MXPA00005677A - Hemiasterlin analogs - Google Patents

Abstract

This invention provides analogs of hemiasterlin, methods of synthesis of the analogs and use of the analogs as a cytotoxic and anti-mitotic agents.

Description

ANALOGS OF HEM1ASTERLINE FIELD OF THE INVENTION This invention relates to biologically active compounds and compositions, their use and derivation.

BACKGROUND OF THE INVENTION As described in Talpir, R. et al. (1994) Tetrahedron Lett. : 4453-6 and in the international patent application PCT / GB96 / 00942 published on October 24, 1996 under the number WO96 / 33211, the compound hemiasterline can be obtained from marine sponges or can be synthesized. As disclosed in PCT / GB96 / 00942, hemiasterlin and the synthetic analogs described therein are cytotoxic and antimitotic. The compounds that differ from hemiasterlin in the region of the indole portion of hemiasterlin are novel. It has now been found that hemiasterlin analogues in which the hemolysin diene portion has been removed or replaced demonstrate potent antimitotic and cytotoxic activity.

BRIEF DESCRIPTION OF THE INVENTION This invention provides a compound or pharmaceutically acceptable salt thereof, which has the formula wherein Ri and R2 are independently selected from the group consisting of: H, R, and ArR-, and where at least one of Ri and R2 is R and neither is ArR-, R1 and R2 together may be optionally a ring of three to seven members; R3 and R4 are independently selected from the group consisting of: H, R, ArR-, and where at least one of R3 and R4 is R and neither is ArR- or Ar, R3 and R4 together may be optional a ring of three to seven members; R5 is selected from the group consisting of: H, R, ArR-, and Ar; Re is selected from the group consisting of: H, R, and ArR-; R7 and Re are independently selected from the group consisting of: H, R, and ArR-; Y and wherein, R is defined as a saturated or unsaturated portion having a linear, branched or cyclic base structure containing one to ten carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms, and zero to four sulfur atoms, and the carbon atoms are optionally substituted with: = O, = S, OH, -OR10, -O2CR? 0, -SH, -SR-io, -SOCR10, -NH2 > -NHR10, -N (R10) 2, -NHCOR10, -NR10COR10, -I, Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, -CONHR-io, -CON (R10) 2, -COSH, -COSR-io, -NO2, -SO3H, -SOR10, -SO2R10, wherein R10 is a saturated or unsaturated alkyl group of one to ten carbon atoms, linear, branched or cyclic; X is defined as a portion selected from the group consisting of: -OH, -OR, = O, = S, -O2CR, -SH, -SR, -SOCR, -NH2, -NHR, -N (R) 2, -NHCOR, -NRCOR, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R, -CHO, -COR, -CONH2 > -CONHR, -CON (R) 2, -COSH, -COSR, -NO2, -SO3H, -SOR, and -SO2R; Ar is defined as an aromatic ring selected from the group consisting of: phenyl, naphthyl, anthracyl, phenanthryl, furyl, pyrrolyl, thiophenyl, benzofuryl, benzothiophenyl, quinolinyl, isoquinolyl, imidazolyl, thiazolyl, oxazolyl, and pyridinyl, optionally substituted with R or X; Y is defined as a portion selected from the group consisting of: an alkyl group of one to six carbon atoms, saturated or unsaturated, linear, optionally substituted with R, ArR-, or X; and, Z is defined as a portion selected from the group consisting of: -OH, -OR; -SH; -MR; -NH2; -NHR; -N (R) 2; -NHCH (Rn) COOH; and - NRCH (Rn) COOH, wherein Rn is a portion having the formula: R, or - (CH2) nNR? 2Ri3, wherein n = 1-4 and R12 and R13 are independently selected from the group consisting of: H; R; and -C (NH) (NH2). Also, this invention provides methods for preparing the aforementioned compound of formula I, and precursors thereof, as described herein. This invention also provides the use of the aforementioned compound of formula I, or a pharmaceutically acceptable salt thereof: (a) for the manufacture of a medicament; (b) in a method by means of which cells, including tumor cells, that are susceptible to the cytotoxic effects of the compound are treated with the compound or a pharmaceutically acceptable salt thereof; and (c) in a method by means of which cells are treated with the compound or a pharmaceutically acceptable salt thereof, to cause mitotic disruption in the cells, or the production of abnormal mitotic spindles in the cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view showing a preferred scheme for synthesis of a compound of this invention.

Fig. 2 is a schematic view showing a preferred scheme for synthesis of the amino acid used in the coupling reaction shown in Fig. 1. Fig. 3 is a schematic view showing the steps in the synthesis of a compound of this invention as shown in Figs. described in the examples herein. Figure 4 is a schematic view showing the steps in the synthesis of the dipeptide shown in Figure 3, as described in the examples herein. Figures 5A and 5B are graphs comparing the cytotoxicity of hemiasterlin with SPA-110, as described in the examples herein. Figure 6 is a graph comparing the antimitotic activity of SPA-1 10 (*) with hemiasterlin (D), as described in the examples herein.

DETAILED DESCRIPTION OF THE INVENTION Except where otherwise indicated, the mention of a compound herein covers all possible salts of the compound, and denotes all possible isomers within the given structural formula for said compound, including geometric and optical isomers. Unless otherwise indicated, the materials described herein that comprise a compound for which isomers exist, should be considered as individual isomers of the shell, and, mixtures of isomers including racemic mixtures. In the compound of formula I above, the bonds drawn in wavy line are carbon atoms that can be optical centers. Preferably, the following absolute configurations predominate: Unless otherwise indicated, any portion referred to herein that is described as "alkyl" will preferably be straight chain or, branched where possible, and preferably have up to eight, preferably up to six and very preferably up to four carbon atoms. Except where otherwise indicated, optionally substituted alkyl groups are preferably unsubstituted. Methyl is the preferred alkyl group. In this specification, reference is made to alkyl portions which are saturated or unsaturated, thereby including within the definition of the portion, alkene and alkyne groups (whether internal, terminal or part of a ring). In a compound of formula I, the following substituents are preferred alone, or in combination: (a) Ri and R2 are independently: H, methyl, ethyl, propyl, n-butyl, acetyl; or, where Ri and R2 are attached: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; preferably Ri and R2 are independently: H or CH3; most preferably Ri is H and R2 is CH3; (b) preferably not more than one of R3 and R4 is H; preferably R 3 and R 4 are independently: methyl, ethyl, n-propyl or n-butyl, or, where R 3 and R 4 are attached β-cyclopropyl, β-cyclobutyl, β-cyclopentyl or β-cyclohexyl; most preferably R3 and R4 are each methyl; (c) R5: Ar in the definition of R5 is preferably phenyl, naphthyl, anthracyl or pyrrolyl; preferably R5 is phenyl, methyl or H; most preferably R5 is phenyl or methyl; (d) R6 and Rs are independently: H or methyl, most preferably R6 is H and R8 is methyl; (e) R7: a branched alkyl group of three to six carbon atoms; most preferably R is -C (CHs) 3; and (f) in Rg, Z is preferably OH, -OR14 (wherein R is an alkyl group of one to six carbon atoms, straight or branched, -NHCH (R) n) COOH or -NCH3CH (Rn) COOH where Rn is R, or - (CH2) n NHC (NH) (NH2); or Rg is preferably ° - C-C = C (R16) C-OH wherein R15 is methyl, ethyl, n-propyl, isopropyl, tertbutyl, isobutyl, or secbutyl and Rie is H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or secbutyl; most preferably Z is OH and Rg as a whole is: O -C-C = C (CH3) COH CH (CH3) 2 Where Rg has a chiral center in the Y portion, the following absolute configuration is preferred, with reference to an example wherein the chiral center has a methyl substituent: The compounds of formula I can be prepared by coupling portions A, B and C as shown below using standard procedures, including methods for coupling amino acids through a peptide bond.

A coupling agent, for example PyBroP, is suitably used in the reaction. The reaction suitably comprises connecting amino acid portions in the presence of the coupling agent, a base such as 4-dimethylaminopyridine and an organic solvent such as methylene chloride. Standard reaction extinction and purification procedures known in the art produce the coupled compound. The preparation of portions B and C as described above can be carried out using methods and starting materials known in the art, for example, following the methods described in PCT / GB96 / 00942 mentioned above. The methods as set forth in the examples herein may be employed, with appropriate modification to the materials and reagents, in accordance with the particular substituents of the A, B and C portions. One aspect of this invention is a method for preparing a compound of formula I in which a compound of the formula: it is coupled with a compound of the formula: HN A compound of formula III can be prepared by known methods (as described in PCT / GB96 / 00942) and by the method described in the examples herein. Another aspect of this invention is a method for preparing a compound of formula II described above in which a compound of the formula: is treated with a base such as sodium hydroxide diluted in a solvent such as methanol for a sufficient time to allow the elimination of OR; followed by acidification at about pH 3. Preferably R in formula IV is a simple alkyl chain such as CH3, and a protective agent such as terbutoxycarbonyl (Boc) can be used to protect the amino group; that is, R-i or R2 is replaced by Boc. The Boc group is suitably removed by a reaction such as TFA / CH2Cl2 for about 1 hour at room temperature. A suitable isolation protocol produces the TFA salt. Subsequently another group (eg Ri or R2) could be introduced into the nitrogen by standard techniques known to any person in the art, which produces the compound of formula IV. Another aspect of this invention is a method for preparing a compound of formula IV described above in which a compound of the formula: it is coupled with a compound of the formula: The preparation of compound V can be carried out by many methods known to the person skilled in the art. An example is described later in Figure 2. The compounds of formula VI can be prepared by methods known to those skilled in the art. A preferred method according to this invention for preparing a compound of formula I, is to prepare a dipeptide comprising portions B and C and coupling the dipeptide to portion A. In this method, a compound of the following formula, wherein Q and T together are a combination of two of the substituents: R1 f R2 and a protecting group: it is coupled with a compound of the formula: A compound of formula VIII can be prepared by coupling a compound of formula III as described above, with a compound of the formula: The compounds of formula IX can be prepared by methods known to those skilled in the art. Another aspect of this invention is a method for preparing a compound of formula VII as described above in which a compound of the formula: it is treated with a base followed by an azide compound. The azide derivative thus produced is reduced to form an amine which is then treated with a group selected from: R ?, Ri and Boc in the presence of a base such as sodium hydride. Figure 1 establishes a preferred scheme for the preparation of a compound of formula I comprising the coupling of the amino acid portion A with a dipeptide comprising portions B and C. In the embodiment shown in the figure, the dipeptide substituents are those of hemiasterlin. The Boc-protected dipeptide represented in the figure can be obtained by methods set forth in the examples herein. In portion A, Boc can replace Ri more than R2 or both Ri and R2 can be present in portion A before coupling.

Figure 2 establishes a preferred scheme for the preparation of portion A as used in the scheme shown in Figure 1. R2 can be added instead of Boc and this can replace R-i. The compounds of formula I are biologically active. The invention includes the use of a compound of formula I. The compounds of formula I may have pesticidal activity, for example insecticidal activity.

However, preferably the use is in the pharmaceutical field for medical or veterinary applications. The compounds described herein have utility as cytotoxic agents, particularly against tumor cells and may have utility as antibacterial or antiviral agents. Therefore, this invention includes a pharmaceutical composition comprising an effective amount of a compound of formula I, in association with a carrier. Also, this invention provides the use of a compound of formula I for the manufacture of a medicament for use in the treatment of cancer or a tumor in a mammal. When using a compound of formula I for medical or veterinary applications, the compound is preferably administered in a pharmaceutical composition also comprising a pharmaceutically acceptable carrier, and optionally, one or more biologically active ingredients. Said compositions may be in any form used to administer pharmaceutical substances, for example any form suitable for oral, topical, vaginal, intravenous, subcutaneous, parenteral, rectal and inhalation application. The compositions may be provided in discrete dose units. The carriers may be formed of particles, the compositions being, for example, tablets or powders, or liquids, the compositions being, for example, oral syrups or injectable liquids, or aerosol, for inhalation. For oral administration, an excipient and / or binder may be present. Examples include sucrose, kaolin, glycerin, starch dextrins, sodium aglinate, carboxymethylcellulose and ethylcellulose. Coloring agents and / or flavorings may be present. A covering covering can be used. For rectal administration, oleaginous bases may be used, for example lanolin or cocoa butter. For an injectable formulation, pH regulators, stabilizers and isotonic agents may be included. The dosage of a compound of formula I may depend on the weight and physical condition of the patient; of the severity and duration of the disease; and of the particular form of the active ingredient, the manner of administration and the composition employed. A daily dose of about 0.0001 to about 100 mg / kg of body weight taken individually or in separate doses of up to 6 times per day, or by continuous infusion, includes the effective amounts that are more typically required. A preferred scale is about 0.001 to about 50 mg / kg of body weight, per day, most preferably about 0.01 to about 30 mg / kg of body weight, per day.

It should be understood that the use of a compound of formula I in chemotherapy may comprise the binding of said compound to an agent, for example a monoclonal or polyclonal antibody, a protein or a liposome, which aids the delivery of said compound to tumor cells. This invention also includes the use of a compound of formula I as an antimitotic agent. Such use may be in procedures that require blocking cells at mitosis, such as the preparation of mitotic smears for karyotype analysis. The compounds of this invention can also be used to probe the function of microtubules in mitotic cells.

EXAMPLES The following examples provide a detailed description of the preferred methods of synthesis of a preferred compound of this invention, SPA-110. Precursor compounds as well as various compounds of this invention are also disclosed. Figures 3 and 4 schematically represent the synthesis of a salt of SPA-1 according to the examples. The reference numerals in the examples correspond to the labeling of compounds in figures 3 and 4 and marking of compounds illustrated in the examples. 3-methyl-3-phenylbutanoic acid (2) 3-Methyl-2-butenoic acid (1, 5.10 g, 50.9 mmol) and AICI3 (20.4 g, 153 mmol) were placed in a one-neck round bottom flask. Benzene (50 mL) was added which produced vigorous bubbling. After the bubbling was over, a blocked condenser was fixed (i.e., closed system), the reaction mixture was stirred and placed in an oil bath at 65 ° C. The pressure in the system was released occasionally. The progress of the reactions continued to monitor the loss of starting material by GC. If the reaction did not end in 1 h, a small amount of AICI3 was added and stirring continued. Diethyl ether was added to the solution and the mixture was cooled to 0 ° C. Slowly add concentrated HCl and water until all the solid dissolved and the pH was less than 2. The aqueous layer was extracted with diethyl ether three times. The organic layer was concentrated to 150 mL and then extracted with a solution of saturated sodium bicarbonate six times. The combined aqueous layer was acidified with concentrated HCl until the pH was less than 2. The aqueous acid layer was extracted with diethyl ether three times and the accumulated organic layer was dried with magnesium sulfate. The solution was filtered and the diethyl ether was removed in vacuo yielding a white solid (8.51 g, 47.7 mmol) in 94% yield, which did not need further purification, mp 55-56 ° C. 1 H NMR (400 MHz, CDCl 3) 10.45 (bs, 1 H, CO 2 H), 7.38 (d, 2 H, J = 7.2 Hz, H-11 and H-7), 7.32 (t, 2 H, J = 7.2 Hz, H-10 and H-8), 7.21 (t, 2H, J = 7.2 Hz, H-9), 2.65 (s, 2H, H-2), 1.47 (s, 6H, H-5 and H-4); mass spectrum (El) 178 (23, M +), 119 (100, [C9H11] +). For previous work on the formation of compound 2 see: F.J. Eijkman (1908) Chem. Kentr. II, p. 110; or A. Hoffman (1929) J. Am. Chem. Soc. 51: 2542. (4S) -3- (3-methyl-3-phenyll-1-oxobutyd-4-isopropyl-2-oxazolidinone (3) 3-Methyl-3-phenylbutanoic acid (2.00 g, 5.61 mmol) was dissolved in 70 mL of THF and cooled to -78 ° C. Triethylamine (1.17 mL, 8.42 mmol) and trimethylacetyl chloride (0.760 mL, 6.17 mmol) were added to the reaction flask to yield a white solid. The resulting mixture was heated at 0 ° C for 1 hour and then cooled again to -78 ° C. In a second flask butyllithium (6.84 mL, 1.6 M in hexanes, 10.9 mmol) was added by dropping with vigorous stirring to a solution of (4S) - (-) - 4-isopropyl-2-oxazolidinone (1.45 g, 11.2 mmol) at -78 ° C in THF (60 mL) producing a white precipitate. The resulting suspension of the oxidized oxazolidinone was added through a cannula to the reaction flask. Stirring was continued for 2 hours, water was added and the reaction mixture was warmed to room temperature, where it was then extracted three times with diethyl ether. The combined organic extracts were dried over magnesium sulfate, and concentrated in vacuo. The product was purified by radial chromatography (4mm plate, 3: 7 diethyl ether-pet ether) yielding compound 3 as a clear, colorless oil in 84% yield (1.37 g, 4.74 mmol). 1 H-NMR (400 MHz, CDCl 3) 7.38 (d, 2 H, J = 7.3 Hz, H-19 and H-15) 7.28 (t, 2 H, J = 7.3 Hz, H-18 and H-16), 7.16 ( t, 1 H, J = 7.3 Hz, H-17), 4.22-4.18 (m, 1 H, H-4), 4.05 (dd, 1 H, J = 9.0 and 2.8 Hz, 1 H-5), 4.00 (t, 1 H, J = 9.0 Hz, 1 H-5), 3.38-3.30 (m, 2H, H-10), 2.16-2.12 (m, 1 H, H-6), 1.48 (s, 3H, H-13 or H-12), 1.47 (s, 3H, H-13 or H-12), 0.79 (d, 3H, J = 7.1 Hz, H-8 or H-7), 0.71 (d, 3H, J = 6.9 Hz, H-8 or H-7); mass spectrum (El) 289 (8, M +), 1 19 (100, [C9Hn] +). The optical rotation obtained was [OC] 25D +69.5 (c 1.16, CHCI3). Compound 3 was prepared according to D.A. Evans et al., (1988) Tetrahedron 44: 5525.

Preparation of 4-isopropyl-2-oxazolidinone 4 Oxazolidinone 3 (472 mg, 1.63 mmol), dried under vacuum for 0.5 h, was dissolved in THF and cooled to -78 ° C (10 mL). Potassium bis (trimethylsilyl) amide (15.6 mL, 0.1 15 M in THF, 1.79 mmol) was added recently titrated and the resulting solution was stirred at -78 ° C for 1 h. A solution of 2, 4,6-trisopropylbenzenesulfonyl azide (6.25 mg, 2.04 mmol) in THF (5 mL) at -78 ° C was added through a cannula and after 2 minutes the orange reaction mixture was treated with glacial acetic acid ( 0.429 mL, 7.50 mmol), was heated to 40 ° C in a water bath and stirred for an additional 1 hour. To the light yellow mixture was added brine (35 mL), water (35 mL) and the aqueous phase was extracted three times with 80 mL of diethyl ether. The combined organic extracts were washed with a saturated sodium bicarbonate solution (20 mL), dried with magnesium sulfate and concentrated in vacuo. The product was purified by radial chromatography (4 mm plate, 3: 7 diethyl ether pet ether, the sample was loaded with diethyl ether) yielding azide 5 as a colorless oil (482 mg, 1.46 mmole) in a yield of 89 %. 1 H-NMR (400 MHz, CDCl 3) 7.39 (d, 2 H, J = 7.2 Hz, H-19 and H-15), 7.31 (t, 2 H, J = 7.2 Hz, H-18 and H-16), 7.23 (t, 1 H, J = 7.2 Hz, H-17), 5.64 (s, 1 H, H-10), 3.95 (dd, 1 H, J = 8.7 and 2.2 Hz, 1 H-5), 3.89- 3.85 (m, 1 H, H-4), 3.56 (t, 1 H, J = 8.7 Hz, 1 H-5), 2.31-2.26 (m, 1 H, H-6), 1.54, 152 (s, 3H, H-13 and H-12), 0.83 (d, 3H, J = 7.0 Hz, H-8 or H-7), 0.79 (d, 3H, J = 6.9 Hz, H-8 or H-7); mass spectrum (INN, NH3) 349 (45, [M + NH5] +), 348 (100, [M + NH4] +), 331 (12, [M + H] +), 303 (57, [MN2 ] +), 119 (94, [C9Hn] +). The optical rotation obtained was [a] 25D +121.5 (c 1.1, CHCl3). Compound 4 was prepared according to the methodology developed by D.A. Evans et al., (1990) J. Am. Chem. Soc. 112: 401 1. 2,4,6-Triisopropylbenzenesulfonyl azide was prepared by the O.C. Dermer et al., (1955) J. Am. Chem Soc. 77:70.

Preparation of 4-isopropyl-2-oxazolidinone 5 Azide 4 (418 mg, 1.26 mmol), 10% palladium on carbon (280 mg), and di-fer-butyl dicarbonate (608 mg, 2.78 mmol) were placed in a 100 mL flask. Ethyl acetate (37 mL) was added and the resulting black suspension was stirred at room temperature. The mixture was flushed with argon, then with hydrogen and stirred in a ball flask with hydrogen overnight (~ 14h). The reaction mixture was filtered through silica gel and the collected material was washed with ethyl acetate. The combined filtrate was concentrated in vacuo and the crude mixture was purified by flash column chromatography (3: 7 diethyl ether-pet ether) to yield compound 5, a colorless viscous oil, in a yield of 78% (400 mg, 0.989 mmol). 1 H-NMR (400 MHz, CDCl 3) 7.40 (d, 2 H, J = 7.4 Hz, H-19 and H-15), 7.29 (t, 2 H, J = 7.4 Hz, H-18 and H-16), 7.21 (t, 1 H, J = 7.4 Hz, H-17), 6.12 (d, 1 H, J = 9.9 Hz, H-10), 5.11 (bs, 1 H, NH), 3.89 (d, 1 H, J = 8.4 and 1.9 Hz, H-5), 3.82-3.79 (m, 1 H, H-4), 3.45 (t, 1 H, J = 8.4 Hz, H-5), 2.26-2.22, (m, 1 H, H-6), 1.41 (s, 9H, H-24, H-23 and H-22), 0.80 (d, 3H, J = 7.0 Hz, H-8 or H-7), 0.76 (d , 3H, J = 6.9 Hz, H-8 or H-7); mass spectrum (INN, mixture CH ^ NHa) 405 (1, [M + H] +), 349 (7, [M-C4H9] +), 230 (100, [C9H14N2O5] +). The optical rotation obtained was [a] 2 D +118.4 (c 0.935, CHCI3). Compound 5 was prepared according to the methodology developed by D.A. Evans et al., (1990) [supra]. (2S) -2- (methyl fer-butyloxycarbonyloamino-3-methyl-3-phenylbutanoate (6) Oxazolidinone 5 (245 mg, 0.605 mmol) was dissolved in a mixture of 7.1 mL of THF and 1.8 mL of water. This solution was cooled to 0 ° C and hydrogen peroxide (0.618 mL, 30% aqueous, 5.45 mmol) and lithium hydroxide (1.82 mL, 1.0 M, 1.82 mmol) were added. The resulting mixture was stirred at room temperature overnight (-15 h). The excess peroxide was quenched by the addition of sodium acid sulfite (7.1 mL, 1.5 M, 10.7 mmol) and stirring was continued for 1 hour. The aqueous phase was acidified with 1.0 M citric acid and the mixture was extracted three times with ethyl acetate. The combined ethyl acetate extracts were dried magnesium sulfate and concentrated in vacuo. TO! remaining crude material was added a solution of diazomethane in diethyl ether until the solution remained yellow. After bubbling argon through the solution for 15 minutes, the remaining volatile components were removed in vacuo to yield crude compound 6. Purification of ester 6 was achieved by radial chromatography (2 mm plate, 3: 7 diethyl ether pet ether, the sample was loaded with CHCl 3), yielding a clear colorless oil (171 mg, 0.555) in a yield of 92%. %. 1 H-NMR (400MHz, CDCl 3) 7.33-7.27 (m, 4H-16, H-15, H-13, H-12), 7.20 (t, 1 H, J = 6.7 Hz, H-14), 4.99 ( bd, 1 H, J = 8.8 Hz, H-2), 4.50 (bd, 1H, J = 8.8 Hz, NH), 3.48 (s, 3H, H-17), 1.41, 1.38 (s, 3H, H- 5 and H-4), 1.37 (s, 9H, H-10, H-9, and H-8); mass spectrum (El) 307 (0.1, M +), 234 (2, [M-Oí-Bu] +), 119 (100, [C9Hn] +). The optical rotation obtained was [a] ^ 5 + 35.2 (c 2.98, CHCI3). Compound 5 was prepared according to the methodology developed by D.A. Evans went to. (1990) [supra]. (2S) -N-iyer-butoxycarbonyl -? / - methyl-3-methyl-3-phenylbutanoic acid ÍZ) To a vigorously stirred solution of ester 6 (43.4 mg, 0.141 mmol) in 2 mL of DMF was added sodium hydride (10.2 mg, 4.24 mmol) followed by methyl iodide (0.088 mL, 1.41 mmol) and the resulting gray suspension was stirred overnight (-20 h) at room temperature. The excess sodium hydride was quenched by careful addition of water and the mixture was acidified by drip addition of 1.0 Af citric acid. The acid mixture was extracted three times with ethyl acetate, the combined organic layer was extracted three times with brine, dried over magnesium sulfate and concentrated in vacuo. The resulting pale orange oil was dissolved in 4 mL of methanol in a 25 mL flask. To the solution was added 1.0 mL of water, followed by 1.13 mL of 1.0 Af lithium hydroxide. The reaction mixture was heated at 60 ° C overnight (-14 h), producing a white precipitate. To the resulting mixture was added saturated sodium bicarbonate solution and water; then the mixture was extracted with ethyl acetate. The aqueous layer was acidified with 1.0 Af citric acid until the pH was ~ 4. The mixture was extracted three times with ethyl acetate. The combined organic layers were dried with magnesium sulfate and concentrated in vacuo. Compound 7 was also found in the first extraction of ethyl acetate so it was also added to the crude product. The purification of acid 7 was carried out by column chromatography on silica gel (1: 2 diethyl ether pet ether with 1% acetic acid) resulting in a yield of 49% (21.2 mg, 0.0670 mmol) of a clear colorless oil. 1 H NMR (400 MHz, CDCl 3) 7.41 (d, 1.3 H, J = 7.6 Hz, H-17 and H-13), 7.37 (d, 1.3 H, J = 7.6 Hz, H-17 and H-13), 7.28 (t, 2H, J = 7.6 Hz, H-16 and H-14), 7.18 (t, 1 H, J = 7.2 Hz, H-15), 5.17 (bs, 0.66 H, H-2), 4.93 (bs, 0.33 H, H-2), 2.75 (s, 1.05H, H-6), 2.62 (s, 1.95H, H-6), 1.55 (s, 3H, H-5 or H-4), 1.49 -1.39 (m, 12H, H-5 or H-4 and H-1 1, H-10 and H-9); mass spectrum (El) 307 (0.1, M +), 234 (3, [M-Oí-Bu] +), 119 (100, [CgHpD, 57 (78, [C4H9] +), exact mass calculated for d7H25NO4 307.1783, Found (El): 307.1793.

Preparation of compound 9 The β-amino-8 ester (71.6 mg, 0.174 mmol) was dissolved in 1 mL CH2Cl2 and 1 mL of TFA was added. The reaction mixture was stirred at room temperature for 0.5 hours. The solvent was removed in vacuo, followed by repeated rinsing of the remaining material with CH2Cl2 (3 x 5 mL) and evaporation of the residual solvent yielding the TFA salt of the amino acid ester 8. In a separate flask, to a solution (or suspension) of the protected amino acid N-Boc 7 (51.5 mg, 0.167 mmol) in 0.5 mL of CH2CI2) was added DIEA (0.0875 mg, 0.0503 mmol), DMAP (0.031 mg, 0.10 mmol) and PyBroP (0.0781 mg, 0.167 mmol). The solution was stirred for a few minutes and then the solution of the TFA salt of compound 8 was added in 1 mL to CH2Cl2 via cannula addition. The reaction mixture was stirred at room temperature for 18 hours. To the mixture was added water, CH2Cl2 and ten drops of 10% aqueous HCl. The resulting biphasic solution was extracted with CH2Cl2 (three times with 20 mL). The organic layer was extracted with saturated aqueous sodium bicarbonate (10 mL), dried with magnesium sulfate and the solvent was removed in vacuo. The product was purified by flash chromatography (silica gel, 1: 1 diethyl ether-pet ether) to give the protected tripeptide 9 as a clear colorless oil in a yield of 27% (0.0272 g, 0.0454 mmole). 1 H-NMR (400 MHz, CDCl 3) 7.84 (bd, 1 H, J = 9.5 Hz, NH), 7.4-7.30 (m, 5H, H-28, H-27, H-25, H-24), 7.21 (bt, 1 H, J = 7.2 Hz, H-26), 6.63 (bd, 1 H, J = 9.6 Hz, H-6), 5.08 (t, 1 H, J = 9.6 Hz, H-7), 4.83 (d, 1 H, J = 9.5 Hz, H-13), 4.17 (q, 2H, J = 7.1 Hz, H-2), 3.02 (s, 3H, H-11), 2.15 (s, 0.66H) , H-29), 2.02 (s, 2.37H, H29), 1.94-1.81 (m, 1 H, H-8), 1.88 (s, 3H, H-5), 1.39-1.38 (m, 9H, H -34, H-33 and H-32), 1.28 (t, 3H, J = 7.1 Hz, H-1), 0.98 (s, 9H, H-17, H-16 and H-15), 0.83, 0.77 (d, 3H, J = 6.6 Hz, H-10 and H-9); PyBroP is described in E. Frérot et al. (1991) Tetrahedron 47: 259.

Trifluoroacetate salt-SPA110 (10) To a solution of ethyl ester 9 (23.0 mg, 0.0382 mmol) in 1. 1 mL of MeOH was added 0.30 mL of water and 0.31 mL of a 1.0 M aqueous solution of lithium hydroxide (0.31 mmol). The reaction mixture was stirred at room temperature overnight (-20 h) after which it was acidified by dropwise addition of 1.0 Af citric acid and then extracted three times with ethyl acetate. The combined organic extracts were dried with magnesium sulfate and concentrated in vacuo. Under an argon atmosphere, the crude oil was dissolved in 1 mL of CH2Cl2 and the solution was treated with TFA (1 mL) and then stirred at room temperature for 0.5 hour. Removal of the excess solvents in vacuo, followed by rinsing the remaining material three times with CH2Cl2 (5 mL) and evaporation of the residual solvent, produced the TFA salt. Purification by HPLC of the crude product using a reversed-phase C-18 Magnum column (H2O (45): MeOH (55) with 0.05% TFA) yielded tripeptide 10 as a white powder. 1 H NMR (400 MHz, CD 3 OD) 7.53 (d, 2 H, J = 7.6, H-25 and H-21), 7.44 (t, 2 H, J = 7.6 Hz, H-24 and H-22), 7.34 (t , 1 H, J = 7.6 Hz, H-23), 6.76 (d, 1 H, J = 9.1 Hz, H-4), 5.04 (t, 1 H, J = 10.1 Hz, H-5), 4.91, 4.34 (s, 1 H, H-17 and H-11), 3.13 (s, 3H, H-9), 2.49 (H-26), 2.08-1.99 (m, 1 H, H-6), 1.90 ( s, 3H, H-3), 1.46, 1.37 (s, 3H, H-19 and H-18), 1.05 (s, 9H, H-15, H-14 and H-13), 0.89 (d, 3H , J = 6.1 Hz, H-8 or H-7), 0.88 (d, 3H, J = 6.5 Hz, H-8 or H-7); mass spectrum (El) 474 (0.1, [M-CF3CO2 -] +), 458 (0.1, [M-16-CF3CO2 -] +), 382 (2), 162 (62), 69 (74), 45 (100) Me O I, I Boc "^ N 'OMe 12 To a cold (0 ° C) solution of? / - Boc -? / - methylvaline (11) (5.0 g, 21.6 mmol),? -hydrochloride, O-dimethylhydroxylamine (2.8 g, 28 mmol) and PyBOP® ( 1.2 g, 22 mmol) in CH2Cl2 (22 mL) was added DIEA (8.4 mL, 75 mmol). After 1 minute, the reaction mixture was warmed to room temperature and stirring was continued for 1 hour. If the pH value of the mixture was less than 7, the mixture could be treated with a few drops of DIEA to allow the reaction to conclude. The mixture was poured into 200 mL of diethyl ether and the resulting mixture was washed successively with 3 N hydrochloric acid (3 x 30 mL), saturated aqueous sodium hydrogen carbonate solution (3 x 30 mL) and saturated aqueous sodium chloride ( 3 x 30 mL). The organic layer was dried with magnesium sulfate and the solvent was evaporated, followed by chromatography of the crude product (silica gel, 1: 3 diethyl ether-pet ether), gave 12 (4.46 g, 75% yield) as a colorless oil. 1 H-NMR (200 MHz, CDCl 3): 0.84 (d, J = 6.6 Hz, 4 H, (CH 3) 2), 0.85 (d, J = 6.6 Hz, 2 H, (CH 3) 2, 1.41 (s, 6 H, Boc - (CH3) 3), 1.44 (s, 3H, Boc- (CH3) 3), 2.15-2.3 (m, 1 H, CH), 2.75 (s, 1 H, NCH3, 2.78 (s, 2H, NCH3, 3.10 (bs, 3H, NCH3), 3.64 (s, 1 H, OCH3, 3.68 (s, 2H, OCH3, 4.66 (d, J = 10 Hz, 0.4H, CH), 4.95 (d, J = 10 Hz, 0.6H, CH); exact mass calculated for C13H27N2O4 (M + H) +: 275.19708. Found (DCI): 275. 19710. The obtained optical rotation was [a] 25D + 128.3 (c 2.9, CHCI3).

? / - Boc- / V-methyl-1-valinal (13) 13 Lithium aluminum hydride (875 mg, 23 mmol) was added to a solution of? / - methoxy? -methylamide? -Boc-? Methyl-L-valine (12) (2.0 g, 7.7 mmol) in dry THF (8 mL) and the reaction mixture was stirred for 20 minutes. The mixture was poured into a stirring solution of potassium hydrogen sulfate (3.14 g, 23 mmol) in water (100 mL). Diethyl ether (75 mL) was added, the layers were separated and the aqueous layer was extracted with diethyl ether (3 x 50 mL). The organic layers were combined and washed sequentially with 3 N hydrochloric acid (3 x 30 mL), saturated aqueous sodium acid carbonate (3 x 30 mL) and saturated aqueous sodium chloride (3 x 30 mL). The organic layer was dried with magnesium sulfate and the solvent was evaporated to yield crude aldehyde 13 (1.52 g, 92% yield). The aldehyde 13 was used without further purification. Note: 13 can be stored under argon for - 2 weeks, but when stored in organic solvents at room temperature it undergoes a slow decomposition. 1 H-NMR (200 MHz, CDCl 3): 0.73 (d, J = 6.9 Hz, 3 H, (CH 3), 0.91 (d, J = 6.9 Hz, 3 H, (CH 3), 1.27 (s, 9 H, Boc- (CH 3 ) 3), 2.02-2.15 (m, 1 H, CH), 2. 63 (s, 1 H, NCH3), 3.44 (d, J = 9.5 Hz, 0.5H, CH), 3.86 (d, J = 10 Hz, 9 Hz, 0. 5H, CH); 9.45 (s, 1 H, CH); exact mass calculated for CnH22NO3 (M + H) +: 216. 15997; found (DCI): 216. 15996; the optical rotation obtained was [a] 25D -104.2 (c 5.5, CHCl3). (2E, 4S) - ethyl / V-Boc- / -methyl-4-amino-2,5-dimethylhex-2-enoate i To a solution of aldehyde 13 (1.75 g, 8.7 mmol) in dry CH 2 Cl 2 (9.0 mL) under an argon atmosphere at room temperature was added (carbetoxyethylene) triphenylphosphorane (4.19 g, 11.3 mmol) and stirring was continued for 4 hours. The reaction mixture was diluted with water (100 mL) and extracted with diethyl ether (3 x 100 mL). The combined organic extracts were washed with saturated aqueous sodium chloride (100 mL), dried with magnesium sulfate and concentrated in vacuo. The crude oil was purified by flash chromatography (silica gel, 2:23 diethyl ether-pet ether) to give the required E-2-alkenoate 14 as a colorless oil (2.13 g, 82% yield) 1H- NMR (200 MHz, CDCl 3 0.74 (d, J = 6 Hz, 3 H, CH 3), 0.79 (d, J = 6 Hz, 3 H, CH 3), 1.17 (t, J = 7 Hz, 3 H, CH 3), 1.34 ( s, 9H, Boc- (CH3) 3, 1.72 (m, 1 H, CH), 1.78 (s, 3H, CH3), 2.60 (bs, 3H, NCH3), 4.08 (q, J = Hz, 2H, CH2 ), 4.15 - 4.20 (m, 0.05H, CH), 4.21 - 4.32 (m, 0.05H, CH), 6.54 (d, J = 8 Hz, 1 H, CH), exact mass calculated for C16H30NO4 (M + H ) +: 300.21750, found (DCI): 300.21754, the optical rotation obtained was [a] 25D + 61.1 (c 9.1, CHCI3).

General Procedure 1: Cutting of N-Boc groups mediated by trifluoroacetic acid Ester of N-Boc-amino acid (1.0 equiv.) Was treated with TFA CH2CI2 (0.1 mmol / 1 mL) at room temperature for 0.5 hours. Removal of the solvent in vacuo, followed by repeated rinsing of the residual material with CH 2 Cl 2 (3 x 5 mL) and evaporation of the remaining traces of solvent produced the TFA salt of the amino acid ester in quantitative yield. The TFA salts were used without further purification.

General procedure 2: peptide coupling mediated by trimethylacetyl chloride To a cold (-78 C) solution and stirred acid (1.1 equiv.) In THF (1 mUmmoles) under an argon atmosphere was added DIEA (1.5 equiv.) and trimethylacetyl chloride (1.2 equiv.). The resulting mixture was heated at 0 ° C for 1 hour and cooled again to -78 ° C. DIEA (2.2 equiv.) Was added with a cannula to the reaction flask followed by the addition by means of a cannula of the TFA salt of the amino acid ester (1.0 equiv., Prepared by general procedure 1) in dry THF (0.5. mL / mmole) at -78 ° C. Stirring was continued for 1 hour and water (40 mL) was added. The mixture was allowed to warm to room temperature and extracted with diethyl ether (3 x 50 mL). The combined organic extracts were washed with saturated aqueous sodium chloride (50 mL), dried over magnesium sulfate and concentrated in vacuo. The crude oil was purified by flash chromatography (silica gel, diethyl ether-pet ether) to give the desired dipeptide as a colorless oil.

Dipeptide 8 Following general procedure 2, dipeptide 8 was prepared with the following amounts of reagents and solvents:? / - Boc-fer-leucine (15), 156 mg (0.52 mmol); trimethylacetyl chloride, 64 mL (0.52 mmol); DIEA, 99 mL (0.57 mmol); ? / - Boc-MHW-OEt (14), 10 mg (0.47 mmol); DIEA, 198 mL (1.14 mmol); THF, 7 mL. Purification of the crude product by flash chromatography (silica gel, 1: 5 diethyl ether-pet ether) gave 121 mg of 8 (62% yield). 1 H-NMR 8200 MHz, CDCl 3) 0.76 (d, J = 6 Hz, CH 3, 0.80 (d, J = 6 Hz, 3 H, CH 3), 0.88 (s, 9 H, (CH 3) 3, 1.22 (t, J = 7Hz, 3H, CH3, 1.33 (s, 9H, Boc- (CH3) 3), 1.79 - 1.89 (m, 1 H, CH), 1.83 (s, 3H, CH3), 2.91 (s, 3H, NCH3, 4.12 (q, J = 7 Hz, 2H, CH2, 4.35 (d, J = 10 Hz, 1 H, CH), 5.03 (t, J = 10 Hz, 1 H, CH), 5.14 (d, J = 10 Hz , 1 H, NH), 6.57 (d, J = 8 Hz, 1 H, CH), exact mass calculated for C22H41N2O5 (M + H) +: 413.30154, found (DCI): 413.30154. ] 25D -76.9 (c 2.43, CHCI3).

Cytotoxicity test The cytotoxicity of SPA-110 compared to hemiasterline as against p53 + and p53- variants of human breast cancer MCF-7 cells and A549 tumor cells was determined according to the methods described in J. Immunol. Methods 65: 55-63 (1983). The results shown in Figures 5A and 5B show that SPA-110 is more cytotoxic in some cases than the naturally occurring compound.

Test for antimitotic activity Antimitotic activity is detected by the enzyme-linked immunosorbent assay using a mitosis specific antibody, TG-3 (from Albert Einstein College of Yeshiva University, Bronx, NY) and see: PCT application published on July 4 of 1996 as WO96 / 20218). Mp53"MCF-7 cells (expressing a dominant-negative p53 mutation such as that described in S. Fan, et al. (1955) Cancer Research 55: 1649-1654) were cultured as monolayers in DMEM supplemented with fetal bovine serum at 10%, 2 mM L-glutamine, 50 units / ml penicillin, 50 μg / ml streptomycin, 1 mM sodium pyruvate, non-essential MEM amino acids, 1 μg / ml bovine insulin, 1 μg / ml hydrocortisone , 1 ng / ml of human epidermal growth factor and 1 ng / ml of ß-estradiol at 37 ° C in 5% humidified CO2.The cells were seeded at 10,000 cells per well of 96-well polystyrene tissue culture plates. cavities (Falcon) in a volume of 200 μl of cell culture medium.The cells were grown for 24 hours and the compounds were added at approximately 1 μg / ml or 10 μg / ml (from 1000-fold groups in sulfoxide). dimethyl) and the cells were incubated for 20 hours.Nocodazole (Sigma) served as a positive control. After treatment with the agent to be tested, the cell culture medium was completely removed and the 96-well tissue culture plates were frozen at -70 ° C for up to 2 hours. Frozen cells were thawed by the addition of 100 μl of ice-cooled lysis pH buffer (0.5 mM phenylmethylsulfonyl fluoride, 1 mM ethylene glycol bis (β-aminoethyl ether) N, N, N, N'- tetraacetic, pH 7.4), and lysed by pipetting up and down 10 times. The used cells were transferred to 96-well PolySorp ELISA plates (Nunc) and dried completely by blowing hot air at about 37 ° C with a hair dryer placed approximately 1 meter above the plates. Protein binding sites were blocked by adding 200 μl per cavity of 10 mM Tris HCl pH 7.4, 150 mM NaCl, 0.1 mM PMSF, 3% (w / v) dehydrated milk dehydrated (Camation) for 1 hour at room temperature. This was removed and replaced with 100 μl of the same solution containing 0.1-0.15 μg / ml monoclonal antibody specific for TG-3 mitosis and goat anti-mouse IgM labeled with horseradish peroxidase (1021-05, Southern Biotechnology Associates ) at a dilution of 1/500. After the nocturnal incubation at 4 ° C the antibody solution was removed and the wells were rinsed 3 times with 200 μl rinse buffer (10 mM Tris HCl pH 7.4, 0.02% Tween 20). 100 μl of 120 mM Na2HPO4, 100 mM citric acid, pH 4.0 containing 0.5 mg / ml of 2,2'-azino-bis (3-ethylbenzthiazole-6-sulfonic acid) and 0.01% hydrogen peroxide was added for 1 hour at room temperature and the plates were read at 405 nm using a BioTek plate reader. The results of the comparison of the antimitotic activity of hemiasterlin with SPA-110 are shown in Figure 6. The SPA-110 exhibited considerably greater antimitotic activity than the naturally occurring compound.

In vivo activity of SPA-1 The compound SPA-110 was evaluated in vivo using standard pharmacological test procedures that measure its ability to inhibit the growth of human tumor xenografts. The LOVO human color carcinoma (American Type Culture Collection, Rockville, Maryland # CCL-229) was grown in tissue culture in RPMI supplemented with 10% FBS. Atomic nu / nu female mice (Charles River, Wilmington, MA) were injected subcutaneously in the lateral area with 7.0 x 106 LOVO cells. When the tumors obtained a mass between 80 and 120 mg, the mice were randomly selected in treatment groups (day zero). The animals were treated intravenously once a day on days 1, 5 and 9 after selection (day zero) with 1 mg / kg / dose of SPA-1 10 prepared in 2.5% ethanol in saline or saline solution as the vehicle control. Some animals were treated intraperitoneally once a day on days 1, 5 and 9 after selection with 1 mg / kg / dose of vincristine as a positive control. The tumor mass was determined every 7 days [(length x width2) / 2] for 28 days after selection. The RTG or relative tumor growth (average tumor mass on days 7, 14, 21 and 28 divided by the average tumor mass on day zero) was determined for each treatment group. % T / C was calculated as RTC (treated group)% RTC (vehicle control group) x 100. Statistical analyzes (student t-test) of logarithmic relative tumor growth were used to compare the treated group versus the control group in every experiment In each case, a p value (p <0.05) was obtained, which indicates a statistically significant reduction in the relative tumor growth. Five of 5 animals treated on day 28 with SPA-110 survived. Nine of 10 animals treated on day 28 with vincristine survived. The results are shown in the following table.

Relative activity of the compounds of this invention A number of SPA-110 analogs have been synthesized and have been characterized for their cytotoxic and antimitotic activity. The high degree of correlation between cytotoxicity and antimitotic activity indicates that the cytotoxicity of the compounds of this invention is due to the antimitotic activity of the compounds. The following structures are analogs that are within the scope of this invention illustrated in an approximately descending order of cytotoxic / antimitotic activity.

As will be apparent to those skilled in the art in light of the foregoing description, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention should be considered in accordance with the subject matter defined by the present claims, which subject matter includes obvious chemical equivalents of the compounds and methods described in the claims.

Claims (21)

NOVELTY OF THE INVENTION CLAIMS

1. - A compound or pharmaceutically acceptable salt thereof, which has the formula wherein Ri and R2 are independently selected from the group consisting of: H, R, and ArR-, and where at least one of Ri and R2 is R and neither is ArR-, Ri and R2 together may be optionally a ring of three to seven members; R3 and R4 are independently selected from the group consisting of: H, R, ArR-, and where at least one of R3 and R4 is R and neither is ArR- or Ar, R3 and R4 together may be optional a ring of three to seven members; R5 is selected from the group consisting of: H, R, ArR-, and Ar; R6 is selected from the group consisting of: H, R, and ArR-; R7 and Rs are independently selected from the group consisting of: H, R, and ArR-; and R9 is: Z- C- Y-; and wherein, R is defined as a saturated or unsaturated portion having a linear, branched or cyclic base structure containing one to ten carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms, and zero to four sulfur atoms, and the carbon atoms are optionally substituted with: = O, = S, OH, -OR10, -O2CR? 0l -SH, -SR10, -SOCR10, -NH2, -NHR10, -N ( R10) 2, -NHCOR10, -NR10COR10, -I, Br, -Cl, -F, -CN, -C02H, -CO2R10, -CHO, -COR10, -CONH2, -CONHR10, -CON (R10) 2, - COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R? 0, wherein R10 is a saturated or unsaturated alkyl group of one to ten carbon atoms, linear, branched or cyclic; X is defined as a portion selected from the group consisting of: -OH, -OR, = O, = S, -02CR, -SH, -SR, -SOCR, -NH2, -NHR, -N (R) 2, -NHCOR, -NRCOR, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R, -CHO, -COR, -CONH2, -CONHR, -CON (R) 2, -COSH, - COSR, -NO2, -SO3H, -SOR, and -SO2R; Ar is defined as an aromatic ring selected from the group consisting of: optionally substituted phenyl, naphthyl, anthracyl, phenanthryl, furyl, pyrrolyl, thiophenyl, benzofuryl, benzothiophenyl, quinolinyl, isoquinolyl, midazolyl, thiazolyl, oxazolyl, and pyridinyl with R or X; Y is defined as a portion selected from the group consisting of: an alkyl group of one to six carbon atoms, saturated or unsaturated, linear, optionally substituted with R, ArR-, or X; and, Z is defined as a portion selected from the group consisting of: -OH, -OR; -SH; -MR; -NH2; -NHR; -N (R) 2; -NHCH (Rn) COOH; and -NRCH (Rn) COOH, wherein Rn is a portion having the formula: R, or - (CH2) nNR12R13, wherein n = 1-4 and R12 and R13 are independently selected from the group consisting of: H; R; and -C (NH) (NH2).

2. The compound according to claim 1, further characterized in that Ar is phenyl, naphthyl, anthracyl, or pyrrolyl.

3. The compound according to claim 2 wherein Rs is phenyl, naphthyl, anthracyl, or pyrrolyl.

4. The compound according to claim 1, 2 or 3, further characterized in that R3 and R4 are independently selected from the group consisting of: methyl, ethyl, n-propyl and n-butyl, or, R3 and R4 together are they are selected from the group consisting of: β-cyclopropyl, β-cyclobutyl, β-cyclopentyl and β-cyclohexyl.

5. The compound according to any of claims 1-4, further characterized in that R1 and R2 are independently selected from the group consisting of: H, methyl, ethyl, propyl, n-butyl, acetyl; or, R1 and R2 are attached and form a portion of the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

6. The compound according to any of claims 1-4, further characterized in that R1 and R2 are independently: H, CH3, or acetyl.

7. The compound according to any of claims 1-4, further characterized in that R \ is H, and R2 is -CH3.

8. The compound according to any of claims 1-7, further characterized in that Z is: OH, -OCH3, -NHCH (Rn) COOH, or, -NCH3CH (Rn) COOH, wherein R is R, or - (CH2) n NHC (NH) (NH2).

9. The compound according to any of claims 1-7, further characterized in that Z is Z is OH or -ORu, wherein Ru is an alkyl group of one to six carbon atoms, linear or branched.

10. The compound according to any of claims 1-7, further characterized in that Rg has the formula: O w -C -C = C (R16) C-OH wherein R15 is selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, tertbutyl, isobutyl, and secbutyl; and R 6 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl and secbutyl.

11. The compound according to claim 10, further characterized in that Ris is isopropyl and Rie is methyl.

12. The compound according to any of claims 1-11, further characterized in that R7 is a branched alkyl group of three to six carbon atoms.

13. The compound according to any of claims 1-12, further characterized in that Re and Rs are independently: H, or CH3.

14. The compound according to any of claims 1-11, further characterized in that Re is H, R7 is: -C (CH3) 3, and R8 is -CH3.

15. - The compound according to any of claims 1-14, further characterized in that R3 and R4 are each R.

16. The compound according to any of claims 1-14, further characterized in that R3 and R4 are each -CH3.

17. The compound according to claim 16, further characterized in that R5 is phenyl.

18. The compound according to claim 17, further characterized in that Rg has the formula: CH (CH3) 2

19. A method for preparing a compound as described in claim 1 comprising the step of: (a) coupling an amino acid having the formula: wherein R3-R5 are as defined in claim 1 and Q and T are selected from the group consisting of: R1 and R2 as defined in claim 1, and a protecting group; with a dipeptide that has the formula: wherein Re-Rg are as defined in claim 1; and where Q or T is a protecting group, the additional step of replacing the protecting group with Ri or R2 to form compound I; or (b) coupling a dipeptide having the formula: wherein R3-R7 are as defined in claim 1 and Q and T are as defined above; with an amino acid that has the formula: R8c HN wherein Rs and Rg are as defined in claim 1; and where Q or T is a protecting group, the additional step of replacing the protecting group with R1 or R2 to form compound I

20. An amino acid suitable for use in the method according to claim 19, having the formula: wherein R3 - R5, Q and T are as defined in claim 19.

21. A suitable dipeptide for use in the method according to claim 19, having the formula: 5 in which R3 - R7, Q and T are as defined in claim 19.