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Publication numberUS3716631 A
Publication typeGrant
Publication dateFeb 13, 1973
Filing dateFeb 24, 1969
Priority dateFeb 23, 1968
Also published asDE1908804A1
Publication numberUS 3716631 A, US 3716631A, US-A-3716631, US3716631 A, US3716631A
InventorsHoutman A, Steggerda J
Original AssigneePhilips Corp
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Use of radioactive compounds to determine fat absorption
US 3716631 A
Abstract
Use of radioactive labeled phenylsubstituted fatty acids to determine disturbances in fat metabolism in animals, the degree of disturbance being determined by measurement of a radioactive degradation product excreted in the urine.
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1:; v g mte States Patent 1 [1 1 3,716,631 Steggerda et al. 1 Feb. 13, 1973 [54] USE OF RADIOACTIVE COMPOUNDS [58] Field of Search ..424/l, 9; 23/230; 128/2 TO DETERMINE FAT ABSORPTION [75 Inventors: Jan Frederik Steggerda, Emmasin- [56] References cued gel, Eindhoven; Al'y Cornelis Houtman, Van Houtenlaan, Weesp, both of Netherlands 3,061,510 10/1962 Numerof et al. ..424/1 4 h [73] Assignee: U.S. Philips Corporation, New Cor- 66'361 9/1969 RIC ards poranon Primary Examiner-Reuben Epstein [22] Filed: Feb. 24, 1969 AttorneyFrank R. Trifari 2 A 1. N 0 771 I 1 PP 0 8 57 ABSTRACT [30] Foreign Application Priority Data Use of radioaotive labeled phenylsubstituted fatiy V acids to determine disturbances m fat metabolism in F6). 23, Netherlands animals tha degree of disturbance being determined by measurement of a radioactive degradation product [52] 11.8. C1. ..424/1, 424/9, 23/230, excreted in the firm 260/408, 260/413 [51] Int. Cl. ..A6lk 27/04 5 Claims, No Drawings USE OF RADIOACTIVE COMPOUNDS TO DETERMINE FAT ABSORPTION The invention relates to the absorption of fats through the intestinal wall and has for its object to find disturbances in the fat absorption and to establish the extent of the disturbance.

A frequently used method is that in which disturbances in the fat absorption are determined by faces examination. For this purpose a known quantity of fat is administered to a patient for a period of a few days, usually 5 days, the faces excreted during the last few days of the above-mentioned period are collected, and the quantity of fat present in the feces is then determined quantitatively. The difference between the administered and excreted quantity of fat is assumed to be absorbed in the body. It is to be noted that the first few days on which the fat diet is administered is considered as a starting period in which no feces examination takes place. Furthermore it is a first requisite for the success of the examination that the quantity of fat determined in the feces originates exclusively from a known quantity of administered fat. In order to establish this with certainty, inert indicators are used which are administered together with -the fat and limit the quantity of feces to be examined. This method of determining the absorption of fats has the following drawbacks:

a. A long observation period in a hospital which makes the method very expensive.

b. The accuracy is low and strongly depends upon human factors. There is no possibility of correction when portions of feces are lost.

c. The therapy can be started comparatively late.

d. Collecting and examining feces is an unpleasant and time-consuming job.

According to another method of determining fat absorption, an accurately determined quantity of radioactive labelled iodine glyceroltrioleate is administered to the patient. At given instants after the administration of the radioactive preparation the patient is bled and the radioactivity of the blood samples is determined.

This method has the drawback that the examination is unpleasant to the patient and the accuracy to be desired in many cases because the method is based on comparative examination and the patient must satisfy certain standard conditions. Moreover precautions must be taken that no supply of radioactive iodine takes place in the body, notably in the thyroid gland. It is therefor desirable that during the examination according to this method the thyroid gland is blocked.

Applicants have now found a method of determining disturbances in the fat absorption which does not have the abovementioned drawbacks. The method according to the invention is based on the recognition of the fact that disturbances in the fat absorption can be determined by means of a urine examination. It has been found more particularly that disturbances in fat equilibrium can be determined by using compounds which, as regards the intestinal absorption, behave as fatty acids or esters of fatty acids, for example, glycerol esters of fatty acids, and after absorption in the body are degradated therein, at least one degradation product coming in the urine.

In order to be able to determine quantitatively the part of the compound according to the invention excreted in the urine, the compounds according to the invention are labelled radioactive and that in such manner that upon degradation of the compound in the body at least the degradation product which has come in the urine is radioactive.

As compared with the known methods, the method according to the invention has the following advantages:

a. It is sufficient to collect the urine during a period of maximally 24 hours after the instant of administration of the radioactive compounds.

b. The method is very accurate. Corrections for any lost portions of urine are possible.

0. Therapy may be started very rapidly.

d. Collecting and processing urine is no unpleasant job.

e. The absorption through the intestinal wall is measured and not, as is the case with the method on feces examination, the sum of the non-absorbed fat, the fat excreted through the intestinal wall out of the body, and the bacterial fat.

f. The urine examination is very simple. It is sufficient to determine the volume of the received urine and the radioactivity in a part thereof.

Radioactive compounds according to the invention which are suitable for use in determining disturbances of the fat equilibrium may be represented by the general formula I in which formula X hydroxy group, an alkoxy group containing one to three carbon atoms or a branched or non-branched alkyl group having one to four carbon atoms,

Hal a halogen atom chosen from the group consisting of a chlorine, bromine and iodine atom,

R carboxyl group, COOMe group, in which Me is a metal atom and preferably an alkali metal atom, or a COOR' group, wherein R is an aliphatic hydrocarbon radical which may be substituted with one to two hydroxy groups, I

m O or 1 n 0, l or 2 p 2-20 (inclusive) and in which formula the broken line denoted by r.a. denotes that the part of the compound enclosed by that line is labelled with radioactivity.

It has been found more particularly that the compounds according to the above formula, in which p has the value 4-12, are very suitable for use in determining the extent of disturbances in the fat absorption.

Very good results are obtained with compounds of the formula are:

Dacronx 3 BQ-wmm-ooox 3 Q-wnm-c o 0 CH3 :11

In these compounds the symbol H means that the phenyl nucleus with tritium is labelled radioactive (generally labelled), the symbol C that the phenyl nucleus contains radioactive C atoms, the symbol I denotes a iodine isotope 1 or I) and X means a hydrogen atom or an alkali metal atom.

The radioactive compounds according to the invention are new and may be prepared in a manner known for similar compounds or in a manner analogous thereto.

for example, the compounds according to the invention may be prepared by: 1

Converting a compound of formula l 1 2 c-(oH1)p-1R Xm I (HaDn in which formula X is an alkoxy group having one to three carbon atoms or a branched or non-branched alkyl group having one to four carbon atoms and Hal, R, r.a., m, n and p have the same meanings as denoted with the general formula I, with a suitable reduction agent into a compound of formula.

in which formula the symbols X, Hal, R, r.a., m, n and p have the above meanings;

b. alkylating a compound of the formula in which formula Hal, R, r.a. n and p have the meanings denoted with formula I and X is a hydrogen atom or an alkyl group having one to four carbon atoms, in which a compound of the formula is obtained, in which formula X, l-Ial, R, r.a., n and p have the above meanings and Alk is an alkyl group having one to three carbon atoms;

c. hydrolyzing a compound of the formula in which formula X, Hal, r.a., m, n and p have the above meanings and R" is a hydrocarbon radial or an alkali metal atom, in which a compound of the formula.

. H l n r.a. L

is obtained, in which formula I-Ial, r.a., m, n and p have the above meanings and X is a hydroxy group or a branched or non-branched alkyl group having one to four carbon atoms;

d. converting a compound of the formula in which formula l-lal, r.a. X, m, n and p have the above meanings, into the corresponding ester or salt which may be represented by the formula (Hah 1 a.r.- L J in which formula X, Hal, r.a., m, n and p have the above meanings and R is a COOMe group, wherein Me is a metal atom and preferably an alkali metal atom, or a group COOR; in which latter group R is an aliphatic hydrocarbon radical which may contain one to two hydroxy groups;

e. halogenating a compound of the formula in which formula X, R, m and p have the above meanings with radioactive halogen, or a halogen compound in which a compound of the formula in which formula X, R and m have the above meanings, (Hal*),, is a radioactive halogen atom and n 1 or 2, is obtained; a

f. reducing a compound of the formula in which formula X is a hydroxy group, an alkoxy group having one to three carbon atoms or a branched or non-branched alkyl group having one to four carbon atoms, Hal is a halogen atom selected from the group consisting of a chlorine, bromine and iodine atom, R is a lower alkyl group and r.a. denotes that the part of the compound enclosed b the broken line is radioactive m 0 or 1' n 0, or 2 and p 2-20 to a compound of the formula in which formula X, Hal, R, r.a., m, n and p have the above meanings;

g. reducing a compound of the formula in which formula X is a hydroxy group, an alkoxy group having one to three carbon atoms or a branched or non-branched alkyl group having one to four carbon atoms, Hal is a halogen atom selected from the group consisting of a chlorine, bromine and iodine atom,

R is a lower alkyl group and r.a. denotes that the part of the compound enclosed by the broken line is radioactive,

m=0orl n=0, 1 or2and p=420 to form a compound of the formula in which formula X, Hal, R, r.a., m, n and p have the above meanings; V g g h. saponifying with alkali a compound of the formula in which a compound of the formula is obtained, in which formula X, Hal, R r.a., m, n and p have the above meanings;

i. reducing a compound of the formula r "1 0 -(C111) CI 2-C-(CHZ)q-2 C I ll Xm 1 0H r.a.-l (HaDu in which formula X is an alkoxy group having one to three carbon atoms or is a branched or non-branched alkyl group having one to four carbon atoms, Hal is a halogen atom selected from the group consisting of a chlorine, bromine and iodine atom, r.a. denotes that the part of the compound enclosed by the broken line is radioactive m l 0 or 1 n 0, l or 2 q 3 or 4 and p 3 20, in which a compound of the formula o 1 E 4 o1n)po 1'. a. E Xm OR is obtained, in which formula X, Hal, R, r.a., m, n and p have the above meanings;

k. reducing a compound of the formula l "1 f f onl t-o-(oHnwo Ex... "l'" 5 OH l'.:t.--- I (llnlh.

in which formula X is an alkoxy group having one to three carbon atoms or a branched or non-branched alkyl group having one to four carbon atoms, Hal is a halogen atom selected from the group consisting of a chlorine, bromine and iodine atom,

r.a. denotes that the part of the compound enclosed by the broken line is radioactive,

m O or 1 n O, l or 2 and p 6 -20, in which a compound of the formula is obtained, in which formula X, Hal, r.a., m, n and p have the above meanings.

The chemical conversions mentioned in the above methods (a -k), for example, reduction of a ketone (methods a, i and k) alkylation (method b), saponification (methods c and h), esterification (method d), halogenation (method e) and reduction of a double bond (methods f and g) may be carried out according to conventional chemical methods and those skilled in the art will experience no difficulties in preparing the compounds according to the invention by the above methods.

A further explanation of the chemical conversions used is given below, the indications a to k referring to the above methods a to k. re a, i and k The reduction may be carried out, for example, by treatmentwith hydrazine and alkali according to the method by Wolff Kishner or the Huang Minlon variation thereto. It is alternatively possible to reduce with zinc amalgam and hydrochloric acid according to the method by Clemmcnsen. A catalytic hydrogenation may also be carried out, for example, with a palladium on carbon catalyst. re b The alkylation of phenols is preferably carried out by reaction with, for example, dimethylsulphate. The phenol is reacted in an alkaline solution with the said reagent, initially at room temperature and then on steam bath. Alkylation may also be carried out by reaction with an alkyl halide in the presence of a base or after the starting product has been converted into the acid chloride, for example, by reaction with thionyl chloride, PCl or PCI S converting this acid chloride with an alcoholate, for example, sodium ethanolate. re c and h For the saponification of esters the conventional methods may be used, for example, heating with an alcoholic NaOH solution succeeded by acidification and pouring in water. re d The m-phenyl fatty acids may be esterified by reacting the acids for a longer period of time with alcohols and preferably with absolute ethanol at a temperature which may vary between room temperature and the boiling point of the reagent. The reaction may be catalyzed by a small quantity of a mineral acid. The esters may also be prepared by first converting the acids with thionyl chloride or a similar acid chlorination agent to the corresponding acid chlorides which are then converted in alkaline medium with an alcohol. When the above m-phenyl fatty acids are esterified with glycerin it is to be preferred to carry out the reaction in an inert solvent, for example, benzene or chloroform.

re e

For the halogenation of the aromatic ring use is preferably made of radioactive N-bromosuccinimide, N-chlorosuccinimide or a solution of KJ-J, in an NaOH-boric acid buffer (J. Org. Chem. 30, 304 (1965 re f and g The reduction of a double bond may be carried out, for example, with zinc amalgam or mercury amalgam according to the method by Clemmensen or catalytically, for example, under the influence of a platinum catalyst or a palladium on carbon catalyst.

The starting products in the preparation of the compounds according to the invention may also be prepared according to known methods or according to methods analogous thereto. For example, these starting substances may be obtained by reacting a compound of the formula in which formula X is a hydroxy group, an alkoxy group having one to three carbon atoms or an alkyl group having one to four carbon atoms and m or 1, with a compound of the formula in which p 2 -20 (inclusive) in the presence of a Friedel Crafts catalyst and at a temperature of 0 5 C, in which a compound of the formula X O H:

. .10 isobtained, in which formula X, m and p have the above meanings.

It is also possible first to hydrolyse the reaction' product with alkali to the corresponding acid, in which an alkoxy group present in the phenyl nucleus is converted into a hydroxy group and then to reduce the resulting product. The resulting compounds in the ester form or as an acid may serve as reaction products for the mode of preparation c.

It will be obvious that if the above-mentioned compounds are halogenated in the acid form, the resulting radioactive acid may serve as a starting product for the methods mentioned sub b and d according to which the phenol group is alkylated (method b) and the radioactive acid is converted into the ester or the salt thereof (method d), respectively. When the above-mentioned compound of the formula m OCH;

is halogenated directly with radioactive halogen or a halogen compound, the resulting radioactive ester be converted subsequently, if desired, by hydrolysis into the corresponding acid (method 0).

Another mode of preparing the starting products consists in acylating radioactive benzene or a radioactive benzene derivative of the formula 1 OCH:

in which formula p 2-20 In the presence of a Friedel- Crafts catalyst, for example AlCl at a temperature of 05 C, a compound of the formula (HaDn OCHa being obtained, in which formula X, Hal, m, n and p have the above meanings and r.a. denotes that the part surrounded by the broken line is labelled radioactive.

The radioactive compounds thus prepared may then be converted into the compounds according to the invention by using the reduction mentioned sub a.

The above radioactive benzene or benzene derivative mentioned as a starting substance contains one or several radioactive atoms, for example, a tritium atom, a C atom or a atom. The position of the radioactive atom in the benzene (derivative) molecule is of no essential importance. For example, the radioactive atom may form part of the benzene ring or be in the substituent of the benzene ring denoted by X. It is alternatively possible that the halogen substituent of the benzene ring is radioactive. If the benzene derivative contains two halogen atoms, both atoms or one of both atoms may be radioactive.

The starting products of the method mentioned sub f may be obtained by reacting a compound of the formula in which formula X, Hal, r.a., m, n and p have the meanings mentioned with the method f, with the methyl ester or ethyl ester of bromoacetic acid, a compound of the formula being obtained, in which formula X, Hal, r.a., m, n and p have the above meanings and R is a methyl group or an ethyl group and then dehydrating the resulting product.

The starting substances of method g are prepared in the same manner as described above, but with this difference that instead of the methyl ester or ethyl ester of bromoacitic acid, the methyl ester or ethyl ester of bromocrotonic acid is used. The above-mentioned aldehyde starting product may be prepared by hydrogenating the corresponding acid chloride catalytically (Rosemund reduction) or reducing the corresponding ester according to the method by Bouveault-Blanc.

The starting products of the method mentioned sub h may be prepared by reducing a compound of the formula 0R u 1 l. 1

in which formula X, Hal, r.a., p, m and n have the meanings mentioned with method and R is a lower alkyl group, with LiAllH and converting the resulting in which formulas X, Hal, r.a., m, n and p have the above meanings and R is a lower alkyl group.

The starting substances used in method i are obtained by reacting a compound of the formula A CH Br, in which A is the group in which group X, Hal, r.a., m, n, p and q have the meanings indicated with method i, with Mg to form a compound of the formula ACh Mg-Br and reacting the resulting product in the presence of CdCl with a compound of the formula O OH in which formula q has the above meaning.

The starting substances of method k can be prepared by reacting a compound of the formula in which A is the group in which group X, Hal, r.a., m, n and p have the meanings denoted with method k, with a compound of the formula is obtained.

This latter compound is hydrolyzed with acid to form a compound of the formula I and then saponified with alkali, the starting product of the method mentioned sub k being obtained.

The cyclohexane morpholine reagent may be prepared according to known methods (Berichte 90,

2833 (1957) and 91,129 (1958)).

The preparation of specific compounds according to the invention is described in detail with reference to examples which are included at the end of the description.

If a compound according to the invention contains a tritium atom a C atom, the compound produces a B radiation, whereas upon application of, for example, a .1 atom, the compounds according to the invention show a radiation. Applicants have found that both compounds having B radiation and 7 radiation may be used in the method according to the invention for determining disturbances of the fat absorption.

The use of compounds labelled with 'y radiators has the advantage that the analysis apparatus is simple, cheap and easy to operate. Notably the latter advantage is of importance since errors of analysis are minimized.

The compounds according to the invention can be processed in the conventional manner to pharmaceutical compositions suitable for oral administration, for example, tablets, coated tablets, capsules, water-soluble powders and oil dispersions. In such pharmaceutical dosage units forms the active substance is mixed with or dissolve or dispersed in a liquid or solid carrier, if desired while adding surface-active substances, for example, emulsifiers, furthermore lubricants, binding agents, flavoring substances and similar substances.

For the preparation of a number of compositions according to the invention the prescriptions below may be used. In these prescriptions the expression active substance means tritiated phenyloctyl-carboxylic acid or the sodium salt thereof.

Prescriptions 1. Tablet five grns of active substance are mixed with 100 grns of lactose, 50 gms of sucrose and gms of a binding agent.

The mixture is granulated with water and 5 percent potato-starch, 2 percent talcum and 1 percent magnesium stearate are added to the granulate.

The resulting product is tabletted in the conventional manner.

2. Capsule five mgms of active substance are mixed with so much microcrystalline lactose (approximately 95 mgms) that a hardened gelatin capsule can be filled with the mixture.

3. Capsule five mgms of active substance are diluted with arachis oil and incorporated in a gelatin capsule.

4. Aqueous solution An aqueous solution is obtained by taking up 5 percent by weight of active substance in water to which approximately 0.1 percent of a preservative, for example, esters of p-hydroxybenzoic acid, has been added.

The success of the method of examination according to the invention depends upon the accuracy with which the quantity of radioactive compound which has come in the urine and hence the quantity of radioactivity occurring in the urine can be determined. The accuracy greatly depends upon the recording power of the analysis apparatus used with regard to the used radioactivity ([3 or 7 radiation). For good results a minimum quantity of radioactivity occurring in the urine is necessary. The dosing of the composition according to the invention is therefore preferably expressed in a quantity of radioactivity to be administered. Taking into account possible differences between administered quantity of radioactivity and quantity of radioactivity coming in the urine, which differences are dependent inter alia on the extent to which a disturbance in fat equilibrium is present and on the type of compound used, it may be said that when using compounds which produce a 7 radiation an administered quantity of radioactivity of 0.1 to 10 p. Ci gives good results and when using [3 radiation a quantity of from 5-100 ,1 Ci.

For the preparation of the composition according to the invention it is desirable to convert the above-men tioned quantity of radioactivity to be administered into a quantity of the radioactive-compound to be administered, or to be processed in pharmaceutical compositions. Since the quantity of radioactivity which is produced by a compound according to the invention depends upon the number of radioactive atoms which the compound contains and the value of the radioactivity of the isotope used (half lifetime), the quantity of active compound to be administered or to be processed, will vary for each individual compound in accordance with specific radioactivity of the compound. In general a dose of from 0.1 to 50 mgms of active compound will give the desired results.

From the investigations which led to the present invention it has been found that after the administration of a composition according to the invention to man or animal, the active compounds present in the composition are taken up quantitatively in the body or for a certain percentage through the small intestine under the influence of secretion products of the liver (emulsifying activity) and the enzyme lipase originating from the pancreas (saponification). It has furthermore been found that the compounds according to the invention are degradated in the body according to the theory by Knoop to benzoic acid derivatives if an even number of CH-,. groups is present between the phenyl group and the carboxylic acid group or to phenyl-acetic acid derivatives if the number of -CH groups is odd. The theory by Knoop involves that upon degradation of fatty acids in the body each time two carbon atoms are split off. In order to obtain the intermediate degradation products, Knoop used fatty acids with an w-phenyl group namely phenylcapronic acid and phenylvaleric acid. The final products of the degradation (benzoic acid and phenyl acid) are coupled in the liver with glycine to phenylacetal glycine and hippuric acid and then secreted in the urine.

Applicants have found that the compounds according to the invention after degradation in the body to the above-mentioned benzoic acid derivatives and phenyl acetic acid derivatives are likewise detoxicated in the liver by coupling with glycine and are then secreted in the urine. It has been found, for example, that a quantity of S-tritiated phenyl-octyl-carboxylic acid-l administered to healthy people comes in the urine for substantially percent as radioactive hippuric acid.

The percentage of active compound which upon administration of a composition according to the invention is taken up in the body through the small intestine is determined by measuring the quantity of radioactivity of the collected urine fractions and expressing it in per cent of the total quantity of administered radioactivity.

In order to establish the correlation between the absorption of a compound according to the invention and of a fat, an accurately determined quantity of fat was administered to a teste and it was investigated by feces examination what part hereof comes in the feces. It is assumed that the remaining part has been absorbed in the body. Comparisons of the results of this experiment with those of an absorption experiment carried out with compounds according to the invention demonstrated that some compounds according to the invention, for example, 8-tritiated phenyl-octyl-carboxylic acid-l are absorbed in the body for the same percentage as an administered fat through the small intestine. Other compounds, for example, 8-(p-hydroxy-m,m-di iodophenyl)-octane-carboxylic acid-l, were found to be absorbed by the body for a smaller percentage as compared with an administered fat. The last-mentioned group of compounds nevertheless is suitable for establishing disturbances of fat absorption since differences in fat absorption are expressed in differences in absorption of the said compounds according to the invention. In order to arrive in this case at a good judgement of disturbances of fat absorption, correlation graphs must be drawn up in which, for example, the percentage of radioactivity secreted in the urine is plotted against the value of the disturbance of the fat absorption. The results obtained in an experiment should be interpreted with reference to these graphs. Furthermore it is pointed out that experiments in rats have demonstrated that the radioactive degradation product of a compound according to the invention is not stored in the body but is excreted quantitatively or substantially quantitatively in feces and or urine.

In order that the invention may be readily carried into effect it will now be described in greater detail with reference to the ensuing specific examples.

1. Determination of disturbances in the fat absorption by using 8-tritiated-phenyl-octano-carboxylic acidfive mgms (40 p. Ci) of S-tritiated phenyl-octano-carboxylic-acid 1 dissolved in arachis oil or as the sodium salt in water was administered to healthy test persons and patients suffering from steatorrhoea. Of the urine obtained at certain instants after administration the volume was measured. Then 0.5 m1 hereof were pipetted and radiation was determined in Mead and Stiglitz counting liquid first without and then with an internal standard by means of the Tri-Carb. By crystallization to constant specific activity the metabolites which were secreted in the urine were determined. Fecal fats were extracted from feces homogenates (approximately 3 gms) after acidification of the homogenates with concentrated HCl (0.1 ml per gram of homogenate) by adding 5 times the volume of methanol.. After centrifuging the residue was washed two times with the same volume of methanol. The combined methanol extracts were evaporated to approximately 8 mls under reduced pressure. Then mils of water were added after which this solution was shaken three times with 20 mls of toluene. The toluene-extracted dyes were removed by passing the combined toluene extracts through a column of silicagel and then recovering them quantitatively by washing the column with toluene. The purified toluene extract was evaporated under reduced pressure and a part of the residue was counted in toluene-POPOP-PPO scintilation liquid.

It has been found that the orally administered quantity of radioactivity, independent of the formulations used, is rapidly excreted for substantially 100 percent in the urine by healthy persons. In the patients under examination the excreted quantity of radioactivity in the urine after oral administration of the same dose is lower than in healthy persons. From at equilibrium experiments it has been found that the difference between the administered quantity of radioactivity and the quantity of radioactivity in the urine, expressed in percent of the administered quantity of radioactivity, corresponds to the percentage of fat not absorbed by the body. A radioactivity determination in the feces demonstrated in addition that no radioactive degradation product is stored in the body since the sum of the quantities of radioactivity found in the feces and in the urine is equal to the administered quantity.

2. Identification of metabolites of S-tritiated phenyloctane-carboxylic acid-l in the urine.

It was investigated whether the H activity which is excreted with the urine after an oral administration of 8-tritiated phenyl-octane-carboxylic acid-l was coupled to hippuric acid.

For this purpose 2.250 gms of hippuric acid as sodium salt were dissolved in a quantity of urine which contained 9.9 X 10 dpm l-I activity. If all the H activity originates from hippuric acid, the specific activity must be 4.40 X 10 dpm/g of hippuric acid. Of the percipitate which was obtained after dissolving 30 gms of NaCl per 100 mls of urine the specific activity was determined as well as of the crystals which were obtained after crystallization of the precipitate from ethanol and of the crystals which were obtained after crystallization from water of the crystals obtained from ethanol. From the results it appeared that substantially all the radioactivity present in the urine was coupled to hippuric acid.

3. Determination of disturbances in the fat absorption by using S-tritiated carboxylic acid.

One mg 15 p. Ci) of l4-tritiated-phenyl-tetradecane carboxylic acid was dissolved in alcohol and, after admixture to food, administered to healthy persons and to patients suffering from steatorrhoea. Of the urine obtained at given instants after administration the volume was measured. 0.5 ml hereof was pipetted and examined in the same manner as described in example 1. It was found that the orally administered quantity of radioactivity was secreted in the urine rapidly and for percent by healthy persons. In patients suffering from steatorrhoea a quantity of radioactivity secreted in the urine was found which varied from 25 to 50 percent of the administered quantity of radioactive composition, dependent upon the severity of the disturbance and slightly dependent upon the patients age.

7-p-methoxybenzoyl-heptane-carboxylic acid-1 fifty three gms of dry AlCl were added while stirring under anhydrous conditions to 23 gms of anisol in 100 ml of tetrachloroethane at a temperature of from 5 to C. 47 gms of monomethyl ester of azelaic acid in 100 mls of tetrachloroethane were then added at a temperature between and 0C. The reaction mixture was stirred at a temperature of from -5 to 0C for 3 to 4 hours, then slowly heated to room temperature and then poured in icy-water. The excess of anisol and the tetrachloroethane were removed by steam distillation and the ester was extracted from the water with 3 X 200 mls of ether. After drying the ether extracts on Na the ether was evaporated and the ester saponified by refluxing in 150 mls of percent ethanolic sodium hydroxide solution. The saponification mixture was made up with water to twice the volume and the alcohol was distilled off. The solution was then neutralized with HCl, treated with norit, filtered and acidified. After cooling the precipitated 7- methoxybenzoyl-heptane-carboxylic acid-1 was flltered off. Yield 40.1 gms, melting point 83-86C.

5. 8-p-methoxyphenyl-octane-carboxylic acid-1 thirty five gms of 7-p-methoxybenzoyl-heptane-carboxylic acid-1, 12.5 mls of hydrazine hydrate 90 percent, 239 gms of KOH and 130 mls of diethylene glycol were mixed and refluxed for 1 hour. The excess of hydrazine hydrate and water was then distilled off and the reaction mixture heated at 200 C for 2 hours. After cooling, again hydrazine hydrate (10 mls) was added, again refluxed for 1 hour and after distillation of the excess of hydrazine hydrate and water again heated at 200 C for 2 hours. After cooling the reaction mix-. -ture was poured out in dilute HCl. The 8-p-methoxyphenyl-octane-carboxylic acid-l separated. Yield of crude product 28.3 gms (86 percent), melting point 50-53.

6. S-p-hydroxyphenyl-octane-carboxylic acid 1 twenty five gms of (crude) 8-p-methoxyphenyl-octane-carboxylic acid 1 were refluxed for 4 hours with 75 mls of H] (s.g. 1.7). After the addition of 50 mls of water the formed CH was distilled off. The distillation residue was then poured out in 4 X the volume of water and the formed precipitate of 8-p-hydroxy'phenyl-octame-carboxylic acid-l was filtered off and processed in the normal manner. Yield 23 gms (96 percent), melting point 98-103.

7. 8-(p-hydroxy-m,mdiiodo)phenyl-octane-carboxylic acid-1.

Two gms of 8-p-hydroxyphenyl-octane-carboxylic acid were dissolved in a buffered medium which consisted of 1.3 gms of NaOl-l, 9.9 gms of boric acid and 270 mls of water. To this solution was added at room temperature a radioactive solution (lmCi 'J, or 10 mCi J) consisting of 4.13 gms of J and 2.7 gms of K] in water. After leaving to stand for a short time the reaction mixture was acidified and the precipitated 8- (p-hydroxy-m,m-diiodo)phenyl-octane-carboxylic acid-l was filtered off, washed, dried and recrystallized from benzene-petroleumether. Yield of crude product 3.6 gms, melting point 8587 C.

8. 8-(p-methox.y-m,m-diiodo)phenyl-octane-carboxylic acid-l. acid-l.

3.5 gms of 8-(p-hydroxy-m,m-diiodo)phenyl-octanecarboxylic acid-1 were dissolved in 150 ml of methanol, the solution was heated and 5 mls of dimethylsulphate were added to the solution. The reaction mixture was kept alkaline by the addition of a KOH solution in water after which another 2 mls of dimethylsulphate were added. After the second addi tion the reaction mixture had an acid reaction and 50 mls of water were added to the mixture. The methanol was distilled off and the precipitated 8-(p-methoxym,m-diiodo)phenyl-octane-carboxylic phenyl-octanecarboxylic acid-l was crystallized from methanolwater. Yield 3.1 gms (84 percent), melting point 3739 C.

9. 83H-phenyl-octane carboxylic acid-1 1.20 gms of anhydrous AlCl were added to a solution of 3.96 gms of tritiated benzene in 22 mls of tetrachloroethane. A solution of 9.90 gms of 7- (chloroformyl)-l-heptane carboxylic acid methyl ester in 15 mls of tetrachloroethane was added dropwise while stirring and cooling with ice. The resulting reaction mixture is slowly heated to 90 C. and stirred at this temperature for 2 hours. After cooling, the reaction mixture is decomposed by the dropwise addition of 30 mls of 10 percent hydrochloric acid.

The tetrachloroethane layer is separated and the water layer is extracted with benzene. The combined organic extracts are washed with water, dried on Na SQ, and evaporated. The resulting ester is saponified by dissolving it in 30 mls of ethanol and ad ding to it a solution of 3.3 gms of NaOH in 19 mls of water. After heating on a steam bath for 30 minutes the solution is cooled, the sodium salt of 7 H-benzoylheptane carboxylic acid crystallizing out. After sucking off and washing with percent ethanol the resulting sodium salt is dissolved in 50 mls of water. This solution is filtered and the filtrate is acidified, the 7 H- benzoyl-heptane-carboxylic acid-l precipitating, melting point 7879 C. 20 mls of diethylene glycol, 8.5 mls of hydrazine hydrate 80 percent, 5.6 gms of KOH and 5.5 gms of 7 l l-benzoyl heptane carboxylic acid- 1 were mixed and refluxed for 2 hours. The excess of hydrazine hydrate and the water formed were distilled off until the reaction mixture reacted a temperature of 220 C. The mixture was heated at this temperature for 3 hours. After cooling, the reaction mixture was dissolved in 200 mls of water and this solution was acidified with hydrochloric acid. The precipitated oil was extracted with ether, the ethoreal extract was washed with water, dried on Na SO and then evaporated. The residue was distilled in vacuo. Yield 4.79 gms of 8 I-I-phenyl-octane carboxylic acid-1. Boiling point 160 C at 0.2 mm. Melting point 35-37 C.

10. 4-Tritiated-phenyl butane carboxylic acid-1 A solution of 3 gms of tritiated bromobenzene in 10 mls of absolute ether was added by stirring to 0.45 mgms of Mg curls in 2 mls of absolute ether and the resulting mixture was stirred for some time. A solution of 1.7 gms of cyclopentanone in 15 mls of absolute ether was then added dropwise and the solution was stirred again. By the addition of 15 ml of 1N HCl the reaction mixture was then decomposed. Yield 2.72 gms of l-phenyl-cyclopentanol (83 percent), boiling point 121/6mm.

2.72 gms of l-phenylcyclopentanol dissolved in ml of glacial acetic acid were oxidized by the addition of CrO The reaction temperature was kept at 30 C and after completion of the reaction the reaction mixture was poured out in approximately 300 mls of water.

The reaction mixture was extracted times with totally 300 mls of ether and the combined ether extracts were washed with water until the color was bright. The ether was then evaporated in vacuo and the residue poured out in 300 mls of water, benzoylbutyric acid precipating. Yield 1.5 gms (43.4 percent), melting point 124-127 C.

1.04 gms of powdered NaOH were dissolved in a mixture of 1.3 ml of 85 percent hydrazine hydrate and 6.5 ml of diethylene glycol. 1.3 grns of benzoylbutyric acid were added to this mixture and the whole was refluxed. The water in the reaction mixture was distilled off and the residue kept at a temperature of 230 C for 3 hours.

After cooling the residue was taken up in mls of water and then neutralized with hydrochloric acid. The separated oil became rapidly crystalline and was recrystallized in ether. Yield 530 mgms. Melting point 59.5-60.5 C.

l l. S-Tritiated-phenyl pentane carboxylic acid-l In the same manner as described in Example 10, 0.45 grns of Mg, 3 gms of tritiated bromobenzene and 2.18 g of cyclohexanone were reacted. Yield 2.38 gms of 1- phenylcyclo hexanol. Melting point 61 .562.5 C.

2.38 grns of phenylcyclohexanol were dissolved in 90 mls of glacial acetic acid and oxidized at 30 C by the addition of 7.2 gms of CrO The reaction mixture was stirred for 1 hour, poured out in water and extracted with ether. The ether solution was then extracted with NaOH and the extract was acidified. Yield 0.85 grns of benzoylvaleric acid. Melting point 75 .576.5 C.

0.8 gms of NaOH were dissolved in a mixture of 5 mls of diethylene glycol and 1 ml of 85 percent hydrazine hydrate. To the mixture was added 1 gm of benzoylvaleric acid and the whole was refluxed for 1 hour. The reaction mixture was distilled at a temperature of 230 C and kept at this temperature for 3 hours. After processing the reaction mixture 0.8 grns of an oil were obtained. The oil was distilled in vacuo. Yield 0.4 gms of 5-tritiated-phenylpentane-carboxylic acid-1. Melting point 9.5l 1 C.

12. 14 l-I-phenyl tetradecane carboxylic acid-1 4.79 grns of 8 H-phenyl-octane carboxylic acid-l were added dropwise to 30 mls of thionyl chloride, after which the mixture was refluxed for 30 minutes. After evaporating thionyl chloride whereby traces of thionyl chloride were removed with benzene the resulting acid chloride was dissolved in 20 mls of dichloromethane and added dropwise, while stirring, to a solution of 3.5 grns of morpholino-cyclohexane-l and 2.5 grns of triethyl-amine in 25 mls of dichloromethane. The temperature was kept at 35C. At this temperature the mixture was stirred for another hour. The next day, 10 mls of 20 percent hydrochloric acid were added and the resulting mixture refluxed for 3 hours while stirring. The layer of dichloromethane was then separated, washed with water, dried and evaporated. The resulting residue was distilled in vacuo. Boiling point 188-192 C at 0.3 mm. 3.45 grns of 2-(9 I-l-phenyl-nonanoyl) cyclohexanone were heated on a steam bath for minutes with a solution of 1.9 gms of KOH in 2.4 mls of water. The solid mass was dissolved by the addition of 60 mls of water. The resulting solution was acidified, the 14 -"H-phenyl-6-oxo-tetradecane carboxylic acid-1 precipitating. After sucking-off and washing with water, there was recrystallized from methanol. Melting point: 98100 C.

10 mls of diethylene glucol, 0.7 mls of hydrazine hydrate percent, 0.50 gms of KOH and 1.24 grns of 14 H-phenyl-6-oxo-tetradecane carboxylic acid-1 were mixed and refluxed for 2 hours. The excess of hydrazine hydrate and the water formed were distilled off until the reaction mixture reached a temperature of 220 C. The it was heated at 220 C for 2 hours. After cooling, the reaction mixture was dissolved in 50 mls of water and this solution was acidified with hydrochloric acid. The precipitated 14 l-l-phenyl-tetradecane carboxylic acid-l was sucked off, washed with water dried. The precipitate was recrystallized from petroleum ether. Melting point 6263 C.

What is claimed is:

What is claimed is:

l. A method of determining the degree of fat or fatty acid absorption in a mammal, said method comprising orally administering to said mammal a measured amount of a radio-active compound of the formula wherein X is a moiety selected from the group consisting of hydroxy, alkoxy of l-3 carbon atoms and alkyl of 14 carbon atoms, Hal is a halogen selected from the group consisting of chlorine, bromine and iodine, R is a moiety selected from the group consisting of carboxyl, COO Me wherein Me is a pharmaceutically acceptable metal and COOR' wherein R' is aliphatic hydrocarbon of up to two carbon atoms unsubstituted or substituted with up to two hydroxy groups, m 0 or 1, n 0, l or 2, p 2-20 inclusive and wherein the broken line denoted by r.a. denotes that the part of the compound enclosed by the broken line is radioactively labeled the degree of absorption of said radioactive compound having a previously determined and constant relationship to the degree of absorption of a fat or fatty acid, said radioactive compound when absorbed through the intestinal wall metabolizing to form at least one radioactive metabolite eliminated in the urine of said mammal, the total amount of said radioactive metabolite in said urine bearing a constant relationship to the total amount of the specific radioactive compound administered and determining by radioactive measurement the total amount of said radioactive metabolite in the urine.

2. The method of claim 1 wherein p 4-12.

3. The method of claim 1 wherein Me is an alkali metal.

4. The method of claim 2 wherein the radioactive compound is of the formula 21 22 wherein r.a. donates that the phenyl nucleus contains at (CH COOR least one radioactive atom, R is a moiety selected from 2 a the group consisting of hydrogen, alkali metal, alkyl of 3H 7 D V 1 to 2 rb n ato d l l d p -4 12 wherein 3' denotes that the phenyl nucleus is labelled 5. The method of claim 1 wherein the radioactive with tritium and R is a moiety Selected from the group compound i f h fo l consisting of hydrogen and alkali metal.

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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3907846 *Apr 10, 1974Sep 23, 1975Lilly Industries LtdPreparation of substituted decenoic acids
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Classifications
U.S. Classification424/1.81, 436/56, 424/1.85, 554/218, 554/220
International ClassificationC07C57/58, A61K51/02, C07B59/00, A61K51/04, C07C51/00, C07C67/00, A61K31/185, A61K31/19, A61K51/00, C07C57/00, C07C51/377, C07C59/00, C07C57/30, C07C59/64, C07C51/347
Cooperative ClassificationA61K51/04, A61K2123/00
European ClassificationA61K51/04
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