WO1994011392A1

WO1994011392A1 - Peptide inhibitors of tyrosine kinases and therapeutic uses thereof

Info

Publication number: WO1994011392A1
Application number: PCT/US1993/010610
Authority: WO
Inventors: Ellen Myra Dobrusin; Dennis Joseph Mcnamara; David W. Fry; Juswinder Singh
Original assignee: Warner-Lambert Company
Priority date: 1992-11-06
Filing date: 1993-11-03
Publication date: 1994-05-26
Also published as: AU5548694A; MX9306930A

Abstract

Novel peptide compounds, and pharmaceutical compositions containing the compounds as useful pharmacological agents in the control of hyperproliferative disease such as cancer and in the treatment of viral, inflammatory, allergic, and cardiovascular disease. The peptides in salt form or nonsalt form comprise a tyrosine phosphorylation site of phospholipase C-η1 (PLC-η1) wherein the tyrosine is modified, such that in a cellular process the peptide inhibits the EGF receptor tyrosine kinase.

Description

PEPTIDE INHIBITORS OF TYROSINE KINASES

AND THERAPEUTIC USES THEREOF

FIELD OF THE INVENTION

The present invention relates to novel peptide compounds, and pharmaceutical compositions containing the compounds as useful pharmacological agents in the control of hyperproliferative disease such as cancer and in the treatment of viral, inflammatory, allergic, and cardiovascular disease. This invention also relates to methods of treatment employing the

compounds.

BACKGROUND OF THE INVENTION

Protein phosphorylation and dephosphorylation are critical mechanisms for regulating the signal

transduction pathways in normal and transformed cells. Protein tyrosine kinases (PTK) are an important class of phosphorylating enzymes which mediate signalling and regulate cell growth and proliferation by catalyzing the transfer of the terminal phosphate from ATP to the phenol of tyrosine in substrate proteins. The over- expression or inappropriate expression of normal or mutant tyrosine kinases (TK) can result in the loss of growth control and the unregulated cell proliferation associated with hyperproliferative disorders such as cancer. Inhibitors of these enzymes have been shown to restore normal regulation to unregulated cell

proliferation attributed to over-expression or

inappropriate expression of normal or mutant TK. It has also been demonstrated that these inhibitors are of therapeutic value, especially as antitumor agents. More specifically, some growth factor receptors, protooncogene and oncogene products associated with malignancy possess PTK activity. In some growth factor dependent tumors, the growth factor signal transduction pathway employs the intrinsic TK activity of the growth factor receptor for autophosphorylation and the

phosphorylation of specific cellular substrate proteins involved in cell cycle events including those involved in mitogenesis and cell proliferation. It has been demonstrated that inhibition of growth factor receptor TK in certain tumors can result in uncoupling the PTK from the signal transduction pathway and that this is sufficient for antitumor activity. This antitumor activity has been demonstrated both in vitro and in vivo. Many known TK inhibitors are activated styrene-like small molecules or other Michael-type acceptors resembling tyrosine itself.

For example, the epidermal growth factor receptor tyrosine kinase (EGFR TK) inhibitor erbstatin is reported to inhibit the growth of human epidermoid carcinoma A431 cells with an IC₅₀ = 3.6 μg/mL [J.

Antibiot., 1986;39:170]. Erbstatin also inhibits the growth of the human mammary carcinoma MCF-7 and some esophageal tumors in nude mice in a dose dependent manner [Eur. J. Cancer 1990;26 (6) : 722 and Japanese patent 03,109,323]. Another class of PTK inhibitor called the tyrphostins also potently inhibited the EGF-dependent growth of A431 cells in vitro [J. Med. Chem. 1989;32:2344; J. Med. Chem. 1991;34:1896]. The antitumor activity of two tyrphostins has been reported in vivo in nude mice bearing human squamous cell carcinoma MH-85 [Cancer Res. 1991;51:4430]. In vitro and in vivo antitumor activity against A431 tumors has also been reported for a series of sulfonylbenzoyl nitrostyrene TK inhibitors [J. Med. Chem. 1991; 34:2328] and for other classes of TK inhibitors [Cancer Res. 1992;52:4492].

In a further specific example, Yoneda, et al.

(Cancer Res. 1991;51:4430) have shown that TK

inhibitors RS-13022 and 14620 suppressed EGF-stimulated proliferation of HER-14 cells (transfected NIH 3T3 cells) and MH-85 tumor cells in vitro. The MH-85 tumor is a well-characterized human squamous cell carcinoma associated with 3 paraneoplastic syndromes:

hypercalcemia, leukocytosis, and achexia. MH-85 cells over- express endogenous EGFR TK and are dependent on the EGFR signal transduction pathway for growth

in vitro and in nude mice. In vivo, the compounds suppressed the growth of HM-85 tumors in nude mice as well as the expression of the paraneoplastic syndromes. An increase in life span of 75% was observed for

RG-13022-treated tumor bearing mice. In tumor cells, the direct inhibition of TK activity can lead to antitumor activity, i.e., the restoration of the normal regulation of cell growth and differentiation.

Phospholipase C-γ1 (PLC γ1) is one of the

substrates for receptor TK and it is believed to play a critical role in growth factor mediated mitogenic signaling [Science 1989;244:546; PNAS : USA

1989,-86:1568; Cell 1989;57:1101; Cell 1989;57:1109]. This protein is one of the substrates of the EGFR TK. The specific tyrosines in PLC γ1 which are

phosphorylated have been identified both in vitro and in vivo [J. Biol. Chem. 1990;265:3940; J. Biol. Chem. 1990;265:3944].

Likewise, it has been demonstrated by those skilled in the art using test systems which model other than hyperproliferative disorders that TK inhibitors can block phosphorylations associated with signaling pathways involved in these disorders. For example: Antiviral

Science 1992;256:542. Participation of tyrosine phosphorylation in the cytopathic effect of human immunodeficiency virus- 1.

Antiinflammatory

J. Biol. Chem. 1992;267:426. Nuclear signaling in human neutrophils.

Blood 1991;78:1842. Involvement of TK in the activation of human peripheral blood neutrophils by granulocyte-macrophage colony stimulating factor.

Antiallergic

Nature 1991;353:855. Phosphorylation and

dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement.

Nature 1992; 355: 78. Engagement of the high affinity IgE receptor activates src protein-related TK. Autoimmune Disease

Int. Immun. 1991;4:1201. The PTK inhibitor herbimycin A specifically inhibits signal transduction by the T cell antigen receptor.

PNAS U.S.A. 1990;87:7722. Inhibition of tyrosine phosphorylation prevents T cell receptor-mediated signal transduction.

Cell 1991;65:281. Regulation of T cell receptor signaling by a src family PTK (p59^fyn).

Cardiovascular Disease

Circulation 1992; 86 (Suppl. I):I-169, Abstract 0675). Platelet derived growth factor receptor (PDGFR) PTK activity in carotid artery is enhanced by injury and inhibited by tyrphostin RG-13291.

Circulation 1992;86 (Suppl. I):I-292, Abstract

1160). TK inhibitor, RG-13291 prevents association of beta-PDGFR with phosphatidylinositol 3'-kinase (PI3K) in vascular smooth muscle cells. Summary and Detailed Description of the Invention

This invention, therefore, pertains in one

preferred aspect to novel peptides that inhibit one or more of the following PTK: abl, bcr-abl, v-src, EGFR, PDGFR, erb B-2/neu growth factor (neu) receptor, fibroblast growth factor receptor (FGFR), or macrophage colony stimulating factor receptor (CSF-1R). The peptides have an amino acid sequence incorporating the tyrosine which is phosphorylated by the PTK, whether that be from an autophosphorylation site in the PTK itself, or from a cellular TK substrate protein

selected from one of the following: PLC γ1, ras GTPase activator protein (GAP), and the p85 subunit of PI3K wherein that tyrosine is replaced with D or L 2,3,5,6- tetrafluorotyrosine or D- tyrosine itself.

This invention also pertains in one preferred aspect to novel peptides which we discovered bind to the EGFR TK and also pertains to synthetic peptides and their substitution analogs derived from the primary amino acid sequence of PLC γ1. The peptides of the present invention are comprised of native (wild type) amino acid sequences from PLC γ1 and derivatives thereof. The amino acid sequences of the peptide inhibitors are derived from the native amino acid sequences incorporating each of the tyrosines of PLC γ1 phosphorylated by the EGFR TK located at tyrosines 472, 771, 783, and 1254 [J. Biol. Chem. 1990;265:3940;

J. Biol. Chem. 1990;265:3944].

In another preferred aspect of this invention, analogs of shorter or longer sequence (N-terminally and C-terminally shortened or lengthened fragments),

N-terminally and C-terminally modified peptides, and those incorporating modified sequences in which one or more amino acids in the native sequence are replaced by natural or unnatural amino acids (in their L or

D forms) are also included in this invention. Specifically included in this invention are analogs incorporating an "unnatural" (modified) tyrosine, especially those bearing electron withdrawing groups such as halogen, nitro, etc. The rationale for replacing tyrosine with modified tyrosines bearing electron withdrawing groups such as tetrafluorotyrosine to convert a tyrosine containing peptide from a TK substrate to an inhibitor has been reported and

successfully demonstrated [J. Biol. Chem.

1990;265:7108; J. Biol. Chem. 1990;265:16205].

Further, the invention includes the aforementioned peptides and their analogs which have undergone a mutation. A "mutation" in a peptide alters its primary structure (relative to the commonly occurring or specifically described peptide) due to changes in its synthetic preparation or the nucleotide sequence of the DNA which encodes it if prepared by recombinant

technology. These mutations specifically include allelic variants. In spite of the mutation, the peptides and their analogs are still characterized by their function which is to bind to EGFR TK or to inhibit the association of a protein tyrosine kinase with a cellular substrate protein selected from one of the following: PLC 71, ras GTPase activator protein (GAP), and the p85 subunit of PI3K wherein that

tyrosine is replaced with D or L 2,3,5,6- tetrafluoro tyrosine or D-tyrosine itself.

Mutational changes in the primary structure of a peptide result from deletions, additions, or

substitutions. A "deletion" is defined as a

polypeptide in which one or more internal amino acid residues are absent. An "addition" is defined as a polypeptide which has one or more additional internal amino acid residues as compared to the wild type. A "substitution" results from the replacement of one or more amino acid residues by other residues. A protein "fragment" is a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the protein to which the polypeptide is related.

Preferred "substitutions" are those which are conservative, i.e., wherein a residue is replaced by another of the same general type. As is well

understood, naturally occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. It is generally preferred that peptides differing from the native form contain

substituted amino acids, or codons for amino acids, which are from the same group as that of the amino acid replaced.

Thus, in general, the basic amino acid Lys, Arg, and His are interchangeable; the acidic amino acids aspartic and glutamic are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, lie, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, lie, and Leu are more closely related), and the aromatic amino acids Phe, Trp, and Tyr are interchangeable.

While proline is a nonpolar neutral amino acid, it represents difficulties because of its effects on conformation, and substitutions by or for proline are not preferred, except when the same or similar

conformational results can be obtained. Polar amino acids which represent conservative changes include Ser, Thr, Gln, Asn; and to a lesser extent, Met. In

addition, although classified in different categories, Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into this group, or may be classified with the polar neutral amino acids. The peptides of this invention which bind to and inhibit TK mediating signal transduction pathways used by hyperproliferating cells are expected to be

therapeutically beneficial in the treatment of diseases such as cancer, viral infection, psoriasis, and the like. The present invention also discloses methods and compositions for the inhibition of PTK involved in the phosphorylation of biologically active proteins

involved in signal transduction pathways.

In another preferred aspect, therefore, the present invention relates to novel peptides inhibiting TK useful as pharmaceutical agents, to pharmaceutical compositions which include these peptides and a

pharmaceutically acceptable carrier for administering an effective dose of the peptide, and to pharmaceutical methods of treatment. More specifically, the novel compounds of the present invention are useful as therapeutic agents for controlling proliferative diseases like cancer and psoriasis. They are also useful for the treatment of viral, inflammatory, allergic, and cardiovascular diseases.

The invention also relates to intermediates used in their preparation, to their use as inhibitors of PTK, and therapeutic compositions thereof for use in hyperproliferative diseases such as cancer. Most preferred peptides are those which through modification of the native sequence have been converted from

substrates to inhibitors of the EGFR TK.

Included among the peptides of this invention are the following peptide sequences (for some of which illustrative preparative examples are given herein below) in Table 1 which follows.

The compounds of the invention are capable of further forming both pharmaceutically acceptable acid addition and/or base salts depending on the particular amino acid sequence. All of these forms are within the scope of the present invention. The compounds of the invention possess chiral centers and each center may exist in the (D) or (L) configuration. The present invention includes, therefore, all enantiomeric and diastereomeric forms as well as the appropriate

mixtures thereof. In addition, this invention also includes peptides truncated at the N-terminus,

C-terminus, or both the N- and C-termini whose total length is no less than 3 amino acids.

The compounds may be administered orally or parenterally or by direct injection into the target organ. The usual human dosage ranges for a 80 kg subject from about 1 mg to about 1 g/day (0.1 mg to 10 mg/kg of weight per day), preferably 10 mg to

100 mg/day (0.1 mg to 1.0 mg/kg of weight per day), optionally in divided portions.

The above employed pharmaceutical compositions are produced formulating a compound of the foregoing formula (active ingredient) in dosage unit form with a pharmaceutical carrier. Some examples of dosage unit forms are tablets, capsules, lozenges, and pills; as well as powders and aqueous and nonaqueous oral

solutions and suspensions and parenteral solutions, packaged in containers containing either one or some larger number of dosage units and capable of being subdivided into individual doses by such means as measurement into a teaspoon or other standard

container. Some examples of suitable pharmaceutical carriers, including pharmaceutical diluents, are gelatin capsules; sugars such as lactose and sucrose; starches such as corn starch and potato starch;

cellulose derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, and cellulose acetate phthalate; gelatin; talc, stearic acid; magnesium stearate; vegetable oils such as peanut oil, cotton seed oil, sesame oil, olive oil, corn oil, and oil of theobroma; propylene glycol; glycol;

glycerine; sorbitol; polyethylene glycol; water; agar; alginic acid; isotonic saline; and phosphate buffer solutions; as well as other compatible substances normally used in pharmaceutical formulations. The compositions of the invention can also contain other components such as coloring agents, flavoring agents, and/or preservatives. These materials, if present, are usually used in relatively small amounts. The

compositions can, if desired, also contain other therapeutic agents.

The percentage of the active ingredient in the foregoing compositions can be varied within wide limits but for practical purposes it is preferably present in a concentration of at least 10% in a solid composition and at least 2% in a primarily liquid composition. The most satisfactory compositions are those in which a much higher proportion of the active ingredient is present. The compositions of the invention preferably contain from 2 mg to 1 g of the active ingredient per dosage unit so that the entire amount to be

administered during a day can be made up from a

reasonable number of dosage units.

Among the preferred compounds in this invention for inhibiting the EGFR TK are those in Table 2.

Among the most preferred compounds in this invention for inhibiting the EGFR TK are those in Table 3.

Biological Assay.

EGF receptors were isolated to homogeneity from A431 human epidermoid cells by the method of Gill and Weber, "Purification of functionally active epidermal growth factor receptor protein using a competitive antagonist monoclonal antibody and competitive elution with epidermal growth factor, " Methods in Enzymology. 1987;146:82-88.

The Biological Assay and resulting test data for representative peptide compounds of the invention show that the peptides inhibit the EGF receptor tyrosine kinase with IC₅₀ values in the micromolar range. The IC₅₀ is defined as the concentration of peptide

necessary to reduce the incorporation of ³²P into a substrate by 50%. Similar assays are widely used by those skilled in the art.

General Tyrosine Kinase Inhibition Assay.

K_i values for peptides were determined by

measuring their inhibitory effect on the

phosphorylation of a substrate peptide, whose sequence was Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr-Glu- Glu-Val (OH). The ATP concentration was held constant at 10 μM and both substrate and inhibitor

concentrations were varied from 25 to 200 μM and 6.25 to 1000 μM, respectively. Specifically, in a small glass tube, 20 μL of EGF receptor (20-40 ng), 20 μL of reaction buffer (20 mM Hepes, pH 7.4, 2 mM MnCl₂, 5 mM MgCl₂, 50 μM NaVO₄) and 20 μL of 50 μM ATP (in reaction buffer) were mixed and allowed to incubate at room temperature for 30 minutes to insure complete

autophosphorylation of the receptor. The tubes were cooled in a water-ice bath to 4°C and substrate and inhibitor were added to the reaction mixture. The tubes were allowed to incubate 30 minutes at 4°C to allow equilibration of the enzyme/substrate/inhibitor mixture. Two μCi of [γ-³²P] -ATP were added to each tube, which were then mixed and incubated for

10 minutes with shaking in a water bath at 25°C. The reaction was stopped by adding 2 mL of 75 mM phosphoric acid to each tube. The reaction mixture was slowly filtered through phosphocellulose paper and washed 5 times with 2 mL of ice cold 75 mM phosphoric acid so as to bind the substrate peptide. Radioactivity retained on the filter was determined by scintillation counting. K_i values for the inhibitory peptides and a K_m value for the substrate peptide were calculated by fitting the data by nonlinear regression analysis to either the Michaelis-Menten equation or equations which describe competitive, noncompetitive, or uncompetitive inhibition.

Biological Data: TABLE 4

The invention and the best mode of practicing the same are described in the following general

nonlimitative example. The compounds of this invention were prepared by the following process using the appropriate amino acids.

EXAMPLE 1

Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu- Val-OH

The title compound was prepared by standard solid phase peptide methodology utilizing a fluorenylmethoxy- carbonyl (Fmoc) / (t-Bu) strategy (Stewart J.M. and

Young J.D., Solid Phase Peptide Synthesis, Pierce

Chemical Co., Rockford, IL, 1984). All protected amino acids and reagents were obtained from commercial sources and were not further purified. The protected peptide resin was prepared on an Applied Biosystems 431A Peptide Synthesizer, using protocols supplied for a 1-hydroxybenzotriazole/dicyclohexylcarbodiimide

(HOBT/DCC) coupling scheme (Standard Fmoc,

Version 1.12). Starting with 0.25 mmol of Wang resin (4-alkoxybenzyl alcohol resin, 0.238 g, 1.05 mmol/g), the protected peptide was prepared by first loading the resin with 0.5 mmol of N-α-Fmoc-Val symmetrical

anhydride with 4-dimethylaminopyridine (DMAP) as catalyst, followed by the sequential coupling of the following amino acids (in order of addition):

N-α- Fmoc-Glu (OtBu), N-α-Fmoc-Glu (OtBu), N-α-Fmoc-Phe, N-α- Fmoc-Ala, N-α-Fmoc-Ser (tBu), N-α-Fmoc-Gly,

N-α- Fmoc-Glu (OtBu), N-α-Fmoc-Ala, N-α-Fmoc-Leu,

N-a- Fmoc-Lys (Boc), N-α-Fmoc-Lys (Boc), N-α-Fmoc- His(Trt), N-α-Fmoc-Lys (Boc).

A typical cycle for the coupling of an individual amino acid residue is illustrated below (reproduced from the ABI manual): 1. Deprotection with 20% piperidine in NMP for

20 minutes

2. Washes with NMP for 9 minutes

3. Coupling of Fmoc amino acid-HOBT activated ester in NMP for 71 minutes

4. Washes with NMP for 7 minutes

After the coupling of the terminal N-α-Fmoc- Lys(Boc), the Fmoc group was removed and the terminal amine was acetylated with N-acetylimidazole (1.0 g, 60 minutes) in 20 mL of dichloromethane (DCM). The resin was washed with DCM (3 x 20 mL) and dried under reduced pressure (0.845 g).

The peptide was liberated from the solid support and the side chains deprotected by treatment of 0.400 g of the resin with a solution of trifluoroacetic acid (TFA, 9.5 mL), ethanedithiol (0.25 mL), and H₂O

(0.25 mL) at room temperature for 1.5 hours. The suspension was filtered and the filtrate was

concentrated under reduced pressure to give a syrup. Treatment with diethyl ether gave a solid which was collected by filtration, dissolved in H₂O, and

lyophilized to give 0.215 g of a white powder. The crude peptide (0.125 g) was dissolved in 1.5 mL of 0.1% TFA/H₂O and 1.0 mL of acetic acid (HOAc), filtered through a 0.45 μM syringe filter, and chromatographed on a Vydac 218TP 1022 column (2.2 x 25.0 cm,

15.0 mL/min, A = 0.1% TFA/H₂O, B = 0.1% TFA/CH₃CN, 0%B for 10 minutes, 0% to 10%B over 10 minutes, and 10% to 30%B over 120 minutes). Individual fractions were collected and combined on the basis of analytical HPLC and capillary zone electrophoresis (CZE). The combined fractions were concentrated under reduced pressure, diluted with H₂O, and lyephilized (0.067 g). The homogeneity and structure of the resulting peptide was confirmed by analytical HPLC, CZE, Proton Nuccear Magnetic Resonance Spectroscopy (H¹-NMR), Amino Acid Analysis, and Electrospray Mass Spectroscopy (ES-MS), MH⁺ found, 1615.4; calcd., 1614.8.

In a process analogous to the above example using the appropriate amino acids and changes where noted, the following compounds in Examples 2-37 were prepared:

EXAMPLE 2

Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Ala-Glu-Glu- Val-OH

Deprotected as in Example 1. ES-MS, MH⁺ found, 1539.2; calcd., 1538.8

EXAMPLE 3

Ac-Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu-OH

This peptide was cleaved from the resin and deprotected by treatment with a solution of TFA

(9.5 mL) and H₂O (0.5 mL). FAB-MS, MH⁺ found, 994.4; calcd., 994.4.

EXAMPLE 4

Ac-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu-Val-OH

Deprotected as in Example 3. ES-MS, MH⁺ found, 980.5; calcd., 980.4.

EXAMPLE 5

Leu-Ala-Glu-Gly-Ser-Ala-D-Tyr-Glu-Glu-Val-OH

The peptide was not acetylated. Deprotected as in Example 3. FAB-MS, MH⁺ found, 1067.6; calcd., 1067.5.

EXAMPLE 6

Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu-Val-OH

This peptide was not acetylated. Deprotected as in Example 3. FAB-MS, MH⁺ found, 1051.3; calcd.,

1051.5. EXAMPLE 7

Leu-Ala-Glu-Gly-Ser-Ala-D-Phe-Glu-Glu-Val-OH

This peptide was not acetylated. Deprotected as in Example 3. FAB-MS, MH⁺ found, 1052.0; calcd., 1051.5.

EXAMPLE 8

Ac-Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu-Val-NH₂

FAB-MS, (M+Na)⁺ found, 1114.2; calcd., 1114.5

EXAMPLE 9

Leu-Ala-Glu-Gly- Ser ( PO ₃H₂ ) -Ala- LTyr (2 , 3 , 5 , 6 - F₄ ) -Glu- Glu-OH

The title peptide was assembled in its

nonphosphorylated form on Wang resin using the

Fmoc/t-Boc strategy described in Example 1 with

2 exceptions from the standard. First, the Ser was incorporated with its hydroxyl group unprotected.

Second, the terminal Leu was incorporated as its

N- α-Boc protected derivative. The Ser was

phosphitylated on the resin with di-t-Bu- N,N-diethylphosphoramidate and subsequently oxidized with aqueous t-Bu-hydroperoxide according to the procedure of Staerkaer, et al. [Staerkaer G.,

Jakobsen M.H., Olsen C.E., and Holm A. Tet. Lett.

1991:5389]. The peptide was cleaved from the resin and deblocked as in Example 3.

EXAMPLE 10

Leu-Ala-Glu-Gly-Ser-Ala-DTyr-Glu-Glu-Val (OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found, 1067.6; calc, 1067.5. EXAMPLE 11

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-I)-Glu- Glu-Val(OH)

Deprotected as in Example 1. ES-MS, MH⁺ found, 1716.2; calc, 1715.7.

EXAMPLE 12

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-F)-Glu- Glu-Val(OH)

Deprotected as in Example 1. FAB-MS, MH⁺ found,

1606.9; calc, 1606.8.

EXAMPLE 13

Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-F)-Glu-Glu-Val(OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found,

1086.2; calc, 1085.5.

EXAMPLE 14

Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-NO₂)-Glu-Glu(OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found,

1013.1; calc, 1013.4.

EXAMPLE 15

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Glu-Glu(OH) Deprotected as in Example 3. FAB-MS, MH⁺ found,

1040.7; calc, 1040.4. EXAMPLE 16

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu-Glu(OH) Deprotected as in Example 3. FAB-MS, MH⁺ found,

1040.1; calc, 1040.4.

EXAMPLE 17

Glu-Gly-Ser-Ala-LTyr(2,a,5,6-F₄)-Glu-Glu(OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found,

856.3; calc, 856.3. EXAMPLE 18

Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu-Glu(OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found, 856.3; calc, 856.3.

EXAMPLE 19

Leu-Ala-Glu-Gly-Glu-Ala-LTyr(2,3,5,6-F₄)-Glu-Glu(OH) Deprotected as in Example 3. FAB-MS, (M+2Na)⁺ found, 1127.9; calc, 1127.4.

EXAMPLE 20

Leu-Ala-Glu-Gly-Glu-Ala-DTyr(2,3,5,6-F₄)-Glu-Glu(OH)

Deprotected as in Example 3. FAB-MS, (M+Na)⁺ found, 1104.7; calc, 1104.4.

EXAMPLE 21

Leu-Ala-Glu-Gly-Ser-Asn-DLTyr(2,3,5,6-F₄)-Glu-Glu(OH) Deprotected as in Example 3. FAB-MS, MH⁺ found, 1083.6; calc, 1083.4

EXAMPLE 22

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Glu-Arg(OH)

Deprotected as in Example 1. FAB-MS, MH⁺ found, 1067.2; calc, 1067.4.

EXAMPLE 23

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu-Arg(OH)

Deprotected as in Example 1. FAB-MS, MH⁺ found, 1067.5; calc, 1067.4.

EXAMPLE 24

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Ile-Glu(OH) Deprotected as in Example 3. ES-MS, MH⁺ found, 1024.3; calc, 1025.0. EXAMPLE 25

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Ile-Glu(OH) Deprotected as in Example 3. ES-MS, MH⁺ found, 1024.4; calc, 1025.0.

EXAMPLE 26

Leu-Ala-Glu-Gly-Ser-Ala-Ala-Glu-Glu(OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found, 876.3; calc, 876.4.

EXAMPLE 27

Leu-Ala-Glu-Gly-Ser-Ala-Ser-Glu-Glu-Val (OH)

Deprotected as in Example 4. FAB-MS, MH⁺ found, 991.7; calc, 991.5.

EXAMPLE 28

Leu-Ala-Glu-Gly-Ser-Ala-Ser-Glu-Glu(OH)

Deprotected as in Example 3. FAB-MS, MH⁺ found, 892.3; calc, 892.4.

EXAMPLE 29

Glu-Ala-LTyr(2,3,5,6-F₄)-Glu(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 583.2; calc, 583.5.

EXAMPLE 30

Glu-Ala-DTyr(2,3,5,6-F₄)-Glu(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 583.2; calc, 583.5.

EXAMPLE 31

Glu-Asn-LTyr(2,3,5,6-F₄)-Ile(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 610.3; calc, 610.5. EXAMPLE 32

Glu-Asn-DTyr(2,3,5,6-F₄)-Ile(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 610.3; calc, 610.5.

EXAMPLE 33

Ala-Asn-LTyr(2,3,5,6-F₄)-Ile(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 552.3; calc, 552.5.

EXAMPLE 34

Ala-Asn-DTyr(2,3,5,6-F₄)-Ile(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 552.3; calc, 552.5.

EXAMPLE 35

Glu-Ala-LTyr(2,3,5,6-F₄)-lle(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 567.3; calc, 567.5.

EXAMPLE 36

Glu-Ala-DTyr(2,3,5,6-F₄)-Ile(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 567.3; calc, 567.5.

EXAMPLE 37

Glu-Asn-Ala-Ile(OH)

Deprotected as in Example 3. ES-MS, MH⁺ found, 446.3; calc., 446.5.

Claims

1. A peptide that inhibits one or more of the

following PTK: abl, bcr-abl, v-src, EGFR, PDGFR, neu receptor, FGFR, or CSF-1R, the peptide having an amino acid sequence incorporating the tyrosine which is phosphorylated by the PTK, the amino acid sequence being from an autophosphorylation site in the PTK or from a cellular TK substrate protein selected from one of the group consisting of:

PLC γ1, ras GAP, and the p85 subunit of PI3K wherein that tyrosine is replaced with D or

L 2,3,5,6-tetrafluorotyrosine or D-tyrosine.

2. A peptide that inhibits one or more of the

following PTK: abl, bcr-abl, v-src, EGFR, PDGFR, neu receptor, FGFR, or CSF-1R, the peptide having an amino acid sequence incorporating at least one tyrosine from the EGFR which is autophosphorylated wherein the tyrosine is replaced with a D or

L 2,3,5,6-tetrafluorotyrosine or D-tyrosine.

3. A peptide that inhibits one or more of the

following PTK: abl, bcr-abl, v-src, EGFR, PDGFR, neu receptor, FGFR, or CSF-1R, the peptide having an amino acid sequence incorporating at least one tyrosine from PLC γ1 which is phosphorylated by the EGFR wherein the tyrosine is replaced with a D or L 2,3,5,6-tetrafluorotyrosine or D-tyrosine itself.

4. A peptide of Claim 1 which is selected from the group consisting of:

Ala-Glu-DTyr-Leu-Arg-Val-Ala-Pro-Gln-Ser (OH); Ala-Glu-DTyr(2,3,5,6-F₄)-Leu-Arg-Val-Ala-Pro- Gln-Serr (OH); Ala-Glu-LTyr(2,3,5,6-F₄)-Leu-Arg-Val-Ala-Pro- Gln-Ser(OH);

Asp-Asn-Pro-Asp-DTyr-Gln-Gln-Asp-Phe-Phe (OH);

Asp-Asn-Pro-Asp-DTyr(2,3,5,6-F₄)-Gln-Gln-Asp- Phe-Phe (OH);

Asp-Asn-Pro-Asp-LTyr(2,3,5,6-F₄)-Gln-Gln-Asp- Phe-Phe(OH);

Gly-Ser-Val-Gln-Asn-Pro-Val-DTyr-His-Asn-Gln- Pro-Leu-Asn(OH);

Gly-Ser-Val-Gln-Asn-Pro-Val-DTyr(2,3,5,6 - F₄ ) -

His-Asn-Gln-Pro-Leu-Asn(OH);

Gly-Ser-Val-Gln-Asn-Pro-Val-LTyr(2,3,5,6-F₄)- His-Asn-Gln-Pro-Leu-Asn(OH);

Phe-Leu-Pro-Val-Pro-Glu-DTyr-Ile-Asn-Gln-Ser- Val (OH);

Phe-Leu-Pro-Val-Pro-Glu-DTyr(2,3,5,6-F₄)-Ile- Asn-Gln-Ser-Val (OH);

Phe-Leu-Pro-Val-Pro-Glu-LTyr(2,3,5,6-F₄)-Ile- Asn-Gln-Ser-Val (OH);

Asp-Ala-Asp-Glu-DTyr-Leu-Ile-Pro-Gln-Gln-

Gly(OH);

Asp-Ala-Asp-Glu-DTyr(2,3,5,6-F₄)-Leu-Ile-Pro- Gln-Gln-Gly(OH);

ASP-Ala-ASP-Glu-LTyr(2,3,5,6-F₄)-Leu-Ile-Pro- Gln-Gln-Gly(OH);

Ala-Arg-Glu-Gly-Ser-Phe-Glu-Ala-Arg-DTyr-Gln- Gln-Pro(OH);

Ala-Arg-Glu-Gly-Ser-Phe-Glu-Ala-Arg- DTyr(2,3,5,6-F₄)-Gln-Gln-Pro (OH);

Ala-Arg-Glu-Gly-Ser-Phe-Glu-Ala-Arg-

LTyr(2,3,5,6-F₄)-Gln-Gln-Pro (OH);

Glu-Gly-Arg-Asn-Pro-Gly-Phe-DTyr-Val-Glu- Ala(OH);

Glu-Gly-Arg-Asn-Pro-Gly-Phe-DTyr(2,3,5,6-F₄)- Val-Glu-Ala(OH); Glu-Gly-Arg-Asn-Pro-Gly-Phe-LTyr(2,3,5,6-F₄)- Val-Glu-Ala(OH);

Ala-Glu-Pro-Asp-DTyr-Gly-Ala-Leu (OH);

Ala-Glu-Pro-Asp-DTyr(2,3,5,6-F₄)-Gly-Ala- Leu(OH);

Ala-Glu-Pro-Asp-LTyr(2,3,5,6-F₄)-Gly-Ala- Leu(OH);

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-DTyr- Glu-Glu-Val (OH);

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-

DTyr(2,3,5,6-F₄)-Glu-Glu-Val (OH): and

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala- LTyr(2,3,5,6-F₄)-Glu-Glu-Val (OH).

5. A peptide of Claim 1 which is selected from the group consisting of:

Ala-Glu-DTyr-Leu-Arg-Val-Ala-Pro-Gln-Ser(OH); Ala-Glu-DTyr(2,3,5,6-F₄)-Leu-Arg-Val-Ala-Pro-Gln- Ser (OH);

Ala-Glu-LTyr(2,3,5,6-F₄)-Leu-Arg-Val-Ala-Pro- Gln-Ser(OH);

Asp-Asn-Pro-Asp-DTyr-Gln-Gln-Asp-Phe-Phe(OH); Asp-Asn-Pro-Asp-DTyr(2,3,5,6-F₄)-Gln-Gln-Asp- Phe-Phe(OH);

Asp-Asn-Pro-Asp-LTyr(2,3,5,6-F₄)-Gln-Gln-Asp- Phe-Phe(OH);

Gly-Ser-Val-Gln-Asn-Pro-Val-DTyr-His-Asn-Gln- Pro-Leu-Asn(OH);

Gly-Ser-Val-Gln-Asn-Pro-Val-DTyr(2,3,5,6-F₄)-

His-Asn-Gln-Pro-Leu-Asn(OH);

Gly-Ser-Val-Gln-Asn-Pro-Val-LTyr(2,3,5,6-F₄)- His-Asn-Gln-Pro-Leu-Asn(OH);

Phe-Leu-Pro-Val-Pro-Glu-DTyr-Ile-Asn-Gln-Ser- Val (OH);

Phe-Leu-Pro-Val-Pro-Glu-DTyr(2,3,5,6-F₄)-Ile- Asn-Gln-Ser-Val(OH); Phe-Leu-Pro-Val-Pro-Glu-LTyr(2,3,5,6-F₄)-Ile- Asn-Gln-Ser-Val(OH);

Asp-Ala-Asp-Glu-DTyl-Leu-Ile-Pro-Gln-Gln-

Gly(OH);

Asp-Ala-Asp-Glu-DTyr(2,3,5,6-F₄)-Leu-lle-Pro- Gln-Gln-Gly(OH); and

Asp-Ala-Asp-Glu-LTyr(2,3,5,6-F₄)-Leu-Ile-Pro- Gln-Gln-Gly(OH).

6. A peptide of Claim 1 which is selected from the group consisting of:

Ala-Arg-Glu-Gly-Ser-Phe-Glu-Ala-Arg-DTyr-Gln- Gln-Pro(OH);

Ala-Arg-Glu-Gly-Ser-Phe-Glu-Ala-Arg-

DTyr(2,3,5,6-F₄)-Gln-Gln-Pro(OH);

Ala-Arg-Glu-Gly-Ser-Phe-Glu-Ala-Arg- LTyr(2,3,5,6-F₄)-Gln-Gln-Pro(OH);

Glu-Gly-Arg-Asn-Pro-Gly-Phe-DTyr-Val-Glu- Ala(OH);

Glu-Gly-Arg-Asn-Pro-Gly-Phe-DTyr(2,3,5,6-F₄)- Val-Glu-Ala(OH);

Glu-Gly-Arg-Asn-Pro-Gly-Phe-LTyr(2,3,5,6-F₄)- Val-Glu-Ala(OH);

Ala-Glu-Pro-Asp-DTyr-Gly-Ala-Leu(OH);

Ala-Glu-Pro-Asp-DTyr(2,3,5,6-F₄)-Gly-Ala- Leu(OH);

Ala-Glu-Pro-Asp-LTyr(2,3,5,6-F₄)-Gly-Ala- Leu(OH);

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-DTyr-

Glu-Glu-Val(OH);

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala- DTyr(2,3,5,6-F₄)-Glu-Glu-Val(OH): and

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala- LTyr(2,3,5,6-F₄)-Glu-Glu-Val(OH).

7. A peptide of Claim 1 which is selected from the group consisting of:

Leu-Ala-Glu-Gly-Ser-Ala-DTyr-Glu-Glu-Val(OH); Leu-Ala-Glu-Gly-Ser-Ala-DTyr-Glu-Glu(OH);

Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-

Phe-Glu-Glu-Val(OH);

Ac-Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu- Val(NH₂);

Ac-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu-Val(OH); Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu-Val(OH);

Leu-Ala-Glu-Gly-Ser-Ala-DPhe-Glu-Glu-Val(OH); Ac-Leu-Ala-Glu-Gly-Ser-Ala-Phe-Glu-Glu(OH); Leu-Ala-Glu-Gly-Ser-Ala-Phe(4-NO₂)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Phe(4-NH₂)-Glu-

Glu(OH);

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala- Tyr(3-I)-Glu-Glu-Val(OH);

Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala- Tyr(3-F)-Glu-Glu-Val(OH):

Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-F)-Glu-Glu- Val(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Tyr(2-F)-Glu-Glu(OH); Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-I)-Glu-Glu(OH); Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-NO₂)-Glu-

Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Tyr(3-NH₂)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Glu- Glu (OH);

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu- Glu(OH);

Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Glu-Glu(OH); Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu-Glu(OH); Leu-Ala-Glu-Gly-Glu-Ala-LTyr(2,3,5,6-F₄)-Glu-

Glu(OH); Leu-Ala-Glu-Gly-Glu-Ala-DTyr(2,3,5,6-F₄)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Asn-LTyr(2,3,5,6-F₄)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Asn-DTyr(2,3,5,6-F_a)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Glu- Arg(OH);

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2.3,5,6-F₄)-Glu- Arg(OH);

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Ile- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5.6-F₄)-Ile- Glu(OH);

Leu-Ala-Glu-Gly-Ser(PO₃H₂)-Ala-LTyr(2,3,5.6- F₄)-Glu-Glu(OH);

Leu-Ala-Glu-Gly-Ser(PQ₂H₂)-Ala-DTyr(2,3.5,6- F₄)-Glu-Glu(OH);

Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala- Ala-Glu-Glu-Val(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Ala-Glu-Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Ser-Glu-Glu-Val(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Ser-Glu-Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Cys-Glu-Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Glu-Glu-Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-Arg-Glu-Glu(OH);

Glu-Ala-LTyr(2,3,5,6-F₄)-Glu(OH);

Glu-Ala-DTyr(2,3,5,6-F₄)-Glu(OH);

Glu-Ala-Ala-Glu(OH);

Glu-Asn-LTyr(2,3,5,6-F₄)-Ile(OH);

Glu-Asn-DTyr(2,3,5,6-F₄)-Ile(OH);

Ala-Asn-LTyr(2,3,5,6-F₄)-Ile(OH);

Ala-Asn-DTyr(2,3,5,6-F₄)-Ile(OH);

Glu-Ala-LTyr(2,3,5,6-F₄)-Ile(OH);

Glu-Ala-DTyr(2,3,5,6-F₄)-Ile(OH);

Glu-Asn-LTyr(2,3,5,6-F₄)-Ala(OH); Glu-Asn-DTyr (2,3,5,6-F₄)-Ala(OH); and

Glu-Asn-Ala-Ile(OH).

8. A peptide of Claim 7 which is selected from the group consisting of:

Leu-Ala-Glu-Gly-Ser-Ala-Ala-Glu-Glu (OH); and Glu-Asn-Als-Ile(OH).

9. A peptide of Claim 7 which is selected from the group consisting of:

Leu-Ala-Glu-Gly-Ser-Ala-LTyr(2,3,5,6-F₄)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu-

Glu(OH);

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu- Arg(OH);

Leu-Ala-Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Ile- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Asn-DTyr(2,3,5,6-F₄)-Glu- Glu(OH);

Leu-Ala-Glu-Gly-Ser-Asn-LTyr(2,3,5,6-F₄)-Glu- Glu-(OH);

Leu-Ala-Glu-Gly-Glu-Ala-DTyr(2,3,5,6-F₄)-Glu-

Glu(OH);

Glu-Gly-Ser-Ala-DTyr(2,3,5,6-F₄)-Glu-Glu(OH); Glu-Ala-DTyr(2,3,5,6-F₄)-Glu(OH);

Glu-Asn-DTyr(2,3,5,6-F₄)-Ile(OH);

Ala-Asn-DTyr(2,3,5,6-F₄)-Ile(OH): and

Glu-Ala-DTyr(2,3,5,6-F₄)-Ile(OH).

10. A peptide as described in Claim 3 wherein the

peptide is derived from PLC γ1 472.

11. A peptide as described in Claim 3 where in the

peptide is derived from PLC γ1 771.

12. A peptide as described in Claim 3 wherein the peptide is derived from PLC γ1 783.

13. A peptide as described in Claim 3 wherein the

peptide is derived from PLC γ1 1254.

14. A peptide according to Claim 1 which is N- or

C-terminally modified derivative thereof.

15. A pharmaceutical composition useful for

controlling proliferative disease and for treating viral, inflammatory, allergic, and cardiovascular disease containing a therapeutically effective amount of a peptide of Claim 1.

16. A method for controlling proliferative disease and for treating viral, inflammatory, allergic, and cardiovascular disease which comprises

administering to a patient in need thereof a therapeutically effective amount of a

pharmaceutical composition of Claim 15.