Pharmaceutical compounds targeted by MIF affinity-tethered moieties

There is disclosed a compound, a pharmaceutical composition and a method of treatment using a pharmaceutical composition comprising a tethering moiety that is capable of binding to a macrophage migration inhibitory factor (MIF) polypeptide, optionally linked to a linker moiety and further covalently bound to a drug moiety or imaging agent. More specifically, there is disclosed a genus of affinity-tethering moieties covalently bound to a drug moiety or imaging agent either directly or optionally via a linker moiety to covalently link the tethering moiety to a drug moiety. Without being bound by theory, the disclosed pharmaceutical compounds are targeted to cancer cells or immune cells via an affinity-tethering moiety that hitch-hikes to or into its target cell while bound to endogenous MIF.

TECHNICAL FIELD

The present disclosure provides compounds, pharmaceutical compositions and methods of treatment using a pharmaceutical composition comprising a tethering moiety that binds to a macrophage migration inhibitory factor (MIF) polypeptide, optionally linked to a linker moiety and further covalently bound to a drug moiety. More specifically, the present disclosure provides a pharmaceutical composition and method of treatment comprising a genus of affinity-tethering moieties covalently bound to a drug moiety either directly or optionally via a linker moiety to covalently link the affinity-tethering moiety to a drug substance. Further still, the present disclosure provides a compound, pharmaceutical composition and method of treatment comprising a tethering moiety that competes with ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester) for binding to the tautomerase site of MIF. Further still, the present disclosure provides a, pharmaceutical composition and method of treatment comprising a teathering moiety that bind to the tautomerase site of MIF with a dissociation constant between 10 mM and 1 pM.

BACKGROUND

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that contains both thiol-protein oxidoreductase activity and tautomerase/isomerase activity. MIF is released by T-cells and macrophages and modulates not only macrophage functions, but also T cell functions (Kitaichi et al.,Immunobiology,2000. 201 (3-4): p. 356-67). MIF is viewed to play a role in a wide range of diseases including, cancer, rheumatoid arthritis, sepsis, atherosclerosis, colitis, lupus, asthma, acute respiratory distress syndrome and acute graft-versus-host disease. MIF is involved in cellular proliferation and differentiation and has been demonstrated to have protumorigenic activity. MIF expression in tumors is thought enhance the aggressiveness and metastatic potential of tumor cells. MIF is overexpressed in many tumors, including breast, ovarian, colon and prostate cancer, melanoma, cervical cancer, gastric cancers, hepatocellular carcinoma, and glioblastomas (Hagemann et al., 2007, Mol. Cancer Ther.,6, 7, 1993-2002; Akbar et al., 2001,Cancer Lett.,171, 2, 125-32, Kamimura et al., 2000,Cancer,89, 2, 334-41; Xu et al., 2008,Cancer Lett.,261, 2, 147-57; Munaut et al., 2002,Neuropathol Appl. Neurobiol.,28, 6, 452-60; Bacher et al., 2003, Am. J. Pathol.,162, 1, 11-7; and Meyer-Siegler et al., 2002,Cancer,94, 5, 1449-56).

Analyses of an MIF knockout mouse model and the use of anti-MIF antibodies to modulate MIF levels have demonstrated MIF involvement in cancer and inflammation. MIF may play a role in the progression to more invasive tumors and MIF may control the tumor spectrum mediating a shift in frequency between T-cell lymphomas, fibrosarcomas and B-cell lymphomas (Bernhagen et alNature Med.,2007. 13(5): p. 587-96 and Taylor et al.BMC Cancer,2007. 7: p. 135.). De Jong and associates showed that Murine colitis is dependent on continuous MIF production by the immune system. Both ulcerative colitis (UC) and Crohn's colitis patients are at increased risk of developing colorectal cancer (De Jong et al.Nature Immunol.,2001. 2(11): p. 1061-6 and Itzkowitz and Yio,Am. J. Physiol Gastrointest. Liver Physiol.,2004. 287(1): p. G7-17) used mice knocked out both for MIF and a second gene causing T-cell deficiency. Colitis was shown to be dependent on MIF produced by non-lymphocyte hematopoetic cells.

MIF circulates normally in human plasma at high levels of 2-6 ng/ml (Stosic-Grujicic et al.Autoimmun. Rev.,2009. 8(3): p. 244-9) and these levels can be increased in disease states including many cancers. In sepsis, levels of MIF may be elevated more than 100 fold over basal level (Emonts et al.,Clin. Infect. Dis.,2007. 44(10): p. 1321-8).

A major problem in most forms of cancer chemotherapy is the severe non-specific toxicity chemotherapeutic drugs may also have against rapidly-dividing cells and healthy tissues. These side effects often result in dose reduction, treatment delay or discontinuance of therapy. Targeted drug delivery systems have been developed to try to circumvent these side effects, using targeting agents such as receptor ligands, sugars, lectins, antibodies, antibody fragments, hormones, and hormone analogues. Therefore, there is a need in the art to better target cytotoxic moieties into those areas of cancer cells where the toxic moiety can better exert its pharmacologic influence.

SUMMARY

The present disclosure provides compounds, pharmaceutical compositions and methods of treatment using a pharmaceutical composition comprising a tethering moiety that is capable of binding to a macrophage migration inhibitory factor (MIF) polypeptide, optionally linked to a linker moiety and further covalently bound to a drug or imaging moiety. Preferably, the tethering moiety comprises a moiety from formula (1)

wherein Q is selected from the group consisting of O, S, N(R9), and C(R9)R10;

X is selected from the group consisting of nothing, O, S, N(R9), N(R9)N(R10), (CH2)k-(OCH2CH2)l, CR9R10-CR11R12, and C(O);

j, m, and n are each integers independently from 0 to 8, k is an integer from 0 to 2, 1 is an integer from 1 to 8; and Z represents the drug or imaging moiety.

Preferably the tether moiety is selected from the group consisting of:

wherein L is an optional linker or spacer unit. More preferably, L comprises a linear or branched chain comprising a plurality of linking groups Lm, wherein m is an integer from 0 to about 50.

Preferably, Q is O; j is 1; X is C(O); Y is nothing; R1, R4, R5, R7, and R8 are hydrogen; R3 is methyl; R6 is hydroxyl and Z is Doxorubicin. Preferably, the compound N-((2R,3R,4R,6R)-6-(((1R,3R)-3,12-dihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)-2-(2-hydroxy-4-((5-methyl-2-oxobenzo[d]oxazol-3(2H)-yl)methyl)phenoxy)acetamide is:

Preferably, Q is O; j is 1; X is nothing; Y is C(O)N(R9)N═; R1, R2, R4, R5, R7, R8, and R9 are hydrogen; R3 is methyl; R6 is hydroxyl; and Z is Doxorubicin. Preferably, the compound is N′—((Z)-1-((2R,4S)-4-(((2S,4S,5S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-5-hydroxy-7-methoxy-2-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-2-(2-hydroxy-4-((5-methyl-2-oxobenzo[d]oxazol-3(2H)-yl)methyl)phenoxy)acetohydrazide:

Preferably, Q is O; j is 1; X is C(O); Y is nothing; R1, R2, R4, R5, R7, and R8 are hydrogen; R3 is methyl; R6 is hydroxyl and Z is artemisinin. Preferably, the compound is:

The present disclosure further provides a genus of novel affinity-tethering moieties covalently bound to a drug moiety or to an imaging moiety, either directly or optionally via a linker moiety, to covalently link the affinity-tethering moiety to a drug substance or to an imaging moiety. Further still, the present disclosure provides a therapeutic compound comprising a tethering moiety that competes with ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester) for binding to a tautomerase site of MIF, covalently bound to a drug moiety or to an imaging moiety, optionally via a linking moiety, wherein the therapeutic compound is able to block at least 50% of the binding of ISO-1 to the tautomerase site of MIF. The dissociation constant of ISO-1 is 14.5 μM.

Further still, the present disclosure provides a therapeutic compound comprising a teathering moiety capable of binding to a tautomerase site of MIF with a dissociation constant of between 10 mM and 1 pM, and covalently bound to a drug moiety, optionally via a linking moiety. Without being bound by theory, the disclosed pharmaceutical compounds are targeted to cancer cells or immune cells via the tethering moiety, wherein the tethering moiety hitch-hikes to or into its target cell while bound to endogenous MIF.

The present disclosure provides compounds, pharmaceutical compositions and methods of treatment using a pharmaceutical composition, comprising a tethering moiety that is capable of binding to a macrophage migration inhibitory factor MIF polypeptide, optionally linked to a linker moiety and further covalently bound to a therapeutic agent or imaging agent. More specifically, the present disclosure provides a genus of novel tethering moieties covalently bound to pharmacologic cytotoxic agents or imaging agents, either directly or optionally via a linker moiety, to covalently link the tethering moiety to the cytotoxic agent. The disclosed cytotoxic pharmaceutical compounds and imaging agents are targeted to preferentially gain cellular access into target cells via the MIF tethering moiety as an express pathway to a cellular nucleus without degradation on cellular lysozymes.

DETAILED DESCRIPTION

The present disclosure often uses names for pharmaceutical compounds that are composed of a novel tether moiety or sometimes also called an affinity reagent or an affinity compound. The disclosed pharmaceutical composition optionally also sometimes contains a linker to link the tether to the toxic moiety or drug payload. Table 1 below provides a key to the compound naming system.

The present disclosure provides compounds, pharmaceutical compositions and methods of treatment using a pharmaceutical composition comprising a tethering moiety that is capable of binding to a macrophage migration inhibitory factor (MIF) polypeptide, optionally linked to a linker moiety and further covalently bound to a drug or imaging moiety. Preferably, the tethering moiety comprises a moiety from formula (1)

wherein Q is selected from the group consisting of O, S, N(R9), and C(R9)R10;

X is selected from the group consisting of nothing, O, S, N(R9), N(R9)N(R10), (CH2)k-(OCH2CH2)l, CR9R10-CR11R12, and C(O);

j, m, and n are each integers independently from 0 to 8, k is an integer from 0 to 2, 1 is an integer from 1 to 8; and Z represents the drug or imaging moiety.

Preferably, Q is O; j is 1; X is nothing; Y is C(O)N(R9)N═; R1, R2, R4, R5, R7, R8, and R9 are hydrogen; R3 is methyl; R6 is hydroxyl; and Z is Doxorubicin. Preferably, the compound is N′—((Z)-1-((2R,4S)-4-(((2S,4S,5S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-5-hydroxy-7-methoxy-2-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-2-(2-hydroxy-4-((5-methyl-2-oxobenzo[d]oxazol-3(2H)-yl)methyl)phenoxy)acetohydrazide:

Preferably, Q is O; j is 1; X is C(O); Y is nothing; R2, R4, R5, R7, and R8 are hydrogen; R3 is methyl; R6 is hydroxyl and Z is artemisinin. Preferably, the compound is:

The present disclosure further provides a genus of novel affinity-tethering moieties covalently bound to a drug moiety or to an imaging moiety, either directly or optionally via a linker moiety, to covalently link the affinity-tethering moiety to a drug substance or to an imaging moiety. Further still, the present disclosure provides a therapeutic compound comprising a tethering moiety that competes with ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester) for binding to a tautomerase site of MIF, covalently bound to a drug moiety or to an imaging moiety, optionally via a linking moiety, wherein the therapeutic compound is able to block at least 50% of the binding of ISO-1 to the tautomerase site of MIF. The dissociation constant of ISO-1 is 14.5 μM.

Further still, the present disclosure provides a therapeutic compound comprising a teathering moiety capable of binding to a tautomerase site of MIF with a dissociation constant of between 10 mM and 1 pM, and covalently bound to a drug moiety, optionally via a linking moiety. Without being bound by theory, the disclosed pharmaceutical compounds are targeted to cancer cells or immune cells via the tethering moiety, wherein the tethering moiety hitch-hikes to or into its target cell while bound to endogenous MIF.

The present disclosure provides compounds, pharmaceutical compositions and methods of treatment using a pharmaceutical composition, comprising a tethering moiety that is capable of binding to a macrophage migration inhibitory factor MIF polypeptide, optionally linked to a linker moiety and further covalently bound to a therapeutic agent or imaging agent. More specifically, the present disclosure provides a genus of tethering moieties covalently bound to pharmacologic cytotoxic agents or imaging agents, either directly or optionally via a linker moiety, to covalently link the tethering moiety to the cytotoxic agent. Without being bound by theory, the disclosed cytotoxic pharmaceutical compounds and imaging agents are targeted to preferentially gain cellular access into target cells, via the MIF tethering moiety as an express pathway to a cellular nucleus without degradation on cellular lysozymes.

The present disclosure further provides a genus of novel affinity-tethering moieties covalently bound to a drug moiety or to an imaging moiety, either directly or optionally via a linker moiety, to covalently link the affinity-tethering moiety to a drug substance or to an imaging moiety. Further still, the present disclosure provides a therapeutic compound comprising a teathering moiety that competes with ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester) for binding to a tautomerase site of MIF, covalently bound to a drug moiety or to an imaging moiety, optionally via a linking moiety, wherein the therapeutic compound is able to block at least 50% of the binding of ISO-1 to the tautomerase site of MIF.

Further still, the present disclosure provides a therapeutic compound comprising a teathering moiety that bind to the tautomerase site of MIF with a dissociation constant between 10 mM and 1 pM, covalently bound to a drug moiety, optionally via a linking moiety. The disclosed pharmaceutical compounds are specifically targeted to cancer cells or immune cells via an affinity-tethering moiety that hitch-hikes to or into its target cell while bound to endogenous MIF.

DEFINITIONS

“Alkyl” is a saturated or unsaturated, straight or branched, hydrocarbon chain. In various embodiments, the alkyl group has 1-18 carbon atoms, i.e. is a C1-C18group, or is a C1-C12group, a C1-C6group, or a C1-C4group. A lower alkyl group has 1-6 carbons. Independently, in various embodiments, the alkyl group has zero branches (i.e., is a straight chain), one branch, two branches, or more than two branches. Independently, in one embodiment, the alkyl group is saturated. In another embodiment, the alkyl group is unsaturated. In various embodiments, the unsaturated alkyl may have one double bond, two double bonds, more than two double bonds, and/or one triple bond, two triple bonds, or more than two triple bonds. Alkyl chains may be optionally substituted with 1 substituent (i.e., the alkyl group is mono-substituted), or 1-2 substituents, or 1-3 substituents, or 1-4 substituents, etc. The substituents may be selected from the group consisting of hydroxy, amino, alkylamino, boronyl, carboxy, nitro, cyano, and the like. When the alkyl group incorporates one or more heteroatoms, the alkyl group is referred to herein as a heteroalkyl group. When the substituents on an alkyl group are hydrocarbons, then the resulting group is simply referred to as a substituted alkyl. In various aspects, the alkyl group including substituents has less then 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 carbons.

“Lower alkyl” means a group having about 1 to about 6 carbon atoms in the chain which chain may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, heptyl, nonyl, and decyl.

“Alkoxy” means an alkyl-O-group wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and heptoxy. The bond to the parent moiety is through the ether oxygen.

“Alkoxyalkyl” means an alkoxy-alkyl-group in which the alkoxy and alkyl are as previously described. Preferred alkoxyalkyl comprise a lower alkyl group. The bond to the parent moiety is through the alkyl.

“Alkylaryl” means an alkyl-aryl-group in which the alkyl and aryl are as previously described. Preferred alkylaryls comprise a lower alkyl group. The bond to the parent moiety is through the aryl.

“Aminoalkyl” means an NH2-alkyl-group, wherein alkyl is as defined above, bound to the parent moiety through the alkyl group.

“Aryl” (sometimes abbreviated “Ar”) is an aromatic carbocyclic hydrocarbon ring system. The ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.). In one embodiment, the aryl group is monocyclic, and is preferably a C6ring system, i.e. a phenyl ring is a preferred aryl ring, where preferred bicyclic aryl rings are C8-C12, or C9-C10. A naphthyl ring, which has 10 carbon atoms, is a preferred polycyclic aryl ring. Unless otherwise indicated herein, the term “aryl” as used herein is meant to include aryl rings optionally substituted by one or more substituents selected from acyl (—C(O)—R), alkoxy (—O—R), alkyl, aryl, alkylamino (—N(H)—R and —N(R)R), alkylthio (—S—R), amino (—NH2), azido (—N3), boronyl (—B(R)R or —B(OH)2or —B(OR)2), carboxy (—C(O)—OH), alkoxycarbonyl (—C(O)—OR), aminocarbonyl (—C(O)—NH2), aminosulfonyl (—S(O)2—NH2), alkylaminocarbonyl (—C(O)—N(H)R and —C(O)—N(R)R), cyano, halo (fluoro, bromo, chloro, iodo), haloalkyl, haloalkoxy, heterocyclyl, heteroalkyl, hydroxyl (—OH), acyloxy (—O—C(O)—R), ketone (—C(O)—R), substituted halomethylketone (—C(O)—CHmXn, where m+n=3, X═F, Cl, Br), mercapto (—SH and —S—R) and nitro (—NO2) where each R group is an alkyl group having less than about 12 carbons, preferably where the R group is a lower alkyl group. Non-limiting examples of suitable aryl groups include: phenyl, naphthyl, indenyl, tetrahydronaphthyl, indanyl, anthracenyl, and fluorenyl.

“Arylalkyl” refers to an alkyl group as defined substituted by one or more aryl groups as defined below. Phenyl and naphthyl are preferred aryl groups in an arylalkyl group. A preferred alkyl group is methyl, so that a preferred arylalkyl group is benzyl or benzyl having one or more substituents on the phenyl ring. Unless otherwise indicated, the term “arylalkyl” as used herein is meant to include arylalkyl groups wherein the aryl ring therein is optionally substituted by one or more substituents selected from acyl (—C(O)—R), alkoxy (—O—R), alkyl, aryl, alkylamino (—N(H)—R and —N(R)R), alkylthio (—S—R), amino (—NH2), azido (—N3), boronyl (—B(R)R or —B(OH)2or —B(OR)2), carboxy (—C(O)—OH), alkoxycarbonyl (—C(O)—OR), aminocarbonyl (—C(O)—NH2), aminosulfonyl (—S(O)2—NH2), alkylaminocarbonyl (—C(O)—N(H)R and —C(O)—N(R)R), cyano, halo (fluoro, bromo, chloro, iodo), haloalkyl, haloalkoxy, heterocyclyl, heteroalkyl, hydroxyl (—OH), acyloxy (—O—C(O)—R), ketone (—C(O)—R), substituted halomethylketone (—C(O)—CHmXn, where m+n=3, X═F, Cl, Br), mercapto (—SH and —S—R) and nitro (—NO2) where each R is an alkyl group having less than about 12 carbons, preferably where the R group is a lower alkyl group.

“Arylalkyl” means an aryl-alkyl-group in which the aryl and alkyl are as previously described. Preferred arylalkyls comprise a lower alkyl group. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl and napthalenylmethyl. The bond to the parent moiety is through the alkyl.

“Aryloxy” means an aryl-O-group in which the aryl group is as previously described. Non-limiting examples of suitable aryloxy groups include phenoxy and naphthoxy. The bond to the parent moiety is through the ether oxygen.

“Carboxyalkyl” means an HOOC-alkyl-group, wherein alkyl is as defined above, bound to the parent moiety through the alkyl group.

“Cycloalkyl” means a non-aromatic mono- or multicyclic ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 7 ring atoms. A multicyclic cycloalkyl substituent may include fused, spiro, or bridged ring structures. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Non-limiting examples of suitable multicyclic cycloalkyls include 1-decalin, norbornyl, adamantly and the like. Cycloalkyl substituents may be substituted or unsubstituted. In one embodiment, the cycloalkyl is unsubstituted. In another embodiment, the cycloalkyl is substituted with, e.g., 1 substituent (i.e., the cycloalkyl group is mono-substituted), or 1-2 substituents, or 1-3 substituents, or 1-4 substituents, etc. In one embodiment, the substituents that may be present on the cycloalkyl aliphatic ring are selected from acyl (—C(O)—R), alkoxy (—O—R), alkyl, aryl, alkylamino (—N(H)—R and —N(R)R), alkylthio (—S—R), amino (—NH2), azido (—N3), boronyl (—B(R)R or —B(OH)2or —B(OR)2), carboxy (—C(O)—OH), alkoxycarbonyl (—C(O)—OR), aminocarbonyl (—C(O)—NH2), aminosulfonyl (—S(O)2—NH2), alkylaminocarbonyl (—C(O)—N(H)R and —C(O)—N(R)R), cyano, halo (fluoro, bromo, chloro, iodo), haloalkyl, haloalkoxy, heterocyclyl, heteroalkyl, hydroxyl (—OH), acyloxy (—O—C(O)—R), ketone (—C(O)—R), substituted halomethylketone (—C(O)—CHmXn, where m+n=3, X═F, Cl, Br), mercapto (—SH and —S—R) and nitro (—NO2) In one aspect the R group in the above substituents is an alkyl group having less than about 12 carbons, while in another aspect the R group is a lower alkyl group.

“Cycloalkylalkyl” means a cycloalkyl group bound to the parent moiety through an alkyl group. Non-limiting examples include: cyclopropylmethyl and cyclohexylmethyl.

“Cycloalkylaryl” means a cycloalkyl group bound to the parent moiety through an aryl group. Non-limiting examples include: cyclopropylphenyl and cyclohexylphenyl.

“Fluoroalkoxy” means an alkoxy group as defined above wherein one or more hydrogen atoms on the alkoxy is or are replaced by a fluoro group.

“Fluoroalkyl” means an alkyl group as defined above wherein one or more hydrogen atoms on the alkyl are replaced by a fluoro group.

“Halo” means fluoro, chloro, bromo, or iodo groups. Preferred are fluoro, chloro or bromo, and more preferred are fluoro and chloro.

“Heteroalkyl” is a saturated or unsaturated, straight or branched, chain containing carbon and at least one heteroatom. The heteroalkyl group may, in various embodiments, have on heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms. In one aspect the heteroalkyl chain contains from 1 to 18 (i.e., 1-18) member atoms (carbon and heteroatoms), and in various embodiments contain 1-12, or 1-6, or 1-4 member atoms. Independently, in various embodiments, the heteroalkyl group has zero branches (i.e., is a straight chain), one branch, two branches, or more than two branches. Independently, in one embodiment, the hetereoalkyl group is saturated. In another embodiment, the heteroalkyl group is unsaturated. In various embodiments, the unsaturated heterolkyl may have one double bond, two double bonds, more than two double bonds, and/or one triple bond, two triple bonds, or more than two triple bonds. Heteroalkyl chains may be substituted or unsubstituted. In one embodiment, the heteroalkyl chain is unsubstituted. In another embodiment, the heteroalkyl chain is substituted. A substituted heteroalkyl chain may have 1 substituent (i.e., by monosubstituted), or may have 1-2 substituents, or 1-3 substituents, or 1-4 substituents, etc. Exemplary heteroalkyl substituents include esters (—C(O)—O—R) and carbonyls (—C(O)—).

“Heterocyclic” (or “heterocycloalkyl” or “heterocyclyl”) refers to a non-aromatic saturated monocyclic or multicyclic ring system comprising 3 to 10 ring atoms (e.g., 3 to 7 ring atoms), or 5 to 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. There are no adjacent oxygen and/or sulfur atoms present in the ring system. Examples of heterocyclics or heterocycloalkyls include rings having 5 to 6 ring atoms. The prefix aza, oxa or thia before the heterocyclic or heterocycloalkyl root name means that at least a nitrogen, oxygen or sulfur atom, respectively, is present as a ring atom. The nitrogen or sulfur atom of the heterocyclic or heterocycloalkyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide. Any nitrogen atoms may be optionally quaternized. Non-limiting examples of monocyclic heterocyclic or heterocycloalkyl rings include: piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophen-yl, and tetrahydrothiopyranyl. The heterocyclyl may be unsubstituted or substituted. In one embodiment, the heterocyclyl is unsubstituted. In another embodiment, the heterocyclyl is substituted. The substituted heterocyclyl ring may contain 1 substituent, or 1-2 substituents, or 1-3 substituents, or 1-4 substituents, etc. In one embodiment, the substituents that may be present on the heterocyclyl ring are selected from acyl (—C(O)—R), alkoxy (—O—R), alkyl, aryl, alkylamino (—N(H)—R and —N(R)R), alkylthio (—S—R), amino (—NH2), azido (—N3), boronyl (—B(R)R or —B(OH)2or —B(OR)2), carboxy (—C(O)—OH), alkoxycarbonyl (—C(O)—OR), aminocarbonyl (—C(O)—NH2), aminosulfonyl (—S(O)2—NH2), alkylaminocarbonyl (—C(O)—N(H)R and —C(O)—N(R)R), cyano, halo (fluoro, bromo, chloro, iodo), haloalkyl, haloalkoxy, heterocyclyl, heteroalkyl, hydroxyl (—OH), acyloxy (—O—C(O)—R), ketone (—C(O)—R), substituted halomethylketone (—C(O)—CHmXn, where m+n=3, X═F, Cl, Br), mercapto (—SH and —S—R) and nitro (—NO2) In one aspect, the R group which is, or is part of the substituent attached to the heterocyclic ring is an alkyl group having less than about 12 carbons, while in another aspect the R group is a lower alkyl group.

“Heterocycloalkylalkyl” means a heterocycloalkyl-alkyl group, wherein said heterocycloalkyl and said alkyl are as defined above, bound to a parent moiety through the alkyl group.

“Heteroaryl” means an aromatic monocyclic or multicyclic ring system comprising 5 to 14 ring atoms, or 5 to 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. Heteroaryls can contain 5 to 6 ring atoms. The prefix aza, oxa or thio before the heteroaryl root name means that at least a nitrogen, oxygen or sulfur atom respectively, is present as a ring atom. A nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide. Any nitrogen atoms may be optionally quaternized. Non-limiting examples of heteroaryls include: pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, and benzothiazolyl. The heteroaryl may be unsubstituted or substituted. In one embodiment, the heteroaryl is unsubstituted. In another embodiment, the heteroaryl is substituted. The substituted heteroaryl ring may contain 1 substituent, or 1-2 substituents, or 1-3 substituents, or 1-4 substituents, etc. In one embodiment, the substituents that may be present on the heteroaryl ring are selected from acyl (—C(O)—R), alkoxy (—O—R), alkyl, aryl, alkylamino (—N(H)—R and —N(R)R), alkylthio (—S—R), amino (—NH2), azido (—N3), boronyl (—B(R)R or —B(OH)2or —B(OR)2), carboxy (—C(O)—OH), alkoxycarbonyl (—C(O)—OR), aminocarbonyl (—C(O)—NH2), aminosulfonyl (—S(O)2—NH2), alkylaminocarbonyl (—C(O)—N(H)R and —C(O)—N(R)R), cyano, halo (fluoro, bromo, chloro, iodo), haloalkyl, haloalkoxy, heterocyclyl, heteroalkyl, hydroxyl (—OH), acyloxy (—O—C(O)—R), ketone (—C(O)—R), substituted halomethylketone (—C(O)—CHmXn, where m+n=3, X═F, Cl, Br), mercapto (—SH and —S—R) and nitro (—NO2). In one aspect, the R group which is, or is part of the substituent attached to the heteroaryl ring is an alkyl group having less than about 12 carbons, while in another aspect the R group is a lower alkyl group.

“Heteroaralkyl” or “heteroarylalkyl” means a heteroaryl-alkyl-group, in which the heteroaryl and alkyl are as previously described. Preferred heteroaralkyls can contain a lower alkyl group. Non-limiting examples of suitable aralkyl groups include pyridylmethyl, 2-(furan-3-yl)ethyl and quinolin-3-ylmethyl. The bond to the parent moiety is through the alkyl.

“Hydroxyalkyl” means an HO-alkyl-group, in which alkyl is previously defined. Preferred hydroxyalkyls contain lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

“Hydrate” is a solvate wherein the solvent molecule is H2O.

The compounds disclosed herein form salts that are also within the scope of this disclosure. Reference to a compound herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, when a compound contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. The salts can be pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts, although other salts are also useful. Salts of the compounds may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.

Classes of compounds that can be used as the chemotherapeutic agent (antineoplastic agent) include: alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogs), and synthetics. Examples of compounds within these classes are given below.

Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the “Physicians' Desk Reference” (PDR), e.g., 2008 edition (Thomson P D R, Montvale, N.J. 07645-1742, 25 USA); the disclosure of which is incorporated herein by reference herein.

As used herein, a microtubule affecting agent is a compound that interferes with cellular mitosis, i.e., having an anti-mitotic effect, by affecting microtubule formation and/or action. Such agents can be, for instance, microtubule stabilizing agents or agents that disrupt microtubule formation.

Particularly, agents can be compounds with paclitaxel-like activity. These include, but are not limited to paclitaxel and paclitaxel derivatives (paclitaxel-like compounds) and analogues. Paclitaxel and its derivatives are available commercially.

Without being bound by theory, the disclosed pharmaceutical compounds are specifically targeted to cancer cells or immune cells via an affinity-tethering moiety that hitch-hikes to or into its target cell via the covalently attached tethering moiety.

Drug Moieties

The following table lists various examples of drug moieties to be covalently attached to a tethering moiety.

In addition, the present invention discloses having a bodipy agent covalently linked to a tethering moiety and having two free amine moieties, wherein the amine moieties form a covalent link to a cytotoxic agent, such as an artemesin moiety.

Lymphoma cells expressing CD74 can import approximately 107molecules of an anti-CD74 mAb (LL1) per cell per day (Hansen et al.Biochem J.320:293-300, 1996). Expression of CD74 and/or MIF was observed in a variety of malignant cells including most B-cell cancers (Burton et al.Clin. Cancer Res.10:6606:6611, 2004; McClelland et al.Am. J. Pathol.174:638-646, 2009; and Cutbert et al.Eur. J. Cancer45:1654-1663, 2009).

Synthesis Example 1

This example illustrates synthesis of preferred teathering moiety product (2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy)acetic acid (compound 7). The last step of this synthesis covalently binds product {2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetic acid (compound 7) to toxic agent doxorubicin.

A solution of 20 g (162 mmol) of 2-amino-4-methylphenol (compound 1) and 20.4 g of vanillin (compound 2) (162 mmol) in 200 mL of EtOH was added Ti(OEt)4(92.2 g, 400 mmol) at 25° C. The reaction mixture was stirred at 25° C. for 2 hrs. The reaction was cooled to 0° C. and 20 g (526 mmol) of NaBH4was added in portions at 0° C. The reaction mixture was stirred for additional 3 hrs as 25° C.

The reaction mixture was diluted with 200 mL of H2O and 200 mL of EtOH. The resulting precipitate was concentrated and concentrated to remove EtOH. Then, 200 mL of EtOAc was added, the mixture was filtered and solid was washed with EtOAc (100 mL*3). Filtrate was separated and the aqueous layer was extracted with EtOAc (100 mL*3). The combined organic layer was washed with brine (250 mL), dried over Na2SO4, filtered and concentrated to afford 30 g of 2-[(4-hydroxy-3-methoxybenzyl)amino]-4-methylphenol (compound 3) as brown oil (in about 70% purity), which was used for next step without further purification.

To a solution of compound 3 (30 g, 0.115 mol) in 300 mL of CH2Cl2was added Et3N (49 mL, 0.35 mol) at 0° C. The mixture was stirred at 0° C. for 15 min. Then a solution of triphosgene (11.44 g, 38.6 mmol) was added into above solution at 0° C. for 1 hr. After addition, the reaction was allowed to warm to room temperature and stirred for 15 hrs.

The reaction mixture was washed with st. NaHCO3aqueous (200 mL) and brine (200 mL), dried over MgSO4, filtered and concentrated to afford 30 g of crude product as brown oil, which was used for next step without further purification.

5.1 g (126 mmol) of NaH (60%) was added into a solution of 3-(4-hydroxy-3-methoxybenzyl)-5-methyl-1,3-benzoxazol-2(3H)-one (compound 4) (30 g crude, 105 mmol) and 21 g (126 mmol) of ethyl 2-bromoacetate in 800 mL of DMF at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred for 15 hrs.

The reaction mixture was diluted with 1 L of EtOAc and 1 L of H2O. The mixture was separated and aqueous layer was extracted with EtOAc (500 mL*3). The combined organic layers was washed with brine (1 L) and dried over Na2SO4, filtered and concentrated to afford 30 g of crude product as brown oil, which was purified by column chromatography (Elute:PE:EA=8:1 to PE:EA=2:1) to afford 10 g of pure product as yellow solid.

1 g (2.7 mmol) of ethyl(2-methoxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy)acetate (compound 5) was dissolved in 20 mL of MeOH, following 20 mL of 2M NaOH aqueous was added. The reaction mixture was stirred at 25° C. for 15 hrs. The reaction mixture was concentrated to remove MeOH. Then 2N HCl was added in to aqueous to pH=2. The reaction mixture was extracted with EtOAc (200 mL*3). The combined organic layer was washed with brine (60 mL) and dried over Na2SO4, filtered and concentrated. The residue was solidified with TBME (100 mL). The resulting precipitate was filtered and solid was washed with TBME to afford 600 mg of compound 6 as brown solid, which was used for next step without further purification.

To a solution of {2-methoxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetic acid (compound 6) (600 mg, 1.75 mmol) in dichloromethane (6 mL) was added BBr3(0.88 g, 3.5 mmol) at −40° C. And then the mixture was stirred at room temperature overnight. LC-MS indicated the complete consumption of the starting material. The mixture was cooled to 0° C. and MeOH (10 mL) was added. The solvent was removed under reduced pressure. The residue was dissolved in water (20 mL) and quenched with saturated NaHCO3solution (20 mL). The mixture was extracted with ethyl acetate (25 mL*3). The organic layers were combined and dried over MgSO4. The solvent was removed under reduce pressure to afford the desired product (2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy acetic acid (compound 7) (200 mg, yield 40%) as a light yellow solid. The crude product was used in the next step without purification.

To a mixture of Compound 7 (1 eq), doxorubicin (compound 8) (1 eq) and DIEA (3 eq) in DMF was added HATU (1 eq) in one portion. The mixture was stirred at 25° C. for 2 h. The reaction mixture was purified by preparative HPLC to give the desired product amine tethered Doxorubicin/N-((2R,3R,4R,6R)-6-(((1R,3R)-3,12-DIHYDROXY-3-(2-HYDROXYACETYL)-10-METHOXY-6,11-DIOXO-1,2,3,4,6,11-HEXAHYDROTETRACEN-1-YL)OXY)-3-HYDROXY-2-METHYLTETRAHYDRO-2H-PYRAN-4-YL)-2-(2-HYDROXY-4-((5-METHYL-2-OXOBENZO[D]OXAZOL-3(2H)-YL)METHYL)PHENOXY)ACETAMIDE (compound 9).

Synthesis Example 2

This example illustrates the covalent linkage of {2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetic acid (compound 7) to artemisinyl propylamine (compound 10) to form teathered artemisinin.

Artemisinyl propylamine (compound 10) (0.32 gram, 1 mmole), {2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetic acid (Compound 7), (0.33 gram, 1 mmole) and HOBt-H2O (0.30 gram, 2 mmole) were dissolved in 10 mL of CH2Cl2-DMF, 1:1 (v/v) mixture. The mixture was cooled to 0° C. in ice-bath, and dicyclohexylcarbodiimide (DCC, 0.22 gram, 1.1 mmole) was added with stirring. The mixture was kept stirring at 0° C. for 2 hr and then at room temperature for overnight. White precipitates, dicyclohexylurea, were removed by suction filtration, and washed with CH2Cl2. The filtrate and washings were combined, and the solvent was removed by Rotavap to yield the crude product. The product, tethered artemisinin 2-(2-hydroxy-4-((5-methyl-2-oxobenzo[d]oxazol-3(2H)-yl)methyl)phenoxy)-N-(3-((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)propyl)acetamide (compound 11) was further purified by silica gel chromatography, using CH2Cl2-methanol as an eluent. Yield 0.5 gram (75%). White powder, MALDI-MS: 635 [M+H]+.

Synthesis Example 3

This example illustrates the covalent linkage of 2-{2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetohydrazide (compound 17) to doxorubicin to form Hydrzone-tethered doxorubicin (compound 18).

3,4-Dihydroxybenzaldehyde (compound 12) (20 g, 152 mmol) was suspended in 200 mL of THF and cooled to 0° C. NaOH solution (12 g in 150 mL of H2O) was added dropwise, followed by a dropwise addition of Ac2O (18 g, 176 mmol). The reaction mixture was stirred at room temperature for 20 mins then it was diluted with EtOAc (200 mL). The reaction mixture was then acidified to pH=6 with 1N HCl and the organic layer were washed with water. After drying and filtration, 31 g crude product 5-formyl-2-hydroxyphenyl acetate (compound 13) was used in the next step without further purification.

The mixture of compound 13 (31 g crude), ethyl 2-bromoacetate (60 g, 359 mmol) and K2CO3(42 g, 300 mmol) in CH3CN (500 mL) was stirred at reflux for 18 h. The reaction mixture was filtered and concentrated. The residue was purified by chromatography on silica gel (petroleum ether to petroleum ether/EtOAc=6/1 as the eluent) to obtain 12 g ethyl[2-(acetyloxy)-4-formylphenoxy]acetate, (compound 14).

The mixture of compound 14 (12 g, 45 mmol), 2-amino-4-methylphenol (5.54 g, 45 mmol), and Et3N (13.66 g, 135 mmol) in CH2Cl2was stirred at 25° C. for 1 h. Then NaBH(OAc)3(28.6 g, 135 mmol) was added. The resulting mixture was stirred at 25° C. for 18 h. The reaction was diluted with CH2Cl2(200 mL), water (400 mL) and Et3N (40 mL). The organic layer was washed with brine, dried over MgSO4, filtered and concentrated to obtain 19 g crude product ethyl[2-(acetyloxy)-4-{[(2-hydroxy-5-methylphenyl)amino]methyl}phenoxy]acetate (compound 15) which was used in the next step without further purification.

To a solution of compound 15 (19 g, crude) in 200 mL of dichloromethane was added Et3N (15.2 g, 150 mmol), followed by triphosgene (4.98 g, 16.7 mmol) in 50 mL of dichloromethane at 0° C. The resulting mixture was stirred at 25° C. for 18 h. The reaction was diluted with water (400 mL) and extracted with dichloromethane. The combined organic layers were concentrated. The residue was suspended in 50 0 mL (EtOAc/PE=1/10:v/v). The solid was collected by filtration and dried under vacuum to obtain 5 g ethyl {2-(acetyloxy)-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetate, (compound 16). LCMS: 14220-029-1E (94.85%, M+23: 421.8)

N2H4—H2O (50 mL, 85%) was added to a mixture of compound 16 in 500 mL of EtOH. The reaction mixture was stirred at reflux for 8 h. Solid was collected and dried under vacuum to obtain 3 g 2-{2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetohydrazide (compound 17).

MeOH (500 mL) was bubbled with N2flow for 30 mins. Then doxorubicin (500 mg, 862 μmol) and compound 17 (444 mg, 1.29 mmol) were added under N2. CF3COOH (0.2 mL) was added. The resulting mixture was stirred at r.t. for 18 h under N2. The reaction mixture was concentrated to about 180 mL, and CH3CN (500 mL) was added. After cooling, the solid was filtrated off. The mother liquid was concentrated to about 50 mL, CH3CN (200 mL) was added. The red solid was purified by HPLC. After lyophilization, 200 mg of Hydrzone-tethered doxorubicin RJS009_1 (compound 18) was obtained.

Synthesis Example 4

This example illustrates the covalent linkage of {2-hydroxy-4-[(5-methyl-2-oxo-1,3-benzoxazol-3(2H)-yl)methyl]phenoxy}acetic acid (compound 7) to Doxorubicin using a Amino-PEG4-acid linker (BroadPharm, San Diego, Calif.).

To a solution of 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (Compound 19) (320 mg 1.2 mmol) in THF (10 mL) CbzCl was added (248 mg, 1.46 mmol) and saturated aqueous Na2CO3(5 mL). The mixture was stirred at 17° C. for 15 hrs. The mixture was then diluted with water and acidified to pH=2. The mixture was extracted with CH2Cl2(40 mL). The organic layer was washed with brine, dried over MgSO4, filtered and concentrated to obtain 3-oxo-1-phenyl-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oic acid (compound 20) (380 mg).

To a solution of 19-benzyl 1-(tert-butyl) 18-amino-4-oxo-7,10,13,16-tetraoxa-2,3-diazanonadecanedioate (compound 22) (100 mg 194 umol) in MeOH (10 mL) 20 mg of Pd/C was added. The mixture was stirred at 20*C for 15 hrs under H2 (40 Psi). The mixture was filtered and concentrated to obtain tert-butyl 18-amino-4-oxo-7,10,13,16-tetraoxa-2,3-diazaoctadecanoate (compound 23) (50 mg).

MeOH (500 mL) is bubbled with N2flow for 30 mins. Then doxorubicin and N-(15-hydrazinyl-15-oxo-3,6,9,12-tetraoxapentadecyl)-2-(2-hydroxy-4-((5-methyl-2-oxobenzo[d]oxazol-3(2H)-yl)methyl)phenoxy)acetamide (compound 25) is added under N2, with CF3COOH. The resulting mixture is bestirred at r.t. for 18 h under N2and concentrated to about 180 mL. CH3CN is added. After cooling, the solid is filtrated off and the mother liquid is concentrated to about 50 mL, CH3CN (200 mL) is added. The red solid is purified by HPLC to obtain N-((E)-2-((2S,4S)-4-(((4S,5 S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-1-hydroxy-5-oxo-8,11,14,17-tetraoxa-3,4-diazanonadec-2-en-19-yl)-2-(2-hydroxy-4-((5-methyl-2-oxobenzo[d]oxazol-3(2H)-yl)methyl)phenoxy)acetamide (Compound 26).

1,4,7,10-TetraaZaCyCIOdOdeCaUe N,N′,N″,N″′-tetraacetic acid (DOTA) forms kinetically stable chelates With metal ions of lanthanide series (such as yttrium and gadolinium) of the periodic table. DOTA N-hydroxysulfosuccinimide ester is prepared following a known procedure (Lewis et al.,Bioconjugate Chem.,5: 565-576, 1994), by mixing 60 mg (128 Mmol) of trisodium DOTA and 27.7 mg. (28 Mmol) of N-hydroxysulfosuccinimide, in 0.96 ml of water, and incubating this solution With 49 #1 of a freshly prepared solution of ‘EDC’ (50 mg/rnl) at 4° C. for 30 min. 1 ml of this solution. Contains 12.68 yrnol (theoretical) of the mono-activated DOTA sulfosuccinirnide.

An excess of this reagent is reacted with any of the arnine-deprotected biotinylating reagent shown in Table 1, and stirred at 4° C. for a period of 18-24 hours. The biotinylated DOTA product is purified on a reverse phase preparative HPLC Column using acetonitrile-water gradient-elution at a HOW rate of 1 rnl/rnin and monitoring the eluent with a refractive indeX detector. The purified material is analyzed by NMR spectroscopy and mass spectrometry.

Synthesis Example 3

Synthesis of RJS012_1

To a solution of Compound 1 (2 g, 9.74 mmol), methyl 4-hydroxy-3-methoxybenzoate (1.95 g, 10.72 mmol) and PPh3(3.07 g, 11.69 mmol) in 30 mL of THF was added DIAD (2.36 g, 11.69 mmol) dropwise at 0° C. under N2. The mixture was stirred overnight and isolated with NaHCO3(50 mL) and EtOAc (50 mL), the aqueous layer was extracted with EtOAc (2*50 mL). The combined organic layer was washed with brine (2*50 mL), dried over MgSO4and concentrated, the residue was purified by column (Petroleum Ether:EtOAc=2:1) to give Compound 2 as white solid.
2. Synthesis of Compound 3:

Compound 2 (2.1 g, 5.68 mmol) in 10 mL of AcOH was added HNO3(3.58 g, 56.8 mmol) dropwaise at 20° C. and stirred for 3 hrs. The mixture was quenched by adding in portions to ice water (30 mL). The resulting mixture was basified with 1M NaOH to pH=10 and extracted with EtOAc (3*40 mL). The combined organic layer was dried and concentrated. The residue was used to next step without purification.
3. Synthesis of Compound 4:

To a solution of Compound 3 (1.9 g crude) in 20 mL of dry CH2C12was added Et3N (2.45 g, 24.18 mmol). To the above solution was added TFAA (2.54 mg, 12.09 mmol) dropwise at 0° C. and stirred for 2 hrs. The mixture was concentrated and the residue was purified by column (Petroleum Ether:EtOAc=2:1) to give compound 4 as oil (1.2 g).
4. Synthesis of Compound 5:

A mixture of compound 4 (1.2 g, 2.92 mmol) and Pd/C (250 mg) in 50 mL of MeOH was stirred at r.t. under 15 psi of H2for 3 hrs. The mixture was filtered. To the filtrate, formimidamide acetate was added (3.01 g, 28.92 mmol) and stirred at 90° C. for 3 hrs. The mixture was concentrated, the residue was washed with H2O (100 mL) to give compound 5 as solid (1 g).
5. Synthesis of Compound 8:

To a solution of compound 5 (1 g, 2.66 mmol) and DMF (1 drop) in 30 mL of CHCl3was added (COCl)2(676 mg, 5.33 mmol) drop wise and stirred at 80° C. for 3 hrs. The mixture was concentrated. To the residue was added i-PrOH (30 mL) and compound 7 (739.4 mg, 5.08 mmol). To the above mixture was added HCl (1 mL, 12 M) and refluxed for 2 hrs. The residue was isolated with NaHCO3(50 mL) and CHCl3(90 mL). The organic layer was washed with brine (50 mL), dried over MgSO4and concentrated. The residue was purified by column (Petroleum ether:EtOAc=1:3) to give compound 8 as oil (600 mg).
6. Synthesis of Compound 9:

To a solution of compound 8 (30 mg, 59.66 umol) in 1 mL of MeOH was added K2CO3(41.2 mg, 298.3 umol). The mixture was stirred at 80° C. for 6 hrs. The mixture was isolated with EtOAc (10 mL) and H2O (10 mL). The aqueous layer was extracted with EtOAc (2*10 mL). The combined organic layer was washed with brine (15 mL), dried over MgSO4and concentrated to give compound 9 as a solid.
7. Synthesis of RJS012_1:

To a solution of compound 9 (110 mg, 270.38 umol), compound 11 (133.55 mg, 405.57 umol) and 4-(pyrrolidin-1-yl)pyridine (80.14 mg, 540.76 umol) in 20 mL of DMF was added EDC HCl (103.66 mg, 540.76 umol). The mixture was stirred overnight and concentrated. The residue was purified by column (EtOAc:MeOH=20:1) to give product as solid. Totally 110 mg was obtained.

Synthesis of RJS012_1

Synthesis of RJS012_3

Synthesis of RJS012_3

To an excess of stirred molten pyridinium hydrochloride at 180° C. was added portionwise 6-acetoxy-7-methoxy-quinazolone (1) (49.5 g, 211.35 mmol) and the resulting solution was stirred at 180° C. for 4 hours. After cooling to room temperature, water (500 ml) was added and the pH adjusted to 7 with aqueous ammonia. The resulting precipitate was collected by filtration, washed with water (5×20 ml), ether (5×20 ml) and dried to a constant weight in a vacuum oven at 40° C. to afford 6,7-dihydroxyquinazolinone (2) (38 g, 100%) as a beige solid:

To a stirred suspension of 6,7-dihydroxyquinazolinone (2) (38.0 g, 213.3 mmol) and TEA (89.1 ml, 640 mmol) in DMF (200 ml), was added dropwise trimethylacetyl chloride (78.8 ml, 640 mmol). The resulting suspension was stirred at room temperature for 1 hour, diluted with EtOAc (500 ml) and washed with water (5×20 ml). The organic phase was concentrated to dryness and the residue triturated with water and the resulting precipitate was collected by filtration, washed with water (5×20 ml) and dried to a constant weight to afford the title compound (52.3 g, 71%) as a pale pink solid: LCMS (retention time=3.65 min., purity=100%), ESI+m/z 347.33 (M+H)+;1H-NMR (DMSO-d6) δ (ppm) 1.32 (s, 18H), 7.59 (s, 1H), 7.92 (s, 1H), 8.14 (s, 1H).

To a stirred suspension of 4-oxo-3,4-dihydroquinazoline-6,7-diyl bis(2,2-dimethylpropanoate) (68.5 g, 197.8 mmol) and TEA (110.3 ml, 791 mmol) in toluene (700 ml) at 0° C., was added neat POCl3(64.5 ml, 692 mmol). The reaction mixture was stirred for 1 h at room temperature, 2 h at 40° C. and concentrated to dryness. The residue was dissolved in DCM (500 ml) and washed with a saturated solution of sodium bicarbonate (50 ml) and water (3×50 ml). The organic phase was dried over magnesium sulfate, filtered and the filtrate concentrated to dryness. The residue was dissolved in dichloromethane and purified by flash chromatography eluting with a mixture of DCM/EtOAc (75/25+3% de DIPEA) to afford 4-chloroquinazoline-6,7-diyl bis(2,2-dimethylpropanoate) (3) (64.7 g, 76%) as a yellow oil, which crystallized upon standing to afford an orange solid: LCMS (retention time=4.32 min., purity=100%), ESI+m/z 365.37 (M+H)+;1H-NMR (DMSO-d6) δ (ppm) 1.42 (s, 18H), 7.26 (s, 1H) 7.89 (s, 1H), 8.04 (s, 1H), 9.03 (s, 1H).

To a stirred slurry of 4-chloroquinazoline-6,7-diyl bis(2,2-dimethylpropanoate) (3) (50.7 g, 139 mmol) at 0° C., was added dropwise a solution of ammonia in methanol (7 N, 500 ml). The reaction mixture was stirred for 1 h at room temperature and evaporated to dryness. The solid was triturated with MeCN (100 ml), collected by filtration and washed with DCM (2×20 ml) and diethyl ether (5×20 ml) to afford 4-chloro-6,7-hydroxyquinazoline (4) (25.4 g, 93%) as a pale yellow solid.

To a stirred suspension of 4-chloro-6,7-hydroxyquinazoline (4) (10.0 g, 50.9 mmol) and TEA (28.3 ml, 203 mmol) in DCM (100 ml) at −10° C. (acetone/ice bath) was added dropwise trimethylacetyl chloride (8.77 ml, 71.2 mmol). The reaction mixture was stirred at room temperature for 2 hours, diluted with dichloromethane (200 ml) and washed with a 10% aqueous solution of critic acid (2×20 ml). The organic phase was dried over magnesium sulfate and concentrated to dryness at room temperature. The resulting pale yellow residue was dissolved in dichloromethane and purified by flash chromatography on silica gel eluting with a gradient of DCM/EtOAc (100/0 to 50/50) to afford 4-chloro-7-hydroxyquinazolin-6-yl pivalate (5) (8.2 g, 57%) as a white solid. The material was used without further characterization.
Synthesis of Compound 6
To a stirred suspension of polymer-supported triphenylphosphine (3 eq, 1.2 mmol/g), the first alcohol (3 eq) in DCM (5 ml/g of resin) at 0° C., was added di-tert-azadicarboxylate (DTAD, 3 eq) followed by 5 (0.36 mmol). The reaction mixture was slowly agitated for 1 hour at room temperature, filtered and the filtrate was concentrated. Column purification may be necessary before next step, if the excess alcohol still present by TLC monitoring.

C16H19ClN2O4, Exact Mass: 338.10. ESI+m/z 339.2 (M+H)+. The compound is used in the next step without further characterization.

C22H30ClN3O6, Exact Mass: 467.18. ESI+m/z 468.4 (M+H)+. The filtrate was concentrated and purified by column chromatography. The resulting pure compound is used in the next step without further characterization.

Synthesis of Compound 7

The residue (containing 6) was dissolved in methanolic ammonia (7N) and stirred for 5 hours, concentrated to dryness, re-dissolved in THF and re-concentrated to dryness to afford phenol 7.

C11H11ClN2O3, Exact Mass: 254.05. ESI+m/z 255.4 (M+H)+. The compound is used directly in the next step without further characterization.

C17H22ClN3O5, Exact Mass: 383.12. ESI+m/z 384.2 (M+H)+. The compound is used directly in the next step without further characterization.

Synthesis of Compound 8

The phenol 7 was subsequently added to a stirred suspension of polymer-supported triphenylphosphine (4 eq), the second alcohol (4 eq), and DTAD (4 eq) at 0° C. The reaction mixture was slowly agitated for 1 hour at room temperature, filtered and concentrated to dryness. The residue was used directly in the next step without further characterization.

Synthesis of Compound 9

The resulting residues (8) were taken up in DMF or ACN and treated with 1.5 equivalents of aniline and 4 equivalents of HCl in dry 1,4-dioxane at 80° C. for 2 hours. The resulting mixture was diluted with water and extracted with EtOAc. The organic layer was separated, dried over MgSO4and concentrated. The residue was then purified by column chromatography to afford compound 9. The white solid obtained was used directly in the next step without further characterization.

Synthesis of Compound 10

Compound 9 was subsequently completely dissolved in 6N HCl (aq) and stirred further until a white precipitate forms. The precipitate was then filtered and washed with Et2O and dried to afford the final compounds 10.

Conjugate Assembly

This example illustrates several in vitro experiments in predictive models of cancer to show the potential therapeutic utility of the disclosed compounds. Human tumor cell lines of a cancer screening panel were 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 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37° C., 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs.

After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition (T=zero). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 g/ml gentamicin. Additionally four, 10-fold serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions were added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations.

Following drug addition, the plates were incubated for an additional 48 h at 37° C., 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and were incubated for 60 minutes at 4° C. The supernatant is discarded, and the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero, (T=zero), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels.

Percentage growth inhibition was calculated as:
[(Ti−Tzero)/(C−Tzero)]×100 for concentrations for whichTi>/=Tzero
[(Ti−Tzero)/Tzero]×100 for concentrations for whichTi<Tzero.
Three dose response parameters were calculated for each experimental agent. Growth inhibition of 50% (GI50) was calculated from [(Ti−Tzero)/(C−Tzero)]×100=50, which was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti=Tzero. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti−Tzero)/Tzero]×100=−50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. These parameters were used to plotFIG. 1throughFIG. 18. The same procedures were used to createFIGS. 19 and 20except a single 10 μM data point was recorded for each cell line.

Assay Example 2

This example illustrates an in vitro experiment using predictive models of cancer to show the cellular specificity and potential therapeutic utility of the inventive compounds. The human leukemic cell line THP-1 was grown in RPMI 1640 medium with penicillin, streptomycin, and containing 25 mM Hepes, 0.05 mM 2-mercaptoethanol, 10% fetal bovine serum and 2 mM L-glutamine. Cells washed 2× with Gibco Life Sciences (Grand Island, N.Y.) AIM-V serum free medium and then were inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 25,000 to 100,000 cells/well. Five to ten million frozen peripheral blood mononuclear cells (PBMCs) (Astarte Biologics, LLC. (Redmond, Wash.)) in a solution of 10% DMSO, 2% human serum albumin in phosphate buffered saline were thawed and immediately washed in AIM-V media. An approximately equal number of these cells (25,000 to 100,000 cells/well) were added to the experimental plate. Doxorubicin was added to a final concentration of 0.8 μM. The microtiter plates were incubated at 37° C., 5% CO2, 95% air and 100% relative humidity for 2-2.5 h then drug uptake was quantified by flow cytometry for example using a BD FACSCanto II (San Jose, Calif.). Doxorubicin uptake was evaluated in the PE channel. Individual cell populations were identified by size, shape and through the use of dye labeled antibodies.

Synthesis of RJS012_1

To a solution of Compound 1 (2 g, 9.74 mmol), methyl 4-hydroxy-3-methoxybenzoate (1.95 g, 10.72 mmol) and PPh3(3.07 g, 11.69 mmol) in 30 mL of THF was added DIAD (2.36 g, 11.69 mmol) dropwise at 0° C. under N2. The mixture was stirred overnight and isolated with NaHCO3(50 mL) and EtOAc (50 mL), the aqueous layer was extracted with EtOAc (2*50 mL). The combined organic layer was washed with brine (2*50 mL), dried over MgSO4and concentrated, the residue was purified by column (Petroleum Ether:EtOAc=2:1) to give Compound 2 as white solid.
2. Synthesis of Compound 3:

Compound 2 (2.1, 5.68 mmol) in 10 mL of AcOH was added HNO3(3.58 g, 56.8 mmol) dropwaise at 20° C. and stirred for 3 hrs. The mixture was quenched by adding in portions to ice water (30 mL). The resulting mixture was basified with 1M NaOH to pH=10 and extracted with EtOAc (3*40 mL). The combined organic layer was dried and concentrated. The residue was used to next step without purification.
3. Synthesis of Compound 4:

To a solution of Compound 3 (1.9 g crude) in 20 mL of dry CH2C12 was added Et3N (2.45 g, 24.18 mmol). To the above solution was added TFAA (2.54 mg, 12.09 mmol) dropwise at 0° C. and stirred for 2 hrs. The mixture was concentrated and the residue was purified by column (Petroleum Ether:EtOAc=2:1) to give compound 4 as oil (1.2 g).
4. Synthesis of Compound 5:

A mixture of compound 4 (1.2 g, 2.92 mmol) and Pd/C (250 mg) in 50 mL of MeOH was stirred at r.t. under 15 psi of H2for 3 hrs. The mixture was filtered. To the filtrate, formimidamide acetate was added
(3.01 g, 28.92 mmol) and stirred at 90° C. for 3 hrs. The mixture was concentrated, the residue was washed with H2O (100 mL) to give compound 5 as solid (1 g).
5. Synthesis of Compound 8:

To a solution of compound 5 (1 g, 2.66 mmol) and DMF (1 drop) in 30 mL of CHCl3was added (COCl)2(676 mg, 5.33 mmol) drop wise and stirred at 80° C. for 3 hrs. The mixture was concentrated. To the residue was added i-PrOH (30 mL) and compound 7 (739.4 mg, 5.08 mmol). To the above mixture was added HCl (1 mL, 12M) and refluxed for 2 hrs. The residue was isolated with NaHCO3(50 mL) and CHCl3(90 mL). The organic layer was washed with brine (50 mL), dried over MgSO4and concentrated. The residue was purified by column (Petroleum ether:EtOAc=1:3) to give compound 8 as oil (600 mg).
6. Synthesis of Compound 9:

To a solution of compound 8 (30 mg, 59.66 umol) in 1 mL of MeOH was added K2CO3(41.2 mg, 298.3 umol). The mixture was stirred at 80° C. for 6 hrs. The mixture was isolated with EtOAc (10 mL) and H2O (10 mL). The aqueous layer was extracted with EtOAc (2*10 mL). The combined organic layer was washed with brine (15 mL), dried over MgSO4and concentrated to give compound 9 as solid.
7. Synthesis of RJS012_1:

To a solution of compound 9 (110 mg, 270.38 umol), compound 11 (133.55 mg, 405.57 umol) and 4-(pyrrolidin-1-yl)pyridine (80.14 mg, 540.76 umol) in 20 mL of DMF was added EDC HCl (103.66 mg, 540.76 umol). The mixture was stirred overnight and concentrated. The residue was purified by column (EtOAc:MeOH=20:1) to give product as solid. Totally 110 mg was obtained.
Synthesis of RJS012_3

1. Synthesis of Compound 2

To a solution of resorcinol (20.0 g, 181.64 mmol) in a mixture (2:1, 900 mL) of chloroform and acetic acid was slowly added a solution of nitric acid (20 mL) in acetic acid (100 mL). After being stirred for 1 h, the reaction mixture was quenched with water (1 L) and extracted with CH2Cl2(1 L*3). The organic layer was dried over MgSO4and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc/Petroleum Ether=1:4 to 1:2) to afford of nitroresorcinol 2 as yellow solid (6 g, 21.3%).
2. Synthesis of Compound 3

AlCl3(15.61 g, 117.07 mmol, 4.00 Eq) was added to the solution of (3-acetoxy-4-nitro-phenyl)acetate (7.00 g, 29.27 mmol, 1.00 Eq) in CHCl3 (300 mL) at 0° C. under N2. The reaction mixture was stirred at 0° C. for 0.5 h and 25° C. for 3 h. The reaction mixture was poured into ice-water, and extracted with CH2Cl2(100 mL*2). The combined organic layers were washed with 1N HCl and brine. After drying over MgSO4and filtering, the filtrate was concentrated. The residue was purified by flash chromatography on silica gel (0-30% EtOAc in petroleum ether) to obtain (3-hydroxy-4-nitro-phenyl)acetate (4.50 g, 22.83 mmol, 77.99% yield) as light yellow solid.
4. Synthesis of Compound 5

LiOH (216.06 mg, 9.02 mmol, 4.00 Eq) in Water (5 mL) was added to the solution of ethyl 2-[2-acetoxy-4-[(6-acetoxy-2-oxo-1,3-benzoxazol-3-yl)methyl]phenoxy]acetate (1.00 g, 2.26 mmol, 1.00 Eq) in THF (10 mL) acidified to pH=3. The mixture was extracted with EtOAc (500 mL*3). The combined organic layers were dried over MgSO4, filtered and concentrated to obtain 2-[2-hydroxy-4-[(6-hydroxy-2-oxo-1,3-benzoxazol-3-yl)methyl]phenoxy]acetic acid (500.00 mg, 1.50 mmol, 66.55% yield, 99.65% purity) which was used in the next step without further purification.
8. Synthesis of Compound RJS012_3

The IC50's of Doxorubicin and Doxorubicin conjugates in large cell immunoblastic lymphoma cell lines (ATCC SR) and Normal Human Adult Dermal Fibroblasts (ATCC PCS-201-012) were compared using a WST-8 cellular proliferation assay (Cayman Chemical Co. (Ann Arbor, Mich.). Large Cell Immunoblastic Lymphoma cells (SR) were grown in TCC-formulated RPMI-1640 Medium plus 10% fetal bovine serum. Primary dermal fibroblasts were grown in Fibroblast Basal Medium (ATCC) plus the contents of the ATCC fibroblast growth kit low serum (rh FGF b, L-glutamine, Ascorbic acid, Hydrocortisone, rh Insulin hemisuccinate, Fetal Bovine Serum). Assays were performed according to the manufactures instructions.

TABLE 3Selectivity of affinity-tethered Artemisinin derivativesIC50Cancer CellsIC501°ATCC SRFibroblastsDrug(μM)(μM)SelectivityART-OH28.6>100>3.8RJS05_11.051.351.3RJS05_24.611926RJS05_38.510312
Table 3—The IC50's of Artemisinin and Artemisinin conjugates in large cell immunoblastic lymphoma cell lines (ATCC SR) and Normal Human Adult Dermal Fibroblasts (ATCC PCS-201-012) were compared using a WST-8 cellular proliferation assay (Cayman Chemical Co. (Ann Arbor, Mich.). Large Cell Immunoblastic Lymphoma cells (SR) were grown in TCC-formulated RPMI-1640 Medium plus 10% fetal bovine serum. Primary dermal fibroblasts were grown in Fibroblast Basal Medium (ATCC) plus the contents of the ATCC fibroblast growth kit low serum (rh FGF b, L-glutamine, Ascorbic acid, Hydrocortisone, rh Insulin hemisuccinate, Fetal Bovine Serum). Assays were performed according to the manufactures instructions.

The IC50's of EGFRi's and EGFRi conjugates in large cell lung cancer cell lines (NCI-H460) and normal human adult dermal fibroblasts were compared using a WST-8 cellular proliferation assay (Cayman Chemical Co. (Ann Arbor, Mich.). NCI-H460 lung cancer cells were grown in TCC-formulated RPMI-1640 Medium plus 10% fetal bovine serum. Primary dermal fibroblasts were grown in Fibroblast Basal Medium (ATCC) plus the contents of the ATCC fibroblast growth kit low serum (rh FGF b, L-glutamine, Ascorbic acid, Hydrocortisone, rh Insulin hemisuccinate, Fetal Bovine Serum). Assays were performed according to the manufactures instructions.

Assay Example 3

This example illustrates an in vitro experiment using predictive models of cancer to show the cellular specificity and potential therapeutic utility of the inventive compounds. The human leukemic cell line THP-1 was grown AIM-V serum free medium Gibco Life Sciences (Grand Island, N.Y.). Doxorubicin or compound 9 were added to the medium (AIM-V serum free media) and used for ISO-1 dilution. ISO-1 was diluted in Doxorubicin or compound 9, also called “tethered-doxorubicin,” to the desired ISO-1 concentration (120 μl per well 96 well dish). Then the washed THP-1 cells were added to the drug dilutions to start the experiment ˜75,000 cells per well. Test compounds were incubated 2 hrs at 37° C., then the plate was read by flow cytometry BD Canto 2 (80 μl˜25,000 cells). A population of Single Round “average” cells was selected by FSC and SSC—constituting %70 of the total cell population examined. The amount of doxorubicin uptake was evaluated for this population in the PE channel.FIG. 22shows that a known MIF tautomerase inhibitor (ISO-1) interferes with the selective uptake of doxorubicin covalently bound to a tethering moiety described herein (compound 9) but not doxorubicin.

Assay Example 4

This example illustrates an in vitro experiment using predictive models of cancer to show the altered intracellular compartmentalization and potential therapeutic utility. THP-1 monocytes were cultured in RPMI-1640 Medium, 2-mercaptoethanol to a final concentration of 0.05 mM and fetal bovine serum to a final concentration of 10%. A549 cells were cultured in F-12K Medium with fetal bovine serum to a final concentration of 10%.

Cancer cell lines were treated with 0.8 μM compound 9 or doxorubicin for 2 hrs at 37° C. The cells were then treated with the nuclear stain Hoechst trihydrochloride, trihydrate, (Invitrogen, Grand Island, N.Y.) for 30′. Images were recorded using a Zeiss LSM 510 meta confocal microscope 40×. Data shown inFIGS. 23 and 24demonstrate efficient transport of (compound 9), into the cytoplasm of treated cancer cell lines.