DE2908032A1 - ACETALS AND HEMIACETALS OF AMINOALDEHYDES AND THERAPEUTIC PREPARATIONS CONTAINING THEM - Google Patents

Description

description

The invention relates to the inhibition of cell proliferation, i.e. cell proliferation, and particularly, but not exclusively the inhibition of lymphocyte mitosis. Among other things, she poses a method of inhibiting cell proliferation in vitro and in vivo by administering an inhibitory amount of certain Aminoaldehyde derivatives are available to the cells and includes therapeutic preparations that use these derivatives as Contain active ingredients, as well as the derivatives as such, insofar as these are new.

Animal growth and life depend, among other things, on cell proliferation, which usually takes place in an orderly manner. The mechanism that controls cell proliferation is not clear, but it is normally found in living tissues homeostatic balance between cell proliferation and cell death. The condition commonly referred to as "cancer" is that Result of a homeostatic imbalance. It can be seen that the inhibition of cell proliferation for cancer therapy matters.

The majority of animal cells are "diploid" cells that are multiply by dividing into two daughter cells that have the same chromosomes as the mother cell from which they originated

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are. This division is known as "mitosis". Diploid cells have a cyclic growth pattern with four recognizable Phases, namely the:

G1 phase - general growth S phase - nucleic acid synthesis G2 phase - preparation for mitosis M phase - mitosis

Lymphocytes generally do not mitosis in vitro, so that it is common practice in research to reduce mitosis in vitro by adding a stimulant ("mitogen") to the culture, in which the cells grow, to stimulate. A common mitogen is phytohaemagglutinin, hereinafter referred to as "PHA" is abbreviated. One way of determining the amount of mitosis is by determining the amount of radioactive labeled substance which is required for nucleic acid synthesis and which is taken up by the cell during the S phase. as such substance is commonly used tritiated thymidine, often referred to by the abbreviation HTdR. This abbreviation is also used below. It will be appreciated that when tritiated thymidine is used, any identified inhibitory effect occurred in the G1 or S phase, so that no conclusions can be drawn about changes in the G2 and M-phase let go if such changes occur.

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Lymphocytes and granulocytes represent diploid leukocytes, i. white blood cells, the main purpose of which is to fight infections by reinforcing what is known as the "immune response" to There is foreign matter in the body. The cancer disease leukemia results from the proliferation of undeveloped leukocytes. Developed and thus functioning leukocytes are responsible for the rejection of foreign tissue from the body after tissue and Organ transplants responsible, e.g. heart or kidney transplants. It follows that the inhibition of leukocyte proliferation is important for the therapy of leukemia and tissue and organ transplants.

There is evidence that putrescine and its polyamine derivatives Spermidine and spermine and the enzymes involved in their biosynthesis are important participants in cell proliferation see e.g. Cohen S.S. (1971) Introduction to the Polyamines, Prentice Hall, New Jersey and Bachrach U. (1973) Function of the Naturally Occurring Polyamines, Academic Press, New York. The advancement of the diploid cells from one dormant to a proliferating state is accompanied by a change in the polyamine content of the cell. The reaction of putrescine with sadenosylmethionine to form Spermidine and spermine are maximal in the middle G1 phase and in the G2-M phase of the cell cycle. The enzyme S-adenosylmethionine decarboxylase, that is required for polyamine biosynthesis,

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is inhibited by methylglyoxal-bis- (guanylhydrazone), however protein synthesis is not affected by this inhibition. An optimal lymphocyte conversion is with increased endogenous polyamine levels, and exposure to either methylglyoxal-bis- (guanylhydrazone) or c ^ .- methylornithine (an inhibitor of putrescine synthesis) decreases theirs Reversibility. This inhibition can be achieved by the addition of exogenous polyamine. It follows that spermidine and spermine participate in the conversion of lymphocytes and thus involved in lymphocyte proliferation.

The in vitro reaction between spermidine or spermine and sera from ruminants has been shown to be highly suppressive the cell proliferation, see e.g. Älarcon and coworkers (1961) Arch. Biochem. Biophys., Volume 94, pages 540 to 541. Initially it was assumed that this phenomenon was due to the formation of a cytotoxic reaction product, how acrolein, i.e. acrylaldehyde, is due. However, evidence of this was found later, see e.g. Tabor and coworkers (1964), J. Biol. Chem. Vol. 239, pp. 2194 to 2203 that polyamine oxidase in the sera of ruminants causes oxidative deamination of spermidine or spermine according to the following scheme to the corresponding aldehydes:

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_ _1o _ 2308032

(CH ₂ J ₄ NH ₂ + O ₂ + H ₂ O

+ NH ₃ +

J ₃ NH ₂ + 2O ₂ + OHC (CH ₂ J ₂ NH (CH ₂ J ₄ NH (Ch ₂ J ₂ CHO + 2NH ₃ +

These aldehydes are commonly referred to in the literature as "oxidized spermidine" and "oxidized spermine". These Designations are also used below. The above-mentioned polyamine oxidase in sera from ruminants does not lead to an oxidative deamination of other biogenic polyamines, such as putrescine and cadaverine, with the formation of corresponding ones Aldehyde and is not found in effective amounts in human sera.

The role of oxidized spermidine and oxidized spermine in cell proliferation has recently been disputed, See Byrd et al., Nature, 1977, vol. 267, p 621, and it is believed that a factor other than polyamine oxidase contributes to the deterioration of these polyamines in the Cell proliferation is involved. Byrd and coworkers showed that spermidine and spermine stimulated the mitosis of PHA Strongly inhibit lymphocytes in vitro in the presence of fetal calf serum in the culture medium, but that in their absence no such inhibitory effect occurs in this serum or in the presence of human serum.

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Oxidized spermidine and / or oxidized spermine have been reported to have the following effects:

(a) Both have antiviral effects (Kremzner and colleagues, Biochem. Pharmacol. 1970, Volume 19 (9), pages 2541-50); '

(b) both inhibit nucleic acid and protein synthesis in Escherichia coli (Kimes and coworkers, Biochem. Biophys. Acta 1971, volume 228 (1), pages 235-44);

(c) both inactivate myxoviruses (Bachrach et al., J. Gen. Virol. 1971, Volume 11 (1), pages 1 to 9);

(d) oxidized spermine has a phagocidal effect (Fukami and Staff, Biochem. Biophys. Res. Commun. 1967, volume 28 (1), pages 19-24 and Nishimura et al., Ägric. Biol. Chem. 1975, vol. 39 (8), pages 1663-6); and

(e) oxidized spermine has a spermicidal effect (Tabor and coworkers, J. Pharmacol. Exp. Ther. 1956, Volume 116, Page 139).

Fukami and co-workers describe a process for the production of oxidized spermine (N, N'-bis- (2-formylethyl) -butane-1,4-diamine) and a homologue (Ν, Ν'-bis- (3-formylpropyl) -butane-1,4-diamine) on one of the corresponding acetals

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closing route (N, N'-bis- (3,3-diethoxypropyl) -butane-1,4-diamine and Ν, Ν'-bis (4,4-diethoxybutyl) -butane-1,4-diamine). The acetals are subjected to acid hydrolysis with an excess of aqueous oxalic acid, whereupon the desired Aminoaldehyde is obtained. The acetals are made by reducing the corresponding succinamide derivative with lithium aluminum hydride made in tetrahydrofuran. This preparation is disclosed in Japanese Patent Publication No. 7124365 (Takeda Chemical Industries Limited). The Japanese patent relates generally to manufacture of acetals of the formula:

R ¹ O (R ² O) CH (CH ₀ ) NH (CH ₀ ) NH (CH ₀ ) CH (OR ² ) OR ¹ , 2. η ζ m 2 η

1 2
in which R and R independently of one another represent lower alkyl groups or together represent a C1- or C1-alkylene group, η = 2 or 3 and m is an integer from 3 to 6. According to the Japanese patent, the acetals are new and useful intermediates. Neither Fukami et al. Nor the Japanese patent suggest or suggest that the acetals have any pharmacological activity.

Israel et al. (J. Med. Chem. 1973, Volume 16 (1), pp 1 to 5) describe the production of oxidized spermine (4,9-diazadodecanedialdehyde) and a homologue (4,14-diaza-

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heptadecandialdehyde) and their evaluation in the inhibition of Tumor growth in vivo. It was assumed that these dialdehydes were cytotoxic metabolites of spermine and an experimental one Antitumor agent, N, N'-bis (3-aminopropyl) -nonane-1,9-diamine are. In vivo, however, they were found to be ineffective against L1210 and P1534 leukemia at non-toxic doses Mice. Both dialdehydes inhibited the growth of human epidermal carcinoma KB cells in vitro in the absence or in Presence of calf serum. They were obtained by acid hydrolysis of the corresponding diethylacetals (N, N'-bis- (3,3-diethoxypropyl) -butane-1,4-diamine and N, N'-bis- (3,3-diethoxypropyl) -nonane-1,9-diamine) manufactured. The acid hydrolysis was carried out with aqueous oxalic acid to form the oxalate salts of the dialdehydes, which were then treated with barium chloride to form the hydrochloride salts. The purification of the oxidized Spermine hydrochloride gave a product containing 2-4% of the corresponding diethyl acetal or ethyl hemiacetal. It is stated, however, that this contamination did not significantly affect the results obtained with the following biological studies with the oxidized sperm salt were obtained. Israel and its staff do not give anything pharmacological effect of the acetals, and the reference to the acetal contamination in the oxidized spermine and the specified Toxicity of both dialdehydes clearly leads away from such a mode of action.

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From this prior art it can be seen that the inhibitory effect of spermidine and spermine on cell proliferation for at least 1961 was known, also that it has been known since at least 1964 that their aminoaldehyde derivatives participate in this inhibitory effect are. In addition, it was known since at least 1967 that aminoaldehydes have pharmacological activity and im In the case of oxidized spermine, they can be obtained synthetically via the corresponding diethylacetal. In addition, in the past 15 years looking for substances that inhibit cell proliferation in order to make progress in the Cancer therapy, tissue transplantation, organ transplantation and to achieve contraception, enormous expenditures in capital and labor are expended. However, it is not known that attempts have hitherto been made to reduce cell proliferation by using stabilized derivatives of the amino aldehydes mentioned to inhibit. Surprisingly, it has been found that acetal derivatives of these amino aldehydes are relatively low Cytotoxicity have a significant inhibitory effect and are likely to be used clinically in the treatment of, among other things, leukemia, tissue rejection after skin grafts and organ transplants and can be used for contraception. However, more research will still be needed before these derivatives can be said to be effective and safe for use in humans. Nevertheless, with the Invention an unexpected and significant technical advance in the field of inhibiting cell proliferation achieved.

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In particular, it has been found that the acetals of oxidized Spermine, oxidized spermidine and certain analogues and homologues of these compounds are persistent and mitosis inhibit at least the following cells in vitro and in the absence of ruminant sera

a) spontaneously dividing lymphocytes,

b) PHA-stimulated lymphocytes,

c) J 111 leukemia cells (presumably originally Were fibroblasts),

d) granulocytes and

e) Burkitt lymphoma cells (malignant lymphocytes).

According to one embodiment of the invention, the cell proliferation inhibited by treating the cells with an inhibitory effective amount of a hydrolyzable acetal or hemiacetal an aminoaldehyde of formula 1 added:

R (NHA ¹ ) _r NHA ² CHO (1)

3 12

in which R denotes hydrogen or -A CHO, where A , A and A independently of one another represent divalent alkylene groups, including cycloalkylene groups, and r = O or an integer.

The term "hydrolyzable" as used herein means that the acetal is in the cells or in the vicinity of the cells

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with formation of the corresponding aminoaldehyde of formula 1 is hydrolyzed.

In addition to the use of hydrolyzable acetals and hemiacetals of amino aldehydes of the general formula 1, if appropriate together with pharmacologically suitable carriers or extenders, for the inhibition of cell proliferatxone in humans and animals, the invention relates to hydrolyzable acetals and hemiacetals of amino aldehydes of the general formula 1, which are new as such.

12 3

A, A and A are preferably, independently of one another, acyclic alkylene groups having 2 to 12 carbon atoms and especially 2 to 6 carbon atoms. Furthermore, r is preferably 0 or an integer from 1 to 3, in particular The acetals of amino aldehydes with the general formula 2 are preferred

R ¹ / NH (CH ₂ ) _n _7 _r NH (CH ₂ ) _m CHO (2)

in which R is hydrogen or - (CH ₂ ) CHO, m, η and ρ independently of one another an integer from 2 to 12 and in particular from 2 to 6 and r = 0 or an integer from 1 to 3, in particular 0 or 1 mean.

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The most preferred compounds are the acetals of amino aldehydes of formula 2, in which r = 1, η = 2 to 6, m = 2 and ρ is preferably also 2.

12 3

The alkylene groups A, A and A can be independent of one another Represent or include cycloalkylene groups, e.g., those having from 5 to 7 carbon atoms. These alkylene groups appropriately have the structural formula

(CH ₉ ) - (3)

in which q = 1, 2 or 3 and in particular 2 and t = O or one is an integer from 1 to 3, in particular 0, 1 or 2.

Above all, acetals of the general formula are preferred

R ² (NHA ¹ ) NHA ² CHC ^ (4)

OR

^r \ 4

OR

2 3 3 -r- ^0R ι

in which R is hydrogen, -A CHO or -A CHC ^. represents, A,

2 3 ^0R

A, A and r are those given above for general formula 1

3 4
Have meanings and R and R, independently of one another, denote alkyl or alkeny groups having 1 to 6 and in particular 1 to 4 carbon atoms which are optionally substituted by hydroxyl groups, alkoxy groups or halogen atoms, or

3 4

R and R together form an alkylene group with 2 to 3 carbon

3 4 atoms form. Examples of R and R groups are the methyl,

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the η- and i-propyl, propenyl and preferably the ethyl

3 4 group. An example of combined R and R groups is the ethylene group. If there are acetal groups at both ends of the molecule, these can be the same or different. Particularly preferred acetals have the general formula 5: _QR 3

R ⁵ / NH (CH _O ) / NH (CH ₀ ) CH. (5)

^u 2. n- r 2 m \ ^ .

OR -.

^is (CH ₀₎ CH ^ T λ "- where R is hydrogen, - (CH ₀₎ CHO or

^{A P Δ P} OR

n, m, ρ and r are the values given above for general formula 2

3 4
NEN meanings and R and R have the meanings given above for the general formula 4.

Most preferred are the acetals of the general formula 6

^{And 7} OC ₂ H ₅

H ₂ N (CH ₂ ) _n NH (CH ₂ ) ₂ CH ^ (6)

OC ₂ H ₅

C _n H ₁ -O. OC ₀ H ₁ -

2 5 N. s e.g.

CH (CH ₂ ) ₂ NH (CH ₂ ) _n NH (CH ₂ J ₂ CII ^ (7)

C ₂ H ₅ O OC ₂ H ₅

used. Other acetals can also be used, e.g., aryl acetals in which R and / or R in general Formula represent 4 or 5 aryl groups such as the phenyl group;

3 4
Aralkylacetals in which R or R in the general formula 4 or 5 denotes an aralky group, for example the benzy group;

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- ι y -

3

and substituted acetals in which R or R in the general formula has 4 or 5 substituted alkyl, aryl or aralkyl groups represent, e.g. hydroxyalkyl, aminoalkyl, alkoxyalkyl and chloroalkyl groups. The acetal can be a hemiacetal

3

where one of the substituents R and R in the general formula 4 or 5 is hydrogen; a thioacetal by one or both oxygen atoms of the acetal group through sulfur are replaced; or a cyclic amino acetal by adding one of the oxygen atoms of the acetal group to a secondary amino group which is connected to the remaining oxygen atom via an alkylene group, e.g. the ethylene group is.

Examples of compounds of general formula 1 that are prepared and inhibited cell proliferation in in vitro tests

H N CCH) NH (CH) CH (OC H) 2 24 22

H N (CH) NH (CH) CH (OC H) 2 22 2 2

H N (CH) NH (CH) CH (OC II) 2 25 22

H N (CH) NH (CH) CH (OC H) 2 26 22

II N (CH) NH (CH) CH (OCH CH OCII) 2 24 22 2232

0- CH

HN (CH) NH (CH) CH ' j 2 2 6 2 2 Ο — CH ·

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- to -

(C II O) CH (CH) NII (CH) NH (CH) CH (OC II) 252 22 24 22 252

CH - O O - CH

2 \ / I 2

.CII (CH) NH (CH) NH (CH) CH. '

CH- (Τ 2 2 2 4 2 2 ^X O-CH

2 2

(C II O) CH (CH) NH (CH) NH (CII) CH (OC H) 252 22 22 2 2. 252

As mentioned above, certain acetals of general formula 5 are disclosed in Japanese Patent Publication No. 7124365 to Fukami and employees as well as Israel and employees. Also in the Organic and Biochemicals Catalog 1975 to 19 76 der Aldrich Chemical Co. Ltd., Gillingham, Dorset, England, compounds of the general formula 5 are given as intermediate products, in which R is hydrogen, r = 0, m = 2 or 4 and

3 4
R and R both represent an ethyl group. It is believed, however, that the remaining acetals of general formula 1 are new.

The acetals can be made by any suitable method. To those skilled in the art of making acetals and / or amino aldehydes, many such processes are known. In particular, those in Japanese Patent No. 71 24365, the procedures or analogous procedures specified by Fukami et al. And Israel et al will. A currently preferred method of manufacturing

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Development of the acetals consists in heating the corresponding acetal of an aldehyde of the general formula 8

Cl A ² CHO (8)

with an amine of the general formula 9

R (NHA ¹ ) _r NH ₂ (9)

1 2

in which R, A and A are those given above for general formula 1 Have meanings or, when R contains an aldehyde group, with the corresponding acetal of the amine reactant. The reaction is preferably carried out in a polar organic solvent at a temperature below 100.degree. If a symmetrical acetal of an aminoaldehyde of the general formula 1 is to be prepared, in which R represents the group -A CHO means, this can be obtained by making the amine reactant replaced by a diamine of the general formula 1O

H (NHA ¹ ) NH ₉ (10)

wherein A and r are as defined above for general formula 1, and a suitable excess of the acetal reactant of the general formula 8 is used. The hemiacetals can be obtained by partial hydrolysis of a corresponding Acetals can be obtained.

In a typical procedure, the acetal reactant of general formula 8 becomes with the amine reactant of the general formulas 9 or 10 for 60 hours in absolute alcohol

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held at reflux temperature. The product comes in a with Water-immiscible organic solvent extracted, which is then dried and then evaporated. The residue is distilled under reduced pressure to obtain the desired acetal.

The acetals according to the invention can be added to the cells in vitro or in vivo. The procedure and nature of the Additions are selected taking into account the particular application of the acetals. When the acetals are used in vivo, they are usually administered in the form of a pharmaceutical preparation that mixes the acetal or another Manner associated with a pharmacologically acceptable carrier or excipient. Appropriate formulations for this Pharmaceutical preparations can easily be produced by the person skilled in the art, if necessary after a simple preliminary test taking into account the route of administration to be used. Similarly, the dosages for the application can be set easily determined in vitro and in vivo by the person skilled in the art by standard methods.

At present, parenteral administration is preferred, especially the intravenous, at a daily dose of 1 to 10 mg / kg body weight. The carrier or diluent It is therefore advisable to use a physiological medium, e.g. isotonic saline solution.

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The pharmaceutical preparations are preferably used in Prepared unit dosage form, each unit dose, for example 5 to 500 mg and especially 10 to 250 mg of the invention contains effective acetals.

The invention also encompasses therapeutic preparations of the known ones Compounds in e.g. pyrogen-free water or a physiological saline solution or in another form adapted for therapeutic use.

The following examples illustrate the invention.

example 1

N- (3,3-diethoxypropyl) -1,4-aminobutane
(CH ₃ CH ₂ O) ₂ CH (CH ₂ ) ₂ NH (CH ₂ J ₄ NH ₃

A mixture of 18.8 g of 1, i-diethoxy-3-chloropropane and 1O.6 g

3
1,4-Diaminobutane in 40 cm of 99.9% abs is heated to reflux temperature for 60 hours.

3
1,4-diaminobutane in 40 cm of 99.9% absolute ethanol was

The ethanol was evaporated and the remaining oil extracted with 100 cm 4 M sodium hydroxide solution. The aqueous solution was then extracted four times with 40 cm of chloroform each time, and the extracts were dried with sodium sulfate and then the solvent removed.

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The residue obtained was distilled. 6 g of N- (3,3-diethoxypropyl) -1,4-aminobutane with a boiling point of 127 bis were obtained 130 C / 1 mm. The compound was initially determined by IR, NMR and mass spectroscopy and then identified by elemental analysis.

Example 2

N, N'-bis (3,3-diethoxypropyl) -1,4-diaminobutane (CH ₃ CH ₂ O) ₂ CH (CH ₂ J ₂ NH (CH ₂ J ₄ NH (CH ₂ J ₂ CH (OCH ₂ CH ₃ ) ₂ A mixture of 18.8 g of 1, i-diethoxy-3-chloropropane and 5.3 g of 1,4-diaminobutane in 40 cm of 99.9% absolute ethanol was heated to reflux temperature for 60 hours.

The ethanol was evaporated and the remaining oil was extracted with 100 cm 4 M sodium hydroxide solution. This aqueous solution was then extracted four times with 40 cm of chloroform each time. The extracts were dried over sodium sulfate, whereupon removed the solvent.

The residue was distilled to give 1 g of N, N'-bis (3,3-diethoxypropyl) -1,4-diaminobutane from boiling point 186 to 196 C / 3 mm. It was first determined by IR, NMR and mass spectroscopy identified. The elemental analysis confirmed the results of the physical measurement methods.

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The degree to which the compounds of Examples 1 and 2 inhibit the mitosis of diploid cells was determined using Methods determined based on those described by Allen and coworkers in Nature Volume 267, pages 623 to 625 (1977) with the exception that the fetal calf serum has been replaced by human serum. Basically, the corresponding cells are cultured in an amount of 10 cells / ml in 200 μl containers in a culture medium which contains the Compound to be investigated contained. The culture medium consisted of RMPI 1640, supplemented by antibiotics, 2 mM L-glutamine, 16 mM sodium bicarbonate buffer and 12.5% human serum. After adding the compound to be investigated, the Medium incubated for 1 to 2 hours before adding cells. After the addition of the cells, the medium was at 37 C in a Atmosphere of 5% carbon dioxide incubated in air. The uptake of 1, O, uCi HTdR (2 Ci / millimole) was in with trichloroacetic acid Using precipitable material from washed harvested cells in a toluene-based scintillator measured by a liquid scintillation counter. The results were calculated from a dose-response curve used for the medium d.p.m. (Background peeled off) from triplicate cultures. Cytotoxicity was determined by standard color exclusion technique established. The results of these investigations are summarized below.

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1. Both compounds inhibited spontaneously dividing thymic lymphocytes of rats. The doses for 50% inhibition in 1 day old cultures were approximately 5 mmol for the compound of Example 1 ("Compound 1") and about 4 mmol for the compound of Example 2 ("Compound 2")

2. Both compounds inhibited PHA stimulated thymic lymphocytes of rats. The doses that resulted in 50% inhibition in 3 day old cultures were about 570 / UM for connection 1 and less than 100 µM for connection 2 (116 / UM of compound 2 resulted in a 90% strength Inhibition).

3. Both compounds inhibited J111 leukemia cells, one of which they were believed to be pibroblasts, but at much higher doses than required to be inhibit the aforementioned lymphocytes. The dose for 50% inhibition is greater than for both compounds 5 mM.

4. Both compounds inhibit rat granulocytes. the Doses for 50% inhibition are about 1 mM for compound 1 and about 2 mM for compound 2.

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5. Both compounds inhibited Burkitt lymphoma cells (malignant Lymphocytes). The doses that lead to a 50% inhibition led were about 500 .mu.M for compound 1 and about 250, µM for compound 2.

6. No compound appears to have significant cytotoxic activity as demonstrated by color exclusion technology. However, higher cytotoxicity was observed in the malignant lymphocytes than in the non-malignant lymphocytes.

7. Spermine and spermidine are inactive in medium containing an additive of human serum.

An in vivo test to examine the effectiveness of compound 1 against leukemia in mice had to be discontinued after about 20 weeks when it became clear that the mice were not of the pure AKR strain as indicated by the supplier. Nearly 90% of the mice from the pure AKR strain had a diagnosed leukemia of 66 to 87% at the age of 400 days have to die. In a control group of 5 male and 5 female mice, only one (female) mouse died during of the test period and in 7 test groups of 5 male and 5 female animals, only a total of 4 mice (all female) Leukemia. Two of these 4 mice with leukemia died approximately 1 week after a single intravenous injection of 10 mg / kg of the

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Compound 1 of this disease, which after diagnosis of the Disease had been administered. The other two recovered however, after treatment as described below.

A mouse with 28 χ 10 white blood cells per cmin, which are mainly consisted of lymphoblasts, received 1 mg / kg intravenously of compound 1 for four consecutive weeks. By the fourth week the white blood cell count had increased

stabilized at 10 χ 10 per cm and there were no lymphoblasts more noted. The animal remained healthy until the end of the experiment approximately 16 weeks later.

2
Another mouse with 36.0 × 10 white blood cells per cmm, which consisted mainly of lymphoblasts, received 10 mg / kg of the compound intravenously on two consecutive days. Immediately before treatment, the animal was in
a very poor clinical condition with a hunched posture and an upright head. It made sounds when touched and the venous pressure appeared very low. To
After one week of treatment, the condition of the blood had changed dramatically. The white blood cell count was up

10.0 χ 10 per cmm fallen. Morphologically, the animal was starting out of acute leukemia reaches normal within a week, which is about 20 weeks later than the trial ended, was still going on.

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The effectiveness of various other compounds according to the invention can be seen from the attached graph emerged. It shows the relationship between the inhibition of cell growth or stimulation as indicated by the ingestion of tritiated thymidine and the Concentration of test compound when spontaneously dividing human lymphoblastic leukemia cells (CCRF-CEM) in one Amount of 0.25 χ 10 cells / ml were cultured using the method described above, but with 20% fetal Calf serum instead of 12.5% human serum. Fetal calf serum was used because it is commonly used for cultivation is applied to these cells and it has proven difficult to achieve satisfactory cell growth, if you replace it with human serum.

In the graphical representation means:

A. N- (3,3-Diethoxypropyl) -1,4-diaminobutane

B. N- (3,3-Diethoxypropyl) -1,4-diaminoethane

C. N- (3,3-Diethoxypropyl) -1,4-diaminohexane

D. N- (3,3-Ethylenedioxypropyl) -1,4-diaminobutane

E. Ν, Ν'-bis- (3,3-ethylenedioxypropyl) -1,4-diaminobutane

F. N- (3,3-Diethoxypropyl) -1,5-diaminopentane.

It is believed that the compound E at low concentrations observed stimulation and the generally

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This compound has little effectiveness on a rate of hydrolysis that is not attributable to. matches the mitosis of the particular cells used in this test.

The results available so far suggest that the compounds according to the invention are more effective against lymphocytes are compared to granulocytes and, for a given cell type, to proliferating cells (i.e., PHA-simulated or malignant cells) are more effective than against non-proliferating cells. Although investigations still need to be carried out, to be able to say that the compounds according to the invention are used in an effective and safe manner in human therapy The results to date indicate that they and certain analogs and homologues of these compounds very likely at least in the treatment of leukemia, the control of tissue rejection after tissue transplantation and organ transplants as well as contraception.

Of course, the acetals described here can be substituted by atoms or groups that reduce toxicity of the substituted molecule do not greatly increase or do not inactivate the acetal to inhibit cell proliferation. It should also be noted that in each formula given here, in which the same symbol is used more than once is included, the atoms or groups represented by this symbol may be the same or different.

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Example 3

N- (3,3-diethoxypropyl) -1, 2-diaminoethane A mixture of 3O g of 1,2-diaminoethane and 91.5 g Λ, 1-diethoxy-3-chloropropane (91% w / w) was dissolved in 100 ml dry ethanol heated under reflux for 60 hours. The ethanol was then evaporated off under reduced pressure and the residue shaken with 200 ml of 4M sodium hydroxide solution. The mixture was then extracted five times with 100 ml of dichloromethane each time, the extracts were dried over magnesium sulfate and then evaporated in vacuo.

The remaining oil was distilled in vacuo. 18.7 g of crude N- (3,3-diethoxypropyl) -1,2-diaminoethane were obtained as yellow Liquid with a boiling point of 74 to 80 ° C / 0.1 mm.

The crude product was distilled twice more in vacuo, whereupon N- (3,3-diethoxypropyl) -1,2-diaminoethane was obtained as a pale yellow liquid obtained from the boiling point 62 to 64 ° C / O, 0.5 mm.

Example 4 N- (3,3-diethoxypropyl) -1,4-diaminobutane

A mixture of 44 g 1,4-diaminobutane and 91.5 g 1,1-diethoxy-3-chloropropane (91% w / w) was refluxed in 120 ml of dry ethanol for 60 hours. The crude product was

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obtained in a manner analogous to that in Example 3. The distillation of the mixture in vacuo with subsequent double distillation the main fraction yielded 32.0 g of N- (3,3-diethoxypropyl) -1,4-diaminobutane as a colorless liquid with a boiling point of 86 to 88 ° C / 0.1 mm.

Example 5 N- (3,3-diethoxypropyl) -1,6-diaminohexane

A mixture of 58 g 1,6-diaminohexane and 91.5 g 1,1-diethoxy-3-chloropropane (91% w / w) was refluxed in 150 ml of dry ethanol for 60 hours. The isolation of the Crude product according to the procedure of Example 3 with subsequent double distillation in vacuo gave 28.4 g of N- (3,3-diethoxypropyl) -1,6-diaminohexane as a colorless liquid with a boiling point of 116 to 118 ° C./0.2 mm.

Example 6 N- (3,3-ethylenedioxypropyl) -1,4-diaminobutane

A mixture of 66.7 g of ß-chloropropionaldehyde ethylene acetal (98.5% w / w) and 44 g of 1,4-diaminobutane became 48 Heated under reflux in dry ethanol for hours. Isolation of the crude product according to the method of Example 3 with subsequent distillation under reduced pressure

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25.6 grams of crude N- (3,3-ethylenedioxypropyl) -1,4-diaminobutane. This Product was made with the equivalent distillate fraction from a similar small-scale reaction combined and distilled again in vacuo. 25 g of N- (3,3-ethylenedioxypropyl) -1,4-diaminobutane were obtained as a colorless liquid with a boiling point of 1O4 ° C / 0.1 mm.

A higher-boiling fraction with a boiling point of 175 to 188 ° C / 0.2 mm was also isolated and identified as the disubstituted derivative of the compound of Example 7 below; Yield 4.7g.

Example 7

N, N'-bis- (3,3-Ethylenedioxypropyl) -1,4-diaminobutane The reaction of Example 6 was carried out using two equivalents of ß-chloropropionaldehyde-ethylene acetal (98.5% w / w; 123.4 g) and 44 g of 1,4-diaminobutane repeated. After two distillation in vacuo, 19.3 g of crude N, N'-bis- (3,3-ethylenedioxypropyl) -1,4-diaminobutane were obtained as a colorless oil with a boiling point of 182 ° C./0.2 mm. By triturating the distillate with diethyl ether and subsequent recrystallization of the solid obtained from diethyl ether and mineral spirits with a boiling point of 60 to 80 ° C., Ν, Ν'-bis (3,3-ethylenedioxypropyl) -1,4-diaminobutane were obtained in the form of white needles from Melting point 59 to 61 ⁰ C.

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Example 8

N- (3,3-bis (2'-methoxyethoxy> propy1) -1,4-diaminobutane A mixture of 44 g 1,4-diaminobutane and 131.7 g ß-propionaldehyde-2-methoxyethyl acetal (86% weight / Weight) was refluxed for 60 hours in 150 ml of dry ethanol.Isolation of the product by the procedure of Example 3 gave, after distillation in vacuo, 21.3 g of crude N- (3,3-bis (2'-methoxy) thoxy} propyl) -1,4-diaminobutane After two distillation in vacuo, N- (3,3-bis- (2'-methoxyethoxy) propyl) -1,4-diaminobutane was obtained as a colorless oil with a boiling point of 88 bis 90 ° C / 0.1 mm.

Example 9 N- (3,3-diethoxypropyl) -1,5-diaminopentane

A mixture of 51 g 1,5-diaminopentane and 91.5 g 1,1-diethoxy-3-chloropropane (91% w / w) in 120 ml of dry ethanol was refluxed for 60 hours. The raw product was isolated as in Example 3. The distillation under reduced pressure with subsequent repeated distillation gave 21.5 g of N- (3,3-diethoxypropyl) -1,5-diaminopentane as a colorless oil with a boiling point of 96 to 98 ° C / O, O2 mm.

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Example 10

Ν, Ν'-bis- (3,3-diethoxypropyl) -1,2-diaminoethane A mixture of 15 g 1,2-diaminoethane and 91.5 g 1,1-diethoxy-3-chloropropane (91% w / w ) was refluxed in 100 ml of dry ethanol for 60 hours. After isolation using the usual procedure, the crude product was distilled. A yellow oil with a boiling point of 140 bis was obtained

160 ° C / 1 to 2 mm, which was then distilled again under reduced pressure. Yield 3.2 g of N, N'-bis (3,3-diethoxypropyl) -1,2-diaminoethane as a light yellow oil.

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Claims (3)

UEXKULL & -5TOLBi = RG BESELERSTI-A = J ^ F 4 2000 HAMBURG 52 PATENT LAWYERS DR. J.-D. FRHR. by UEXKÜLL DR. ULRICH GRAF STOLBERG DIPL.-ING. JÜRGEN SUCHANTKE Nicholas International Ltd. 33 Albert Road
Melbourne, Victoria 3004 Australia (Priority: March 3, 1978
GB 8656/78 - 15570) Hamburg, February 28, 1979 Acetals and hemiacetals of amino aldehydes and containing them therapeutic preparations Claims 1 .J Therapeutic preparation for inhibiting cell proliferation, characterized in that it contains one or more hydrolyzable acetals and hemiacetals of amino aldehydes of general formula 1 R (NHA ¹ ) _r NHA ² CHO (D 12 3 in which R denotes hydrogen or -A CHO, A, A and A independently of one another are divalent alkylene groups and r = 0 or an integer, and optionally pharmacologically suitable carriers or extenders. 909837/0682 2. Therapeutic preparation according to claim 1 , characterized in that it contains acetals of the general formula 4 OR ³ R ² (NHA ¹ ) NHA ² CH <; (4), ^X OR ⁴ 2 3 3 / ^Or3 in which R is hydrogen, -A CHO or -A CH <^ . means, 12 3 ^0R A, A and A independently of one another are divalent alkylene groups represent, r = O or an integer, 3 4
R and R independently of one another represent alkyl or alkylene groups with 1 to 6 carbon atoms, which are optionally sub- 3 4
are substituted, or R and R together form an alkylene group having 2 or 3 carbon atoms. 3. Therapeutic preparation according to claim 2, characterized in that that it contains acetals of the general formula 5 OR ³ R ⁵ / NH (CH ₀ ) 7 ,, NH (CH ₀ ) O <(5), OR ς ^ OR ³ in which R is hydrogen or - (CH ₂ ) CH ^, OR ⁴ n, m and ρ are independently integers of 2 to 12, r = 0 or an integer from 1 to 3 4
3 is, R and R independently of one another alkyl or 909837 / 06St Denote alkylene groups with 1 to 6 carbon atoms, optionally by hydroxyl groups, alkoxy groups with 1 to 6 carbon atoms or by halogen atoms 3 4 are substituted, or R and R together are an alkylene group with 2 or 3 carbon atoms. 4. Therapeutic preparation according to claim 3, characterized in that 3 4
indicates that R and R independently of one another are unsubstituted alkyl groups having 1 to 4 carbon atoms 3 4 mean, or R and R together form the ethylene group . 5. Hydrolyzable acetals and hemiacetals of amino aldehydes according to claim 1, excluding those of the general Formula 5 according to claim 3, in which 5 3 4 (a) R is hydrogen, r = O, m = 2 or 4, and R and R both represent the Ethy! group; (b) R ⁵ = - (CH ₀ ) -CH ^ ² P \ 4
OR r = 1, m = 3 to 6, and m and ρ are both 2 or 3 or OC ₂ H ₅ (c) R ⁵ = - (CH ₉ ) ^ - CHC; OC ₂ H ₅ r = 1, n = 9, m = 2 and R ³ and R ⁴ both represent the ethyl group. 909837/0652 6. Hydrolyzable acetals and hemiacetals of amino aldehydes according to claim 1 with the general formula 1A H ₂ NA ¹ NHA ² CHO (1A) 1 2
in which A and A are, independently of one another, divalent alkylene groups. 7. Acetals according to claim 6 of the general formula 4A 1 2 ^ - ^Or3
Η, ΝΑ NHA oilers . (4A) 1 2
in which A and A are independently divalent 3 4
Alkylene groups and R and R, independently of one another, denote alkyl or alkylene groups having 1 to 6 carbon atoms, which are optionally substituted by hydroxyl groups, alkoxy groups having 1 to 6 carbon atoms or by 3 4 Halogen atoms are substituted, or R and R together are an alkylene group having 2 or 3 carbon atoms represent. 8. Acetals according to claim 7 with the general formula 5A H ₂ N (CH ₂ ) _n NH (CH ₂ J _1n CHC ^ ₄ (5A) OR in which η and m are independently integers of 3 4
2 to 12 and R and R, independently of one another, represent alkyl or alkylene groups with 1 to 6 carbon atoms, which are optionally replaced by hydroxyl groups, 909837/0052 Alkoxy groups with 1 to 6 carbon atoms or by 3 4 halogen atoms are substituted, or R and R together mean an alleylene group with 2 or 3 carbon atoms « 9. acetals according to claim 8, characterized in that R 4th
and R independently of one another unsubstituted alkyl groups 3 4 pen with 1 to 4 carbon atoms or R and R together represent the ethylene group. 909837 / 06S2