Note: Descriptions are shown in the official language in which they were submitted.
CA 02244673 1998-07-22
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:
IA2 1A3
N--CH--R3--CH--R4--CH--R5--A4--R6
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;
CA 02244673 1998-07-22
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. 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;
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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
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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
CA 02244673 1998-07-22
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.
CA 02244673 1998-07-22
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
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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,
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. 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
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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
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- -- 10 --
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%).
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WO 97/28183 PCT~B97/00221
-- 11 --
(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
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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
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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
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W O97/28183 PCT~B97/00221
- 14 -
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).
(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
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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
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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
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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
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¦ 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:
