JPH02215711A - N-methyl-sphingosine derivative, protein kinase inhibitor,and stereoisomer thereof - Google Patents

Abstract

PURPOSE: To obtain a protein kinase activity inhibitor containing an N- methylsphingosine derivative. CONSTITUTION: This medicine having a strong inhibiting activity against protein kinases such as C-kinase, EGF-receptor kinase and SRC tumor gene kinase contains N-methyl-D-erythro-sphingenine or N-methyl-L-erythro-sphingenine. The medicine can also be applied as a growth inhibitor effective against tumor cells, especially applied to the inhibition of IL-2 dependent T-cell growth, and is useful for the therapy of autoimmune diseases. The medicine is prepared by suitably compounding the sphingosine derivative or its salt as the active ingredient with a conventional medicinal carrier, a conventional diluent, a conventional excipient, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to inhibitors of protein kinases, and more particularly to recent discoveries by the inventors showing that N-methyl-sphingosine is a strong inhibitor of protein kinases. . "N-Methyl-sphingosine" represents a D- or L-/or erythro- or threo configuration; N,N'-dimethyl-sphingosine or N-
means monomethylsphingosine. These compounds have the potential to be used as effective growth inhibitors against tumor cells.

BACKGROUND OF THE INVENTION Sphingosine and sphingoids are known inhibitors of tyrosine kinases involving C-kinase and EGF receptors. [Hanoun and Be/L/
(Hannun and Be1l), Science (S
science), 235.670-674, l 987
; Hannun et al., JBC, 261, 1
2604-12609.1986; Kreuter
utter) et al., JBC, 262, 1632-1
637.1987"l. However, in these studies, the sphingosine used was a commercially available product [Sigma Che+n1cal
y) ). Commercially available sphingosine is produced by metathurisis of sphingomyelin or cerebroside, so it contains various impurities generated by the process.
For example 3-0.5-0 methylsphingosine and N-
Contains methylsphingosine.

Furthermore, the plug structure of sphingosine, ie, the D-erythro configuration, may be partially converted to the D-)reo configuration.

Therefore, claims pointing to sphingosine as an important inhibitor of C-kinase and EGF receptor kinase remain unclear due to the heterogeneity of these structures present in commercially available sphingosine.

However, since sphingosine and sphingoid bases have strong proliferation-modifying activity and it is possible to apply these compounds to the regulation of cell proliferation, research in this area is still ongoing. It is actively practiced.

Therefore, it is important to establish, using structurally accurate sphingosine and sphingosine bases or their derivatives, that these compounds indeed have a strong effect on cell growth regulation in a variety of cells. This is highly desirable.

SUMMARY OF THE INVENTION One object of the present invention is to provide information regarding the growth-modifying activity of sterically defined sphingosine and sphingoid bases and/or their derivatives synthesized via stereoselective routes. be.

Yet another object of the present invention is to provide in vitro application of derivatives of these compounds with strong growth inhibitory activity against a wide range of cells.

The above object of the present invention was achieved by measuring the degree of inhibition of protein kinase activity in the presence of N-methyl-D-losefingenin or N-methyl-L-erythro-sfingenin.

The present invention also provides agents for inhibiting the proliferation of human and animal cells, which include (1) N-methyl-D-erythro-sphingenin, N-methyl-L-erythro-sphingenin, and pharmaceutically acceptable pharmaceutically thereof. and (2) a pharmaceutically acceptable carrier, diluent or excipient. It is.

The present invention further provides a method for inhibiting the proliferation of human and animal cells in vivo, comprising:
contacting with a cell growth-inhibiting amount of one or more growth inhibitors selected from the group consisting of disolidosfingenin N-methyl-L-erythro-sfingenin and pharmaceutically acceptable salts thereof. Provide a method for including.

The most preferred substance as an inhibitor of protein kinase activity and as a cell growth inhibitor is N-methyl-D-di'losefingenin.

As the protein kinase, C-kinase, EGF kinase or SRC@tumor gene kinase is preferred.

DETAILED DESCRIPTION OF THE INVENTION To show that sphingosine has inhibitory activity against protein kinases such as C-kinase, EGF receptor kinase and 5RC1li tumor gene kinase,
As shown in FIG. 1, the present inventors investigated the protein kinase inhibitory activity of these compounds by using D- and L-erythropoietin and threosphingosine and their N-dimethyl derivatives obtained by precision chemical synthesis. was evaluated.

A comparison of the effects of sphingosine and its derivatives with clear stereochemistry on C-kinase activity obtained from A413 cells (human epidermal carcinoma cells) is shown in Figure 2, right and Figure 3, as well as Tables ■ and Table ■. (See Example 2)
.

The following conclusions can be drawn: (1) D-erythro and L-erythro-sphingenin showed comparable weak inhibitory activity.

(2) N-methyl-〇-disolidrosefingenin showed strong inhibitory activity against C-kinase. N
-Methyl-L-erythro-sphingenin showed a weaker, yet stronger inhibitory activity than unsubstituted sphingenin with a free amino group.

(3) The inhibitory activity of N-methyl-D-erythro-sphingenin was higher than gangliosides such as N-acetyl-GM, , N-glycolyl-GM, . It showed inhibitory activity comparable to that of the inhibitory ganglioside GT.

(4) From this, it is expected that N-methyl-D-erythro-sphingenin and N-methyl-L-erythro-sphingenin exhibit similar activities against EGF receptor kinase.

The inventor also discovered that a commercially available product [Sigma Chemical Co. (Si)] containing N-methyl-sphingosine as an impurity
found that sphingosine from GMA Chemical Co.) inhibited SRC oncogene kinase activity. However, SRC oncogene kinase activity was not inhibited by synthetic (+) erythrosphingenin or L (−) erythrosphingenin. Subsequently, the inventors determined that N-methyl-
I was certain that it was a sphingosine.

As a result of these discoveries by the inventors, the present invention provides N-methyl-D-erythro-sphingenin or N-methyl-
Protein kinase containing L-erythro-sphingenin
The present invention provides inhibitors of enzyme activity.

The protein kinase inhibitor is preferably N-methyl-D-erythrosfingenin, and the kinase is preferably C-kinase, EGF receptor kinase or SRC oncogene kinase.

N-methyl-D-erythro-sphingenin and N-
Methyl-L-erythro-sphingenin was synthesized as D(+)erythro-sphingenin and L(-) in the presence of formaldehyde and sodium borohydride, respectively.
It can be produced by known methods including reductive methylation of erythro-sphingenin [Means and Feeney (M
eans and feeney), Biochemistry, 2192.196
8; Chemical modification of proteins - (Chemical Mo
dification of Proteins)
, Holden Fist Day Company () Io! den-Day,
Inc., San Francisco), p.
217.1971]. The produced N,N-dimethyl-sphingosine derivative and N-methylsphingosine derivative can be separated by thin layer chromatography. D(+)erythro-sphingenin and L
(-)Erythro-sphingenin can also be produced by known methods [Koike, K.
), as well as Carbohydrate Research (Carb
ohydrate Research).

158.113-123.1986; Koik
E, K) and others, efficient synthesis of ceramide from D-glucose, Glycoconjugate Journal (Gl
ycoconjugate Journal), ■,
107-109.1984). The structure of the N-methyl derivative can be confirmed by NMR and FAB mass measurements.

in vitro or in v
Concentrations suitable for inhibiting protein kinase activity in vitro) are shown in Figure 2 and Table 1, for example.
o data (see Example 2).

Furthermore, as a result of the discovery of the inhibitory effects of N-methyl-D-erythro-sphingenin and N-methyl-L-erythro-sphingenin on protein kinase activity, the present invention provides the ability to inhibit the proliferation of human and animal cells. a drug selected from the group consisting of (1) N-methyl-D-erythrosfingenin, N-methyl-d-erythrosfingenin, and pharmaceutically acceptable salts thereof; A medicament is provided that includes a cell growth inhibiting amount of one or more growth inhibitors; and (2) a pharmaceutically acceptable carrier, diluent or excipient.

Similarly, the present invention also provides a method for inhibiting the proliferation of human and animal cells in vivo, comprising:
- erythro-sphingenin and a pharmaceutically acceptable salt thereof.

For both the drug and the method, N-methyl-0-1000 glydrosfingenin is preferred.

N-methyl-D-erythro-sphingenin and N-
Suitable pharmaceutically acceptable salts of methyl-L-erythro-sphingenin can be readily determined by those skilled in the art.

Said agents and methods for inhibiting the proliferation of human and animal cells are particularly applicable to the treatment of mammalian cells, in particular to malignant or non-malignant tumor cells for preventing or retarding tumor growth and for preventing or retarding tumor metastasis. Applicable. They are also particularly applicable to inhibiting IL-2 dependent T cell proliferation. Based on these results, it can be expected that these substances will be useful in the treatment of autoimmune diseases.

It is also possible to use appropriate pharmaceutically acceptable carriers, diluents, excipients, etc. depending on the respective medical use of the drug of the present invention.

The method of administering the drug of the present invention can be easily determined by those skilled in the art depending on the individual medical use.

Appropriate doses of the agents of the invention will depend on the particular medical application and the subject's weight, sex, etc., and can be readily determined by a disintegrator, for example, by supplementing in vitro data.

EXAMPLES The present invention will now be described with respect to specific examples, but the invention is not limited thereto.

Unless otherwise indicated, all percentages and ratios are based on weight.

Example 1 Synthesis of sphingosine derivatives (+) Erythrosphingenin, L (-) Erythrosphingenin, L (-) Threosphingenine, L (-)) Rheosphingenine were synthesized by the method described in the literature. Synthesis [Koike, K
), as well as Carbohydrate Research (Carb
158.11
3~123.1986; Koike 0 (oike, K)
In addition, efficient synthesis of ceramide from D-glucose,
Glycocon Journal
jugate Journal) 1.107~1
09.1984). The N-methyl derivative is erythroxylated in the presence of formaldehyde and sodium borohydride.
[Means & Feen] produced by reductive methylation of sphingenin
ey), Biochemist
ry87.2192.196; Chemical Modification of Proteins
PProtein), Holde Y-Day
n-Day, Inc. San Francisco)
, p. 217.1971).

Crude sphingosine was first produced by Ziehlfelder and
Tierfelder and Klenk
) [Chemistry of Cerebrosides and Phosphatides (DieC
hem'ie de Cerebrosida and
Phosphatide), Springer Verlag.

It was prepared by metathurisis of seroprossite according to the method described in (Berlin, 1932).

The N-dimethylsphingosine derivative was confirmed by NMR spectroscopy and fast atom bombardment mass spectroscopy.

Example 2 A43 by sphingosine and sphingosine derivatives
1 cell in DM supplemented with 10% fetal calf serum (FC3)
The cells were cultured in a mixed medium of E. and Ham's F-121:1 (weight ratio). Cells collected from petri dishes with a diameter of 50 to 150 mm were collected using a Kreutter
et al. (JBC, 262, 1633-1637.198
7) was prepared for partial purification of C-kinase. To describe it more clearly, cells were scraped off with a rubber policeman and treated with 20mM Tris-HCl (
pH7.5), 2mM-EDTA, 0.5mM-EG
TA, 0.15U/mj! Aprotinin and 0.25
Suspend in 50-mL of sucrose, homogenizer (Dounce) C40ml size, Wheat 7 (W)
Heaton) at 4°C 50 times to homogenize. The homogenized cells were ultracentrifuged at 100,000×g for 60 minutes.

The supernatant was diluted with 20mM) Lis-HCl! (pH 7,5), DE equilibrated with 2mM-E'DTA and 0.5mM-EGTA (buffer B).
52 column and washed thoroughly with this buffer.

The C-kinase active fraction was eluted with buffer B containing 0.1 M NaCi. The activity in both parts is 200-50
The quality was 0 pmol P/min 7 mg protein.

Fractions free of A-kinase and other kinases were separated and stored at -80°C.

Determination of the inhibitory effect of sphingosine and sphingosine derivatives on C-kinase Bell and Hannun
(Science, 235.670
~674.1987: The results obtained in mixed micelle systems as described by I Kletter et al. CJ BC,:jj) are highly variable.
112605979-5986.1985), and the effects of sphingosine and sphingosine derivatives (non-glycosylated and glycosylated) were studied under these conditions.

Phosphatidylserine (
5 μg/tube) and 1,2-diolein (0,0
5 μg/tinib) with or without an appropriate amount of sphingosine, its derivatives or sulfatide in an organic solvent (ethanol or chloroform-methanol).
The mixture was evaporated under a stream of N2. Lipid mixture 2QrnM)'JSuHCj7 (pH 7,5) 30
Sonicate for 30 minutes in μβ.

25mM) Lis-HCβ (p
H7,5), 10mM MgCj22.400! j
M-EDTA, 50μM-EGTA, 500μM-Ca
Cj'2.20 O11g7ml Histo
n) I[[-8 and 20 μM gamma [”Pl
The reaction mixture was supplemented with -ATP (2X 10'cpm); the final volume was 90[mu][beta].

The reaction was carried out using 10 μn of the C-kinase fraction prepared as described above.
(containing 1-2 μg of protein) and the reaction mixture was incubated at 30° C. for 10 minutes. The reaction was carried out in 25% TCA l- and 1mM ATP solution (p
It was terminated by the addition of 200 μβ of 1% B5A in H7,5). The precipitate was centrifuged, washed twice with 1 ml' of 25% TCA, and washed with 1 ml' of 1N-Na containing 0.1% deoxycholate.
Dissolved in OH with some heating (80° C., 10 minutes) and counted in a scintillation counter. Values without phosphatidylserine, 1,2-diolein or Ca'+ were used as controls. The effects of sphingosine and sphingosine drama conductor are shown in Figures 2 and 3 and Table 1
and are summarized in Table ■.

Table ■ Sphingosine and various sphingosynths Mid No inhibition Sphingosine (D-Eli) Toro, Sigma SM) sphingosine sphingosine sphingenin sphingenin (0-erythro) (Shi-Eritro) (L-Threo) (Shi-Threo) O N-acetylsphingosine No inhibition Lactosylsphingosine GM, (N-acetyl) GM3 (N-glycolyl) G.D.

GT.

No inhibition From the data shown in Tables 1 and 2 and the results in Figures 2 and 3, the following conclusions were drawn.

(1) D-erythro and L-erythro-sphingenin showed comparable weak inhibitory activity.

(2) N-methyl-D-erythro-sphingenin showed extremely strong inhibitory activity against C-kinase.

Although the inhibitory activity of N-methyl-L-erythro-sphingenin was weak, it still had stronger inhibitory activity than unsubstituted sphingenin, which has a free amide 7 group.

(3) The inhibitory activity of N-methyl-D-erythro-sphingenin is higher than N-acetyl-GM, N-glycolylGM3 and GD, and is comparable to ganglioside GT, which has the strongest inhibitory activity. Met.

Although the invention has been described in detail and with respect to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

[Brief explanation of the drawing]

FIG. 1 shows the structure of sphingosine and various sphingosine derivatives. FIG. 2 is a graph showing the inhibitory effect of sphingosine and various sphingosine derivatives on the activity of C-kinase. The horizontal axis indicates the concentration of sphingosine, sphingosine derivative, or sulfatide in μM. The vertical axis is C-
Relative activity (%) of kinases is shown. Open: N-methyl-D-erythrose sfingenin (N-Me-D-erythro); Open: N-methyl-L-erythrose sfingenin (N-Me-L-erythro): Closed square; L-erythro Figgenin (L-erythro); open square: D-erythro-sphingenin (D-erythro); open triangle: sulfatide. FIG. 3 is a graph showing the inhibitory effects of gangliosides and sulfatides on C-kinase activity. The horizontal axis shows the concentration of ganglioside or sulfatide in μM. The vertical axis shows the relative activity (%) of C-kinase. Open use: G T +b; Kaida: GM3; open triangle; sulfitide. Glycolipids are Svennerform (Svennerh)
olm) system [Svennerh olm]
Olm, L.), Journal of Neurochemistry (J, Neurochem,), 10.613~
623.1963]. Figure 2 μM

Claims (17)

[Claims] (1) An inhibitor of protein kinase activity containing N-methyl-D-erythro-sphingenin or N-methyl-L-erythro-sphingenin. (2) The inhibitor according to claim (1), which contains N-methyl-D-erythro-sphingenin. (3) The inhibitor according to claim (1) or (2), wherein the protein kinase is selected from the group consisting of C-kinase, EGF-receptor kinase and SRC oncogene kinase. (4) A drug that inhibits the proliferation of human and animal cells, and (i) N-methyl-D-erythrose sfingenin refers to N-methyl-L-erythrose sfingenin and its pharmaceutical 1 selected from the group consisting of acceptable salts
and (ii) a pharmaceutically acceptable carrier, diluent or excipient. (5) the one or more growth inhibitors are N-methyl-
The drug according to claim (4), comprising D-erythrosfingenin or a pharmaceutically acceptable salt thereof. (6) The drug according to claim (4) or (5), wherein the cells are mammalian cells. (7) The drug according to claim (4) or (5), wherein the cells are malignant or non-malignant tumor cells. (8) Claims that the drug prevents or slows tumor growth (
4) or the drug described in (5). (9) The drug according to claim (4) or (5), wherein the drug prevents or delays tumor metastasis. (10) The drug according to claim (4) or (5), wherein the drug treats an autoimmune disease by inhibiting IL-2-dependent T cell proliferation. (11) A method for inhibiting the proliferation of human and animal cells in vivo, the cells being treated with N-methyl-D-erythrosfingenin, N-methyl-L-erythrosfingenin and their pharmaceutical agents. 1. A method comprising contacting a cell with a growth inhibiting amount of one or more growth inhibitors selected from the group consisting of publicly acceptable salts. (12) The method according to claim (11), wherein the one or more growth inhibitors comprises N-methyl-D-erythrosfingenin or a pharmaceutically acceptable salt thereof. (13) The method according to claim (11) or (12), wherein the cell is a mammalian cell. (14) The method according to claim (11) or (12), wherein the cells are malignant or non-malignant tumor cells. (15) The method of claim (11) or (12), wherein the method prevents or slows tumor growth. (16) The method of claim (11) or (12), wherein the method prevents or delays tumor metastasis. (17) Claim (11) or (12) wherein the method treats an autoimmune disease by inhibiting IL-2 dependent T cell proliferation.
) method described.