MC1692A1 - PROCESS FOR THE PREPARATION OF PHENYLTETRAENYL DERIVATIVES PHENYLTETRAENYL DERIVATIVES AND THEIR APPLICATION - Google Patents

Description

1

The present invention relates to compounds having the following formula:

*7 R8

,7 ,8

CH=CH-C =CH-CH=CH-C =CH-R

8 7 6 5

3 2 1

X-R,

10

15

20

where n is a chosen integer from 6 or 7; R

1

represents a hydrogen, a lower alkyl, a chlorine, a fluorine, or a trifluoromethyl; R2 represents a hydrogen, a lower alkoxy, a lower trifluoromethyl alkoxy, a hydroxy, a lower alkyl, a chlorine, a trifluoromethyl, or a fluorine; R^ represents a hydrogen, a lower alkyl, a chlorine, or a fluorine; R^ represents an alkyl containing 4 to 10 carbon atoms or -Ch^( )Ch^OH; X represents

- C H - 0 - , R10

-CH-, R10

, -C = CH-R1 0

,-0-

Rq represents — C — 0 R Q ; and R-,, > H y i

0

Or

'10 r9 and R10

individually represent a lower alkyl or a hydrogen;

and their salts where R^ represents a hydrogen; a process for their preparation and pharmaceutical compositions containing them.

Figures 1, 2, 3, 4, 5, 6 and 7 schematically represent the steps in the process of preparing the compounds of formula I above.

In the compounds of this invention, the term "halogen" encompasses the four halogens, namely chlorine, bromine, iodine, and fluorine, with chlorine and bromine being preferred. The term "lower alkyl," as used here, denotes an alkyl group with a straight or branched chain containing from 1 to 7 carbon atoms. Examples include methyl, ethyl, isopropyl, n-butyl, etc.

are among the preferred lower alkyl groups, with methyl and ethyl being especially preferred. The term "lower alkoxy" refers to lower alkoxy groups containing 1 to 7 carbon atoms, such as methoxy, ethoxy, isopropoxy, isobutoxy, etc. The term trifluoromethyl-lower alkoxy refers to a lower alkoxy substituent substituted with a trifluoromethyl group, where the lower alkoxy is defined as above. The term alkylidene refers to a saturated acycli-based hydrocarbon group where the terminal carbon atom is divalent.

The term "aryl" refers to mononuclear aromatic hydrocarbon groups that may be unsubstituted or substituted at one or more positions by a lower alkyl group, such as phenyl or tolyl, etc., and to polynuclear aromatic groups that may be unsubstituted or substituted at one or more positions by a lower alkyl group, such as naphthyl, phenantryl, or anthryl. The preferred aryl group is phenyl.

In one embodiment of the compounds of Formula I, X represents -O- and Ry and Rg represent lower alkyls, preferably methyls. In another preferred embodiment, represents -(Cl^) H, y representing 6 to 9, more particularly 8 to 9, 9 being especially preferred. In this embodiment of the invention,

R^, R2 and R-j preferably represent hydrogen, or R,j and R^ can represent hydrogen and R2 can represent a lower alkoxy such as methoxy or eth'oxy. Furthermore, in this embodiment, R2 and R^ can represent hydrogen, representing chlorine or fluorine, or R,j and R2 can represent hydrogen, representing chlorine or fluorine.

In another embodiment of compounds of formula I, R_n represents -CH₂(CH₂)nCH₂OH. In this embodiment of compounds of formula I, R_n, R₂, and R_n represent hydrogen, or R_n represents chlorine or fluorine, and R_n represents hydrogen. Furthermore, in this embodiment of compounds of formula I, R_n and R_n represent hydrogen, and R_n represents a lower alkoxy, preferably a methoxy or ethoxy. Preference is also given to compounds of this embodiment of compounds of formula I where R_n and R_n represent hydrogen, and R_n represents chlorine or fluorine.

Also included in this invention are salts of the compounds of formula I above with non-toxic, pharmaceutically acceptable inorganic or organic bases, for example, salts of alkali and alkaline earth metals. Preferred salts include sodium, potassium, magnesium, or calcium salts, as well as salts with ammonia or suitable non-toxic amines, such as lower alkyl amines, for example, triethylamine, lower hydroxy-1-alkyl amines, for example, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, or tris-(2-hydroxyethyl)amine.

Amino acids, cycloalkylamines, for example, dicyclohexyl1-amine, or benzylamines, for example, N,N'-dibenzyl1-ethylene diamine, and dibenzylamine. These salts can

4

to be prepared by treating the compounds of formula I, where represents a hydrogen, with inorganic or organic bases by a classical method well known in the trade.

5 The compounds of formula I as well as their salts are effective as disease-modifying agents for the treatment of rheumatoid arthritis and related conditions such as osteoarthritis.

The compounds of formula I can be used to treat patients with rheumatoid arthritis and related conditions. In these cases, the compounds alleviate the effects of these diseases by reducing the joint destruction caused by them and by alleviating the inflammation, burning sensation, and pain in the joints caused by rheumatoid arthritis and related conditions. The compounds of formula I and their salts are also useful for treating conditions due to immune hyperactivity, such as transplant autoimmunity, autoimmune disease, and graft-versus-host disease. The unexpected lack of toxicity of the compounds of this invention is demonstrated by the fact that the compound—(all-trans)-(nonyloxy-2-phenyl)-9-dimethyl-3,7-2,4,6,8-nonatetraene-oiq—exhibits an LD50 greater than 1000 mg/kg in mice, whether administered intraperitoneally or orally.

The efficacy of the compounds of this invention as anti-arthritic agents is shown in the results obtained when these compounds are administered to rats according to the adjuvant-induced chronic arthritis test system described in Billingham and Davies

<h

10

15

20

25

“Handbook of Experimental Pharmacology” (editors J.R.

Vane and S.H. Ferreira) Vol. 50/11, pp.108-144, Springer-Verlag, Berlin, 1979).

In this experiment, arthritis was induced by the injection on day 0

0.05 ml of adjuvant [Mycobacterium butyricum suspension, 0.5% (w/v), in heavy mineral oil containing 0.2% digitonin] was injected into the plantar pad of the right hind paw of male Charles River Lewis rats (120–140 g) kept in individual cages and given ad libitum food and water. The volume of the paws (both hind paws) was measured immediately after the adjuvant injection. To monitor the development of inflammation-induced edema in arthritic paws, paw volume was also measured at 3–7 day intervals by immersing the paw up to the level of the lateral malleolus in a mercury plethysmograph.

The products were administered once daily (starting on the day of adjuvant injection) via feeding tube using Tween 80 (polyoxyethylene scTirbitan monooleate) as the excipient at a dose of

0.25 ml/100 g body weight. Control arthritic rats received only daily doses of excipient. On days 23-25, the rats were sacrificed, plasma was collected, and fibrinogen levels were determined (ammonium sulfate turbidimetric method), as described by Exner et al., Amer. J. Clin. Path, 7J_: 521-527 (1979). The anti-inflammatory activity of the tested products was determined by comparing the degree of leg edema (leg volume on a particular day, i.e., from day 4 to day 25, minus the leg volume) to the volume of the legs on the entire day.

6

of day 0) in arthritic rats treated with the products, with the significance of leg edema in arthritic rats treated with the excipient. The reduction induced by the products in plasma fibrinogen 5 levels, an acute-phase protein whose level is elevated in the plasma of rats with adjuvant-induced arthritis, was also used to quantitatively establish anti-inflammatory activity.

The results of different compounds of this invention compared with those of indomethacin and cis-1.3 vitamin A are presented in the following table (TABLE I).

TABLE I

Structure

Oral dose µmol/kg

1

Percentage reduction in leg volume on day 23 (no uroite).

Plasma fibrinogen, variation in % (reduction)

Body weight gain (g)

Excipient

—

...

. —

28 - 31

. Indo.netbâcine

3

-66

-67

-30

i 46

CO2 « 1

60

-33

• -49

-18

1

-7.

^^^^^OCgHig -

76

-43

-58

-55

* 21

^^OCgHjg

75 •

-27

-24

-21

* 27

_ JL 0?And

■ OCT^

— -KIICBHI 7

75

-53

-53 "

♦ 24

ée^"*

75

-29

-32

♦ 35

ofCH^JaOH

95

-37

-49

-35

♦ 27

X^A^A^"

^^OCgHjg

'75

-56 1

-68

-51

+46

8

In Table I above, the progressive reduction in leg volume demonstrates the ability of the compounds of this invention to reduce edema caused by adjuvant-induced arthritis. As can be seen from the results presented in this table, the compounds of this invention effectively reduced adjuvant-induced edema. Furthermore, the compounds of this invention were effective in reducing fibrinogenemia, which is commonly associated with rheumatoid arthritis. In addition, as can be seen from the animals' weight gain, the compounds of this invention, at the tested dose, did not cause any significant reduction in the animals' weight gain. This indicates the absence of toxicity exhibited by the compounds of this invention.

Formula I compounds and their pharmaceutically acceptable salts can be used in various pharmaceutical preparations. In these preparations, the compounds are administrable in oral unit dosage forms such as tablets, pills, powders, and capsules, as well as in forms such as injectable solutions, solutes, suppositories, emulsions, dispersions, and other suitable forms. Pharmaceutical preparations containing formula I compounds are conveniently formed by mixing them with a non-toxic pharmaceutical organic excipient or a non-toxic pharmaceutical inorganic excipient. Typical pharmaceutically acceptable excipients include, for example,

Water, gelatin, lactose, starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, petrolatum, and other conventionally used pharmaceutical excipients. Pharmaceutical preparations may also

9

10

15

20

contain non-toxic auxiliary substances, including emulsifying agents, preservatives and wetting agents and similar substances such as, for example, sorbitan monolaurate, triethanolamine oleate,

sodium dioctylsulfosuccinate and similar products.

The daily dose administered of the compounds will obviously vary depending on the specific new compound used, the chosen route of administration, and the size of the recipient. The administered dose is not subject to precise limits, but it will usually fall within the active amounts of the pharmaceutical function of the compounds of this invention. Oral administration is representative of a characteristic method for administering the compounds of formula I or their salts. By this route, the compounds of formula I or their salts can be administered at a dose of 0.5 mg/kg per day orally to 100 mg/kg per day orally. These compounds can preferably be administered daily to patients in unit-dose oral dosage forms at daily doses of 1 to 30 mg/kg of body weight, with doses of 1 to 10 mg/kg being especially preferred.

The compound of formula I, where X represents -O-, can be prepared from compounds having the formula

III

where R,j, and Rj are as per the reaction scheme of the above, figure 1.

10

In the reaction scheme of Figure 1, n, R^,

R2, Rj, R4>. Ry and Rg are as above and R' represents a lower alkyl, Z represents a leaving group; Y represents an aryl, preferably phenyl; Z' represents a halo.

Compound of formula III is transformed into compound of formula IV by reaction step (a) by reducing the aldehyde group to an alcohol. This reaction is carried out using a common reducing agent that converts aldehydes to alcohols. Any common reducing agent for this purpose can be used in the reaction of step (a). When carrying out this reaction, it is generally preferred to use an alkali metal borohydride such as sodium borohydride as the reducing agent. Any of the classical conditions in such reduction reactions can be used to carry out the reaction of step (a). If R^ represents a hydroxyl group, it is generally preferred to protect the hydroxyl group designated by R^ during the reduction of compound of formula III and its subsequent transformation into compound of formula I. Any classical hydrolyzable hydroxyl-protecting group, such as a lower alkanoyl group, can be used to protect the hydroxyl group when R^ represents a hydroxyl group. This ester protecting group can be cleaved by classical ester hydrolysis after the formation of Wittig salts of formulas XIII and XVI or after the formation of ether of formula X.

The compound of formula IV is transformed into the compound of formula VI by reaction step (b) by treating the compound of formula IV with a tri-arylphosphine hydrohalogen. In this way, the phos-phonium salt of formula VI is obtained. Any conventional method for making

11

Reacting an allylic alcohol with a triarylphosphine hydrate halide can be used to carry out this reaction. The phosphonium salt of formula VI is reacted by a Wittig reaction with the compound of formula VII in step (c) to form the compound of formula VIII. Any of the conditions commonly used in Wittig reactions can be used to carry out the reaction in step (c).

Furthermore, compound III can be directly transformed into compound VIII by reaction with the phosphonium salt of compound IX, as in reaction step (e). The reaction of the phosphonium salt IX with compound III to obtain compound VIII is carried out using the same conditions as those described in relation to reaction step (c).

The compound of formula VIII is transformed into the compound of formula X by esterification or alkylation of the compound of formula VIII with a compound of formula V as in reaction step (d). In the compound of formula V, Z can represent any usual leaving group such as mesyloxy, tosyloxy, or a halide.

Any classical method of esterification of a hydroxy group by reaction with a halide or leaving group can be used to carry out the reaction in step (d).

According to another embodiment of this invention, the compound of formula X, where, when R^ represents a hydroxyl group, the hydroxyl group is protected by a hydrolyzable ester, can be obtained from the compound of formula III by alkylation or esterification of the

1 2

Compound of formula III is reacted with compound of formula V to obtain compound XI. This reaction is carried out by alkylation of compound of formula III with compound of formula V, as in step (d). In the reaction of steps (f) and (d), where R represents an alkyl hydroxyl group, the hydroxyl group in R does not need to be protected. This is true because, under the conditions used in this reaction step, compound of formula V will react with either compound of formula III or compound of formula VIII to give compound of formula XI or compound of formula X without any need to protect the hydroxyl group in the alkyl chain. The alkylation or esterification will take place directly with the hydroxyl portion of the phenyl group on either compound of formula III or compound of formula VIII, and there will be at most only a weak reaction with the hydroxyl group in the alkyl chain designated by R. The compound of formula XI is transformed into the compound of formula XII by reaction step (g) through reduction. The same conditions as those described in relation to reaction step (a) can be used to transform the compound of formula XI into the compound of formula XII.

Compound XII is transformed, by the fecation step (h), into compound XIII by treating compound XII with a triarylphosphine hydrate halogen in the manner described previously in relation to step (b). Compound XIII is transformed into compound X by reacting compound XIII with compound VII by the reaction step (i).

This reaction step is carried out in the same way as that described previously in relation to reaction step (c).

13

According to another embodiment of this invention, the compound of formula X is obtained by first transforming the compound of formula XI into the compound of formula XIV. The compound of formula XI is transformed into the compound of formula XIV by the condensation of aldol with the compound of formula XX. Any conventional method of aldol condensation can be used to react the compound of formula XI with the compound of formula XX to form the compound of formula XIV. In the next step, the compound of formula XIV is condensed either by a Grignard reaction with magnesium vinyl halide or by a lithium condensation reaction with lithium vinyl to obtain the compound of formula XV. The reaction in step (k) can be carried out using any of the usual conditions in lithium condensations or Grignard condensation reactions. The compound of formula XV is transformed into the compound of formula XVI by reacting the compound of formula XV with a triarylphosphine hydrate halogen in the manner described previously in relation to the reaction in step (b). The compound of formula XVI is then transformed into the compound of formula X, by reaction step (m), through reaction with the compound of formula XVII. The reaction in step (m) is carried out using a standard Wittig reaction as described in relation to the reaction in step (c). The compound of formula XVI, reacting with the compound of formula XVII, gives the compound of formula X. The compound of formula X can be transformed into the free acid, that is, the compound of formula I where R_i represents COOH, by hydrolysis of the ester. Any conventional method of ester hydrolysis will give the compound of formula I where R_i represents COOH.

14

The compounds of formula I where X represents -N- are i

R1 0

prepared from compounds having the formula cii2oh

XXII

where R^, R^ and Rj are as above by the reaction scheme in Figure 2.

5 In Figure 2, R1, R2, R^, R^, R?, RQ, R'g, Z'

and Y are as above. In Figure 2, R^ is the same as R^ with one less carbon than the alkyl group contained by R^. This is why R^ is an alkyl group containing 3 to 9 carbon atoms or 10 -CH2(CH2)mCH20H where m is a number equal to n minus one,

- that is to say, m is an integer from 5 to 6. In Figure 2, R^g represents a hydrogen or a lower alkyl group containing from 1 to 7 carbon atoms, and R-jg1 represents a lower alkyl group containing from 1 to 15 carbon atoms. In this embodiment, R^g" re represents a lower alkyl group having one less carbon atom than the alkyl group designated by R-| g ' •

In Figure 2, the compound of formula XXII is reacted with the acid chloride of formula XIX, where 20 - Z' represents a halogen, to obtain the compound of formula XXIII, which is then transformed either into the compound of formula XXVI or into the compound of formula XXXI. Where R in the compound of formula XIX represents -CH^-(CH2)j^CH^OH, where m is an integer from 5 to 6 carbon atoms.

15

The presence of the hydroxyl group on the substituent will not influence the reaction to give the compound with formula XXVI or the compound with formula XXXI. It has been found that this hydroxyl group is not affected throughout the entire reaction sequence shown in Figure 2.

Furthermore, this hydroxyl group can be protected by forming a functional hydrolyzable ether group that protects the hydroxyl group during all these reactions. Any common ether protecting group can be used to protect the hydroxyl group during all these reactions. Preferred ether protecting groups include tetrahydropyranyloxy, t-butoxy, and tri(lower alkyl)-silyloxy, for example, trimethylsililoxy.

Any usual ether protecting group can be used to protect the terminal hydroxy group which may be present as R-j^*. Furthermore, the reactions shown in Figure 2 can be carried out without any protection of the terminal hydroxy group.

In the first step of the reaction, compound XXII reacts with compound XIX to give compound XXIII. Any conventional method for condensing an amine with a hydrohalic acid can be used to carry out this reaction. In the next step, compound XXIII is transformed into compound XXIV by reaction step (n), by treating compound XXIII with a reducing agent. Any common alkali metal aluminum hydride reducing agent can be used to carry out this reaction, with lithium aluminum hydride being the preferred reducing agent. Any of the usual conditions for reduction with an alkali metal aluminum hydride reducing agent can be used to carry out this reaction.

16

The compound of formula XXIV can be transformed into the compound of formula XXVI via XXV. In the first step of this process, step (o), the compound of formula XXIV is transformed into the phosphonium salt of formula XXV by reaction with a triarylphosphine hydrate halide as described previously in relation to the reaction in step (b). The compound of formula XXV is transformed into the compound of formula XXVI by reaction step (p), by reaction with the aldehyde of formula VII (see Figure 1). The reaction in step (p) to obtain the compound XXVI is carried out by a Wittig reaction in the manner described in reaction step (c) above. The compound of formula XXVI, where Rg represents a lower alkyl group, can, if desired,

to be transformed into a free acid by conventional basic hydrolysis. Any conventional basic hydrolysis method for hydrolyzing esters can be used to transform the compound of formula XXVI into a free acid. Furthermore, the compound of formula XXVI, where R^ contains a terminal hydroxyl group esterified with a typical ether protecting group, can be transformed into a free alcohol by acid hydrolysis. Any of the conventional ether hydrolysis methods can be used to carry out this reaction. The hydrolysis of the ether of the compound of formula XXVI can be performed either before or after the acid hydrolysis used to hydrolyze the ester protecting Rg'. On the other hand, if R^ in the compound of formula X in Figure 1 contains an esterified hydroxyl group, the compound of formula X can be hydrolyzed in the same way as the compound of formula XXVI.

Furthermore, the compound of formula XXIV can be transformed into a tertiary amine compound of formula XXXI.

17

In this reaction, the compound of formula XXIV is reacted, by reaction step (q), with the hydracid of formula XIX-A, to obtain the compound of formula XXVII in the same way as that described previously in relation to the reaction to transform the compound of formula XXII and compound of formula XXIII.

The compound of formula XXVII is transformed into formula XXIX, by reaction step (r), by treating the compound of formula XXVII with a reducing agent, lithium aluminium hydride, as described in relation to the reaction of step (n).

In the next step of this reaction scheme, compound XXIX is transformed into compound XXX by reaction step (s) with a triarylphosphine hydrate halide. This reaction is carried out in the same manner as described in relation to reaction step (b) previously. Compound XXX is transformed into compound XXXI by reaction step (t) with compound VII (Figure 1). A Wittig reaction is used in the reaction of step (t). The reaction in step (t) can be carried out in the same manner as described previously in relation to the reaction in step (c). Compound XXXI, where Rg' represents a lower alkyl group, can be transformed, if desired, into the corresponding compound XXXI containing the free acid group by basic hydrolysis. Furthermore, if it contains a terminal hydroxyl group protected by the formation of a hydrolyzable ether, the compound of formula XXXI can be transformed into the corresponding compound where R represents a free hydroxyl group, by classical ether hydrolysis. This hydrolysis of the ether

18

can be carried out before or after hydrolysis of the ester group designated by Rg'.

In the reaction scheme of Figure 2, where R2 represents a hydroxyl group, it is preferable for this hydroxyl group to be protected by the formation of a protecting ester group. The protecting ester group can be removed after the formation of Wittig salts of formula XXX.

Compounds with the formula I or X representing -CH- are

I

R1 0

prepared from a compound having the formula:

“I

<7lt

XXXV

where Z" represents either bromo or iodo;

R^, R2 and Rj are as above; and represents a hydrogen or a lower alkyl as shown in Figure 3. In Figure 3, R^, R^, R-j, R^, R'7, Rg, R-, R1q, R13, V, Z' and Z" are as above.

In Figure 3, compound XXXV is first reacted with compound XXXIV via reaction step (u) to obtain compound XXXVI. This reaction is carried out by a Wittig reaction. In compound XXXVI, R^ can represent, if desired, -CH^-CH^i-OH where the free hydroxyl group can, if desired, be protected by the

19

formation of any of the classical ether groups mentioned previously. Furthermore, it has been found that this OH group can be a free hydroxyl group and does not need to be protected by an ether protecting group. By carrying out the reactions in Figure 3, this free hydroxyl group is not affected by the reactions that transform the compound of formula XXXVI into the compound of formula XXXXI. However, for better yields, it is generally preferable to protect this hydroxyl group by the formation of a hydrolyzable ether.

The reaction in step (u) is carried out by a Wittig reaction between the compounds of formula XXXV and the compound of formula XXXIV using the same reaction conditions as those described below in relation to reaction step (c).

The compound of formula XXXVI can be transformed into the compound of formula XXXVII by a reaction step (v) via hydrogenation. Any conventional hydrogenation method can be used to carry out this reaction. Among the conventional hydrogenation methods is the treatment of the compound of formula XXXVI, in an inert organic solvent, with hydrogen gas in the presence of a catalyst. Any conventional hydrogenation catalyst can be used to carry out this reaction. Palladium is a preferred catalyst. In carrying out this reaction, any conventional inert organic solvent can be used. Furthermore, any of the usual conditions in catalytic hydrogenation can be used in the reaction step (v).

20

In the next step of this reaction, compound XXXVII is transformed into compound XXXIX by reaction step (w), by treating compound XXXVII with formaldehyde or a formaldehyde-releasing compound. In this reaction, compound XXXVII is first metallized with an alkali metal alkyl, for example, n-butyllithium. This reaction is usually carried out in an inert organic solvent such as an ether. Preferred solvents include diethyl ether and tetrahydrofuran. Temperature and pressure are not critical in this reaction; it can be carried out at room temperature and atmospheric pressure. Higher and lower temperatures can be used if desired. After treating the compound of formula XXXVIII with an alkali metal alkyl, formaldehyde or a formaldehyde-releasing compound is added to the reaction medium. Any conventional formaldehyde-releasing compound, such as paraformaldehyde, can be used to carry out this reaction. This reaction is performed in the same reaction medium and using the same conditions as those under which the metallation of the compound of formula XXXVIII was carried out.

The compound of formula XXXIX is transformed into the compound of formula XXXX by reaction step (x) by treating the compound of formula XXXIX with triarylphosphine hydrate halide. This reaction is carried out in the same manner as that described in relation to reaction step (b) as previously described. The compound of formula XXXX is transformed, by reaction step (y), into the compound of formula XXXXI.

21

by reaction with the compound of formula VII (see Figure 1). This reaction in step (y) is carried out by a Willig reaction using the same conditions as those described in relation to reaction step (c). The compound of formula XXXXI can be transformed into the corresponding compound containing the free carboxyl group instead of Rg'. This reaction is carried out by classical ester hydrolysis in the manner described previously. Any classical method of ester hydrolysis can be used. If it contains a terminal hydroxyl group protected by an ether protecting group, this ether group can be hydrolyzed to give the free hydroxyl group by classical ether hydrolysis, particularly using an aqueous inorganic acid. Any classical method of ether hydrolysis can be used. The ether-protected hydroxyl group can be hydrolyzed either before or after hydrolysis of the ester group to form the free acid of the compound of formula XXXXI.

If we wish to obtain compounds of formula I and where X represents -C=CH-, we react the compound of

R10

formula XXXV of figure 3, by reaction step (u), with the compound of formula XXXIV where R^ represents R^ to obtain the compound of formula XXXVI where re represents R^. This compound of formula XXXVI where R^ represents R^ is then subjected to the same sequence of reactions as the compound of formula XXXVII, i.e. reaction steps (w), (x) and (y), to obtain the compounds of formula I where X represents -C=CH-,

L

22

In the reaction scheme of Figure 3, where R2 in the compound of formula XXXV represents a hydroxyl group, it is preferable to protect this hydroxyl group by esterification with a lower alkanoic acid. This protecting ester group can be cleaved after the formation of the Wittig salt of formula XXXX.

The compound formula can be prepared from

VfHZ'

R:

where R^, R2> R3 and Z" are as above by the reaction scheme in Figure 4. In Figure 4, -R.j, R2> R-jj R y, Rg, R Q, Rg', Y and Z" are as above and R^ represents R^ where a hydroxyl group contained in is protected in the form of a group

hydrolyzable ether such as tetrahydropyranyl as well as the ether groups mentioned previously.

The compound of formula L is transformed into the compound of formula LI by reaction with the alkali metal alkoxide of formula L-A. This reaction is carried out by reacting the compound of formula L with the compound of formula L-A using the usual conditions in the reaction of an alkali metal alkoxide with a halide.

In this step, the compound with formula LI is transformed into a compound with formula LII by first processing

I and II where X represents -CH-0

R10

of a compound having the formula

23

The compound of formula LI is treated with an alkyl lithium such as n-butyl lithium to metallize the compound of formula LI. The metallized compound of formula LI is then reacted with formaldehyde or a formaldehyde-releasing compound. In transforming the compound of formula LI into the compound of formula LII, the same reaction conditions as those described in relation to reaction step (w) are used. The compound of formula LII is transformed into a phosphonium salt of formula LIII by treating the compound of formula LII with a triarylphosphine hydrate halide in the manner described in reaction step (b) above. The phosphonium salt of formula LIII is reacted by a Wittig reaction with the compound of formula VII (see Figure 1) to form a compound of formula LIV. This reaction to form the compound of formula LIV is carried out in the same manner as described in relation to step (c) above.

When R^,. of the compound of formula LIV contains a protected hydroxyl substituent, said substituent can be hydrolyzed to form the free hydroxyl compound by conventional methods of hydrolysis of readily removable ether groups. Any conventional method of hydrolysis of protecting ether groups can be used. The usual conditions for hydrolyzing protecting ether groups will not affect the other ether group contained in the compound of formula LIV. The compound of formula LIV can be transformed into a free acid by conventional ester hydrolysis.

If R_2 in the compounds of formulas L, LI, LII and LUI represents a hydroxyl group, it is preferable that

24

The hydroxy group is protected by a hydrolyzable ester group such as a lower alkanoyloxy. The hydrolyzable ester protecting group can be cleaved after formation of the Wittig salt of formula LIII.

If desired, the double bonds in compound I at positions 2-3, 4-5, 6-7, and 8-9 can be in the cis or trans configuration. Furthermore, these compounds can represent a mixture of various cis and trans isomers. In compound VII, the double bonds it contains can be in the cis or trans configuration, according to the desired stereo configuration of the double bonds in compounds I. The Wittig reaction carried out to obtain compounds I and II, as in steps (c), (e), (i), etc., gives the double bond at position 8-9 as a mixture of the cis and trans isomers 8-9. These cis and trans isomers can be separated by conventional methods, such as fractional crystallization, etc.

Furthermore, where compounds of formula I have a double bond in the trans configuration at the 2-3 position, this isomer can be transformed into the corresponding cis double bond using conventional isomerization methods known in the field. Among these methods is the treatment of compound I with iodine in an inert organic solvent. Isomerization with iodine yields compound I with a 2-3 double bond in the cis position.

The compounds of formula I include all its geometric isomers including mixtures of these geometric isomers.

25

The compound of formula XI where represents a fluoro is a new compound and it can be prepared from a compound having the formula where R^ and are as above by the reaction scheme in Figure 5. In Figure 5, R^ and R^ are as above.

In Figure 5, the compound with formula LV is alkylated by reaction with an allyl bromide. If the compound where R^ represents a hydroxyl group is desired, the compound with formula LV where the hydroxyl group designated by R^ is protected by esterification is used as the starting material, i.e., the compound with formula LV

where R^ represents a protected hydroxyl group. Any conventional method for alkylating a hydroxyl group with an allyl bromide can be used to carry out the reaction transforming the compound of formula LV into the compound of formula LVI. The compound of formula LVI is rearranged into the compound of formula LVII by heating the compound of formula LVI to a temperature of 190 to 230 degrees Celsius. This rearrangement can take place without the use of any solvent or in the presence of a high-boiling hydrocarbon solvent. If R^ represents a hydrogen, the compound of formula LVII is formed by

26

as a mixture with the isomer of the compound of formula LVII where the allyl group is in a para position relative to the fluorine substituent on the benzyl ring.

This isomer can be separated or used in subsequent reactions and separated from the reaction mixture at a more advanced stage.

The compound of formula LVII is then transformed into the compound of formula LVIII by reaction with the compound of formula V (Figure 1), as described in reaction step (d) above. In the next step of this reaction scheme, the compound of formula LVIII is transformed into the compound of formula LIX by isomerization with a strong base, in particular an alkali metal alkoxide, in the presence of an inert organic solvent, preferably potassium tert-butoxide in dimethyl sulfoxide. The compound of formula LIX is transformed into the compound of formula LX by treating the compound of formula LIX with ozone gas. Temperatures of -70°C to -20°C are used when carrying out this reaction. Furthermore, this reaction is performed in an inert organic solvent. Any conventional inert organic solvent can be used, preferably halogenated hydrocarbons such as methylene chloride.

A compound with formula LX is the compound with formula XI, where R represents a fluoro group. This compound can be transformed into a compound with formula X according to the reaction scheme shown in Figure 1.

Where R2 represents one hydroxyl group in compound III, there are two hydroxyl groups. Therefore, it is generally preferable to prepare compound XI, where R2 represents a protected hydroxyl group.

27

starting from a compound of formula LXI as shown in Figure 6. In this way, compounds of formula I can be prepared where X represents -O-; represents a hydroxyl group, and R^ and R^ are as above. In Figure 6, R^ and R^, taken with its bonded oxygen atom, represent a hydroxyl group protected by a conventional hydrolyzable protecting group, preferably a lower aleanoyl group.

In Figure 6, the compound of formula LXI is transformed into a compound of formula LXII using the same reaction as that described in relation to the transformation of the compound of formula LV into a compound of formula LVI (see Figure 5). The compound of formula LXII is then transformed into a compound of formula LXIII using the same process as that described in relation to the transformation of the compound of formula LVI into LVII. In the following steps, the compound of formula LXIII is transformed into a compound of formula LXIV by the same process as that described in relation to step (g') of Figure 5, and then into a compound of formula LXV by the process described in relation to step (h') of Figure 5. The transformation of the bromobenzene compound of formula LXV into the phenol compound of formula LXVI is carried out by processes well known in the art, as described by Kidwell and Darling, Tetrahedron Letters, (1966) pp. 531-535.

In the next step of preparing the intermediate of formula XI, where R^ represents a protected hydroxyl group, i.e., the compound of formula XI-A, the hydroxyl group is protected on the compound of formula LXVI by esterification with any conventional hydrolyzable ester group to form the compound of formula

28

LXV11 where R^ ^ taken with its bonded oxygen forms a hydrolyzable ester group. Any classical method of esterifying a hydroxyl group with an organic acid such as a lower alkanoic acid containing 1 to 7 carbon atoms can be used to prepare the compound of formula LXVII. The compound of formula XI-A is formed from the compound of formula LXVII by the reaction described previously with regard to the transformation of a compound of formula LIX to LX (see Figure 5).

When transforming a compound of formula XI-A into a compound of formula XVII, as in Figure 1, it is generally preferable to hydrolyze the ester substituent that forms R^ after the formation of the Wittig salt of formula XIII or formula XVI

According to another embodiment of this invention, the compound of formula XI, where R^ represents CF^ (the compound of formula XI-B), can be formed by the reaction shown in Figure 7 from a compound of formula LXX. In the first step of this reaction, the compound of formula LXX is transformed into a compound of formula LXXI using the same process as previously described in relation to the reaction, by step (d), where the compound of formula VIII is reacted with a compound of formula V to obtain a compound of formula X. In this reaction, where R^ represents OH, alkylation occurs very slowly on the hydroxyl group in the ortho position relative to the CF^ group. Therefore, protection of this group may not be necessary since alkylation occurs preferentially with the meta hydroxyl group. All mixtures of alkylated products obtained from this reaction can be separated.

29

by usual separation processes. The compound of formula LXXI is transformed into the compound of formula XII-B by usual processes of formylation of a benzene ring, such as by treatment with an alkyl lithium and dimethylformamide.

The following examples are representative but limiting of the invention. In the examples, the ether is diethyl ether and the solvents have been removed under vacuum.

Example 1

[[[(nonyloxy)-2 phenyl]methyl]triphenylphos-phoniu bromide]

2-Hydroxybenzaldehyde (110 g) was alkylated by mixing this compound with 1-bromononane (180 g), anhydrous potassium carbonate, and dimethylformamide (800 mL). This mixture was heated at 80°C for 14 hours. Hexane and water were then added, and the hexane extract was concentrated. The residue was distilled to obtain 2-nonyloxybenzaldehyde (210 g), at 121°C (0.3 mm kl g). A solution of 2-nonyloxybenzaldehyde prepared above (100 g) in ethanol (1000 mL) at 10°C was reduced by treatment with an excess of sodium borohydride (6 g). After stirring the mixture for a further 15–20 minutes at room temperature, the compound 2-nonyloxybenzyl alcohol was isolated by extraction in hexane. Removal of the hexane under vacuum gave crude 2-nonyloxybenzyl alcohol (98 g). The obtained 2-nonyloxybenzyl alcohol was added to a mixture of triphenylphosphine brorahydrate (144 g) in acetone nitrile (500 mL), and the resulting solution was heated under reflux for 14 hours. The solvents were removed.

30

under vacuum and crystallization of the residue from a tetrahydrofuran/ethyl ether mixture gave pure C[ (nonyloxy )-2 phenyl]methyl]triphenyl-phosphonium bromide (208 g).

5 Example 2

[(2-hydroxyphenyl)methyl1-]triphenylphosphonium bromide

Benzyl hydroxy alcohol was treated with triphenylphosphine hydrobromide in aceto-nitrile as described in Example 1 to give 10[(2-hydroxypheny1)methyl]triphenylphos- bromide

p h <5 n i u m .

Example 3

Ethyl ester of (all-trans)-(2-hydroxyphenyl)-9-dimethyl-3,7-2,4,6,8-nonatetraene-oic acid 15 A solution of [2-hydroxyphenyl)methyl bromide

triphenylphosphonium (1 mol) in tetrahydrofuran was converted to the ylide at -35°C with a solution of n-butyllithium in hexane (2.1 mol Eq.), then exposed to the ethyl ester of 7-formyl-20,3-methyl-2,4,6-octatrieneoic acid (1 mol), and then shaken at -0°C.

for another 15 minutes. Isolation of the organic products with a hexane/ethyl acetate mixture (4:1 parts by volume) and dilute mineral acid (aqueous 2M HCl) gave the ethyl ester of (all-trans) 25-(2-hydroxyphenyl)-9-dimethyl-3,7-2,4,6,8-nonatetraene

pure icic acid (yield 90?o) after chromatography followed by crystallization from a dichloromethane/hexane mixture.

31

Example 4

Ethyl ester of (all-trans)-(2-hydroxyphenyl)-9-dimethyl-3,7-nond:2,4,6,8-ethrene

A mixture of 2-hydroxybenzaldehyde (0.5 mol),

(7-carboxy-2,6-dimethyl-2,6-heptatriene-2,4,6-yl-1)triphenylphosphonium bromide (0.6 mol) in 1,2-epoxy-butane (750 mL) was heated under reflux for 30 minutes, cooled, poured into an ether/hexane mixture (1:1 parts by volume), filtered, and concentrated. The residue was then crystallized from a hexane/ether mixture to obtain the ethyl ester of (all-trans)-(2-hydroxyphenyl)-9-3,7-dimethyl-2,4,6,8-nonatetraeneoic acid (yield 38%), at 143-146°C.

Example 5

(All-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene

A solution of [[(nonyloxy)-2-phenyl]methyl]triphenylphosphonium bromide (150 g) in tetrahydrofuran (1100 mL) was cooled to -50°C to give a fine suspension of the solid salt. A solution of n-butyllithium in hexane (180 mL, 1.6 molar) was added to this mixture to obtain a solution of the ylide. The mixture was then stirred for a further 15 minutes at -40°C, cooled to -70°C, and treated with the ethyl ester of 7-formyl-3-methyl-2,4,6-octatriene (65 g) dissolved in tetrahydrofuran (250 mL). The addition of hexane and aqueous methanol (40?£) to the reaction mixture followed by concentration of the extract with hexane gave the ethyl ester of (all-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid (64 g, yield 5.8 µo ) ,

32

At a boiling point of 52-53°C, this ester was hydrolyzed, forming a solution of 70 g in 1000 mL of ethanol. This solution was treated with aqueous potassium hydroxide (80 g in 400 mL of water) and heated under reflux for 1 hour. Water and aqueous mineral acid were then added, and the solids were extracted in chloroform. Concentration of this organic extract and crystallization of the residue from an ethyl acetate/hexane mixture yielded (all-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraeneoic acid (38 g), at a boiling point of 102-103°C.

Example 6

(All-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-

3,7 nonatetraene-2,4,6,8 ic

A mixture of sodium hydride (24 g, 50% by weight in mineral oil) and dimethylformamide

(1000 mL) at 10°C was treated with the ethyl ester of (all-trans)-(2-hydroxy)1-9-phenyldimethyl-3,7-2,4,6,8-nonatetraeneoic acid (0.4 Eq.). The resulting mixture was then stirred at room temperature until complete cessation of hydrogen evolution to obtain the sodium salt of the ethyl ester of (all-trans)-(2-hydroxyphenyl)-9-dimethyl-3,7-2,4,6,8-nonatetraeneoic acid. A solution of 1-nonyl tosylate (0.5 Eq.) in dimethylformamide (200 mL) was then added to this salt solution, and the reaction mixture was stirred at 45°C for 14 hours. Hexane and water were then carefully added, and the hexane extract was concentrated. The residue was purified by silica gel chromatography. Crystallization from hexane then yielded the ethyl ester of (all-trans)-[(nonyloxy)-2](all-trans) acid.

33

pure phenyl]-9-3,7-dimethyl-2,4,6,8-nonatetraene-oic acid. Hydrolysis of this ester as in Example 5 gave (all-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-2,4,6,8-nonatetraene-oic acid.

Example 7

(all-trans)-3,7-dimethyl-[[(2,2-dimethyl-octyl)

oxy]-2 phenyl]-9 2,4,6,8-nonatetraene ic

2-Hydroxybenzaldehyde was condensed with 2,2-dimethyliodo-1-octane to obtain 2-(dimethyl-2,2-octyloxy)benzaldehyde, which was reduced to 2-(dimethyl-2,2-octyloxy)benzyl alcohol, and then converted to [[(2,2-dimethyl-2,2-octyloxy)-2-phenyl]methyl]triphenylphosphonium bromide as in Example 1. Condensation of this phosphonium bromide with the ethyl ester of 7-formyl-3-methyl-2,4,6-octatriene-oic acid as described in Example 3, followed by hydrolysis, as in Example 5, gave (all-trans)-3,7-dimethyl-2,7-[[(2,2-dimethyl-2,2-octyl)oxy]-2-phenyl]-9-2,4,6,8-nonatetraene-oic acid, p.f. 113-117° (from the mixture of hydrogen ethane/hexane).

Example 8

(All-trans) 3,7-dimethyl[[(octyloxy)-methyl]-2-phenyl]-9-2,4,6,8-nonatetraene-oic acid

Lithium octanoate, prepared from octanol and n-butyllithium, in a tetrahydrofuran/hexane/dimethylformamide mixture, was condensed with 2-bromobenzyl bromide to obtain 1'-(octyloxy)-2-methylbromobenzene. This material was treated with n-butyllithium in an ether/hexane mixture and then treated with paraformaldehyde to obtain the alcohol.

34

(octyloxy)-Z methylbenzyl. This material was then treated with triphenylphosphine bromide to obtain [[(octyloxy)-1-methyl]-2-phenylmethyl]triphenylphosphonium bromide. Condensation of this material 5 with the ethyl ester of 7-formyl-3-methyl octa-

Z,4,6-triene-oic acid, as in Example 3, followed by hydrolysis, as in Example 5, gave (all-trans)-dimethyl-3,7 [[(octyloxy)-methyl]-Z phenyl]-9 nonatetraene-Z,4,6,8-oic acid, w.p. 120—1Z10 10 (from the dichloromethane/hexane mixture).

Example 9

(All-trans)-[chloro-Z (nonyloxy)-6-phenyl]-9-dimethyl-3,7-Z,4,6,8-nonatetraene-oic acid

Chloro-Z 6-hydroxybenzaldehyde was alkylated 15 with 1-bromo-nonane as in Example 1 to give chloro-Z 6-nonyloxybenzaldehyde. Reduction with sodium borohydride as in Example 1 gave chloro-Z 6-nonyloxybenzyl alcohol which, after treatment with triphenylphosphine hydrobromide in acetonitrile, as in Example 1, gave [[chloro-Z 6-nonyloxy]phenyl]methyl]triphenylphosphonium bromide. The condensation of 7-formyl-3-methyl-octatriene-Z,4,6-oic acid with the ethyl ester, as described in Example 3, gave the ethyl ester of (all-trans)-[chloro-Z (nonyloxy)-6-phenyl]-9-dimethyl-3,7-nonatetraene-Z,4,6,8-oic acid. The ester was hydrolyzed, as in Example 3, to obtain (all-trans)-[chloro-2 (nonyloxy)-6-phenyl]-9-dimethyl-3,7-nonatetraene-Z,4,6,8-oic acid, p.f. 30 1Z9-1310 (from the ethyl acetate/hexane mixture).

here

35

Example 10

(All-trans)-(5-methoxy-2-nonyloxy-9-phenyl)-3,7-dimethyl-2,4,6,8-noratetraene

5-Methoxy-2-Hydroxybenzaldehyde was alkylated with nonyl bromide and reduced with sodium borohydride, as in Example 1, to give 5-Methoxy-2-Nonyloxybenzyl alcohol, which, after exposure to triphenylphosphine bromide, gave [(5-Methoxy-2-Nonyloxy-Phenyl)methyl-1]triphenylphosphonium bromide. Condensation of this material with the ethyl ester of 7-Formyl-3-Methyl-2,4,6-Octatrieneoic acid, as in Example 3, followed by hydrolysis, as in Example 5, gave (all-trans)(5-Methoxy-2-Nonoxy-2-Phenyl)-9-Dimethyl-2,4,6,8-Nonatetraeneoic acid, p.f. 125–126° (from methanol).

Example 1 1

(All-trans)-[[8-hydroxy-octyl)oxy]-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene

The sodium salt of the ethyl ester of (all-trans)-(2-hydroxyphenyl)-9-dimethyl-3,7-nona-tetraene-2,4,6,8-oic acid in dimethylformamide prepared as described in Example 6 was treated with dihydroxy-1,8-octane monotosylate, as previously described in Example 6, and gave the ethyl ester of (all-trans)-[[8-hydroxyocty1)oxy].-2-phenyl-9-diethyl-3,7-nonatetraene-2,4,6,8-oic acid after silica gel chromatography. Hydrolysis, as in Example 5, gave (all-trans)-[[(8-hydroxy-octyl)oxy]-2-phenyl-9-dimethyl-3,7-2,4,6,8-nonatetraene-oic acid, p.f. 122-123° (from ethyl acetate).

36

Example 12

(All-trans) bromide of [(2-nonyloxy-2-phenyl)-5-methyl-3-pentadien-2,4-yl]triphenylphosphonium

2-(nonyloxy)-benzaldehyde (62 g) dissolved in acetone (500 mL) was treated with aqueous sodium hydroxide (100 mL, 1 M) at room temperature for 18 hours. Salt water and ethyl acetate/hexane (1:1 by volume) were then added. Concentration of the organic phase followed by crystallization from hexane yielded 2-(nonyloxy-4-phenyl)-3-butene-2-one (53 g).

A solution of 2-(nonyloxy-2-phenyl)-4-butene-2-one (58 g) in tetrahydrofuran (200 mL) was added to a solution of vinylmagnesium bromide in tetrahydrofuran (200 mL, 1.6 M diluted to 1 L with more tetrahydrofuran) at -30°C. After complete addition, the mixture was stirred at 0°C for 30 minutes, fixed with saturated aqueous ammonium chloride (100 mL) and ether (2 L), and filtered to remove solids. Concentration of the organic extract and purification by silica gel chromatography yielded 1'(E)-(2-nonyloxy-2-phenyl)-5-hydroxy-73-3-methyl-1,4-pentadiene (40 g) as an oil.

A solution of (E)-(nonyloxy-2 phenyl)-5-hydroxy-3-methyl-1,4-pentadiene (66 g) in acetonitrile (250 mL) was added to a slurry of triphenylphosphine hydrobromide (66 g) in a larger quantity of acetonitrile (300 mL) at 10°C. After heating to room temperature, the mixture was stirred at this temperature for 2 hours to obtain a solution.

37

This solution was then extracted with hexane (2 x 250 mL) and the acetonitrile layer was concentrated (approximately 400 mL) and cooled to -10°C. The solids were separated by filtration, washed with acetonitrile, hexane, and dried to give pure (all-trans)-[(nonyloxy-2-phenyl)-5-methyl-3-pentadien-2,4-yl]triphenylphosphonium bromide (21 g).

Example 13

Starting with (all-trans)-[(nonyloxy-2-phenyl)-5-methyl-3-pentadiene-2,4-yl]triphenylphosphonium bromide and using the process of Example 5, the ylide was reacted with the ethyl ester of 3-formyl-2-butene-oic acid to obtain (all-trans)-(nonyloxy-2-phenyl)-9-dimethyl-3,7-nona-tetraene-2,4,6,8-oic acid after purification by silica gel chromatography and hydrolysis with an ethanolic solution of aqueous potassium hydroxide, as in Example 5.

Example 14

(Z,E,E,E)-[(2-nonyloxyphenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene-oic acid

The ethyl ester of (all-trans)-[(nonyl-oxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraeneic acid (10 g) was dissolved in hexane (200 mL) containing iodine (0.5 g) and stirred at room temperature for 30 minutes. The hexane was washed to remove the iodine with an aqueous solution of sodium thiosulfate (T0'?o by weight), dried, and concentrated to obtain a mixture of double-bonded isomers. Separation by silica gel chromatography

38

given the ethyl ester of the acid (Z,E,E,E-[(nonyl-oxy)-2 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid (1.5 g). Reflux hydrolysis with an aqueous ethanoic solution of potassium hydroxide gave pure (Z,E,E,E)-[(nonyloxy)-2 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid: w.p. 135-136°C.

Example 15

(E,E,E,Z)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatraene

The mother liquor material resulting from the crystallization of the ethyl ester of (all-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid in Example 5 was a mixture containing various isomers. Purification by chromatography gave a pure ethyl ester at 80% which, after hydrolysis, as in Example 5, gave pure (E,E,E,Z)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid: wt. 105-109°.

Example 16

Decyl-2-bromo-1-benzene

Nonylmethyltriphenyl phosphonium bromide (0.1 mol) in tetrahydrofuran (200 mL) was transformed into the ylide with n-butyllithium (0.1 mol.Eq.; 1.6M in hexane) at -10°C.

2-Bromobenzaldehyde (0.09 mol) was then added to tetrahydrofuran (25 mL), and after the mixture had been thoroughly stirred for 30 minutes at 0°C, hexane and aqueous methanol (40:60) were

39

added. The hexane extract was concentrated and the residue was distilled to obtain (1-deceneyl)-2-bromo-1-benzene (90).

This material was dissolved in hexane containing a palladium-on-carbon catalyst (10%) and hydrogenated at ambient temperature and pressure until the olefinic bond was saturated. The solids were separated by filtration, and removal of the hexane and distillation of the residue gave pure 2-decyl-1-bromobenzene (80 µe): w.f. 120° (0.001 mm Hg).

Example 17

Decyl-2-hydroxymethyl-1-1-benzene

Decyl-2 bromo-1 benzene (0.1 mol) dissolved in ether (150 mL) was treated with n-butyllithium (0.11 Eq. 1.6M in hexane) and the mixture was stirred at room temperature for 2 hours.

Dry paraformaldehyde (0.2 molEq.) was then added and the mixture was stirred for a further 18 hours at room temperature.

Water and a supplement of 1 ether were then added, and the ether extracts were dried and concentrated. The residue after chromatography gave 2-decyl-1-hydroxymethylbenzene (yield 15%).

Example 18

(All-trans)-(decylpheny3)r9-dimethyl-3,7-nona-tetraene-2,4,6,8-oic acid

Decyl-2-hydroxymethyl-1-benzene was transformed

40

in phosphonium salt with triphenylphosphonium hydrobromide in acetonitrile by the process of Example 1. This salt was then exposed to n-butyllithium in tetrahydrofuran as before and then treated with formyl-7 methyl ester

3-methyl-2,4,6-heptatriene-oic acid as before.

Purification of the crude condensation product by silica gel chromatography followed by basic hydrolysis gave (all-trans)-(decylphenyl)-9 10 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid: p.f. 107-108° (from hexane ether).

Example 19

Ethyl ester of (all-trans)-(octylamino-2-phenyl)-9-Dimethyl-1-3,7-2,4,6,8-nonatetraene-oic acid

Benzyl 2-amino alcohol (1 mol) was treated with octanoyl chloride (2.2 mol) in a dichloromethane-triethylamine mixture at 0°C. After 30 minutes at 10°C, the mixture was washed with water and the ether was removed by distillation. The crude residue was dissolved in tetrahydrofuran (2000 mL), treated with aqueous sodium hydroxide (1 N, 1500 mL), and stirred at room temperature for 3 hours.

The addition of water and ether gave crude hydroxymethyl-octylamide. Purification by chromatography gave pure octylamide (85 µg).

This material (100 g) was dissolved in tetrahydrofuran (500 mL) and added to a slurry of lithium aluminum hydride (2 mol Eq.) in tetrahydrofuran (1000 mL). The mixture was then heated under reflux.

15

20

41

for 8 hours, cooled to 0°C and fixed with an aqueous solution of sodium sulfate (100 mL).

The solids were separated by filtration, the solvents were removed under vacuum and the residue was purified by silica gel chromatography to obtain pure 1'hydroxymethyl-2-N-octylamine (75 g).

This material was dissolved in acetonitrile (300 mL) containing triphenylphosphine hydrobromide (1.1 Eq.) and the mixture was heated under reflux for 24 hours, then concentrated. The residue was digested with ether to give the phosphonium salt as a white solid.

This material was converted to the corresponding ylide with n-butyllithium (1.5 ml eq.) and stirred at 0°C for 1 hour. The ethyl ester of 7-formyl-3-methyl-2,4,6-heptatrieneoic acid (1.6 mol eq.) was then added in excess to the tetrahydrofuran and the mixture was stirred at 10°C for 1 hour.

The addition of hexane and aqueous methanol (2:3) and the removal of hexane under vacuum gave the crude coupled product. Purification by silica gel chromatography and crystallization from hexane gave the ethyl ester of pure (25%) (all-trans)-(octy1-amino-2-phenyl)-9-dimethyl-3,7-2,4,6,8-nonatetraene-oic acid: w.p. 38-40°C.

Example 20

Fluoro-2-nonyloxy-6-benzyl alcohol

A solution of fluorine-3 phenol (100 g) in the

42

dimethylformamide (1000 mL) containing potassium carbonate (165 g) was treated with allyl bromide (115 g) and heated at 80° for 18 hours.

Water and hexane were then added, and the hexane extract was washed with aqueous sodium hydroxide (5% saline) solution and concentrated to obtain allyl ether (155 g). This material (134 g) was heated at 220°C for 16 hours to obtain a mixture of 3-fluoro(2-butene-2-yl)-2-phenol and 5-fluoro(2-butene-2-yl)-2-phenol. This mixture was dissolved in dimethylformamide (2000 mL) containing 1-bromononane (170 g) and potassium carbonate (15 g) and heated at 80°C for 16 hours. Dilution with water and hexane extraction yielded a concentrated mixture of products. Distillation gave a mixture of [(fluoro-2 nonyloxy-6)phenyl ]-3 butene and [(fluoro-3 nonyloxy-2)phenyl]-3 butene (186 g): p.e. 120-125° at 0.1 mm.

This mixture of isomers (185 g) in dimethyl sulfoxide (1000 mL) containing potassium tert-butoxide (1.5 g) was left at room temperature for 6 hours. The addition of water and hexane extraction gave a mixture of [(2-fluoro-6-nonyloxy)phenyl]-1-butene and [(3-fluoro-2-nonyloxy)phenyl]-1-butene.

This mixture of isomers (175 g) was dissolved in a mixture of dichloromethane and methanol (9:1,

2000 mL) and exposed to an ozone stream at -40° for 8 hours. After this time, the reaction mixture was poured into a mixture of water, hexane and dimethyl sulfide (100 mL) and stirred at room temperature for 1 hour.

43

The hexane extract was washed (water), dried (MgS0^), treated with a dimethyl sulfide supplement (50 m|_) and left at room temperature for 16 hours.

The removal of solvents gave the mixture of aldehydes fluoro-2 nonyloxy-6 benzaldehyde and fluor-4 nonyloxy-2 benzaldehyde (155 g).

This mixture of aldehydes (150 g) in ethanol (2000 mL) was exposed to sodium borohydride (15 g) at 5° and then stirred at room temperature for 30 minutes. Water (1500 mL) and salt water (500 mL) were then added, and the alcohol mixture was extracted in hexane. Solvent removal and silica gel chromatography of the residue (a 5% ethyl acetate-hexane mixture) yielded pure 2-fluoro-6-nonyloxybenzyl alcohol (76 g).

Example 21

(All trans)-[2-fluoro-6-nonyloxyphenyl]-9-3,7-dimethyl-2,4,6,8-nonatetraene

A mixture of fluoro-2-nonyloxy-6-benzyl alcohol (19 g) and triphenylphosphine hydrobromide (26 g)

The solution in acetonitrile (250 mL) was heated under reflux for 14 hours, then concentrated to dryness to obtain [[(2-fluoro-6-nonyloxyphenyl]methyl]tri-phenylphosphonium bromide (42 g). This phosphonium salt was dissolved in tetrahydrofuran (600 mL), cooled to -50°C, and treated with n-butyllithium (45 mL, 1.6 M in hexane). After stirring for a further 15 minutes at -50°C, the ethyl ester of 7-formyl-3-methyl-2,4,6-octatrieneoic acid (8.4 g) was added, and the reaction mixture was heated to the temperature

44

The mixture was left at room temperature and stirred for another 15 minutes. Hexane was then added, and the mixture was washed with water, 40% aqueous methanol, and dried (MgSO₄). Concentration of the hexane extract and purification by chromatography (5% ether-hexane) yielded the pure trans isomer (11 g).

Crystallization from ethyl hexane-acetate gave the ethyl ester of (all-trans)-[fluoro-2 nonyloxy-6 phenyl]-9 dimethyl 3,7 nonatetraene-2,4,6,8 oic acid (9.5 g).

A solution of the ester (6.5 g) in ethanol (150 mL) was treated with a solution of potassium hydroxide (7 g) in water (40 mL) and heated under reflux for 1 hour. The cooled reaction mixture was poured into cold aqueous hydrochloric acid, and the acid was extracted in chloroform. Removal of the solvents and crystallization from ethyl hexaneacetate gave (all-trans)-[fluoro-2-nonyloxy-6-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraeneoic acid; p.f. 107-109°.

45

Example 22 CAPSULE COMPOSITIONS:

Item Constituents mg/capsule mg/capsule mg/capsule

1. (all-trans)-15[(nonyloxy)-2-phenyl]dimethyl-3,7-2,4,6,8-nonatetraene-oic acid

2. Lactose 239

3. Starch 30

4. Talc 15

5. Magnesium 1

30

60

224 30 15 1

194 30 15 1

Weight of capsule contents:

300 mg

300 mg

300 mg

OPERATING PROCEDURE:

1) Mix steps 1-3 in a suitable blender

2) Add the talc and magnesium stearate and mix for a short period

3) Encapsulate on a suitable encapsulating machine

46

Example 23

The capsules are prepared by the process of Example 22 except that the active constituent (item 1) was (all-trans)-[(fluoro-2 (nonyloxy)-6 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid.

Example 24

TABLET COMPOSITION (Granulation in wet state)

Point Constituents mg/tablet mg/tablet mg/tablet

1. Acid (all-trans)- 100 250 500 [(nonyloxy)2 phenyl]-9

3,7-dimethyl-2,4,6,8-nonatetraene

2. Lactose 98.5 147.5 170

3. Polyvinylpyrrolidone

(PVP) 15 30 40

4. Modified starch 15 30 40

5. Corn starch 15 30 40

6. Magnesium stearate 1.5 2.5 5

Tablet weight: 245 mg, 490 mg, 795 mg

47

Operating procedure:

1. Mix items 1, 2, 4 and 5 in a suitable mixer, granulate with PVP and dissolve in water/alcohol. Dry the granules. Grind the dry granules using a suitable grinder.

2. Add the magnesium stearate and compress on a suitable press.

Example 25

The tablets are prepared in the same manner as in Example 24 except that the active constituent (item 1) was (all-trans)-[fluoro-2 (nonyloxy)-6 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid.

Example 26

TABLET COMPOSITION: (Direct compression)

Constituent Point mg/tablet mg/tablet mg/tablet

1. (All-trans)-15 30 60 [(nonyloxy)-2 phe-

nyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene

2. Lactose 207,192,162

3. Avicel 45 45 45

4. Starch for direct compression 30 30 30

5. Magnesium 3 3 3

Tablet weight

300 mg

300 mg

300 mg

48

OPERATING PROCEDURE:

1. Mix the product from point 1 with an equal amount of lactose. Mix well.

2. Mix with points 3, 4 and the remainder of point 2. Mix well.

3. Add the magnesium stearate and mix for 3 minutes.

4. Compress onto a suitable matrix.

Example 27 CAPSULE COMPOSITIONS:

Point Constituents mg/capsule

1. (All-trans)-15-dimethyl-3,7-[[(8-hydroxy-oxtyl)oxy]-2-phenyl]-9-2,4,6,8-nonatetraene-oic acid

2.

3.

4.

5.

Lactose

Starch

Talc

Magnesium

239 30 15 1

mag/capsule mg/capsule 30 60

224 30 15 1

194 30 15 1

Weight of capsule contents:

OPERATING PROCEDURE:

300 mg

300 mg

300 mg

1) Mix points 1-3 in a suitable blender.

2) Add the talc and magnesium stearate and mix for a short time.

3) Encapsulate on a suitable encapsulating machine.

49

TABLET COMPOSITION: (Granulation in the wet state)

Item Constituents mg/tablet mg/tablet mg/tablet

1. Acid (all-trans)-100 250 500 dimethyl-3,7

[[(octyloxy)methyl]-2-phenyl]-9-2,4,6,8-nonatetraene icic

2. Lactose 98.5 147.5 170

3. Polyvinyl pyrrolidone 15 30 40

4. Modified starch 15 30 40

5. Corn starch 15 30 40

6. Magnesium stearate 1.5 2.5 5

Tablet weight: 245 mg, 490 mg, 795 mg

Operating procedure:

1. Mix items 1, 2, 4 and 5 in a suitable mixer, granulate with PVP and dissolve in water/alcohol. Dry the granules. Grind the dry granules using a suitable grinder.

2. Add the magnesium stearate and compress on a suitable press.

Example 29

[[trifluoromethyl1-2 (nonyloxy)-6 phenyl]methyl]triphenylphosphonium bromide

A mixture of α,α,α-trifluoro-μ-creso1 (51 g), α-bromo-β-nonane (70 g), and potassium carbonate (100 g) in dimethylformamide was heated at 85°C for 48 hours. The addition of water and hexane gave α-(3-trifluoro)phenylnonyl ether (89 g).

50

Pure: evaporated at 15°C at 0.1 mmHg. This product (89 g) in ether (1.5 L) at -20°C was mixed with n-butyllithium (1.5 H in hexane; 233 mL), then stirred for 2 hours at room temperature. This mixture was then cooled to -40°C, treated with an excess of dry dimethylformamide (40 mL) in ether (100 mL), heated to 0°C, and then treated with water. Extraction with hexane and silica gel chromatography (5 α-ether-hexane) gave (trifluoromethyl-2.6-nonyloxy)benzaldehyde (35 g). The reduction of this product with sodium borohydride in ethanol by the process described in Example 1 gave methyl alcohol of (2-trifluoromethyl-6-nonyloxy)benzene (32 g) after silica chromatography. This material (31 g) was converted to [[2-trifluoromethyl](nonyloxy)-6-phenyl]methyl1]triphenylphosphonium bromide by reaction with triphenylphosphine hydrobromide by the process indicated in Example 1.

Example 30

(All-trans)-[(trifluoromethyl1-2 (nonyloxy)-6 phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid. [[trifluoromethyl-2 nonyloxy)-6 phenyl]methyl]triphenylphosphonium bromide (97 mmol) in tetrahydrofuran (600 mL) was transformed by reaction with the ethyl ester of 7-formyl-3-methyl-2,4,6-octatriene-oic acid to the ethyl ester of (all-trans)-[(trifluoromethyl1-2 (nonyloxy)-6 phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid by the process shown in Example 3. Purification by chromatography and crystallization from hexane were

51

given the pure ethyl ester (41%). Hydrolysis (5.2 g), as in Example 5, gave pure (all-trans)-[(trifluoromethyl1)-2 (nonyloxy)-6 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid (3 g): wt. 135-136° (from ethyl hexane acetate).

Example 31

(All-trans)-[(hexyloxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene

The bromide of [[(hexyloxy)-2phenyl]methyl]triphenyl phosphonium, prepared by the process of Example 1 by reacting 2-hydroxybenzaldehyde and 1-bromohexane, was converted to (all-trans)-[(hexyloxy)-2-phenyl]-9-diethyl-3,7-nonatetraene-2,4,6,8-oic acid; p.f. 137-138° (from ethanol) by the process of Example 3.

Example 32

[[[(nonyloxy )-2 (hydroxy)-5 phenylmethyl ] bromide]

t r i p h é n y 1 p h o s p h o n i u m

A solution of 4-bromophenol (1 mol) in tetrahydrofuran (500 mL) was added to a slurry of sodium hydride (1.17 mol) in dimethylformamide (1.2 L) at 25°C. After complete reaction, allyl chloride (1.32 mol) was added, and after further stirring for 3 hours at 45°C, the product was isolated with water and hexane. Distillation gave allyl-(4-bromophenyl)ether, boiling point 65-67°.

0.1 mm (82%). This material was heated at 195°C with dimethylaniline for 4 hours, then distilled to obtain 2-allyl-4-bromophenol (0.81 mol). A solution of this material (0.81 mol) in tetrahydrofuran

52

(200 mL) was added to a mixture of 1-bromo-nonane (0.8 mol), sodium hydride (0.92 mol), and potassium iodide (1 g) in dimethylformamide (1 L) at 25°C. After the end of hydrogen evolution, the mixture was heated to 50°C for 14 hours, cooled, added to excess water, and extracted with hexane. Distillation gave nonyl-(2-allyl-4-bromo-phenyl)ether (256 g): e.g. 147–156°C at 0.1 mm. This material (255 g) in dimethyl sulfoxide (1 L) and tetrahydrofuran (0.5 L) was heated to 35–40°C with potassium tert-butoxide (2 g) for 2 hours, then fixed with acetic acid (5 mL) in water. Isolation of the reaction products with hexane gave [(nonyloxy)-2-bromo-5-phenyl]-1-propene (234 g): e.g. 145-155° at 0.1 mm. A solution of the above material (0.56 mol) in tetrahydrofuran (600 mL) was converted to a Grignard reagent with magnesium (1 mol) at 55°C for 3 hours. After complete reaction, the mixture was cooled to 0°C and treated with trimethyl borate (0.75 mol) in ether (200 mL). After stirring for a further 30 minutes at 25°C, the mixture was cooled to 0°C and exposed to a mixture of ammonium chloride (10 µg) and hydrogen peroxide (10%, 500 mL) and stirred for a further hour at 25°C. The addition of water and hexane gave the crude material after removal of the hexane under vacuum. The crude product was passed through a silica gel buffer to obtain para[[[(1-propene-1-yl)-2-(nonyloxy)-4-phenyl]phenol (73 g). Acetylation of this material (0.8 g) with acetyl chloride and triethylamine in dichloromethane gave [(1-propene-1-yl)-2-(nonyloxy)-4-acetoxy]benzene (89 µg). This material (99 g) was dissolved in a mixture of methanol (150 mL) and dichloromethane (1.5 L) and treated with ozone at -40°C until

53

that all the starting materials had been used. Dimethyl sulfide (50 mL) and water (500 mL) were then added, and after vigorous stirring for 30 minutes at 25°C, the organic phase was dried (MgSO₄) and concentrated to obtain [(nonyloxy)-2(acetoxy)-5]benzaldehyde (83 g). Reduction of this material (80 g) with sodium borohydride (6 g) in ethanol (1 L) at 20°C for 2 hours gave [(nonyloxy)-2(acetoxy)-5]benzene methyl alcohol, which was immediately exposed to aqueous potassium hydroxide (300 mL, 40%) in ethanol (1 L) for 30 minutes at 60°C. Acidification with aqueous acid (6 M hydrogen chloride) and chloroform extraction gave the crude product after concentration. Digestion of the residue with hexane gave 11[(hydroxymethyl1)-3 (nonyloxy)-4]pheno1 (63 g) as a solid. A solution of this material (62 g)

in a mixture of acetonitrile (0.5 L) and triphenylphosphine hydrobromide (86 g) was heated under reflux for 14 hours and concentrated to dryness at 50°C to obtain [[(nonyloxy)-2 (hydroxy)-5 phenyl]methyl]triphenylphosphonium bromide in glass form.

Example 33

(All-trans)-[5-hydroxy-2-(nonyloxy)phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene

The bromide of [[(nonyloxy)-2 (hydroxy)-5 phenyl] methyl] triphenylphosphonium (0.23 mol) in tetrahydrofuran (1.5 L) at -70°C was treated with n-butyllithium (1.6 M in hexane; 315 mL), then treated with ethylformyl-8-dimethyl-3,7-2,4,6-octatriene oate (59 g) in tetrahydrofuran. The mixture was then

54

was warmed to -15°C, acidified with acetic acid, and extracted in ether and aqueous methanol (40%). Purification by silica gel chromatography gave the ethyl ester of pure (all-trans)-5-[hydroxy-5(nonyloxy)-2-phenyl]-9-dimethyl-3,7-nona-2,4,6,8-tetraeneoic acid. Hydrolysis of this ester (6 g) by the process described in Example 5 gave (all-trans)-[hydroxy-5(nonyloxy)-2-phenyl]-9-dimethyl-3,7-nona-2,4,6,8-tetraeneoic acid (3.5 g): w.f.

10 170-173° (from ethyl acetate).

Example 34

(all-trans)-[(nonyloxy)-2 (trifluoro-2,2,2

ethoxy)-5 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 o igu e

The ethyl ester of (all-trans)-[5-hydroxy-(nonyloxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraeneoic acid (4.4 g) was heated at 90°C for 72 hours with potassium carbonate (7 g), 2,2,2-trifluoroethyl-p-toluene sulfonate (6 g) in dimethylformamide (200 mL). Treatment with water and hexane followed by purification over silica gave the pure ethyl ester (0.75 g). Hydrolysis of this ester (0.9 g) by the process of Example 5 gave (all-trans)-[(nonyloxy)-2 (trifluoro-2,2,2 ethoxy)-5 phenyl ]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid (0.6 g) after crystallization from a mixture of tetrahydrofuran and hexane: w.p. 121°C.

Example 35

(Z)-[[(1-decene-yl)-2-phenyl]methyl]tri-30 phenylphosphonium bromide and (E)-[[[1-decene-yl)-2

phenyl] m ethyl]triphenylphosphonium

15

20

25

d

55

A mixture (E,Z) (1-deceneyl)-2-bromo-1-benzene, such as that prepared in Example 16, (1:4) was transformed into a mixture (E,Z) of (1-deceneyl)-2-hydroxymethyl-1-benzene by the process of Example 17. This mixture was separated by silica gel chromatography to obtain the pure alcohols (J1) and (Z). The reaction of each of these isomers with triphenylphosphine hydrobromide, as in Example 1, gave the corresponding phosphonium salts, namely (Z)-[[1-deceneyl)-2-phenyl]methyl]triphenylphosphonium bromide and (E)-[[1-deceneyl)-2-phenyl]methyl]triphenylphosphonium bromide.

Example 36

(All-trans)-[(1-decene-2-yl)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene-oic acid

(E)-[[(1-decene-2-yl)-2-phenyl]methyl]triphenylphosphonium bromide was converted to the ethyl ester of (all-trans)-[(1-decene-2-yl)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraeneoic acid by the process described in Example 1. Hydrolysis with a base,

as in example 1, and the crystallization of the crude acid from acetonitrile gave (all-trans)-[(decene-1 yl)-2 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid, p.f. 105-107°.

Example 37

(E,E,E,E,Z)-[(1-decene yl)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene acid

The compound in the labeling was prepared in the same way as in Example 36 using bromide

56

of (Z)-[[(decene-1 yl)-2 phenyl]methyl]tryphenylphos-phonium. Hydrolysis of the ethyl ester and crystallization of the crude acid from the ether gave pure (E,E,E,E,Z)-[(decene-1 yl)-2 phenyl]-9 5 dimethyl-3,7 nonatetraene-2,4,6,8 oic acid: wt. 103-105°.

In the following examples, Compound A is (all-trans)-[(nonyloxy)-2-phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraeneoic acid. In the following examples, Compound A was tested by various assays regarding its anti-inflammatory activity in animal models of inflammation and in certain chronic models of adjuvant-induced arthritis.

In all tests, Compound A and the other 15 retinoids tested simultaneously were prepared in peanut oil containing 0.05% propyl gallate as an antioxidant. The injected volumes used

-1 -1

The doses were 5 ml/kg for rats and 10 ml/kg for mice. The control group received the corresponding volume of excipient - peanut oil.

Example 38

Effect of Compound A on delayed hypersensitivity to methylated bovine serum albumin (MBSA).

Animals: M'FI mice, E33 substrain, males and females. 25. Initial weight approximately 25 g.

Material Methylated bovine serum albumin (MBSA) (Sigma). Complete Freund's adjuvant (Difco).

57

Method: Groups of 10 mice were sensitized (Cjûur 0) by intradermal injection at two points on the abdomen of 0.05 ml of water-in-oil emulsion of MBSA and Freund's complete adjuvant. On day 9, the mice were challenged by injecting 20 µl of 1 µl MBSA solution into one paw and 20 µl of water into the contralateral paw. Paw volume was measured 24 hours later by mercury displacement plethysmography. The mean percentage increase in the volume of the paw challenged with MBSA compared to that of the paw challenged with water was calculated for each treated group. Administration of the excipient and retinoid began on day 0 and ended on day 9.

Results The results are presented in the following table (Table II).

O,

58

Table II. Effects of Compound A in the delayed hypersensitivity test to MBSA

Treatment application Increase % Decrease % Variation in mg.kg" vol. legs (v. control body weight)

5 peanut oil) medium (g)

10

15

Peanut Oil Etretinate Compound A Compound A Compound A 100

10

10

30

109 + 11

59 + g**

102 + 12nS

50 + 7***

40 + 5***

46

54-

63

m 3.8 f 0.2 m -0.8 f -2.0 m 3.3 f -0.2 m 2.8 f 0.5 m 3.3 f -0.2

15

Each group consisted of 4 male mice and 6 female mice.

(kept in individual cages). The products have

_ -]

were administered orally in a volume of 10 ml.kg (10 doses).

ns. Not significant * * p < 0.01 * * * p < 0.001 compared to the control receiving the excipient using Student's two-sided t-test.

59

Example 39

Effect of Compound A on the progression of adjuvant-induced arthritis in rats

Animals Female AHH/H rats (PVG derivatives) with an initial weight of 110 to 140 g were used.

Material Adjuvant for injection. A homogenized suspension of heat-killed M. tuberculosis (human strains C, DT and PN), 5 mg.Ml ^ in liquid paraffin was prepared.

Method: Rats were randomly divided into groups of five animals, and arthritis was induced by injecting 0.1 ml of adjuvant suspension into the footpad of each rat's right hind paw. The test compounds were administered via tube each morning, beginning on the day of the adjuvant injection. Two groups of control rats received the excipient, as did a group of three normal rats included for comparison. The administration took place

6 ms each day until the end of the test on the 15th

day except for the first end of e-

/th m e , ,th . \ .

Maine (5 and 6 days). The treated groups are presented in Table III

and etretinate is included as a standard retinoid.

60

Measurements of the volume of the right hind legs were taken at the beginning and the

_th , ,,th . , , . . , . , , , ,

2 and 4 days after adjuvant injection (primary phase). The volume of the right and left hind legs was then measured.

on day 8 and every two or three days until the end of the experiment on day 15 (secondary phase). At that time the mobility of each ankle joint and the incidence and severity of secondary lesions on the nose, ears, front paws, left hind paw and tail were also assessed with regard to the degrees of possible flexion and using an arbitrary scoring system.

Evaluation of results

The time evolution curves for the injected legs were integrated from day 6

Days 0-4 were used to represent primary edema, and days 8-15 were used for secondary edema. Secondary edema in the non-injected leg was integrated in the same way from day 9-15 to day 150. Calculations were performed using a specific computer program that calculated the mean SE for each integrated area. The significance of differences compared to controls was determined using a two-tailed Student's t-test, and percentage reductions compared to control areas were calculated. Percentage improvements in joint mobility and percentage reductions were also calculated.

61

The lesion scores were also determined. In the latter case, the Wilcoxon rank-sum test (two-sided) was used to express the difference from the control score using the raw data. The variation in mean body weight in each group was recorded.

Results The results are presented in the following table (Table III).

TABLE III

Effect of Compound A and lertrenate on adjuvant-induced arthritis in rats

Treatment

Normal T control

Adjuvant T Witness

Compound A

Ertretinate

Dose mg.kg-1 p.o.

15

45

15

Reduction

Primary

(RIGHT)

-6

16

% leg edema1

Secondary (right)

30

51

30

Secondary (left)

94

75

.67

Score 2 lesions (% RDN)

72

65

56"

Joint mobility (% INC)

66"

• 66

50

Change in body weight g

Days 0-15

+ 13.3

- 0.6

+ 2.2

+ 5.4

+ 1.0

Days 8-15

+ 3.7

5.5

- 0.4

+ 1.2

- 5.0

ON N3

1. Statistical analysis using the two-tailed Student's t-test

2. Statistical analysis using the Wilcoxon rank-sum test (two-tailed) * = p < 0.05 ** = p < 0.02 *** = p < 0.01

'v.

63

Example 40

Effect of Compound A on established type II collaqene arthritis

Animals: Rats, Alderley Park, Strain 1, males and females,

Material: Type 2 collagen (prepared from bovine nasal cavity cartilage), Freund's incomplete adjuvant (Difco).

Method: Rats were sensitized to type 2 collagen by intradermally injecting 1 ml of a water-in-oil emulsion consisting of equal parts collagen solution.

_ -]

Gene type 2, 1 mg/ml, in 0.45 M NaCl, 0.02 M Tris, pH 7.4, and Freund's incomplete adjuvant. Rats that developed arthritis were assigned on day 15 after sensitization to either a control group treated with peanut oil (6 males, 4 females) or the group treated with Compound A (6 males, 5 females). Hind leg volume measurements were taken to ensure even distribution of rats between the groups. An overnight urine collection was performed.

the 15th/-] day e^ the administration has

©

The study began on day 16. These urine samples were analyzed to measure glucosaminoglucans (GAGs). Compound A was

_ -]

administered at a dose of 100 mg/kg orally. On the 9th day, a second overnight urine collection was carried out.

64

These urine samples were analyzed to measure glucosaminoglucans (GAGs), s m 6

On day 20, a second measurement of the hind legs was taken. The rats were then anesthetized with sodium pentobarbital.

The rats were bled, sacrificed, and radiographs were taken of their hind and forelegs. They were treated from day 16 to day 19 inclusive (4 doses).

10

Results The results are presented in the following table (Table IV).

î

TABLE IV.

Effects of compound A in the type II collagen-induced arthritis test

DAY

16

20

Leg volume 2.44 + 0.08 2.66 + 0.06

Compound A 100 mg.kg-^GAG (^g)

Weight (g) Volume paw

1586 + 307 247 + 14 2.48 + 0.07

634 + 72229 + 10 2.46 + 0.07

Peanut Oil Witness

GAG (pg) Pds (g)

1538 + 192

249 + 19

729 + 134,258 + 19

ON VI

Note: Both male and female rats lost weight during treatment with compound A.

•v

66

Example 41

Effect of Compound A on non-immune inflammation

Animals: Alderley Park Strain 1 female rats weighing 170-205 g at the start of the experiment were used.

Lambda-carrageenan material. Prepared as a solution in physiological saline and sterilized by autoclaving.

Method: Compound A was administered orally

to groups of 8 rats once a day for 10 days at doses of 10, 30 and _"]

100 mg/kg. Control animals received the excipient. One hour after the last dose, the animals were anesthetized with methoexitone.

-1

(Brietal, 50 mg/kg) and 0.2 ml of lambda-carrageenan at 1% concentration were injected into the pleural cavity. Four hours later, the animals were sacrificed with an excessive dose of pentobarbital (Sagatal), the pleural exudate was collected, and the pleural cavity was washed with 2 ml of phosphate-buffered saline (PBS-A, Oxoid). The exudate volume was recorded, and blood cell counts were determined using an automatic counter (Coulter). Differential counts were performed on Giemsa-stained exudate smears to separately determine the counts of polymorphonuclear leukocytes (PMNs) and mononuclear cells.

67

Immediately after the recovery of pleural fluid, the animals' tibias were excised and their tensions at the fracture were determined.

5 The body weight of the animals was recorded

. _ recorded daily. Statistical analyses were performed using Student's t-test.

Results The results are presented in Table 10 below (Table V). In the following table, the dose is given in mg per kg per day.

I

TABLE V

Effect of Compound A on the evolution of carrageenan-induced pleurisy over 4 hours

Compound

Dose-

Exudate volume

Early blood cell count.

total PMN

MN totals

—Hi; -r

Tibial fracture tension

(p.o.)

(ml) % change x106% change x106% change x106% change

(right+left) Average %

(kg) variation

Witness

(peanut oil)~

[ 5ml

1.40 +0.16

129.3 +3.1

119.1 +2.9

10.2 +0.3

6.7 +0.3

Compound A (solution)

OMG

0.94* -32.8 +0.08

120.8N*S* -6.5

±7-3

112.4N*S* + 6.7

-5.6

8.4N,S,'-17.4 +0.8

6.5N,S* -3.4 +0.1

Compound A

30mg

0.88* -36.9 +0.09

112.1* -13.3 +6.0

104.2* +5.6

-12.4

7.9* -23.1 +0.7

6.8N*S* +1.5 +0.3

Compound A

lOOmg

0.80** -42.9 +0.08

109.1N*S* -15.7 +8.1

101.6N*S* +7.1

-14.7

7.5* -26.7 + 1.!

6.7N,S* -0.3 +0.2

N.S. - not significant

* = p < 0.05; ** = p < 0.01 compared to the control treated with the excipient using Student's two-tailed t-test.

69

Example 42

Effect of Compound A on granuloma caused by an impregnated sponge in rats

Animals Female AHH/R rats (PVG derivatives) with

. . an initial weight of 120-140 g were used.

Materials: Sponge preparation. Tablets (6.5

mm in diameter) were cut from a fabric

cellulose sponge ("Wettex") and 0.1 ml of

_ 1

A suspension containing 0.5 mg/ml of heat-killed M. tuberculosis (human strains C, DT, and PN) in sterile saline solution was applied to each lozenge. The lozenges were dried, weighed, and sterilized by autoclave.

Method: The rats were randomly divided into groups of five animals and daily administration of the tested compounds was started.

After the fifth dose, the rats were

_ "|

The rats were anesthetized with Sagatal (45 mg/kg i.p.), their backs were shaved, and two pellets were implanted under the skin (one on each side) in each rat through a small midline incision on the back. The incision was closed, and the rats were allowed to recover from the anesthesia.

Seven days after implantation, the rats were sacrificed and the pellets were removed, along with the tissue adhering to them.

70

The pellets were weighed. Each pellet was then placed in a 4 ml aliquot of distilled water, crushed into very small fragments with fine scissors, and treated ultrasonically. After centrifugation, the Na+ and K+ content of the supernatant was determined by flame photometry. In addition, the adrenal glands and thymus of each rat were removed and weighed, and the lower parts of the hind legs were removed for tibial fracture tension measurement. Body weight was recorded throughout the experimental period.

Results The results are presented in the following table (Table VI).

Evaluation of results

The mean sequencing (SE) for each parameter was calculated, and differences from control values were determined using a two-tailed Student's t-test. Percentage reductions in granuloma weight, Na+ and K+ content, and percentage changes in adrenal glands and thymus, weight, and tibial fracture tension were determined.

I

TABLE VI

Effect of Compound A, 11-etretinate and dexamethasone in the granuloma test induced by an impregnated sponge in rats

Treatment

Dose Number mg.kg-1 of rats

% reduction in granulome wet weight .Na+ K+

% Variation

of the pds of the pds of the of the tensor adrenals thymus of fracture of the tibias

1. Variation in body weight

(g)

♦

T Witness Compound A

Etretinate

8

-

-

+9.6+1.3

15 p.o 5 45 p.o 5

15.1** 14.5** -6.9**

îo.B ii o.a

+17.6** -4.4 -4.0 +29.0** +14.3 -1.12

+7.2 +0.74 +11.4 +0.98

15 p.o 4

2.7 5.9 -3.9

+22.9** +15.2 -4.6

+13.3+2.6

Dexamethasone

0.5 se 5

65.5*** 51.2*** 75.2***

-49.5*** -79*** -11.5*

-10.2+1.59

* p < 0.05; ** p < 0.02; *** p < 0.01

Note: 2 control rats and 1 rat treated with etretinate died under anesthesia

'v

( TABLE VII

Effect on the course of established arthritis caused by an adjuvant 3

Leg variation 0

C

Weight variation

Dose Volume

Plasma fibrinogen

1 bodily

Band

Treatment

(mg/kg) (ml)

(mg/dl)

(g)

Arthritis (10)D

Excipient

+0.53 + 0.08

1773 + 30

7.5 + 1.2

Arthritis (IO)

Compound A

100 - 0.96 + 0.10*

874 + 57

5.2 + 2.1

Arthritis (IO)

Indomethacin

1 - 1.22 + 0.15

978 + 100

25.6 + 3.1

+ M

Averages - S.E.

The products were administered once daily for 7 days a week via tube, starting on day 22 after arthritis induction. Tweeen 80 was used as an excipient.

The change in leg volume/body weight is equal to the leg volume/body weight on day 28 minus the leg volume/body weight on day 22. Given that the adjuvant was

injected at the base of the tail, the variation in leg volume is a combined value for both hind legs.

Number of animals per group

Val. significantly different from the value found for arthritic animals treated with the excipient (Student's t-test, p 0.05)

FV 4070/66

73

Claims (15)

Demands: 1. A process for preparing compounds having the following formula R7 Rfl I I8 ch=ch-c=ch-ch=ch-c=ch-r 5 7 6 5 4 3 2 1 Where R\j represents a hydrogen, a lower alkyl, a chlorine, a fluorine or a trifluoromethyl; R^ represents a chlorine, a trifluoromethyl, a lower alkyl, a fluorine, a hydroxy, a lower alkoxy, a lower trifluoromethyl-alkoxy, a hydrogen; R^ represents a hydrogen, a lower alkyl, a chlorine or a fluorine; R^ represents an alkyl group having a straight chain length of 4 to 9 carbon atoms or -■ C H 2 (. C H_2 ) n 0 H ; X represents -CHO-, -CH-, -0-, R10 R10 -C=CH-, or -N-; R- represents C0QRq; i i 5 y R1 0 R1 0 and Ry, Rg, Rçj and R^g represent a lower hydrogen or alkyl; n is 6 or 7; and their pharmaceutically acceptable salts where R^ represents a hydrogen, which includes a) the reaction of a compound of formula 74 with a compound of the formula • w np u I7 I8 II ohc-c=ch-ch=ch-c=ch-c-or,9 VIT or b) the reaction of a compound of formula with a compound of formula FV 4070/66 75 ib n 0hc-c=ch-c-0rq xvn 9 or c) the reaction of a compound of formula with a compound of formula R^Z, where in the above formulas R^, R2, R, R^, Ry and Rg are as in claim 1; R^ represents a lower alkyl; Y represents an aryl, Z represents a leaving group and Z' represents a halide ion; and if desired, the transformation of an alkyl ester group of the carboxylic acid -C00R^, contained in the reaction product into a free acid and if desired further the transformation of the acid into a pharmaceutically acceptable salt. 2. A process according to claim 1 for the preparation of compounds of formula I where R^ represents a hydrogen, a chlorine, or a fluorine; R^ represents a hydrogen, a lower alkoxy, a chlorine, or a fluorine; R^ represents a hydrogen, a lower alkyl, a chlorine, or a fluorine; R^ represents an alkyl containing 8 to 10 carbon atoms with a length of FV 4070/66 76 straight chain of 8 or 9 carbon atoms; X represents -CH-O-, -CH-, -O- or -N-; R c , , Il | ^ represents R10 R10 R10 COORg and n, R^, Rg, R^ and R^g are as in claim 1; and their salts where R^ represents a hydrogen. 3. A process according to claims 1 or 2 for the preparation of compounds of formula I where R^ represents an alkyl group as defined in these claims. 4. A process according to claim 3 for the preparation of compounds of formula I where X represents -0-. 5. A process according to claim 4 for the preparation of compounds of formula I where R^ represents a chlorine or a fluorine. '6. A process according to claim 4 for the preparation of compounds of formula I where R2 represents a lower alkoxy. 7. A process according to claim 1 for the preparation of [chloro-2 (nony1oxy)-6-pheny1]-9 dimethyl-3,7 nonatetraene-2,4,6,8 icic acid. 8. A process according to claim 1 for the preparation of (all-trans)-[fluoro-2 (nonyloxy)- acid 6 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 oic. 9. A process according to claim 1 patation of dimethyl-3,7 (methoxy-5 phenyl)-9 nonatetraene-2,4,6,8 icic acid. for pre-nonyloxy-2 FV 4070/66 77 10. A process according to claim 1 for the preparation of [(nonyloxy)-2 phenyl]-9 dimethyl-3,7 nonatetraene-2,4,6,8 icic acid. 11. A process according to claim 1 for the preparation of (all-trans)-dimethyl-3,7 (octyl-amino-2-pheny-1)-9 nonatetraene-2,4,6,8 oic acid. 12. A process according to claim 1 for the preparation of (all-trans)-dimethyl-3,7[[(hydroxy-8 octyl)oxy]-2 phenyl]9 nonatetraene-2,4,6,8 oic acid. 13. A process according to claim 1 for the preparation of: (all-trans)-[(trifluoromethyl)-2(nonyl-oxy)-6-phenyl]-8-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid, dimethyl-3,7-[(octyloxy)-2-phenyl]-9-nonatetraene-2,4,6,8-oic acid, dimethyl-3,7-[[(dimethyl-2,2-octyl)oxy]-2-phenyl]-9-nonatetraene-2,4,6,8-oic acid, ethyl ester of [(nonyloxy)-2-phenyl]-9-dimethyl- 3.7 nonatetraene-2,4,6,8 oic acid, (all-trans)- [(hexyloxy)-2 phenyl]-9 dimethyl-3,7 nonatetraene-2,4, 6.8-oic acid, (all-trans)-[5-hydroxy-2-(nonyloxy)phenyl]-9-dimethyl-3,7-2,4,6,8-nonatetraene, (all-trans)-[(nonyloxy)-2 (trifluoro-2,2,2/ethoxy)-5-phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid, dimethyl-3,7-[[(octyloxy)-methyl]-2-phenyl]-9-nonatetraene-2,4,6,8-oic acid, (decylphenyl)-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid, ethyl ester of dimethyl-3,7-(octylamino-2-phenyl)-9-nonatetraene-2,4,6,8-oic acid, and (all-trans)-[(decene-1-yl)-2-phenyl]-9-dimethyl-3,7-nonatetraene-2,4,6,8-oic acid FV 4070/66 78 14. A process for the preparation of pharmaceutical compositions having anti-rheumatic, anti-arthritic or immunosuppressive properties which comprises mixing a compound of formula I or its salt where represents a hydrogen with a pharmaceutically acceptable excipient. FV 4070/66 79 15. The new compounds, intermediates, formulations, processes and methods in substance as described herein. ORIGINAL pages conien ni ttenvpis .n-joî C u R A U Industrial Property Attorney 26 b,sf Bout. Princess Charlotte MONTE-CARLO ..r ■ % r/y '■ ■} r 'Vn f ■' m,/a M* SA 9t>