CA2244673A1 - Peptide inhibitors of hematopoietic cell proliferation - Google Patents
Peptide inhibitors of hematopoietic cell proliferation Download PDFInfo
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- CA2244673A1 CA2244673A1 CA002244673A CA2244673A CA2244673A1 CA 2244673 A1 CA2244673 A1 CA 2244673A1 CA 002244673 A CA002244673 A CA 002244673A CA 2244673 A CA2244673 A CA 2244673A CA 2244673 A1 CA2244673 A1 CA 2244673A1
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- lys
- asp
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- pro
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention features derivatives of the tetrapeptide Ser-Asp-Lys-Pro and the use thereof to inhibit the proliferation of hematopoietic cells.
Description
W O97/28183 PCT~B9710022 PEPTIDE INHIBITORS OF HEMATOPOIETIC CELL PROLIFERATION
.
Backqround of the Invention The tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) 5 was originally isolated from fetal calf bone marrow.
Lenfant, et al., Proc. Natl. Acad. Sci. USA 86:779-782 (1989). AcSDKP is a negative regulator of hematopoietic stem cell proliferation, preventing stem cell recruitment into the S-phase. Frindel, et al., Exp. Hematol. 5:74-76 (1977). AcSDKP appears to exert this function by blocking the action of stem cell-specific proliferation stimulators. Robinson, et al., Cell Proliferation 25:623-32 (1992). Phase-specific antic~ ~ drugs (e.g., Ara-C or cisplatin) or radiation act on cells committed 15 to proliferation, irrespective of whether the cell is malignant. Thus, a~~ ; n; ~tration of AcSDKP in conjunction with cytotoxic therapy, protect normal hematopoietic progenitor cells in the quiescent state.
S, ~y of the Invention In one aspect, the invention features compounds of the formula:
.
Backqround of the Invention The tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) 5 was originally isolated from fetal calf bone marrow.
Lenfant, et al., Proc. Natl. Acad. Sci. USA 86:779-782 (1989). AcSDKP is a negative regulator of hematopoietic stem cell proliferation, preventing stem cell recruitment into the S-phase. Frindel, et al., Exp. Hematol. 5:74-76 (1977). AcSDKP appears to exert this function by blocking the action of stem cell-specific proliferation stimulators. Robinson, et al., Cell Proliferation 25:623-32 (1992). Phase-specific antic~ ~ drugs (e.g., Ara-C or cisplatin) or radiation act on cells committed 15 to proliferation, irrespective of whether the cell is malignant. Thus, a~~ ; n; ~tration of AcSDKP in conjunction with cytotoxic therapy, protect normal hematopoietic progenitor cells in the quiescent state.
S, ~y of the Invention In one aspect, the invention features compounds of the formula:
2~ R
wherein Al is the identifying group of the D- or L- isomer of Ser;
A2 is the identifying group of the D- or L- isomer of Asp or Glu;
A3 is the identifying group of the D- or L- isomer of Lys, Arg, or Orn;
W O 97/28183 P~T~B97100221 . 2 -A4 is the D- or L- isomer of Pro;
R1 is H~ C1-12 alkyl, C7_20 arylalkyl, R7CO, or R70C(O), where R7 is C1_12 alkyl, C7_20 arylalkyl, or C1_12 alkyl or C7_20 arylalkyl substituted, e.g., one to three 5 times, with OH, C02H, or NE2;
R2 is H~ Cl-12 alkyl, or C7_20 arylalkyl;
each of R3 and R4, independently, is CO-NH, CH2-NH, CH2-S, CH2-O, Co-CH2, CH2-CO, O-CH2-CH2;
R5 is CO or CH2; and R6 is OH~ NH2~ C1-12 alkoxy, or NH-Y-CH2-z, where Y
is a branched or straight chain Cl_l2 hydrocarbon, e.g., branched or straight chain, moiety and Z is H, OH, C02H, or CONH2; provided that if R6 is OH, R3 is CO-NH, and R4 is CO-N~, then R5 is CH2; or a pharmaceutically accepta~le ~5 salt thereof.
Examples of compounds of the invention are the ~ollowing:
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH (Analog 1);
CH3CO-Ser-Asp-~tCH2NH)-Lys-Pro-OH (Analog 2);
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-OH (Analog 3);
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
CH3CO-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-NH2;
H-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
H-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
H-Ser-Asp-Lys-~(CH2N)-Pro-OH;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-OH;
H-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
H-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
H-Ser-Asp-Lys-~(CH2N)-Pro-NH2;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-NE2;
HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-NH2;
CA 02244673 l998-07-22 W O 97/28183 PCTnB97JOD221 CH3~0--Ser--Asp--Lys--Pro--NH2;
H-Ser-Asp-Lys-Pro-NH2;
CH3CO-Ser-~sp-Lys-Pro-NHCH3;
H-Ser-Asp-Lys-Pro-NHCH3;
HOOCCH2C~2C~-Ser-Asp-Lys-Pro-NHCH3; and HOOCCH2CH2CO-Ser-Asp-Lys-Pro-NH2 With the exception o~ the N-terminal amino acid and Pro, all abbreviations (e.g., Asp) of amino acids in this disclosure stand for the structure of -NH-CH(~)-CO-, lo wherein R is a side chain "identifying group" of an amino acid (e.g., CH20H for Ser, CH2COOH for Asp, CH2C~2COOH for Glu, CH2CH2CH2NHC(NH2)NH2 for Arg, (CH2)3NH2 for Orn, and (CH2)4NH2 for Lys). For the N-terminal amino acid, the abbreviation stands for the structure of =N-CH(R)-CO- or 15 -NH-CH(R)-Co-, wherein R is the identifying group of the amino acid.
Pro is the abbreviation of prolyl. By non-peptide bond or pseudopeptide bond is meant that, where the ~-amino group of proline is not involved, the peptide CO-NH bond 20 between two amino acid residues is replaced with a no~-peptide bond, e.g., CH2-NH, CH2-S, CH2-0, CO-CH2t CH2-CO, or CH2-CH2 (symbolized by ~(CH2-NH) or the like); or that~
where the ~-amino group of proline is involved, the carbonyl group of the peptide bond is replaced with CH2 (symbolized by ~(CH2-N)). Cl_12 alkyl and Cl_12 alkoxy may be straight chained or branched, e.g., methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, or isopropoxy. C7_20 arylalkyl may be straight c-h~; ne~ or branched, e.g., benzyl, napthyl, or phenylethyl.
The compounds of the present invention can be used to inhibit the proliferation of hematopoietic cells. The compounds of the invention can be used to protect hematopoietic cells (e.g., stem cells) during treatment with cytotoxic agents (e.g., chemotherapy) or radiation (e.g., radiotherapy). The compounds of the invention may W O 97/28183 PCT~B97/00221 be administered prior to the administration of the cytotoxic agent or radiation and continued through the duration of the cytotoxic treatment or radiation.
The compounds of this invention can be provided in 5 the form of pharmaceutically acceptable salts.
Acceptable salts include, but are not limited to, acid addition salts of inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate; or salts of organic acids such as acetate, 10 maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, palmoate, salicylate, oxalate, and stearate. Also within the scope of the present invention, where applicable, are salts formed from bases such as sodium or potassium hydroxide.
15 For further examples of pharmaceutically acceptable salts see, "Berge et al.", J. Pharm. Sci. 66:1 (1977).
A therapeutically effective amount (e.g., an amount effective to reduce the proliferation of hematopoietic cells) of a compound of this invention and 20 a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle) together form a therapeutic composition (e.g., a pill, tablet, capsule, or liquid) for a~ ;n;~tration (e.g., 25 orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, ionphoretically, or intratracheally) to a subject in need of the compound.
The pill, tablet, or capsule can be coated with a substance capable of protecting the composition from the 30 gastric acid or intestinal enzymes in the subject's stomach for a period of time sufficient to allow the composition to pass undigested into the subject's small intestine. The therapeutic composition can also be in the form of a biodegradable or sustained release 35 formulation for subcutaneous or intramuscular WO 97128183 PCT~B97/00221 -~ 5 -administration. See, e.g., U.S. Patents 3,773,919 and 4,767,628 and PCT Application No. WO 94/00148.
Continuous administration can also be obtained using an implantable or external pump (e.g., INFUSAIDTM pump) to 5 administer the therapeutic composition.
The dose of a compound of the present invention for protecting hematopoietic cells varies depending upon the manner of a~ n; ~tration, the age and the body weight of the subject, and the condition of the subject to be 10 treated, and ultimately will be decided by the attending physician or veterinarian. such an amount of the compound as determined by the at~n~;ng physician or veterinarian is referred to herein as a l'therapeutically effective amount." The compound of the present invention 15 may also be administered with a cytotoxic agent or radiation. Examples of cytotoxic agents include, but are not limited to, daunorubicine, cyclophosphamide, taxol, 5-fluorouracil, dioxorubicine, cisplatin, methotrexate, cytosine, arabinoside, mitomycin C, prednisone, 20 vindesine, carboplatinum, vincristine, or 3'-azido-3'deoxythymidine (AZT). The compound of the present invention may also be a~ i n; ~tered with an angiotensin converting enzyme (ACE) inhibitor. Examples of ACE
inhibitors are listed in Jackson, et al., Renin and 25 Angiotensin, in Goodman & Gillman's, The Pharmacological Basis of Therapeutics, 9th ed., eds. Hardiman, et al.
(McGraw Hill, 1996).
Also contemplated within the scope of this invention is a compound covered by the above generic 30 formula for use in protection of hematopoietic cells during cytotoxic treatment, e.g., chemotherapy, viral treatment, or radiation treatment.
Other features and advantages of the present invention will be apparent from the detailed description 35 and ~rom the claims.
W O 97128183 PCT~B97/00221 Detailed DescriPtion of the Invention It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its ~ullest extent. The following specific 5 embodiments are, therefore, to be construed as merely illustrative, and not limitative of the r~in~er of the disclosure in any way whatsoever.
Unless defined otherwise, all t~-hn; cal and scientific terms used herein have the same -Aning as 10 commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
SYnthesis The synthesis of short peptides are well ~ined in the art. The peptides of the invention were synthesized using the following general synthesis procedure.
All protected amino acids were purchased from 20 Bachem (Bobendorf, Switzerland), Calbiochem (San Diego, CA), or Nova Biochem (ha Jolla, CA). Mass spectra were obtained using a mass spectrometer (MS50) using a xenon fast atom bombardment (FAB) gun using glycerol, thioglycerol, or nitrobenzyl alcohol as a matrix. Thin-25 layer chromatography (TLC) was performed on silica gelprecoated plates (60F 254, Merck, Darmstadt, Germany).
The following solvent systems were used: A) dichloromethane/methanol, 95/5; B) dichloromethane/methanol 9/1; C) ethyl acetate/heptane, 30 1/1; D) n-butanol/acetic acid/water, 4/1/1; and E) n-butanol/acetic acid/water/pyridine, 1/1/l/1. W light, ninhydrin, and/or Pataki reagent were used ~or detection.
Protected peptides were purified by chromatography on Merck silica gel 60 (40-60~m) columns. All reagents and WO 97/28183 PCT~B97100221 solvents were of analytical grade and used as supplied except for tetrahydrofuran (THF) which was either distilled from sodium/benzophenone or filtered through a column of basic alumina immediately prior to use and 5 dimethylformamide (DMF) which was distilled from ninhy~rin under reduced pressure and stored over 4 angstrom molecular sieves. Protected peptides were characteriZed by their ~ast atom bombardment ~FAB) or secondary ion mass spectra (SIMS~. High pressure liquid 10 chromatography (HPLC) purifications were performed on a reverse phase B.~c3 ~nn Ultrasphere C--18 column (5,u particle size, 10 x 250 mm; Beckman, Fullerton, CA) using either a gradient or an isocratic elution with a mixture of acetonitrile and water cont~;n;ng 0.1% trifluoroacetic 15 acid (TFA) at 3 ml/min flow rate. Elution was monitored by recording absorbance at 210 nm. Pure peptides were characterized by their FAB or SI mass spectrum. HPLC
analysis for purity control was performed on a Novapak column C-18, 5~m (3.9 x 150 mm; Waters, Milford, MA) with 20 a solvent system consisting of a binary system of water and acetonitrile cont~;n;ng 0.1% TFA at 1 ml/min flow rate with monitoring at 210 nm. The solvent program involved the following linear gradients: 1) 0~ to 50%
acetonitrile over 50 min, 2) 0% to 80% acetonitrile over 25 40 min. k values are reported in the two solvent systems.
The following is the description of the synthesis of N-Ac-Ser-Asp-~(CH2NH)-Lys-Pro-OH (Analog 2). The abbreviations Ac, Z, Boc, t-But, and Bzl mean, 30 respectively, acetyl, benzyloxycarbonyl, tert-butoxycarbonyl, tert-butyl, and benzyl.
(1) N-~-(Z)-N-~-(Boc)-L-lysyl-L-proline-tert-butylester To a stirred solution of Z-Lys(Boc)-OH (2.66 g, W O 97/28~83 PCT~B97/00221 . 8 -7 mmol) in THF (35 ml), cooled to -15~C, was added N-methylmorpholine (0.77 ml, 7 mmol) followed by isobutylchloroformate (0.98 ml, 7 mmol). The solution was stirred at -15~C for 5 min and then cooled to -20~C.
5 Proline tert-butyl ester (1-32 g, 7.7 mmol), dissolved in 't DMF, was added. The temperature was maintained at -10~C
for 1 h, and the solution was then allowed to warm up to room temperature. After stirring for 5 h, the reaction mixture was concentrated under reduced pressure. The 10 residue was then dissolved in ethyl acetate (200 ml) and 5% citric acid (50 ml). The aqueous layer was extracted with ethyl acetate (50 ml). The pooled organic layers were washed with water, 5% sodium bicarbonate, and brine, dried over Na2S04, and concentrated under reduced pressure 15 to afford the protected dipeptide as an oil (3.6 g;
yield: 96~).
(2) N-~-(Boc)-L-lysyl-L-proline-tert-butylester The oil from step (1) (1.08 g; 2 mmol) was dissolved in ethanol (40 ml). 10% palladium on carbon 20 catalyst (0.120 g) was added, and the suspension was stirred for 4 hours 30 min under an atmosphere of hydrogen. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.672 g; yield:
25 84%).
(3) N-~-(Z)-~-(t-But)-~-aspartyl N,0-dimethyl hydroxamate This compound was prepared and converted to the corresponding aldehyde as previously described by 30 Martinez, et al., J. Med. Chem., 28:1878 (1985).
(4) N-~-(Z)-~-(0-t-But)-L-aspartyl-~(CH2NH)-N-~-(Boc)-L-lysyl-~-proline-tert-butylester W O 97128183 PCT~B97/00221 -_ 9 _ The aldehyde obtained in step (3) (2 mmol) was added to a solution of the dipeptide from step (2) (1 mmol) in methanol (MeOH) cont~in;ng 1 percent of acetic acid (7 ml). Sodium cyanoborohydride (0.094 g) was added J 5 in portions over 30 min. After 2 hours 30 min, the reaction mixture was cooled on a ice-water bath and under stirring, and a cool saturated sodium bicarbonate solution was added at o~C followed by ethyl acetate. The a~ueous phase was extracted with ethyl acetate. The 10 pooled organic layers were washed with water, dried over Na2SO4, and then concentrated under reduced pressure. The crude product was chromatographed on silica gel using CH2Cl2/MeOH (99/1) and CH2Cl2/MeOH (98/2) as eluents to give the desired product (yield: 56%).
15 (5) ~-(O-t-But)-L-aspartyl-~(CH2NH)-N-~-(t-Boc)-L-lysyl-L-proline-tert-butylester The compound obtained in step (4) (0.5 mmol) was dissolved in ethanol (13 mL). 10% Palladium on carbon catalyst (0.040 g) was added, and the suspension was 20 stirred for 24 hours under an atmosphere of hydrogen.
Additional catalyst in water (1 ml) was added. After 24 hours, the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.277 g; yield: 100~).
25 (6) N-~-(Z)-~-(O-t-But)-L-seryl-~-(O-t-But)-L-aspartyl-~(C~2NH)-N-~-(Boc)-L-lysyl-L-proline-tert-butylester To a stirred solution of Z-(O-t-But)-Ser-OH (0.132 g, 0.45 mmol) in THF (2.5 ml) cooled to -15~C, was added N-methylmorpholine (0.050 ml, 0.45 mmol) followed by 30 isobutylchloroformate (0.063 ml, 0.45 mmol). The solution was stirred at -15~C for 5 min then cooled to -20~C. The tripeptide of step (5) dissolved in the minimum amount of dichloromethane was added. The W O 97/28183 PCT~B97/00221 temperature was maintained at -10~C for 1 hour, then allowed to warm up to room temperature. After stirring for 5 hours, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl 5 acetate and 5% citric acid. The aqueous layer was v extracted with ethyl acetate (AcOEt) (50 ml). The pooled organic layers were washed with water, 5% sodium bicarbonate, and brine, and then dried over Na2SO4 and concentrated under reduced pressure to afford a white 10 foam (0.351 g). The crude product was chromatographed on silica gel using AcOEt/Hexane (1/l) as an eluant (0.233 g; yield: 70%).
(7) ~-(O-t-But)-L-seryl-~-(O-t-But)-L-aspartyl-~(CH2NH)-N-~-(Boc)-L-lysyl-L-proline-tert-butylester The compound obtained in step (6) (0.2 mmol) was dissolved in 10% ethanol (4.4 ml). 10% Palladium on carbon catalyst (0.035 g) was added, and the suspension was stirred under an atmosphere of hydrogen overnight.
The catalyst was removed by filtration, and the filtrate 20 was concentrated under reduced pressure (0.110 g).
(8) N-~-(acetyl)-~-(O-t-But)-L-seryl-~-(O-t-But)-L-aspartyl--Y! (cH2NH)-N-~- (Boc)--L--lysyl--L--proline--tert--butyl ester The amine of step (7) (0.110 g, 0.16 mmol) was 25 dissolved in DMF (0.4 ml) and reacted with acetylimidazole (0.026 g, 0.24 mmol). After stirring for 3 hours, the reaction mixture was diluted with ethyl acetate. The organic phase was then washed with water and brine, dried over Na2SO4, and concentrated under 30 reduced pressure. The crude product (0.120 g) was chromatographed on silica gel using AcOEt/MeOH(99/1) as an eluent (0.090 g; yield: 76%).
WO 97/28183 PCT~B97/00221 (g) N-~-(acetyl)-L-seryl-~-aspartyl-~(CH2NH)-L-lysyl-L-proline-OH
The compound obtained in step (8) (0.086 g, 0.12 mmol) was dissolved in TFA/CH2Cl2 (0.4 ml). The solution 5 was stirred for 2 hours 30 min at room temperature. The reaction mixture was concentrated under reduced pressure.
The residue was triturated with dry ether and dried under vacuum after removal of ether. Purification by HPLC on a C18 column using the following gradient solvent system:
10 0% at 3% acetonitrile over 10 min, 3% acetonitrile over 15 min with a flow rate of 3 ml/min (k'(1)= 8.17; k'(2)=
8) yielded the desired N-acetylated reduced tetrapeptide.
The following is the synthesis of CH3CO-Ser-Asp-Lys-Pro-NH2. (Analog g) (1) N-~-Benzyloxycarbonyl-N-~-tert-Butoxycarbonyl-L-lysyl-~-proline-benzyl-ester To a stirred solution of Z-Lys(Boc)-OH (1.54 g, 4 mmol) in THF (20 mL), cooled to -15QC, was added 0.5 mL
(4 mmol) N-Methylmorpholine followed by 0.44 mL (4 mmol) 20 isobutylchloroformate. The solution was stirred at -15QC
for 5 min and then cooled to -20QC. Benzylester proline hydrochloride (1.06 g; 4.4 mmol), in suspension in DMF (6 mL), was added followed by N-Methylmorpholine (0.48 mL, 4.4 mmol). The temperature was maintained below -10~C
25 ~or one hour and then allowed to warm up to room temperature. After 5 hours of stirring, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (120 mL) and 5%
citric acid (60 mL). The aqueous phase was extracted 30 with ethyl acetate (60 mL). The combined organic layers were washed with water, 5~ sodium bicarbonate, and brine, then dried over Na2 SO4, and concentrated under reduced pressure to afford the product as a syrup. The crude W O97/28183 PCT~B97/00221 ' - 12 -product was chromatographed on silica gel using CH2C12/MeOH (99/1) as an eluant to give 1. Yield: 1.76 g (77%), Rf(CH2C12/MeOH, 98/2) - 0.22; Rf (AcOEt/ Heptane, 1/1) = 0.24, MS (FAB)m/z = 590 (MNa+), 568 (MH+), 512 (MH+-But), 468 (MH+-Boc~, 434 MH+-Z), 378 (MH+-Boc-Bzl), 334 (MH+-Boc-Z).
(2) N-~-tert-Butoxycarbonyl-L-lysyl-L-proline Product from step (1) (1 g; 1.75 mmol) was dissolved in 10% aqueous methanol (33 mL). 10% palladium 10 on carbon catalyst (0.200 g) was added, and the suspension was stïrred under an atmosphere of hydrogen overnight. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure.
Yield: 0.565 g (94%); Rf (n-Butanol/Acetic acid/Water, 15 4/1~1) = 0.55. MS (FAB) m/z = 366 (MNa+), 344 (MH~), 288 (MH+-BUt), 244 (MH~-Boc).
(3) N-~-Benzyloxycarbonyl-~-O-tert-butyl-L-aspartyl-N-~-tert-Butoxycarbonyl-L-lysyl-~-proline To a stirred solution of Z-L-Asp(O-t-But)-OH
(0.323 g, 1 mmol) in THF (5 mL), cooled to -15QC, was added 0.11 mL (1 mmol) of N-Methylmorpholine, followed by 0.14 ml (1 mmol) isobutylchloroformate. The solution was stirred at -15QC for 5 minutes and then cooled to -20QC.
The product of step (2) was added in solution in DMF (2.5 25 mL). After 5 hours stirring, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and 5% citric acid (25 mL). The a~ueous phase was extracted with ethyl acetate (25 m~). The combined organic layers were washed with 30 water and brine, then dried over Na2SO4, and concentrated, under reduced pressure, to afford a white foam. The crude product was chromatographed on silica gel using CH2C12/MeOH/AcOH (97/3/0.5) as an eluant. Yield: 0.450 g (60%), Rf (CH2C12/MeOH/AcOH, 97/3/0.5) = 0.11 MS (FAB) m/z W O 97/28183 PCTAB97~0221 = 693 (M2Na+-H), 671 (MNa+), 615 (MNa+-But), 549 (MH+-Boc), 537 (MNa+-Z), 515 (MNa+-But-Boc), 493 (MH+-But-Boc), 437 (MNa+-Boc-Z).
(4) N-~-Benzyloxycarbonyl-~-O-tert-butyl-L-aspartyl-N-E-tert-Butoxycarbonyl-L-lysyl-L-proline amide To a stirred solution of the product of step (3) (0.129 g, 0.2 mmol) in THF (5 mL), cooled to -15QC, was added 0.022 mL (0.2 mmol) of N-Methylmorpholine followed by 0.028 mL (0.2 mmol) isobutylchloroformate. The 10 solution was stirred at -15QC for 5 minutes, and then cooled to -20QC. 0.2 mL of a cold 34% ammonia solution was added.
After one hour stirring, at a temperature below -10QC, a further hour at a temperature below OQC, the 15 reaction mixture was concentrated under reduced pressure.
The residue was dissolved in ethyl acetate and 5% citric acid. The aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water and brine, then dried over Na2SO4, and concentrated 20 under reduced pressure to afford a white foam. The crude product was chromatographed on silica gel using CH2Cl2/MeOH (95/5) as an eluant. Yield: 0.105 g (81~.
Rf (CH2Cl2/MeOH, 95/5) = 0.24; Rf (AcOEt/MeOH, 99/1) 0.45. MS (FAB) m/z = 670 (MNa+), 648 (MH+), 614 (MNa+-25 But), 548 (MH+-Boc), 534 (MH+-ProNH2), 514 (MH+-Z), 492 (MH+-But-Boc).
(5) ~-O-tert-butyl-L-aspartyl-N-~-tert-Butoxycarbonyl-L-lysyl-L-proline amide The product of step (4) (0.152 g; 0.23 mmol) was 30 dissolved in methanol (6 mL), 10% palladium in carbon catalyst (0.030 g) was added, and the suspension was stirred under an atmosphere of hydrogen for 2 hours. The catalyst was removed by filtration on a Celite pad, and W O97/28183 PCT~B97/00221 the filtrate was concentrated under reduced pressure.
Yield: 0.111 g (94%); R~ (CH2C12/MeOH, 95/5) = 0.08; Rf (CH2C12/MeOH, 9/1) = 0.35. MS (FAB) m/z = 536 (MNa+), 514 (MH~), 480 (MNa+--But), 458 (MH+--But), 414 (MH+-Boc), 5 400(MH+-ProNH2).
wherein Al is the identifying group of the D- or L- isomer of Ser;
A2 is the identifying group of the D- or L- isomer of Asp or Glu;
A3 is the identifying group of the D- or L- isomer of Lys, Arg, or Orn;
W O 97/28183 P~T~B97100221 . 2 -A4 is the D- or L- isomer of Pro;
R1 is H~ C1-12 alkyl, C7_20 arylalkyl, R7CO, or R70C(O), where R7 is C1_12 alkyl, C7_20 arylalkyl, or C1_12 alkyl or C7_20 arylalkyl substituted, e.g., one to three 5 times, with OH, C02H, or NE2;
R2 is H~ Cl-12 alkyl, or C7_20 arylalkyl;
each of R3 and R4, independently, is CO-NH, CH2-NH, CH2-S, CH2-O, Co-CH2, CH2-CO, O-CH2-CH2;
R5 is CO or CH2; and R6 is OH~ NH2~ C1-12 alkoxy, or NH-Y-CH2-z, where Y
is a branched or straight chain Cl_l2 hydrocarbon, e.g., branched or straight chain, moiety and Z is H, OH, C02H, or CONH2; provided that if R6 is OH, R3 is CO-NH, and R4 is CO-N~, then R5 is CH2; or a pharmaceutically accepta~le ~5 salt thereof.
Examples of compounds of the invention are the ~ollowing:
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH (Analog 1);
CH3CO-Ser-Asp-~tCH2NH)-Lys-Pro-OH (Analog 2);
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-OH (Analog 3);
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
CH3CO-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-NH2;
H-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
H-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
H-Ser-Asp-Lys-~(CH2N)-Pro-OH;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-OH;
H-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
H-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
H-Ser-Asp-Lys-~(CH2N)-Pro-NH2;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-NE2;
HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-NH2;
CA 02244673 l998-07-22 W O 97/28183 PCTnB97JOD221 CH3~0--Ser--Asp--Lys--Pro--NH2;
H-Ser-Asp-Lys-Pro-NH2;
CH3CO-Ser-~sp-Lys-Pro-NHCH3;
H-Ser-Asp-Lys-Pro-NHCH3;
HOOCCH2C~2C~-Ser-Asp-Lys-Pro-NHCH3; and HOOCCH2CH2CO-Ser-Asp-Lys-Pro-NH2 With the exception o~ the N-terminal amino acid and Pro, all abbreviations (e.g., Asp) of amino acids in this disclosure stand for the structure of -NH-CH(~)-CO-, lo wherein R is a side chain "identifying group" of an amino acid (e.g., CH20H for Ser, CH2COOH for Asp, CH2C~2COOH for Glu, CH2CH2CH2NHC(NH2)NH2 for Arg, (CH2)3NH2 for Orn, and (CH2)4NH2 for Lys). For the N-terminal amino acid, the abbreviation stands for the structure of =N-CH(R)-CO- or 15 -NH-CH(R)-Co-, wherein R is the identifying group of the amino acid.
Pro is the abbreviation of prolyl. By non-peptide bond or pseudopeptide bond is meant that, where the ~-amino group of proline is not involved, the peptide CO-NH bond 20 between two amino acid residues is replaced with a no~-peptide bond, e.g., CH2-NH, CH2-S, CH2-0, CO-CH2t CH2-CO, or CH2-CH2 (symbolized by ~(CH2-NH) or the like); or that~
where the ~-amino group of proline is involved, the carbonyl group of the peptide bond is replaced with CH2 (symbolized by ~(CH2-N)). Cl_12 alkyl and Cl_12 alkoxy may be straight chained or branched, e.g., methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, or isopropoxy. C7_20 arylalkyl may be straight c-h~; ne~ or branched, e.g., benzyl, napthyl, or phenylethyl.
The compounds of the present invention can be used to inhibit the proliferation of hematopoietic cells. The compounds of the invention can be used to protect hematopoietic cells (e.g., stem cells) during treatment with cytotoxic agents (e.g., chemotherapy) or radiation (e.g., radiotherapy). The compounds of the invention may W O 97/28183 PCT~B97/00221 be administered prior to the administration of the cytotoxic agent or radiation and continued through the duration of the cytotoxic treatment or radiation.
The compounds of this invention can be provided in 5 the form of pharmaceutically acceptable salts.
Acceptable salts include, but are not limited to, acid addition salts of inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate; or salts of organic acids such as acetate, 10 maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, palmoate, salicylate, oxalate, and stearate. Also within the scope of the present invention, where applicable, are salts formed from bases such as sodium or potassium hydroxide.
15 For further examples of pharmaceutically acceptable salts see, "Berge et al.", J. Pharm. Sci. 66:1 (1977).
A therapeutically effective amount (e.g., an amount effective to reduce the proliferation of hematopoietic cells) of a compound of this invention and 20 a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle) together form a therapeutic composition (e.g., a pill, tablet, capsule, or liquid) for a~ ;n;~tration (e.g., 25 orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, ionphoretically, or intratracheally) to a subject in need of the compound.
The pill, tablet, or capsule can be coated with a substance capable of protecting the composition from the 30 gastric acid or intestinal enzymes in the subject's stomach for a period of time sufficient to allow the composition to pass undigested into the subject's small intestine. The therapeutic composition can also be in the form of a biodegradable or sustained release 35 formulation for subcutaneous or intramuscular WO 97128183 PCT~B97/00221 -~ 5 -administration. See, e.g., U.S. Patents 3,773,919 and 4,767,628 and PCT Application No. WO 94/00148.
Continuous administration can also be obtained using an implantable or external pump (e.g., INFUSAIDTM pump) to 5 administer the therapeutic composition.
The dose of a compound of the present invention for protecting hematopoietic cells varies depending upon the manner of a~ n; ~tration, the age and the body weight of the subject, and the condition of the subject to be 10 treated, and ultimately will be decided by the attending physician or veterinarian. such an amount of the compound as determined by the at~n~;ng physician or veterinarian is referred to herein as a l'therapeutically effective amount." The compound of the present invention 15 may also be administered with a cytotoxic agent or radiation. Examples of cytotoxic agents include, but are not limited to, daunorubicine, cyclophosphamide, taxol, 5-fluorouracil, dioxorubicine, cisplatin, methotrexate, cytosine, arabinoside, mitomycin C, prednisone, 20 vindesine, carboplatinum, vincristine, or 3'-azido-3'deoxythymidine (AZT). The compound of the present invention may also be a~ i n; ~tered with an angiotensin converting enzyme (ACE) inhibitor. Examples of ACE
inhibitors are listed in Jackson, et al., Renin and 25 Angiotensin, in Goodman & Gillman's, The Pharmacological Basis of Therapeutics, 9th ed., eds. Hardiman, et al.
(McGraw Hill, 1996).
Also contemplated within the scope of this invention is a compound covered by the above generic 30 formula for use in protection of hematopoietic cells during cytotoxic treatment, e.g., chemotherapy, viral treatment, or radiation treatment.
Other features and advantages of the present invention will be apparent from the detailed description 35 and ~rom the claims.
W O 97128183 PCT~B97/00221 Detailed DescriPtion of the Invention It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its ~ullest extent. The following specific 5 embodiments are, therefore, to be construed as merely illustrative, and not limitative of the r~in~er of the disclosure in any way whatsoever.
Unless defined otherwise, all t~-hn; cal and scientific terms used herein have the same -Aning as 10 commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
SYnthesis The synthesis of short peptides are well ~ined in the art. The peptides of the invention were synthesized using the following general synthesis procedure.
All protected amino acids were purchased from 20 Bachem (Bobendorf, Switzerland), Calbiochem (San Diego, CA), or Nova Biochem (ha Jolla, CA). Mass spectra were obtained using a mass spectrometer (MS50) using a xenon fast atom bombardment (FAB) gun using glycerol, thioglycerol, or nitrobenzyl alcohol as a matrix. Thin-25 layer chromatography (TLC) was performed on silica gelprecoated plates (60F 254, Merck, Darmstadt, Germany).
The following solvent systems were used: A) dichloromethane/methanol, 95/5; B) dichloromethane/methanol 9/1; C) ethyl acetate/heptane, 30 1/1; D) n-butanol/acetic acid/water, 4/1/1; and E) n-butanol/acetic acid/water/pyridine, 1/1/l/1. W light, ninhydrin, and/or Pataki reagent were used ~or detection.
Protected peptides were purified by chromatography on Merck silica gel 60 (40-60~m) columns. All reagents and WO 97/28183 PCT~B97100221 solvents were of analytical grade and used as supplied except for tetrahydrofuran (THF) which was either distilled from sodium/benzophenone or filtered through a column of basic alumina immediately prior to use and 5 dimethylformamide (DMF) which was distilled from ninhy~rin under reduced pressure and stored over 4 angstrom molecular sieves. Protected peptides were characteriZed by their ~ast atom bombardment ~FAB) or secondary ion mass spectra (SIMS~. High pressure liquid 10 chromatography (HPLC) purifications were performed on a reverse phase B.~c3 ~nn Ultrasphere C--18 column (5,u particle size, 10 x 250 mm; Beckman, Fullerton, CA) using either a gradient or an isocratic elution with a mixture of acetonitrile and water cont~;n;ng 0.1% trifluoroacetic 15 acid (TFA) at 3 ml/min flow rate. Elution was monitored by recording absorbance at 210 nm. Pure peptides were characterized by their FAB or SI mass spectrum. HPLC
analysis for purity control was performed on a Novapak column C-18, 5~m (3.9 x 150 mm; Waters, Milford, MA) with 20 a solvent system consisting of a binary system of water and acetonitrile cont~;n;ng 0.1% TFA at 1 ml/min flow rate with monitoring at 210 nm. The solvent program involved the following linear gradients: 1) 0~ to 50%
acetonitrile over 50 min, 2) 0% to 80% acetonitrile over 25 40 min. k values are reported in the two solvent systems.
The following is the description of the synthesis of N-Ac-Ser-Asp-~(CH2NH)-Lys-Pro-OH (Analog 2). The abbreviations Ac, Z, Boc, t-But, and Bzl mean, 30 respectively, acetyl, benzyloxycarbonyl, tert-butoxycarbonyl, tert-butyl, and benzyl.
(1) N-~-(Z)-N-~-(Boc)-L-lysyl-L-proline-tert-butylester To a stirred solution of Z-Lys(Boc)-OH (2.66 g, W O 97/28~83 PCT~B97/00221 . 8 -7 mmol) in THF (35 ml), cooled to -15~C, was added N-methylmorpholine (0.77 ml, 7 mmol) followed by isobutylchloroformate (0.98 ml, 7 mmol). The solution was stirred at -15~C for 5 min and then cooled to -20~C.
5 Proline tert-butyl ester (1-32 g, 7.7 mmol), dissolved in 't DMF, was added. The temperature was maintained at -10~C
for 1 h, and the solution was then allowed to warm up to room temperature. After stirring for 5 h, the reaction mixture was concentrated under reduced pressure. The 10 residue was then dissolved in ethyl acetate (200 ml) and 5% citric acid (50 ml). The aqueous layer was extracted with ethyl acetate (50 ml). The pooled organic layers were washed with water, 5% sodium bicarbonate, and brine, dried over Na2S04, and concentrated under reduced pressure 15 to afford the protected dipeptide as an oil (3.6 g;
yield: 96~).
(2) N-~-(Boc)-L-lysyl-L-proline-tert-butylester The oil from step (1) (1.08 g; 2 mmol) was dissolved in ethanol (40 ml). 10% palladium on carbon 20 catalyst (0.120 g) was added, and the suspension was stirred for 4 hours 30 min under an atmosphere of hydrogen. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.672 g; yield:
25 84%).
(3) N-~-(Z)-~-(t-But)-~-aspartyl N,0-dimethyl hydroxamate This compound was prepared and converted to the corresponding aldehyde as previously described by 30 Martinez, et al., J. Med. Chem., 28:1878 (1985).
(4) N-~-(Z)-~-(0-t-But)-L-aspartyl-~(CH2NH)-N-~-(Boc)-L-lysyl-~-proline-tert-butylester W O 97128183 PCT~B97/00221 -_ 9 _ The aldehyde obtained in step (3) (2 mmol) was added to a solution of the dipeptide from step (2) (1 mmol) in methanol (MeOH) cont~in;ng 1 percent of acetic acid (7 ml). Sodium cyanoborohydride (0.094 g) was added J 5 in portions over 30 min. After 2 hours 30 min, the reaction mixture was cooled on a ice-water bath and under stirring, and a cool saturated sodium bicarbonate solution was added at o~C followed by ethyl acetate. The a~ueous phase was extracted with ethyl acetate. The 10 pooled organic layers were washed with water, dried over Na2SO4, and then concentrated under reduced pressure. The crude product was chromatographed on silica gel using CH2Cl2/MeOH (99/1) and CH2Cl2/MeOH (98/2) as eluents to give the desired product (yield: 56%).
15 (5) ~-(O-t-But)-L-aspartyl-~(CH2NH)-N-~-(t-Boc)-L-lysyl-L-proline-tert-butylester The compound obtained in step (4) (0.5 mmol) was dissolved in ethanol (13 mL). 10% Palladium on carbon catalyst (0.040 g) was added, and the suspension was 20 stirred for 24 hours under an atmosphere of hydrogen.
Additional catalyst in water (1 ml) was added. After 24 hours, the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.277 g; yield: 100~).
25 (6) N-~-(Z)-~-(O-t-But)-L-seryl-~-(O-t-But)-L-aspartyl-~(C~2NH)-N-~-(Boc)-L-lysyl-L-proline-tert-butylester To a stirred solution of Z-(O-t-But)-Ser-OH (0.132 g, 0.45 mmol) in THF (2.5 ml) cooled to -15~C, was added N-methylmorpholine (0.050 ml, 0.45 mmol) followed by 30 isobutylchloroformate (0.063 ml, 0.45 mmol). The solution was stirred at -15~C for 5 min then cooled to -20~C. The tripeptide of step (5) dissolved in the minimum amount of dichloromethane was added. The W O 97/28183 PCT~B97/00221 temperature was maintained at -10~C for 1 hour, then allowed to warm up to room temperature. After stirring for 5 hours, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl 5 acetate and 5% citric acid. The aqueous layer was v extracted with ethyl acetate (AcOEt) (50 ml). The pooled organic layers were washed with water, 5% sodium bicarbonate, and brine, and then dried over Na2SO4 and concentrated under reduced pressure to afford a white 10 foam (0.351 g). The crude product was chromatographed on silica gel using AcOEt/Hexane (1/l) as an eluant (0.233 g; yield: 70%).
(7) ~-(O-t-But)-L-seryl-~-(O-t-But)-L-aspartyl-~(CH2NH)-N-~-(Boc)-L-lysyl-L-proline-tert-butylester The compound obtained in step (6) (0.2 mmol) was dissolved in 10% ethanol (4.4 ml). 10% Palladium on carbon catalyst (0.035 g) was added, and the suspension was stirred under an atmosphere of hydrogen overnight.
The catalyst was removed by filtration, and the filtrate 20 was concentrated under reduced pressure (0.110 g).
(8) N-~-(acetyl)-~-(O-t-But)-L-seryl-~-(O-t-But)-L-aspartyl--Y! (cH2NH)-N-~- (Boc)--L--lysyl--L--proline--tert--butyl ester The amine of step (7) (0.110 g, 0.16 mmol) was 25 dissolved in DMF (0.4 ml) and reacted with acetylimidazole (0.026 g, 0.24 mmol). After stirring for 3 hours, the reaction mixture was diluted with ethyl acetate. The organic phase was then washed with water and brine, dried over Na2SO4, and concentrated under 30 reduced pressure. The crude product (0.120 g) was chromatographed on silica gel using AcOEt/MeOH(99/1) as an eluent (0.090 g; yield: 76%).
WO 97/28183 PCT~B97/00221 (g) N-~-(acetyl)-L-seryl-~-aspartyl-~(CH2NH)-L-lysyl-L-proline-OH
The compound obtained in step (8) (0.086 g, 0.12 mmol) was dissolved in TFA/CH2Cl2 (0.4 ml). The solution 5 was stirred for 2 hours 30 min at room temperature. The reaction mixture was concentrated under reduced pressure.
The residue was triturated with dry ether and dried under vacuum after removal of ether. Purification by HPLC on a C18 column using the following gradient solvent system:
10 0% at 3% acetonitrile over 10 min, 3% acetonitrile over 15 min with a flow rate of 3 ml/min (k'(1)= 8.17; k'(2)=
8) yielded the desired N-acetylated reduced tetrapeptide.
The following is the synthesis of CH3CO-Ser-Asp-Lys-Pro-NH2. (Analog g) (1) N-~-Benzyloxycarbonyl-N-~-tert-Butoxycarbonyl-L-lysyl-~-proline-benzyl-ester To a stirred solution of Z-Lys(Boc)-OH (1.54 g, 4 mmol) in THF (20 mL), cooled to -15QC, was added 0.5 mL
(4 mmol) N-Methylmorpholine followed by 0.44 mL (4 mmol) 20 isobutylchloroformate. The solution was stirred at -15QC
for 5 min and then cooled to -20QC. Benzylester proline hydrochloride (1.06 g; 4.4 mmol), in suspension in DMF (6 mL), was added followed by N-Methylmorpholine (0.48 mL, 4.4 mmol). The temperature was maintained below -10~C
25 ~or one hour and then allowed to warm up to room temperature. After 5 hours of stirring, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (120 mL) and 5%
citric acid (60 mL). The aqueous phase was extracted 30 with ethyl acetate (60 mL). The combined organic layers were washed with water, 5~ sodium bicarbonate, and brine, then dried over Na2 SO4, and concentrated under reduced pressure to afford the product as a syrup. The crude W O97/28183 PCT~B97/00221 ' - 12 -product was chromatographed on silica gel using CH2C12/MeOH (99/1) as an eluant to give 1. Yield: 1.76 g (77%), Rf(CH2C12/MeOH, 98/2) - 0.22; Rf (AcOEt/ Heptane, 1/1) = 0.24, MS (FAB)m/z = 590 (MNa+), 568 (MH+), 512 (MH+-But), 468 (MH+-Boc~, 434 MH+-Z), 378 (MH+-Boc-Bzl), 334 (MH+-Boc-Z).
(2) N-~-tert-Butoxycarbonyl-L-lysyl-L-proline Product from step (1) (1 g; 1.75 mmol) was dissolved in 10% aqueous methanol (33 mL). 10% palladium 10 on carbon catalyst (0.200 g) was added, and the suspension was stïrred under an atmosphere of hydrogen overnight. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure.
Yield: 0.565 g (94%); Rf (n-Butanol/Acetic acid/Water, 15 4/1~1) = 0.55. MS (FAB) m/z = 366 (MNa+), 344 (MH~), 288 (MH+-BUt), 244 (MH~-Boc).
(3) N-~-Benzyloxycarbonyl-~-O-tert-butyl-L-aspartyl-N-~-tert-Butoxycarbonyl-L-lysyl-~-proline To a stirred solution of Z-L-Asp(O-t-But)-OH
(0.323 g, 1 mmol) in THF (5 mL), cooled to -15QC, was added 0.11 mL (1 mmol) of N-Methylmorpholine, followed by 0.14 ml (1 mmol) isobutylchloroformate. The solution was stirred at -15QC for 5 minutes and then cooled to -20QC.
The product of step (2) was added in solution in DMF (2.5 25 mL). After 5 hours stirring, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and 5% citric acid (25 mL). The a~ueous phase was extracted with ethyl acetate (25 m~). The combined organic layers were washed with 30 water and brine, then dried over Na2SO4, and concentrated, under reduced pressure, to afford a white foam. The crude product was chromatographed on silica gel using CH2C12/MeOH/AcOH (97/3/0.5) as an eluant. Yield: 0.450 g (60%), Rf (CH2C12/MeOH/AcOH, 97/3/0.5) = 0.11 MS (FAB) m/z W O 97/28183 PCTAB97~0221 = 693 (M2Na+-H), 671 (MNa+), 615 (MNa+-But), 549 (MH+-Boc), 537 (MNa+-Z), 515 (MNa+-But-Boc), 493 (MH+-But-Boc), 437 (MNa+-Boc-Z).
(4) N-~-Benzyloxycarbonyl-~-O-tert-butyl-L-aspartyl-N-E-tert-Butoxycarbonyl-L-lysyl-L-proline amide To a stirred solution of the product of step (3) (0.129 g, 0.2 mmol) in THF (5 mL), cooled to -15QC, was added 0.022 mL (0.2 mmol) of N-Methylmorpholine followed by 0.028 mL (0.2 mmol) isobutylchloroformate. The 10 solution was stirred at -15QC for 5 minutes, and then cooled to -20QC. 0.2 mL of a cold 34% ammonia solution was added.
After one hour stirring, at a temperature below -10QC, a further hour at a temperature below OQC, the 15 reaction mixture was concentrated under reduced pressure.
The residue was dissolved in ethyl acetate and 5% citric acid. The aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water and brine, then dried over Na2SO4, and concentrated 20 under reduced pressure to afford a white foam. The crude product was chromatographed on silica gel using CH2Cl2/MeOH (95/5) as an eluant. Yield: 0.105 g (81~.
Rf (CH2Cl2/MeOH, 95/5) = 0.24; Rf (AcOEt/MeOH, 99/1) 0.45. MS (FAB) m/z = 670 (MNa+), 648 (MH+), 614 (MNa+-25 But), 548 (MH+-Boc), 534 (MH+-ProNH2), 514 (MH+-Z), 492 (MH+-But-Boc).
(5) ~-O-tert-butyl-L-aspartyl-N-~-tert-Butoxycarbonyl-L-lysyl-L-proline amide The product of step (4) (0.152 g; 0.23 mmol) was 30 dissolved in methanol (6 mL), 10% palladium in carbon catalyst (0.030 g) was added, and the suspension was stirred under an atmosphere of hydrogen for 2 hours. The catalyst was removed by filtration on a Celite pad, and W O97/28183 PCT~B97/00221 the filtrate was concentrated under reduced pressure.
Yield: 0.111 g (94%); R~ (CH2C12/MeOH, 95/5) = 0.08; Rf (CH2C12/MeOH, 9/1) = 0.35. MS (FAB) m/z = 536 (MNa+), 514 (MH~), 480 (MNa+--But), 458 (MH+--But), 414 (MH+-Boc), 5 400(MH+-ProNH2).
(6) N-~-Benzyloxycarbonyl-L-seryl-~-O-tert-butyl-L-aspartyl-N-~-tert-Butoxycarbonyl-L-lysyl-L-proline amide To a stirred solution of Z-L-Ser-OH (0.045 g, 0.19 10 mmol) in THF (1 mL), cooled to -15QC, was added 0.021 mL
(O.19 mmol) N-Methylmorpholine followed by 0.026 mL (0.19 mmol) isobutylchlo~oformate. The solution was stirred at -15QC for 5 minutes and then cooled to -20QC. The product of step (5) (0.105 g, 0.2 mmol) was added in 15 solution in DMF (1 mL).
After 5 hours stirring, the reaction mixture was concentrated under reduced pressure. The reaction mixture was dissolved in ethyl acetate (50 mL) and 5%
citric acid (25 mL). The aqueous phase was extracted 20 with ethyl acetate (25 mL). The combined organic layers were washed with water and brine, then dried over Na2SO4, and concentrated, under reduced pressure. The crude product was purified on a silica gel column using CH2C12/MeOH (94/6) as an eluant. Yield: 0.140 g (80%).
Rf (CH2C12/MeOH, 95/5) = 0.18; Rf (CH2C12/MeOH, 9/1) --0.43. MS (FAB) m/z = 757 (MNa~), 735 (MH+), 701 (MNa+-But), 635 (MH+-Boc), 601 (MH+-Z), 579 (MH+-But-Boc), 501 (MH+-Boc-Z), 465 (MH+-Boc-But-ProNH2).
(O.19 mmol) N-Methylmorpholine followed by 0.026 mL (0.19 mmol) isobutylchlo~oformate. The solution was stirred at -15QC for 5 minutes and then cooled to -20QC. The product of step (5) (0.105 g, 0.2 mmol) was added in 15 solution in DMF (1 mL).
After 5 hours stirring, the reaction mixture was concentrated under reduced pressure. The reaction mixture was dissolved in ethyl acetate (50 mL) and 5%
citric acid (25 mL). The aqueous phase was extracted 20 with ethyl acetate (25 mL). The combined organic layers were washed with water and brine, then dried over Na2SO4, and concentrated, under reduced pressure. The crude product was purified on a silica gel column using CH2C12/MeOH (94/6) as an eluant. Yield: 0.140 g (80%).
Rf (CH2C12/MeOH, 95/5) = 0.18; Rf (CH2C12/MeOH, 9/1) --0.43. MS (FAB) m/z = 757 (MNa~), 735 (MH+), 701 (MNa+-But), 635 (MH+-Boc), 601 (MH+-Z), 579 (MH+-But-Boc), 501 (MH+-Boc-Z), 465 (MH+-Boc-But-ProNH2).
(7) N-~-acetyl-L-seryl-~-O-tert-butyl-L-aspartyl-N-~-tert-Butoxycarbonyl-L-lysyl-L-proline amide The product of step (6) (0.080 g, 0.011 mmol) was dissolved in AcOEt ( 2 mL). 10% palladium carbon catalyst (0.016 g) and acetylimidazole (0.014 g, 0.013 mmol) was added, and the suspension was stirred under an atmosphere CA 02244673 l998-07-22 WO 97/28183 PCTnB97/OV221 of hydrogen overnight. The catalyst was removed by filtration on a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was purified on a gel column using CH2Cl2/MeOH (9/1) as an 5 eluant. Yield: 0.050 g (71%~; Rf (CH2Cl2/MeOH, 9/1) =
0.29. MS (FAB) m/z = 665 (MNa+), 643 (MH+), 609 (MNa+-But), 543 (MH+-Boc), 487 (MH+-But-Boc), 373 (MH+-Boc-But-ProNH2 ) -(8) N-~-acetyl-L-seryl-L-aspartyl-L-lysyl-L-proline 10 amide The product of step (7) (0.039 g, 0.06 mmol) in solution in 200 ~l trifluoroacetic acid, containing 20 ~l of water, was stirred at room temperature for 95 minutes.
The reaction mixture was concentrated under reduced 15 pressure, and the residue was triturated twice with dry ether. After removal of ether, the solid white residue was taken up in 1.5 ml water and lyophilized. The crude peptide was purified by HPLC on C-18 column (Beckman Ultrasphere ODS (10 x 250 mm)) using an elution 20 consisting of two solvents (A: H20/0.1% TFA, B:
acetonitrile 0.1% TFA; 100% to over 20 minutes; tR = 13 minutes) with a flow rate of 3 ml.min-1. The collected fraction was lyophilized and analyzed by HPLC on a Waters Nova-Pak C-18 column (Waters, Milford, MA), 4 ~, 80 A
(3.9 x 150 mm) with two different elution programs using the same solvent system as above and a 1 ml.min-l flow rate. k = 8.4, 100 to 50% A over 50 min.; k = 7.1, 100 to 20% A over 40 min.; MS (FAB)m/z = 509 (MNa+), 487 (MH+) .
Other substitutions may similarly be added to the N-terminus of the peptide by similar methods known in the art. For example, N-~-(HOOCCH2CH2CO)-~-(O-t-But)-L-Ser-~-(O-t-But)-L-aspartyl-~(CH2NH)-N-~-(Boc)-L-lysyl-L-proline-OH may be synthesized by ;~i ng the amine of step W O 97/28183 PCT~B97/00221 (7) above dissolved in the minimum amount of CH2C12 with a solution of succinic anhydride dissolved in THF. The reaction is stirred at room temperature and then the mixture is evaporated under reduced pressure. The 5 residue is dissolved in AcoEt and washed with 5% citric acid, water, brine, and then dried over Na2SO4. The resulting compound may then be deprotected to yield the desired product.
Other peptides of the invention can be prepared in 10 an analogous manner by a person of ordinary skill in the art.
Biological activitY of AcSDKP analogues The activity of the compounds of the invention was evaluated by their ability to inhibit the in vitro entry 1~ into S-phase of murine primitive hematopoietic cells:
"HPP-CFC". In order to trigger the quiescent stem cells into cycle, normal murine bone marrow cells (5 x 106 cells/ml in Dulbecco's medium) were incubated with the same volume of either stimulatory medium (conditioned 20 medium of bone marrow cells obt~;~e~ from sublethally irradiated mice, 4.5 GY whole body X-1 irradiated upon dose), or with Dulbecco's medium as control. Test compounds were added at the beginning of the incubation at a final concentration of 2 x 10-9 M. Incubations were 25 performed in pair tubes at 37~C for 3 h. One hour before the end of the incubation, cells in S-phase were killed by adding cytosine arabinoside (Ara-C) at a final concentration of 25 ~g/ml in the first set of tubes.
Dulbecco's medium is added in the other tubes as control.
30 Incubation with Ara-C leads to the death of cells which have been triggered into S-phase. Therefore, cells which have been prevented to cycle by the action of analogues will be insensitive to the phase-specific toxicity of W O 97/28183 PCTnB97100221 Ara-C. Cells were washed twice prior to subsequent HPP-CFC assay.
HPP-CFC were studied using a bilayer semi-solid agar assay as described by Robinson, et al., Cell Prolif.
5 25:623-632, 1992. Two milliliters of Dulbecco's medium containing 20% horse serum, 10% conditioned medium from the WEHI 3B myelomonocytic leukaemic cell line (as a source o~ IL-3/multi-CSF), 10% conditioned medium from L929 fibroblast cell line (as a source of M-CSF/CSF1), 10 0.5% melted agar, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ~gtml streptomycin were ali~uoted into 55 mm diameter non-tissue culture grade plastic petri-dishes as the underlayer. Two milliliters of Dulbeccos's medium supplemented with 20~ horse serum, 0.3~ melted agar, 2 mM
15 L-glutamine, 100 U/ml penicillin, and 100 ~g/ml streptomycin contAining 4 x 104 bone marrow cells were then aliquoted over prepared underlayers. Quadruplicate cultures were incubated for 14 days at 37~C in a fully humidified atmosphere with 5% C02. Twelve hours before 20 the end of the culture, 1 ml of a colorless 1 mg/ml 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) solution in saline was added, allowing the st~; n; ng of viable cells by INT processing into a red derivative which precipitates inside cells. HPP-CFC
25 derived macroscopic colonies were defined as those above 2 mm in diameter and scored. Table I lists the percent decrease of ~PP-CFC derived macroscopic colonies entering the S-phase induced by the test compounds.
TABLE I
TEST ~K~ D~R~ OF
CO11~UN~ HPP-CFC IN S-PHASE
Analog 1 61.2 Analog 2 67.6 W O 97/28183 PCT~B97/00221 ¦ Analog 3 ¦ 70.8 Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed 5 description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the claims.
What is claimed is:
0.29. MS (FAB) m/z = 665 (MNa+), 643 (MH+), 609 (MNa+-But), 543 (MH+-Boc), 487 (MH+-But-Boc), 373 (MH+-Boc-But-ProNH2 ) -(8) N-~-acetyl-L-seryl-L-aspartyl-L-lysyl-L-proline 10 amide The product of step (7) (0.039 g, 0.06 mmol) in solution in 200 ~l trifluoroacetic acid, containing 20 ~l of water, was stirred at room temperature for 95 minutes.
The reaction mixture was concentrated under reduced 15 pressure, and the residue was triturated twice with dry ether. After removal of ether, the solid white residue was taken up in 1.5 ml water and lyophilized. The crude peptide was purified by HPLC on C-18 column (Beckman Ultrasphere ODS (10 x 250 mm)) using an elution 20 consisting of two solvents (A: H20/0.1% TFA, B:
acetonitrile 0.1% TFA; 100% to over 20 minutes; tR = 13 minutes) with a flow rate of 3 ml.min-1. The collected fraction was lyophilized and analyzed by HPLC on a Waters Nova-Pak C-18 column (Waters, Milford, MA), 4 ~, 80 A
(3.9 x 150 mm) with two different elution programs using the same solvent system as above and a 1 ml.min-l flow rate. k = 8.4, 100 to 50% A over 50 min.; k = 7.1, 100 to 20% A over 40 min.; MS (FAB)m/z = 509 (MNa+), 487 (MH+) .
Other substitutions may similarly be added to the N-terminus of the peptide by similar methods known in the art. For example, N-~-(HOOCCH2CH2CO)-~-(O-t-But)-L-Ser-~-(O-t-But)-L-aspartyl-~(CH2NH)-N-~-(Boc)-L-lysyl-L-proline-OH may be synthesized by ;~i ng the amine of step W O 97/28183 PCT~B97/00221 (7) above dissolved in the minimum amount of CH2C12 with a solution of succinic anhydride dissolved in THF. The reaction is stirred at room temperature and then the mixture is evaporated under reduced pressure. The 5 residue is dissolved in AcoEt and washed with 5% citric acid, water, brine, and then dried over Na2SO4. The resulting compound may then be deprotected to yield the desired product.
Other peptides of the invention can be prepared in 10 an analogous manner by a person of ordinary skill in the art.
Biological activitY of AcSDKP analogues The activity of the compounds of the invention was evaluated by their ability to inhibit the in vitro entry 1~ into S-phase of murine primitive hematopoietic cells:
"HPP-CFC". In order to trigger the quiescent stem cells into cycle, normal murine bone marrow cells (5 x 106 cells/ml in Dulbecco's medium) were incubated with the same volume of either stimulatory medium (conditioned 20 medium of bone marrow cells obt~;~e~ from sublethally irradiated mice, 4.5 GY whole body X-1 irradiated upon dose), or with Dulbecco's medium as control. Test compounds were added at the beginning of the incubation at a final concentration of 2 x 10-9 M. Incubations were 25 performed in pair tubes at 37~C for 3 h. One hour before the end of the incubation, cells in S-phase were killed by adding cytosine arabinoside (Ara-C) at a final concentration of 25 ~g/ml in the first set of tubes.
Dulbecco's medium is added in the other tubes as control.
30 Incubation with Ara-C leads to the death of cells which have been triggered into S-phase. Therefore, cells which have been prevented to cycle by the action of analogues will be insensitive to the phase-specific toxicity of W O 97/28183 PCTnB97100221 Ara-C. Cells were washed twice prior to subsequent HPP-CFC assay.
HPP-CFC were studied using a bilayer semi-solid agar assay as described by Robinson, et al., Cell Prolif.
5 25:623-632, 1992. Two milliliters of Dulbecco's medium containing 20% horse serum, 10% conditioned medium from the WEHI 3B myelomonocytic leukaemic cell line (as a source o~ IL-3/multi-CSF), 10% conditioned medium from L929 fibroblast cell line (as a source of M-CSF/CSF1), 10 0.5% melted agar, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ~gtml streptomycin were ali~uoted into 55 mm diameter non-tissue culture grade plastic petri-dishes as the underlayer. Two milliliters of Dulbeccos's medium supplemented with 20~ horse serum, 0.3~ melted agar, 2 mM
15 L-glutamine, 100 U/ml penicillin, and 100 ~g/ml streptomycin contAining 4 x 104 bone marrow cells were then aliquoted over prepared underlayers. Quadruplicate cultures were incubated for 14 days at 37~C in a fully humidified atmosphere with 5% C02. Twelve hours before 20 the end of the culture, 1 ml of a colorless 1 mg/ml 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) solution in saline was added, allowing the st~; n; ng of viable cells by INT processing into a red derivative which precipitates inside cells. HPP-CFC
25 derived macroscopic colonies were defined as those above 2 mm in diameter and scored. Table I lists the percent decrease of ~PP-CFC derived macroscopic colonies entering the S-phase induced by the test compounds.
TABLE I
TEST ~K~ D~R~ OF
CO11~UN~ HPP-CFC IN S-PHASE
Analog 1 61.2 Analog 2 67.6 W O 97/28183 PCT~B97/00221 ¦ Analog 3 ¦ 70.8 Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed 5 description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the claims.
What is claimed is:
Claims (20)
1. A compound of the formula:
wherein A1 is the identifying group of the D- or L-isomer of Ser;
A2 is the identifying group of the D- or L-isomer of Asp or Glu;
A3 is the identifying group of the D- or L-isomer of Lys, Arg, or Orn;
A4 is the D- or L- isomer of Pro;
R1 is H, C1-12 alkyl, C7-20 arylalkyl, R7CO, or R7OC(O), where R7 is C1-12 alkyl, C7-20 arylalkyl, or C1-12 alkyl or C7-20 arylalkyl substituted with OH, C02H, or NH2;
R2 is H, C1-12 alkyl, or C7-20 arylalkyl;
each of R3 and R4, independently, is CO-NH, CH2-NH, CH2-S, CH2-O, CO-CH2, CH2-CO, or CH2-CH2;
R5 is CO or CH2; and R6 is OH, NH2, C1-12 alkoxy, or NH-Y-CH2-Z, where Y is a C1-12 hydrocarbon moiety and Z is H, OH, CO2H, or CONH2; provided that if R6 is OH, R3 is CO-NH, and R4 is CO-NH, then R5 is CH2; or a pharmaceutically acceptable salt thereof.
wherein A1 is the identifying group of the D- or L-isomer of Ser;
A2 is the identifying group of the D- or L-isomer of Asp or Glu;
A3 is the identifying group of the D- or L-isomer of Lys, Arg, or Orn;
A4 is the D- or L- isomer of Pro;
R1 is H, C1-12 alkyl, C7-20 arylalkyl, R7CO, or R7OC(O), where R7 is C1-12 alkyl, C7-20 arylalkyl, or C1-12 alkyl or C7-20 arylalkyl substituted with OH, C02H, or NH2;
R2 is H, C1-12 alkyl, or C7-20 arylalkyl;
each of R3 and R4, independently, is CO-NH, CH2-NH, CH2-S, CH2-O, CO-CH2, CH2-CO, or CH2-CH2;
R5 is CO or CH2; and R6 is OH, NH2, C1-12 alkoxy, or NH-Y-CH2-Z, where Y is a C1-12 hydrocarbon moiety and Z is H, OH, CO2H, or CONH2; provided that if R6 is OH, R3 is CO-NH, and R4 is CO-NH, then R5 is CH2; or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1, wherein A2 is the identifying group of the D- or L-isomer of Asp and A3 is the identifying group of the D- or L-isomer of Lys.
3. A compound of claim 2, wherein A1 is the identifying group of L-Ser;
A2 is the identifying group of L-Asp;
A3 is the identifying group of L-Lys; and A4 is the identifying group of L-Pro.
A2 is the identifying group of L-Asp;
A3 is the identifying group of L-Lys; and A4 is the identifying group of L-Pro.
4. A compound of claim 3, wherein each of R3 and R4, independently, is CO-NH or CH2-NH.
5. A compound of claim 4, wherein R1 is H or R7CO
(where R7 is C1-12 alkyl or C1-12 substituted with OH) and R2 is H.
(where R7 is C1-12 alkyl or C1-12 substituted with OH) and R2 is H.
6. A compound of claim 5, further provided that if R3 is CO-NH and R4 is CO-NH, then R5 is CH2.
7. A compound of claim 6, wherein R1 is H, CH3CO, or HOOCCH2CH2CO and R6 is OH or NH2.
8. A compound of claim 1 of the formula:
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.
9. A compound of claim 1 of the formula:
CH3CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.
CH3CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.
10. A compound of claim 1 of the formula:
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-OH;or a pharmaceutically acceptable salt thereof.
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-OH;or a pharmaceutically acceptable salt thereof.
11. A compound of claim 1 of the formula:
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
CE3CO-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-NH2;
H-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
H-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
H-Ser-Asp-Lys-~(CH2N)-Pro-OH;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-OH;
H-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
H-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
H-Ser-Asp-Lys-~(CH2N)-Pro-NH2;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-NH2; and HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-NH2; or a pharmaceutically acceptable salt thereof.
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
CE3CO-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
CH3CO-Ser-Asp-Lys-~(CH2N)Pro-NH2;
H-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
H-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
H-Ser-Asp-Lys-~(CH2N)-Pro-OH;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-OH;
H-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
H-Ser-Asp-~(CH2NH)-Lys-Pro-NH2;
H-Ser-Asp-Lys-~(CH2N)-Pro-NH2;
HOOCCH2CH2CO-Ser-~(CH2NH)-Asp-Lys-Pro-NH2;
HOOCCH2CH2CO-Ser-Asp-~(CH2NH)-Lys-Pro-NH2; and HOOCCH2CH2CO-Ser-Asp-Lys-~(CH2N)Pro-NH2; or a pharmaceutically acceptable salt thereof.
12. A compound of claim 5, wherein R6 is NH2 or NH-Y-CH2-Z (where Y is a C1-12 hydrocarbon moiety and Z is H).
13. A comppound of claim 12, wherein R3 and R4 are CO-NH and R5 is CO.
14. A compound of caim 13, wherein R1 is H, CH3CO, or HOOCCH2CH2CO.
15. A compound of claim 14 of the formula:
CH3CO-Ser-Asp-Lys-Pro-NH2; or a pharmaceutically acceptable salt thereof.
CH3CO-Ser-Asp-Lys-Pro-NH2; or a pharmaceutically acceptable salt thereof.
16. A compound of claim 14, of the formula:
H-Ser-Asp-Lys-Pro-NH2;
CH3CO-Ser-Asp-Lys-Pro-NHCH3;
H-Ser-Asp-Lys-Pro-NHCH3;
HOOCCH2CH2CO-Ser-Asp-Lys-Pro-NHCH3; and HOOCCH2CH2CO-Ser-Asp-Lys-Pro-NH2; or a pharmaceutically acceptable salt thereof.
H-Ser-Asp-Lys-Pro-NH2;
CH3CO-Ser-Asp-Lys-Pro-NHCH3;
H-Ser-Asp-Lys-Pro-NHCH3;
HOOCCH2CH2CO-Ser-Asp-Lys-Pro-NHCH3; and HOOCCH2CH2CO-Ser-Asp-Lys-Pro-NH2; or a pharmaceutically acceptable salt thereof.
17. A method of protecting hematopoietic cells in a subject undergoing chemotherapy or radiotherapy, said method comprising administering to said subject a therapeutically effective amount of a compound of claim 1, said amount being effective to reduce the proliferation of hematopoietic cells during said chemotherapy or radiotherapy.
18. A method of claim 17, wherein said compound is of the formula:
CE3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
CH3CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
CH3CO-Ser-Asp-Lys-~(CH2NH)-Pro-OH;
CH3CO-Ser-Asp-Lys-Pro-NH2; or a pharmaceutically acceptable salt thereof.
CE3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH;
CH3CO-Ser-Asp-~(CH2NH)-Lys-Pro-OH;
CH3CO-Ser-Asp-Lys-~(CH2NH)-Pro-OH;
CH3CO-Ser-Asp-Lys-Pro-NH2; or a pharmaceutically acceptable salt thereof.
19. A method of inhibiting the proliferation of hematopoietic cells in a patient, said method comprising administering to said patient a compound of claim 1.
20. A method of claim 19, wherein said compound is of the formula:
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.
CH3CO-Ser-~(CH2NH)-Asp-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US59536196A | 1996-02-01 | 1996-02-01 | |
| US08/595,361 | 1996-02-01 | ||
| US75451196A | 1996-11-19 | 1996-11-19 | |
| US08/754,511 | 1996-11-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2244673A1 true CA2244673A1 (en) | 1997-08-07 |
Family
ID=27082249
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002244673A Abandoned CA2244673A1 (en) | 1996-02-01 | 1997-01-31 | Peptide inhibitors of hematopoietic cell proliferation |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0877753A1 (en) |
| JP (1) | JP2002515864A (en) |
| AU (1) | AU721261B2 (en) |
| CA (1) | CA2244673A1 (en) |
| IL (1) | IL125509A0 (en) |
| PL (1) | PL328081A1 (en) |
| WO (1) | WO1997028183A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2814076B1 (en) | 2000-09-21 | 2002-12-20 | Centre Nat Rech Scient | ANGIOGENIC AGENT AND USES THEREOF |
| FR2872040B1 (en) | 2004-06-23 | 2006-09-22 | Centre Nat Rech Scient Cnrse | COSMETIC USE OF AT LEAST ONE AC-N-SER-ASP-LYS-PRO NATURAL TETRAPEPTIC OR ONE OF ITS ANALOGUES AS AN ANTI-AGING AND RESTRUCTURING AGENT OF THE SKIN |
| FR2882256B1 (en) | 2005-02-18 | 2007-05-25 | Centre Nat Rech Scient | COSMETIC USE OF AT LEAST ONE AC-N-SER-ASP-LYS-PRO NATURAL TETRAPEPTIDE OR ONE OF ITS ANALOGUES AS AN AGENT TO SLOW DOWN THE FALL OF HAIR AND / OR STIMULATE THEIR GROWTH |
| KR101335203B1 (en) * | 2010-03-26 | 2013-11-29 | 숙명여자대학교산학협력단 | Peptides for Promotion of Angiogenesis and the use thereof |
| WO2014078787A1 (en) | 2012-11-19 | 2014-05-22 | Wellstat Therapeutics Corporation | Stem cell mobilization and tissue repair and regeneration |
| FR3120794B1 (en) | 2021-03-16 | 2023-11-24 | Rpm Dermatologie | Composition for injection comprising a peptide conjugate |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2601678B1 (en) * | 1986-07-18 | 1989-11-24 | Inst Nat Sante Rech Med | PEPTIDES COMPRISING THE SERYL-ASPARTYL-LYSYL-PROLYLE SEQUENCE, PROCESS FOR THE EXTRACTION OF THE CORRESPONDING TETRAPEPTIDE, AND APPLICATIONS, IN PARTICULAR FOR THE PROTECTION OF THE BONE MARROW DURING CHEMOTHERAPY ANTI-CANCER TREATMENTS |
| DE4224509C2 (en) * | 1992-07-24 | 1995-04-20 | Ruhenstroth Bauer Gerhard Prof | Active ingredient for inhibiting the proliferation rate of liver cells |
-
1997
- 1997-01-31 EP EP97903550A patent/EP0877753A1/en not_active Withdrawn
- 1997-01-31 JP JP52745097A patent/JP2002515864A/en active Pending
- 1997-01-31 CA CA002244673A patent/CA2244673A1/en not_active Abandoned
- 1997-01-31 PL PL97328081A patent/PL328081A1/en unknown
- 1997-01-31 AU AU18075/97A patent/AU721261B2/en not_active Ceased
- 1997-01-31 WO PCT/IB1997/000221 patent/WO1997028183A1/en not_active Application Discontinuation
- 1997-01-31 IL IL12550997A patent/IL125509A0/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0877753A1 (en) | 1998-11-18 |
| JP2002515864A (en) | 2002-05-28 |
| AU1807597A (en) | 1997-08-22 |
| WO1997028183A1 (en) | 1997-08-07 |
| IL125509A0 (en) | 1999-03-12 |
| AU721261B2 (en) | 2000-06-29 |
| PL328081A1 (en) | 1999-01-04 |
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Legal Events
| Date | Code | Title | Description |
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| FZDE | Discontinued |