WO2011011865A1

WO2011011865A1 - Anticancer agents based on amino acid derivatives

Info

Publication number: WO2011011865A1
Application number: PCT/CA2010/001125
Authority: WO
Inventors: Gervais Berube; Caroline Descoteaux; Eric Asselin
Original assignee: 3R VALO SEC
Current assignee: 3R VALO SEC
Priority date: 2009-07-29
Filing date: 2010-07-16
Publication date: 2011-02-03
Anticipated expiration: 2012-01-29

Abstract

The present invention provides compounds of formula (I): in which T is L or -A-NH-C(O)-L-; L is -(CH₂)n- or -(CH₂)p-Z-; A is -(CH₂)m- or an alkyl component of a naturally occurring amino acid; R is -CO₂R₁, -CH₂OH, -C(O)NH(hydroxyphenyl) or C(S)NH(hydroxyphenyl); R₁ is H or is C₁-C₁₂ alkyl; X is -OH, OSO₂R₂, -Cl, -Br, or -I; R₂ is C₁-C₁₂ alkyl or -CF₃; Z is phenyl or naphthyl; m is an integer having a value of 1 to 20; n is an integer having a value of 1 to 20; and p is an integer having a value of 1 to 20 or an enantiomer, diastereoisomer, racemic mixture, pharmaceutically acceptable salt, solvate or prodrug thereof. These compounds can be useful as anticancer agents.

Description

ANTICANCER AGENTS BASED ON AMINO ACID DERIVATIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority on U.S. provisional application

No. 60/229,632 filed on July 29, 2009, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] This disclosure relates to the field of amino acid derivatives as active agents. More particularly, this dicslosure relates to amino acid derivatives which are usefull as anticancer agents for treating cancer such as melanoma, breast cancer, uterine cancer and ovarian cancer.

BACKGROUND OFTHE DISCLOSURE

[0003] There are several types of polyfunctional alkylating agents. The three major clinically useful classes have a structure containing a bis(chloroethyl)amine, an aziridine, or a nitrosourea moiety.¹ The general and characteristic structural features of the alkylating moiety are presented in scheme 1.

Bis(chloroethyl)amine moiety Aziridine moiety Nⁱtrosourea moⁱety

Scheme 1: Structures of major classes of alkylating agents [0004] There are four bis(chloroethyl)amines used clinically; mechlorethamine (Mustargen ) ' , cyclophosphamide (Cytoxan ) ' , melphalan (Alkeran ) ' and chlorambucil (Leukeran ) ' (scheme 2). Mechlorethamine is effective in Hodgkin's disease.⁴ Cyclophosphamide is active orally which is a great advantage over other alkylating agents. It is active against multiple myeloma, chronic lymphocytic leukemia, and acute leukemia in children.⁴ Melphalan is used against multiple myeloma and ovarian cancers.⁴ Chlorambucil is a slow acting nitrogen mustard which can be administered orally. It is mainly used in chronic lymphocytic leukemia and primary macro globulinemia. It is also useful in treating lymphosarcoma and Hodgkin's disease.⁴ All nitrogen mustard anticancer drugs can be used as a single agent or in combination therapy with other antineoplastic agents.³

M echloTethamine Cyclophosphamide

Melphalan Chlorambucil

Scheme 2: Structures of four bis(chloroethyl)amines used clinically

[0005] In general, alkylating agents are both mutagenic and genotoxic.⁵ The alkylating agents form adducts with DNA, inhibiting protein synthesis in fast replicating malignant cells. However, they also form adducts with RNA and protein and this is likely to contribute to the overall cytotoxicity.⁵ There are several DNA repair mechanisms which protects the cells from the lesions caused by alkylating agents.⁵

[0006] The main side effects of alkylating agents are bone marrow suppression, anaemia and weakened immune systems.⁶ SUMMARY OF THE DISCLOSURE

[0007] According to one aspect, there are included compounds of formula I

(I)

wherein

T is L or -A-NH-C(O)-L-;

L is -(CH₂)_H- or -(CH₂)_P-Z- ;

A is -(CH₂)_m- or an alkyl component of a naturally occurring amino acid

R is -CO₂R₁, -CH₂OH,

Ri is H or is C₁-Ci₂ alkyl;

X is -OH, -OSO₂R₂, -Cl, -Br, or -I;

R₂ is Ci-C₁₂ alkyl or -CF₃;

Z is phenyl or naphthyl;

m is an integer having a value of 1 to 20;

n is an integer having a value of 1 to 20; and

p is an integer having a value of 1 to 20, or enantiomers, diastereoisomers or tautomers of the compounds of formula I in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula I or enantiomers, diastereoisomers or tautomers of the compounds of formula I in any ratio, or mixtures of any of the above.

[0008] According to another aspect, there is included a composition comprising a pharmaceutically acceptable carrier and at least one compound of the present disclosure.

[0009] According to another aspect, there is included a method for treating cancer or at least one cancer chosen from breast cancer, uterine cancer and ovarian cancer, said method comprising administering to a subject in need thereof an effective amount of at least one compound of the present disclosure.

[0010] According to another aspect, there is included a method for reducing the risks of developing cancer or for reducing the risk of developingat least one cancer in a subject, the cancer being, for example, chosen from melanoma, breast cancer, uterine cancer and ovarian cancer, said method comprising administering to the subject an effective amount of at least one compound of the present disclosure.

[0011] According to another aspect, there is included a method for inhibiting cancer cell growth, the method comprising administering to a subject in need thereof an effective amount of at least one compound of the present disclosure. For example, the cancer can be chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0012] According to another aspect, there is included the use of at least one compound of the present disclosure for treating cancer or for treating at least one cancer chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0013] According to another aspect, there is included the use of at least one compound of the present disclosure for reducing the risks of developing cancer or for reducing the risks of developing at least one cancer chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0014] According to another aspect, there is included the use of at least one compound of the present disclosure in the manufacture of a medicament for treating cancer or for treating at least one cancer chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0015] According to another aspect, there is included the use of at least one compound of the present disclosure in the manufacture of a medicament for reducing the risks of developing cancer or for reducing the risks of developing at least one cancer chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0016] According to another aspect, there is included the use of at least one compound of the present disclosure for inhibiting cancer cell growth. For example, the cancer can be chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0017] According to another aspect, there is included the use of at least one compound of the present disclosure in the manufacture of a medicament for inhibiting cancer cell growth. For example, the cancer can be chosen from melanoma, breast cancer, uterine cancer and ovarian cancer.

[0018] According to another aspect, there is provided a method for preparing a compound of formula I :

(I) wherein

T is L or -A-NH-C(O)-L-;

L is -(CH₂)_n- or -(CH₂)_P-Z- ;

A is -(CH₂)_In- or an alkyl component of a naturally occurring amino acid

R is -CO₂R₁₁ -CH₂OH,

R₁ is H or is C₁-C₁₂ alkyl;

X is -OH, -OSO₂R₂, -Cl, -Br, or -I;

R₂ is C₁-C₁₂ alkyl or -CF₃;

Z is phenyl or naphthyl;

m is an integer having a value of 1 to 20;

n is an integer having a value of 1 to 20; and

p is an integer having a value of 1 to 20,

or an enantiomer, diastereoisomer, tautomer or racemic mixture thereof. said method comprising :

(i) reacting a compound of formula (VIII) or a derivative thereof and a compound of formula (IX) or a derivative thereof:

(VIII) (IX)

wherein

E is -NH₂, or -NH₃ ⁺X₁ ^";

Xf is a suitable counter anion;

R₄Is -OH, Cl, or -Q(C=O)R₅; R₅ is a C₁-C₅ alkyl; and

R, T, and X are as previously defined for formula I,

under conditions to form said compound of formula I,

or

(ii) reacting a compound of formula (X) or a derivative thereof and a compound of formula (XI) or a derivative thereof:

wherein

E iS -NH₂, Or -NH₃ ⁺X₁ ^" ;

Xf is a suitable counter anion;

R₄ is -OH, Cl, or -O(C=O)R_5;

R₅ is a Ci-C₅ alkyl; and

R, A, L and X are as previously defined for formula I,

under conditions to form said compound of formula I. DETAILED DESCRIPTION OF THE DISCLOSURE

[0019] The term a "therapeutically effective amount", "effective amount" or a

"sufficient amount " of a compound of the present disclosure is a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount" or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating cancer, for example, it is an amount of the compound sufficient to achieve such treatment of the cancer as compared to the response obtained without administration of the compound. The amount of a given compound of the present disclosure that will correspond to an effective amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a "therapeutically effective amount" , "effective amount" or a "sufficient amount" of a compound of the present disclosure is an amount which inhibits, suppresses or reduces a cancer (e.g., as determined by clinical symptoms or the amount of cancerous cells) in a subject as compared to a control.

[0020] The term "subject" as used herein includes all members of the animal kingdom including human. According to one embodiment, the subject is a human.

[0021] The term "alkyl" as used herein means straight and/or branched chain, saturated alkyl groups containing from one to n carbon atoms and includes (depending on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t- butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl and the like, wherein n is the maximum number of carbon atoms in the group.

[0022] The expression "an alkyl component of a naturally occurring amino acid" as used herein refers to the portion of a naturally occurring amino acid that is comprised between the carbon of the carbonyl group of the amino acid and the nitrogen atom of the amino acid. [0023] The expression "compound(s) of the present disclosure" as used in the present document refers to compounds of formulae I, II, Ha, III, Ilia, IV, IVa, V, Va, VI, Via, VII, Vila, VIIb, and VIIc, presented in the present disclosure, isomers thereof, such as stereoisomers (for example, enantiomers, diastereoisomers, including racemic mixtures) or tautomers, or to pharmaceutically acceptable salts, solvates, hydrates and/or prodrugs of these compounds, isomers of these latter compounds, or racemic mixtures of these latter compounds. The expression "compound(s) of the present disclosure" also refers to mixtures of the various compounds or variants mentioned in the present paragraph.

[0024] It is to be clear that the present disclosure includes isomers, racemic mixtures, pharmaceutically acceptable salts, solvates, hydrates and prodrugs of compounds described therein and mixtures comprising two or more of such compounds.

[0025] The compounds of the disclosure may have at least one asymmetric centre. Where the compounds according to the present document possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present disclosure. It is to be understood that while the stereochemistry of the compounds of the present disclosure may be as provided for in any given compound listed herein, such compounds of the disclosure may also contain certain amounts (for example less than 30%, less than 20%, less than 10%, or less than 5%) of compounds of the present disclosure having alternate stereochemistry.

[0026] The term "suitable", as in for example, "suitable counter anion" or

"suitable reaction conditions" means that the selection of the particular group or conditions would depend on the specific synthetic manipulation to be performed and the identity of the molecule but the selection would be well within the skill of a person trained in the art. All process steps described herein are to be conducted under conditions suitable to provide the product shown. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so. [0027] The expression "pharmaceutically acceptable" means compatible with the treatment of subjects such as animals or humans.

[0028] The expression "pharmaceutically acceptable salt" means an acid addition salt or basic addition salt which is suitable for or compatible with the treatment of subjects such as animals or humans.

[0029] The expression "pharmaceutically acceptable acid addition salt" as used herein means any non-toxic organic or inorganic salt of any compound of the present disclosure, or any of its intermediates. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the present disclosure are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g. oxalates, may be used, for example, in the isolation of the compounds of the present disclosure, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt, hi embodiments of the present disclosure, the pharmaceutically acceptable acid addition salt is the hydrochloride salt.

[0030] The term "pharmaceutically acceptable basic addition salt" as used herein means any non-toxic organic or inorganic base addition salt of any acid compound of the invention, or any of its intermediates. Acidic compounds of the invention that may form a basic addition salt include, for example, where R is CO₂H. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art. Other non- pharmaceutically acceptable basic addition salts, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0031] The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.

[0032] The term "solvate" as used herein means a compound of the present disclosure, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate". The formation of solvates of the compounds of the present disclosure will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.

[0033] Compounds of the present disclosure include prodrugs. In general, such prodrugs will be functional derivatives of these compounds which are readily convertible in vivo into the compound from which it is notionally derived. Prodrugs of the compounds of the present disclosure may be conventional esters formed with available hydroxy, or amino group. For example, an available OH or nitrogen in a compound of the present disclosure may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (Cg-C₂₄) esters, acyloxymethyl esters, carbamates and amino acid esters, hi certain instances, the prodrugs of the compounds of the present disclosure are those in which one or more of the hydroxy groups in the compounds is masked as groups which can be converted to hydroxy groups in vivo. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985.

[0034] Compounds of the present disclosure include radiolabeled forms, for example, compounds labeled by incorporation within the structure ²H, ³H, ¹⁴C, ¹⁵N, or a radioactive halogen such as ¹²⁵I. A radiolabeled compound of the compounds of the present disclosure may be prepared using standard methods known in the art.

[0035] As used herein, and as well understood in the art, "treatment" or

"treating" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" or "treating" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0036] "Palliating" a disease or disorder, means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.

[0037] The expression "derivative thereof as used herein when referring to a compound of formula (VIII) means a derivative of the compound of formula (VIII) that has a similar reactivity and that could be used as an alternative to the compound of formula (VIII) in order to obtain the same desired result i.e. formation of a compound of formula (I) when reacting this derivative with a compound of formula (IX) or a derivative thereof under conditions to form the compound of formula I.

[0038] The expression "derivative thereof as used herein when referring to a compound of formula (IX) means a derivative of the compound of formula (IX) that has a similar reactivity and that could be used as an alternative to the compound of formula (IX) in order to obtain the same desired result i.e. formation of a compound of formula (I) when reacting this derivative with a compound of formula (VIII) or a derivative thereof under conditions to form the compound of formula I.

[0039] The expression "derivative thereof as used herein when referring to a compound of formula (X) means a derivative of the compound of formula (X) that has a similar reactivity and that could be used as an alternative to the compound of formula (X) in order to obtain the same desired result i.e. formation of a compound of formula (I) when reacting this derivative with a compound of formula (XI) or a derivative thereof under conditions to form the compound of formula I.

[0040] The expression "derivative thereof as used herein when referring to a compound of formula (XI) means a derivative of the compound of formula (XI) that has a similar reactivity and that could be used as an alternative to the compound of formula (XI) in order to obtain the same desired result i.e. formation of a compound of formula (I) when reacting this derivative with a compound of formula (X) or a derivative thereof under conditions to form the compound of formula I.

[0041] The expression "coupling reagent" as used herein refers to a reagent effective for coupling together an amine or a derivative thereof and an organic acid or a derivative thereof in order to obtain an amide. Non-limitative examples of such coupling reagents include DCC (dicyclohexylcarbodiimide), CDI (N,N'- carbonyldiimidazole), BOP (benzotriazole- 1 -yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate), DEPBT (3-(diethoxy-phosphoryloxy)-3H- benzo[d][l,2,3] triazin-4-one), EDCHCl (l-ethyl-3-(3- dimethyllaminopropyl)carbodiimide hydrochloride), HATU (2-(lH-7- azabenzotriazol- 1 -yl)~ 1,1,3 ,3 -tetramethyl uronium hexafluorophosphate methanaminium), HOBt (1-hydroxybenzotriazole or N-hydroxybenzotriazole), HBTU (O-benzotriazole-N,N,N' ,N'-tetramethyl-uronium-hexafluoro-phosphate), HOAt (1 - hydroxy-7-azabenzotriazole), HOOBt (hydroxy-3,4-dihydro-4-oxo-l ,2,3- benzotriazine), HCTU (lH-benzotriazolium l-[bis(dimethylamino)methylene]-5- chloro-hexafluorophosphate (l-),3 -oxide), Cl-HOBt (6-chloro-l- hydroxybenzotriazole), PyBOP (benzotriazol-1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromo-tris-pyrrolidino phosphoniumhexafluorophosphate), TATU, TBTU (O-(benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate), TCTU, TDBTU, TSTU, 4,5-dicyanoimidazole, etc.

[0042] hi understanding the scope of the present disclosure, the term

"comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0043] In an embodiment of the present disclosure, there are included compounds of formula II :

(H) wherein:

R is -CO₂Ri or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

X is -OH, -OSO₂R₂ or -Cl;

R₁ is chosen from H, straight alkyl group having from 1 to 5 carbon atoms, and branched alkyl group having from 3 to 5 carbon atoms;

R₂ is -CH₃ or -CF₃; and

n is 1, 2, 3, 4, 5, 6, 7 or 8,

or enantiomers, diastereoisomers or tautomers of the compounds of formula II in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula II or enantiomers, diastereoisomers or tautomers of the compounds of formula II in any ratio, or mixtures of any of the above.

[0044] In another embodiment of the present disclosure, there are included compounds of formula Ha :

(Ha) wherein:

R is -CO₂R₁ or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂),,-;

A is -(CH₂)_m-;

X is -OH, -OSO₂R₂ or -Cl;

Ri is chosen from H, straight alkyl group having from 1 to 5 carbon atoms, and branched alkyl group having from 3 to 5 carbon atoms;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or enantiomers, diastereoisomers or tautomers of the compounds of formula Ha in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of forrmila Ha or enantiomers, diastereoisomers or tautomers of the compounds of formula Ha in any ratio, or mixtures of any of the above.

[0045] In a further embodiment of the present disclosure, there are included compounds of formula III :

wherein:

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂),,-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃; and

n is 1, 2, 3, 4, 5, 6, 7 or 8,

or enantiomers or tautomers of the compounds of formula III in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula III or enantiomers or tautomers of the compounds of formula III in any ratio, or mixtures of any of the above.

[0046] In still a further embodiment of the present disclosure, there are included compounds of formula Ilia :

(Ilia) wherein:

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂),,-;

A is -(CH₂)_m-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or enantiomers or tautomers of the compounds of formula Ilia in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula

Ilia or enantiomers or tautomers of the compounds of formula IHa in any ratio, or mixtures of any of the above.

[0047] In yet a further embodiment of the present disclosure, there are included compounds of formula IV :

(IV) wherein:

R is -CO₂R₁ or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃; and

n is 1, 2, 3, 4, 5, 6, 7 or 8,

or enantiomers, diastereoisomers or tautomers of the compounds of formula IV in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula IV or enantiomers, diastereoisomers or tautomers of the compounds of formula IV in any ratio, or mixtures of any of the above.

[0048] In another embodiment of the present disclosure, there are included compounds of formula IVa :

(IVa)

wherein:

R is -CO₂R₁ or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

A is -(CH₂)_m-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or enantiomers, diastereoisomers or tautomers of the compounds of formula IVa in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula IVa or enantiomers, diastereoisomers or tautomers of the compounds of formula IVa in any ratio, or mixtures of any of the above.

[0049] In another embodiment of the present disclosure, there are included compounds of formula V :

(V) wherein:

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃; and

n is i, 2, 3, 4, 5, 6, 7 or 8,

or enantiomers or tautomers of the compounds of formula V in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula

V or enantiomers or tautomers of the compounds of formula V in any ratio, or mixtures of any of the above.

[0050] In another embodiment of the present disclosure, there are included compounds of formula Va :

(Va) wherein:

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂)_n-;

A is -(CHa)_1n-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or enantiomers or tautomers of the compounds of formula Va in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula

Va or enantiomers or tautomers of the compounds of formula Va in any ratio, or mixtures of any of the above.

[0051] In another embodiment of the present disclosure, there are included compounds of formula VI :

(VI) wherein L is -CH₂CH₂CH₂-C₆H₄-,

or enantiomers or tautomers of the compounds of formula VI in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula VI or enantiomers or tautomers of the compounds of formula VI in any ratio, or mixtures of any of the above. [0052] In another embodiment of the present disclosure, there are included compounds of formula Via :

(Via) wherein L is -CH₂CH₂CH₂-C₆H₄-, A is -(CH₂)_m-, and m is 5 or 10, or enantiomers or tautomers of the compounds of formula Via in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula Via or enantiomers or tautomers of the compounds of formula Via in any ratio, or mixtures of any of the above.

[0053] In another embodiment of the present disclosure, there are included compounds of formula VII :

(VII)

wherein L is -CH₂CH₂CH₂-C₆H₄-, or enantiomers or tautomers of the compounds of formula VII in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula VII or enantiomers or tautomers of the compounds of formula VII in any ratio, or mixtures of any of the above.

[0054] In another embodiment of the present disclosure, there are included compounds of formula Vila :

(Vila) wherein L is -CH₂CH₂CH₂-C₆H₄-, A is -(CH₂)_m-, and m is 5 or 10, or enantiomers or tautomers of the compounds of formula Vila in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula Vila or enantiomers or tautomers of the compounds of formula Vila in any ratio, or mixtures of any of the above.

[0055] In another embodiment of the present disclosure, there are included compounds of formula VIIb :

(VIIb)

wherein L is -CH₂CH₂CH₂-C₆H₄-, or enantiomers or tautomers of the compounds of formula VIIb in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula VIIb or enantiomers or tautomers of the compounds of formula VIIb in any ratio, or mixtures of any of the above.

[0056] In another embodiment of the present disclosure, there are included compounds of formula VIIc :

(VIIc) wherein L is -CH₂CH₂CH₂-C₆H₄-, A is -(CH₂)_m-, and m is 5 or 10,

or enantiomers or tautomers of the compounds of formula VIIc in any ratio, or pharmaceutically acceptable salts, solvates or prodrugs of the compounds of formula VIIc or enantiomers or tautomers of the compounds of formula VIIc in any ratio, or mixtures of any of the above.

[0057] In another embodiment of the present disclosure, A is the alkyl component of a hydrophobic naturally occurring amino acid.

[0058] In another embodiment of the present disclosure, the naturally occuring amino acid is chosen from alanine, phenylalanine, leucine, isoleucine, tryptophan, valine and proline.

[0059] In another embodiment of the present disclosure, non natural amino acids are also used to make derivatives of formula Ha, Ilia, IVa, Va, Via, Vila, VIIb, and VIIc. [0060] In another embodiment of the present disclosure, A is chosen from

[0061] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, X is Cl and R is -CO₂CH₃.

[0062] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, X is Cl and R is -CH₂OH.

[0063] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, A is -(CH₂V-, X is -Cl and R is^■ CO₂CH₃.

[0064] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, A is -(CH₂V-, m is 5 or 10, X is -Cl and R is -CO₂CH₃.

[0065] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, A is -(CH₂V-, X is -Cl and R is -CH₂OH.

[0066] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, A is -(CH₂V-, m is 5 or 10, X is -Cl and R is -CH₂OH.

[0067] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄- and X is Cl.

[0068] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, A is -(CH₂)_m-, and X is Cl. [0069] In another embodiment of the present disclosure, L is -CH₂CH₂CH₂-

C₆H₄-, A is -(CH₂J_m-, m is 5 or 10, and X is Cl.

[0070] In another embodiment of the present disclosure, the conditions to form a compound of formula I comprise the presence of at least one coupling reagent and a base.

[0071] In another embodiment of the present disclosure, the least one coupling reagent is chosen from DCC (dicyclohexylcarbodiimide), CDI (N₅N'- carbonyldiimidazole), BOP (benzotriazole- 1 -yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate), DEPBT (3-(diethoxy-phosphoryloxy)-3H- benzo[d][l,2,3] triazin-4-one), EDCHCl (l-ethyl-3-(3- dimethyllaminopropyl)carbodiimide hydrochloride), HATU (2-(lH-7- azabenzotriazol- 1 -yl)~ 1,1,3 ,3 -tetramethyl uronium hexafluorophosphate methanaminium), HOBt (1-hydroxybenzotriazole or N-hydroxybenzotriazole), HBTU (O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate), HOAt (1- hydroxy-7-azabenzotriazole), HOOBt (hydroxy-3,4-dihydro-4-oxo-l ,2,3- benzotriazine), HCTU (lH-benzotriazolium l-[bis(dimethylamino)methylene]-5- chloro-hexafluorophosphate (l-),3-oxide), Cl-HOBt (6-chloro-l- hydroxybenzotriazole), PyBOP (benzotriazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromo-tris-pyrrolidino phosphoniumhexafluorophosphate), TATU, TBTU (O-(benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate), TCTU, TDBTU, TSTU, and 4,5- dicyanoimidazole.

[0072] In another embodiment of the present disclosure, the conditions to form a compound of formula I comprise the presence of dicyclohexylcarbodiimide and N-hydroxybenzotriazole.

[0073] In another embodiment of the present disclosure, Xf is chosen from,

Cl-, Br-, F-, I-, CF₃CO₂ ^", and CH₃CO₂-.

[0074] Schemes 3 to 5 represent examples of synthetic routes used for the preparation of the compounds of the present disclosure. The reaction conditions of each step are presented directly in the schemes.

[0075] Scheme 3 illustrates the preparation of first generation tyrosine- nitrogen mustard hybrids of formulae types II, Ha, IV and IVa. [0076] As shown in scheme 3, L-tyrosine (1) (or D-tyrosine) was treated with thionyl chloride in methanol to give derivative 2 (100%). Compound 2 was transformed into tyrosine-nitrogen mustard 3 (81%) upon treatment with chlorambucil (CHL), 1-hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC) and triethylamine (TEA) in dimethylformamide (DMF). Compound 3 was reduced to L- tyrosinol-nitrogen mustard derivative 4 with lithium borohydride (LiBH₄) in dry diethylether (Et₂O) with 88% yield.

[0077] 6- Amino caproic acid and 11 -amino undecanoic acid were protected with a t-butyloxycarbonyl group (Boc) upon treatment with Boc-ON™ and TEA in a mixture of dioxane and water. Note: Other hydrophobic amino acids (natural and non- natural) can be protected and used in the same manner as presented herein. The Boc- amino acids (Boc-aa) were then coupled with derivative 2 upon treatment with HOBt, DCC, TEA in DMF to give compounds 5 (m = 5 or 10) with 96% yield. Then, the Boc protective group was removed upon treatment with trifluoroacetic acid (TFA) in dichloromethane (DCM). The reaction mixture was evaporated and the intermediate was immediately coupled with chlorambucil as described above for the synthesis of derivative 3 to give tyrosine-nitrogen mustards 6 (m = 5 or 10) with 60% yield. Finally, reduction of 6 with LiBH₄ gave the tyrosinol-nitrogen mustards 7 (m = 5 or 10) with 57% yield. The synthesis presented in scheme 3 shows the feasibility of synthesis of the tyrosine-nitrogen mustard hybrids and could give easily access to more sophisticated hybrids. For example, one could easily use commercially available L-rø-tyrosine, D-m-tyrosine or DL-o-tyrosine as well as other known tyrosine derivatives.

Scheme 3. Synthesis of tyrosine-nitrogen mustard hybrids first generation.

I

\^^LI

Boc-aa, m = 5 or 10 (97%)

performed with D-p-, DL-O- and L-m- the starting material.

Reagents: (a) MeOH, SOCl₂. Δ (100%); (b) Chlorambucil, HOBt, DCC, Et₃N, DMF (81%); (c) LiBH₄ Et₂O, O⁰C to 22⁰C (4, 88%; 7, 57%); (d) Boc-ON, 1,4-dioxane, Et₃N, H₂O (97%); (e) BocNH(CH₂)_mCO₂H, HOBt, DCC, Et₃N, DMF (96%); (f) TFA, CH₂Cl₂ 10 min, (100%).

[0078] In order to verify if some epimerization occurred during the formation of derivatives S-2 or R-2, the corresponding Mosher's amides were synthesized as illustrated in scheme 4. Thus, independent treatment of S-2 and R-2 with commercially available ,S-(+)-α-methoxyphenyl acetic acid, HOBt, DCC, TEA in DMF gave the corresponding amides S,S-8 and R₁S-S with about 70% yield. Proton nuclear magnetic resonance (¹H-NMR) spectroscopic analysis of the two diastereoisomers showed no trace of epimerization in the final products. Further confirmation of this was done by the preparation of a 1 :2 mixture of S, S-S and R,S-S which shows by ¹H-NMR many distinct signals assigned to each specific diastereoisomers. This confirms that no epimerization occurred during the first step of the synthesis of the tyrosine-nitrogen mustard hybrids described above.

Scheme 4. Synthesis of diastereoisomeric Mosher's amide derivatives.

L-Tyrosine (S-I) S-2 (96%) S.S-S (₅4%)

D-Tyrosine (R-I)

Reagents: (a) MeOH, SOCl₂, Δ (96%); (b) 5-(+)-α-Methoxyphenyl acetic acid, HOBt, DCC, Et₃N, DMF (₅4-82%)

[0079] Scheme 5A illustrates the preparation of second generation tyrosine- nitrogen mustard hybrids of formulae types III, Ilia, V, Va, V, Va, VII, Vila, VIIb, and VIIc.

[0080] As shown in scheme 5A, commercially available iV-Boc-L-tyrosine (9) is coupled with aminophenol (o-, m- and p-) upon treatment with HOBt, DCC in DMF to give derivative 10 with 96% yield. This compound was readily deprotected and coupled with chlorambucil using standard reaction conditions (see scheme 5A for the reactions conditions) to give derivative 11 with 44% yield. Similarly, derivative 10 was deprotected with TFA to give trifluoroacetic ammonium salt intermediate 12 which was easily coupled with N-Boc-6-amino caproic acid or iV-Boc- 11 -amino undecanoic acid (Boc-aa, m = 5 or 10) to give derivative 13 with 46% yield. The final tyro sine-nitrogen mustard hybrids 15 were obtained in a two-step reaction sequence. First, derivative 13 was deprotected with TFA in dichloromethane to give the trifluoroacetic ammonium salt intermediate 14. Secondly, derivative 14 was coupled with chlorambucil using HOBt, DCC, triethylamine in DMF to give hybrid 15 with 19% yield. Scheme 5A. Synthesis of tyrosine-nitrogen mustard hybrids second generation.

JV-Boc-L-Tyrosine (9)

13 (46% with^-aminophenol, m = 5 or m = 10)

chemical

could be

D- starting

m = 5)

Reagents (a) aminophenol, HOBt DCC, DMF (96%), (b) TFA, CH₂Cl₂ 10 mm (100%), (c) CHL, HOBt, DCC, TEA, DMF (11, 44%, 15 19%), (d) BocNH(CH₂)_mCO₂H, HOBt, DCCTEA, DMF (19%)

[0081] Alternatively, the hybrids 15 were synthesized using a convergent and more efficient approach. 6-Amino caproic acid and 11 -amino undecanoic acid were initially coupled with chlorambucil as shown in scheme 5B to give 16 with 98% yield. This transformation was performed upon treatment of the amino acid with 1,1,1,3,3,3- hexamethyldisilazane (HMDS) in the presence trimethylchlorosilane (TMSCl) and catalytic sulfuric acid in DCM and TEA to give the sililated amino acid intermediate. The latter was then added to activated chlorambucil to produce derivative 16 with excellent yields. Note: Other hydrophobic amino acids (natural and non-natural) can be coupled to chlorambucil and used in the same manner as presented herein. Then, derivative 10 was treated with TFA and the trifluoroacetic ammonium salt was coupled with 16 using standard reaction conditions (see scheme 5B) to give hybrid 15 with 51% yield. The overall yield for the synthesis of 15 n = 5 was 8% via the direct synthesis (see scheme 5A) and 24% via the convergent synthesis (see scheme 5B). The convergent synthesis is, as anticipated, more efficient than the direct synthesis. Indeed, it is 3 times more efficient.

Scheme 5B. Preparation of amino acid-linked chlorambucil for convergent synthesis

of tyrosine-nitrogen mustard hybrids second generation.

*

_HO2CTU₂

m = 5 and 10

16, m = 5 or 10 (98%)

₁₀ (b) TFA, CH₂Cl₂, 10 min

^STSS⁰ (016, HOBt₁ DCC TEA¹DMF " ^

Reagents: (a) 1. HMDS, H₂SO₄ cat., TMSCl, DCM, TEA; 2. CHL, HOBt, DCC, TEA, DMF (98%);

(b) TFA, CH₂Cl₂, 10 min (100%); (c) 16, HOBt, DCC, TEA, DMF (51%) .

* Note: Any hydrophobic amino acids (natural or non-natural) could be used for this specific transformation.

[0082] Of note, the derivatives of the first generation seen in scheme 3 can also be obtained by convergent synthesis. This was done for the first generation D- tyrosine series as described in the experimental section. Of note, solid phase synthesis can be used to make these tyrosine-nitrogen mustard hybrids.

[0083] As it can be appreciated by the skilled artisan, the above synthetic schemes are not intended to be a comprehensive list of all means by which the compounds described and claimed in this application may be synthesized. Further methods can also potentially be used to prepare the compounds of the present disclosure.

[0084] The compounds of the present disclosure may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

[0085] The compounds of the present disclosure may contain one or more asymmetric carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomer, diastereomeric mixtures and individual diastereoisomers. All such isomeric forms of these compounds are expressly included in the present disclosure. Each stereo genie carbon may be of the R or S configuration.

[0086] In the present disclosure, the following abbreviations are used:

Abbreviation Meaning

AP Aminophenol

Boc t-Butyloxycarbonyl

Boc-aa Boc protected amino acid

cat Catalytic

CHL Chlorambucil

DCC Dicyclohexylcarbodiimide

DCM Dichloroniethane

DMF Dimethylformamide

DTP Developmental therapeutics program

ER+ Estrogen receptor positive

ER- Estrogen receptor negative

Et₂O Diethyl ether

EtOAc Ethyl acetate

GI₅₀ Growth inhibition of 50%

h Hour

HMDS 1 , 1 , 1 ,3 ,3 ,3 -Hexamethyldisilazane

HCl Chlorhydric acid

HOBt 1-hydroxybenzotriazole or N-hydroxybenzotriazole

LC₅o Net loss of cells 50% (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)

LiBH₄ Lithium borohydride m Meta

MeOH Methanol

min Minute

mmol Millimole

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NCI/NIH National Cancer Institute / U.S. National Institutes of

Health

NMR Nuclear magnetic resonance

O Ortho

P Para

Phe Phenyl

SRB Sulforhodamine B colorimetric assay

TGI Total growth inhibition

TLC Thin layer chromatography

TEA Triethylamine

TMSCl Trimethylchlorosilane

TFA Trifluoroacetic acid

tyr Tyrosine

EXAMPLES

[0087] This section also describes the synthesis of several compounds that are presented in this document. These examples are not to be construed as limiting the scope of the present disclosure in any way.

Materials and Methods

[0088] Anhydrous reactions were performed under an inert atmosphere, the set-up assembled and cooled under dry nitrogen. Unless otherwise noted, starting material, reactant and solvents were obtained commercially and were used as such or purified and dried by standard means.⁷ Organic solutions were dried over magnesium sulfate, evaporated on a rotatory evaporator and under reduced pressure. All reactions were monitored by UV fluorescence, or staining with iodine. Commercial TLC plates were Sigma T 6145 (polyester silica gel 60 A, 0.25mm). Flash column chromatography was performed according to the method of Still and co-workers on Merck grade 60 silica gel, 230-400 mesh.⁸ All solvents used in chromatography had been distilled

[0089] The infrared spectra were taken on a Nicolet Impact 420 FT-IR. Mass spectral assays were obtained using a MS model 6210, Agilent technology instrument. The high resolution mass spectra (HRMS) were obtained by TOF (time of flight) using ESI (electrospray ionization) using the positive mode (ESI+). (Plateforme analytique pour molecules organiques de l'Universite du Quebec a Montreal).

[0090] Nuclear magnetic resonance (NMR) spectra were recorded on a Varian

200 MHz NMR apparatus. Samples were dissolved in deuterochloroform (CDCl₃), deuteroacetone (acetone-ιi₆) or deuterodimethylsulfoxide (DMSO-J₆) for data acquisition using tetramethylsilane or chloroform as internal standard (TMS, δ 0.0 ppm for ¹H-NMR and CDCl₃ δ 77.0 ppm for ¹³C-NMR). Chemical shifts (δ) are expressed in parts per million (ppm), the coupling constants (J) are expressed in hertz (Hz). Multiplicities are described by the following abbreviations: s for singlet, d for doublet, dd for doublet of doublets, t for triplet, dt for doublet of triplets, q for quartet, dq for doublet of quartets, m for multiplet, #m for several multiplets, br for a broad signal.

[0091] The following compounds were prepared from amino acid using the procedures summarized in schemes 3, 4, 5 A or 5B.

Example 1. Preparation of iV-Boc-amino acid alkyl chains (Boc-aa, m = 5 or

10)

Step A. Synthesis of jY-Boc-amino acid alkyl chains (Boc-aa, m = 5 or 10)

[0092] A solution of a commercially available aminoacid alkyl chain (7.77 mmol) in water and dioxane was stirred at room temperature. Trielthyamine (11.65 mmol) was added slowly followed by Boc-ON (8.54 mmol). The solution was kept for 23 h under nitrogen. Afterwards, the mixture was diluted with diethyl ether and water and then washed with water (6x). The aqueous layers were then combined and a few drops of 10% aqueous HCl solution were slowly added in order to reach pH 2.5. The resulting solution was then washed with dichloromethane (3x). The organic layers were washed with saturated sodium chloride salt solution. The organic phase was dried with magnesium sulfate, filtered and evaporated. Flash chromatography (hexanes: acetone, 4:1) was performed to yield a colorless yellow viscous oil in 97% yield.

6-Boc-amino-hexanoic acid (Boc-aa, m = 5)

IR (NaCl, v_max, cm^"1) : 3339 (N-H), 2860-3100 (O-H), 1711 (2x C=O), 1527 and 1250 (C-N-H), 1173 (C-O-C).

¹H-NMR (CDCl₃, Jppm) : 8.91 (IH, br s, COOH), 4.74 (IH, s, NH), 3.02 (2H, m, CH₂NH), 2.27 (2H, t, J = 7.4 Hz, CH₂COOH), 1.24-1.68 (6H, #m, 3 x CH₂), 1.37 (s, 9H, 3 x CH₃).

¹³C-NMR (CDCl₃, £ppm) : 178.7 (COOH), 156.4 (OCONH), 79.4 ((CH₃)₃C), 40.5 (NHCH₂), 34.1 (CH₂COOH), 29.8 (NHCH₂CH₂), 28.6 (3 x CH₃), 26.4 (CH₂CH₂CH₂CH₂CH₂), 24.5 (CH₂CH₂COOH).

ESI+ HRMS: [M + Na]⁺ calculated for C₁₁H_2JNNaO₄ = 254.1363; found = 254.1360.

ll-Boc-amino-undecanoic acid (Boc-aa, m = 10)

IR (NaCl, v_max, cm^'1) : 3365 (N-H), 2850-3100 (O-H), 1704 (C=O, COOH), 1683

(C=O OCONH), 1526 (C-N-H), 1171 (C-O-C).

¹H-NMR (CDCl₃, £ppm) : 10.98 (IH, br s, COOH), 4.55 (IH, s, NH), 3.06 (2H, m,

CH₂NH), 2.32 (2H, t, J = 7.4 Hz, CH₂COOH), 1.61 (2H, m, CH₂), 1.43 (s, HH, 3 x

CH₃ and Ix CH₂), 1.26 (br s, 12H, 6 x CH₂).

¹³C-NMR (CDCl₃, £ppm) : 179.7 (COOH), 156.3 (OCONH), 79.3 ((CH₃)₃C), 40.8

(NHCH₂), 34.3 (CH₂COOH), 30.2, 29.6, 29.5, 29.4 (2C), 29.2, 28.6 (3 x CH₃), 27.0,

24.9.

ESI+ HRMS: [M + Na]⁺ calculated for C₁₆H₃₁NNaO₄ = 324.2145; found = 324.2145. Example 2. Preparation of iV-chlorambucil-amino acid alkyl chain (16, m = 5 or 10)

Step A. Synthesis of JV-chlorambucil-amino acid derivative (16, m = 5 or

10)

[0093] The amino acid (6.57 mmol) was dissolved in dichloroniethane.

Hexamethyldisilazane (21.69 mmol) and concentrated sulfuric acid (cat) were added. The mixture was stirred and heated to reflux under nitrogen atmosphere until complete dissolution. The solution was kept to reflux for 0.5 h. After cooling down, benzene, triethylamine (4.91 mmol) and chlorotrimethylsilane (4.91 mmol) were added. The resulting mixture was stirred at room temperature for 12 h. In another flask, chlorambucil (3.29 mmol) was dissolved in dichloromethane at 0⁰C. Triethylamine (3.62 mmol) and isobutylchloroformate (3.62 mol) were added and the mixture was kept at 0°c for 1 h. Then, the chlorambucil solution was added to the activated amino acid solution and the mixture was stirred at room temperature for 4 h. Work-up was done by diluting with ethyl acetate and by washing the organic phase with HCl 10% solution (2x) and with a saturated sodium chloride solution (2x). The organic phase was dried with anhydrous magnesium sulfate, filtered and evaporated. The product was purified by flash chromatography (hexanes: acetone, 7:3) to give the desired material in 98 % yield.

N-6-chlorambUcil-caproic acid (16, m = 5)

IR (NaCl, v_max, cm^"1) : 3300 (N-H), 3200-2600 (O-H), 1716 (C=O, COOH), 1622

(C=O, NHCOO), 1522 and 1255 (C-N-H).

¹H-NMR (Acetone-rf₆, <?ppm) : 7.26 (IH, br s, NH), 7.06 (2H, d, J = 8.6 Hz, 3-CH

CHL), 6.70 (2H, d, J = 8.6 Hz, 2-CH CHL), 3.73 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.19 (2H, m, CH₂NHCO), 2.51 (2H, t, J = 7.4 Hz, CH₂CH₂Ph), 2.27 (2H, t, J = 7.2

Hz, CH₂COOH), 2.18 (2H, t, J = 7.4 Hz, CH₂NHCOCH₂), 1.78-1.96 (2H, m,

CH₂CH₂CH₂Ph), 1.28-1.67 (6H, m, 3x CH₂), (IH, COOH hidden).

¹³C-NMR (Acetone-rf₆, £ppm) : 174.3 (COOH), 173.0 (CONH), 144.8 (1-C CHL),

130.9 (4-C CHL), 129.7 (2C, 3-C CHL), 112.5 (2C, 2-C CHL), 53.3 (2C, 2x

NCH₂CH₂Cl), 41.0 (2x C, 2x NCH₂CH₂Cl), 39.1, 35.6, 34.3, 33.6, 29.4, 27.9, 26.5,

24.7.

ESI+ HRMS: [M + H]⁺ calculated for C₂₀H₃₁Cl₂N₂O₃ = 417.1706; found = 417.1707.

N-6-chlorambucil~undecanoic acid (16, m = 10)

IR (NaCI, v_max, cm-¹) : 3300 (N-H), 3200-2600 (O-H), 1714 (C=O, COOH), 1618

(C=O, NHCOO), 1526 and 1250 (C-N-H).

¹H-NMR (Acetone-<*₆, δppm) : 7.07 (2H, d, J = 8.6 Hz, 3-CH CHL), 7.03 (IH, br s,

NH), 6.72 (2H, d, J = 9.0 Hz, 2-CH CHL), 3.75 (8H, m, 2x CH₂Cl and 2x NCH₂),

3.17 (2H, m, CH₂NHCO), 2.90 (IH, br s, COOH), 2.51 (2H, t, J = 7.4 Hz,

CH₂CH₂Ph), 2.27 (2H, t, J = 7.4 Hz, CH₂COOH), 2.14 (2H, t, J = 7.2 Hz,

CH₂NHCOCH₂), 1.80-1.92 (2H, m, CH₂CH₂CH₂Ph), 1.41-1.62 (4H, m, 2x CH₂),

1.30 (12H, 6x CH₂).

¹³C-NMR (Acetone-^, δppm) : 174.0 (COOH), 172.1 (CONH), 144.9 (1-C CHL),

131.0 (4-C CHL), 129.7 (2C, 3-C CHL), 112.5 (2C, 2-C CHL), 53.3 (2C, 2x

NCH₂CH₂Cl), 41.0 (2C, 2x NCH₂CH₂Cl), 39.0, 35.5, 34.2, 33.6, 29.8, 29.5 (2C), 29.3

(3C), 27.9, 27.0, 25.0.

ESI+ HRMS: [M + H]⁺ calculated for C₂₅H₄₁Cl₂N₂O₃ = 487.2489; found = 487.2485.

Example 3. Preparation of tyrosine nitrogen mustard hybrids first generation

(3 and 4)

Step A. Synthesis of L-p-tyrosine-methyl ester hydrochloride (2) and various isomers of (2) (D-p-tyr, DL-σ-tyr, L-m-fyr))

[0094] A solution of tyrosine 1 (D-p-, DL-o-, L-m- or L-p-, 1.67 mmol) in anhydrous methanol was stirred at 0⁰C. Thionyl chloride (5.00 mmol) was then added dropely. The mixture was heated to reflux for 12 h. Then, the solvent was evaporated and recristalized in diethyl ether. The desired compound was filtered, washed with cold ether and dried in a dessicator for a 1 day. The crude hydrochloric acid salt, obtained in a 100% yield, was pure enough to be used as such in the next step.

L-p-tyrosine methyl ester hydrochloride (2)

IR (KBr, v_max, cm^"1) : 3354 (N-H), 3078 (O-H), 1742 (C=O ester), 1224 (CO-O-C).

¹H-NMR (DMSCM₆, £ppm) : 9.46 (IH, br s, OH), 8.60 (3H, br s, NH₃), 7.01 (2H, d, J = 8.6 Hz, 3-CH tyr), 6.73 (2H, d, J = 8.60 Hz, 2-CH tyr), 4.16 (IH, m, CHNH₃),

3.67 (3H, s, OCH₃), 3.03 (2H, m, CH₂CH).

¹³C-NMR (DMSO-^₆, £ ppm) : 170.1 (COOCH₃), 157.4 (1-C tyr), 131.0 (2C, 3-C tyr), 125.0 (4-C tyr), 116.1 (2C, 2-C tyr), 54.1 (CHNH₃), 53.2 (OCH₃), 35.7

(CH₂CH).

ESI+ HRMS: [M + H]⁺ calculated for C₁₀H₁₄NO₃ = 196.0968; found = 196.0968.

L-m-tyrosine methyl ester hydrochloride (isomer of 2)

IR (ATR, v_max, cm^"1) : 3243 (N-H), 3194 (O-H), 1745 (C=O ester), 1231 (CO-O-C).

¹H-NMR (DMSO, δppm) : 9.53 (IH, br s, OH), 8.66 (3H, br s, NH₃), 7.08 (IH, t, J =

7.7 Hz, 5-CH tyr), 6.69-6.59 (2H, 2x dd hidden, 4-CH tyr and 6-CH tyr), 6.62 (IH, s,

2-CH tyr), 4.16 (IH, t, J = 6.25 Hz, CHNH₃), 3.65 (3H, s, OCH₃), 3.14-2.92 (2H, m,

CH₂CH).

¹³C-NMR (DMSO, δppm) : 170.0 (COOCH₃), 158.0 (1-C tyr), 136.2 (3-C tyr),

130.0 (5-C tyr), 120.3 (4-C tyr), 116.7 (2-C tyr), 114.8 (6-C tyr), 53.7 (CHNH₃), 53.0

(OCH₃), 36.3 (CH₂CH).

ESI+ HRMS : [M + H]⁺ calculated for C₁₀H₁₄NO₃ = 196.0968; found = 196.0964.

DL-o-tyrosine methyl ester hydrochloride (isomer of 2)

IR (ATR, v_max, cm^"1) : 3258 (N-H), 3076 (O-H), 1734 (C=O ester), 1246 (CO-O-

C).

¹H-NMR (DMSO, δppm) : 8.74 (3H, br s, NH₃), 7.05 (IH₅ 1, J = 10.9 Hz, 5-CH tyr),

7.03 (IH, d, hidden, 3-CH tyr), 6.84 (IH, d, J = 7.4 Hz, 6-CH tyr), 6.71 (IH, t, J = 6.9

Hz, 4-CH tyr), 4.08 (IH, t, J = 6.7 Hz, CHNH₃), 3.58 (3H, s, OCH₃), 3.02 (2H, d, J =

6.7 Hz, CH₂CH), 2.48 (IH, s, OH). ¹³C-NMR (DMSO, <? ppm) : 170.0 (COOCH₃), 156.2 (1-C tyr), 131.5 (3-C tyr), 129.1 (2-C tyr), 121.2 (5-C tyr), 119.4 (4-C tyr), 115.4 (6-C tyr), 52.9 (CHNH₃), 52.4 (OCH₃), 32.0 (CH₂CH).

ESI+ HRMS : [M + H]⁺ calculated for C₁₀H₁₄NO₃ = 196.0968; found = 196.0966.

D-p-tyrosine methyl ester hydrochloride (isomer of 2)

IR (KBr, v_max, cm^'1) : 3341 (N-H), 3100 (O-H), 1743 (C=O ester), 1227 (CO-O-C).

¹H-NMR (DMSO-</₆, <?ppm) : 9.44 (IH, s, OH), 8.34 (3H, br s, NH₃), 7.00 (2H, d, J

= 8.6 Hz, 3-CH tyr), 6.72 (2H, d, J = 8.20 Hz, 2-CH tyr), 4.19 (IH, t, J = 6.4 Hz,

CHNH₃), 3.69 (3H, s, OCH₃), 2.99 (2H, m, CH₂CH).

¹³C-NMR (DMSO-</₆, £ ppm) : 170.3 (COOCH₃), 157.3 (1-C tyr), 131.1 (2C, 3-C tyr), 125.0 (4-C tyr), 116.1 (2C, 2-C tyr), 54.2 (CHNH₃), 53.3 (OCH₃), 35.9

(CH₂CH).

ESI+ HRMS: [M + H]⁺ calculated for C₁₀Hi₄NO₃ = 196.0968; found = 196.0968.

Step B. Synthesis of N-chlorambucil-L-p-tyrosine methyl ester (3) and various isomers of (3) (D-p-tyr, DL-ø-tyr, h-m-tyr))

[0095] The tyrosine methyl ester 2 (0.32 mmol) was dissolved in dimethylformamide and triethylamine (0.32 mmol) was added. At the same time, a solution of chlorambucil (0.49 mmol) in dimethylformamide was activated using DCC (0.52 mmol) followed by HOBt (0.52 mmol). The tyrosine solution was then added to the activated acid chlorambucil solution. The mixture was stirred at room temperature under nitrogen at room temperature for 24 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 4:1) to give a pure compound in 81 % yield.

N-chlorambucil-L-p-tyrosine methyl ester (3)

IR (KBr, v_max, cm^"1) : 3100-3450 (O-H and N-H), 1741 (C=O, COOCH₃), 1650

(C=O, NHCO), 1517 and 1221 (C-N-H). ¹H-NMR (Acetone-^, δ ppm) : 8.27 (IH, s, OH), 7.25 (IH, d, J = 8.2 Hz, CHNHCO), 7.05 and 7.03 (4H, 2d, J = 8.6 Hz and J = 9.0 Hz, 3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 9.0 Hz, 2-CH tyr), 6.70 (2H, d, J = 9.0 Hz, 2-CH CHL), 4.63 (IH, m, CHNH), 3.74 (8H, dt, J = 3.5 Hz and J = 1.8 Hz, CH₂Cl and NCH₂), 3.65 (3H, s, OCH₃), 2.82-3.08 (2H, m, CH₂CHNH), 2.46 (2H, t, J - 7.0 Hz, CH₂CH₂Ph), 2.18 (2H, d, J = 7.0 Hz, NHCOCH₂), 1.76-1.87 (2H, m, CH₂CH₂CH₂).

1³C-NMR (Acetone-rf₆, δ ppm) : 172.43 (NHCOCH₂), 172.38 (COOCH₃), 156.5 (1- C tyr), 144.8 (1-C CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 127.9 (4-C tyr), 115.4 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 54.0 (CHNH), 53.3 (2C, 2x NCH₂CH₂Cl), 51.5 (OCH₃), 41.0 (2x C, 2x NCH₂CH₂Cl), 36.9 (CH₂CHNH), 35.0 (CH₂CH₂Ph), 34.0 (NHCOCH₂), 27.7 (CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₄H₃₁Cl₂N₂O₄ = 481.1655; found = 481.1650.

N-chlorambucil-L-m-tyrosine methyl ester (isomer of 3)

IR (ATR, v_max, cm^"1) : 3100-3450 (O-H and N-H), 1748 (C=O, COOCH₃), 1643

(C=O, NHCO), 1519 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 8.31 (IH, s, OH), 7.28 (IH, d, J = 8.2 Hz,

CHNHCO), 7.06 (IH, t, J = 8.4 Hz, 5-CH tyr), 7.03 (2H, d, J = 9.0 Hz, 3-CH CHL),

6.72 (IH, s, 2-CH tyr), 6.70 (4H, 3d, J = 9.0 Hz, 4-CH tyr, 6-CH tyr and 2-CH CHL),

4.72 (IH, m, CHNH), 3.74 (8H, dt, J = 4.7 Hz and J = 1.2 Hz, CH₂Cl and NCH₂),

3.66 (3H, s, OCH₃), 2.85-3.12 (2H, m, CH₂CHNH), 2.46 (2H, t, J = 7.7 Hz,

CH₂CH₂Ph), 2.18 (2H, t, J = 7.4 Hz, NHCOCH₂), 1.76-1.88 (2H, m, CH₂CH₂CH₂).

¹³C-NMR (Acetone-d₆, <?ppm) : 172.18 (NHCOCH₂), 172.10 (COOCH₃), 157.4 (1-

C tyr), 144.6 (1-C CHL), 138.7 (3-C tyr), 130.6 (4-C CHL), 129.5 (2C, 3-C CHL),

129.3 (5-C tyr), 120.2 (4-C tyr), 116.0 (2-C tyr), 113.7 (6-C tyr), 112.2 (2C, 2-C

CHL), 53.4 (CHNH), 53.0 (2C, 2x NCH₂CH₂Cl), 51.3 (OCH₃), 40.8 (2x C, 2x

NCH₂CH₂Cl), 37.3 (CH₂CHNH), 34.8 (CH₂CH₂Ph), 33.7 (NHCOCH₂), 27.4

(CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₄H_3ICl₂N₂O₄ = 481.1655; found = 481.1650.

N-chlorambucil-DL-o-tyrosine methyl ester (isomer of 3)

IR (ATR, v_max, cm^'1) : 3150-3400 (O-H and N-H), 1745 (C=O, COOCH₃), 1652

(C=O, NHCO), 1524 (C-N-H). ¹H-NMR (Acetone-d₆, δ ppm) : 8.74 (IH, s, OH), 7.39 (IH, d, J = 7.4 Hz, CHNHCO), 7.01-7.13 (2H, m, 3-CH and 5-CH tyr), 7.03 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.67-6.88 (2H, m, 4-CH and 6-CH tyr), 6.69 (2H, d, J - 9.0 Hz, CH CHL), 4.75 (IH, m, CHNH), 3.74 (8H, dt, J = 4.7 Hz and J = 1.2 Hz, CH₂Cl and NCH₂), 3.63 (3H, s, OCH₃), 2.92-3.20 (2H, m, CH₂CHNH), 2.46 (2H, t, J = 7.7 Hz, CH₂CH₂Ph), 2.17 (2H, t, J = 7.4 Hz, NHCOCH₂), 1.76-1.87 (2H, m, CH₂CH₂CH₂). ¹³C-NMR (Acetone-dβ, £ppm) : 172.48 (NHCOCH₂), 172.3 (COOCH₃), 155.4 (1-C tyr), 144.6 (1-C CHL), 131.1 (3-C tyr), 130.6 (C, 4-C CHL), 129.5 (2C, 3-C CHL), 128.0 (2-C tyr), 123.5 (5-C tyr), 119.5 (4-C tyr), 115.1 (C, 6-C tyr), 112.2 (2C, 2-C CHL), 53.0 (2C, 2x NCH₂CH₂Cl), 52.8 (CHNH), 51.2 (OCH₃), 41.8 (2x C, 2x NCH₂CH₂Cl), 34.9 (CH₂CHNH), 33.7 (CH₂CH₂Ph), 32.4 (NHCOCH₂), 27.4 (CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₄H₃₁Cl₂N₂O₄ = 481.1655; found = 481.1652.

N-chlorambucil-D-p-tyrosine methyl ester (isomer of 3)

IR (KBr, v_max, cm-¹) : 3100-3450 (O-H and N-H), 1742 (C=O, COOCH₃), 1648

(C=O, NHCO), 1516 and 1220 (C-N-H).

¹H-NMR (Acetone-rf₆, δ ppm) : 7.18 (IH, d, J = 7.8 Hz, CHNHCO), 7.05 and 7.04

(4H, 2d, J = 8.6 Hz and J = 8.6 Hz, 3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 8.2 Hz,

2-CH tyr), 6.71 (2H, d, J = 8.2 Hz, 2-CH CHL), 4.66 (IH, m, CHNH), 3.75 (8H, dt, J

= 1.2 Hz and J = 3.7 Hz, CH₂Cl and NCH₂), 3.65 (3H, s, OCH₃), 2.84-3.00 (2H, m,

CH₂CHNH), 2.47 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.17 (2H, d, J = 7.4 Hz,

NHCOCH₂), 1.76-1.87 (2H, m, CH₂CH₂CH₂), (IH, OH hidden).

¹³C-NMR (Acetone-rfή, δ ppm) : 172.4 (NHCOCH₂), 172.1 (COOCH₃), 156.3 (1-C tyr), 144.8 (1-C CHL), 131.1 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL),

128.0 (4-C tyr), 115.3 (2C, 2-C tyr), 112.6 (2C, 2-C CHL), 53.9 (CHNH), 53.3 (2C,

2x NCH₂CH₂Cl), 51.5 (OCH₃), 40.9 (2x C, 2x NCH₂CH₂Cl), 36.8 (CH₂CHNH), 34.9

(CH₂CH₂Ph), 34.0 (NHCOCH₂), 27.6 (CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₄H_3ICl₂N₂O₄ = 481.1655; found = 481.1647. Step C. Synthesis of N-chlorambucil-L-p-tyrosinol (4) and various isomers of (4) (D-p-tyr, DL-ø-tyr, L-m-fyr))

[0096] The N-chlorambucil-tyrosine methyl ester 3 (0.15 mmol) was dissolved in diethyl ether and DCM and was stirred under nitrogen atmosphere. The resulting solution was cooling down with an ice and water bath, afterwards lithium borohydride (0.90 mmol) was added. The mixture was kept at 0°c for 3 h. Sodium sulfate decahydrate (0.3 g) was added. Work-up was done by diluting with diethyl ether and the organic phase was washed with saturated NH₄Cl solution (2x) and with water (4x). The organic phase was dried with anhydrous sodium sulfate, filtered and evaporated. The product was purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 88 % yield.

N-chlorambucil-L-p-tyrosinol (4)

IR (NaCl, v_max, cm^"1) : 3100-3400 (O-H and N-H), 1646 (C=O, NHCO), 1516 and

1250 (C-N-H).

¹H-NMR (Acetone-^, Sppm) : 8.16 (IH, s, OH), 7.08 and 7.04 (4H, 2d, J = 9.0 Hz and J = 9.0 Hz, 3-CH tyr and 3-CH CHL), 6.87 (IH, d, J = 8.2 Hz, CHNHCO), 6.75

(2H, d, J = 7.0 Hz, 2-CH tyr), 6.70 (2H, d, J = 7.4 Hz, 2-CH CHL), 4.08 (IH, m,

CHNH), 3.74 (8H, dt, J = 1.8 Hz and J = 3.5 Hz, 2x CH₂Cl and 2x NCH₂), 3.51 (2H, t, J = 5.3 Hz, CH₂OH), 2.62-2.88 (2H, m, CH₂CHNH), 2.46 (2H, t, J = 7.6 Hz,

CH₂CH₂Ph), 2.14 (2H, d, J = 7.4 Hz, NHCOCH₂), 1.76-1.87 (2H, m, CH₂CH₂CH₂).

¹³C-NMR (Acetone-^, δ ppm) : 172.4 (NHCOCH₂), 156.0 (1-C tyr), 144.8 (1-C

CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.9 (4-C tyr), 129.7 (2C, 3-C CHL),

115.2 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.5 (CH₂OH), 53.3 (3C, CHNH and 2x

NCH₂CH₂Cl), 41.0 (2C, 2x NCH₂CH₂Cl), 36.2 (CH₂CHNH), 35.5 (CH₂CH₂Ph), 34.1

(NHCOCH₂), 27.8 (CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₃H₃₁Cl₂N₂O₃ = 453.1706; found = 453.1703.

N-chlorambucil-L-m-tyrosinol (isomer of 4)

IR (ATR, v_max, cm ¹) : 3100-3450 (O-H and N-H), 1619 (C=O, NHCO), 1526 and

1254 (C-N-H). ¹H-NMR (Acetone-d₆, £ρpm) : 8.34 (IH, s, OH), 7.08 (IH, t, J = 7.4 Hz, 5-CH tyr), 7.03 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.97-7.01 (IH, d, hidden, CHNHCO), 6.70 (2H, d, J = 8.6 Hz, 2-CH CHL), 6.64-6.79 (3H, #m, hidden, 2-CH tyr, 4-CH tyr and 6-CH tyr), 4.14 (IH, m, CHNH), 3.74 (8H, dt, J = 1.2 Hz and J = 4.7 Hz, 2x CH₂Cl and 2x NCH₂), 3.53 (2H, d, J - 4.7 Hz, CH₂OH), 2.67-2.91 (2H, m, CH₂CHNH), 2.46 (2H, t, J = 7.5 Hz, CH₂CH₂Ph), 2.16 (2H, t, J = 7.4 Hz, NHCOCH₂), 1.77-1.88 (2H, m, CH₂CH₂CH₂).

¹³C-NMR (Acetone-de, δ ppm) : 172.4 (NHCOCH₂), 157.4 (1-C tyr), 144.6 (1-C CHL), 140.6 (3-C tyr), 130.7 (4-C CHL), 129.5 (2C, 3-C CHL), 129.1 (5-C tyr), 120.3 (4-C tyr), 116.1 (2-C tyr), 113.1 (6-C tyr), 112.2 (2C, 2-C CHL), 63.2 (CH₂OH), 53.0 (2C, 2x NCH₂CH₂Cl), 52.8 (CHNH), 40.8 (2C, 2x NCH₂CH₂Cl), 36.8 (CH₂CHNH), 35.3 (CH₂CH₂Ph), 33.9 (NHCOCH₂), 27.6 (CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₃H_3ICl₂N₂O₃ = 453.1706; found = 453.1707.

N-chlorambucil~DL-o-tyrosinol (isomer of 4)

IR (ATR, v_max, cm^"1) : 3100-3450 (O-H and N-H), 1638 (C=O, NHCO), 1517 and

1242 (C-N-H).

¹H-NMR (Acetone-dβ, S ppm) : 9.03 (IH, s, OH), 7.35 (IH, d, J = 6.3 Hz,

CHNHCO), 7.02-7.11 (2H, m, 3-CH tyr and 5-CH tyr), 7.07 (2H, d, J = 8.6 Hz, 3-CH

CHL), 6.71 (2H, d, J = 9.0 Hz, 2-CH CHL), 6.69-6.85 (2H, m, hidden, 4-CH tyr and

6-CH tyr), 4.18 (IH, t, J = 5.7 Hz, CHNH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz,

2x CH₂Cl and 2x NCH₂), 3.58 (2H, dt , J = 1.2 Hz and J = 5.5 Hz, CH₂OH), 2.92 (IH, s, CH₂OH), 2.78-2.91 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.4 Hz, CH₂CH₂Ph), 2.25

(2H, t, J = 7.3 Hz, NHCOCH₂), 1.83-1.94 (2H, m, CH₂CH₂CH₂).

¹³C-NMR (Acetone-d₆, S ppm) : 173.8 (NHCOCH₂), 156.0 (1-C tyr), 144.7 (1-C

CHL), 130.9 (4-C CHL), 130.6 (3-C tyr), 129.5 (2C, 3-C CHL), 127.7 (5-C tyr), 124.6

(2-C tyr), 119.3 (4-C tyr), 115.8 (6-C tyr), 112.2 (2C, 2-C CHL), 62.3 (CH₂OH), 54.1

(CHNH), 53.0 (2C, 2x NCH₂CH₂Cl), 40.7 (2C, 2x NCH₂CH₂Cl), 35.1 (CH₂CHNH),

33.9 (CH₂CH₂Ph), 31.9 (NHCOCH₂), 27.5 (CH₂CH₂CH₂).

ESI+ HRMS: [M + H]⁺ calculated for C₂₃H₃₁Cl₂N₂O₃ = 453.1706; found = 453.1702. N-chlorambucil-D-p-tyrosinol (isomer of 4)

IR (KBr, v_max, cm-¹) : 3100-3400 (O-H and N-H), 1620 (C=O, NHCO), 1516 and

1243 (C-N-H).

¹H-NMR (Acetone-i₆, £ppm) : 8.21 (IH, s, OH), 7.08 and 7.04 (4H, 2d, J - 8.6 Hz and J = 9.0 Hz, 3-CH tyr and 3-CH CHL), 6.90 (IH, d, J = 7.4 Hz, CHNHCO), 6.74

(2H, d, J = 8.2 Hz, 2-CH tyr), 6.70 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.06 (IH, m,

CHNH), 3.74 (8H, t, J = 4.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.52 (2H, t, J = 4.9 Hz,

CH₂OH), 2.67-2.85 (2H, m, CH₂CHNH), 2.46 (2H, t, J = 7.4 Hz, CH₂CH₂Ph), 2.14

(2H, d, J = 7.4 Hz, NHCOCH₂), 1.76-1.84 (2H, m, CH₂CH₂CH₂).

¹³C-NMR (Acetone-*/*, δ ppm) : 172.5 (NHCOCH₂), 156.0 (1-C tyr), 144.8 (1-C

CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.8 (4-C tyr), 129.7 (2C, 3-C CHL),

115.3 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.4 (CH₂OH), 53.30 (2C, 2x

NCH₂CH₂Cl), 53.25 (CHNH), 41.0 (2C, 2x NCH₂CH₂Cl), 36.2 (CH₂CHNH), 35.6

(CH₂CH₂Ph), 34.1 (NHCOCH₂), 27.8 (CH₂CH₂CH₂).

Example 4. Preparation of tyrosine nitrogen mustard hybrids first generation

(6 and 7) (direct synthesis; using L-p-tyr)

Step A. Synthesis of N-((iV-Boc-amino)alcanoyl)-L-p-tyrosine methyl ester

(5)

[0097] To a solution of Boc-aminoacid alkyl chain (Boc-aa, m = 5 or 10)

(1.57 mmol) in dimethylformamide, were added DCC (1.67 mmol), followed by HOBt (1.67 mmol). In another flask, the L-tyrosine ester hydrochloride salt 2 (1.05 mmol) was put in solution with dimethylformamide. Triethylamine (1.05 mmol) was added in order to get neutralization of the basic salt. The L-tyrosine solution was then added to the activated acid solution. The mixture was stirred at room temperature under nitrogen at room temperature for 20 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with magnesium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 96% yield.

5, m = 5 or 10

N-((6-N-Boc-amino)hexanoyl)-L-p-tyrosine methyl ester (5, m = 5)

IR (NaCl, v_max, cm^"1) : 3200-3500 (O-H and N-H), 1747 (C=O, COOCH₃), 1690 (2 x

C=O, NHCOCH₂ and NHCOO), 1521 and 1255 (C-N-H).

¹H-NMR (Acetone-^, δ ppm) : 8.65 (IH, br s, OH), 7.57 (IH, d, J = 8.2 Hz,

CHNHCO), 7.04 (2H, d, J = 8.6 Hz, 3-CH tyr), 6.75 (2H, d, J = 8.6 Hz, 2-CH tyr),

6.08 (IH, t, J = 5.5 Hz, NH-Boc), 4.69 (IH, m, CHNH), 3.64 (3H, s, OCH₃), 2.80-

3.09 (4H, #m, CH₂CHNH and CH₂NHBoc), 2.19 (2H, t, J = 7.0 and 7.4 Hz, NHCOCH₂), 1.46-1.61 (4H, #m, 2 x CH₂), 1.40 (s, 9H, 3 x CH₃), 1.07-1.28 (2H, m, CH₂).

¹³C-NMR (Acetone-4, δ ppm) : 173.4 (NHCOCH₂), 172.4 (COOCH₃), 156.6

(NHCOO), 156.4 (1-C tyr), 130.4 (2C, 3-C tyr), 127.7 (4-C tyr), 115.5 (2C, 2-C tyr),

78.1 ((CH₃)₃C), 54.1 (CHNH), 51.7 (OCH₃), 40.4, 36.8, 35.7, 29.9, 28.1 (3 x CH₃),

26.3, 25.5.

ESI+ HRMS: [M + Na]⁺ calculated for C₂₁H₃₂N₂NaO₆ = 431.2153; found =

431.2149; [M + H]⁺ calculated for C₂iH₃₃N₂O₆ = 409.2333; found = 409.2335.

N-((ll-N-Boc-amino)undecanoyl)-L-p-tyrosine methyl ester (5, m = 10)

IR (NaCl, v_max, cm^'1) : 3200-3500 (O-H and N-H), 1741 (C=O, COOCH₃), 1686

(C=O, NHCOO), 1649 (C=O, NHCOCH₂), 1516 and 1253 (C-N-H).

¹H-NMR (Acetone-rf₆, δ ppm) : 8.43 (IH, br s, OH), 7.37 (IH, d, J = 8.2 Hz,

CHNHCO), 7.03 (2H, d, J = 8.2 Hz, 3-CH tyr), 6.75 (2H, d, J = 8.6 Hz, 2-CH tyr),

5.98 (IH, br s, NH-Boc), 4.67 (IH, m, CHNH), 3.64 (3H, s, OCH₃), 2.80-3.07 (4H,

#m, CH₂CHNH and CH₂NHBoc), 2.18 (2H, t, J = 7.4 Hz, NHCOCH₂), 1.46-1.53

(4H, #m, 2 x CH₂), 1.40 (s, 9H, 3 x CH₃), 1.26 (12H, br m, 6 x CH₂).

¹³C-NMR (Acetone-^, δ ppm) : 173.0 (NHCOCH₂), 172.4 (COOCH₃), 156.6

(NHCOO), 156.2 (1-C tyr), 130.4 (2C, 3-C tyr), 127.8 (4-C tyr), 115.4 (2C, 2-C tyr), 77.8 ((CH₃)₃C), 54.0 (CHNH), 51.6 (OCH₃), 40.5, 36.9, 35.8, 30.2, 29.7, 29.5, 29.4 (2C), 29.2, 28.1 (3 x CH₃), 26.9, 25.7.

ESI+ HRMS: [M + Na]⁺ calculated for C₂₆H₄₂N₂NaO₆ = 501.2935; found = 501.2936; [M + H]⁺ calculated for C₂₆H₄₃N₂O₆ = 479.3116; found = 479.3114.

Step B. Synthesis of 7V-('(7V-chlorambucilainino)alcanoyl)-L-p-tyrosine methyl ester (6, m = 5 or 10)

[0098] The N-Boc-aminoalcanoyl-L-p-tyrosine methyl ester 5 (m = 5 or 10)

(0.28 mmol) was dissolved in dichloromethane. Trifluoroacetic acid (2.80 mmol) was added. The solution was stirred at room temperature for 1 h. Then the solvent was evaporated under vacuum. The resulting trifluoroacetic salt (0.28 mmol) was dissolved in dimethylformamide and triethylamine (0.28 mmol) was added in order to neutralize the compound. At the same time, a solution of chlorambucil (0.42 mmol) in dimethylformamide was activated using DCC (0.45 mmol) followed by HOBt (0.45 mmol). The tyrosine solution was then added to the activated acid chlorambucil solution. The mixture was stirred at room temperature under nitrogen for 23 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 3:2) to give a pure compound in 60% yield.

N-((6-N-chlorambucilamino)hexanoyl)-L-p-tyrosine methyl ester (6, m = 5)

IR (NaCI, v_max, cm^"1) : 3150-3500 (O-H and 2x N-H), 1745 (C=O, COOCH₃), 1646

(2 x C=O, 2xNHCO), 1523 and 1253 (C-N-H). ¹H-NMR (Acetone-^, δ ppm) : 8.48 (IH, br s, OH), 7.27 (IH, d, J = 8.2 Hz, CHNHCO), 7.16 (IH, br t, J = 5.9 Hz, CH₂NHCO), 7.07 and 7.04 (4H, 2d, J = 8.6 Hz and J = 8.6 Hz, 3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.71 (2H, d, J = 9.0 Hz, 2-CH CHL), 4.65 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.65 (3H, s, OCH₃), 2.85-3.17 (4H, #m, CH₂CHNH and CH₂NHCO), 2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.16 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.89 (2H, m, CH₂CH₂CH₂Ph), 1.19-1.54 (6H, Hm, 3x CH₂). ¹³C-NMR (Acetone-rf₆, δ ppm) : 172.5 (CONH), 172.4 (CONH), 172.1 (COOCH₃), 156.5 (1-C tyr), 144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.0 (4-C tyr), 115.4 (2C, 3-C tyr), 112.4 (2C, 3-C CHL), 53.8 (CHNH), 53.3 (2C, 2x NCH₂CH₂Cl ), 51.5 (OCH₃), 41.0 (2C, 2x NCH₂CH₂Cl), 39.0, 36.9, 35.7, 35.5, 34.2, 27.9, 26.4, 25.4, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₀H₄₂Cl₂N₃O₅ = 594.2496; found = 594.2490.

N-((l]-N-chlorambucilamino)undecanoyl)-L-p-tyrosine methyl ester (6, m = 10) IR (NaCl, v_raax, cm^'1) : 3250-3450 (O-H and 2x N-H), 1745 (C=O, COOCH₃), 1656 and 1614 (2x C=O, 2x NHCO), 1516 and 1216 (C-N-H).

¹H-NMR (Acetone-rf₆, δ ppm) : 8.49 (IH, s, OH), 7.23 (IH, d, J = 7.8 Hz, CHNHCO), 7.12 (IH, br t apparent, partly hidden, CH₂NHCO), 7.06 and 7.03 (4H, 2d, J = 8.6 Hz and J = 8.2 Hz, 3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 8.2 Hz, 2- CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.65 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.65 (3H, s, OCH₃), 3.19 (2H, q, J = 6.3 Hz, CH₂NHCO), 2.81-3.07 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.16 (4H, 2d overlapped, J = 7.4 Hz, CH₂NHCOCH₂ and CHNHCOCH₂), 1.78-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.26-1.52 (16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-^, £ppm) : 172.5 (2C, 2x CONH), 172.4 (COOCH₃), 156.6 (1- C tyr), 144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C₅ 3-C tyr), 129.7 (2C, 3-C CHL), 127.8 (4-C tyr), 115.4 (2C, 3-C tyr), 112.4 (2C, 3-C CHL), 53.9 (CHNH), 53.3 (2C, 2x NCH₂CH₂Cl ), 51.5 (OCH₃), 41.0 (2C, 2x NCH₂CH₂Cl), 39.0, 36.9, 35.7, 35.6, 34.3, 27.9, 26.4, 25.4, (6C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₅₂Cl₂N₃O₅ = 664.3279; found = 664.3273. Step C. Synthesis of N-((N-chlorambucilamino)alcanoyl)-L-p-tyrosinol (7, m = 5 or 10)

[0099] The appropriate N-((7V-chlorambucilamino)alcanoyl)-L-/?-tyrosine methyl ester 6 (m = 5 or 10) (0.11 mmol) was dissolved in diethyl ether and was stirred under nitrogen atmosphere. The resulting solution was cooled down with an ice and water bath, afterwards lithium borohydride (0.67 mmol) was added. The mixture was kept at 0°c for 3 h. Sodium sulfate decahydrate (0.3 g) was added. Work-up was done by diluting with diethyl ether and the organic phase was washed with saturated ammonium chloride solution (2x) and with water (4x). The organic phase was dried with anhydrous sodium sulfate, filtered and evaporated. The product was purified by flash chromatography (hexanes: acetone, 1:1) to give a pure compound in 57 % yield.

N-((6-N-chlorambucilamino)hexanoyl)-L-p-tyrosinol (7, m = 5))

IR (NaCl, v_max, cm^"1) : 3100-3400 (2x O-H and 2x N-H), 1639 (2x C=O, 2x NHCO), 1523 and 1246 (C-N-H).

¹H-NMR (Acetone-^, δ ppm) : 8.33 (IH, s, OH), 7.12 (IH, br t, partly hidden, CH₂NHCO), 7.07 and 7.06 (4H, 2d, J = 8.6 Hz and J = 8.2 Hz, 3-CH tyr and 3-CH CHL), 6.87 (IH, d, J = 8.2 Hz, CHNHCO), 6.74 (2H, d, J = 8.2 Hz, 2-CH tyr), 6.71 (2H, d, J = 9.0 Hz, 2-CH CHL), 4.06 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.51 (2H, d, J = 5.1 Hz, CH₂OH), 3.14 (2H, m, CH₂NHCO), 2.88 (IH, s, CH₂OH), 2.58-2.87 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.17 (2H, t, J = 7.4 Hz, CH₂NHCOCH₂), 2.10 (2H, t, J = 6.3 Hz, CHNHCOCH₂), 1.78- 1.93 (2H, m, CH₂CH₂CH₂Ph), 1.38-1.55 (4H, m, CH₂CH₂NHCO and CHNHCOCH₂CH₂), 1.22 (2H, m, CH₂CH₂CH₂CH₂CH₂). ¹³C-NMR (Acetone-rf₆, £ppm) : 172.54 (CONH), 172.48 (CONH), 156.1 (1-C tyr), 144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.9 (4-C tyr), 129.7 (2C, 3-C CHL), 115.3 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.8 (CH₂OH), 53.3 (2C, 2x NCH₂CH₂Cl), 53.2 (CHNH), 41.0 (2x C, 2x NCH₂CH₂Cl), 39.0 (CH₂NHCO), 36.2 (CH₂CHNH), 36.1 (CHNHCOCH₂), 35.6 (CH₂CH₂Ph), 34.3 (CH₂NHCOCH₂), 27.9 (COCH₂CH₂CH₂Ph), 26.4 (CH₂CH₂CH₂CH₂CH₂), 25.6

(NHCOCH₂CH₂CH₂CH₂CH₂), (IX CH₂ hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₂₉H₄₂Cl₂N₃O₄ = 566.2547; found = 566.2541.

N-((ll-N-chlorambuciamino)undecanoyl)-L-p-tyrosinol (7, m = 1O))

IR (KBr, v_mas, cm^"1) : 3200-3500 (2x O-H and 2x N-H), 1649 (2x C=O, NHCO),

1540 and 1212 (C-N-H).

¹H-NMR (Acetone-*/*, S ppm) : 8.28 (IH, s, OH), 7.15 (IH, br t apparent, partly hidden, CH₂NHCO), 7.07 (4H, d, J = 8.6 Hz, 3-CH tyr and 3-CH CHL), 6.85 (IH, d,

J = 8.2 Hz, CHNHCO), 6.74 (2H, d, J = 8.2 Hz, 2-CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-

CH CHL), 4.10 (IH, m, CHNH), 3.75 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.50 (2H, t,

J = 4.9 Hz, CH₂OH), 3.18 (2H, m, CH₂NHCO), 2.81 (IH, s, CH₂OH), 2.64-2.84 (2H, m, CH₂CHNH), 2.51 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.16 (2H, t, J = 7.4 Hz,

CH₂NHCOCH₂), 2.10 (2H, t, J = 7.4 Hz, CHNHCOCH₂), 1.81-1.92 (2H, m,

CH₂CH₂CH₂Ph), 1.25-1.54 (16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-^, £ ppm) : 172.5 (CONH), 172.3 (CONH), 156.1 (1-C tyr),

144.9 (1-C CHL), 131.0 (4-C CHL), 130.4 (2C, 3-C tyr), 129.8 (4-C tyr), 129.7 (2C,

3-C CHL), 115.2 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.7 (CH₂OH), 53.3 (3C, 2x

NCH₂CH₂Cl and CHNH), 41.0 (2x C, 2x NCH₂CH₂Cl), 38.9, 36.2, 35.6, 34.2, 26.8,

25.8, (8x CH₂ hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₄H₅₂Cl₂N₃O₄ = 636.3329; found = 636.3327.

Example 5. Preparation of tyrosine nitrogen mustard hybrids first generation

(isomers of 6 and 7) (convergent synthesis using D-p-tyr, DL-ø-tyr and L-»ι-tyr)

Step A. Synthesis of _J/V-((Λ^/-chlorambucilamino)alcanoyl)~tyrosine methyl ester (isomers of 6, made with D-p-tyr, DL-ø-tyr, and L-m-tyr) [00100] The tyrosine methyl ester hydrochloride 2 (0.41 mmol) was dissolved in dimethylformamide and triethylamine (0.41 mmol). The latter was added in order to neutralize the hydrochloride salt. At the same time, a solution of N-chlorambucil- amino acid derivative 16 (0.62 mmol) in dimethylformamide was activated using DCC (0.66 mmol) followed by HOBt (0.66 mmol). The tyrosine solution was then added to the activated iV-chlorambucil-amino acid derivative solution. The mixture was stirred at room temperature for 24 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 71% yield.

N-ό-chlorambucil-hexanoyl-L-m-tyrosine methyl ester (isomer of 6, m = 5)

IR (ATR, v_max, cm^"1) : 3150-3450 (O-H and 2x N-H), 1745 (C=O, COOCH₃), 1638

(2x C=O, 2x NHCO), 1517 and 1217 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 8.66 (IH, s, OH), 7.24 (IH, d, J = 7.8 Hz,

CHNHCO), 7.16 (IH, t, hidden, CH₂NHCO), 7.09 (IH, t apparent, J = 8.2 Hz, 5-CH tyr), 7.07 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.71 (2H, d, J = 8.6 Hz, 2-CH CHL), 6.64-

6.75 (3H, m apparent, 2-CH tyr, 4-CH tyr and 6-CH tyr), 4.71 (IH, m, CHNH), 3.75

(8H, dt, J = 1.2 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.66 (3H, s, OCH₃), 3.17

(2H, m, CH₂NHCO), 2.84-3.10 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.7 Hz,

CH₂CH₂Ph), 2.18 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.94

(2H, m, CH₂CH₂CH₂Ph), 1.20-1.59 (6H, #m, 3x CH₂).

¹³C-NMR (Acetone-dβ, δ ppm) : 172.5 (CONH), 172.1 (CONH), 172.0 (COOCH₃),

157.6 (1-C tyr), 144.6 (1-C CHL), 138.6 (3-C tyr), 130.6 (4-C CHL), 129.5 (2C, 3-C

CHL), 129.2 (5-C tyr), 120.1 (4-C tyr), 116.3 (2-C tyr), 113.8 (6-C tyr), 112.2 (2C, 2-

C CHL), 53.3 (CHNH), 53.0 (2C, 2x NCH₂CH₂Cl ), 51.3 (OCH₃), 40.7 (2C, 2x

NCH₂CH₂Cl), 38.7, 37.4, 35.4 (2C), 34.0, 27.7, 26.1, 25.1, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₀H₄₂Cl₂N₃O₅ = 594.2496; found = 594.2491.

N-ό-chlorambucil-hexanoyl-DL-o-tyrosine methyl ester (isomer of 6, m = 5)

IR (ATR, v_max, cm^"1) : 3100-3400 (O-H and 2x N-H), 1745 (C=O, COOCH₃), 1634

(2x C=O, 2x NHCO), 1520 and 1253 (C-N-H). ¹H-NMR (Acetone-d₆, S ppm) : 9.04 (IH, s, OH), 7.36-7.20 (2H, 2d apparent, CHNHCO and CH₂NHCO), 7.06-7.10 (2H, m, hidden, 3-CH and 5-CH tyr), 7.08 (2H, d, J = 7.0 Hz, 3-CH CHL), 6.75-6.92 (2H, 2d apparent, 4-CH and 6-CH tyr), 6.72 (2H, d, J = 6.6 Hz, 2-CH CHL), 4.67 (IH, m, CHNH), 3.75 (8H, s, 2x CH₂Cl and 2x NCH₂), 3.63 (3H, s, OCH₃), 3.17 (2H, br m, CH₂NHCO), 2.92-3.17 (2H, m, CH₂CHNH), 2.53 (2H, t, J = 7.0 Hz, CH₂CH₂Ph), 2.15 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂), 1.84-1.95 (2H, m, CH₂CH₂CH₂Ph), 1.28-1.58 (6H, #m, 3x CH₂).

1³C-NMR (Acetone-d₆, <5ppm) : 172.4 (CONH), 172.2 (2C, CONH and COOCH₃),

155.5 (1-C tyr), 144.7 (1-C CHL), 131.1 (3-C tyr), 130.7 (4-C CHL), 129.5 (2C, 3-C CHL), 128.0 (2-C tyr), 123.6 (5-C tyr), 119.5 (4-C tyr), 115.1 (6-C tyr), 112.2 (2C, 2- C CHL), 53.2 (CHNH), 53.0 (2C, 2x NCH₂CH₂Cl ), 51.2 (OCH₃), 40.7 (2C, 2x NCH₂CH₂Cl), 38.6, 35.4, 35.3, 34.0, 32.2, 29.0, 27.7, 25.8, 24.9.

ESI+ HRMS: [M + H]⁺ calculated for C₃₀H₄₂Cl₂N₃O₅ = 594.2496; found = 594.2488.

N-((6-N-chlorambucilamino)hexanoyl)-D-p-tyrosine methyl ester (isomer of 6, m =

5)

IR (KBr, v_max, cm^"1) : 3150-3450 (O-H and 2x N-H), 1743 (C=O, COOCH₃), 1640

(2x C=O, 2x NHCO), 1517 and 1223 (C-N-H).

¹H-NMR (Acetone-rf₆, <?ppm) : 7.29 (IH, d, J = 8.2 Hz, CHNHCO), 7.19 (IH, br t, J

= 5.3 Hz, CH₂NHCO), 7.06 and 7.04 (4H, 2d, J - 8.6 Hz and J = 8.6 Hz, 3-CH tyr and 3-CH CHL), 6.76 (2H, d, J - 9.0 Hz, 2-CH tyr), 6.71 (2H, d, J = 9.0 Hz, 2-CH

CHL), 4.67 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.65 (3H, s,

OCH₃), 3.16 (2H, m, CH₂NHCO), 2.78-3.11 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.6

Hz, CH₂CH₂Ph), 2.17 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂), 1.78-

1.93 (2H, m, CH₂CH₂CH₂Ph), 1.16-1.64 (6H, #m, 3x CH₂).

¹³C-NMR (Acetone-rf₆, δ ppm) : 172.8 (CONH), 172.5 (CONH), 172.4 (COOCH₃),

156.6 (1-C tyr), 144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 127.8 (4-C tyr), 115.5 (2C, 3-C tyr), 112.4 (2C, 3-C CHL), 53.9 (CHNH), 53.3 (2C, 2x NCH₂CH₂Cl ), 51.5 (OCH₃), 41.0 (2C, 2x NCH₂CH₂Cl), 39.1, 36.9, 35.7, 35.6, 34.3, 27.9, 26.4, 25.4, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₀H₄₂Cl₂N₃O₅ = 594.2496; found = 594.2483. N-ll-chlorambucil-undecanoyl-L-m-tyrosine methyl ester (isomer of 6, m = 10)

IR (ATR, v_max, cm^"1) : 3150-3400 (O-H and 2x N-H), 1745 (C=O, COOCH₃), 1642 and 1613 (2x C=O and 2x NHCO), 1517 and 1253 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 8.44 (IH, br s, OH), 7.26 (IH, d, J = 7.8 Hz,

CHNHCO), 7.10 (IH, br t apparent, hidden, CH₂NHCO), 7.04-7.13 (IH, hidden, 5-

CH tyr), 7.06 (2H, d, J = 8.2 Hz, 3-CH CHL), 6.71 (2H, d, J = 9.0 Hz, 2-CH tyr),

6.65-6.75 (3H, hidden, 2-CH tyr, 4-CH tyr and 6-CH tyr), 4.69 (IH, m, CHNH), 3.74

(8H, dt, J = 1.6 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.65 (3H, s, OCH₃), 3.19

(2H, q, J = 5.9 Hz, CH₂NHCO), 2.83-3.10 (2H, m, CH₂CHNH), 2.51 (2H, t, J = 7.6

Hz, CH₂CH₂Ph), 2.16 (4H, 2t overlapped, J = 7.2 Hz, CH₂NHCOCH₂ and

CHNHCOCH₂), 1.81-1.92 (2H, m, CH₂CH₂CH₂Ph), 1.25-1.55 (16H, #m and s, 8x

CH₂).

¹³C-NMR (Acetone-d₆, δ ppm) : 172.2 (CONH), 172.1 (2C, COOCH₃ and CONH),

157.5 (1-C tyr), 144.6 (1-C CHL), 138.6 (3-C tyr), 130.7 (4-C CHL), 129.5 (2C, 3-C

CHL), 129.2 (5-C tyr), 120.1 (4-C tyr), 116.1 (2-C tyr), 113.6 (6-C tyr), 112.2 (2C, 3-

C CHL), 53.4 (CHNH), 53.0 (2C, 2x NCH₂CH₂Cl ), 51.3 (OCH₃), 40.7 (2C, 2x

NCH₂CH₂Cl), 38.8, 37.4, 35.5, 35.3, 34.0, 29.6, 29.2, 29.1, 28.9, 27.7, 26.7, 25.4,

(2C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₅₂Cl₂N₃O₅ = 664.3279; found = 664.3270.

N-ll-chlorambucil-undecanoyl-DL-o-tyrosine methyl ester (isomer of 6, m = 10) IR (ATR, v_max, cm^"1) : 3200-3400 (O-H and 2x N-H), 1749 (C=O, COOCH₃), 1645 and 1602 (2x C=O and 2x NHCO), 1520 and 1213 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 8.83 (IH, br s, OH), 7.31 (IH, d, J = 7.4 Hz, CHNHCO), 7.03-7.12 (3H, m, hidden, CH₂NHCO, 3-CH tyr and 5-CH tyr), 7.07 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.69-6.89 (2H, m, hidden, 4-CH tyr and 6-CH tyr), 6.71 (2H, d, J = 9.0 Hz, 2-CH CHL), 4.69 (IH, m, CHNH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.62 (3H, s, OCH₃), 3.19 (2H, q, J = 5.9 Hz, CH₂NHCO), 2.90-3.16 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.5 Hz, CH₂CH₂Ph), 2.17 (4H, m, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.90 (2H, m, CH₂CH₂CH₂Ph), 1.25-1.54 (16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-d₆, £ppm) : 172.3 (2C, 2x CONH), 172.1 (COOCH₃), 155.4 (1- C tyr), 144.7 (1-C CHL), 131.1 (3-C tyr), 130.7 (4-C CHL), 129.4 (2C, 3-C CHL), 128.0 (2-C tyr), 123.6 (5-C tyr), 119.4 (4-C tyr), 115.1 (6-C tyr), 112.2 (2C, 3-C CHL), 53.0 (2C, 2x NCH₂CH₂Cl ), 52.8 (CHNH), 51.1 (OCH₃), 40.7 (2C, 2x NCH₂CH₂Cl), 38.8, 35.5, 35.3, 34.0, 33.7, 32.4, 29.5, 29.2, 29.1, 29.0, 28.8, 27.7, 26.6, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₅₂Cl₂N₃O₅ = 664.3279; found = 664.3271.

N-((ll-N-chlorambucilamino)undecanoyl)-D-p-tyrosine methyl ester (isomer of 6, m = 10)

IR (NaCl, v_max, cm^"1) : 3150-3400 (O-H and 2x N-H), 1742 (C=O, COOCH₃), 1645 and 1619 (2x C=O and 2x NHCO), 1518 and 1244 (C-N-H).

¹H-NMR (Acetone-d₆, δppm) : 7.18 (IH, d, J = 7.8 Hz, CHNHCO), 7.10 (IH, br t apparent, partly hidden, CH₂NHCO), 7.06 and 7.03 (4H, 2d, J = 8.6 Hz and J = 8.6

Hz, 3-CH tyr and 3-CH CHL), 6.75 (2H, d, J = 7.8 Hz, 2-CHtyr), 6.71 (2H, d, J = 8.6

Hz, 2-CH CHL), 4.65 (IH, m, CHNH), 3.75 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.65

(3H, s, OCH₃), 3.19 (2H, q, J = 6.5 Hz, CH₂NHCO), 2.81-3.07 (2H, m, CH₂CHNH),

2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.16 (4H, 2d overlapped, J = 7.6 Hz,

CH₂NHCOCH₂ and CHNHCOCH₂), 1.78-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.26-1.59

(16H, #m and s, 8x CH₂), (IH, OH, hidden).

¹³C-NMR (Acetone-d₆, δppm) : 172.41 (2C, 2x CONH), 172.35 (COOCH₃), 156.6

(1-C tyr), 144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.7 (2C, 3-C

CHL), 127.8 (4-C tyr), 115.3 (2C, 3-C tyr), 112.4 (2C, 3-C CHL), 53.9 (CHNH), 53.3

(2C, 2x NCH₂CH₂Cl ), 51.5 (OCH₃), 41.0 (2C, 2x NCH₂CH₂Cl), 39.0, 36.9, 35.7,

35.6, 34.2, 29.4 (2C), 27.9, 26.8, 25.7, (4C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₅₂Cl₂N₃O₅ = 664.3279; found = 664.3269.

Step B. Synthesis of Λ^/-((Λ^r-chlorambuciIamino)alcanoyl-tyrosinol (isomers of 7, made with D-/»-tyr, DL-ø-tyr, and L-m-tyr)

[00101] The Λ^/-((7Y-chlorambucilamino)alcanoyl)-tyrosine methyl ester 6 (0.12 mmol) was dissolved in diethyl ether and DCM and was stirred under nitrogen atmosphere. The resulting solution was cooling down with an ice and water bath, afterwards lithium borohydride (0.73 mmol) was added. The mixture was kept at 0°c for 3 h. Sodium sulfate decahydrate (0.3 g) was added. Work-up was done by diluting with diethyl ether and washing the organic phase with saturated ammonium chloride solution (2x) and with water (4x). The organic phase was dried with anhydrous sodium sulfate, filtered and evaporated. The product was purified by flash chromatography (hexanes: acetone, 3:2) to give a pure compound in 82 % yield.

N-ό-chlorambucil-hexanoyl-L-m-tyrosinol (isomer of 7, m = 5)

IR (ATR, v_max, cm^"1) : 3100-3400 (2x O-H and 2x N-H), 1683 (2x C=O, 2x NHCO),

1520 and 1253 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 8.59 (IH, s, OH), 7.20 (IH, br t, J = 5.3 Hz, CH₂NHCO), 7.07 (IH, t, J = 7.4 Hz, 5-CH tyr), 7.06 (2H, d, J = 8.6 Hz, 3-CHCHL), 6.98 (IH, d, J = 8.5 Hz, CHNHCO), 6.64-6.76 (3H, m, hidden, 2-CH tyr, 4-CH tyr and 6-CH tyr), 6.71 (2H, d, J = 8.5 Hz, 2-CH CHL), 4.13 (2H, m, CHNH and CH₂OH), 3.74 (8H, dt, J = 1.2 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.52 (2H, t, J = 5.1 Hz, CH₂OH), 3.16 (2H, m, CH₂NHCO), 2.64-2.91 (2H, m, CH₂CHNH), 2.51 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.14 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.89 (2H, m, CH₂CH₂CH₂Ph), 1.20-1.58 (6H, #m, 3x CH₂). ¹³C-NMR (Acetone-d₆, S ppm) : 172.51 (CONH), 172.46 (CONH), 157.5 (1-C tyr), 144.6 (1-C CHL), 140.5 (3-C tyr), 130.6 (4-C CHL), 129.5 (2C, 3-C CHL), 129.0 (5- C tyr), 120.3 (4-C tyr), 116.3 (2-C tyr), 113.1 (6-C tyr), 112.2 (2C, 2-C CHL), 63.4 (CH₂OH), 53.0 (2C, 2x NCH₂CH₂Cl), 52.8 (CHNH), 40.8 (2x C, 2x NCH₂CH₂Cl), 38.7 (CH₂NHCO), 36.8 (CH₂CHNH), 35.9 (CHNHCOCH₂), 35.6 (CH₂CH₂Ph), 35.4 (CH₂NHCOCH₂), 34.0, 27.7, 26.2, 25.3.

N-ό-chlorambucil-hexanoyl-DL-o-tyrosinol (isomer of 7, m = 5)

IR (ATR, v_max, cm^"1) : 3100-3400 (2x O-H and 2x N-H), 1638 (2x C=O, 2x NHCO),

1520 and 1242 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 9.16 (IH, br s, OH), 7.37 (IH, d, J = 6.6 Hz, CHNHCO), 7.17 (IH, br t, J = 5.1 Hz, CH₂NHCO), 7.02-7.11 (2H, m apparent, 3- CH tyr and 5-CH tyr), 7.06 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.70-6.88 (2H, m apparent, 4-CH tyr and 6-CH tyr), 6.71 (2H, d, J = 8.5 Hz, 2-CH CHL), 4.01 (IH, m, CHNH), 3.89 (IH, br s, hidden, CH₂OH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.51 (2H, d apparent, J = 5.8 Hz, CH₂OH), 3.19 (2H, dq, J = 1.8 Hz and J = 6.8 Hz, CH₂NHCO), 2.71-2.93 (2H, m, CH₂CHNH), 2.51 (2H, t, J =

7.4 Hz, CH₂CH₂Ph), 2.19 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂),

1.78-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.27-1.67 (6H, #m, 3x CH₂).

¹³C-NMR (Acetone-d₆, £ ppm) : 173.8 (CONH), 172.4 (CONH), 156.0 (1-C tyr),

144.7 (1-C CHL), 131.0 (4-C CHL), 130.7 (3-C tyr), 129.5 (2C, 3-C CHL), 127.7 (5-

C tyr), 124.7 (2-C tyr), 119.3 (4-C tyr), 115.7 (6-C tyr), 112.2 (2C, 2-C CHL), 62.6

(CH₂OH), 53.2 (CHNH), 53.0 (2C, 2x NCH₂CH₂Cl), 40.8 (2x C, 2x NCH₂CH₂Cl),

38.6, 35.7, 35.4, 34.0, 31.8, 29.2, 27.7, 26.1, 25.1.

ESI+ HRMS: [M + H]⁺ calculated for C₂₉H₄₂Cl₂N₃O₄ = 566.2547; found = 566.2540.

N-ό-chlorambucil-hexanoyl-D-p-tyrosinol (isomer of 7, m = 5)

IR (KBr, v_max, cm^"1) : 3100-3450 (2x O-H and 2x N-H), 1684 (2x C=O, 2x NHCO),

1516 and 1239 (C-N-H).

¹H-NMR (Acetone-rf₆, £ ppm) : 8.31 (IH, s, OH), 7.12 (IH, br t apparent, partly hidden, CH₂NHCO), 7.07 and 7.06 (4H, 2d, J = 8.6 Hz and J = 8.2 Hz, 3-CH tyr and

3-CH CHL), 6.84 (IH, d, J - 8.2 Hz, CHNHCO), 6.74 (2H, d, J = 8.6 Hz, 2-CH tyr),

6.72 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.05 (IH, m, CHNH), 3.99 (IH, s, partly hidden,

CH₂OH), 3.75 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.51 (2H, t, J - 5.1 Hz, CH₂OH),

3.14 (2H, m, CH₂NHCO), 2.58-2.88 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.6 Hz,

CH₂CH₂Ph), 2.17 and 2.13 (4H, 2t apparent, J = 7.4 Hz and J = 8.2

HZ₅CH₂NHCOCH₂ and CHNHCOCH₂), 1.81-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.18-

1.55 (6H, #m, 3x CH₂).

¹³C-NMR (Acetone-</₆, <?ppm) : 172.52 (CONH), 172.43 (CONH), 156.1 (1-C tyr),

144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.9 (4-C tyr), 129.7 (2C,

3-C CHL), 115.3 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 63.8 (CH₂OH), 53.3 (2C, 2x

NCH₂CH₂Cl), 53.2 (CHNH), 41.0 (2x C, 2x NCH₂CH₂Cl), 39.0 (CH₂NHCO), 36.2

(CH₂CHNH), 36.1 (CHNHCOCH₂), 35.6 (CH₂CH₂Ph), 34.2 (CH₂NHCOCH₂), 27.9

(COCH₂CH₂CH₂Ph), 26.4 (CH₂CH₂CH₂CH₂CH₂), 25.6

(NHCOCH₂CH₂CH₂CH₂CH₂), (IX CH₂ hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₂₉H₄₂Cl₂N₃O₄ = 566.2547; found = 566.2537. N-ll-chlorambucil-undecanoyl-L-m-tyrosinol (isomer of 7, m = 10)

IR (ATR, v_max, cm^"1) : 3100-3400 (2x O-H and 2x N-H), 1634 (2x C=O, NHCO),

1520 and 1246 (C-N-H).

¹H-NMR (Acetone-d₆, S ppm) : 8.45 (IH, s, OH), 7.14 (IH, br t apparent,

CH₂NHCO), 7.07 (IH, t, J = 7.6 Hz, 5-CH tyr), 7.06 (2H, d, J = 8.6 Hz, 3-CH CHL),

6.97 (IH, d, J = 7.8 Hz, CHNHCO), 6.65-6.77 (3H, m, hidden, 2-CH tyr, 4-CH tyr and 6-CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.08 (IH, m, CHNH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.52 (2H, t, J = 5.1 Hz,

CH₂OH), 3.19 (2H, q, J = 6.5 Hz, CH₂NHCO), 2.92 (IH, s, CH₂OH), 2.65-2.91 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.4 Hz, CH₂CH₂Ph), 2.13 (4H, m apparent,

CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.25-1.56

(16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-d₆, δ ppm) : 172.5 (CONH), 172.2 (CONH), 157.5 (1-C tyr),

144.6 (1-C CHL), 140.6 (3-C tyr), 130.7 (4-C CHL), 129.5 (2C, 3-C CHL), 129.0 (5- C tyr), 120.3 (4-C tyr), 116.2 (2-C tyr), 113.1 (6-C tyr), 112.2 (2C, 2-C CHL), 63.3 (CH₂OH), 53.0 (2C, 2x NCH₂CH₂Cl), 52.8 (CHNH), 40.7 (2C, 2x NCH₂CH₂Cl), 38.8, 36.8, 36.0, 35.4, 34.0, 29.6, 29.3, 29.2, 29.1, 29.0, 28.9, 27.7, 26.7, 25.6.

ESI+ HRMS: [M + H]⁺ calculated for C₃₄H₅₂Cl₂N₃O₄ = 636.3329; found = 636.3326.

N-ll-chlorambucil-undecanoyl-DL-o-tyrosinol (isomer of 7, m = 10)

IR (ATR, v_max, cm^"1) : 3100-3400 (2x O-H and 2x N-H), 1631 (2x C=O, NHCO),

1524 and 1246 (C-N-H).

¹H-NMR (Acetone-d₆, δ ppm) : 9.11 (IH, s, OH), 7.36 (IH, d, J = 5.9 Hz,

CHNHCO), 7.02-7.10 (3H, m, hidden, 3-CH tyr, 5-CH tyr and CH₂NHCO), 7.07

(2H, d, J = 8.6 Hz, 3-CH CHL), 6.68-6.86 (2H, m, hidden, 4-CH tyr and 6-CH tyr),

6.71 (2H, d, J = 8.6 Hz, 2-CH CHL), 4.11 (IH, m, CHNH), 3.74 (8H, dt, J = 1.6 Hz and J = 5.1 Hz, 2x CH₂Cl and 2x NCH₂), 3.58 (2H,br d, J = 5.1 Hz, CH₂OH), 3.18

(2H, q, J = 5.8 Hz, CH₂NHCO), 2.70-2.93 (3H, m and br s, CH₂CHNH and CH₂OH),

2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.22 (2H, t, J = 7.2 Hz, CH₂NHCOCH₂), 2.16

(2H, t, J = 7.2 Hz, CHNHCOCH₂), 1.81-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.29-1.59

(16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-d₆, £ ppm) : 173.9 (CONH), 172.0 (CONH), 156.0 (1-C tyr),

144.7 (1-C CHL), 130.9 (4-C CHL), 130.7 (3-C tyr), 129.4 (2C₅ 3-C CHL), 127.7 (5- C tyr), 124.6 (2-C tyr), 119.2 (4-C tyr), 115.8 (6-C tyr), 112.2 (2C, 2-C CHL), 62.3 (CH₂OH), 53.1 (CHNH), 53.0 (2C, 2x NCH₂CH₂Cl), 40.7 (2x C, 2x NCH₂CH₂Cl),

38.8, 35.7, 35.3, 34.0, 31.9, 29.6, 29.2 (3C), 29.0 (2C), 26.7, (2C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₄H₅₂Cl₂N₃O₄ = 636.3329; found = 636.3318.

N-((ll-N-chlorambucilamino)undecanoyl)-D-p-tyrosinol (isomer of 7, m = 10)

IR (NaCl, v_max, cm^"1) : 3200-3400 (2x O-H and 2x N-H), 1641 (2x C=O, NHCO),

1517 and 1248 (C-N-H).

¹H-NMR (Acetone-rf₆, δ ppm) : 8.31 (IH, s, OH), 7.10 (IH, br t, J = 8.6 Hz,

CH₂NHCO), 7.07 (4H, d, J = 8.6 Hz, 3-CH tyr and 3-CH CHL), 6.84 (IH, d, J = 8.6

Hz, CHNHCO), 6.74 (2H, d, J = 8.2 Hz, 2-CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH

CHL), 4.06 (IH, m, CHNH), 4.02 (IH, br s, partly hidden, CH₂OH), 3.75 (8H, m, 2x

CH₂Cl and 2x NCH₂), 3.50 (2H,br d, J = 4.7 Hz, CH₂OH), 3.18 (2H, q, J = 6.4 Hz,

CH₂NHCO), 2.61-2.86 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph),

2.16 (2H, t, J = 7.4 Hz, CH₂NHCOCH₂), 2.14 (2H, t, J = 7.4 Hz, CHNHCOCH₂),

1.81-1.92 (2H, m, CH₂CH₂CH₂Ph), 126-1.55 (16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-*^, δ ppm) : 172.6 (CONH), 172.3 (CONH), 156.1 (1-C tyr),

144.9 (1-C CHL), 130.9 (4-C CHL), 130.4 (2C, 3-C tyr), 129.8 (4-C tyr), 129.7 (2C,

NCH₂CH₂Cl), 53.2 (CHNH), 41.0 (2x C, 2x NCH₂CH₂Cl), 39.0, 36.2, 35.6, 34.2,

27.9, 26.8, 25.8, (7x CH₂ hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₄H₅₂Cl₂N₃O₄ = 636.3329; found = 636.3319.

Example 6. Preparation of tyrosine nitrogen mustard hybrids second generation (11) (using L-p-tyr)

Step A. Synthesis of TV-Boc-hydroxyphenyl-L-p-tyrosinamide (10)

[00102] The aminophenol (3.09 mmol) was dissolved in dimethylformamide and triethylamine (2.94 mmol) was added. Separately, iV-Boc-tyrosine 9 (2.94 mmol) derivative was dissolved in dimethylformamide, and DCC (3.24 mmol) followed by HOBt (3.24 mmol) were added to activate the acid function. Then, the aminophenol solution was added to the activated tyrosine solution. The resulting mixture was stirred at room temperature for 25 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 96% yield.

10 (made with/?αrø-aminophenol)

N-Boc-p-hydroxyphenyl-L-p-tyrosinamide (10 (made with jrøra-aminophenol)

IR (KBr, v_max, cm^"1) : 3100-3600 (2x O-H and 2x N-H), 1669 (2x C=O, NHCOO and NHCO), 1516 and 1246 (C-N-H).

¹H-NMR (Acetone-^, Jppm) : 9.05 (IH, s, OH AP), 8.24 (2H, br s, OH tyr and NH

AP), 7.41 (2H, d, J = 9.0 Hz, 3-CH AP), 7.10 (2H, d, J = 8.2 Hz, 3-CHtyr), 6.76 (2H, d, J = 9.0 Hz, 2-CH AP), 6.74 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.11 (IH, d, J = 8.2 Hz,

NH-Boc), 4.42 (IH, m, CHNH),2.80-3.16 (2H, m, CH₂CHNH), 1.36 (9H, s, 3x CH₃).

¹³C-NMR (Acetone-4, <?ppm) : 170.2 (CONH), 156.3 (1-C tyr), 155.7 (NHCOO),

154.0 (1-C AP), 131.1 (4-C AP), 130.6 (2C, 3-C tyr), 128.4 (4-C tyr), 121.8 (2C, 3-C

CHL), 115.3 (4C, 2-C tyr and 2-C CHL), 78.8 (C(CH₃)₃), 57.0 (CHNH), 37.8

(CH₂CHNH), 27.9 (3C, 3x CH₃).

ESI+ HRMS: [M + Na]⁺ calculated for C₂₀H₂₄N₂NaO₅ = 395.1577; found =

395.1572; [M + H]⁺ calculated for C₂₀H₂₅N₂O₅ = 373.1758; found = 373.1756.

10 (made with metø-aminophenol)

N-Boc-m-hydroxyphenyl-L-p-tyrosinamide (10 (made with meto-aminophenol)

IR (KBr, v_max, cm-¹) : 3100-3600 (2x O-H and 2x N-H), 1671 (2x C=O, NHCOO and NHCO), 1514 and 1240 (C-N-H). ¹H-NMR (Acetone-^, <?ppm) : 9.20 (IH, s, OH AP), 8.39 (IH, br s, OH tyr), 8.27 (IH, br s, NH AP), 7.34 (IH, t, J = 2.0 Hz, 4-CH AP), 7.10 (2H, d, J = 8.2 Hz, 3-CH tyr), 7.06 (IH, d, J = 7.8 Hz, 2-CH AP), 6.97 (IH, dt, J = 1.2 Hz and J = 8.6 Hz, 5-CH AP), 6.74 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.56 (IH, dq, J = 1.2 Hz and J = 7.8 Hz, 6- CH AP), 6.16 (IH, d, J - 7.4 Hz, NH-Boc), 4.45 (IH, m, CHNH),2.85-3.15 (2H, m, CH₂CHNH), 1.36 (9H, s, 3x CH₃).

¹³C-NMR (Acetone-rf₆, £ ppm) : 170.8 (CONH), 158.0 (1-C AP), 156.3 (1-C tyr), 155.8 (NHCOO), 140.1(3-C AP), 130.6 (2C, 3-C tyr), 129.6 (5-C AP), 128.2 (4-C tyr), 115.4 (2C, 2-C tyr), 111.1 (2C, 4-C AP and 6-C AP), 107.2 (2-C AP), 78.6 (C(CH₃)₃), 57.2 (CHNH), 37.7 (CH₂CHNH), 27.9 (3C, 3x CH₃).

ESI+ HRMS: [M + Na]⁺ calculated for C₂₀H₂₄N₂NaO₅ = 395.1577; found = 395.1578; [M + H]⁺ calculated for C₂₀H₂₅N₂O₅ = 373.1758; found = 373.1760.

10 (made with ort/20-aminophenol)

N-Boc-o-hydroxyphenyl-L-p-tyrosinamide (10 (made with ortto-aminophenol)

IR (KBr, v_max, cm^"1) : 3100-3500 (2x 0-H and 2x N-H), 1686 (2x C=O, NHCOO and NHCO), 1519 and 1257 (C-N-H).

¹H-NMR (Acetone-^, £ ppm) : 9.21 (2H, br s, 2x OH), 8.26-9.19 (IH, br s, NH

AP), 7.73 (IH, dd, J = 1.6 Hz and J = 7.8 Hz, 3-CH AP), 7.15 (2H, d, J = 8.6 Hz, 3-

CH tyr), 6.78 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.74-7.04 (3H, #m, 4-CH AP, 5-CH AP and 6-CH AP), 6.41 (IH, d, J = 7.0 Hz, NH-Boc), 4.55 (IH, m, CHNH), 2.92-3.26

(2H, m, CH₂CHNH), 1.37 (9H, s, 3x CH₃).

¹³C-NMR (Acetone-^, <?ppm) : 171.8 (CONH), 156.4 (1-C tyr), 156.0 (NHCOO),

148.1 (1-C AP), 130.6 (2C, 3-C tyr), 128.3 (4-C tyr), 126.5 (2-C AP), 125.6 (5-C AP),

121.9 (3-C AP), 120.0 (4-C AP), 116.9 (6-C AP), 115.5 (2C, 2-C tyr), 79.3 (C(CH₃)₃),

57.3 (CHNH), 37.2 (CH₂CHNH), 27.9 (3C, 3x CH₃).

395.1582. Step B. Synthesis of iV-chlorambucil-hydroxyphenyl-L-p-tyrosinaniide (11)

[00103] The N-Boc-hydroxyphenyl-L-p-tyrosinamide 10 (0.16 mmol) was dissolved in dichloromethane and trifluoro acetic acid (1.60 mmol) was added. The solution was stirred at room temperature for 24 h. After evaporation, the resulting trifluoroacetic salt was dissolved in dimethylformamide and neutralized with triethylamine (0.16 mmol). Simultaneously, chlorambucil (0.24 mmol) was dissolved in dimethylformamide, and DCC (0.25 mmol) followed by HOBt (0.25 mmol) were added. Then, the hydroxyphenyltyrosamide solution was added to the activated chlorambucil solution. The resulting mixture was stirred at room temperature for 27 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 44% yield.

11 (made with ^αra-aminophenol)

N-chlorambucil-p-hydroxyphenyl-L-p-tyrosinamide (11 (made with para- aminophenol)

IR (KBr, v_max, cm^"1) : 3100-3600 (2x O-H and 2x N-H), 1654 (2x C=O, NHCO),

1517 and 1244 (C-N-H).

¹H-NMR (Acetone-rf₆, £ppm) : 9.15 (IH, s, OH AP), 8.18 and 8.20 (2H, 2s, OH tyr and NH AP), 7.41 (IH, d, partly hidden, NH tyr), 7.40 (2H, d, J = 9.0 Hz, 3-CH AP),

7.11 (2H, d, J = 8.6 Hz, 3-CH tyr), 7.01 (2H, d, J = 9.0 Hz, 3-CH CHL), 6.66-6.77

(6H, 3d, J = 9.0 Hz, J = 8.6 Hz and J = 9.0 Hz, 2-CH AP, 2-CH tyr and 2-CH CHL), 4.78 (IH, m, CHNH), 3.73 (8H, m, 2x CH₂Cl and 2x NCH₂), 2.84-3.15 (2H, m,

CH₂CHNH), 2.44 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.21 (2H, t, J = 7.2 Hz,

NHCOCH₂), 1.76-1.87 (2H, m, CH₂CH₂CH₂Ph).

¹³C-NMR (Acetone-^, <?ppm) : 172.9 (CO CHL), 170.0 (CO tyr), 156.3 (1-C tyr),

154.0 (1-C AP), 144.8 (1-C CHL), 131.2 (4-C AP), 130.9 (4-C tyr), 130.6 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.5 (4-C CHL), 121.6 (2C, 3-C AP), 115.3 (4C, 2-C tyr and 2-C AP), 112.4 (2C, 2-C CHL), 55.6 (CHNH), 53.3 (2C, NCH₂CH₂Cl), 41.0 (2C,

NCH₂CH₂Cl), 37.5, 35.2, 34.1, 27.7.

ESI+ HRMS: [M + H]⁺ calculated for C₂₉H₃₄Cl₂N₃O₄ = 558.1921; found = 558.1929.

11 (made with metø-aminophenol)

N-chlorambucil-m-hydroxyphenyl-L-p-tyrosinamide (11 (made with meta- aminophenol)

IR (NaCl, v_max, cm-¹) : 3100-3500 (2x O-H and 2x N-H), 1656 (2x C=O, NHCO),

1516 and 1239 (C-N-H).

¹H-NMR (Acetone-*/,;, £ppm) : 9.30 (IH, s, OH AP), 8.40 (IH, s, OH tyr), 8.22 (IH, s, NH AP), 7.44 (IH, d, J = 8.2 Hz, NH tyr), 7.34 (IH, t, J = 2.1 Hz, 4-CH AP), 7.11

(2H, d, J = 8.6 Hz, 3-CH tyr), 7.01 (2H, d, J = 9.0 Hz, 3-CH CHL), 6.96-7.14 (2H, m apparent, partly hidden, 2-CH and 5-CH AP), 6.73 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.68

(2H, J = 8.6 Hz, 2-CH CHL), 6.55 (IH, dq, J = 1.2 Hz and J = 7.8 Hz, 6-CH AP),

4.80 (IH, m, CHNH), 3.73 (8H, m, 2x CH₂Cl and 2x NCH₂), 2.84-3.17 (2H, m,

CH₂CHNH), 2.44 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.22 (2H, t, J = 7.2 Hz,

NHCOCH₂), 1.73-1.87 (2H, m, CH₂CH₂CH₂Ph).

¹³C-NMR (Acetone-rf₆, £ppm) : 173.1 (CO CHL), 170.4 (CO tyr), 158.0 (1-C AP),

156.3 (1-C tyr), 144.8 (1-C CHL), 140.2 (3-C AP), 130.8 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 129.6 (5-C AP), 128.4 (4-C CHL), 115.3 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 110.9 (2C, 4-C AP and 6-C AP), 107.0 (2-C AP), 55.8 (CHNH), 53.3 (2C, NCH₂CH₂Cl ), 41.0 (2C, NCH₂CH₂Cl ), 37.3, 35.2, 34.0, 27.7. ESI+ HRMS: [M + H]⁺ calculated for C₂₉H₃₄Cl₂N₃O₄ = 558.1921; found = 558.1925.

11 (made with or^o-aminophenol)

N-chlorambucil-o-hydroxyphenyl-L-p-tyrosinamide (11 (made with ortho- aminophenol)

IR (NaCl, v_max, cm^"1) : 3100-3400 (2x O-H and 2x N-H), 1704 and 1656 (2x C=O,

NHCO), 1523 and 1257 (C-N-H).

¹H-NMR (Acetone-rf₆, £ppm) : 9.16 (IH, s, OH AP), 8.29 (IH, d, J = 7.5 Hz, NH tyr), 8.16 (IH, s, OH tyr), 7.58 (IH, d, J = 7.5 Hz, 3-CH AP), 6.97-7.17 (7H, #m apparent, 3-CH CHL, 3-CH tyr, 4-CH AP, 5-CH AP and NH AP), 6.66-6.80 (5H, #m apparent, 2-CH tyr, 2-CH CHL and 6-CH AP), 4.72 (IH, m, CHNH), 3.73 (8H, m, 2x

CH₂Cl and 2x NCH₂), 2.79-3.24 (2H, m, CH₂CHNH), 2.63 (2H, t, J = 7.6 Hz,

CH₂CH₂Ph), 2.20 (2H, t, J = 7.4 Hz, CHNHCOCH₂), 1.76-1.87 (2H, m,

CH₂CH₂CH₂Ph).

¹³C-NMR (Acetone-rf₆, <? ppm) : 174.0 (CONH), 171.6 (CO tyr), 156.3 (1-C tyr),

145.0 (1-C AP), 144.9 (1-C CHL), 130.7 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-

C CHL), 128.6 (4-C CHL), 126.0 (2-C AP), 123.8 (5-C AP), 122.9 (3-C AP), 121.5

(4-C AP), 115.4 (2C, 2-C tyr), 112.5 (6-C AP), 112.4 (2C, 2-C CHL), 55.9 (CHNH),

53.3 (2C, NCH₂CH₂Cl ), 41.0 (2C, NCH₂CH₂Cl ), 35.2, 34.1, 34.0, 27.8.

ESI+ HRMS: [M + H]⁺ calculated for C₂₉H₃₄Cl₂N₃O₄ = 558.1921; found = 558.1925.

Example 7. Preparation of tyrosine nitrogen mustard hybrids second generation (15) (direct synthesis using />-aminophenol and m = 5)

Step A. Synthesis of iV-ffiV-Boc-amino)alcanoyl)-/7-hydroxyphenyl-L-p- tyrosinamide (13)

[00104] The N-Boc-p-hydroxyphenyl-L-p-tyrosinamide derivative 10 (0.57 mmol, see example 6) was dissolved in dichloromethane and trifluoroacetic acid (5.70 mmol) was added. The solution was stirred at room temperature for 29 h. After evaporation, the resulting trifluoroacetic salt was dissolved in dimethylformamide and neutralized with triethylamine (0.57 mmol). Separately, iV-Boc-amino acid alkyl chain (Boc-aa, m = 5, 0.60 mmol) was dissolved in dimethylformamide, and DCC (0.63 mmol) followed by HOBt (0.63 mmol) were added. Then, the p- hydroxyphenyltyrosamide solution was added to the activated amino acid chain solution. The resulting mixture was stirred at room temperature for 28 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 46% yield.

13, m— 5 or 10 (made with pαra-aminophenol)

N-((N-Boc-amino)hexanoyl)-p-hydroxyphenyl-L-p-tyrosinamide (13, m = 5)

IR (KBr, v_max, cm^"1) : 3150-3450 (2x O-H and 3x N-H), 1666 (3x C=O, NHCOO and 2x NHCO).

¹H-NMR (Acetone-*^, Jppm) : 9.36 (IH, s, OH AP), 8.44 (IH, br s, OH tyr), 7.71

(2H, d, J = 8.2 Hz, NH AP), 7.41 (2H, d, J = 8.4 Hz, 3-CH AP), 7.10 (2H, d, J = 8.2

Hz, 3-CH tyr), 6.76 and 6.74 (4H, 2d, J = 9.0 Hz and J = 8.6 Hz, 2-CH AP and 2-CH tyr), 5.97 (IH, br t, J = 5.9 Hz, NH-Boc), 4.86 (IH, m, CHNH), 2.85-3.18 (4H, 2m, CH₂CHNH and CH₂NHCO), 2.19 (2H, t apparent, partly hidden, NHCOCH₂), 1.41

(9H, s, 3x CH₃), 1.21-1.51 (6H, #m, 3x CH₂).

¹³C-NMR (Acetone-d₆, δppm) : 173.4 (CONH), 170.3 (CONH), 156.3 (2C, 1-C tyr and NHCOO), 154.1 (1-C AP), 131.0 (4-C AP), 130.6 (2C, 3-C tyr), 128.4 (4-C tyr),

121.8 (2C, 3-C CHL), 115.4 (4C, 2-C tyr and 2-C CHL), 78.1 (C(CH₃)₃), 59.9

(CHNH), 40.4 (CH₂NH), 37.8 (CH₂CHNH), 35.9 (NHCOCH₂), 29.6, 28.1 (3C, 3x

CH₃), 26.4, 25.5.

ESI+ HRMS: [M + Na]⁺ calculated for C₂₆H₃₅N₃NaO₆ = 508.2418; found =

508.2422; [M + H]⁺ calculated for C₂₆H₃₆N₃O₆ = 486.2599; found = 486.2606.

N-((N-Boc-amino)undecanoyl)-p-hydroxyphenyl-L-p-tyrosinamide (13, m = 10) IR (KBr, v_max, cm^"1) : 3100-3400 (2x O-H and 3x N-H), 1667 and 1681 (3x C=O, NHCOO and 2x NHCO).

1H-NMR (Acetone-rf₆, £ppm) : 9.05 (IH, s, OH AP), 8.18 (2H, s, OH tyr and NH),

7.40 (2H, d, J = 9.0 Hz, 3-CH AP), 7.28 (IH, br d, J = 7.8 Hz, NH), 7.09 (2H, d, J = 8.6 Hz, 3-CH tyr), 6.73 and 6.75 (4H, 2d, J = 8.6 Hz and J = 9.0 Hz, 2-CH tyr and 2- CH AP), 5.93 (IH, br s, NH-Boc), 4.70 (IH, m, CHNH), 2.94-3.10 (4H, 2m, CH₂CHNH and CH₂NHCO), 2.19 (2H, t, J = 7.2 Hz, NHCOCH₂), 1.40 (9H, s, 3x CH₃), 1.26-1.50 (16H, #m, 8x CH₂).

¹³C-NMR (Acetone-d₆, δppm) : 172.7 (CONH), 169.6 (CONH), 156.3 (2C, 1-C tyr and NHCOO), 153.9 (1-C AP), 131.3 (4-C AP), 130.5 (2C, 3-C tyr), 128.5 (4-C tyr), 121.5 (2C, 3-C CHL), 115.2 (4C, 2-C tyr and 2-C CHL), 77.7 (C(CH₃)₃), 55.5 (CHNH), 40.4 (CH₂NH), 37.3 (CH₂CHNH), 35.9 (NHCOCH₂), 32.0, 26.8, 25.7, 22.7 (7C hidden).

ESI+ HRMS: [M + Na]⁺ calculated for C₃₁H₄₅N₃NaO₆ = 578.3201; found = 578.3200; [M + H]⁺ calculated for C₃iH₄₆N₃O₆ = 556.3381; found = 556.3381.

Step B. Synthesis of Λ^L((Λ^Lchlorambucilamino)hexanoyl)-p- hydroxyphenyl-L-p-tyrosinamide (15)

[00105] The TV-Boc-p-hydroxyphenyl-L-p-tyrosinamide 13 (0.17 mmol) was dissolved in dichloromethane and treated with trifluoroacetic acid (1.70 mmol). The solution was stirred at room temperature for 22.5 h. After evaporation, the resulting trifluoroacetic salt was dissolved in dimethylformamide and neutralized with triethylamine (0.18 mmol). Simultaneously, chlorambucil (0.17 mmol) was dissolved in dimethylformamide and treated with DCC (0.19 mmol) and with HOBt (0.19 mmol). Then, the tyrosamide solution was added to the activated chlorambucil solution. The resulting mixture was stirred at room temperature for 44 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purifted by flash chromatography (chloroform: methanol, 97:3) to give a pure compound in 19% yield.

15, m = 5 or 10 (made with pαra-aminophenol)

N-((N-chlorambucilamino)hexanoyl)-p-hydroxyphenyl-L-p-tyrosinamide (15, m =

5)

IR (KBr, v_max, cm^'1): 3100-3500 (2x O-H and 3x N-H), 1639-1613 (3x C=O,

NHCO), 1516 and 1230 (C-N-H).

¹H-NMR (Acetone-</₆, δppm) : 9.13 (IH, s, OH AP), 8.24 and 8.34 (2H, 2s, OH tyr and NH AP), 7.41 (2H, d, J = 9.0 Hz, 3-CH AP), 7.34 (IH, d, J = 8.20 Hz, NH tyr),

7.13 (IH, br t, J = 6.0 Hz, CH₂NHCO), 7.09 (2H, d, J = 8.6 Hz, 3-CH tyr), 7.06 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.69-6.78 (6H, 3d, J = 9.0 Hz, J = 8.6 Hz and J - 8.6 Hz,

2-CH AP, 2-CH tyr and 2-CH CHL), 4.74 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 2.80-3.20 (4H, 2m, CH₂CHNH and CH₂NHCO), 2.51 (2H, t, J = 7.6

Hz, CH₂CH₂Ph), 2.17 (2H, t, J = 7.6 Hz, CH₂NHCOCH₂), 2.11-2.19 (2H, t partly hidden, CHNHCOCH₂), 1.81-1.92 (2H, m, CH₂CH₂CH₂Ph), 1.12-1.54 (6H, #m, 3x

CH₂).

¹³C-NMR (Acetone-rf₆, £ ppm) : 172.74 (CONH), 172.70 (CONH), 170.0 (CONH tyr), 156.4 (1-C tyr), 154.0 (1-C AP), 145.0 (1-C CHL), 131.2 (4-C AP), 130.9 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.5 (4-C CHL), 121.6 (2C, 3-C AP), 115.4 (2C, 2-C tyr), 115.3 (2C, 2-C AP), 112.5 (2C, 2-C CHL), 55.5 (CHNH), 53.3 (2C, NCH₂CH₂Cl), 41.0 (2C, NCH₂CH₂Cl), 39.1, 37.5, 35.9, 35.6, 34.2, 27.9, 26.4, 25.5, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₄₅Cl₂N₄O₅ = 671.2762; found = 671.2754.

N-ffN-chlorambucilaminoJundecanoylJ-p-hydroxyphenyl-L-p-tyrosinamide (15, m

= 10)

IR (KBr, v_max, cm^'1) : 3100-3500 (2x O-H and 3x N-H), 1643 (3x C=O, NHCO),

1517 and 1227 (C-N-H).

¹H-NMR (Acetone-4, <?ppm) : 9.11 (IH, s, OH AP), 8.34 (2H, br s, OH tyr and NH AP), 7.41, (2H, d, J = 9.0 Hz, 3-CH AP), 7.36 (IH, d, partly hidden, NH tyr), 7.15 (IH, br t, J = 6.0 Hz, CH₂NHCO), 7.09 (2H, d, J = 8.6 Hz, 3-CH tyr), 7.06 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.69-6.78 (6H, 3d, J = 9.0 Hz, J = 8.6 Hz and J = 8.6 Hz, 2-CH AP, 2-CH tyr and 2-CH CHL), 4.73 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 2.83-3.25 (4H, 2m, CH₂CHNH and CH₂NHCO), 2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.18 (4H, 2t overlapped, J = 7.2 Hz, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.20-1.56 (16H, m, 8x CH₂). ¹³C-NMR (Acetone-^, J ppm) : 172.98 (CONH), 172.71 (CONH), 169.8 (CONH tyr), 156.4 (1-C tyr), 154.0 (1-C AP), 145.0 (1-C CHL), 131.2 (4-C AP), 130.9 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.5 (4-C CHL), 121.5 (2C, 3-C AP), 115.3 (4C, 2-C tyr and 2-C AP), 112.5 (2C, 2-C CHL), 55.6 (CHNH), 53.3 (2C, NCH₂CH₂Cl), 41.0 (2C, NCH₂CH₂Cl), 39.0, 37.3, 35.9, 35.6, 34.2, 29.8 (2C), 29.4 (3C), 27.9, 26.8, 25.7, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₄₀H₅₅Cl₂N₄O₅ = 741.3544; found = 741.3548.

Example 8. Preparation of tyrosine nitrogen mustard hybrids second generation (15) (convergent synthesis using o, m, and p- aminophenol, m = 5 or 10)

Step A. Synthesis of Λ^r-((Λ^/-chlorambucilamino)alkaloyl)-hydroxyphenyl-L-

/j-tyrosinamide (15) [00106] The appropriate N-Boc-hydroxyphenyl-L-p-tyrosinamide 10 (0.38 mmol) was dissolved in dichloromethane and treated with trifhioroacetic acid (3.80 mmol). The solution was stirred at room temperature for 24 h. After evaporation, the resulting trifluoroacetic salt was dissolved in dimethylformamide and neutralized with triethylamine (0.40 mmol). At the same time, the N-chlorambucil-amino acid derivative 16 (0.40 mmol) was dissolved in dimethylformamide, and DCC (0.42 mmol) followed by HOBt (0.42 mmol) were added. Then, the hydroxyphenyltyrosamide solution was added to the activated chlorambucil-amino acid derivative (16) solution. The resulting mixture was stirred at room temperature for 29 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 7:3) to give a pure compound in 51% yield.

N-((N-chlorambucilamino)hexanoyl)-p-hydroxyphenyl-L-p-tyrosinamide (15, m =

5)

[00107] The spectral data of this derivative were identical to those described in example 7 for which the direct synthesis was used to provide the same derivative.

N-tfN-chlommbucilaminoJundecanoyty-p-hydroxyphenyl-L-p-tyrosinamide (15, m = 10)

[00108] The spectral data of this derivative were identical to those described in example 7 for which the direct synthesis was used to provide the same derivative.

N-((N-chlorambucilamino)hexanoyl)-m-hydroxyphenyl-L-p-tyrosinamide (isomer of l5, m = 5)

IR (NaCl, v_max, cm^"1) : 3100-3600 (2x O-H and 3x N-H), 1619 (3x C=O, NHCO),

1515 and 1239 (C-N-H).

¹H-NMR (Acetone-^, £ppm) : 9.20 (IH, s, OH AP), 8.49 (IH, s, OH tyr), 8.34 (IH, s, NH AP), 7.29-7.35 (2H, d, J = 8.2 Hz, NH tyr and t apparent, J - 2.34 Hz, 4-CH AP), 7.13 (IH, br t, J = 5.1 Hz, CH₂NHCO), 7.09 (2H, d, J = 8.2 Hz, 3-CH tyr), 7.07 (2H, d, J = 8.6 Hz, 3-CH CHL), 6.99-7.11 (2H, m apparent, partly hidden, 2-CH and 5-CH AP), 6.74 (2H, d, J = 8.2 Hz, 2-CH tyr), 6.71 (2H, J = 9.0 Hz, 2-CH CHL), 6.54 (IH, dq, J = 1.2 Hz and J = 7.8 Hz, 6-CH AP), 4.73 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 2.79-3.21 (2H, m, CH₂CHNH), 3.15 (2H, m, CH₂NHCO), 2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.18 (4H, 2t overlapped, J = 7.2 Hz, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-1.93 (2H, m, CH₂CH₂CH₂Ph), 1.18-1.59 (6H, #m, 3x CH₂).

¹³C-NMR (Acetone-^, £ ppm) : 172.9 (CONH), 172.8 (CONH), 170.3 (CO tyr), 158.1 (1-C AP), 156.4 (1-C tyr), 144.9 (1-C CHL), 140.2 (3-C AP), 130.9 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 129.6 (5-C AP), 128.4 (4-C CHL), 115.4 (2C, 2-C tyr), 112.4 (2C, 2-C CHL), 110.9 (2C, 4-C AP and 6-C AP), 107.0 (2-C AP), 55.7 (CHNH), 53.3 (2C, NCH₂CH₂Cl ), 41.0 (2C, NCH₂CH₂Cl ), 39.1, 37.1, 35.8, 35.6, 34.2, 27.9, 26.3, 25.5, (1C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₄₅Cl₂N₄O₅ = 671.2762; found = 671.2757.

N-^N-chlorambucilaminojundecanoyty-m-hydroxyphenyl-L-p-tyrosinamide (isomer of l5, m = 10)

IR (NaCl, v_max, cm^"1) : 3100-3500 (2x O-H and 3x N-H), 1704, 1649 and 1622 (3x

C=O, NHCO), 1526 and 1222 (C-N-H).

¹H-NMR (Acetone-*/*, £ppm) : 9.34 (IH, s, OH AP), 8.55 (IH, s, OH tyr), 8.45 (IH, s, NH AP), 7.47 (IH, d, J = 8.2 Hz, NH tyr), 7.33 (IH, t, J = 2.1 Hz, 4-CH AP), 7.21

(IH, br t, J = 5.7 Hz, CH₂NHCO), 7.10 (2H, d, J = 8.2 Hz, 3-CH tyr), 7.06 (2H, d, J =

8.2 Hz, 3-CH CHL), 6.96-7.12 (2H, m apparent, partly hidden, 2-CH and 5-CH AP),

6.74 (2H, d, J = 8.2 Hz, 2-CH tyr), 6.70 (2H, J = 8.6 Hz, 2-CH CHL), 6.56 (IH, dq, J

= 1.2 Hz and J = 7.8 Hz, 6-CH AP), 4.78 (IH, m, CHNH), 3.73 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.20 (2H, q, J = 5.9 Hz, CH₂NHCO), 2.83-3.15 (2H, m, CH₂CHNH),

2.52 (2H, t, J = 7.6 Hz, CH₂CH₂Ph), 2.19 (4H, 2t overlapped, J = 7.2 Hz,

CH₂NHCOCH₂ and CHNHCOCH₂), 1.79-1.96 (2H, m, CH₂CH₂CH₂Ph), 1.22-1.59

(16H, #m and s, 8x CH₂).

¹³C-NMR (Acetone-rf₆, δ ppm) : 173.3 (CONH), 172.9 (CONH), 170.4 (CO tyr),

158.1 (1-C AP), 156.5 (1-C tyr), 144.9 (1-C CHL), 140.3 (3-C AP), 130.9 (4-C tyr),

130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 129.6 (5-C AP), 128.3 (4-C CHL), 115.3

(2C, 2-C tyr), 112.4 (2C, 2-C CHL), 110.9 (2C, 4-C AP and 6-C AP), 107.1 (2-C AP),

55.8 (CHNH), 53.3 (2C, NCH₂CH₂Cl ), 41.0 (2C, NCH₂CH₂Cl ), 39.1, 35.9, 35.7,

34.3, 29.4 (3C), 29.1 (2C), 28.0, 28.0 (2C), 26.8, 25.7.

ESI+ HRMS: [M + H]⁺ calculated for C₄₀H₅₅Cl₂N₄O₅ = 741.3544; found = 741.3541.

N-((N-chlorambucilamino)hexanoyl)-o-hydroxyphenyl-L-p-tyrosinamide (isomer of

15, m = 5)

IR (NaCl, v_max, cm^"1) : 3100-3400 (2x O-H and 3x N-H), 1646 (3x C=O, NHCO),

1526 and 1246 (C-N-H).

¹H-NMR (Acetone-rf₆, £ ppm) : 9.60 (IH, br s, OH AP), 9.17 (IH, br s, OH tyr), 7.90 (IH, d, J = 7.8 Hz, NH tyr), 7.68 (IH, d, J = 7.8 Hz, 3-CH AP), 7.45 (IH, br t, J = 5.7 Hz, CH₂NHCO), 7.13 (2H, d, J = 8.6 Hz, 3-CH tyr), 7.06 (2H, d, J = 8.6 Hz, 3- CH CHL ), 6.96 (2H, 2x d overlapped, J - 3.9 Hz, 4-CH AP and 5-CH AP), 6.78 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.70 (2H, d, J = 8.6 Hz, 2-CH CHL), 6.68-6.83 (2H, m apparent, partly hidden, 6-CH AP and NH AP), 4.86 (IH, m, CHNH), 3.72 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.16 (2H, m, CH₂NHCO), 2.86-3.29 (2H, m, CH₂CHNH), 2.51 (2H, t, J = 7.4 Hz, CH₂CH₂Ph), 2.22 (4H, m apparent, CH₂NHCOCH₂ and CHNHCOCH₂), 1.80-1.95 (2H, m, CH₂CH₂CH₂Ph), 1.11-1.63 (6H, #m, 3x CH₂). ¹³C-NMR (Acetone-^, δ ppm) : 173.7 (CONH), 173.6 (CONH), 170.8 (CO tyr), 156.5 (1-C tyr), 147.7 (1-C AP), 144.8 (1-C CHL), 130.9 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.4 (4-C CHL), 126.7 (2-C AP), 125.0 (5-C AP), 121.2 (3-C AP), 120.0 (4-C AP), 116.9 (6-C AP), 115.5 (2C, 2-C tyr), 112.5 (2C, 2-C CHL), 55.9 (CHNH), 53.3 (2C, NCH₂CH₂Cl ), 41.0 (2C, NCH₂CH₂Cl ), 39.3, 36.7, 35.9, 35.6, 34.2, 33.6, 27.9, 26.3, 25.6.

ESI+ HRMS: [M + H]⁺ calculated for C₃₅H₄₅Cl₂N₄O₅ = 671.2762; found = 671.2763.

N-tfN-chlorambucilaminoϊundecanoyty-o-hydroxyphenyl-L-p-tyrosinamide (isomer of 15, m = 10)

IR (NaCl, v_max, cm^"1) : 3100-3600 (2x O-H and 3x N-H), 1690, 1646 and 1615 (3x

C=O, NHCO), 1526 and 1250 (C-N-H).

¹H-NMR (Acetone-</₆, £ppm) : 9.21 (IH, br s, OH tyr), 7.70 (IH, dd, J = 1.4 Hz and

J = 7.8 Hz, 3-CH AP), 7.49 (IH, d, J = 8.2 Hz, NH tyr), 7.13 (2H, d, J = 8.6 Hz, 3-CH tyr), 7.11-7.15 (IH, hidden, CH₂NHCO), 7.06 (2H, d, J = 8.6 Hz, 3-CH CHL ), 6.83-

7.02 (2H, #m, 4-CH AP and 5-CH AP), 6.76 (2H, d, J = 8.6 Hz, 2-CH tyr), 6.71 (2H, d, J = 9.0 Hz, 2-CH CHL), 6.68-6.80 (2H, m apparent, partly hidden, 6-CH AP and

NH AP), 4.83 (IH, m, CHNH), 3.74 (8H, m, 2x CH₂Cl and 2x NCH₂), 3.20 (2H, m,

CH₂NHCO), 2.88-3.25 (2H, m, CH₂CHNH), 2.52 (2H, t, J = 7.4 Hz, CH₂CH₂Ph),

2.22 (4H, 2x d, J = 7.2 Hz and J = 7.4 Hz, CH₂NHCOCH₂ and CHNHCOCH₂), 1.82-

1.93 (2H, m, CH₂CH₂CH₂Ph), 1.24-1.56 (16H, #m and s, 8x CH₂), (IH, OH AP, hidden).

¹³C-NMR (Acetone-^, S ppm) : 173.2 (CONH), 172.7 (CONH), 171.2 (CO tyr),

156.5 (1-C tyr), 148.1 (1-C AP), 144.9 (1-C CHL), 130.9 (4-C tyr), 130.5 (2C, 3-C tyr), 129.7 (2C, 3-C CHL), 128.3 (4-C CHL), 126.7 (2-C AP), 125.4 (5-C AP), 121.7

(3-C AP), 119.8 (4-C AP), 116.9 (6-C AP), 115.4 (2C, 2-C tyr), 112.5 (2C, 2-C CHL),

55.6 (CHNH), 53.3 (2C, NCH₂CH₂Cl ), 41.0 (2C, NCH₂CH₂Cl ), 39.0, 36.7, 35.9,

35.8, 35.6, 34.2, 33.6, 29.0 (2C), 28.0, 26.7, 25.7, (2C hidden).

ESI+ HRMS: [M + H]⁺ calculated for C₄₀H₅₅Cl₂N₄O₅ = 741.3544; found = 741.3531. Example 9. Preparation of 7V-(2-methoxy-2-phenyl-acetylamino)-L-p-tyrosine methyl ester (S,S-S and R₁S-S)

[00109] The appropriate tyrosine methyl ester hydrochloride salt 2 (0.25 mmol) was dissolved in dimethylformamide and neutralized with triethylamine (0.27 mmol). Simultaneously, methoxyphenyl acetic acid (0.27 mmol) was dissolved in dimethylformamide to which solution DCC (0.28 mmol) and HOBt (0.28 mmol) were added. Then, the tyrosine solution was added to the activated methoxyphenyl acetic acid solution. The resulting mixture was stirred at room temperature for 17.5 h. The solution was diluted with ethyl acetate and water, and then washed with water (4x). The organic phase was dried with sodium sulfate, filtered and evaporated. The product was further purified by flash chromatography (hexanes: acetone, 4:1) to give a pure compound in 82% yield.

7V-(2-methoxy-2-phenyl-acetylamino)-L-/7-tyrosine methyl ester (S,S-8)

IR (NaCl, v_max, cm^'1) : 3150-3400 (O-H and N-H), 1742 (C=O, COOCH₃),

1661(C=O, NHCO), 1516 and 1224 (C-N-H).

¹H-NMR (Acetone-rf₆, S ppm) : 8.23 (IH, s, OH), 7.44 (IH, d, J = 7.8 Hz,

CHNHCO), 7.22-7.34 (5H, #m, 2-CH Phe, 3-CH Phe and 4-CH Phe), 6.93 (2H, d, J

= 8.6 Hz, 3-CH tyr), 6.71 (2H, d, J = 8.6 Hz, 2-CH tyr), 4.70 (IH, m, CHNH), 4.61

(IH, s, CHPhe), 3.71 (3H, s, COOCH₃ tyr), 3.33 (3H, s, OCH₃), 2.93-3.12 (2H, m,

CH₂CHNH).

¹³C-NMR (Acetone-</₆, δ ppm) : 172.0 (COOCH₃), 169.9 (NHCOCH₂), 156.5 (1-C tyr), 137.9 (1-C Phe), 130.5 (2C, 3-C tyr), 128.3 (2C, 2-C Phe), 128.2 (4-C tyr), 127.5

(3C, 3-C Phe and 4-C Phe), 115.4 (2C, 2-C tyr), 84.0 (CHPhe), 56.8 (CHNH), 53.1

(OCH₃), 51.8 (OCH₃), 36.5 (CH₂CHNH).

ESI+ HRMS: [M + Na]⁺ calculated for Ci₉H₂₁NNaO₅ = 366.1312; found = 366.1316;

[M + H]⁺ calculated for Ci₉H₂₂NO₅ = 344.1492; found = 344.1493. iV-(2-methoxy-2-phenyl-acetylamino)-D-/?-tyrosme methyl ester (R,S-S)

IR (NaCl, v_max, cm^"1) : 3150-3400 (O-H and N-H), 1743 (C=O, COOCH₃),

1661(C=O, NHCO), 1517 and 1226 (C-N-H). ¹H-NMR (Acetone-rf_fi, δ ppm) : 8.46 (IH, br s, OH), 7.54 (IH, d, J = 8.2 Hz,

CHNHCO), 7.28-7.43 (5H, #m, 2-CH Phe, 3-CH Phe and 4-CH Phe), 7.05 (2H, d, J

= 8.6 Hz, 3-CH tyr), 6.77 (2H, d, J - 8.6 Hz, 2-CH tyr), 4.63 (IH, m, CHNH), 4.62

(IH, s, CHPhe), 3.65 (3H, s, COOCH₃ tyr), 3.28 (3H, s, OCH₃), 3.04-3.11 (2H, m,

CH₂CHNH).

¹³C-NMR (Acetone-*^, <? ppm) : 171.8 (COOCH₃), 170.2 (NHCOCH₂), 156.6 (1-C tyr), 138.1 (1-C Phe), 130.5 (1C, 3-C tyr), 128.4 (2C, 2-C Phe), 128.3 (4-C tyr), 127.7

(2C, 3-C Phe), 127.6 (4-C Phe), 115.5 (2C, 2-C tyr), 83.8 (CHPhe), 57.0 (CHNH),

53.5 (OCH₃), 51.7 (OCH₃), 36.5 (CH₂CHNH).

ESI+ HRMS: [M + Na]⁺ calculated for C₁₉H₂iNNaO₅ = 366.1312; found = 366.1313;

[M + H]⁺ calculated for C₁₉H₂₂NO₅ = 344.1492; found = 344.1490.

IN VITRO CYTOTOXIC ACTIVITY OF THE AMINO ACID-LINKED

NITROGEN MUSTARD HYBRIDS

Cell proliferation with the MTT assay (uterine, ovarian and breast cancer cell lines)

[00110] Several human uterine, ovarian and breast cancer cell lines were used to evaluate the antitumor activities of the new amino acid-linked nitrogen mustard hybrids. The cytotoxicity of the nitrogen mustard derivatives was done along with chlorambucil as the control on both ER⁺ and ER" human female cancers. Cell proliferation was done with the MTT assay as reported in literature.⁹'¹⁰ The MTT assay is based on the ability of viable cells to reduce a soluble colorless tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), into an insoluble blue formazan derivative.

[00111] The D-p-, DL-ø-, L-m- and L-p-tyrosine nitrogen mustard 3, 4, 6, 7, 11 and 15 (o, m and />, with m = 5 or 10) were evaluated with the MTT assay on breast cancer cell lines. The structures of the compounds are presented in scheme 6. The results are presented on Table 1 below. Scheme 6. Structures of tyrosine-nitrogen mustard hybrids used for the MTT assays

6 and isomer of 6, R = CO₂CH₃

3 and isomer of 3, R = CO₂CH₃

7 and isomer of 7, R = CH₂OH

4 and isomer of 4, R = CH₂OH

11, made with ortho-, meta- andj∞ra-aminophenol 15, made with ortho-, meta- andpαra-aminophenol

Table 1 : Cell proliferation with the MTT assay on breast carcinomas obtained for L-tyrosine-nitrogen mustard hybrids and for chlorambucil (CHL).

* IC₅₀ (Inhibitory concentration ₅0%), data in μM;

NR = IC₅₀ not reached at the concentrations tested

Experiments were performed in duplicates and the results represent the mean ± SEM of three independent experiments [00112] The D- and L-tyrosine nitrogen mustard hybrids with m = 5 were evaluated with the MTT assay on uterine, ovarian and breast cancer cell lines. The results are presented on Table 2 below.

Table 2: Cell proliferation with the MTT assay on breast, ovarian and uterine carcinomas obtained for L- or D-tyrosine-nitrogen mustard hybrids and for

chlorambucil (CHL).

* IC₅₀ (Inhibitory concentration ₅0%), data in μM

NR = IC₅0 not reached at the concentrations tested

Experiments were performed in duplicates and the results represent the mean + SEM of three independent experiments

[00113] As it can be seen in Tables 1 and 2, the compounds tested are significantly more active than chlorambucil on certain cell lines related to uterin cancer, ovarian cancer and breast cancer.

[00114] Table 3 below presents the results obtained for the National Cancer

Institute (NCI) for derivative 6, m = 5 tested on its 60 cancer cell line screen (NCI-60 DTP Human Tumor Cell Line Screen).¹¹ This table indicates that compound 6, m = 5 possesses a higher activity than chlorambucil (CHL) itself on the different cell lines tested, further demonstrating the usefulness of the compounds of the present disclosure. Furthermore, compound 6, m = 5 show much higher activity than chlorambucil on melanoma cancer cell lines. Regarding certain cell lines related to ovarian cancer, the activity of compound 6, m = 5, is considerably higher than the activity of chlorambucil. Table 3 : Log GI₅₀ as measured by SRB staining for compound 6, m = 5 and for chlorambucil (CHL) performed on the NCI-60 cancer cell line screen.

*Data obtained from the NCI/NIH

[00115] Table 4 summarizes all the results obtained on the NCI-60 cancer cell line screen for compound 6, m =5 and CHL. As defined by the NCI, three dose response parameters are calculated for each experimental agent. Growth inhibition of 50 % (GI₅₀) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is 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) is calculated from Ti = Tz. 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 is calculated from [(Ti-Tz)/Tz] x 100 = -50.

Table 4: GI₅₀, Log GI₅₀, TGI, Log TGI, LC₅₀ and Log LC₅₀ for derivatives 6, m = 5 and CHL. The table gives the average value over all cell lines tested and the GI₅0, TGI and LC₅₀ ratios CHL/6.

Compounds GI₅₀ Log GI₅₀ TGI Log TGI LC₅₀ Log LC₅₀

(μM) (uM) (μM)

Chlorambucil (CHL) 9.1 -5.04 35.4 -4.45 79.4 -4.10

(NCI #3088)

6, m = 5 5.7 -5.24 21.3 -4.67 61.6 -4.21

Ratio CHL/6 1.6 1.7 1.3

*Data obtained from the NCI/NIH [00116] Based on the results provided in Table 4, it can be seen that compound

6, m = 5 is about 37 % (1/1.6) more efficient than chlorambucil for growth inhibition and about 41 % (1/1.7) more efficient than chlorambucil for total growth inhibition. Finally, compound 6, m = 5 is about 23 % (1/1.3) more efficient that chlorambucil for

LC₅₀.

[00117] In view of the results provided in the present disclosure, it can be said that the compounds of the present disclosure have an activity on various types of cancer and can thus be used accordingly as indicated in the present document.

[00118] It was thus shown that the compounds of the present disclosure are able to target cancers such as, for example, melanoma, breast, uterine and ovarian cancers, but also to display increased efficacy and overall decreased systemic toxicity. They are also active against various types of cancer cell lines.

[00119] The present disclosure comprises a novel class of nitrogen mustard anticancer compounds including their pharmaceutically acceptable derivatives. These molecules demonstrated in vitro cytotoxic activity on human breast cancer. Therefore, these compounds can be, for example, used to provide medicaments with anticancer activity against hormono-dependent breast, uterine, melanoma as well as ovarian cancers as well as various other types of cancer These compounds can be used, for example, alone or in combination with other therapeutic or prophylactic agents for the treatment of melanoma breast, uterine and ovarian cancers.

REFERENCES

1. Salmon, SE, Sartorelli, AC. Cancer chemotherapy, In Basic and clinical pharmacology, 4^th Ed., Katzung BG. Ed., Appleton & Lange, Norwalk, Chapter 56, pp. 686-689 (1989)

2. Bergel F, Stock JA. J. Chem. Soc. 2409 (1954); Deglin JH, Vallerand AH. In Guide des medicaments, ERPI, pp. 1095-1098 (1995)

3. Phillips AP, Mentha JW. US Patent 3,046,301 (October 29, 1959); Deglin JH, Vallerand AH. In Guide des medicaments, ERPI, pp. 424-427 (1995)

4. Remers, WA. Antineoplastic agents, In Wilson and Gisvold's Textbook of organic Medicinal and pharmaceutical chemistry, 9ⁿ Ed., Delgado JN, Remers, WA. J.B. Eds, Lippincott, New York, Chapter 8, 321-322 (1989) 5. Drabløs F, Feyzi E, Aas PA, Vaagbø CB, Kavli B, Bratlie MS, Pefia-Diaz J, Otterlei M, Slupphaug G, Krokan HE. DNA Repair, 31389 (2004)

6. Dorr RT, Fritz WL. Cancer Chemotherapy Handbook; Elsevier Science: New York, 1982; p 486.

7. Perrin DD, Armarego CF. In Purification of Laboratory Chemicals, 3^rd Ed., Pergamon Press, Oxford, New York (1988)

8. Still WC, Kahn M, Mitra A. Rapid chromatographic technique for preparative separations with moderate resolution, J. Org. Chem., 43, 2923-2925 (1978)

9. Carmichael J, Degraff WG, Gazdar AF, Minna JD, Mitchell JD, Evaluation of a tetrazolium-based semiautmated colorimetric assay : Assessment of radiosensitivity. Cancer Res., 47, 943-946 (1987)

10. Ford CHJ, Richardson VJ, Tsaltas K, Comparaison of tetrazolium colorimetric and [³H]-uridine assays for in vitro chemosensitivity testing. Cancer Chemother. Pharmacol, 24, 295-301 (1986)

11. National Cancer Institute, 9000 Rockville Pike, Bethesda, Maryland 20892

[00120] The present disclosure has been described with regard to specific examples. The description was intended to help the understanding of the present disclosure, rather than to limit its scope. It will be apparent to one skilled in the art that various modifications may be made to the present disclosure without departing from the scope of the present disclosure as described herein, and such modifications are intended to be covered by the present document.

Claims

CLAIMS:

1. A compound of formula I:

(I) wherein

T is L or -A-NH-C(O)-L-;

L is -(CHj)_n- or -(CH₂)_P-Z- ;

A is -(CH₂)_m- or an alkyl component of a naturally occurring amino acid

R is -CO₂R₁₅ -CH₂OH,

R₁ is H or is C₁-Cj₂ alkyl;

X is -OH, -OSO₂R₂, -Cl, -Br, or -I;

R₂ is C₁-C₁₂ alkyl or -CF₃;

Z is phenyl or naphthyl;

m is an integer having a value of 1 to 20;

n is an integer having a value of 1 to 20; and

p is an integer having a value of 1 to 20, or an enantiomer, diastereoisomer, racemic mixture, pharmaceutically acceptable salt, solvate or prodrug thereof.

2. The compound of claim 1, wherein said compound is a compound of formula II :

wherein

R is -CO₂R₁ or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂)_n-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃; and

n is 1, 2, 3, 4, 5, 6, 7 or 8.

3. The compound of claim 1, wherein said compound is a compound of formula Ha :

(Ha) wherein

R is -CO₂R₁ or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂)_n-;

A is -(CH₂)_m-;

X is -OH, -OSO₂R₂ or -Cl; R₁ is chosen from H, straight alkyl group having from 1 to 5 carbon atoms, and branched alkyl group having from 3 to 5 carbon atoms;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

mis 1,2, 3,4, 5, 6, 7, 8, 9 or 10.

4. The compound of claim 1, wherein said compound is a compound of formula III :

wherein

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂)_n-;

Xis-OH,-OSO₂R₂or-Cl;

R₂ is -CH₃ or -CF₃; and

nis 1,2, 3,4, 5, 6, 7 or 8.

5. The compound of claim 1, wherein said compound is a compound of formula Ilia :

(Ilia) wherein

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂),,-;

A is -(CH₂)_m-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃;

n is i, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

6. The compound of claim 1, wherein said compound is a compound of formula IV :

(IV) wherein

R is -CO₂Ri or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃; and

n is i, 2, 3, 4, 5, 6, 7 or 8.

7. The compound of claim 1, wherein said compound is a compound of formula IVa :

(IVa) wherein

R is -CO₂Ri or -CH₂OH;

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

A is -(CH₂)_m-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

8. The compound of claim 1, wherein said compound is a compound of formula V:

(V)

wherein

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃; and

n is 1, 2, 3, 4, 5, 6, 7 or 8.

9. The compound of claim 1, wherein said compound is a compound of formula Va:

(Va) wherein

L is -CH₂CH₂CH₂-C₆H₄- or -(CH₂V;

A iS -(CH₂V-;

X is -OH, -OSO₂R₂ or -Cl;

R₂ is -CH₃ or -CF₃;

n is 1, 2, 3, 4, 5, 6, 7 or 8; and

m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

10. The compound of any one of claims 1, 3, 5, 7, and 9 wherein A is the alkyl component of an hydrophobic amino acid.

11. The compound of any one of claims 1, 3, 5, 7, and 9 wherein said naturally occurring amino acid is chosen from alanine, phenylalanine, leucine, isoleucine, tryptophan, valine and proline.

12. The compound of any one of claims 1, 3, 5, 7, and 9 wherein A is chosen from

13. The compound of claim 2, wherein L is -CH₂CH₂CH₂-C₆H₄-, X is Cl and

14. The compound of claim 2, wherein L is -CH₂CH₂CH₂-C₆H₄-, X is Cl and R is -CH₂OH.

15. The compound of claim 3, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂V-, m is 5 or 10, X is -Cl and R is -CO₂CH₃.

16. The compound of claim 3, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, m is 5 or 10, X is -Cl and R is -CH₂OH.

17. The compound of claim 4, wherein L is -CH₂CH₂CH₂-C₆H₄- and X is Cl.

18. The compound of claim 4, wherein said compound is a compound of formula (VI) :

(VI) wherein L is -CH₂CH₂CH₂-C₆H₄-.

19. The compound of claim 5, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is (CH₂)_m-, and m is 5 or 10.

20. The compound of claim 5, wherein said compound is a compound of formula (Via) :

(Via) wherein L is -CH₂CH₂CH₂-C₆H₄-, A is -(CH₂V. and m is 5 or 10.

21. The compound of claim 6, wherein L is -CH₂CH₂CH₂-C₆H₄-, X is Cl and R is -CO₂CH₃.

22. The compound of claim 6, wherein L is -CH₂CH₂CH₂-C₆H₄-, X is Cl and R is -CH₂OH.

23. The compound of claim 7, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, X is Cl and R is -CO₂CH₃.

24. The compound of claim 7, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, m is 5 or 10, X is Cl and R is -CO₂CH₃.

25. The compound of claim 7, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, X is Cl and R is -CH₂OH.

26. The compound of claim 7, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, m is 5 or 10, X is Cl and R is -CH₂OH.

27. The compound of claim 8, wherein L is -CH₂CH₂CH₂-C₆H₄-, and X is Cl.

28. The compound of claim 8, wherein said compound is a compound of formula VII :

(VII) wherein L is -CH₂CH₂CH₂-C₆H₄-.

29. The compound of claim 9, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, and X is Cl.

30. The compound of claim 9, wherein L is -CH₂CH₂CH₂-C₆H₄-, A is - (CH₂)_m-, m is 5 or 10, and X is Cl.

31. The compound of claim 9, wherein said compound is a compound of formula Vila :

(Vila) wherein L is -CH₂CH₂CH₂-C₆H₄-, and A is -(CH₂)_m-.

32. The compound of claim 9, wherein said compound is a compound of formula Vila :

(Vila) wherein L is -CH₂CH₂CH₂-C₆H₄-, A is -(CH₂)_m-, and m is 5 or 10.

33. The compound of claim 9, wherein said compound is a compound of formula VIIb :

(VIIb)

wherein L is -CH₂CH₂CH₂-C₆H₄-.

34. The compound of claim 9, wherein said compound is :

35. The compound of claim 9, wherein said compound is a compound of formula VIIc :

(VIIc)

wherein L is -CH₂CH₂CH₂-C₆H₄-, and A is -(CH₂)_m-.

36. The compound of claim 9, wherein said compound is a compound of formula VIIc :

(VIIc)

wherein L is -CH₂CH₂CH₂-C₆H₄-, A is -(CH₂)_m-, and m is 5 or 10.

37. The compound of claim 9, wherein said compound is of formula

38. The compound of claim 9, wherein said compound is of formula

wherein m is 5 or 10.

39. A composition comprising a pharmaceutically acceptable carrier and at least one compound as defined in any one of claims 1 to 38.

40. A method for treating cancer, said method comprising administering to a subject in need thereof an effective amount of at least one compound as defined in any one of claims 1 to 38.

41. A method for reducing the risks of developing cancer in a subject, said method comprising administering to the subject an effective amount of at least one compound as defined in any one of claims 1 to 38.

42. A method for inhibiting cancer cell growth, said method comprising administering to a subject in need thereof an effective amount of at least one compound as defined in any one of claims 1 to 38.

43. The method of any one of claims 40 to 42, wherein said cancer is breast cancer.

44. The method of any one of claims 40 to 42, wherein said cancer is uterine cancer.

45. The method of any one of claims 40 to 42, wherein said cancer is ovarian cancer.

46. The method of any one of claims 40 to 42, wherein said cancer is melanoma.

47. A method for treating at least one cancer chosen from breast cancer, uterine cancer and ovarian cancer, said method comprising administering to a subject in need thereof an effective amount of at least one compound as defined in any one of claims 1 to 38.

48. A method for reducing the risks of developing at least one cancer in a subject, said cancer being chosen from breast cancer, uterine cancer and ovarian cancer, said method comprising administering to the subject an effective amount of a compound as defined in any one of claims 1 to 38.

49. Use of at least one compound as defined in any one of claims 1 to 38 for treating cancer.

50. Use of at least one compound as defined in any one of claims 1 to 38 for reducing the risks of developing cancer.

51. Use of at least one compound as defined in any one of claims 1 to 38 for inhibiting cancer cell growth.

52. Use of at least one compound as defined in any one of claims 1 to 38 in the manufacture of a medicament for treating cancer.

53. Use of at least one compound as defined in any one of claims 1 to 38 in the manufacture of a medicament for reducing the risks of developing cancer.

54. Use of at least one compound as defined in any one of claims 1 to 38 in the manufacture of a medicament for inhibiting cancer cell growth.

55. The use of any one of claims 49 to 54, wherein said cancer is breast cancer.

56. The use of any one of claims 49 to 54, wherein said cancer is ovarian cancer.

57. The use of any one of claims 49 to 54, wherein said cancer is uterine cancer.

58. The use of any one of claims 49 to 54, wherein said cancer is melanoma.

59. Use of at least one compound as defined in any one of claims 1 to 38 for treating at least one cancer chosen from breast cancer, uterine cancer and ovarian cancer.

60. Use of at least one compound as defined in any one of claims 1 to 38 for reducing the risks of developing at least one cancer chosen from breast cancer, uterine cancer and ovarian cancer.

61. Use of at least one compound as defined in any one of claims 1 to 38 in the manufacture of a medicament for treating at least one cancer chosen from breast cancer, uterine cancer and ovarian cancer.

62. Use of at least one compound as defined in any one of claims 1 to 38 in the manufacture of a medicament for reducing the risks of developing of at least one cancer chosen from breast cancer, uterine cancer and ovarian cancer.

63. A method for preparing a compound of formula I :

(I) wherein

T is L or -A-NH-C(O)-L-;

L is -(CH₂V- or -(CH₂)_P-Z- ;

A is -(CH₂)_m- or an alkyl component of a naturally occurring amino acid R is -CO₂R₁₅ -CH₂OH,

R₁ is H or is Ci-C₁₂ alkyl;

X is -OH, -OSO₂R₂, -Cl, -Br, or -I;

R₂ is C₁-C₁₂ alkyl or -CF₃;

Z is phenyl or naphthyl;

m is an integer having a value of 1 to 20;

n is an integer having a value of 1 to 20; and

p is an integer having a value of 1 to 20,

(VIII) (IX)

wherein

E is chosen from -NH₂, -NH₃ ⁺Xf;

Xi is a suitable counter anion; R₄Is chosen from -OH, Cl, or -O(G=O)R_5;

R₅ is a Ci-C₅ alkyl;

R, T, and X are as previously defined for formula I,

under conditions to form said compound of formula I,

or

wherein

E iS -NH₂₅ Or -NH₃ ⁺Xf ;

X₁ is a suitable counter anion;

R₄ is -OH, Cl, or -0(C=O)R₅;

R₅ is a C₁-C₅ alkyl;

R, A, L and X are as previously defined for formula I,

under conditions to form said compound of formula I.

64. The method of claim63, wherein said conditions comprise the presence of at least one coupling reagent and a base.

65. The method of claim 64, wherein said at least one coupling reagent is chosen from DCC (dicyclohexylcarbodiimide), CDI (N,N'-carbonyldiimidazole), BOP (benzotriazole- 1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), DEPBT (3 -(diethoxy-phosphoryloxy)-3H-benzo[d] [ 1 ,2,3] triazin-4-one), EDCHCl ( 1 -ethyl-3-(3 -dimethyllaminopropyl)carbodiimide hydrochloride), HATU (2-( 1 H-7-azabenzotriazol- 1 -yl)~ 1 , 1 ,3 ,3 -tetramethyl uronium hexafluorophosphate methanaminium), HOBt (1-hydroxybenzotriazole or N-hydroxybenzotriazole), HBTU (O-benzotriazole-N,N,N',N'-tetramethyl- uronium-hexafluoro-phosphate), HOAt (l-hydroxy-7-azabenzotriazole), HOOBt (hydroxy-3,4-dihydro-4-oxo-l,2,3-benzotriazine), HCTU (lH-benzotriazolium 1- [bis(dimethylamino)methylene]-5-chloro-hexafluorophosphate (1 -),3 -oxide), Cl- HOBt (6-chloro- 1-hydroxybenzotriazole), PyBOP (benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromo-tris- pyrrolidino phosphoniumhexafluorophosphate), TATU, TBTU (O-(benzotriazol- l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), TCTU, TDBTU, TSTU, and 4,5-dicyanoimidazole.

66. The method of claim 64, wherein said conditions comprise the presence of dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (or N- hydroxybenzotriazole).