DE2209595A1 - Low molecular weight prostaglandin antagonists and processes for their preparation - Google Patents

Description

PATENT APPROVAL

PATENTANWÄLTE LICHT, HANSMANN, HERRMANN a MÖNCHEN 2 THERESIENSTRASSE 33

DipUig. MARTIN LICHT Dr. REINHOLD SCHMIDT Dipl.-Wirtsch.-Ing. AXEL HANSMANN Dipl.-Phys. SEBASTIAN HERRMANN

Munich, the 2 ?. February 1972

Your sign Our sign

ALLERGAN PHARMACEUTICALS

Santa Ana, California 1000, South Grand Avenue V. St. A.

Low molecular weight prostaglandin antagonists and processes for making the same.

The invention relates to low molecular weight prostaglandin antagonists and / or adenosine -3 ", 5'- monophosphate antagonists (cyclic AMP) and in particular low molecular weight polymers of certain polymeric phosphoric acid esters, processes for producing the same and processes for separating them from high molecular weight polymers.

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The prostaglandins are a group of long-chain unsaturated hydroxy and hydroxyketocarboxylic acids. These are in human and animal tissues are widespread and have a wide variety of pharmacological effects. that the prostaglandins play a fundamental biochemical role in most and perhaps all abnormal cells. The biochemical and pharmacological effects of prostaglandins are dealt with in various references. The current state of knowledge regarding the functions of prostaglandins is from the article "Hypothesis on Physiological Roles of Protaglandins "(E.W., Horton, Physiological Review, Vol. 49, No. 1, January 1969).

Naturally occurring prostaglandins are fatty acids containing twenty carbon atoms and containing a cyclopentane ring. The saturated parent acid has been described as Prostanoicsäure (P ^r o ~ stanoic acid) and has the following formula:

COOH

Prostanoic acid

Four series of natural prostaglandins have been described so far, which are designated below with the capital letters B, E, F and A are designated. The differences in the cyclopentane ring are listed in the following sub-formulas:

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Ο- Ο OH

All prostaglandins have a hydroxyl substituent in the 15 position. The number of unsaturated bonds in the side chains are indicated by the lower numbers. B. the prostaglandin E has a trans double bond in the 13-position, E also has a cis double bond in the 5-position, E has a third double bond in the 17-

Position, etc. The structural formula of 14 naturally occurring prostaglandins are shown in Figure 3 of the above Reference in Physiological Review, Volume 49, given.

The prostaglandins have a wide variety of pharmacological effects in humans and animals and are for example active in the following very different areas: fertility, sperm transport, menstruation, childbirth (parturition), blood flow in the placenta, gastric or gastric secretion, muscle contraction (including the vascular smooth muscles, respiratory smooth muscles, the smooth gastrointestinal muscles, the smooth muscles of the uterus and the smooth capsular spleen muscles), the development of the epidermal tissue and the central nerve transmitters. It is assumed that the prostaglandins also influence the permeability of, for example, the skin and the eyes. Hence it is also believed that the prostaglandins too

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can act as accelerators or mediators of various forms of inflammation. The prostaglandins The attributed role in inflammation is reinforced by the involvement of these compounds in the inflammatory processes in joints, skin and eyes.

In the reproductive field, for example, the control of prostaglandins could lead to direct inhibition or Promoting fertility in males or females as the prostaglandins normally result in high levels Concentrations are found in semen and are known to affect the smooth muscles of the female reproductive tract influence significantly. The association or connection of sub-normal prostaglandin levels with the infertility in males supports the hypothesis that the prostaglandins play an essential role to support conception or conception. Hence there could be specific control of these prostaglandins lead to a change or regulation of male or female fertility or infertility.

It is known that the amniotic fluid at birth during labor has a significantly higher amount of prostaglandins contains than the amniotic fluid before the onset of labor. Therefore, it is believed that the prostaglandins during the Childbirth contribute to uterine contractions. Specific control of these prostaglandins can be anti-abortive or lead therapeutically abortive agents that would be of great value in obstetrics.

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Prostaglandins have been identified in the central nervous system and are known to respond to nerve stimulation be cleared of the brain and the spinal cord and that they are normal components of the cerebrospinal fluid are. In the above cited reference by Horten in Physiological Review, Volume 49, page 136, is indicated that the discovery of a specific prostaglandin antagonist would be of great help in understanding the role of the To elucidate prostaglandins in the central nervous system.

It was also found that certain prostaglandins Strong vasodilators in most parts of the vascular system (vascular beds) are possible local mediators or mediators of the blood flow are to be considered taking into account their wide distribution in the tissue. The identification of a vasodilatory prostaglandin in the renal medula ascribes a role to the prostaglandins in the Control of the kidney blood flow and as etiological factors in kidney hypertension or in increased blood pressure in the kidneys.

Prostaglandins have also been found in the lungs and are believed to have certain functions in bronchial asthma to have. The prostaglandins are present in the stomach and intestines and are spontaneous by different parts of the gastrointestinal tract released. It is also believed that circulating prostaglandins cause diarrhea.

Taking into account the versatile functions listed above of prostaglandins it is clear that a specific control of prostaglandin activity leads to a control of several Body functions that were previously not possible.

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Adenosine-3 ', ö'-monophosphate (cyclic AMP) is hormonal sensitive, i.e. when a hormone reaches the cell, the level of cyclic AMP associated with the cell builds up is on and arouses or enhances hormonal activity. The cyclic AMP is found in most animal cells and plays an essential role in muscle function, gastric and enzymatic secretion, in the central nervous system, in cellular metabolism and in production and in releasing hormones. Cyclic AMP is derived from adenosine triphosphate (ATP), which is a key substance The generation of energy in cells is due to a reaction that is catalyzed by adenyl cyclase. It is believed, that the prostaglandins act as a feedback regulator for cyclic AMP, as the prostaglandins appear to do often released due to hormones that stimulate adenyl cyclase. In many cases the prostaglandins work so as to suppress the accumulation of cyclic AMP. This can be done like with the prostaglandins Controlling the activity of the cyclic AMP similarly leads to the control of bodily functions that previously did not had been achieved. Taking into account the above, the designation used in the description relates to "Prostaglandin Antagonists" means the prostaglandin antagonists and / or the antagonists of cyclic AMP.

In U.S. Patent Nos. 2,962,495, 2,962,515 and 3,317,383, phosphoric acid ester high molecular weight polymeric substances are disclosed (Molecular weight 2,000 - 50,000) described by phosphorylation of flavones, flavanones, chalcones and Öihydrochalconen be obtained, the at least one phenolic hydro> ^ oil group contain. Such connections are for example

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Polyphloretin phosphate, polymethylphloretin phosphate, polyquercetin phosphate, Polynaringenin phosphate and polyhesperetin phosphate. The patent specifications given above show that that these substances have an increased anti-enzymatic effect on hyaluronidase.

Some of these polymeric phosphoric acid esters, including the polyphloretin phosphate, selectively inhibit the Effects of prostaglandin. It was found that this prostaglandin inhibitory activity was not related relates to the ability of these polymeric phosphoric acid substances to inhibit hyaluronidase.

It has meanwhile been established that the prostaglandin is inhibited with certain polymeric phosphoric acid esters is actually only the property of a specific low molecular weight fraction of these polymers, i.e. the dimer, the trimer and the tetramer and especially the dimer. As can also be seen from the examples given below can, the low molecular weight fraction has anti-prostaglandiriactivity, which is 26 times the activity of the same concentration of the unseparated material. Farther it can be seen from the examples that the low molecular weight fraction has essentially anti-hyaluronidase activity owns.

The invention relates to polymeric phosphoric acid esters, in which the phosphoric acid radicals through ester bonds by means of organic Groups are linked to one another, each organic group being a glycoside residue of a polyhydro> o / compound like the flavones, flavanones, chalcones and dihydrochalcones,

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the polymeric phosphoric acid esters being prostaglandin antagonists that have essentially no anti-hyaluronidase activity and a molecular weight of less than about 1,400 own.

The invention also relates to a method for separating low molecular weight prostaglandin antagonists from high molecular weight ones polymeric phosphoric acid esters ^ in which the phosphoric acid radicals through ester bonds by means of organic Groups are linked together, each organic group being a glycoside residue of a poly hydroxy compound such as a flavone, flavanone, chalcone and / or dyhydrochalcone, which is characterized in that the high molecular weight polymeric phosphoric acid ester are passed through a material that allows molecular fractionation, and the part recovering the polymeric phosphoric ester having a molecular weight of less than about 1,400.

The present method according to the invention further relates to a method for blocking the effects of prostaglandins in humans and animals, which is characterized in that humans or animals are given an effective amount the low molecular weight compounds described above in combination with a suitable pharmaceutical carrier is administered.

The present invention also relates to methods of chemical Synthesis of low molecular weight prostaglandin antagonists of the type described above, characterized in that a suitable amount of a polymer forming monomeric compound, as described above, in a solution

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agents, such as pyridine, quinoline or benzene, dissolved that are capable of phosphorylation by a phosphorylating agent such as phosphorus oxychloride, Methylphosphoramide dichloride or JD-nitrophenylphosphorodichloridate, To promote the desired phosphorylating agent with or without the addition of conventional phosphorylation promoting agents is added, the reaction mixture is reacted until the desired degree of polymerization has been reached and the reaction product is hydrolyzed will.

Figures 1-6 are graphical representations of those in the following Examples obtained experimental results.

According to the process of the invention, compounds having the following formulas are prepared:

(1) AB-PO-BAj

(2) AB-PO-BA-PO-BAj ^K 4 4

(3) AB-PO-BA-PO-AB-PO-BAj and

^{v J} 4 4 4

(4) PO ₄ (AB) _g

where A is a group of the formula:

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in this are:

R OH or OX;

R ₀ H, Cl, F ₀ C, F or X;

R, R or R any substituent that is not too large, to cause steric hindrance to the rest of the molecule. In the R, R and R positions, the following

o 4 5

de substituents are present: halogen substituents such as F or Cl, a nitro group, H, OH, X or OX.

The X can be a methyl group, ethyl group, ni-propyl group or_l · -propyl group.

B is a substituent of the following formula:

therein R_ is H or F or a phenyl group and the group AB ο

is formed by a bond of the 4-position of B with the terminal carbon atom of the alkyl chain of A. The AB units thus formed are linked by a phosphate group which is located at the 1-position of each B ring.

The 2- or 3-positions in the B-ring can be substituted by a group which must not be too large in order not to cause steric hindrance. Such a group X can, for example,

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be H, F, Cl, OH, CH OH and C H OH. The 5 position of the B-ring can be substituted by groups such as alkyl groups up to CH and preferably by straight chain

Ό IO

term groups up to the y-carbon atom such as OCOCH, CH Ph, C H Ph and C H Ph. A particularly suitable substituent

2 2 4 ο Ο

ent in the 5-position is C_H ^ Ph. The benzene ring can in the meta-

O O

Position be substituted by a hydroxyl group. Indole or another lipophilic ring can replace the benzene ring as a substituent in the 5-position.

In general, every AB group in the polymer is the same Way substituted. Nonetheless, you can do in any AB group of the polymer sit different substituents.

Particularly advantageous compounds in the context of the present invention are dimeric, trimeric and tetrameric compounds with the following substituents:

(1) R is OH or OX, where X is as above

is defined;
and R, R, R, R and R are all H.

(2) R is OX, where X is as defined above

is, or preferably OHj

^R 2 is H; ^R 3
^R 4 is
is OCH ₃ ; ^R 5 is H; and ^R 6 is H or F.

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(3) The compound defined in (2), wherein R _{4 is} OH, CH ₂ OH or CHOH.

A preferred compound according to the invention has the following formula:

in which R is OH or OX, where X is defined and R ^, R, R,

^ and R "are hydrogen substituents. 5 6

Other suitable compounds may be substituted as follows:

0) R ₁ , R _Q and R _c are OX, where X is like

Ί O

defined above and R, R and R are hydrogen atoms.

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(2) R ₄ , R "and R,. are OX, where X is like

14 ο

is defined above and R, R and R are hydrogen atoms _o

(3) R. and R ₁ ^ are OX, where X is as above

1 5

is defined and R is X and R, R and R_ are hydrogen atoms.

The substituents can be the same or different in both parts of the molecule. If R is OH or OX, then R is preferably H. For reasons of symmetry, R is preferably H when R is OX.

Another compound according to the invention has the following formula:

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in which Z is a phenyl group or a Ben2yl group and R bis

R are substituents as described above. The index letters 6th

R and S indicate the stereochemical configuration.

The benzene ring of Z can be substituted by t, t becomes ^a η η

defined below.

Other compounds according to the invention are diners, trimers and tetramers having the following repeating rrlonomere formula:

where Y ,, Y_ and Y_ are OH-substituents or H-substituents

15 6>

wherein Y, Y and Y can be OH or H substituents or

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- CH,

where t is H, CH _n , F, Cl or OCH_ and η 1-5 or 3 ο

(2)

- 0- CH

where t is as defined above. or η

(3)

0- C

where t is as defined above, or η

(4) -0-C-CH, or

(5)

, or

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(6) - O -CH ₂ ~ ( ()>, or

(7) alkyl groups with 1-6 carbon atoms (straight or branched), and

(8) - (CH) -NR, where η 1-6 and R is H, CH

or CH and wherein Y, Y, Y 2 5 1 2 ο

and Y ,, Y_ and Y ^ equal or different-

4 5 D.

can be borrowed.

The low molecular weight polymers according to the invention can be modified be, in which the PO group by another, similar linking group, such as SO or

0 = P-O-

is replaced.

The different groups of reagents can have different ring substituents.

The production methods of the high molecular weight polymeric phosphoric acid esters are not part of the present invention. A complete description of these polymeric compounds and the

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Methods of making the same are given in U.S. Patents 2,962,515, 2,962,495, and 3,317,383. The parts of the Processes relating to high molecular weight polymeric phosphoric acid esters with molecular weights above 2,000 will be used included in the present application.

The low molecular weight polymers (molecular weight below about 1,400) according to the invention are prepared as follows:

(1) Physical fractionation

(a) The low molecular weight prostaglandin

High molecular weight polymeric phosphoric acid esters are antagonists through a physical fractionation process separated using gel filtration. In the case of gel filtration, a solution of the to be fractionated Material carried through an appropriate amount of a hydrophilic gel. Molecules of the material to be fractionated, the are larger than the largest pores of the gel, cannot penetrate the gel and therefore pass through the acid in the gel Liquid phase, outside of the gelatinous. Therefore, the larger molecules (the higher molecular weight polymers) eluted first. The light molecules, on the other hand, penetrate the gel to a varying extent, depending on their size and their Shape. This elutes the remaining polymers from the acid to lower the molecular weight. Typical Materials that are suitable for gel filtration are spherical, hydrophilic polyacrylamide beads, which are known as Bio-Gel P are known and sold by Bio-Rad Laboratories and the modified hydrophilic cross-linked polymeric dextran beads commercially available under the name Sephadex and from Pharmacia Fine Chemicals, Uppsala, Sweden,

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to be expelled.

(b) Low molecular weight polymers can

of high molecular weight polymers by means of physical fractionation are separated, by means of dialysis, in conventional dialysis machines, dialysis methods and dialysis materials can be used.

(2) Chemical synthesis

(a) The compounds according to the invention

can by controlled or controlled polymerization of suitably substituted flavones, flavanones, chalcones or dihydrochalcones in the presence of methylphosphoramide dichloride and triethylamine or ethyldiisopropylamine in an inert solvent such as benzene. The procedural responses are as follows Side shown: "

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^C 2 ^H 5

NC ₃ H ₇ ISO

3 7

ft

+ CH-NH-P-Cl

I.

Cl

ISO or

N (CH ₃ ) 3 BENZENE

NHCH,

R.

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The hydrolysis of the phosphorus amine produces the phosphate dimer. The trimer and the tetramer can be made by the coupling reaction is repeated, however, in these cases

necessary that R is OH or OX, as defined above. 3

(b) To the appropriately substituted

To produce chalcone can be a substituted acetophenone with a substituted benzaldehyde in the presence of a basic

Catalyst can be condensed in the following way: CH

CHO

NaOH

C-CH = CH

The condensation product can be selectively hydrogenated in the following manner to form the corresponding dihydrochalcone:

C - CH =

Il

C-CH ₂ -CH

KD

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The molecules of the substituted dihydrochalcone thus formed can be linked as described in (2 a) above.

(c) Similarly, others can

Coupling compounds are used, such as phosphorus oxychloride or jD-nitrophenylphosphorus dichloridate instead the methylphosphoramide dichloride used in (a) above.

It is believed that the low molecular weight phosphorized polymers of the invention are particularly effective and strong Prostaglandin blocking agents are, by their configuration, i.e. the phenolic group and the attached carbon chain are not hindered, which leads to the formation of a helix configuration comes, which makes it possible that the low molecular weight phosphorized polymers selectively the prostaglandins To block.

The effective amount of the low molecular weight polymeric phosphoric acid esters to be administered varies with the type of prostaglandin activity which is to be blocked, with the mode of administration of the blocking agent and depends on whether the blockage is to be in vivo or in vitro . The mode of administration of the blocking agent is understood to mean whether it is to be administered locally or systematically. For example, about 0.1 to 200 mg / ml of a bath liquid in an organ bath of a low molecular weight material derived from polyphloretin phosphate block the action of prostaglandin in vitro on smooth muscle tissue. About 0.01 ml or a 1-10% solution, administered locally in vivo to a rabbit eye, counteracts the effect of the prostaglandin on intraocular pressure,

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The in vivo effects of prostaglandins on blood pressure and on faeces (formation of diarrhea) in mice are blocked by intraperitoneal injections in amounts of 1-2 (X) mg / kg body weight.

The compounds and the method according to the invention are illustrated in detail in the following examples.

example 1

Making the dinner from 3-hydroxy-4 '' nnethoxydihydrochalcone.

(a) Compound III was prepared in the following way:

0 Il

+ CHNHP 3

Cl Cl

II

CH ₃ O

C, -CH-CH 0

III

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To 2.2 g (0.008 mol) of the compound (I) in 50 ml of dry benzene was 0.6 g (0.004 mol) of methylphosphoramide dichloride added. The mixture was stirred and refluxed for 3 hours. The solution was then cooled and three times with Washed 30 ml of water. The organic layer was dried with Na SO and evaporated to dryness. One received 1.4 g of a yellow oil obtained by N.M. R. identified as the compound (III).

(b) The compound IV * was then by the hydrolysis of the

Phosphoramidate dimer (III) produced:

CH ₃ O

.C -

P- NH-CH,

75% HCOOH

Ο, 8 g of the compound III were in 25 ml of a 75% aqueous Formic acid solution dissolved and stirred at reflux temperature for minutes. The solution was then under vacuum and at

ο
a water bath temperature of 65 C reduced to an oil. 30 ml of benzene were then added to the oil and the benzene solution was washed four times with 20 ml of water each time. The Bsnzol extracts were

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dried over Na SO and obtained after evaporation of the benzene 0.6 g of a dark oil obtained by means of N.M. R. identified as Compound IV.

Example 2

Additional compounds analogous to that prepared according to Example 1 Compounds were synthesized by varying the position and nature of the substituents on the rings.

Example 3

Manufacture of the sodium salts of diphloretin phosphate (DPP).

The following reactions (1) to (6) are carried out in the following manner carried out:

HO

WC-

ft

CH ₃ + C Hg-C-Cl

OH

IF

OH

dry HCl gas' CH ₃ COOC ₂ H ₅

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^H 2 ^{/ R} > B0

- 25 -

IF

g-CH-CH O

OH

IF

IF

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CH-CH
2 2

DPP sodium salt

Example 4

(a) Examples 1 and 2 were repeated with the exception

that POCl orjD-nitrophenyl phosphorochloridate instead of CH NHPOCl, were used. Similar results were obtained.

(b) The sodium salt of the diphloretin phosphate of Example 3 was converted to the diphloretin phosphate and treated with hydrochloric acid at acidic pH.

Example 5

The trimer of Phloretinphosphat was formed by Diphloretinphosphat was reacted under the conditions described in Example 3 reaction conditions with the compound is indicated in Step 3 of Example 3. FIG.

Example 6

The phloretin phosphate tetranner was prepared by adding two molecules of diphloretin phosphate under the conditions described in Example 3.

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special conditions have been implemented "
Example 7

The stimulating or stimulating activities of prostaglandins are known in various types of intestinal smooth muscles, both in vitro and in vivo. Semi-liquid feces and diarrhea have been observed as side effects after intravenous injection of prostaglandins for abortion or induction of labor. The purpose of this example is to demonstrate that polyphloretin phosphate (PPP) and diphloretin phosphate (DPP) can counteract prostaglandin-induced diarrhea in mice and that DPP is a more potent antagonist than PPP.

Methods

Swiss albino mice, both sexes, with an average Weights between 20-25 g were used in these studies. The animals had free access to the food and water. The diarrhea was caused in groups of 12 animals by intraperitoneal or intravenous (tail vein) Injection of prostaglandin E (PGE). The appearance of semi-liquid feces within 30 minutes of the injection of the Prostaglandins were considered a positive consequence. The smallest dose / on PGE j that was necessary to achieve an ED -positive

2. 1 ov)

speak to effect was 50 µg / kg for intraperitoneal injection and be · 250 µg / kg for intravenous injection. Varying

AWAY. Leo, Haisinborg, Sweden

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Dose amounts of PPP and DPP were given in a dose volume of 0.5 ml 15 to 20 minutes before the administration of PGE The following Tables 1-3 show the results obtained.

TABLE I. PPP (mgAg) dose

Saline

solution J50 100 200

Caused

Diarrhea 11/12 8/12 3/12 0/12

After the intraperitoneally applied PPP were 15 minutes later given 50 µg / kg PGE intraperitoneally.

TABLE II

PPP (mgAg) dose

Saline solution 25 50 100

Caused

Diarrhea 11/12 10/12 9/12 3/12 0/12

After the intraperitoneally applied PPP were 15 minutes later 250 µg / kg PGE given intravenously.

TABLE III

DPP (mgAg) dose

Saline

solution 10 15 20 25 30

Caused

Diarrhea 11/12 11/12 7/12 6/12 3/12 3/12 2/12

After the intraperitoneally applied DPP were 20 minutes later

! peritoneal donor

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50 µgAg of PGE given intraperitoneally.

Results

200 mgAg PPP completely blocked the diarrhea that would otherwise have been caused by intraperitoneal or intravenous administration of PGE (see Tables I and II). Lower doses of PPP were also effectively proportional to the concentrations administered, with the ED dose ranging between 50 and 100 mgAg. DPP was considerably more effective than PPP in blocking diarrhea for an ED dose in the range of 15-25 mgAg (see Table III). A control group given DPP alone reacted at 20 mgAg with one animal that developed diarrhea and three other animals that showed lesser signs such as stimulated defect. The mice responded similarly to PGE® administration regardless of the route of administration. They became inactive, exhibited a piloerection, and usually exhibited an ^M stretch "posture, which was consistently followed by diarrhea. The diarrhea in the saline-injected control groups occurred within five and a half minutes after administration of PGE. If those with PPP or If diarrhea occurred in DPP treated groups, it was usually detected a little later, and if the diarrhea was blocked in an animal, no signs of inactivity or piloerection could be seen.

discussion

The studies and results described above showed that PPP and DPP are able to counteract diarrhea which is caused by PGE. DPP is approximately five times stronger than PPP, but DPP is slightly agonistic Effect as it stimulates the defect.

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In the following examples, the following pharmacological Test methods applied:

Jirds (Meriones Shawwi) weighing between 50-150 g were killed with a blow to the head. The middle part of the ascending colon was removed and placed in a 5 ml organic Set up a bath containing Jalon's rat intestinal solution,

which had been treated with oxygen gas at 28 ° C. A dose cycle of 3–4 minutes and a contact time of 1–2 Minutes applied. The contractions were isotonic with a brush-isotonic muscle transducer or sound generator (Brush isotonic muscle transducer) measured in conjunction with a Brush 220 recording device.

Before the experiment, the fabric was soaked in the organic for 30 minutes Leave the bath in place, the bath liquid being changed several times. During this period and after the introduction the first few doses of prostaglandin became the remaining Tension adjusted for quick return to baseline with minimal leverage. The sensitivity was electronically adjusted so that the maximum contraction was slightly less than a full scale reading Needle. The inhibitory activity of prostaglandin from polyphloretin phosphate fractions was determined against prostaglandin F oC, a polyphloretin phosphate was used as the standard, the possessed an average molecular weight of 1,500.

Example 8

A sample was prepared by adding 0.5 parts of polyphloretin phosphate (PPP) with an average molecular weight of

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15,000 were dissolved in five parts of distilled water. the Solution was neutralized to pH7 with normal sodium hydroxide. An 80 cm long glass column 1.5 cm in diameter was filled with hydrated Bio-Gel P-2 polyacrylamide gel (50-100 mesh; sieve opening 0.297-0.149 mm) packed full. The sample was applied to the top of the load and eluted with 5 ml fractions using purified water to remove the Rinse the column. The washed fractions were collected and examined for pharmacological activity.

After eluting all of the previous samples, a compound with a known molecular weight at the top of the column packing and eluted in 5 ml fractions by purified Water was used to rinse the column. The compound used was idoxuridine having a molecular weight from 354.

FIG. 1 is a graph showing the percentage of ultraviolet absorption and the relative prostaglandin-absorbing activity of the fractions collected in Example 8. FIG. The percentage ultraviolet absorption (UV) graph is a measure of the amount of organic material in each of the 5 ml fractions that was washed from the column. The percentage of UV light absorption of the 5 ml fraction between 40 and 44 ml is at eb / va 60 % and thus indicates a relatively high concentration of organic material. In contrast, the percentage of UV light absorbed for the 5 ml fraction between 95 and 99 ml is around 15% and thus indicates a relatively low concentration of organic material.

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The graph of prostaglandin inhibitory activity is plotted on a scale showing the relative activity of consecutive 5 ml samples, where 1 shows the activity of the unfractionated polymer. For example, the activity of the 5 ml fraction between 115 and 119 ml is about 13 and the activity of the 5 ml fraction between 80 and 84 at about 18.

The graph in FIG. 3 shows the relative activity of the fractions collected according to Example 8. The relative Activity is calculated by taking the prostaglandin inhibitory activity map divided by the mapping of the percent UV absorption. The resulting number (relative activity) is a measure for the prostaglandin inhibitory activity of the contents of each fraction, the different concentrations of organic material be taken into account in each political group. This enables the activity of each fraction to be compared by means of the relative activity as if the fractions have the same concentrations of organic material. From Fig. 3 are also to see the points at which compounds of known molecular weight are eluted from the column, thereby also the theoretical calculations given below are confirmed.

In table 1 a theoretical calculation is given, which the Indicates the order of magnitude of the molecular weights expected using the conventional calculation method as it is is specified by the manufacturer of the gel used in Example 8.

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Table 1 Milliliters Eluted Molecular weight 107
93
77
64 350
700
1050
1400

Based on the analysis of Table 1 and Figures 1 and 3, it can be concluded that the anti-prostaglandinactivity is almost completely is in the molecular weight range of less than 1400. From Fig. 1 it can be seen that the anti-prostagland is inactivity starts at the 60-64 ml elution fraction. The table 1 indicates that 64 ml is the point at which the molecular weight of 1400 should appear and this is the molecular weight of the Is tetramer of polyphloretin phosphate. This theoretical calculation is roughly confirmed from Fig. 3, from which it can be seen that a known compound having a molecular weight of 350 eluted between the 110 and 120 ml fractions. The table Figure 1 also shows that a compound with a molecular weight of 350 is eluted between the 110 and 120 ml points. The table 1 further indicates that a compound with a molecular weight of 350 should be eluted at the 107 ml point.

Example 9

The experiment according to Example 8 was repeated with the exception that instead of the polyak rylamide gel Sephadex G-25 medium (transverse

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crosslinked dextran polymerj particle diameter 50-150 li) used became. 5 ml fractions were collected and checked for UV absorption analyzed. The fractions were combined into 10 ml samples and checked for pharmacological activity. Two consecutive 5 ml samples were always combined.

Figure 2 is a graph showing the percent ultraviolet absorption and prostaglandin inhibitory activity of the fraction collected according to Example 9 shows.

Figure 4 is a graph of the relative activity of fractions collected according to Example 9. It should be noted that the relative activity of some fractions of the Example 9 is very high. Fraction 20 has an activity of 26.7 and shows that the material in this fraction is 26.7 times more active is than the same concentration of the polyphloretin phosphate itself.

FIG. 5 is an overlay of FIGS. 3 and 4 and shows the great qualitative similarity between the relative activity of the fractions in Experimental Examples 8 and 9.

Example 10

A sample was prepared by dissolving 500 mg of polyphloretin phosphate with an average molecular weight of 15,000 in 2.5 ml of a phosphate buffer with a pH of 7. A 90 cm long glass column with a diameter of 1.5 cm

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was with Sephadex G-25 Fine (particle diameter 20-80 u) cross-linked dextran prepackaged. The sample was applied to the hydrated column and eluted with distilled water. the Fractions were collected. 6 shows the separation of the biological activity in different molecular weight fractions. From this it can be clearly seen that the prostaglandin inhibitory activity is localized in the low molecular weight fractions.

Different fractions were pooled as indicated in FIG. 6, Freeze-dried and pharmacological activity of the freeze-dried powder from three combinations were determined compared with the original sample of the polyphloretin phosphate. As shown in Fig. 6, the freeze-dried powder has the fractions 15-21 approximately half the activity of the original sample of the PPP. In contrast, the freeze-dried material from fractions 26-29 was 7.4 ml stronger than the original sample of the PPP (on a weight basis).

Example 11

This example shows values indicating that the anti-hyaluronidase activity the polymeric phosphoic acid ester is localized in the high molecular weight fractions, which have very low prostaglandin inhibitory activity own. Conversely, the low molecular weight fractions with the high prostaglandin activity have a low one or no anti-hyaluronidase activity at all. It surrenders from the following values:

The turbidimetric investigation or the turbidity measurement of the Hyaluronidase (Meyer, Hoffmann and Liner, The Enzymes, 4, 2nd Edition, 447, Academic Press, Inc. New York, 1960)

209839/1221

based on the studies by Tolksdorf et al, (J. Lab. CHn.Med. 34, 74, 1949) was used to determine the anti-hyaluronidase activity of the polyphloretin phosphate and the three described in Example 10 Investigate factions. PPP and the three fractions thereof were examined at concentrations of 0.01 mg / ml. Her Starch was compared with 0.0005 mg hyaluronidase which was allowed to act on 0.2 mg substrate (hyaluronic acid). Table 2 shows the results obtained:

Table 2

Anti-hyaluronidase anti-prostaglandin

Compound activity% inhibition activity from Fig. 6

PPP 75.0 1

Fractions 1-11 90.0 0

Fractions 15-21 8.0 0.6

Fractions 26-29 3.5 7.5

The prostaglandin inhibitory activity of the polyphloretin phosphate can be better distinguished from the anti-hyaluronidase activity by expressing it in terms of molecular weight. 90 % of the anti-hyaluronidase activity is found in the high molecular weight region of the PPP, which has no anti-prostaglandin activity, while the low molecular weight fraction of the PPP, which has an anti-prostaglandin activity that shows 7.5 times the activity of the PPP, an anti- Has hyaluronidase activity of only 3.5%.

209839/1 221

Example 12

Example 8 was repeated except for the following polymeric phosphoric acid esters were used instead of the polyphloretin phosphate:

Polymethylphloretin phosphate, polyquercetin phosphate, polynaringenin phosphate and polyhesperetin phosphate.

Corresponding results were obtained.

209839/1221

Claims (3)

Claims 1. Process for the production of polymeric phosphoric acid esters, in which the phosphoric acid radicals are linked together by ester bonds through organic groups, and each organic Group is a glucoside residue of a polyhydroxy compound, such as flavones, flavanones, chalcones and dihydrochalcones, where the polymeric phosphoric acid esters are prostaglandin antagonists which have essentially no anti-hyaluronidase activity and have a molecular weight of less than about 1,400, characterized in that polymer-forming compounds, such as substituted chalcones, dihydrochalcones, flavones and flavanones with a phosphorus-containing coupling agent reacted under conditions sufficient to form a polymer and the polymer is hydrolyzed, to form a low molecular weight phosphoric acid ester. 2. The method according to claim 1, characterized in that the polymer-forming compound is a dimer and the polymer formed is a tetramer. 3. The method according to claim 2, characterized in that the dimer is diphloretin phosphate. 4. The method according to claim 1, characterized in that the polymer-forming compounds are monomers and dimers and the polymer formed is the trimer. 5. The method according to claim 4, characterized in that the Monomer phloretin phosphate and the dimer diphloretin phosphate 209839/1221 6. The method according to claim 1, characterized in that the polymeric phosphoric acid esters polyphloretin phosphate, polymethylphloretin phosphate, Are polyquercetin phosphate, polynaringenin phosphate and polyhesperetin phosphate. 7. The method according to claim 1, characterized in that the synthesized compounds have the following formulas: AB-PO-BA; AB-PO-BA-PO-BA:
4 4 AB-PO-BA-PO-AG-PO -BA: and 4 4 4 PO ₄ (AB) ₃ wherein A represents a group having the following formula: in which R is an OH group and X, R is H, Cl, FC, F and X, 1 2 3 and R, R and R are each H, Cl, F, NO, OH, X and OX, and wherein X is a methyl, ethyl, n-propyl or i-propyl group is, and where B is a group of the following formula, 209839/1221 where R_ is H, F and a phenyl group, and the group AB 6th is formed by connecting the 4-position of B with the terminal carbon atom of the alkyl chain of A, the AB units thus formed being linked by means of a phosphate group located in the 1 position on each B-ring. 8. The method according to claim 1, characterized in that the phosphorus-containing coupling compound of phosphorus oxychloride, Methyl phosphoramide dichloride and p-nitrophenyl phosphorus dichloridate consists. 9. The method according to claim 7, characterized in that R OH and OX and R, R, R, R and R are H. 10. The method according to claim 7, characterized in that R OX and OH, R H, R a methyl group, R a methoxy group, R ^ H and R "are H or F.
5 6 . Process according to Claim 10, characterized in that R is a 0H group, CH OH group and C H 0H group. 12. The method according to claim 1, characterized in that the synthesized compounds pharmaceutically acceptable salts the low molecular weight phosphoric acid ester. 13. The method according to claim 1, characterized in that the polymer-forming compound is phloretin and the polymer formed is diphloretin phosphate. 209839/1 221 14. The method according to claim 13, characterized in that the polymer formed is a pharmaceutically acceptable salt of diphloretin phosphate. 15. The method according to claim 1, characterized in that the synthesized compounds having the formula where R is composed of OH and OX groups, R, R , R, R and R_ from H, X from methyl, ethyl, n-propyl and i-propyl- 6 groups and Z and Z consist of H. 16. The method according to claim 15, characterized in that R, R "and R ₁ . OX groups and R ^, R "and R ^ are H, do 2 4 6 17. The method according to claim 15, characterized in that R, R "and R_ OX groups and R ^, R" and R are H. 4 5 ^ 2 3 6 209839/1221 18. The method according to claim 15, characterized in that R and R are OX groups, R X and R, R and R are H. 19. The method according to claim 15, characterized in that Z and Z are phenyl and benzyl groups. 20. The method according to claim 1, characterized in that the synthesized compound has the following formula: Il O - P OH - 0 - where η is a positive integer from 2-4, Y, Y and Y 15 Θ consist of OH and H and Y, Y and Y consist of OH, H and one Group with the following formula: tn -f- NS , and -0-CH _r 209839/1221 where t is H, CH ₀ , F, Cl and OCH, η is a positive integer from 1-5 and Y, Y, Y ₀ , Y, Y and Y_ equal or less than 1 el ο 4 5 6 can be different. 21. The method according to claim 20, characterized in that Y, Y ₃ and Y OH, H, -0-Q-CH, and substituents with the following formulas can be: 0-CH ₂ -
and 22. The method according to claim 20, characterized in that Y2, Y ₀ and Y exist. Y and Y from OH, H and alkyl groups with 1-6 carbon atoms 23. Process for separating low molecular weight polymeric phosphoric acid esters of high molecular weight polymeric phosphoric acid esters, in which the phosphorus radicals by means of ester bonds are linked by organic groups, each organic group being a glycoside residue of a poly hydroxy compound, such as Flavones, flavanones, chalcones and dihydrochalcones are characterized in that the high molecular weight polymeric phosphoric acid esters be guided through a material, with JÖem a physical Molecular fractionation can be carried out, 209839/1221 and recovering that portion of the polymeric phosphoric acid esters having a molecular weight less than about 1,400 which material can be used in dialysis. j q ^ z. γ L (» tts"' ^IV1OT '" ³ ^ ¹¹ ^ ¹ '*"" ⁱ ^ ^r - ^ ei ^ _e sii2__5 ^: ' ^ ^c ' ^u ' ^ ^c '^ 9 ^e f ^<e h'ISe ** ^ ⁿ ^ ' ^: > that people / \ or animals a wirH <samäj ^ S358 ^ Äe ^^ edermolecular pro- i I njl iiliii "Jjj ^ >> fTTTTii 11 n 11 claim-! _Γ -2, S, -4 -, - Compounds consisting of polymeric phosphoric acid esters, in which the phosphoric acid radicals through ester bonds organic groups are connected, each organic group being a glycoside residue of a polyhydroxy compound, such as flavones, flavanones, Chalcones and dihydrochalcones are characterized in that the polymeric phosphoric acid esters are prostaglandin antagonists which have essentially no anti-hyaluronidase activity and a molecular weight of less than about 1,400. Compound according to claim 25, characterized in that the polymeric phosphoric acid esters are made from phospholyphloretin phosphate, polymethylphloretin phosphate, Polyquercetin phosphate, polynaringenin phosphate and polyhesperetin phosphate exist. Polyphloretin phosphate having a molecular weight less than about 1,400. '. Diphloretin phosphate. Pharmaceutically acceptable salts of the compound of claim 28. _{according to input #} ORIGINAL INSPECTED rfngegongen on ^ 209839/1221 A compound of Claim 29 together with a suitable pharmaceutical carrier. Compounds according to claim 1, characterized by a formula those from the groups AB-PO-BA;
AB-PO-BA-PO-BA; AB-PO-BA-PO-AB-PO-BA; and 4 4 4 ' PO ₄ (AB) ₃ consists, ^ wherein A is a group of the formula consists in which R is made up of OH and X, R is made up of H, Cl, FC, F and 1 2 3 X, R, R and R from H, Cl, F, NO, OH, X and OX, X from ο 4 ο 2 Methyl, ethyl, n-propyl or i-propyl groups and wherein B is a group of the formula is where R ^ consists of H, F and a phenyl group, the b Group AB is formed by connecting the 4-position of B with the terminal carbon atom of the alkyl chain of A and the AB units thus formed by a phosphate 209839/1221 group connected to the 1-position of each B-ring hangs. Pharmaceutically applicable salts of the compounds according to claim 31. Compounds according to claim 1, characterized by the formula: O 1 where R is made up of OH and OX, R, R, R, R and R is made up of H, X Ί c. w 4 O w a methyl, ethyl, n-propyl and i-propyl group and Z and Z consist of H. 3§. Compounds according to claim 1, characterized by the formula: 209839/1221 where η is a positive integer from 2-4, Y, Y and Y_ QH- 15 6 Groups and H, and where Y, Y and Y are OH, H and a group of the following formulas, -CH .tn , and -0-CH wherein t is a group such as H, CH, F, Cl and OCH and wherein 3 3 η is a positive integer from 1-5 and Y, Y, Y, Y, Y and Y can be the same and different. 6th 3ψ. Connection according to Claim 34, characterized in that Y, Y and Y groups such as OH., H ₅ -0-C-CH ₅ 209339/12 M and 0-CH - // V could be. Compounds according to claim 34, characterized in that Y, Y and Y are OH, H and alkyl groups with 1-6 carbon atoms 209839/1 221