with improved human blood stabilities.
• camptothecin in the A and B rings with the effect of: 1) reducing protein binding; 2) enhancing lipophilicity; and 3) producing both a concomitant reduction in carboxylate binding to human albumin while also enhancing lipophilicity.
• Our studies have led to the design of novel A, B, E-ring modified camptothecins which are the most blood- stable and intrinsically potent camptothecin analogs yet to be identified, with blood stability parameters outcompeting the prior art compound homocamptothecin as well A,B-ring modified camptothecin analogs containing a conventional ⁇ - hydroxylactone functionality.
• the novel camptothecin analogs of the presnet invention display unique properties such as superior human blood stabilities in combination with high anticancer activities.
• Figures 3 depicts the fluorescence emission spectra of 1 ⁇ M DB-38 in phosphate buffered saline (PBS) and in lipid bilayers.
• PBS phosphate buffered saline
• FIG. 3 depicts the fluorescence emission spectra of 1 ⁇ M DB-38 in phosphate buffered saline (PBS) and in lipid bilayers.
• the data indicate that upon introduction of lipid bilayers into the sample there is an increase in the fluorescence emission of the compound, indicative of an interaction between the drug and the membrane.
• the fluorescence Upon changing the solvent to ethanol the fluorescence also changes .
• membranes there is a marked increase in fluorescence intensity as the drug partitions into the lipid bilayer microenvironment .
• a steady-state fluorescence anisotropy (a) measurement is related to the rotational rate of the fluorescent molecule through the Perrin Equation:
• a 0 is the limiting fluorescence anisotropy in the absence of depolarizing rotations
• ⁇ is the excited-state lifetime
• ⁇ is the rotational correlation time of the fluorophore.
• the excited-state lifetime values of camptothecin in PBS, glycerol, and methanol were examined at 37°C.
• the lifetime values were determined to be 4.7 ns, 3.8 ns, and 3.5 ns, respectively.
• the lifetime value of camptothecin when associated with DMPC bilayers was measured at 37°C, and the average value for membrane-bound drug was found to be 3.7 ns .
• the lifetime measurements described above indicate that the excited-state lifetime of camptothecin is relatively insensitive to alterations in microenvironment (for example, a change in solvent or fluorophore relocation from an aqueous milieu to a phospholipid membrane) .
• a fluorophore having a ⁇ value that remains relatively constant during a transition which strongly impacts on its rotational motion such as a change in solvent viscosity or fluorophore binding to large macromolecular assemblies such as liposomal particles
• the Perrin equation indicates a direct relationship between a and ⁇ values will exist (that is, as the ⁇ value of the fluorescent compound increases, then so too does its steady-state anisotropy value) .
• topotecan has an a value of 0.008 in PBS, but its a value increases 9-fold and 40-fold in the viscous solvents octanol and glycerol, respectively.
• a 21- fold enhancement in the a value of camptothecin is observed upon binding of drug to vesicles composed of either DMPC or DMPG.
• the method of fluorescence anisotropy titration was employed to study the equilibrium binding of camptothecin analogs with lipid bilayers.
• the experiment includes determining the a values for a set of samples where the drug concentration in each was held constant (typically 1 or 2 ⁇ M) , while the lipid concentration among the members of a set was varied from 0 to 0.29 M.
• anisotropy values of DB-38, DB-90 and DB-91 titrated much more rapidly than those of camptothecin or topotecan, indicating that the novel homosilatecans have much stronger interactions with these membranes than camptothecin and topotecan. Because of the potential of the lactone ring of the homosilatecans and camptothecins to hydrolyze in PBS, anisotropy values at each lipid concentration were determined immediately (approx. 1 min.) following the addition of the lactone form of each agent to the liposome suspension as to minimize any possibility of conversion to the carboxylate form. Using drug concentrations of 1 ⁇ M and long pass filters to isolate emitted light from background signal
• [A B ] represents the concentration of bound drug
• [A F ] represents the concentration of free drug
• [L] represents the total lipid concentration in the vesicle suspension.
• K may be determined from the inverse of the slope of a double reciprocal plot. In such a double reciprocal plot, 1/fraction of the total drug bound is plotted vs. 1/ lipid concentration, with a y- intercept value of 1 (for a system displaying binding site homogeneity) .
• binding isotherms were constructed using the method of » fluorescence anisotropy titration as discussed above, and K values were determined from the slopes of the double-reciprocal plots. The K values are subject to 10% uncertainty.
• One of the most striking features of the data contained in Table 2 is the strong modulation which can be achieved through the creation of A, B, E-ring modified camptothecins (for example, the homosilatecans known as DB-38, DB-90, and DB-91) or B, E-ring modified camptothecins (for example, the homosilatecan known as DB-81) .
• Homosilatecans containing either a sole substitution at the 7 position or dual substitution at the 7 and 10 positions have been created and found to display very high lipophilicities . Included in Table 2 are camptothecin compounds (topotecan and camptothecin) . For DB-81, the lipophilicity for DMPC membranes relative to corresponding value for topotecan increases over 1,400-fold. Data for these agents were included to show the highly Hpophilic nature of the new homocamptothecins relative to compounds such as topotecan and camptothecin. From Table 2, it is clear that the compounds of the present invention are much more Hpophilic than either camptothecin or topotecan.
• the homosilatecans not only display markedly enhanced lipophilicity but the levels of differential binding between the lactone and carboxylate forms appear to be significantly greater (10-fold versus 3- fold) relative to camptothecins containing ⁇ -hydroxylactone ring systems.
• the two considerations described above are contributing factors to the optimized blood stabilities which the homosilatecans display over camptothecin and homocamptothecin.
• Figure 5 illustrates the fluorescence emission spectra of 1 ⁇ M 7-t-butyldimethylsilyl-10-hydroxy- homocamptothecin (DB-91) in solutions of phosphate-buffered saline (PBS) at pH 7.4, ethanol, >and admixtures thereof. All spectra were recorded using exciting light of 394 nm at 37°C.
• the emission maxima for DB-91 in PBS is 554 nm, but this value shifts significantly to a ⁇ ., ⁇ of approximately 410 nm in anhydrous ethanol. Because DB-91 contains a 10- hydroxy functionality, the possibility exists that fluorescence can occur from two distinct species.
• a protonated (with respect to the 10 -hydroxy functionality) species predominates, while in protic solvents such as water a deprotonated excited-state complex predominates.
• the 554 nm peak is correlated with the deprotonated excited-state complex while the ⁇ ., ⁇ of approximately 410 nm correlates with the protonated excited-state complex.
• the formation of the deprotonated excited-state complex is greatly facilitated by the presence of water; even at small amounts of water such as 1% a peak is apparent around 550 nm which correlates with the water-facilitated formation of the deprotonated excited-state complex.
• the spectral sensitivity of DB-91, and other members of the camptothecin family containing the 10 -hydroxy functionality provides a useful approach for studying the partitioning of drug from an aqueous environment into a hydrophobic environment such at the surface of a red blood cell.
• Figure 6 shows the fluorescence emission spectra of 1 ⁇ M 7-t-butyldimethylsilyl-10-hydroxy-homocamptothecin
• DB91 in solutions of phosphate-buffered saline (PBS) at pH 7.4 and in PBS at pH 7.4 containing albumin-free red blood cells at a concentration of (10 ⁇ 1) x 10 6 cell/ ⁇ L and provides direct evidence of the extensive interactions of a homosilatecan with red blood cells- Spectra were recorded in front- face cuvettes (to optimize fluorescence versus scatter levels) at 37 °C using a DB-91 concentration of 10 ⁇ M and exciting light of 370 nm.
• PBS phosphate-buffered saline
• the emission maxima for DB-91 in PBS is 554 nm.
• a peak with a significantly lower ⁇ . ⁇ value is observed indicating that the agent is capable of partitioning into the red blood cell membranes.
• the membranes of the red blood cells provide a hydrophobic microenvironment where the protonated excited- state complexes can form and fluoresce from.
• Homosilatecans Display Improved Stabilities in Aqueous Solution Relative to Camptothecins Containing ⁇ -hydroxyLactone Pharmacophores .
• FIGS 8 through 11 illustrate the pH dependence of the stability of 1 ⁇ M solutions of DB-38, DB-81, DB-90, and DB-91 in solutions of phosphate-buffered saline (PBS) at pH values of 5.0, 7.4, 8.0, and 9.0.
• PBS phosphate-buffered saline
• the stability parameters for each drug were determined using HPLC methods. All experiments were conducted at 37°C. Hydrolysis is observed at pH values of 7.4, 8.0, and 9.0 with more extensive hydrolysis being noted at the higher pH values.
• Figure 12 contrasts the improved stabilities of four novel homosilatecans of the present invention with their corresponding silatecan structure containing the ⁇ -hydroxylactone functionality. All the experiments of
• FIG. 12 were conducted in PBS at 37°C. Panels A through D each contain stability profiles for a novel homosilatecan
• the agents containing the expanded E-ring or homosilatecan structures displayed markedly enhanced stability.
• the stability parameters for the homosilatecans are summarized in Table 3.
• the data indicate that the lactone ring of homosilatecans is less labile (that is, significantly slower in undergoing hydrolysis) relative to the ⁇ -hydroxylactone ring moiety contained in both silatecans and camptothecins.
• silatecans and camptothecins such as topotecan and camptothecin
• approximately 12% of lactone remains at equilibrium after 3 hours, whereas greater than 80% lactone remains for each of the homosilatecans under identical incubation conditions. Determination of the Superior Stabilities of Homosilatecans in the Presence of Human Serum Albumin.
• Figure 13 depicts the improved stabilities of four novel homosilatecans of the current invention following incubation in PBS containing 30 mg/ml human serum albumin at 37°C.
• Panels A through D each contain stability profiles for a novel homosilatecan and its corresponding silatecan containing the conventional ⁇ -hydroxylactone ring moiety found in camptothecin and other clinically relevant camptothecin analogs such as topotecan, SN-38, CPT-11 and 9- aminocamptothecin.
• the agents containing the expanded E-ring structures displayed markedly enhanced stabilities in the presence of HSA.
• the stability parameters for the homosilatecans are summarized in Table 3.
• the homosilatecans of the current invention also displayed superior stabilities in human plasma than camptothecins such as topotecan, SN-38, and CPT- 11.
• camptothecins such as topotecan, SN-38, and CPT- 11.
• DB-81 displayed the highest stability in human plasma, followed by DB-90 and DB-91, with DB-38 (the least Hpophilic of the homosilatecans studied) displaying the lowest stablility in human plasma.
• All the experiments of Figure 14 were conducted in human plasma at 37°C. Plasma samples were continuously aerated by a stream of blood gas resulting in the maintenance of pH at values of 7.5 ⁇ 0.1.
• the agents containing the expanded E-ring or homosilatecan structures displayed markedly enhanced stabilities relative to the parent drug camptothecin containing the conventional ⁇ -hydroxylactone ring moiety.
• the stability parameters for the homosilatecans are summarized in Table 3.
• Our studies demonstrate that both Hpophilic as well as more water-soluble homosilatecans display improved stabilities over homocamptothecin that contains no substitutions in the A and B rings. See Lavergne et al . "Homocamptothecins : Synthesis and Antitumor Activity of Novel E-Ring-Modified Camptothecin Analogues" J. Med. Chem. 41 : 5410-5419 (1998) .
• the present invention indicates that substitution of the A and B ring of homocamptothecin is a favorable factor with respect to blood stabilities.
• the likely explanation is that the unsubstituted homocamptothecin carboxylate, like camptothecin carboxylate, binds HSA preferentially in the carboxylate form and effectively shifts the lactone-carboxylate equilibria to the right .
• B-ring modification such as a silyl or an silylalkyl functionality at position 7 (e.g. DB-81)
• A-ring modification such as the structural modifications contained in topotecan (water-solublizing changes such as inclusion of 9-dimethylaminomethyl and 10- hydroxy functionalities disfavor carboxylate binding to HSA
• Figure 15 depicts the stabilities of four novel homosilatecans of the current invention in PBS suspensions containing physiologically-relevant concentrations [(5 ⁇ 1) x 10 6 cell/ ⁇ L] of albumin-free red blood cells. Stability characteristics were determined at 37°C using HPLC methods. In all cases, the agents containing the expanded E-ring or homosilatecan structures displayed markedly enhanced stabilities in the presence of red blood cells relative to published literature values for camptothecin analogs containing the conventional ⁇ -hydroxylactone ring moiety (such as the clinically relevant agents SN-38, 9- aminocamptothecin, 9-nitrocamptothecin, GI-147211C, topotecan, etc. The stability parameters for the homosilatecans are summarized in Table 3.
• Figure 16 and Figure 17 depict the improved human blood stabilities of four novel homosilatecans of the present invention. All experiments were conducted at pH 7.4 and 37°C.
• panels A through D each contain stability profiles for a novel homosilatecan (open circles) and its corresponding silatecan (solid circles) containing the conventional ⁇ -hydroxylactone ring moiety found in camptothecin and other clinically relevant and camptothecin analogs such as topotecan, SN-38, CPT-11 and 9- aminocamptothecin and experimental agents as well .
• the agents containing the expanded E-ring or homosilatecan structures displayed markedly enhanced human blood stabilities relative to camptothecin analogs such as topotecan and SN-38.
• Figure 17 illustrates the improved human blood stabilities of the novel homosilatecans of the present invention compared to current clinically-relevant agents including 9-aminocamptothecin (9AC) , camptothecin
• the human blood stability values noted for DB-81, DB-90 and DB-91 are the highest yet to be measured for a intrinsically potent camptothecin analog.
• the greater than 80% lactone values following 3 hrs . of incubation compare very favorably relative to the corresponding percent lactone levels in whole human blood for 9-aminocamptothecin (approx. 0.3%), camptothecin (approx. 6%) topotecan (approx. 15%), CPT-11 (approx. 21.0%), and SN-38 (approx. 30%).
• the Novel Homosilatecans of the Current Invention Overcome the Marked Interspecies Variations With Respect to Blood Stabilities that Have Been Observed In the Past for Clinically Relevant Camptothecins Such as 9- Aminocamptothecin, 9-Nitrocamptothecin and Camptothecin.
• the drugs contain the lactone ring moiety which, at pH 7.4, hydrolyzes to yield biologically-inactive carboxylate forms.
• Serum albumins from other species were found to bind camptothecin carboxylate not nearly as tightly as HSA. Due to the unique capacity of human albumin to bind camptothecin carboxylate and 9-aminocamptothecin carboxylate resulting m extensive conversion of the drug to its biologically inactive form, it appears that the success of these agents m eradicating cancer m animal models may be inherently more difficult to duplicate humans.
• the data for the novel homosilatecans of the current invention show essentially only minor variations between lactone levels m mouse blood versus human blood.
• the noted changes mouse versus animal blood are very small relative to the 100-fold difference m lactone levels observed for 9-aminocamptothecin.
• the lactone levels actually observed m human blood are modestly underestimated by values of 6% and 20%, respectively.
• 9- ammocamptothec mouse blood overestimates by 100-fold the lactone levels actually observed human blood.
• cytotoxicities of various camptothecins against MDA-MB-435 tumorigenic metastatic human breast cancer cells are summarized in Table 5.
• the cytotoxicity values are for 72 hr. exposure times.
• DB-38 we found DB-38 to be the most potent of the four novel homosilatecans which we studied, with an IC 50 value of 20 nM, while the IC 50 values for the other homosilatecans ranged from 20 nM to 115 nM.
• Our results clearly indicate that through novel homosilatecan development the stability of the agents in human and animal blood can be markedly improved and equilized without compromising the high intrinsic potency and cytotoxicity of this important class of anticancer drugs .
• Camptothecin and topotecan were in their
• Drug Stock Solution Preparation.
• Stock solutions of the drugs were prepared in dimethylsulfoxide (A.C.S. spectrophotometric grade, Aldrich, Milwaukee, WI) at a concentration of 2 x 10 "3 M and stored in the dark at 4°C.
• L- ⁇ -Dimyristoylphosphatidylcholine (DMPC) and L- ⁇ - dimyristoylphosphatidylglycerol (DMPG) were obtained from Avanti Polar Lipids, Alabaster, AL, and were used without further purification. All other chemicals were reagent grade and were used without further .purification.
• Vesicle Preparation Small unilamellar vesicle (SUV) suspensions were prepared the day of an experiment by a methodology reported previously Burke and Tritton, Biochemistry 24 5972-5980 (1985); and Burke, T. G. , Mishra, A. K. , Wani, M. C. and Wall, M. E. "Lipid bilayer partitioning and stability of camptothecin drugs," Biochemistry. 32: 5352-5364 (1993), the disclosures of which are incorporated herein by reference.
• lipid suspensions containing known amount of lipid 200 mg/mL lipid or less in phosphate buffered saline (PBS, pH 7.4) were prepared by Vortex mixing for 5-10 min above the T M of the lipid.
• the lipid dispersions were then sonicated using a bath-type sonicator (Laboratory Supplies Co., Hicksville, NY) for 3-4 h until they became optically clear.
• a decrease in pH from 7.4 to 6.8 was observed for the SUV preparations of DMPG; therefore, the pH of these SUV suspensions was adjusted to 7.4 using small quantities of 2.5 M NaOH in PBS, " followed by additional sonication.
• Each type of vesicle suspension was annealed for 30 min at 37°C and then used in an experiment .
• Steady-state fluorescence measurements were obtained on a SLM Model 9850 spectrofluorometer with a thermostated cuvette compartment. This instrument was interfaced with an IBM PS/2 model 55 SX computer. Excitation and emission spectra were recorded with an excitation resolution of 8 nm and an emission resolution of 4 nm. In all cases spectra were corrected for background fluorescence and scatter from unlabeled lipids or from solvents by subtraction of the spectrum of a blank. Steady-state fluorescence intensity measurements were made in the absence of polarizers. Steady-state anisotropy (a) measurements were determined with the instrument in the "T- format" for simultaneous measurement of two polarized intensities.
• polarizer orientation was checked using a dilute solution of glycogen in water.
• Anisotropy measurements for homosilatecans and camptothecins were conducted using exciting light of 370 to 400 nm and long pass filters on each emission channel to isolate the drug's fluorescence signal from background scatter and/or residual fluorescence. All emission filters were obtained from Oriel Corp (Stamford, CT) . The combination of exciting light and emission filters allowed adequate separation of fluorescence from background signal. The contribution of background fluorescence, together with scattered light, was typically less than 1% of the total intensity. Since the lactone rings of camptothecin and related congeners undergo hydrolysis in aqueous medium with half-lives of approximately 20 min., all measurements were completed within the shortest possible time ( ca .
• mice Blood from mice was collected in heparinized tubes and stored at 5-10°C until use.
• HPLC assays were performed either on a Waters
• the supernatant was removed and placed in an autosampler vial and the vial was quickly added to the autosampler maintained at 4°C.
• the sample was analyzed by HPLC as soon as possible. The data analysis was as described for drug in PBS only samples as described above. Blood gas was continuously bubbled through the plasma samples to pH at 7.5 +/ - 1.0.
• red blood cells obtained from the Central Kentucky Red Cross and were counted using a
• RBCs were incubated at 37°C for 30 min and the pH remeasured to ensure that it was within the same range as before the assays were started. Aliquot of RBCs (2 ml each) were removed and placed in three disposable glass test tubes and the tubes were incubated at 37°C. Aliquots of drug in DMSO were added to RBC suspensions in PBS to result in final drug concentration of 1 ⁇ M. Incubation at 37°C was continued and
• DMPC negatively-charged dimy ⁇ stoylphosphatidylglycerol
• DMPG negatively-charged dimy ⁇ stoylphosphatidylglycerol
• DB-38 which occur m the presence of membranes indicate that the agent is capable of binding both electroneutral and negatively-charged membranes. Spectral recordings were initiated and completed shortly after the addition of the lactone form of the agent to solution or suspension, thereby assuring that the detected signal originates predominantly from the lactone form of the agent (and not the ring-opened form) .
• Fluorescence emission spectra of 1 ⁇ M 7-t- butyldimethylsilylhomocamptothecm (DB-81) were also examined. Lipid concentrations of 10 mM were used. All spectra were recorded using exciting light of 380 nm at 37°C.
• the emission maxima for DB-81 m PBS buffer is 452 nm and this value shifts to lower values in the presence of membranes ( ⁇ max of 443 nm m the presence of DMPC vesicles and ⁇ - ⁇ of 442 nm in the presence of DMPG vesicles) .
• the spectral shifts of DB-81 which occur in the presence of membranes indicate that the agent is capable of binding both electroneutral and negatively- charged membranes.
• the fluorescence emission spectra of 1 ⁇ M 7-t- butyldimethylsilyl-10-aminohomocamptothecin (DB-90) was also examined. All spectra were recorded using exciting light of 430 nm at 37°C.
• the emission maxima for DB-90 in PBS is 535 nm and this value shifts to lower values in the presence of membranes ( ⁇ . ⁇ of 513 nm in the presence of DMPC vesicles and ⁇ max of 512 nm in the presence of DMPG vesicles) .
• the fluorescence emission spectra of 1 ⁇ M 7-t- butyldimethylsilyl-10-hydroxy-homocamptothecin (DB-91) was also studied. Lipid concentrations of 10 mM were used. All spectra were recorded using exciting light of 394 nm at 37°C.
• the emission maxima for DB-91 in PBS is 554 nm and this value shifts to lower values in the presence of membranes ( ⁇ , of 441 nm in the presence of DMPC vesicles and ⁇ ⁇ mx of 434 nm in the presence of DMPG vesicles
• the fluorescence emission' spectra of 1 ⁇ M of the carboxylate form of 7-t-butyl-dimethylsilyl-10- aminohomocamptothecin (DB90 carboxylate) was also studied. Lipid concentrations of 0.15 mM were used. The concentration of lipid used in these experiments was greater than in the experiments conducted using the corresponding lactone form of DB-90; the higher lipid concentrations were used because of the reduced membrane associations of the opened-ring form of the drug relative to the closed-ring lactone form of the drug. All spectra were recorded using exciting light of 430 nm at 37°C.
• the emission maxima for DB-90 carboxylate in PBS is 529 nm and this value shifts to lower values in the presence of membranes ( ⁇ max of 512 nm in the presence of DMPC vesicles and ⁇ - j - j . of 512 nm in the presence of DMPG vesicles
• the fluorescence emission spectra of 1 ⁇ M of the ring-opened or carboxylate form of 7-t-butyldimethylsilyl- 10-hydroxyhomocamptothecin (DB-91 carboxylate) were also acquired. Lipid concentrations of 0.15 M were used (the higher concentration of lipid required in these experiments to promote binding was necessitated by the reduced membrane associations of the opened-ring form of the agent relative to the closed-ring lactone form of the agent.
• the emission maxima for DB-91 carboxylate in PBS is 549 nm and this value shifts to lower values ( ⁇ . ⁇ of 450 nm in the presence of DMPC vesicles and ⁇ . ⁇ of 446 nm in the presence of DMPG vesicles) .
• a protonated (with respect to the 10-hydroxy functionality) species predominates, while in protic solvents such as water a deprotonated excited-state complex predominates.
• the 554 nm peak is correlated with the deprotonated excited-state complex while the ⁇ -. ⁇ of approximately 410 nm correlates with the protonated excited-state complex.
• the formation of the deprotonated excited-state complex is greatly facilitated by the presence of water; even at small amounts of water such as 1% a peak is apparent around 550 nm which correlates with the water-f cilitated formation of the deprotonated excited-state complex.
• Figures 6 and 7 we study the extent of protonated excited-state complex formation and use this parameter as a relative measure of lipophilicity for two 7 -modified campotothecins (DB-91 and
• the novel homosilatecan DB-91 is a more Hpophilic, erythrocyte-interactive agent relative to the known compound 7-ethyl-10-hydroxycamptothecin (SN-38) .
• the emission maxima for SN-38 in PBS is approximately 550 nm.
• the SN-38 agent like DB-91, also contains a 10-hydroxy functionality and, as a consequence, SN-38 also displays fluorescence spectral characteristics which are sensitive to the presence of water.
• lactol (4) (0.100 g, 0.34 mmol) followed by AcOH (9 mL) and lead tetraacetate (0.18 g, 0.406 mmol). After 3 hours at 50°C the mixture was poured into ice cold sat. NaHC03 n extracted with ether (3x75 mL) . The organic layer was dried
• keto-formate ester (5) (0.5 g, 1.69 mmol) followed by dioxane (20 mL) .
• ⁇ -Bromo- tert-butylacetate (0.9 mL, 6.08 mmol) was added followed by activated Zn (0.59 g, 9.1 mmol) .
• the Zn was activated by the Cava method as set forth in the J. Organic. Chem. , 47, p. 5030 (1982), the disclosure of which is incorporated herein by reference.
• 12 (0.16 g, 0.63 mmol) was added and the mixture was sonicated for 3.2 hours.
• iodopyridone (10a) (30 mg, 0.065 mmol) was reacted with para-bocaminophenylisonitrile (57 mg, 0.26 mmol) and hexamethylditin (32.2 mg, 0.1 mmol). in benzene (0.5 mL) and t-BuOH (1 mL) .
• iodopyridone (10b) (45 mg, 0.089 mmol) was reacted with para-bocaminophenylisonitrile (58 mg, 0.27 mmol) and hexamethylditin (45 mg, 0.13 mmol) in benzene (1.3 mL) .
• Trifluoroacetic acid (0.1 mL) was added to a solution containing CH2CI2 (0.5 mL) and compound (la) (8.1 mg, 0.014 mmol) and the contents were stirred at 22 °C.
• 491.2240 found 491.2242 LRMS (El) m/ z 491 (M + ) , 434, 392, 376, 319, 279, 262, 223, 178, 167, 149, 136, 121, 107, 91, 77, 57.
• Trifluoroacetic acid (0.1 mL) was added to a solution containing CH2CI2 (0.5 mL) and compound (lc) (6.6 mg, 0.012 mmol) and the contents were stirred at 22°C.
• iodopyridone (10c) (16 mg, 0.033 mmol) was reacted with para-Bocaminophenylisonitrile (21.6 mg, 0.1 mmol) and hexamethylditin (16.7 mg, 0.051 mmol) in benzene (0.5 mL) .
• iodopyridone (10c) (16 mg, 0.033 mmol) was reacted with para-acetoxyphenylisonitrile (16 mg, 0.1 mmol) and hexamethylditin (16.7 mg, 0.051 mmol) in benzene (0.5 mL) .
• Trifluoroaceticacid (0.1 mL) was added to a solution containing CH2CI2 (0.5 mL) and compound (li) (10.7 mg, 0.018 mmol) and the contents were stirred at 22° C.

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