WO2019169017A1

WO2019169017A1 - Chemotherapeutic agents

Info

Publication number: WO2019169017A1
Application number: PCT/US2019/019874
Authority: WO
Inventors: Gerald F. SWISS; Mansour Bassiri; Richard D. Gless
Original assignee: Bioxiness Pharmaceuticals Inc
Current assignee: Bioxiness Pharmaceuticals Inc
Priority date: 2018-02-28
Filing date: 2019-02-27
Publication date: 2019-09-06
Anticipated expiration: 2020-08-28

Abstract

This invention is directed to chemotherapeutic conjugates comprising a an amino acid mimetic and gemcitabine.

Description

CHEMOTHERAPEUTIC AGENTS

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] This invention is directed to chemotherapeutic agents that block in a target specific manner the intracellular peptide synthesis of cancer cells ln particular, this invention is directed to amino acid mimetics bound to the glutamic acid residue of folic acid. This invention is further directed to pharmaceutical compositions and methods of use for the treating folate dependent tumors.

State of the Art

[0002] Rapidly dividing cancer cells consume disproportionate amounts of the sugar glucose and the amino acid glutamine. However, when the total carbon generated by new cancer cells was evaluated, only about 10 to 15 percent of the carbon found in these cells was associated with glucose whereas only about 10% was associated with glutamine. The remaining carbon found in these cells arose from amino acids absorbed disproportionally by the cancer cells. M1T News, March 7, 2016,

httpi//news.mit.edu/2016/how-cancer-cells-fuel-their-growth-

[0003] While the treatment of cancer has evolved from small molecule chemotherapy to immunotherapy, the use of chemotherapy as a frontline defense against cancer remains a critical part of any treatment regimen whether used alone or in combination with immunotherapy. However, one particular problem with many mainstays of chemotherapy are their systemic uptake rather than targeted therapy.

This has lead to toxicity, adverse side effects and, in many cases, limits on the amount of the chemotherapeutic agent that can be administered either in a single or aggregate dosing regimen.

[0004] Thus, there remains an ongoing need to define new chemotherapeutic agents ln particular, there is an ongoing need to define such agents that are

preferentially absorbed by cancer cells in a target specific manner. SUMMARY OF THE INVENTION

[0005] This invention is directed to chemotherapeutic conjugates comprising a targeting agent and a chemotherapeutic agent ln particular, the targeting agent is folic acid and the chemotherapeutic agent is an amino acid mimetic. These conjugates readily target folate dependent cancer cells whereupon they are absorbed into the intracellular domain. Once absorbed, intracellular enzymes cleave the amino acid mimetic from the conjugate and, once released, the mimetic interferes with cancer cell peptide synthesis. Such interference results in cancer cell death.

[0006] Accordingly, in one embodiment, this invention provides for compounds of the formula 1:

where n is an integer equal to zero, one or two;

p is an integer equal to zero, one, two, three, four, or five;

R¹ is selected from the group consisting of hydrogen, alkyl or substituted alkyl; R² is selected from the group of hydrogen, alkyl, substituted alkyl, aryl, substituted aiyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, cycloalkenyl, and substituted cycloalkenyl; and

or pharmaceutically acceptable salts and/or solvates thereof.

[0007] ln one of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of a compound of formula 1. [0008] ln one of its method aspects, this invention is directed to a method for treating cancer in a patient which method comprises administering to said patient an effective amount of a compound of formula 1 or an effective amount of the

pharmaceutical composition defined above.

DETAILED DESCRIPTION OF THE INVENTION

[0009] This invention is directed to compounds, compositions and methods for treating cancer with a chemotherapeutic conjugate comprising a chemotherapeutic peptide mimetic and a targeting agent covalently linked to each other.

[0010] Throughout this application, the text refers to various embodiments of the present compounds, compositions, and methods. The various embodiments described are meant to provide a variety of illustrative examples and should not be construed as descriptions of alternative species. Rather, it should be noted that the descriptions of various embodiments provided herein may be of overlapping scope. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of this invention.

1. Definitions

[0001] As used herein, the following definitions shall apply unless otherwise indicated. Further, if any term or symbol used herein is not defined as set forth below, it shall have its ordinary meaning in the art.

[0002] As used herein and in the appended claims, singular articles such as“a” and“an” and“the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language [e.g.,“such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non- claimed element as essential.

[0003] As used herein,“about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used lf there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term.

[0004] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³² and S³⁵ are thus within the scope of this invention. Procedures for inserting such labels into the compounds of this invention will be readily apparent to those skilled in the art based on the disclosure herein.

[0005] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³² and S³⁵ are thus within the scope of this invention. Procedures for inserting such labels into the compounds of this invention will be readily apparent to those skilled in the art based on the disclosure herein.

[0006] “Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CF ^CH-), n-butyl

(CH3CH2CH2CH2-), isobutyl ((CF ^CHCF -), sec-butyl ((CH₃)(CH₃CH₂)CH-), t- butyl ((CH3)3C-), n- pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CF JsCCFh-). C_x alkyl refers to an alkyl group having x number of carbon atoms.

[0007] “Substituted alkyl” refers to an alkyl group having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, amino, substituted amino, aryl, substituted aryl, aryloxy, substituted aiyloxy, carboxyl, carboxyl ester, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaiyloxy, substituted heteroaiyloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, and nitro, wherein said substituents are defined herein.

[0008] ln some embodiments the substituted alkyl groups include halogenated alkyl groups and particularly halogenated methyl groups such as trifluoromethyl, difluromethyl, fluoromethyl and the like.

[0009] “Alkoxy" refers to the group -O-alkyl wherein alkyl is defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

[0010] “Substituted alkoxy” refers to the group -0-(substituted alkyl) wherein substituted alkyl is defined herein. Preferred substituted alkyl groups in -0- (substituted alkyl) include halogenated alkyl groups and particularly halogenated methyl groups such as trifluoromethyl, difluromethyl, fluoromethyl and the like ln one embodiment, substituted alkoxy is an -oxy-alkylene-carboxyl or an oxy-alkylene- carboxyl ester. Such substituted alkoxy groups are represented by the formula -0- alkylene-COOH and -O-alkylene-carboxyl ester.

[0011] “Acyl" refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl- C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaiyl-C(O)-, substituted heteroaryl- C(O)-, heterocyclic-C(O)-, and substituted heterocyclic-C(O)-, wherein alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, aiyl, substituted aryl, heteroaiyl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein. Acyl includes the“acetyl” group

CHsCfO)-. ln some embodiments, acyl includes

[0012] “Acyloxy" refers to the group -O-acyl wherein acyl is defined herein. [0013] “Acylamino” refers to the group -acyl-amino, wherein acyl is defined herein and amino is defined herein.

[0014] “Amino” refers to the group -NH₂.

[0015] “Substituted amino” refers to the group -NR³¹R³² where R³¹ and R³² are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aiyl, cycloalkyl, substituted cycloalkyl, heteroaiyl, substituted heteroaiyl, heterocyclic, substituted heterocyclic, and wherein R³¹ and R³² are optionally joined, together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, provided that R³¹ and R³² are both not hydrogen, and wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aiyl, heteroaiyl, substituted heteroaiyl, heterocyclic, and substituted heterocyclic are as defined herein. When R³¹ is hydrogen and R³² is alkyl, the substituted amino group is sometimes referred to herein as alkylamino. When R³¹ and R³² are alkyl, the substituted amino group is sometimes referred to herein as dialkylamino. When referring to a monosubstituted amino, it is meant that either R³¹ or R³² is hydrogen but not both.

When referring to a disubstituted amino, it is meant that neither R³¹ nor R³² are hydrogen.

[0016] “Aryl" or“Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring ( e.g ., phenyl (Ph)) or multiple condensed rings ( e.g ., naphthyl or anthryl) which condensed rings may or may not be aromatic [e.g., 2- benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the point of attachment is at an aromatic carbon atom. Preferred aryl groups include phenyl and naphthyl.

[0017] “Substituted aiyl” refers to aiyl groups which are substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, amino, substituted amino, aiyl, substituted aiyl, aiyloxy, substituted aryloxy, carboxyl, carboxyl ester, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, halo, hydroxy, heteroaryl, substituted heteroaiyl, heteroaryloxy, substituted

heteroaiyloxy, heteroarylthio, substituted heteroaiylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, and nitro, wherein said substituents are defined herein.

[0018] “Aryloxy" refers to the group -O-aryl wherein aryl is defined herein.

[0019] “Substituted aiyloxy” refers to the group -0-(substituted aiyl) wherein substituted aiyl is defined herein.

[0020] “Carbonyl” refers to the divalent group -C(O)- which is equivalent to - C(=0),

[0021] “Carboxy” or“carboxyl” refers to -COOH or salts thereof.

[0022] “Carboxyl ester” or“carboxy ester” refers to the groups -C(0)0-alkyl, - C(0)0-substituted alkyl, -C(0)0-aryl, -C(0)0-substituted aryl, -C(0)0-cycloalkyl, C(0)0-substituted cycloalkyl, -C(0)0-heteroaryloxy, -C(0)0-substituted heteroaryl, - non-heterocyclic, and -C(0)0-substituted heterocyclic wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aiyl, substituted aryl, heteroaiyl, substituted heteroaiyl, heterocyclic, and substituted heterocyclic are as defined herein.

[0023] “Cyano” refers to the group -CºN

[0024] “Cycloalkyl" refers to a saturated or unsaturated but nonaromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. C_x cycloalkyl refers to a cycloalkyl group having x number of ring carbon atoms. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

One or more rings can be aiyl, heteroaryl, or heterocyclic provided that the point of attachment is through the non-aromatic, non-heterocyclic ring saturated carbocyclic ring.

[0025] “Substituted cycloalkyl” refers to a cycloalkyl group having from 1 to 5 or preferably 1 to 3 substituents selected from the group consisting of oxo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,

substituted amino, aiyl, substituted aiyl, aiyloxy, substituted aryloxy, carboxyl, carboxyl ester, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, halo, hydroxy, heteroaryl, substituted heteroaiyl, heteroaryloxy, substituted heteroaiyloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, and nitro, wherein said substituents are defined herein.

[0026] “Cycloalkyloxy” refers to -O-cycloalkyl wherein cycloalkyl is defined herein.

[0027] “Substituted cycloalkyloxy” refers to -0- (substituted cycloalkyl) wherein substituted cycloalkyl is defined herein.

[0028] “Halo” or“halogen” refers to fluoro, chloro, bromo and iodo and preferably is fluoro or chloro.

[0029] “Hydroxy” or“hydroxyl” refers to the group -OH.

[0030] “Heteroaiyl” refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring. Such heteroaiyl groups can have a single ring ( e.g ., pyridinyl or furyl) or multiple condensed rings [e.g., indolizinyl or benzothienyl) wherein the condensed rings may or may not be aromatic and/or contain a heteroatom provided that the point of attachment is through an atom of the aromatic heteroaryl group ln one embodiment, the nitrogen and/or the sulfur ring atom(s) of the heteroaiyl group are optionally oxidized to provide for the N-oxide (N®0), sulfinyl, or sulfonyl moieties. Preferred heteroaryls include 5 or 6 membered heteroaryls such as pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. ln some embodiments, preferred heteroaryls include 5- or 6-membered non-oxygen containing heteroaiyls. ln some embodiments, preferred heteroaiyls include 5- or 6-membered heteroaryls that exclude oxazoles. ln some embodiments, a nitrogen-containing heteroaryl group is attached through a nitrogen on the heteroaiyl group.

[0031] “Substituted heteroaryl” refers to heteroaiyl groups that are substituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.

[0032] “Heteroaiyloxy” refers to -O-heteroaryl and“substituted heteroaiyloxy” refers to -O-substituted heteroaiyl. [0033] “Heterocycle” or“heterocyclic” or“heterocycloalkyl” or“heterocyclyl” refers to a saturated or partially saturated, but not aromatic, group having from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatoms selected from the group consisting of nitrogen, sulfur, or oxygen. Heterocycle encompasses single ring or multiple condensed rings, including fused bridged and spiro ring systems ln fused ring systems, one or more the rings can be cycloalkyl, aiyl or heteroaiyl provided that the point of attachment is through the non-aromatic ring ln one embodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N- oxide, sulfinyl, sulfonyl moieties.

[0034] “Substituted heterocyclic” or“substituted heterocycloalkyl” or

“substituted heterocyclyl” refers to heterocyclyl groups that are substituted with from 1 to 5 or preferably 1 to 3 of the same substituents as defined for substituted cycloalkyl.

[0035] Examples of heterocycle and heteroaiyl include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4, 5, 6, 7- tetrahydrobenzo[b] thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, and tetrahydrofuranyl.

[0036] “Heterocyclyloxy” or“heterocycloalkyloxy” refer to the group -O- (heterocyclyl) wherein heterocyclyl is defined herein.

[0037] “Substituted heterocyclyloxy” or“substituted heterocycloalkyloxy” refer to the group -0-(substituted heterocyclyloxy) wherein substituted heterocyclyloxy is defined herein.

[0038] “Heterocyclylthio” or“heterocycloalkylthio” refer to the group -S- (heterocyclyl) wherein heterocyclyl is defined herein. [0039] “Substituted heterocyclylthio” or“substituted heterocycloalkylthio” refer to the group -S- (substituted heterocyclyl) wherein heterocyclyl is defined herein.

[0040] “Heteroaiylthio” refers to the group -S-(heteroaryl) wherein heteroaryl is defined herein.

[0041] “Substituted heteroarylthio” refers to the group -S-(substituted heteroaiyl) wherein substituted heteroaryl is defined herein.

[0042] “Nitro” refers to the group -N0₂.

[0043] “Oxo” refers to the atom (=0) or (-O ).

[0044] “Spiro ring systems” refers to bicyclic ring systems that have a single ring carbon atom common to both rings.

[0045] Stereoisomers of compounds (also known as optical isomers) include all chiral, d or 1, stereoisomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in this invention include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of this invention.

[0046] The compounds of this invention may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of this invention may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.

[0047] “Subject” refers to a mammal. The mammal can be a human or non human animal mammalian organism.

[0048] “Tautomer” refers to alternate forms of a compound that differ in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaiyl groups containing a ring atom attached to both a ring -NH- moiety and a ring =N- moiety such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

[0049] “Treating” or“treatment” of a disease or disorder in a subject refers to 1) preventing the disease or disorder from occurring in a subject that is predisposed or does not yet display symptoms of the disease or disorder; 2) inhibiting the disease or disorder or arresting its development; or 3) ameliorating or causing regression of the disease or disorder.

[0050] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the

functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent“alkoxycarbonylalkyl” refers to the group (alkoxy)-C(O)- (alkyl)-.

[0051] lt is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aiyl having a substituted aryl group as a substituent which is itself substituted with a substituted aiyl group, etc.) are not intended for inclusion herein ln such cases, the maximum number of such substituents is 3. That is that each of the above definitions is constrained by a limitation that, for example, substituted aryl groups are limited to -substituted aiyl-(substituted aryl) -substituted aryl.

[0052] lt is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluoro groups).

Such impermissible substitution patterns are well known to the skilled artisan.

2. Compounds of the Invention

[0053] The compounds of this invention includes compounds of the formula 1 as follows:

where

n is an integer equal to zero, one or two;

p is an integer equal to zero, one, two, three, four, or five;

each R¹ is independently selected from the group consisting of hydrogen, alkyl or substituted alkyl;

each R² is independently selected from the group of hydrogen, alkyl, substituted alkyl, aryl, substituted aiyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, cycloalkenyl, and substituted

cycloalkenyl; and

or pharmaceutically acceptable salts and/or solvates thereof.

[0064] ln one embodiment, the compounds of this invention include those of formula 11 as follows:

11 where R¹, R², n and p are as defined above as well as pharmaceutically acceptable salts and/or solvates thereof. [0065] Examples of compounds of Formula 1 include the following:

Table 1

as well as pharmaceutically acceptable salts and solvates thereof.

[0066] Examples of compounds of this invention within Formula 11 include the following:

Table 2

as well as pharmaceutically acceptable salts and solvates thereof.

[0067] Preferred compounds of this invention include formula 111 and IV:

IV

where R¹, R² and n are as defined herein.

[0054] The compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures lt will be appreciated that where typical or preferred process conditions (i.e., reaction

temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0055] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

[0056] lf the compounds of this invention contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

[0057] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as SigmaAldrich (St. Louis, Missouri, USA), Bachem (Torrance, California, USA), or Emka-Chemce. Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd’s Chemistry of Carbon Compounds, Volumes 1-5, and Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March’s

Advanced Organic Chemistry, (John Wiley, and Sons, 5^th Edition, 2001), and Larock’s Comprehensive Organic Transformations (VCH Publishers lnc., 1989).

4. Synthesis Methods for Representative Compounds of the Invention

[0058] ln one embodiment, the synthesis of the compounds of this invention involve conventional ester formation between a carboxyl group of glutamic acid or aspartic acid either alone or in an oligopeptide extending an additional of 1-5 amino acid groups where the C-terminus of the amino acid is terminated in -C(0)NHOH group. Such esterification reactions are exceptionally well known in the art as shown below in Scheme 1 using p =1 for exemplary purposes:

Scheme 1

[0059] Specifically, gemcitabine, compound 2, is combined with a dipeptide mimetic, compound 1, in an inert solvent under esterification conditions such as in the presence of an excess of DCC (dicyclohexylcarbodiimide) and DMAP (dimethylamino pyridine). As necessary, the amino group of compound 1 may need to be protected with conventional amino protecting groups such as Boc, Cbz, and the like. The reaction is typically conducted at a temperature of from about 0° to about 60°C for a period of time sufficient for substantial completion of the reaction. Typically, the reaction will be completed within 1 to 72 hours and reaction completion can be monitored by thin layer chromatography (TLC) or other conventional means such as by mass spectroscopy (MS). After the reaction is terminated, the resulting product, compound 3, is recovered and, as necessary, the amino protecting group removed by conventional methods.

Purification proceeds via methods well known in the art such as chromatography, high performance chromatography, precipitation, crystallization, and the like.

[0060] Alternative, if compounds of formula 11 are to be prepared, the reaction proceeds as above with the exception that the side chain carboxyl group is employed to form the ester bond ln addition, it is under stood that additional amino acids can be inserted between gemcitabine and the terminal amino acid mimetic by conventional amidation procedures well known in the art. These addition amino acids can be natural or unnatural amino acids. Examples of natural amino acids include all 20 naturally occurring amino acids as well as other less known amino acids such as 4- hydroxyproline, 3,4-dihydroxyphenylalanine, 5-hydroxytryptophane, and the like.

Examples of unnatural amino acids include phenylglycine, alpha- hydroxyglycine, 4- thiophenylalanine.

[0061] Amino acid mimetics include those that are peptide chain terminating compounds where the carboxyl group has been replaced with an N-hydroxyamide. These mimetics are recognized by the amino acid transferases (enzymes that

specifically add amino acids to initiate or expand upon a peptide chain) but when added, do not allow for the addition of further amino acids. This prevents the cell from properly preparing peptides and, hence, kills the cell. One preferred class of amino acid mimetics is represented by the formula:

where t is an integer equal to 1 or 2; and R’ is hydrogen or C1-C5 alkyl. See, e.g., US Patent No. 9,789,171 which is incorporated herein by reference in its entirety.

5. Pharmaceutical Compositions

[0062] ln general, the compounds of this invention will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. The actual amount of the compound of this invention, i.e., the active ingredient, will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors well know to the skilled artisan. The drug can be administered at least once a day, preferably once or twice a day.

[0063] An effective amount of such agents can readily be determined by routine experimentation, as can the most effective and convenient route of administration, and the most appropriate formulation. Various formulations and drug delivery systems are available in the art. See, e.g., Gennaro, A.R., ed. (1995) Remington’s Pharmaceutical Sciences, 18^th ed., Mack Publishing Co.

[0064] A therapeutically effective dose can be estimated initially using a variety of techniques well-known in the art. lnitial doses used in animal studies may be based on effective concentrations established in cell culture assays. Dosage ranges

appropriate for human subjects can be determined, for example, using data obtained from animal studies and cell culture assays.

[0065] An effective amount or a therapeutically effective amount or dose of an agent, e.g., a compound of this invention, refers to that amount of the agent or compound that results in amelioration of symptoms or a prolongation of survival in a subject. Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50 % of the population), and the Maximum tolerated Dose (MTD), and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 or MTD/ ED50. Agents that exhibit a high therapeutic index are preferred.

[0066] The effective amount or therapeutically effective amount is the amount of the compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Dosages particularly fall within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary within this range depending upon the dosage form employed and/or the route of administration utilized. The exact formulation, route of administration, dosage, and dosage interval should be chosen according to methods known in the art, in view of the specifics of a subject’s condition.

[0067] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to achieve the desired effects; i.e., the minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from, for example, in vitro data and animal experiments. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration ln cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0068] The amount of agent or composition administered may be dependent on a variety of factors, including the sex, age, and weight of the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.

[0069] This invention is not limited to any particular composition or

pharmaceutical carrier, as such may vary ln general, compounds of this invention will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository), or parenteral (e.g., intramuscular, intravenous or subcutaneous) administration. The preferred manner of administration is oral using a convenient daily dosage regimen that can be adjusted according to the degree of affliction. Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions. Another preferred manner for administering compounds of this invention is inhalation.

[0070] The choice of formulation depends on various factors such as the mode of drug administration and bioavailability of the drug substance. For delivery via inhalation the compound can be formulated as liquid solution, suspensions, aerosol propellants or dry powder and loaded into a suitable dispenser for administration.

There are several types of pharmaceutical inhalation devices-nebulizer inhalers, metered dose inhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices produce a stream of high velocity air that causes the therapeutic agents (which are formulated in a liquid form) to spray as a mist that is carried into the subject’s respiratory tract. MDl’s typically are formulation packaged with a compressed gas. Upon actuation, the device discharges a measured amount of therapeutic agent by compressed gas, thus affording a reliable method of administering a set amount of agent. DP1 dispenses therapeutic agents in the form of a free flowing powder that can be dispersed in the subject’s inspiratory air-stream during breathing by the device ln order to achieve a free flowing powder, the therapeutic agent is formulated with an excipient such as lactose. A measured amount of the therapeutic agent is stored in a capsule form and is dispensed with each actuation.

[0071] Pharmaceutical dosage forms of a compound of this invention are manufactured by any of the methods well-known in the art, for example, by

conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tableting, suspending, extruding, spray-drying, levigating, emulsifying, (nano/micro-)

encapsulating, entrapping, or lyophilization processes. These compositions can include one or more physiologically acceptable inactive ingredients that facilitate processing of active molecules into preparations for pharmaceutical use.

[0072] Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Patent No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability.

[0073] The compositions are comprised of, in general, a compound of this invention in combination with at least one pharmaceutically acceptable excipient.

Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the claimed compounds. Such excipient may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.

[0074] Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers,

particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols.

[0075] Compressed gases may be used to disperse a compound of this invention the in aerosol form lnert gases suitable for this purpose are nitrogen, carbon dioxide, etc. Other suitable pharmaceutical excipients and their formulations are described in Remington’s Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing

Company, 18th ed., 1990).

[0076] The compositions of this invention may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass, and rubber stoppers such as in vials. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of this invention can be formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0077] The amount of the compound in a formulation can vary within the full range employed by those skilled in the art. Typically, the formulation will contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt % of a compound of this invention based on the total formulation, with the balance being one or more suitable pharmaceutical excipients. Preferably, the compound is present at a level of about 1-80 wt %. Representative pharmaceutical formulations are described below.

6. Formulation Examples

[0078] The following are representative pharmaceutical formulations containing a compound of Formula 1, 11, 111, and/or IV.

Formulation Example 1 - Tablet formulation

[0079] The following ingredients are mixed intimately and pressed into single scored tablets.

Quantity per

Ingredient_ tablet, mg compound of this invention 400

Cornstarch 50

croscarmellose sodium 25

Lactose 120

magnesium stearate_ 5

Formulation Example 2 - Capsule formulation

[0080] The following ingredients are mixed intimately and loaded into a hard shell gelatin capsule.

Quantity per lngredient capsule, mg compound of this invention 200 lactose, spray-dried 148 magnesium stearate 2

Formulation Example 3— Suspension formulation

[0081] The following ingredients are mixed to form a suspension for oral administration.

Ingredient_ Amount compound of this invention 1.0 g fumaric acid 0.5 g sodium chloride 2.0 g methyl paraben 0.15 g propyl paraben 0.05 g granulated sugar 25.0 g sorbitol (70% solution) 13.00 g

Veegum K (Vanderbilt Co.) 1.0 g

Flavoring 0.035 mL

Colorings 0.5 mg distilled water_ q.s. to 100 mL

Formulation Example 4 - lnjectable formulation

[0082] The following ingredients are mixed to form an injectable formulation. lngredient Amount compound of this invention 0.2 mg-20 mg sodium acetate buffer solution, 0.4 M 2.0 mL

HC1 (IN) or NaOH (IN) q.s. to suitable pH water

., sterile) q.s. to 20 mL

Formulation Example 5— Suppository Formulation [0083] A suppository of total weight 2.5 g is prepared by mixing the compound of this invention with Witepsol® H-15 (triglycerides of saturated vegetable fatty acid; Riches-Nelson, lnc., New York), and has the following composition:

Ingredient _ Amount

Compound of this invention 500 mg

Witepsol® H-15 _ balance

7. Methods

[0084] The methods of this invention include administration of an effective amount of a compound of formula 1, 11, 111 or IV or a mixture thereof to a patient suffering from a cancer for which gemcitabine is typically prescribed. Such cancers include, but are not limited to, pancreatic, ovarian and breast cancers.

[0085] Treatment typically proceeds by administration of an effective amount of a compound of formula 1, 11, 111 or IV or a mixture thereof (by itself or preferably in a pharmaceutical composition such as an intravenous formulation) to the patient for a sufficient period of time to effect treatment of the cancer ln one embodiment, the treatment protocol includes three weeks of treatment as per above followed by a week without treatment.

[0086] The amount of the compound administered depends, again, on the age, weight, sex, and condition (among other factors) of the patient and the extent of the cancer. Such factors are well known to attending clinicians ln one embodiment, the amount of compound administered is from about 0.1 mg/kg/day to about 100 mg/kg/day and preferably from about 0.5 mg/kg/day to about 50 mg/kg/day. As is well recognized in the art, the attending clinician may increase or decrease the dose depending upon the response achieved by the patient so treated.

[0087] The methods of this invention may be combined with other

chemotherapeutic drugs, with immunotherapy including CAR-T-cells, NK cells, anti- fugetactic agents such as NK-92 (see, US Patent No. 9,789,171 which is incorporated herein by reference in its entirety) and the like. [0088] Without being limited to any theory, this invention entails covalent ester linkage of antimicrobial peptide mimetics and gemcitabine, a known chemotherapeutic agent. Once internalized into the cancer either by the gemcitabine pathway and/or the oligopeptide pathway, hydrolysis of the ester bond occurs spontaneously. The resulting oligopeptide mimetic acts as an antimicrobial agent that invades the intracellular bacteria and kills it; whereas the gemcitabine is then allowed to effective treat the cancer.

8. Examples

[0089] This invention is further understood by reference to the following examples, which are intended to be purely exemplary of this invention. Any methods that are functionally equivalent are within the scope of this invention. Various modifications of this invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

Example 1 Synthesis of glutamic acid methionine mimetic conjugate

[0090] ln an inert diluent such as DMF, approximately 1 equivalent of compound 4, a commercially available reagent from SigmaAldrich, St. Louis, Missouri, USA, is combined with approximately 1 equivalent of N-hydroxyamide of methionine in the presence of an excess of dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP). The reaction is maintained at room temperature for approximately 24 hours. The resulting product, compound 6, is recovered by conventional methods including column chromatography. Subsequent removal of the benzyl and Cbz groups by conventional methods provides for compound 7 as shown below.

Example 2 Synthesis of Gemcitabine esters

[0091] ln an inert diluent such as DMF, approximately 1 equivalent of compound 2 is combined with approximately 1 equivalent of compound 8 in the presence of an excess of dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP). The reaction is maintained at room temperature for approximately 24 hours. The resulting product, compound 9, is recovered by conventional methods including column chromatography.

[0092] 10 micrograms of compound 9 dissolved in 10 microliters of distilled water is dropwise added to 200 microliters of a suitable growth medium containing 1 x 10⁶ pancreatic cancer cells (Pane 02.27, ATTC CRL-2549). The growth medium is maintained at 37°C and after 8 hours, the cells are evaluated as remaining viable or not. Upon examination, it is contemplated that a majority of these cells will no longer be viable.

[0093] While some embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of this invention or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof as set forth herein. Each aspect and

embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[0094] This invention is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of this invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art.

Functionally equivalent methods within the scope of this invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims lt is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary lt is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of this invention indicated only by the appended claims, definitions therein and any equivalents thereof. [0095] The embodiments, illustratively described herein, may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms“comprising,”“including,”“containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase“consisting essentially of will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase“consisting of” excludes any element not specified.

[0096] ln addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of this invention. This includes the generic description of this invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0097] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc.

As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”“at least,”“greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. [0098] All publications, patent applications, issued patents, and other documents (for example, journals, articles and/or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0099] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.

Claims

WHA T IS CLAIMED IS:

1. A compound according to formula 1:

where

n is an integer equal to zero, one or two;

p is an integer equal to zero, one, two, three, four, or five;

R¹ is selected from the group consisting of hydrogen, alkyl or substituted alkyl;

R² is selected from the group of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, cycloalkenyl, and substituted cycloalkenyl; and

or pharmaceutically acceptable salts and/or solvates thereof.

2. A pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of a compound of claim 1.

3. A method for treating cancer in a patient which method comprises administering to said patient an effective amount of a compound of claim 1.

4. A method for treating cancer in a patient which method comprises administering to said patient an effective amount of the pharmaceutical composition of claim 2.

5. A compound according to formula 11:

where

n is an integer equal to zero, one or two;

p is an integer equal to zero, one, two, three, four, or five;

R² is selected from the group of hydrogen, alkyl, substituted alkyl, aryl, substituted aiyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, cycloalkenyl, and substituted cycloalkenyl; and

or pharmaceutically acceptable salts and/or solvates thereof.

6. A pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of a compound of claim 5.

7. A method for treating cancer in a patient which method comprises administering to said patient an effective amount of a compound of claim 5.

8. A method for treating cancer in a patient which method comprises administering to said patient an effective amount of the pharmaceutical composition of claim 6.