# Patent

## Title
Anti-cancer compounds and methods related thereto

## Abstract

The present invention provides compounds useful to inhibit tumor growth and to induce apoptosis. In general, the anti-cancer agents (ACA) are described by the formula:

[ACA]n-X[Formula I]

wherein X is a linker group having 2–5 functional groups or is absent, n=1, and ACA is selected from the group consisting of Formula II, Formula III, Formula IV, Formula V, and Formula VI, as described herein. Other compounds described herein are defined by the Formula VII, as described herein.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 09/378,019, filed Aug. 19, 1999, now U.S. Pat. No. 6,388,054, which is hereby incorporated by reference in its entirety, and which claims priority to U.S. Provisional Application Ser. No. 60/097,210, filed Aug. 20, 1998, and U.S. Provisional Application Ser. No. 60/141,169, filed Jun. 25, 1999.


GOVERNMENT RIGHTS
This invention was made in part with government support under grant number NIH HL-26284, awarded by National Institutes of Health. The government has certain rights to this invention.


FIELD OF THE INVENTION
The present invention relates generally to the field of cancer treatments, as well as to the field of peptide and non-peptide pharmaceutical compounds.
BACKGROUND OF THE INVENTION
Many lung and prostate cancers, of which small cell lung cancer (SCLC) is a prime example, have a neuroendocrine phenotype, and their growth is stimulated by neuropeptides. Antagonists of several peptides (e.g. bradykinin, substance P. bombesin) have been used in experimental treatment of models of SCLC in animals. Among the most potent of the peptides examined thus far, crosslinked dimers of certain bradykinin antagonist peptides have been efficacious both in vitro and in vivo against strains of SCLC and other tumors (Chan et al., Immunopharmacology 33: 201–204, 1996; Stewart et al., Can. J. Physiol. Pharmacol. 75: 719–724, 1997; Stewart et al., U.S. patent application Ser. No. 5,849,863, issued Dec. 15, 1998). Prostate cancers show a similar neuroendocrine phenotype and are susceptible to neuropeptide antagonists.
SUMMARY OF THE INVENTION
The present invention provides anti-cancer agents (ACA) comprised of a range of novel amino acid derivatives and small peptides having the ability to inhibit growth of SCLC and certain other tumor cell lines (such as non-small cell lung cancer (NSCLC) and prostate cancer) in standard in vitro tests, as well as certain monomeric peptides that show inhibition of tumor growth in vivo. Certain of the peptides have a general structural relationship to carboxy-terminal fragments of bradykinin antagonists, but the non-peptides show no such general relationship. Monomers, dimers, trimers, tetramers, pentamers and cyclized analogs of the novel molecules are described. The new compounds are tested for bradykinin antagonist activity in standard assays, but there is no apparent relationship between bradykinin antagonist activity and cytolytic potency. All of the molecules described possess both hydrophobic (usually aromatic) and basic groups in their structures. Without being held to one particular theory, it appears that the compounds function by stimulation of cell death (apoptosis) in the tumor cells.
The present invention also provides compounds and methods for inhibiting cancer by administering to a subject afflicted with cancer (ie. of the lung or prostate) a therapeutically effective amount of one or more of the compounds herein described.
In general, the anti-cancer compounds are described by the formula:

[ACA]n-X  [Formula I]

wherein X is a linker having 2–5 functional groups or is absent, n=1–5, and ACA is selected from the group consisting of Formula II, Formula III, Formula IV, Formula V, and Formula VI. Other compounds described herein are defined by the Formula VII. The specifics regarding structure are enumerated in the Detailed Description, Examples and Claims. Certain physical charateristics are enumerated in the Examples as well as the Detailed Description, Examples and Claims.




BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows inhibition of growth in vivo of SCLC strain SHP-77 by B10054.
FIG. 2 shows inhibition of growth in vivo of NSCLC strain A-549 by M620.
FIG. 3 shows inhibition of growth in vivo of SCLC strain SHP-77 by B9430.
FIG. 4 shows inhibition of growth in vivo of SCLC strain SHP-77 by B1023
FIG. 5 shows inhibition of growth in vivo of SCLC strain SHP-77 by M570, both as the trifluoroacetate salt and as the hydrochloride salt.
FIG. 6 shows inhibition of growth in vivo of SCLC strain SHP-77 by M822.
FIG. 7 shows inhibition of growth in vivo of SCLC strain SHP-77 by M638.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a range of monomeric, dimeric, trimeric, tetrameric, pentameric and cyclic small peptides and peptide mimics that are effective as anti-cancer agents.
In general, the anti-cancer agents (ACA) are described by the formula:

[ACA]n-X  [Formula I]

wherein X is a linker group having 2–5 functional groups or is absent, n=1, and ACA is selected from the group consisting of Formula II, Formula III, Formula IV, Formula V, and Formula VI, as described herein. Other compounds described herein are defined by the Formula VII, as described herein.

X can be any linking group which does not interfere with the inhibitory activity of the monomer-linker or oligomerized product using bis-imido-esters, bis-maleimidoalkanes such as bis-maleimidohexane, dicarboxylic acids, tricarboxylic acids, tetracarboxylic acids and multi carboxylic acids. Alkane groups may be substituted with alkyl, amino, carboxyl, halogen, hydroxy, mercapto or methoxy groups. Aminoalkyl, aromatic or cycloalkyl polycarboxylic acids, heterocyclic polycarboxylic acids, carboxylic anhydrides and polyoxyethylene linkers may also be used. For C-terminal crosslinking, X may be any diamino or polyamino alkane, cycloalkane, aromatic, heterocyclic diamine, polyamine or other substituted chelating agent (for example: diethylenetriaminepentaacetic dianhydride, ethylenediaminetetraacetic dianhydride, etc.). Polyamino-polycarboxylic acids may also be used to make heteromers (such as ethylenediamine-N,N′-diacetic acid, etc.).
The linkage may be at the N-terminal or the C-terminal or at any position of the ACA sequence through side-chain functional groups. The linker may have any chain length.
For dimers, there is a correspondence between linker length and cytotoxicity. Alkyl chains of 8 carbons or more are preferred, with those of 8 to 18 carbons being most preferred. Examples of preferred dimer linkers for the α-amino at the N-terminal or for a basic side-chain group at any position of ACA include ADA, BTAC, DDD, DDS, DTP, EGS, EOPC, HDD, HFG, PFS, SBEC, SUB, SUIM and TDIM. For dimerization through the C-terminal carboxyl or any side-chain carboxyl in ACA, the preferred linkers include DDA, DEA, EDA, EDP and HAD. Any di-functional molecule can be used.
For trimers, linkers for basic groups include BTAC, BTC, CHTC, CTAC and TREN-(Suc)3; for carboxyl groups, TREN. Any tri-functional molecule can be used.
For tetramers, linkers can be BAPTA, CPTA, EDTA, EGTA, ETTA, or any tetra-functional molecule.
For pentamers, the linker can be DTPA or any pentameric functional molecule. Compounds formed by ACA and a linker X may be homo or hetero multimers.
[Formula II] comprises:

R-A−1-B0-C1-D2-E3-F4-G5-H6-I7-J8K9 

wherein R, A, B, C, D, E, F, G, H, I, J, and K are selected from the following or may be absent, and wherein K is Arg or an Arg derivative:























A
B
C
D
E
F
G
H
I
J
K


R
−1
0
1
2
3
4
5
6
7
8
9







Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent


or
or
or
or
Or
or
or
or
or
or
or
or


3,3DP
DmK
Apc
ApC
MeP
Hyp
Ava
Add
Arg
2Nal
2Nal
Arg


Aaa
Lys
Arg
Arg
Nig
Pro
BAla
Aud
Gly
DCpG
2Nal-
Arg(H)


Ac
Lys(εF
DArg
DmK
NMF

Dpr
CpG
Pac
DF5F
NH2
Arg-



lu)









CH2O













H


Aca
NiK
DLys
NiK
Pro

Eac
DDMF
Pac
DIgl
3,4F2F
Arg-


BApg
PzO
DmK
NiO


Gly
DMF
Ser
DPFF
3Pal
Arg(N













O2)


Cca

DniK
PaF



Eac
Thr
DPhe
Ac6c
Arg-













OMe


Cin

DpaF
PzO



Igl

DTic
Aic
DArg


Dca

DPZK




Lys

Gly
Ana
DArg-













NH2


Dcg

DPzO




Pac

mABz
Apb
DArg(













NO2)


Dhq

Lys




Phe

pABz
Apb


Dmac

NiK



Thi

Pac
Atpc


Dpa

PaF





PaF(Dc
Bip










g)


F5bz

PzO





pAmb
Cmp


F5c

DArg-
Arg-





CpG




(NO2)
(NO2)


F5pa








DhPhe


Gun








Dpr(Fb











z)


Hxa








Dpr(Pa











a)


Mca








FSF


Mcg








F5F-











NH2


Moti








Hphe


Pcc








Ica


Ppa








Igl


Pya








Igl-











NH2


Saa








Ileu


Ste








Lys(C











H3)3


Tfmc








Lys(F5











bz)











Mapa











MBC











MFF











Nc6G











Nc7G











NMF











OBS











OBT











OBY











OC2Y











Oic











Oic-











NH2











PABz











Pac











PaF(F5











c)











PaF(Fb











z)











PaF(M











cg)











PaF(Pp











a)











PaF(Si











n)











pAmb











pAPa











PCF











PdF











PFF











PFF-











NH2











Phe











PNF











Thi











Tic











Trp











Trx











Tyr









[Formula III] comprises:

R-A1-B2-C3-D4-E5-F6 

wherein R, A, B, C, D, E, and F are selected from the following or may be absent, and wherein F is not Arg or an Arg derivative:


















A
B
C
D
E
F


R
1
2
3
4
5
6







Absent or
Absent or
Absent or
Absent or
Absent or
Absent or
Absent or


2,2Dp
DArg
Arg
Add
2Nal
1Nal
2Nal


3,3Dp
DArg(NO2)

Aud
3Pal
2Nal
3Pal


Aaa


Ava
Arg
2Nap
ABza


Ac


Eac
Arg(Tos)
3Pal
ABza


Aca


Lys
Atcp
Apa
Ama


Boc


Pac
D2NaI
Arg
Ampy


Chc



DArg
Arg-NH2
Ampz


Cin



DArg(Tos)
Asp
Apa


Ctim



DFSF
Atc
Api


Dca



DIgI
Atcp
Aptp


Dcg



DPFF
Bip
Aqd


Dhq



Eac
BtA
Aqu


Dmac



F5F
Cys(Meb)
Arg(H)


Dns



Gly
Cys(SO3H)
Arg-








CH2OH


Dpa



His
D2Na1
Arg-NH2


F5c



Ig1
DArg
Arg-OMe


F5pa



mABz
DArg-NH2
Asp


F5po



OC2Y
F5F
Asp(Aqu)


Gbc



Pac
Glu
Atcp


Gun



PFF
Gly
Atmp


Hxa




Igl
AtmpO


Mcg




Inp
Atpm


Mse




Iqa
Cyh


Pya




mABz
Dmab


Seb




MC2Y
Dmm


Sin




N-Dmb-
Dmp







Tyr(Bz)-







OMe


Sul




OC2Y
Dpea


Tfmc




OCIY
Dpma


Tha




Oic
Dpr(Dcg-2-








Nap)







pABz
Ecap







PaF(Mes)
F5F-NH2







PFF
GaP







Tic
mA2Bz







tLeu
mA2Bz(Dc








g)







Trp
mA2Bz(Gu








n)







Try
mABz







Try(Bzl)
Mapp







Tyr
Matp







Arg(NO2)
MatpO








pABz








PaF








PaF(Dcg)








PaF(Mcg)








PaF-NH2








PFF-NH2








PgF








PzO








Sud








Thm








Thm








Tpac








Tpac








Tyr(Bz)O








Me









[Formula IV] comprises:

A0-B1-C2-D3-E4-F5-G6-H7-I8-J9-K10-L11 

wherein A, B, C, D, B, F, G, H, I, J, K and L are selected from the following or may be absent:






















A
B
C
D
E
F
G
H
I
J
K
L


0
1
2
3
4
5
6
7
8
9
10
11







Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent


or
or
or
or
or
or
or
or
or
or
or
or


DArg
Arg
Pro
Lys
Pro
DTrp
Gln
DTrp
Phe
DTrp
Leu(r)
Leu-



DArg




DNMF




NH2













Leu









[Formula V] comprises:

X-c[A−1-B0-C1-D2-E3-F4-G5-H6-I7-J8-K9]

wherein X, A, B, C, D, E, F, G, H, I, J, and K are selected from the following or may be absent:























A
B
C
D
E
F
G
H
I
J
K


X
−1
0
1
2
3
4
5
6
7
8
9







Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent


or
or
or
or
or
or
or
or
or
or
or
or


α-Aca
Ava
DArg
Arg
Pro
Hyp
Gly
Add
DArg
DDab
DTrp
Arg


3,3Dp
BAla
DNik
NiK



Add
Ser
DDpr
F5F
Leu



DmK
DPaF
PzO



Ava
Thr
DF5F
Lys
NiK



Glt
DPZK




BAla

DIgl
Nc7G
PaF



Lys
DPzO




DNMF

DLys
Oic
3Pal



Suc





Eac

DOm
PaF









Igl

DPaF
PFF









Thi

Nig
Phe











Pac











Phe









[Formula V] also comprises:

X-c[A−1-B0-C1-D2-E3-F4-G5-H6I7-J8]-K9 

[Formula V] also comprises:

X-c[A−1-B0-C1-D2-E3-F4-G5-H6-I7]-J8-K9 

wherein the cyclization is via a side chain functional group other than the C-terminal residue and the residues are as described in the immediately preceding table.

[Formula VI] comprises the following cyclic peptides:














B9458-2






 


B9462













ACA can also be those compounds in Table 4.

[Formula VII] comprises:

[ACA]1-Eac-Eac-[ACA]2 

wherein [ACA] is defined by Formula I or the compounds in Table 4.

The in vivo inhibitory effects of antagonists may be studied using tumor-bearing nude mice. A tumor model employing nude mice orthotopically implanted with human lung cancer cells wherein the ACA is delivered by intratracheal instillation and aerosol inhalation may be used to evaluate the efficacy and feasibility of these antagonists as a means of treating human lung cancers. Control animals without tumor implantation may also be used to study the general side effects or cytotoxicity of the compounds. It is believed that aerosolized delivery or intratracheal instillation of the agents can produce effective dose accumulation in the area of lesion and reduce the overall systemic toxicity of the compounds in the animals more than when the compound is delivered by systemic administration.
The compounds may be administered topically, or by injection or infusion or as an oral suspension in an appropriate vehicle or as tablets, pills, capsules, caplets or the like, or preferably via intratracheal instillation or aerosol inhalation. The dosage and manner of administration will be defined by the application of the ACA and can be determined by routine methods of clinical testing to find the optimum dose. These doses are expected to be in the range of 0.001 mg/Kg to 100 mg/Kg of active compound.
The compounds are composed of amino acids which may form salts due to their acidic or basic nature, and any pharmacologically acceptable salt derived from the compounds described in this invention such as hydrochlorides, acetates, phosphates, maleates, citrates, benzoates, salicylates, succinates, ascorbates and the like, including HCl, trifluoroacetic acid (TFA), and HOAc, are considered an extension of this invention. A common tactic in medicinal chemistry is to modify known drug substances which are peptide based to form esters or amides which exhibit greater bioavailability. Prodrugs derived from the compounds disclosed here are therefore considered an obvious extension of this invention. Methods for designing and preparing prodrugs are described in detail in the medicinal chemical literature.
Structures and biological activities of peptides and peptide mimics related to bradykinin (BKR) are given in Table 1. Structures and biological activities of compounds not related to bradykinin (BKU) are given in Table 2. Structures and biological activities of cyclic peptides are given in Table 3. Structures of previously described known peptides which we have found to be active against cancers in vivo are included in Table 4. Actions of selected compounds on prostate cancer cell lines are given in Table 5. Abbreviations used are as defined in Table 6.
EXAMPLES
In general, Anti-bradykinin activity was determined by the classical guinea pig ileum assay and on Chinese hamster ovary (CHO) cells expressing cloned human bradykinin B2 receptors. Anti-tumor activity was determined on cultured human cancer cell lines using the standard tetrazolium (MTT) assay. No correlation between anti-bradykinin and cytolytic activity was found among the compounds, indicating that cells are not killed due to inhibition of an essential bradykinin function. Potent compounds were found to stimulate apoptosis in SCLC cells, probably by abnormal activation of the intracellular MEKK pathway.
Example I
Synthesis of Peptides
Peptides were synthesized using standard solid phase synthesis methods, well known in the art (Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984) and were purified by HPLC and were characterized by amino acid analysis (AAA), thin layer chromatography (TLC) and laser desorption mass spectrometry (LDMS). Peptide amides were synthesized on methylbenzhydrylamine (MBHA) resin, which yields amides directly. Peptide methyl esters (OMe) were synthesized by reaction of peptides with 2,2-dimethoxypropane (Rachele, J. Org. Chem. 28: 2898, 1963). Cyclic peptides were prepared on resin or in solution with PyAOP and HOAt.
Example II
Synthesis of Non-peptides
Non-peptides were synthesized by standard organic chemistry procedures well known in the art. Compounds were purified by HPLC and were characterized by analytical HPLC, TLC, and LDMS.
Example III
Synthesis of DDD and SUB Dimers
Synthesis on resin: Neutralized peptide-resin (0.05 mmole) was treated with 0.15 mmole diisopropylethyl amine (DIEA) and 0.026 mmole dodecanedioyl dichloride or suberoyl dichloride in 2.5 mL dichloromethane (DCM). The suspension was mixed for 5 h, washed with DCM and ethanol and dried. The peptide dimer was cleaved from the resin with HF, and the peptide was extracted and purified
Synthesis in solution: Carboxyl-derivatized amino acids or dipeptides were dissolved in dimethyl formamide (DMF) and treated with 10 equivalents of DIEA and 0.55 equivalent of dodecanedioyl dichloride or suberoyl dichloride overnight. The DMF was evaporated in vacuo and the resulting dimer was purified by HPLC.
Example IV
Synthesis of EGS, DTP, SBEC and SUB Dimers in Solution
Dimerization in solution proceeded by reacting 1 equivalent of peptide monomer trifluoroacetate, an excess of DIEA and 0.55 equivalent of cross-linking reagent overnight in DMF. The cross-linking agents were purchased from Pierce (EGS dimer, ethylene glycol bis-(succinimidylsuccinate); DTP dimer, dithiobis (succinimidyl propionate); SBEC dimer, bis[(2(succinimidooxycarbonyloxy)ethyl]sulfone; SUB dimer, disuccinimidyl suberate).
Example V
Synthesis of Boc-N-cycloheptylglycine (Nc7G)
N-Cycloheptylglycine was synthesized by reductive amination of cycloheptanone with glycine methyl ester following the procedure described in Gera et al., Immunopharmacology. 33:174–177 (1996). The crude product was converted to the N-Boc derivative (mp, 89–90° C.).
Example VI
Synthesis of TDIM Dimers
Dimethyl tetradecyldiimidate was synthesized from tetradecanedinitrile by the method of De Abreu et al. (Eur. J. Biochem. 97: 379–387, 1979. One equivalent of peptide TFA salt or other molecule having a free amino group was dissolved in DMF and stirred with 10 equivalents of DIEA and 0.7 equivalent of dimethyl tetradecyldiimidate dihydrochloride overnight at room temperature. DMF was evaporated in vacuo and the dimer was purified. SUIM dimers were prepared similarly, using dimethyl suberimidate.
Example VII
Synthesis of B10238: F5C-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg (F5c-B9430)
B10238 was made by standard solid phase synthesis procedures, or by the acylation of B9430 with 2,3,4,5,6-pentafluorocinnamic acid in DMF, using BOP coupling in presence of excess DIEA. The product was purified by HPLC.
EXAMPLE VIII
Synthesis of M822: DDD-(DArg-F5F-Arg)2 
Following standard solid phase synthesis procedures, Boc-Arg(Tos) Merrifield synthesis resin was coupled with Boc-F5F, followed by coupling with Boc-DArg(Tos), using HATU as coupling agent. The peptide-resin was deprotected with TFA-DCM and neutralized with TEA. The peptide-resin was then treated with 0.55 equivalent of dodecanedioyl dichloride and 5 equivalents of DIEA in DCM overnight at room temperature. After washing and drying, the resin was cleaved with anhydrous HF, using standard conditions. The peptide was extracted from the resin with 90% acetic acid and purified by preparative HPLC.
Example IX
Synthesis of M570 Hydrochloride: F5c-OC2Y-Atmp.HCl
4-Amino-2,2,6,6-tetramethylpiperidine (Aldrich) was coupled with Boc-(O-2,6-dichlorobenzyl)-tyrosine, using BOP in DMF solution. The Boc protecting group was removed by TFA and the product coupled with 2,3,4,5,6-pentafluorocinnamic acid in DMF, using BOP in the presence of excess DIEA at room temperature for 3 h. The DMF was removed in vacuo, the product was extracted into ethyl acetate and the solvent was evaporated. The residue was treated with 0.1–1.0 N HCl or 20% ethanolic HCl. The solvent was removed by evaporation in vacuo at room temperature. The residue was lyophilized from water-dioxane or crystallized from ethanol-ether.
Example X
Synthesis of M630: Dmac-OC2Y-Matp.TFA
4-Methylamino-2,2,6,6-tetramethylpiperidine (Matp) was synthesized from 2,2,6,6-tetramethyl-4-piperidone (Aldrich) and methylamine by reductive amination with NaCNBH3. The Matp was coupled with Boc-(O-2,6-dichlorobenzyl)-tyrosine, using BOP in DMF solution. The Boc protecting group was removed by TFA and the product was coupled with 4-(dimethylamino)cinnamic acid in DMF, using BOP in the presence of excess DIEA at room temperature for 3 h. The DMF was removed in vacuo. The product was extracted into ethyl acetate and the solvent was evaporated in vacuo. The crude product was purified by HPLC, giving the TFA salt. The Dmac-OCTY-Matp.TFA salt can be converted to its HCL salt as in Example IX above.
Example XI
Synthesis of M638: DDD-(DArg-Igl-Arg-Matp)2 
In sequence, Boc-Arg(Tos), Boc-Igl and Boc-DArg(Tos) were coupled to 4-methylamino-2,2,6,6-tetramethylpiperidine (Matp), using BOP as coupling agent in DMF in the presence of excess DIEA at room temperature for 3–5 h. After removal of DMF in vacuo, the product was extracted into ethyl acetate. After evaporation of the solvent, the residue was treated with TFA-DCM to remove the Boc group. TFA was removed in vacuo. The DArg(Tos)-Igl-Arg(Tos)-Matp.TFA was treated with dodecanedioyl dichloride (0.55 equiv) and DIEA (5 equiv) in DCM for 5 h. The protecting groups were cleaved by HF and the lyophilized product was purified by HPLC. The M638.TFA salt was converted to its HCl salt, using 0.1–1.0 N HCl or 20% ethanolic HCl as in Example IX above.
Example XII
Synthesis of M590: Atmp-Igl-Pac-α-Sbl-Lys-B9430
In sequence, Boc-Igl, Boc-Pac and mono-methyl sebacate were coupled to 4-amino-2,2,6,6-tetramethylpiperidine (Atmp), using BOP coupling agent in DMF in presence of excess DIEA at room temperature for 3–5 h. DMF was removed in vacuo and the product was extracted into ethyl acetate. After evaporation of the solvent, the methyl ester was hydrolyzed in methanol by 1N NaOH. The crude product (0.025 mmol Atmp-Igl-Pac-Sbl) was coupled to the peptide resin (0.02 mmol Lys(2-ClZ)-DArg(Tos)-Arg(Tos)-Pro-Hyp-Gly-Igl-Ser(Bzl)-DIgl-Oic-Arg(Tos)-Merrifield resin) using BOP/DIEA activation in DMF. The heterodimer peptide was cleaved from the resin with HF, using standard conditions. The peptide was extracted from the resin with acetic acid and purified by preparative HPLC.
Example XIII
Synthesis of M872: c[DArg-Arg-Eac-Ser-DF5F-Oic-Arg]
Following standard solid phase synthesis procedures, Boc-DArg(Tos) was coupled to Boc-Arg(Tos) Merrifield synthesis resin, followed in sequence by Boc-Arg(Tos), Boc-Oic, Boc-DF5F, Boc-Ser(Bzl), and Boc-Eac, using HATU as coupling agent. After deprotection with TFA-DCM, the resin was cleaved with anhydrous HF using standard conditions. The peptide was extracted from the resin with 0.1% TFA-H2O/dioxane and lyophilized. The peptide trifluoroacetate was cyclized with three equivalents of PyAOP and HOAt and 20 equivalents of DIEA in DMF at a concentration of 10−3 M. After removal of the solvent under reduced pressure, the product was lyophilized from dioxane-H2O and purified by HPLC.
Example XIV
Synthesis of M678: (Dns-DArg-Igl-Arg)2-DDA
In sequence, Boc-Arg(Tos), Boc-Igl and Boc-DArg(Tos) (2 equivalents) were coupled to 1,10-decanediamine using BOP as a coupling agent in DMF in presence of excess DIEA at room temperature for 3–5 h. DMF was removed in vacuo and the product was extracted into ethyl acetate. The solvent was evaporated in vacuo and the residue was treated with TFA/DCM to remove the Boc group. TFA was removed in vacuo, and the product was treated with dansyl chloride (2 equivalents) and an excess of DIEA in DCM for 5 h. The Tos groups were cleaved by HF and the crude product was purified by HPLC.
Example XV
Synthesis of M290: BTAC-(2-Nal-Atmp)3 
The benzene-1,3,5-tris-carbamido-ε-caproic acid linker was made from 1,3,5-benzenetricarboxylic acid and N-Boc-ε-caproic acid methyl ester, using the BOP coupling method. The methyl ester was hydrolyzed in methanol by 1N NaOH. The product (1 equivalent BTAC) was coupled to 2-Nal-Atmp (3 equivalents) in DMF, using HATU as coupling agent. The solvent was removed in vacuo, and the residue was purified by HPLC. The BTAC-(2-Nal-Atmp)2-OH was also isolated as a by-product.
Example XVI
Synthesis of M1040: EDTA-(OC2Y-ATMP)4 
Boc-(O-2,6-dichlorobenzyl)-tyrosine was coupled with 4-amino-2,2,6,6-tetramethylpiperidine overnight in DMF, using BOP as coupling agent in the presence of DIEA. After removal of DMF in vacuo, the residue was extracted into ethyl acetate and treated with TFA/DCM to cleave the Boc group. The TFA/DCM was evaporated in vacuo and the product (OCTY-ATMP) was lyophilized from dioxane/water. Ethylenediaminetetraacetic acid (0.25 equivalent EDTA) was coupled with OC2Y-ATMP trifluoroacetate (1 equivalent) in DMF, using BOP as coupling agent in the presence of DIEA. The solvent was removed in vacuo and the residue was purified by HPLC.
Example XVII
Assay of Anti-bradykinin Activity on Guinea Pig Ileum
Male Hartley guinea pigs that had been deprived of food overnight were sacrificed, and sections of terminal ileum, 25 mm in length, were dissected, attached to tissue holders and immersed in 10 ml tissue baths containing Krebs' solution bubbled with 95% O2/5% CO2. Tissues were placed under 1 g tension and incubated for 1 h equilibration. Concentration-effect curves were constructed to bradykinin in the absence and presence of new compounds. Bradykinin showed pD2=7.4, and antagonist B9430 showed pA2=7.9.
Example XVIII
Assay of Anti-bradykinin Activity on Cloned Human B2 Receptors
Chinese hamster ovary cells containing cloned and expressed human bradykinin B2 receptors were grown in cell cups of the Cytosensor microphysiometer in Ham's F-12 medium supplemented with sodium pyruvate and 10% FBS (Gibco 11765-054). For assay the cells were transferred to Ham's F-12 without bicarbonate or serum (Gibco 21700-075) and placed in the Cytosensor. Concentration-response curves were constructed to bradykinin in the presence or absence of new compounds. Bradykinin showed pD2=11, and antagonist B9430 showed pA2=10.5.
Example XIX
Colorimetric Tetrazolium Assay for Cell Survival
Cell growth and survival were measured by a rapid colorimetric assay based on the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Mosmann, J Immunol. Methods 65: 55–63, 1983, with minor modifications). Briefly, 1,000 normal lung fibroblasts or normal epithelial BEAS-2B cells, 1,000 or 5,000 viable non-SCLC cells or 10,000 viable SCLC cells were plated in 100 μL of growth medium in 96-well flat-bottomed microtiter plates. Cells were incubated overnight to allow recovery. Compounds to be tested were added to the cells in triplicate in a range of concentrations and the cells were incubated at 37° C., 5% CO2, with 100% humidity. Control cells were treated in the same way without antagonists. All wells had a final volume of 200 μL. Plates were incubated for 4 days, allowing sufficient time for cell replication and compound-induced cell death to occur. On day 5, 25 μL of a 2 mg/mL solution of MTT (Sigma) dissolved in RMPI-1640was added to each well. The plate was incubated for 4 h at 37° C. The supernate was removed and the blue formazan complex was dissolved by adding 100 μL of 0.02 N HCl in 75% isopropanol to all wells. Absorbance was immediately determined using a scanning multiwell plate reader. B9870 caused 50% cell death at a concentration of 0.15 μM under these conditions.
Example XX
Measurement of Apoptosis in Cultured Cells
Apoptosis, also known as programmed cell death, is the phenomenon by which a cell dies following a series of gene-mediated events, in response to a wide range of intracellular and extracellular agents. Apoptosis, a counterpart of mitosis, plays an important role in the development and homeostasis of many organisms and tissues. It serves to regulate cell numbers, to shape developing organisms and as a defense against potentially harmful agents. Apoptosis is not the only mode of cell death. Necrosis is a type of cell death which is nonspecific and frequently occurs when cells are exposed to high doses of toxic agents. Such exposure usually results in the loss of ionic homeostasis. Unlike apoptosis, necrosis does not seem to be genetically influenced.
Apoptotic and necrotic cells have different appearances which allow them to be distinguished microscopically. Necrotic cells and their mitochondria swell, the cell membrane eventually ruptures, and internal organelles become distended. As a result of the membrane rupture, inflammation occurs in the surrounding tissue. In contrast, the nuclei of apoptotic cells become fragmented into several smaller nuclear bodies, which are quickly recognized by phagocytes and engulfed, and no inflammatory response occurs. Therefore, it is useful to develop chemotherapeutics which induce apoptosis, rather than necrosis, in order to avoid inflammation and the toxic agents which are often released from necrotic tumor cells.
We have used differential fluorescent dye uptake and cellular morphology to distinguish viable and dead cells with apoptotic and/or necrotic morphologies. We have used Rhodamine 123 to stain active mitochondria in viable cells, Hoechst 33324 to stain DNA in both viable and dead cells, and Propidium Iodide to stain DNA in dead cells. These cell subpopulations may be distinguished by the different manners in which they take up the fluorescent probes. The dead apoptotic and necrotic subpopulation, which has lost its membrane potential and organelle function, takes up Propidium Iodide and Hoechst 33324. Since the cells in this subpopulation are dead, the mitochondria are not active and thus there is little or no uptake of Rhodamine 123. Under the fluorescence microscope with a DAPI filter, nuclei in these cells appear pinkish in color due to the mixing of both Propidium Iodide and Hoechst 33324 dyes. Necrotic cells have intact nuclei while apoptotic cells have fragmented multi-nucleated bodies. In contrast, the viable apoptotic subpopulation has an intact membrane but inactive mitochondria. As a result, the fragmented multi-nucleated bodies (a hallmark of apoptotic cells) in these cells take up only Hoechst 33324, which gives them a blue appearance under the fluorescence microscope, but are unable to take up Propidium Iodide or Rhodamine 123. The subpopulation of viable cells has both intact cell membranes and active mitochondria. These cells take up both Hoechst 3324 and Rhodamine 123. Microscopically these cells appear to have single normal blue nuclei when examined with a DAPI filter and bright green mitochondria when examined with a FITC filter.
Example XXI
Inhibition of Tumor Growth in vivo in Nude Mice
Representative peptide and non-peptide compounds having high in vitro cytotoxic activity were tested against implanted tumors in vivo. Athymic nude mice were implanted subcutaneously with either single cell suspensions (2 million SCLC cells or 1 million NSCLC cells) or with small fragments (3×3 mm) of tumors minced from previously grown nude mouse heterotransplants. On the seventh day after tumor implantation groups of 5 mice bearing implants were injected intraperitoneally daily with the compounds being tested at 1, 5, or 10 mg/kg/day; control animals were injected with an equal volume of isotonic saline. Tumor size was measured with a caliper three times per week. Tumor volume was calculated by the formula:

Volume (cc)=π×(length)×(width)2/6

Results of representative in vivo tests are given in FIGS. 1–7. For comparison, bradykinin antagonist peptide dimers B9870 and B10054 caused marked inhibition of growth of the SCLC line SHP-77 at a dose of 5 mg/kg/day.
Example XXII
Data
Examples of peptides and peptide mimics related to the C-terminal part of bradykinin antagonist peptides and their biological activities on cancer cells and bradykinin responses are given in Table 1.
Many compounds not directly related to the structure of bradykinin were synthesized and tested for anti-tumor and anti-bradykinin activity. These are listed in Table 2.
Cyclic peptides related to bradykinin and bradykinin mimics are reported in Table 3, along with their biological activity on cancer cells and anti-bradykinin activity.
Structures of previously described known peptides which have been found to be active against cancers in vivo are included in Table 4.
Cytotoxic activity in vitro of compounds M570 and M590 against various standard strains of prostate cancer is reported in Table 5.
Standard abbreviations were used for natural amino acids. For non-natural amino acids, derivatizing groups and other chemicals, the abbreviations listed in Table 6 are used.







TABLE 1







ACTIVITIES OF FEPTIDES


RELATED TO BRADYKININ STRUCTURE











NUMBER
STRUCTURE
MTTa
GPIb
HUMANc














BKd
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg






(SEQ ID NO: 1)


B9430d
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
120
8.2


B9870-2d
SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-
0.15
8.4



Arg)


B8838
DArg-Arg-Pro-Hyp-Gly-CpG-Ser-DCpG-CpG-Arg 
—
7.0


B8840
DArg-Arg-Pro-Hyp-Gly-Phe-Ser-DCpG-CpG-Arg
—
6.8


B8858
DArg-Arg-Pro-Hyp-Gly-Thi-Ser-CpG-DCpG-DArg
—
5.2


B8994
DArg-Arg-Pro-MeP-Gly-CpG-Ser-DCpG-CpG-Arg
—
—


B9074
Dhq-DArg-Arg-Pro-Hyp-Gly-CpG-Ser-DCpG-CpG-
—
6.3



Arg


B9126
Aaa-DArg-Arg-Pro-Hyp-Gly-(D,L)DMF-Ser-DTic-
—
6.4



Oic-Arg


B9126-2
Aaa-DArg-Arg-Pro-Hyp-Gly-(D,L)DMF-Ser-DTic-
—
7.3



Oic-Arg


B9224-2
Aca-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-(D,L)Igl-Oic-
—
8.4



Arg


B9882
α-Sub-Lys(εFlu)-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-
—
—



Oic-Arg



 └DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
—
—


B9914
Oic-Arg
—
Wk


B9916
DIgl-Oic-Aig
—
Wk


B9490
Dcg-Digl-Oic-Arg
>60
Wk


B9918
Ser-Digl-Oic-Arg
—
—


B9920
Igl-Ser-DIgl-Oic-Arg
—
—


B9922
Gly-Igl-Ser-DIgl-Oic-Arg
—
Wk


B9924
Hyp-Gly-Igl-Ser-DigI-Oic-Arg
—
—


B9926
Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
—
—


B9950
α-Lys-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
8
—



  |



  Sub-Arg-DNMF-DTrp-Phe-DTrp-Leu


B9956
α-DDD-(Lys-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-
—
—



Oic-Arg)2


B9960
DArg-Arg-Nig-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
—
7.7


B9966
DArg-Arg-NMF-Hyp-Gly-Thi-Ser-DIgl-Oic-Arg
—
6.9


B10010
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Nc7G-Arg
—
7.7


B10014
DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Nc6G-Arg
—
7.6


B10054
DDD-(Lys-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-
0.3
7.1



Arg)2


B10062
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg-NH2
Inact
7.1


B10082
SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg-
0.7
7.2



NH2)2


B10084
BApG-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
>20
8.1


B10088
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg-Eac-
4
7.1



Eac-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg


B10092
(Gun)2-BApG-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-
>20
8.7



Oic-Arg


B10098
(DArg-Arg-Pro-Hyp)2Dpr-Igl-Ser-DIgl-Oic-Arg
20
5.3


B10100-2
TDIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-
1
8.0



Arg)2


B10100-1
Moti-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
4
7.8


B10104-2
TDIM-(DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DIgl-Oic-
4
8.0



Arg)2


B10104-3
Moti-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DIgl-Oic-Arg
20
8.1


B10160
Leu-DTrp-Phe-DTrp-DNMF-Eac2-DArg-Arg-Pro-
10
6.3



Hyp-Gly-Igl-Ser-DIgl-Oic-Arg


B10162
Leu-Leu-DTrp-Phe-DTrp-DNMF-Eac2-DArg-Arg-
7
6.3



Pro-Hyp-Gly-Igl-Ser-Digl-Oic-Arg


B10198-1
DDD-(Eac-Arg-Digl-Oic-Arg)2
—
5.7


B10198-2
DDD-(Eac-Arg-Digl-Oic-Arg)2
15
—


B10200
DDD-(Eac-Eac-Arg-DIgl-Oic-Arg)2
16
5.8


B10238
F5c-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
150
8.1


B10252
EGS-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg)2
25
8.3


B10282
Arg-Pro-Pro-Gly-Phe-Thr-DTic-Oic-Arg
—
7.3


B10284
Arg-Pro-Pro-Gly-Phe-Thr-DTic-Oic-NH2
—
7.7


B10382
DArg-PzO-Pro-Hyp-Gly-Igl-Ser-DF5F-Oic-Arg
—
—


B10384
DNiK-PzO-Pro-Hyp-Gly-Igl-Ser-DF5F-Oic-Arg
—
—


B10386
DDD-(DmK-PzO-Pro-Hyp-Gly-Igl-Ser-DF5F-Oic-
—
—



Arg)2


B10388
DNiK-PzO-Pro-Hyp-Gly-Igl-Ser-DF5F-Oic-Arg
—
—


B10390
DNiK-PzO-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg
—
—


B10392
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-PFF-Arg
—
—


B10394
F5c-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DF5F-PFF-Arg
—
—


B10396
F5c-DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-PFF-Arg
—
—


M2
Dcg-D-2-Nal-Arg
—
—


M10
Dcg-2Nal-Arg
—
4.8
—


M20
Gun-2-Nal-Arg
—
4.6


M42
Gun-Eac-DIgl-Oic-Arg
—
5.0


M68
Dcg-BtA-Arg
—
4.9


M70
Dcg-Igl-Arg
—
4.8


M78
Dcg-Apa-Arg
—
5.6


M84
Dcg-Ile-Arg
—
5.1


M86
Dcg-Ac6c-Arg
—
5.0


M88
Gun-Ica-Arg
—
4.7


M94
Dcg-Aic-Arg
—
Wk


M96
Dcg-(D,L)Ata-Arg
—
4.7


M118-1
Ac-PaF(Mcg)-Arg
—
4.9


M118-3
Ac-PdF-Arg
—
5.4


M124
Dcg-Gly-Cmp-Arg
—
4.7


M128
Dcg-Gly-Oic-Arg
—
Wk


M130
Dcg-F5F-Arg
20
4.8


M132
F5bz-F5F-Arg
60
Wk


M134
Dcg-Trx-Arg
—
4.9


M142
Ac-PaF(Sin)-Arg
—
Wk


M146-1
Ac-PaF(Mcg)-p-ABz-Arg
—
Inact


M146-2
Ac-PaF(Dcg)-p-ABz-Arg
—
4.9


M148
F5c-p-ABz-Arg
—
5.1


M160
Ste-2-Nal-Arg
—
Wk


M176
F5c-pABz-2Nal-Arg
Inact
5.4
—


M196
F5c-Gly-mABz-2Nal-Arg
Inact
5.1
—


M198
Ac-Pac-Gly-m-Abz-2-Nal-Arg
—
5.1


M200-1
Mcg-Pac-Gly-m-ABz-2-Nal-Arg
—
Inact


M200-2
Dcg-Pac-Gly-m-ABz-2-Nal-Arg
—
4.9


M216
F5c-p-APa-Arg
>180
—


M226
DDD-(Arg-DIgl-Oic-Arg)2
35
5.7
Inact


M232-1
Dcg-Atpc-Arg
—
4.7


M232-3
Dcg-2-Nal-Atpc-Arg
—
5.1


M346
Dcg-p-Amb-Arg
—
4.6


M348
F5c-p-Amb-Arg
—
4.7


M352
F5c-p-Amb-APa-Arg
—
4.7


M370
F5c-Arg
—
4.8


M372
F5c-APb-Arg
—
4.6


M374
Tfmc-Arg
—
4.6


M380
F5c-Tyr-Arg
—
Inact


M382
F5c-Tic-Arg
—
4.7


M388
F5c-Lys{(CH3)3}-Arg
—
4.9


M392
F5c-Ana-Arg
—
4.5


M394
F5c-Bip-Arg
—
4.7


M398
F5c-Pac-Arg
—
Inact


M400
DDD-(pABz-2Nal-Arg)2
22
5.1
11.5


M406
Arg-Eac-DIgl-Ana-Arg
—
Inact


M410
F5c-Phe-Arg
—
5.4


M412
F5c-m-APa-Arg
—
5.8


M416
F5c-3-Pal-Arg
—
Wk


M420
F5c-hPhe-Arg
60
7.0
10.9


M424
F5c-Thi-Arg
—
4.6


M426
F5c-Trp-Arg
—
Inact


M442
F5c-Oic-Arg
—
—


M446
F5c-2Nal-Arg
60
4.7
9


M450
F5c-2Nal-Arg-NH2
26
4.9
Inact


M484
DDD-(Pac-2Nal-Arg)2
25
Inact
Inact


M494
DDD-(Lys-Pac-Gly-mABz-2Nal-NH2)2
33
5.1
Inact


M498
DDD-(Pac-2Nal-Arg-NH2)2
24
4.9
Inact


M500
DDD-(pABz-2Nal-Arg-NH2)2
40
0
11.4


M504
DDD-(Pac-2Nal-DArg-NH2)2
11
5.4
Wk


M508
DDD-(DArg-2Nal-Arg)2
23
Inact
—


M510
DDD-(DArg-2Nal-Arg-NH2)2
8
Inact
11


M512
F5c-OC2Y-Arg
70
5.7
11


M516
DDD-(DArg-Arg-Aud-Pac-2Nal-Arg)2
1.4
0
Ag


M518
DDD-(DArg-OC2Y-Arg)2
15
Wk
10


M520
F5c-OBS-Arg
Inact
6.1
7


M528
F5c-MBC-Arg
Inact
Inact


M540
Pya-hPhe-Arg
>100
Wk


M542
Dca-hPhe-Arg
80
Wk


M550
F5c-OBT-Arg
80
Inact
Ag


M552
DDD-(p-ABz-hPhe-Arg)2
Inact
Inact


M554
DDD-(DArg-hPhe-Arg)2
—
5.1


M558
Dcg-hPhe-Arg
100
Wk


M560
DDD-(DArg-hPhe-Arg-NH2)2
50
Wk


M564
DDD-(DArg-OBS-Arg)2
—
Wk


M590
Atmp-Igl-Pac-α-Sbl-Lys-B9430
4.5
7.5
Inact


M598
DDD-(Arg-DIgl-Oic-Arg-OMe)2
13
—
10


M600
α-DDD-(Lys-B9430-OMe)2
1.2
6.4
Ag


M608
DDD-(Eac-Arg-DIgl-Oic-Arg-OMe)2
13
—
Inact


M612
F5c-DArg-hPhe-Arg
Inact
Wk


M676
DDD-(DArg-Arg-Eac-Ser-DF5F-Nc7G-Arg)2
—
—


M682
F5c-Lys(F5bz)-Arg
—
4.9


M686
F5c-NMF-Arg
29
5.6


M688
F5c-Dpr(Fbz)-Arg
—
Inact


M690
F5c-Dpr(Paa)-Arg
—
4.8


M692
DDD-(DArg-Arg-Aud-Pac-hPhe-Arg)2
—
Wk


M696
F5c-DArg-Eac-2Nal-Arg
Inact
5.1
—


M698
F5c-DArg-Arg-Aud-Pac-2Nal-Arg
7.1
Wk
—


M706
Cin-hPhe-Arg
Inact
Wk


M708
Ppa-hPhe-Arg
Inact
5.8


M710
DDD-(DArg-Arg-Aud-Pac-2Nal-DArg-NH2)2
1.7
Wk


M714
F5c-PCF-Arg
Inact
Wk


M718
F5c-PFF-Arg
9
5.1


M720
F5c-PaF(Ppa)-Arg
Inact
4.8


M726
D-Arg-Arg-Aud-PaF(F5c)-Arg
Inact
5.6


M728
DDD-(DArg-Arg-Aud-PaF(F5c)-Arg)2
4
5.3


M730
F5c-DhPhe-Arg
—
4.7


M732
F5c-PNF-Arg
—
4.9


M734
DDD-(DArg-Arg-Aud-Pac-PaF(Fbz)-Arg)2
1.8
5.3


M738
F5c-DArg-Eac-hPhe-Arg
Inact
5.3


M746
DDD-(Pac-hPhe-Arg)2
Inact
Inact


M752-2
Pac-hPhe-Arg
Inact
Wk


M752-5
Aaa-Ser-Pac-hPhe-Arg
Inact
Wk


M752-6
Aaa-Pac-hPhe-Arg
Inact
Wk


M754
Aaa-DPhe-hPhe-Arg
Inact
4.6


M756
DDD-(DPhe-hPhe-Arg)2
18
5.2


M758
Saa-hPhe-Arg
—
Wk


M764
Aaa-DTic-hPhe-Arg
—
4.8


M766
F5c-DArg-Arg-Aud-DTic-hPhe-Arg
—
5.1


M770
DDD-(DArg-Arg-Aud-DTic-hPhe-Arg)2 
8
Inact


M772
Aaa-DIgl-hPhe-Arg
—
4.9


M774
F5c-DArg-Arg-Aud-DIgl-hPhe-Arg
8
Inact


M776
DDD-(DIgl-hPhe-Arg)2
30
5


M778-1
Pcc-hPhe-Arg
—
Wk


M780
Mca-hPhe-Arg
—
Wk


M782
Cca-hPhe-Arg
—
Wk


M784
Ac-OC2Y-Arg
Inact
Wk


M786
DDD-(DArg-Arg-Aud-DIgl-hPhe-Arg)2
3.2
Wk


M788
F5c-DArg-Arg-Aud-DTic-Oic-Arg
9
5


M790
DDD-(DArg-Arg-Aud-DTic-Oic-Arg)2
1.7
Inact


M792
F5c-DArg-Arg-Eac-Ser-DTic-Oic-Arg
>100
4.9


M794
DDD-(DArg-Arg-Eac-Ser-DTic-Oic-Arg)2
21
Inact


M796
F5c-DArg-Arg-Eac-Ser-DF5F-Oic-Arg
31
6.5


M802
F5c-Lys-Ser-DF5F-Oic-Arg
Inact
6.3


M804
DDD-(DArg-Arg-Eac-Ser-DF5F-Oic-Arg)2
7.3
7.7


M806
Ava-Igl-Ser-DF5F-Oic-Arg
Inact
5.6


M808
DDD-(Lys-Ser-DF5F-Oic-Arg)2
30
6.9


M810
F5c-F5F-Arg
40
4.6


M812
F5c-PFF-Arg-NH2
15
Wk


M814
Ppa-PFF-Arg
Inact
Wk


M816
Dpa-PFF-Arg
52
4.6


M818
DDD-(DArg-PFF-Arg-NH2)2
60
Wk


M820
DDD-(DArg-PFF-Arg)2
43
Inact


M822
DDD-(DArg-F5F-Arg)2
25
Mixed


M826
F5c-MFF-Arg
76
Inact


M828
F5c-3,4F2F-Arg
—
Wk


M838
F5c-DArg-Arg-Aud-DIgl-PFF-Arg
7.4
5.1


M842
DDD-(DArg-Arg-Aud-DIgl-PFF-Arg)2
1.4
Inact


M844
DArg-Arg-Aud-DIgl-PFF-Arg
12
Wk


M846
DDD-(DArg-Arg-Aud-DF5F-Oic-Arg)2
2
7.1


M852
F5c-DArg-Arg-Eac-Ser-DIgl-Oic-Arg
Inact
5.9


M854
DDD-(DArg-Arg-Eac-Ser-DIgl-Oic-Arg)2
7.3
5.9


M856
F5c-DArg-Arg-Aud-Ser-DIgl-Oic-Arg
21
5.4


M858
DDD-(DArg-Arg-Aud-Ser-DIgl-Oic-Arg)2
4
6.3


M860
F5c-DArg-Arg-Add-Ser-DIgl-Oic-Arg
6
5.4


M862
DDD-( DArg-Arg-Add-Ser-DIgl-Oic-Arg)2
1.3
5.6


M864
DDD-(DArg-Arg-Add-Ser-DIgl-PFF-Arg)2
1.8
Inact


M868
Ac-Darg-Arg-Aud-DF5F-Oic-Arg
55
6.5


M888
F5c-DArg-Arg-Aud-Ser-D5F5-Oic-Arg
12.5
6.6


M890
DDD-(DArg-Arg-Aud-Ser-DF5F-Oic-Arg)2
1.7
5.5


M922
DDD-(DNiK-Arg-Eac-Ser-DF5F-Oic-Arg)2 
—
—


M926
ζ-SUB-(ApC-F5F-Arg)2
Inact
—


M930
α-DDD-(ApC-F5F-Arg)2
Inact
—


M932
DDD-(DArg-Arg-Eac-Ser-DIgl-PFF-Arg)2
6.0
—


M936
DDD-(DNiK-PzO-Eac-Ser-DF5F-Oic-Arg)2
—
—


M944
DDD-(DArg-Arg-Eac-Ser-DF5F-PFF-Arg)2
6.7
—


M946
F5c- DArg-Arg-Eac-Ser-D5F5-PFF-Arg
—
—


M950
α-DDD-(K-DArg-Arg-Eac-Ser-DF5F-Oic-Arg)2
6.7
—


M952
DDD-(DmK-DArg-Arg-Eac-Ser-D5F5-Oic-Arg)2
—
—


M954
Aaa-DArg-Arg-Eac-Ser-D5F5-Oic-Arg
10
—


M956
Aaa-DArg-Arg-Aud-Ser-D5F5-Oic-Arg
14
—


M958
F5bz-DArg-Arg-Aud-Ser-D5F5-Oic-Arg
18
—


M960
Aca-DArg-Arg-Aud-Ser-D5F5-Oic-Arg
21
—


M964
33Dp-DArg-Arg-Aud-Ser-D5F5-Oic-Arg
4–8
—


M968
Dmac-DArg-Arg-Aud-Ser-D5F5-Oic-Arg
15
—


M972
F5pa-DArg-Arg-Aud-Ser-DF5F-Oic-Arg
—
—


M974
DDD-(PzO-F5F-Arg)2
—
—


M976
DDD-(DNiK-F5F-Arg)2
—
—


M978
DDD-(DPzK-F5F-Arg)2
—
—


M980
DDD-(DPzO-F5F-Arg)2
—
—


M1024
SUB-(DArg-Arg-Eac-Ser-DF5f-Nc7G-Arg)2
14
7.2


M1026
DTP-(DArg-Arg-Eac-Ser-DF5F-Nc7G-Arg)2
70
6.9


M1028
SBEC-(DArg-Arg-Eac-Ser-DF5F-Nc7G-Arg)2
28
6.7


M1030
EGS-(DArg-Arg-Eac-Ser-DF5F-Nc7G-Arg)2
51
7.0


M1034
DDD-(DArg-F5F-DArg-NH2)2
—
—


M1036
DDD-(DArg-F5F-DArg)2
40
5.4


M1038
ε-SUB-(Lys-DArg-Arg-Eac-Ser-DF5F-Nc7G-Arg)2
—
6.4


M1042
Aca-DArg-Arg-Eac-Ser-DF5F-Oic-Arg
—
—


M1044
Gun2-BApg-DArg-Arg-Eac-Ser-DF5F-Oic-Arg
—
—


M1046
(F5c-DArg-Igl-Arg)2-DDA
—
—





Footnotes:


aED50 for killing of SCLC strain SHP-77 in vitro, μM.


bpA2 for bradykinin antagonist activity on isolated guinea pig ileum. The pD2 of bradykinin is 7.4 on ileum. Higher numbers indicate higher potency.


cpA2 for bradykinin antagonist potency on cloned human B2 receptors, pM. The pD2 for bradykinin is 11. Higher numbers indicate higher potency.


dData included for comparison


Inact = inactive; Mixed = showing both agonist and antagonist activity; Wk = weak













TABLE 2







ACTIVITIES OF COMPOUNDS NOT RELATED TO BRADYKININ











NUMBER
STRUCTURE
MTTa
GPIb
HUMANc














B9948
Arg-DNMF-DTrp-Phe-DTrp-
2.8
Wk




Leu


B10222
DNMF-DTrp-Phe-DTrp-LeuΨ
6.0
5.2



(CH2NH)Leu-NH2


B10224-1
α-DDD-(Lys-DNMF-DTrp-Phe-
13
—



DTrp-



LeuΨ(CH2NH)Leu-NH2)2


B10224-2
α-DDD-(Lys-DNMF-DTrp-Phe-
7
—



DTrp-LeuΨ(CH2NH)Leu-NH2)2


B10228
DDD-(DNMF-DTrp-Phe-DTrp-
40
Wk



LeuΨ(CH2NH)Leu-NH2)2


B10242
Arg-Pro-Lys-Pro-DTrp-Gln-
40
5.6



DTrp-Phe-DTrp-



LeuΨ(CH2NH)Leu-NH2


B10244
DArg-Arg-Pro-Lys-Pro-DTrp-
12
5.4



Gln-DTrp-Phe-



DTrp-LeuΨ(CH2NH)Leu-NH2


B10246
DArg-Pro-Lys-Pro-DTrp-Gln-
12
6.1



DTrp-Phe-DTrp-



LeuΨ(CH2NH)Leu-NH2


>278
F5c-Iqa-Atmp
9
5.3
—


M8
Gun-Eac-D2Nal-PgF
—
Inact


M12
Dcg-Igl-Aqu
20
5.0
—


M18
Dcg-2Nal-Aqu
30
6.6
—


M26
Gun-2Nal-GaP
—
4.9


M30
Dcg-2Nal-Apa
—
5.4


M32
Gun-2Nal-Apa
—
4.8


M36
Dcg-D2Nal-Apa
—
5.0


M38
Gun-D2Nal-Apa
—
4.9


M62
Dcg-2Nal-Ama
—
4.8


M64
Dcg-2Nal-APa-Sud
—
Ag


M72-1
Dcg-Igl-Apa
—
4.7


M72-2
Dcg-Igl-APa(anisyl)
—
4.6


M76
Dcg-2Nal-mABz
—
4.9


M92-1
Dcg-2Nal-mA2Bz
—
5.0


M92-2
Dcg-2Nal-mA2Bz(Gun)
—
4.8


M92-4
Dcg-2Nal-mA2Bz(Dcg)
—
5.0


M104
Dcg-2Nal-3Pal
—
4.9


M112
Dcg-D2Nal-mABz
—
5.1


M120
Dcg-2Nal-pABz
—
4.7


M122-1
Mcg-APa-mABz
—
5.0


M122-2
Dcg-Apa-mABz
—
4.6


M136
Sin-F5F-3Pal
—
Inact


M162
Dcg-2Nal-Asp
—
4.9


M168-1
2Nap-PaF(Mcg)
—
4.8


M168-2
2Nap-PaF(Dcg)
—
4.7


M172
Inp-Dpr(Dcg-2Nal)
—
4.9


M174
Dcg-Asp-Aqu
—
Inact


M180
F5c-pABz-2Nal
—
5.1


M188B
Dcg-2Nal-Asp(Aqu)
—
5.5


M202
F5c-Gly-mABz-2Nal
—
5.1


M204
Ac-Pac-Gly-mABz-Nal
—
5.0


M218
2Nal-Atmp
Inact
4.8


M222
Dcg-2Nal-Atmp
15
6.8
Inact


M228-2
Dcg(Me)-2Nal-Atmp(Me)
15
7.6
—


M236
Dcg-Igl-Atmp
>50
4.7
—


M240
Dcg-F5f-Atmp
32
5.1
—


M244A
Dcg-2Nal-Atpm
12
5.0


M244B
Dcg-2Nal-Atpc
Inact
4.9


M246
Dcg-D2Nal-Atmp
>50
5.7


M248
F5c-2Nal-Atmp
3.2
6.2
—


M250
Aca-2Nal-Atmp
—
5.2


M252
Dhq-2Nal-Atmp
Inact
4.8


M254
TDIM-(2Nal-Atmp)2
5
5.1
—


M254-1
TDIM-(2Nal-Atmp)2
5
5.1


M254-2
TDIM-(2Nal-Atmp)2
5
5.8


M258
Dcg-Igl-Aptp
—
5.0


M262
Dcg-D2Nal-Atmp
6
5.3


M264
Dcg-Trp-Atmp
16
4.7
—


M266
Dcg-Apa-Atmp
42
4.8
—


M268
F5c-2Nal-Tpac
10
5.2
—


M270
Dcg-2Nal-Tpac
—
6.1


M272
Dpa-2Nal-Atmp
9
5.2
—


M274
Sin-2Nal-Atmp
36
4.7
—


M276
Dca-2Nal-Atmp
4.6
5.3
—


M280
TDIM-(Igl-Atmp)2
6
5.2
—


M280-1
Ctim-Igl-Atmp
21
Wk


M280-2
TDIM-(Igl-Atmp)2
6
5.2


M286
Dtp-(2Nal-Atmp)2
24
5.1
—


M288
Boc-2Nal-Atmp
Inact
5.2
—


M288A
Boc-2Nal-Atmp
>85
5.2


M290-1
Btac-(2Nal-Atmp)2
>60
Wk


M290-2
Btac-(2Nal-Atmp)3
20
Wk


M292
Pac-Igl-Atmp
40
Wk


M294
DDD-(Pac-Igl-Atmp)2
1.8
Inact
10.3


M296
Pya-Bip-Atmp
15
Wk


M302
Atcp-2Nal-Atmp
3.5
5.2


M304
TDIM-(2Nal-Dmm)2
4.2
5.9
—


M306
Gbz-2Nal-Atmp
>100
—


M308
Pac-2Nal-Atmp
>75
5.0


M310
DDD-(Pac-2Nal-Atmp)2
1.2
5.1
Inact


M312
Tfmc-2Nal-Atmp
3.2
5.5
—


M314
F5c-2Nal-Aqd
25
4.6


M316
F5c-Tyr-Atmp
50
—


M318
F5c-Tyr(Bzl)-Atmp
3.6
5.1
—


M320
F5c-Oic-Atmp
13
—
—


M322
F5c-Tic-Atmp
7.6
—
—


M324
Dmac-2Nal-Atmp
3
5.2
—


M336-1
Dcg-2Nal-Asp-(R,S)Aqu
—
5.1


M336-2
Dcg-2Nal-Asp-(R,S)Aqu
—
5.4


M340
Dcg-Pac-Gly-mABz-2Nal
—
4.8


M342
Dcg-2Nal-Asp-Atmp
—
5.4


M350
Dcg-2Nal-Glu-Atmp
—
5.0


M354
Dcg-2Nal-PgF
—
5.2


M362
Dcg-pAPa-Asp-Atmp
—
4.9


M364
F5c-pAPa-Asp-Atmp
—
4.7


M368
Tfmc-pAPa-Asp-Atmp
—
4.7


M396
F5c-2Nal-Cys(SO3H)-Atmp
—
5.0


M408
Pya-2Nal-Cyh
22
4.6
11.5


M418
F5c-BtA-Atmp
3.8
7.0
10.3


M422
Pya-pABz-2Nal
52
—
Inact


M428
Pya-Gly-mABz-Aqd
>300
—


M430
DDD-(BtA-Atmp)2
18
5.3
11.3


M432
DDD-(2Nal-Asp-Atmp)2
70
—
10


M436-1
TDIM-BtA-Atmp
8
4.8


M436-2
TDIM-(BtA-Atmp)2
4.5
5.8
Ag


M438
F5c-3Pal-Atmp
26
4.8
10


M440
Dcg-BtA-Atmp
30
—


M448
Dmac-BtA-Atmp
2.7
5.7
Ag


M456
F5c-Cys(Meb)-Atmp
4.7
5.3
Inact


M460
DDD-(3Pal-Nal-Cyh)2
15
Wk


M466
F5c-2Nal-3Ampy
Inact
Wk


M470
F5c-2Nal-Ampz
11
5.2
Inact


M472
Dmac-2Nal-Ampz
25
5.3
Inact


M474
Pya-2Nal-3Abza
35
Wk
Inact


M476
Tha-BtA-Atmp
15
4.6
Inact


M478
Dmac-2Nal-Thm
30
5.1
Inact


M480-1
HOOC-DDD-Pac-2NaL-Ampz
45
5.1


M480-2
DDD-(Pac-2Nal-Ampz)2
—
5.6


M492
F5c-mABz-2Nal-Ampz
45
5.1
Inact


M506
Mse-Pac-BtA-Atmp
11
4.9
—


M526
F5c-2Nal-Dmp
10
5.4
Inact


M536
F5c-2Nal-Dmab
4
Wk
Inact


M538
DDD-(Pac-2-Nal-Dmp)2
>80
Wk


M568
F5po-2Nal-Atmp
10
5.8
Ag


M570
F5c-OC2Y-Atmp
1.8
5.6
Ag


M572
Dca-2Nal-Acep
2.6
Wk
Wk


M574
Dns-Tyr(Bzl)Atmp
4.5
—
Inact


M582
Dmac-OC2Y-Atmp
3
5.4
9.5


M584-A
DDD-[DArg(Tos)-2Nal-Atmp]2
5
Inact
10.3


M584-B
DDD-(DArg-2Nal-Atmp)2
5
5.7
11.3


M586-A
Mse-Pac-Igl-Atmp
15
5.3
12


M586-B
Seb-Pac-Igl-Atmp
40
Wk
12.3


M588
α-DDD-(Lys-DArg-2Nal-
9.4
Wk
10



Atmp)2


M592
F5c-OC2Y-Matp
1.5
4.9
Ag


M594
F5c-MC2Y-Atmp
3.7
5.0
8


M594
F5c-MC2Y-Atmp
3.7
5.0
8


M596-A
DDD-[Arg(Tos)-2Nal-Atmp]2
15
5.0


M596-B
DDD-(Arg-2Nal-Atmp)2
8.2
Wk
Inact


M602
Chc-OC2Y-Atmp
12
—
10.8


M604
Pac-2Nal-Ecap
43
4.5


M606
DDD-(Pac-2Nal-Api)2
30
5.0
10


M614
F5c-(N-Dmb)-Tyr(Bzl)-OMe
9.1
Wk


M616
DDD-(Pac-1Nal-Atmp)2
1.4
5.4
—


M618
F5c-DArg-2Nal-Arg-Matp
18
—
—


M620
DDD-(DArg-2Nal-Arg-Matp)2
2.0
5.5
—


M622
F5c-OC2Y-Mapp
1.2
5.7
—


M624
Dns-OC2Y-Matp
1.4
5.1
—


M626
Pya-OC2Y-Matp
3.7
4.8
—


M628
Cin-OC2Y-Matp
1.6
5.2
—


M630
Dmac-OC2Y-Matp
1.6
5.0
—


M632
Atcp-OC2Y-Matp
1.4
5.4
—


M636
DDD-(DArg-Arg-Aud-Pac-
1.7
5.8
—



2Nal-Atmp)2


M638
DDD-(DArg-Igl-Arg-Matp)2
0.6
Inact


M640
DDD-(DArg-BtA-Arg-Matp)2
3.0
5.9


M648
F5c-PaF(Mes)-Atmp
Inact
5.0


M650
Atcp-OC2Y-Mapp
3.7
—
—


M652
Ppa-OC2Y-Mapp
7.5
5.7
—


M654
Sul-Atmp
Inact
4.5


M656
Sul-2Nal-Atmp
13
5.4


M660
DDD-(His-1Nal-Atmp)2
30
Wk
—


M662
F5c-tLeu-Atmp
Inact
5.2
—


M664
F5c-OCIY-Matp
1.2
5.0


M666
Dns-OCIY-Matp
1.3
5.0


M668
SBEC-(DArg-2Nal-Arg-Matp)2
3.4
5.2


M670
DTP-(DArg-Igl-Arg-Matp)2
Inact
5.1


M672
HDD-(DArg-Igl-Arg-Matp)2
—
—


M674
DDD-(DArg-F5F-Arg-Matp)2
3.5
Wk


M678
(Dns-DArg-Igl-Arg)2-DDA-
1.1
5.3


M724
F5c-DArg-Aud-OC2Y-
12
5.4



Gly-Atmp


M744
DDD-(DArg-2Nal-Arg-Dmab)2
3.4
5.3


M798
F5c-OC2Y-Dmab
37
—


M800
DDD-(DArg-OC2Y-Dmab)2
27
5.3


M832
F5c-PFF-Dmab
47
4.6


M834
DDD-(DArg-PFF-Arg-Dpea)2
1.6
5.3


M848
DDD-(DArg-F5F-Arg-Dmab)2
—
—


M880
DDD-(DArg-F5F-Arg-Dpea)2
—
—


M886-1
DDD-DArg-PFF-Arg-NH2
3.2
Wk



 └DArg-PFF-Arg-Dpma


M886-2
DDD-(DArg-PFF-Arg-Dpma)2
—
Inact


M892
DDD-(DArg-PFF-Arg-PFF-
8.5
Wk



NH2)2


M900
DDD-(DArg-F5F-Arg-PaF-
6.3
—



NH2)2


M916
F5c-DArg-PFF-Arg-PFF-
5.7
4.9



NH2


M1032
DDD-(DArg-Igl-Mapp)2
15
5.4


M1040
EDTA-(OC2Y-Atmp)4
0.73
—





Footnotes:


aED50 for killing of SCLC strain SHP-77 in vitro, μM.


bpA2 for bradykinin antagonist activity on isolated guinea pig ileum. The pD2 of bradykinin is 7.4 on ileum. Higher numbers indicate higher potency.


cpA2 for bradykinin antagonist potency on cloned human B2 receptors, pM. The pD2 for bradykinin is 11. Higher numbers indicate higher potency.


Ag = agonist; Inact = inactive; Wk = weak













TABLE 3







ACTIVITIES OF CYCLIC PEPTIDES










NUMBER
STRUCTURE
MTTa
GPIb





B9458-2




—
6.1


 


B9462




7.3
6.0


 


B10302
c[DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg]
Inact
5.2


B10304
Aca-c[DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Nig-Oic-Arg]
Inact
6.4


B10306
c[Arg-DNMF-DTrp-Phe-DTrp-Leu]
Inact
Wk


B10312
α-DDD-(c[Lys-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DF5F-
3.8
—



Oic-Arg])2


M680
c[DArg-Arg-Eac-Ser-DF5F-Nc7G-Arg]
—
—


M824
c[Ava-Igl-Ser-DF5F-Oic-Arg]
Inact
5.2


M850
c[DArg-Arg-Aud-DIgl-PFF-Arg]
1.4
5.1


M868-2
c[DArg-Arg-Aud-DF5F-Oic-Arg]
9.2
6.1


M870
c[DArg-Arg-Add-DF5F-Oic-Arg]
5.5
5.3


M872
c[DArg-Arg-Eac-Ser-DF5F-Oic-Arg]
2.2
Inact


M874
c[DArg-Arg-Add-Ser-DF5F-Oic-Arg]
11
5.0


M876
c[DArg-Arg-Aud-Ser-DF5F-Oic-Arg]
22.5
5.4


M878
c[DArg-Arg Add-DIgl-PFF-Arg]
7
Wk


M882
c[DArg-Arg-Add-Ser-DIgl-PFF-Arg]
4.5
Inact


M896
c[DArg-Arg-Eac-DIgl-PFF-Arg]
65
Wk


M902
c[DArg-Arg-Ava-Ser-DIgl-PFF-Arg]
30
5.5


M906
c[DArg-Arg-Eac-DF5F-Oic-Arg]
45
Wk


M908
c[DArg-Arg-Ava-Ser-DF5F-Oic-Arg]
40
4.9


M910
c[Bala-DArg-Arg-Eac-Ser-DF5F-Oic-Arg]
42
5.2


M924
c[Suc-DArg-Arg-Eac-Ser-DIgl-PaF]-Arg
37
Wk


M934
c[DNiK-Arg-Eac-Ser-DF5F-Oic-Arg]
—
—


M940
c[DNiK-PzO-Eac-Ser-DF5F-Oic-Arg]
—
—


M986
c[Add-DArg-F5F-Arg]
—
—





Footnotes:


aED50 for killing of SCLC strain SHP-77 in vitro, μM.


bpA2 for bradykinin antagonist activity on isolated guinea pig ileum. The pD2 of bradykinin is 7.4 on ileum. Higher numbers indicate higher potency.


Inact = inactive; Wk = weak













TABLE 4







PREVIOUSLY DESCRIBED


KNOWN PEPTIDES THAT NEWLY


SHOW IN VIVO ANTI-CANCER ACTIVITY










NUMBER
STRUCTURE







B9430
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg



B9330
DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Nig-Arg



B10044
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DF5F-Oic-Arg



B10050
Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-DTic-ChG



B10206
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DF5F-Nc7G-Arg



B10288
DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg










These compounds showed anti-tumor activity in vivo when tested by the procedure of Example XXI.







TABLE 5







CYTOTOXICITY IN


VITRO AGAINST STRAINS OF PROSTATE CANCER









COMPOUND
PROSTATE CANCER CELL LINE
SCLC













NUMBER
DU14
TSU
LNCa
PC-3
PPC1
SHP-77
















B9870
0.08
6.5
3.7
3.2
4.3
0.15


M570
1.2
2.8
3.0
1.6
3.0
1.8


M590
0.01
7.0
7.0
6.3
12
4.5





Numbers are ED50 (μM) for cytotoxic activity. Activity against SCLC strain SHP-77 is included for comparison.













TABLE 6





ABBREVIATIONS USED FOR COMPOUNDS
















B9430 =
DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg


B9870 =
SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-



Oic-Arg)2


Aaa =
1-Adamantaneacetyl


AAA =
amino acid analysis


ABz =
Aminobenzoic acid


ABza =
Aminobenzyl alcohol


Ac =
Acetyl


Ac3c =
1-Amino-1-cyclopropanecarboxylic acid


Ac5c =
l-Amino-1-cyclopentanecarboxylic acid (“cyclo-Leu”)


Ac6c =
1-Aminocyclohexanecarboxylic acid


Aca =
1-Adamantanecarboxyl


Acep =
4-Amino-1-carbethoxymethyl-2,2,6,6-



tetramethylpiperdine


ADA =
1,3-Adamantanediacetyl


Add =
12-Aminododecanoic acid


Aib =
α-Aminoisobutyric acid


Aic =
2-Aminoindane-2-carboxylic acid


AlG =
α-Allylglycine (2-amino-4-pentenoic acid)


Ama =
Aminomethylanthranilic acid


Amb =
Aminomethylbenzoic acid


Ampy =
3-Aminomethylpyridine


Ampz =
1-Amino-4-methylpiperazine


Ana =
Anthranilic acid


APa =
p-Aminophenylacetic acid


APb =
p-Aminophenylbutyric acid


ApC =
S-3-Aminopropylcysteine


Api =
4-Aminopiperidine


Apmp =
4-Amino-1,2,2,6,6-pentamethylpiperidine


Aptp =
4-Amino-1-phenylmethyl-2,2,6,6-tetramethylpiperidine


Aqd =
4-Aminoquinaldine


Aqu =
3-Aminoquinuclidine


Arg(NO2) =
Arginine(Nitro)


Atc =
2-Aminotetralin-2-carboxylic acid


Atcp =
4-Amino-3,5,6-trichloropicolinic acid


Atmp =
4-Amino-2,2,6,6-tetramethylpiperidine


AtmpO =
4-Amino-2,2,6,6-tetramethylpiperidinyloxy


Atpc =
4-Amino-2,2,6,6-tetramethyl-4-



piperidinecarboxylic acid


Atpm =
4-Amino-4-methoxycarbonyl-2,2,6,6-



Tetramethylpiperidine (4-Amino-2,2,6,6-



tetramethyl-4-piperidinecarboxylic acid methyl



ester)


Aud =
11-Aminoundecanoic acid


Ava =
5-Aminovaleric acid


Azt =
Azetidine-2-carboxylic acid


BAla =
β-Alanine


BApG =
N,N-bis(3-aminopropyl)-glycine


BAPTA =
1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N′,-tetraacetyl


Bip =
Biphenylalanine


Boc =
(tert-Butoxycarbonyl); [(1,1-dimethylethoxy)carbonyl]


BOP =
Benzotriazoyloxytris(dimethylamino)phosphonium



hexafluorophosphate


BPHD =
N,N′-bis(2,2,6,6-tetramethyl-4-piperidinyl)-



1,6-hexanediamine


BSH =
1,6-Bissuccinimidohexane


BtA =
3-Benzothienylalanine


BTAC =
Benzene-1,3,5-tris-carboxamido-6-caproyl


BTC =
1,3,5-Benzenetricarboxyl


Bz =
Benzoyl


Bzl =
Benzyl


CAcH =
cis-2-Amino-1-cyclohexanecarboxylic acid


Cca: 
2-Chlorocinnamic acid


CDF =
p-Chloro-D-phenylalanine


ChA =
α-Cyclohexylalanine


Chc =
α-Cyano-4-hydroxycinnamoyl


ChG =
α-Cyclohexylglycine


CHO =
Chinese hamster ovary


CHTC =
1,3,5-Cyclohexanetricarboxyl


CHyp =
cis-4-Hydroxy-proline


Cin =
Cinnamoyl


CMeb =
S-(4-Methylbenzyl cysteine


CmF =
(Z) p-Chloro-2,3-methanophenylalanine


Cmp =
4-Carboxymethylpiperazine


CpA =
α-Cyclopropylalanine


CpG =
α-Cyclopentylglycine


CpGΨ
CpG pseudo(CH2NH) Arg


(CH2N)Arg =


CPTA =
trans-1,2-Diaminocyclohexane- N,N,N′,N′-tetraacetyl


CTAC =
Cyclohexane-1,3,5-tris-carbamido-ε-caproyl


Ctim =
13-Carboxytridecanimidyl


Cyh =
Cyclohexylamine


Dabz =
Diaminobenzoic acid


DArg(NO2) =
Nitro-Arginine


Dca =
Dicyclohexylacetyl


Dcg =
N,N′-Dicyclohexylguanidyl


DCM =
Dichloromethane


DDA =
1,10-Decanediamine


DDD =
Dodecanedioyl


DDS =
2-Dodecen-1-ylsuccinyl


DEA =
N,N′-Diethylethylenediamine


DhP =
3,4-Dehydroproline


Dhq =
2,3-Dehydroquinuclidine-3-carboxyl


DIC =
Decahydroisoquinoline-3-carboxylic acid


DIEA =
Diisopropylethylamine


Dmab =
4-Dimethylaminobenzylamine


Dmac =
4-Dimethylaminocinnamyoyl


Dmb =
4-(Dimethylamino)benzyl


DmF =
2,4-Dimethylphenylalanine


DMF =
Dimethyl formamide


DmK =
ε-Dimethyllysine


Dmm =
2,6-Dimethylmorpholine


Dmp =
3-Dimethylaminopropylamine


DmtP =
5,5-Dimethyl-4-thiaproline


Dns =
Dansyl (5-dimethylamino-1-naphthalenesulfonyl)


22Dp =
2,2-Diphenylpropionyl


33Dp =
3,3-Diphenylpropionyl


Dpa =
Diphenylacetyl


Dpea =
Diphenylethylamine


Dpma =
Diphenylmethylamine


Dpr =
2,3-Diaminopropionic acid


DTP =
Dithiobis-propionyl


DTPA =
Diethylenetriaminepentaacetyl


Eac =
ε-Aminocaproic acid


Ecap =
N-Ethoxycarbonyl-4-amino-piperidine



(Ethyl 4-amino-1-piperidinecarboxylate)


EDA =
4,4′-Ethylenedianiline


EDP =
4,4′-Ethylenedipiperidine


EDTA =
Ethylenediaminetetraacetyl


EDTP =
Ethylenediaminetetrapropionic acid


EGS =
Ethylene glycol-bis-succinyl


EGTA =
Ethylene glycol-bis(β-aminoethyl ether)-



N,N,N′,N′-tetraacetyl


EOPC =
1,1′-Ethylenebis(5-oxo-3-pyrrolidinecarboxyl)


ETTA =
2,2′2′′,2′′′-[Ethanediylidenetetrakis(thio)tetrakisacetyl


F2F =
Difluorophenylalanine


F5bz =
Pentafluorobenzoyl


F5c =
2,3,4,5,6-Pentafluorocinnamoyl


F5F =
Pentafluorophenylalanine


F5pa =
2,3,4,5,6-Pentafluorophenylacetyl


F5po =
2,3,4,5,6-Pentafluorophenoxyacetyl


Fbz =
para-Fluorobenzoyl


Flu =
Fluorescein thiourea


Gaa =
Guanidinoacetyl


GaP =
2-Guanidyl-3-(4-aminophenyl)propionic acid


Gbz =
4-Guanidinobenzoyl


Glt =
Glutaryl


Gun =
Guanidyl


HATU =
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium



hexafluorophosphate


HbQ =
4-Hydroxybutylglutamine


HDA =
1,6-Hexanediamine


HDD =
Hexadecanedioyl


HF =
Hydrogen fluoride


HFG =
Hexafluoroglutaroyl


HiG =
Hexahydro-2-indanylglycine


HOAt =
1-Hydroxy-7-azabenzotriazole


hPhe =
Homo-phenylalanine


HPLC =
high performance liquid chromatography


Hxa =
Hexanoic acid


Hyp =
trans-4-Hydroxyproline


Ica =
Indoline-2-carboxylic acid


Igl =
α-2-Indanylglycine


Ing =
α-1-Indanylglycine


Inp =
Isonipecotic acid


Iq2a =
6,7-Dimethoxy-3,4-dihydro-1-isoquinolineacetic Acid


Iq4a =
6,7-Dimethoxy-1,2,3,4-tetrahydro-1-



isoquinolineacetic acid


Lau =
Lauroyl


Leu(r)Leu =
Leu-pseudo(CH2NH)Leu


LeuΨ(CH2NH)
Leu-pseudo(CH2NH)Leu


Leu =


LDMS =
laser desorption mass spectrometry


mA2Bz =
3,5-Diaminobenzoic acid


MaG =
α-Methallylglycine (2-amino-3-methyl-



4-pentenoic acid)


Mapp =
4-(Methylamino)-1,2,2,6,6-pentamethylpiperidine


Matp =
4-(Methylamino)-2,2,6,6-tetramethylpiperidine


MatpO =
4-(N-methylamino)-2,2,6,6-tetramethylpiperidinyloxy


MBC =
S-(4-methylbenzylcysteine


MBHA =
Methylbenzhydrylamine


MC2Y =
N-Methyl-O-2,6-dichlorobenzyl-tyrosine


Mca =
2-Methylcinnamic acid


Mcg =
Monocyclohexylguanidyl


Meb =
Methylbenzyl


MeP =
2,4-Methanoproline


Mes =
Methanesulfonyl


MFE =
(E)-2,3-Methanophenylalanine


MFF =
meta-Fluorophenylalanine


Mosi =
Methoxy-suberimido


Moti =
14-Methoxytetradecanediimidoyl


Mse =
Methoxysebacyl


MTT =
(3-(4,5)-Dimethyltriazol-2-yl)-2,5-diphenyl



tetrazolium bromide


Nal =
β-Naphthylalanine


Nap =
Naphthoyl


Nba =
Norbornane-2-acetyl


Nbc =
Norbornenedicarboxyl


Nbi =
Norbornenedicarboximide


Nbn =
2-Aminonorbornane-2-carboxylic acid


Nc5G =
N-Cyclopentylglycine


Nc6G =
N-Cyclohexylglycine


Nc7G =
N-Cycloheptylglycine


Nc8G =
N-Cyclooctylglycine


Nig =
N-2-Indanylglycine


NiK =
ε-Nicotinoyllysine


NMF =
N-Methylphenylalanine


NSCLC =
non-small cell carcinoma


OBS =
O-Benzylserine


OBT =
O-Benzylthreonine


OBY =
O-Benzyltyrosine


OC2Y =
O-2,6-Dichlorobenzyltyrosine


OCIY =
O-2,6-Dichlorobenzyl-3,5-diiodotyrosine


Oct =
Octanoyl


Oic =
Octahydroindole-2-carboxylic acid


OMe =
O-Methyl


OMY =
O-Methyltyrosine


OSY =
Tyrosine O-sulfate ester


Paa =
Phenylacetyl


Pac =
4-Aminocinnamic acid


PaF =
p-Aminophenylalanine


Pal =
β-Pyridylalanine


Pba =
Phenylbutyryl


Pcc =
trans-2-Phenyl-1-cyclopropanecarboxylic acid


PCF =
p-Chlorophenylalanine


Pcpa =
α-Phenylcyclopentaneacetyl


PdF =
p-Dicyclohexylguanidylphenylalanine


PFF =
p-Fluorophenylalanine


PFS =
Perfluorosuberoyl


PgF =
p-Guanidinophenylalanine


PheOL =
Phenylalaninol


PhG =
Phenylglycine


Pip =
Pipecolic acid (“homo-Pro”)


PipA =
β-3-Piperidylalanine


PNF =
p-Nitrophenylalanine


Ppa =
Phenylpropionyl


Pya =
trans-3-(3-Pyridyl)acryloyl


PyAoP =
7-Azabenzotriazol-1-yloxytris(pyrrolidino)



phosphonium hexafluorophosphate


PzK =
ε-Pyrazinoyllysine


PzO =
4-Pyrazinoylornithine


Saa =
trans-Styrylacetic acid


SBEC =
Sulfo-bis-ethoxycarbonyl


Sbl =
Sebacoyl


SCLC =
small cell lung carcinoma


Seb =
Sebacyl


Sin =
Sinapinyl (3,5-dimethoxy-4-hydroxycinnamoyl-)


Ste =
Stearoyl


Sua =
Sulfanilamide (4-Aminobenzenesulfonamide)


SUB =
Suberyl


Suc =
Succinyl


Sud =
Sulfadiazine


SUIM =
Suberimidyl


Sul =
Sulindac


Tba =
t-Butyl-acetyl


TDIM =
Tetradecanediimidyl


TEA =
Triethylamine


TFA =
Trifluoroacetic acid


Tfmc =
trans-4-(Trifluoromethyl)cinnamoyl


Tha =
3-(2-Thienyl)acryloyl


Thi =
β-2-Thienylalanine


Thm =
Thiomorpholine


Thz =
Thiazolidine-4-carboxylic acid (4-thiaproline)


Tic =
1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid


TLC =
thin layer chromatography


TLeu =
tert-Leucine


TMF =
2,4,6-Trimethylphenylalanine


Tos =
p-Toluenesulfonyl


Tpac =
2,2,5,5-Tetramethyl-3-(aminoethyl)-pyrroline-



3-carboxamide


TREN =
tris(2-Aminoethyl)amine


Trx =
Tranexamic acid (trans-4-((Aminomethyl))



cyclohexanecarboxylic acid)

## Claims

1. The compound trans-3-(3-pyridyl)acryloyl-biphenylalanine-4-amino-2,2,6,6-tetramethylpiperidine (Pya-Bip-Atmp), or a pharmaceutically acceptable salt thereof.

2. A method to treat small cell lung cancer in an animal in need of such treatment comprising administering an effective amount of the compound or salt of claim 1 to the animal.

3. The method of claim 2, wherein the animal is a human.

4. A method of inhibiting the growth of a tumor cell comprising contacting said tumor cell with the compound trans-3-(3-pyridyl)acryloyl-biphenylalanine-4-amino-2,2,6,6-tetramethylpiperidine (Pya-Bip-Atmp), or a pharmaceutically acceptable salt thereof.