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AU694428B2 - Use of natural products and related synthetic compounds for the treatment of cardiovascular disease - Google Patents

Use of natural products and related synthetic compounds for the treatment of cardiovascular disease Download PDF

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AU694428B2
AU694428B2 AU68390/94A AU6839094A AU694428B2 AU 694428 B2 AU694428 B2 AU 694428B2 AU 68390/94 A AU68390/94 A AU 68390/94A AU 6839094 A AU6839094 A AU 6839094A AU 694428 B2 AU694428 B2 AU 694428B2
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Prior art keywords
formula
compound
bis
methyl
dihydroxy
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AU6839094A (en
Inventor
Colin Charles Duke
Qian Li
Basil Don Roufogalis
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University of Sydney
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University of Sydney
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Description

WO 94/28886 PCT/AU9400297 USE OF NATURAL PRODUCTS AND RELATED SYNTHETIC COMPOUNDS FOR THE TREATMENT OF CARDIOVASCULAR DISEASE Technical Field The present invention relates to the use of naturally occurring phenolic compounds and related synthetic compounds in the treatment or prophylaxis of cardiovascular disease and to novel phenolic compounds and the use thereof in the treatment or prophylaxis of cardiovascular disease.
Background Art Cardiovascular disease is a serious health problem and a major cause of death in Australia and most developed countries. It has been reported that calcium is central to cardiovascular function, in that the calcium ion controls the contraction of heart muscle and the tone of blood vessels. Certain drugs have been used to increase intracellular calcium in order to stimulate the failing heart (cardiotonic agents). The major drugs used for congestive heart failure in the past are derived from digitalis, found naturally in plants such as foxglove. Their action to raise intracellular calcium, however, is indirect, as they inhibit Na+,K+-ATPase which results in an increase in intracellular Na+, which then in turn stimulates the inflow of extracellular calcium and in turn stimulates the failing heart. These drugs are not ideal as they are toxic at doses only slightly higher than therapeutic cardiotonic concentrations.
There has been an active search for alternative cardiotonic agents in recent years and there is still a need for effective drugs to treat and prevent various aspects of cardiovascular disease.
Ca 2 has a variety of functions in most animal cells.
The concentration of free calci- ion (Ca 2 in the cytoplasmic space acts as an intracellular messenger in both electrical and non-electrical excitable cells. The important role of Ca 2 is in relation to cellular contraction, and proliferation especially contraction and relaxation of the heart.
Ilqe WO 94/28886 PCTMU4/00297 2 The movement of Ca 2 across cells is regulated by number of mechanisms. If there are means that can pharmacologically manipulate these processes then the level of free intracellular Ca 2 may be altered, resulting in a change in cellular response.
There are number of calcium pools which contribute to the concentration of Ca 2 in the cytoplasmic space. Two major important pools are namely, the extracellular pool and the internal store, the so-called sarcoplasmic reticulum (SR) store.
The entry of extracellular Ca 2 down its electrochemical gradient, not only raises the level of intracellular Ca 2 but also initiates the release of Ca 2 from the SR store. This phenomenon explains the rapid contraction of cells. The rise of intracellular Ca 2 is compensated by a number of mechanisms to remove Ca 2 from the cytoplasmic space, either by extruding the Ca 2 out of the cell through the Ca 2 pump, which is biochemically coupled to Ca 2 +-ATPase, and the Ca 2 /Na* exchanger, and by sequestering of Ca 2 back into the SR store through SR Ca2+-ATPase. These removal mechanisms are energy-dependent processes that utilise ATP as the energy source.
The present inventors have found that a range of naturally occurring phenols and related synthetic compounds manipulate the plasma membrane Ca2+-ATPase process named, hereafter, as the plasma membrane Ca 2 ATPase. It is anticipated that they may also alter the SR Ca 2-ATPase, given the similarity of this enzyme to this plasma membrane Ca 2 +-ATPase. Compounds discovered can inhibit the plasma membrane Ca 2 +-ATPase causing an increased level of free Ca 2 inside cell. At the same time, some of the compounds may be chosen to stimulate the SR Ca2+-ATPase, thereby increasing Ca 2 uptake into the internal SR store and making more Ca 2 available for release from the SR. The overall effect of these compounds is to increase the rate of contraction as well as the force of contraction of the heart cells, and particular of the failing heart.
-3 It has been reported that a number of reagents inhibit plasma membrane Ca2+-ATPase nonspecifically. It has also been reported that a number of long chain alcohols, hemin and nonhemin iron and fatty acids partially inhibit Ca 2 ATPase of erythrocyte membrane. The retinoids have been shown to have anti-calmodulin effects and therefore indirect effects on the Ca 2 +-ATPase (plasma-membrane Ca 2 pump) enzyme. The sesquiterpene lactone thapsigargin was found to be a specific inhibitor of Ca2+-ATPase of skeletal muscle endoplasmic (sarcoplasmic) reticulum.
Disclosure of the Invention In one aspect, the present invention provides the use of a compound of formula (I) R aAr(OH) wherein Ar is a ring system comprising one or more optionally substituted phenyl rings optionally linked to and/or .used with one or more other optionally substituted pheryl rings or one or more 5 or 6-membered, optionally o substituted heterocyclic rings wherein the heteroatom is oxygen; wherein Ar comprises 1-4 phenyl rings; wherein Ar can be linked to another Ar via a group X or directly linked to another Ar, wherein the two Ar groups are independently selected; when Ar is linked to another Ar via a group X, the two Ar groups can also be directly linked to each other; where X is optionally substituted C1- 2 0 alkylene, C 2 2 0 alkenylene or C 2 2 0 alkynylene; when Ar is linked to another Ar via a group X, R is hydrogen; C1- 2 0 alkyl, C2- 20 alkenyl, C2- 20 alkynyl, C 2 2 0 alkanoyl, C 2 20 alkenoyl, C2_ 2 0 alkynoyl, each of which can be optionally substituted; when Ar is not linked to another Ar via a group X, R is C5- 2 0 alkyl, C5- 2 0 alkenyl, C 5 2 0 alkynyl, C5- 2 0 alkanoyl, 20 alkenoyl, C5- 2 0 alkynoyl, each of which can be optionally \substituted; 9 4
R
1 is independently selected and is hydrogen; optionally substituted C1- 12 alkyl, C2_ 12 alkenyl, C2- 1 2 alkynyl; -COOR' halogen, -COR', -CONR'R', -S03R', -SO 2 NR'R', -SOR', S0 2
-NO
2
-CN,
glycoside, silyl; where R' is independently hydrogen; alkyl, alkenyl or alkynyl each optionally substituted; and where two qroups R 1 can be joined; wherein the optional substituents are one more independently selected from CI_10alkyl, C2- 1 0 alkenyl, C2_- 0 alkynyl; -COOR" halogen, -COR", -CONR"R", -S0 3
-SO
2 NR"R", -SOR", -SO 2
-NO
2
-CN;
wherein R" is independently hydrogen, alkyl, alkenyl, or alkynyl; r n 1, 2 or 3 m 1, 2, 3 or 4 or a pharmaceutically acceptable derivative thereof to inhibit the action of plasma membrane Ca2+-ATPase enzyme.
In a second aspect, the present invention provides the S use of a compound of formula or a pharmaceutically acceptable derivative thereof in the treatment or prophylaxis of cardiovascular disease related to the action of asma membrane Ca2+-ATPase enzyme.
25 In a third aspect, the present invention provides novel compounds of formulae (II) (III), (VI) .or pharmaceutically acceptable derivatives thereof: SRz -(CH2) I I R2 where
R
2 is 2-hydroxy 2-hydroxy and 4'-hydroxy 2-hydroxy-3-methyl 4-hydroxy-3-methyl 2,4-dihydroxy II ~d ~I P I 3, 5- dihydroxy- 4-methyl 2, 6-dihydroxy-4-methyl 2,4-dihydroxy-3-methyl 3-hydroxy-4-methyl when R 2 is above, r=7-14; when R 2 is above, r=8- 16; provided that when R2 is 2-hydroxy then when then when then when then when then r is not 7-10 and 13;
R
2 is 3,5-dihydroxy-4-rnethyl r is not 14;
R
2 is 2-hydroxy-3-rnethyl r is not
R
2 is 2,4-dihydroxy r is not 8-10 and 13 and
R
2 is 4-hydroxy-3-methyl r is not 0OH O (CH 2 )s where s 8-16
(III)
(IV)
where t 6-15
CH=CH(CH
2 )q where p q 12
VV
7-01RA( AMENDED SHEET
IPEA/AU
E EU 109 -6
(VI)
(CH
2 )q
CH=CH
where p q 12 In a fourth aspect, the present invention provides a method of preparing compounds of formula (II) which comprises where R 2 is one OH group treating the corresponding diacid with a suitable agent to provide the acid 0 dichloride as follows HOOC- (CH 2 r 2 -COOH C dcC- (CH 2 r 2 COC1 (ii) treating the corresponding acid dichloride with phenol as follows ClOC- (CH 2 )r.-2COCl PhOH PhOCO -(CH 2 r- 2 -COOPh (iii) rearrangement of the diacyl groups as follows OH
HO
O CO-fO
H
2 PhOCO -(CH 2 r- 2 -COOPh AMENDED SHEET -1PEA/AU RECEI;VED I 1 T. 7 (iv) followed by reduction of the acyl groups to provide compounds of formula (II); where R 2 is two OH groups treating the corresponding diacid with zinc chloride and resorcinol; and (ii) followed by reduction of the acyl groups to provide compounds of formula (II); where R 2 is 2-hydroxy-3-methyl carrying out steps in above except in (ii) phenol is replaced with a-cresol; where R 2 is 3-hydroxy-4-methyl nitrating the corresponding diketo compound of formula
CH
3 CO4CH 2 )2 CH 3 to give the corresponding bis-3-nitro compound (ii) reducing the bis-3-nitro compound to give the bis-3-amino compound followed by (iii) diazotisation and hydrolysis to give the bis-3-hydroxy compound (iv) followed by reduction of the keto groups to give the desired bis-3-hydroxy compound; where R 2 is 2,6-dihydroxy-4-methyl treatment of a compound of formula
OCH
2 0CH 3
COOH
CH
3 OCH 2 0CH 3 with LDA to give the dianion followed by (ii) treatment with the desired protected AMENDED SHEET
APEA/AU
PE C IV4 U L~ 2 9? 8alkane aldehyde of formula
HC-(CH
2 r6CH 2 0CH 2 Ph 0 to give
OCH
2 0CH 3
OH
OH
3
CH
2 CHCH(0H 2 )r CH 2 0HP
COCH
OCH
2 0CH 3 (iii) dehydrative decarboxylation followed by reduction to give an intermediate product of formula
,OCH
2 0CH 3 )r3CH 2 0H (iv) oxidation to give
OCH
2 00H 3
OH
3
Q(OH
2
CHO
OCH
2 0CH 3 followed by treatment AMENDED SHEET
IPEA/AIJ
-9 with the dianion from step to give
OCH
2
OCH
3
CH
3 0CH 2 0
OH
OH
3 Q (OH 2 CH -HH O-H 3
OCH
2 00H 3 CH 3 0CH 2 0 (vi) dehydrative decarboxylation followed by deprotection and reduction to give the desired product; where R 2 is 3,5-dihydroxy-4-methyl treatment of ca-N,N-dimethylanino-ca-cyano- (3,5-dimethoxy-4-methyl)benzylidene in tetrahydrofuran and hexamethyiphosphoramide (HM'PA) with lithium diisopropylamide (LDA) to give the anion followed by 1s (ii) treatment with ca,c-dibroroalkanes to give
CH
3 0 00H 3 0H 3 0 O O0H 3
H
3 0 CD O(H)r~i-C CD H 3
CH
3 0 oUov (iii) refluxing with 30! aqueous oxalic acid to give the corresponding diacyl compound (iv) reduction of the acyl groups followed by demethylation with hydrogen bromide in acetic acid to provide compounds of formula (II); where R 2 is 2,4-dihydroxy-3-methyl Ci) carrying out steps and (ii) in (b) except in resorcinol is replaced with 2-methylresorcinol; where R 2 is 4-hydroxy-3-methyl treating the corresponding diacid with AMENDED SHEET
,PEA/AIJ
UIAI IRO*I~RUIVPIII~ PII~LX~p~l- P&1 *U /O C Airh~~ 9/1 ortho-cresol in the presence of polyphosphoric acid to give the corresponding diacyl compound (ii) followed by reduction the acyl groups to provide compounds of formula (II).
In a fifth aspect, the present invention provides a method of preparing compounds of formula (III) which comprises treating the corresponding diacid with a AMENDED SHEaf
IPEA/AU
Isc~-- 1 rr~lli s WO 94/28886 4'Cf/AJ94/00297 10 suitable agent to provide the acid dichloride (ii) treating the corresponding acid dichloride with 2-naphthol followed by (iii) rearrangement of the diacyl groups and (iv) followed by reduction of the acyl groups to provide compounds of formula (III) In a sixth aspect, the present invention provides a method of preparing compounds of formula (IV) which compr".ses treatment of 4-alkylresorcinols with ethyl acetocetate in the presence of an acid catalyst to give compounds of formula (IV).
Preferably, Ar is phenyl, naphthalene, anthracene, naphthacene or phenanthrene. More preferably, Ar is phenyl.
All alkyl, alkenyl or alkynyl carbon chains can be straight or branched chain.
Halogen includes bromo, chloro, fluoro or iodo.
The 5 or 6-membered heterocyclic ring can be saturated, partially unsaturated or unsaturated.
Pharmaceutically acceptable derivatives include pharmaceutically acceptable ethers, esters and acid addition salts.
In the preparation of compounds of formula (II), preferably, the acid dichloride is formed by treating the corresponding diacid with thionyl chloride. However, any other suitable agent can be used.
Preferably, in the preparation of compounds of formula (IV) the acid catalyst is boron trifluoride etherate or the like.
Preferably, the rearrangement of the acyl groups to the required positions on the phenyl ring is carried out using CS 2 and AlCl1 as catalyst. The catalyst can generally be any Lewis acid such as BF 3 ZnCl 2 FeBr 3 or the like.
The reduction of the acyl group is preferably carried out using amalgamated zinc and a mixture of hydrochloric acid and optionally acetic acid.
Nitration is preferably carried out in the usual way I I 1. s~ I~e ~DIII*IIRaU" sP"~-sl~ 11 using a combination of nitric acid and sulfuric acid (HN03/HSO 4 The reduction of the nitrate to the amine is preferably carried out using stannous chloride and hydrochloric acid (SnCl 2 /HC1).
Diazotisatioi, is preferably carried out by treatment with aq. H 2 S0 4 /NaNO 2 and hydrolysis is usually carried by using 10% H 2 S0 4 Dehydrative decarboxylation is preferably carried out by using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and deprotection by using p-TsOH/MeOH.
In another aspect, the present invention provides a method for inhibiting the action of plasma membrane Ca ATPase enzymes in a subject in need of such inhibition which comprises administering to the subje-t an effective amount of a compound of formula or a pharmaceutically acceptable derivative thereof.
In yet another aspect, the present invention provides a method of treatment or prophylaxis of cardiovascular S: disease related to the action of plasma membrane Ca2+-ATPase enzyme in a subject in need of such treatment or prophylaxis which comprises administering to the subject an 20 effective amount of a compound of formula or a pharmaceutically acceptable derivative thereof.
Brief Description of the Drawings Figure 1 is a graph showing concentration dependency of inhibition of erythrocyte plasma membrane of synthetic 25 alkyl phenols.
2-nonylphenol A 3-nonylphenol 0 2-octylphenol O 4-octylphenol v 2-decylphenol B 4-nonylphenol 4-decylphenol Figure 2 is a graph showing concentration dependency of inhibition of erythrocyte plasma membrane of synthetic bis (hydroxyphenyl) alkanes.
S1, 10-bis (2-hydroxyphenyl)decane O 1,12-bis(2-hydroxyphenyl) dodecane O 1,14-bis(2-hydroxyphenyl)tetradecane
S
7 A 1- (2-hydroxyphenyl) -10- (4-hydroxyphenyl)decane v hydroxyphenyl) -12- hydroxyphenyl) dodecane TO/ Spec: P04828ZT I YP11 ~9111 WO 94/28886 1,I/Q 9/28886 CTIAU.,94/00297 12 1 1- (2 -hydroxyphenyl) 14 (4 -hydroxyphenyl) tetradecane 1, 10-bis (4-hydroxyphenyl) decane v 1, 14 -bis (4 -hydroxyphenyl) tetrade cane Figure 3 is a graph showing concentration dependency of inhibition of erythrocyte plasma membrane of resorcinol derivatives.
LI ethyl 3, 5-dibrono-2, 4-dihydroxy- 6-nonylbenzoate ethyl 3,5- dibromo-2,4-dihydroxy- 6-decylbenzoate A ethyl 2,4-dihydroxy-6-nonylbenzoate A ethyl 2, 4-dihydroxy-6-decylbenzoate Figure 4 is a graph showing concentration dependency of inhibition of erythrocyte plasma membrane of tertbutylphenols.
2,3-di-tert-butyl-4-met"hoxyphenol ElI 2, 6-di-tert-butyiphenol A 2,4, 6-tri-tert-butyip-henol Figure 5 is a graph showing concentration dependence of Ca 2 1-ATPase inhibition of 2-nonyiphenol derivatives.
0 2-nonylphenol/ 0 (+CaM) v3-methyl-6-nonylphenol/ v (+Ca.M) N 4-methyl-6-nonylphenol/ Ml (+CaD4M) 4 -nitro- 2- nonylphenol/ 0 (.iCaN) v 4 -bromo- 2--nitro- 6-nonylphenol (+CaN) [3 2 -bromo-4 -nitro- 6-nonylphenol A 4-bromo-2-nonylphenol/ A (+CaM) N 4- nonylresorcinol Figure 6 is a graph showing concentration dependence of Ca 2 +-ATPase inhibition of c,wi-bis[2-hydroxyC3,4 and methyl)phenylj alkanes.
1, 8-bis (2-hydroxyphenyl) octane/ 0 (+CaMv) v 1, 9-bis (2-hydroxyphenyl)nonane/ v (+CaM) 1, lO-bis (2-hydroxyphenyl)decane/ El (+CaM) A 1, 12-bis (2-hydroxyphenyl)dodecane/ A (+CaM) I CI O 10 02 914 13 1 1,10-bis(2-hydroxy-3-rethylphenyl)decane/ O (+CaM) 1,10-bis(2-hydroxy-4-methylphenyl)decane/ 0 (+CaM) 1,10-bis(2-hydroxy-5-methylphenyl)decan/ 0I (+CaM) Figure 7 is a graph showing concentration dependence of Ca 2 -ATPase inhibition of u,w-bis[2-4dihydroxyphenyl] alkanes.
(P1,8-bis(2,4-dihydroxyphenyl)octane! 0 (+Ca.i) v 1,10-bis(2,4-dihydroxyphenyl)decane/ 7 (i-CaM) N ,l11-bis(2,4-dihydroxyphenyl)undecane/ El (+CaM) A 1,12-bis(2,4-dihydroxyphenyl)dodecane/ A (+CaM) Figure 8 is a graph showing concentration dependence of Ca 2 +-ATPase inhibition of u,c-bis[hydroxy(methyl)pheny1 and naphthyl] decanes.
1,10-bis(3-hydroxyphenyl)decane/ 0 (+CaM) 1,l0-bis(3-hydroxy-4-methylphenyl)decane/ 0 (+CaM) v 1,10-bis(4-hydroxy-3-methylphenyl)decane/ v (+CaM) 1,l0-bis(2-hydroxy-1-naphthyl)decane/ n (+CaM) 1,l-bis(2-hydroxyphenyl)decane/ a (+CaM) Figure 9 is a graph showing concentration dependence of Ca 2 +-ATPase inhibition of phenolic natural products.
5,7,21,6 -tetrahydroxy-8-lavandulylflavanone M 5,7,2-trihydroxy-8-lavandulylflavanone A 5,21,6t-trihydroxy-8-lavandulyl-7-methoxyflavanone Modes for Carrving Out the Invention Compounds of formula encompasses naturally occurring compounds as well as synthetically prepared related compounds. The naturally occurring compounds of formula were extracted fro.i endemic Australian plants of the Proteaceae family according to standard literature procedure, for example, see Ritchie E, Taylor W C and Vautin S T K (1965), Chemical studies of the Proteaceae I Aust.J.Chem., 18, 2015-2020; Rasmussen M, et al (1968) Chemical studies of the Proteaceae III. Aust.J.Chem., 21 2989-3000 and Ridley D D, et al (1970) Chemical stu'-es AMENDED SHEET IPF/AI I /VT 0 c--IPEA/Aal I~ P/Au 9 4 0 0 2 9 RECEIVED 0 2 MlAR 19 14 of the Proteaceae IV. Aust.J.Chem., 23, 147-183.
Long-chain alkyl phenols were extracted from Grevillea and Persoonia. Novel compounds having the structural formulae and (VI) were extracted and isolated from Grevillea robusta collected in Sydney, Australia. A voucher specimen is available for inspection in the Department of Pharmacy, at the University of Sydney. Briefly, a sample of two kilograms was extracted by percolation with chloroform/ethanol for three days. After concentration of the extract in vacuo, the residue was chromatographed using silica gel short column vacuum chromatography.
Other compounds falling within the scope of formula were prepared according to literature procedures or are commercially available. Starting materials for the syntheses are commercially available or are prepared according to literature procedures.
The 2- and 4-substituted alkylphenols and c,wbis(hydroxyphenyl)alkanes were prepared by published methods (see E Miller and W H Hartung, Organic syntheses (1943) collective volume II, 543-545 and R R Read and J Wood, Organic syntheses (1955), collective volume III, 444-446). The syntheses were performed in three consecutive stages: firstly, the formation of the ester from an acid and phenol; secondly, the rearrangement of the acyl group, using AlC1 3 as catalyst, to the 2- and 4posiati-ons relative to the hydroxy group on the phenol ring and thirdly, the reduction of the acyl group using amalgamated zinc and hydrochloric acid. The rearrangement is a time dependent reaction and generally a short reaction period provides the acyl group at the 4position relative to the hydroxy group.
Short column vacuum chromatography was used at each stage of synthetic reactions to separate and purify the products from the reaction mixture. TLC methods were also employed to identify the products and to determine Pc I the eluent solvent required for column chromatography.
SThe products at each stage were also characterised by NMR AMENDED SHEET
IPEA/AU
WO 94/28886 PCT/AU94100297 15 and CI-MS analysis.
The 3-substituted phenol derivatives were prepared according to another method, described by Itokawa H, et al (1989), A quantitative structure activity relationship for antitumor long-chain phenols from Ginkgo biloba L.
Chem. Pharm.Bull. 37, 1619-1621. This method was used to prepare 3-nonylphenol with the aim of comparing its Ca 2 ATPase inhibitory activity with those of the 2-and 4substituted isomers. Hence, the importance of substitution on the phenol ring for Ca2+-ATPase inhibition could be determined.
The bisphenol compounds were prepared, isolated and purified using procedures similar to those described above for alkylphenol, except that the preparation of esters was carried out in two separate steps.
For compounds of formula (II) where R 2 is 2-hydroxy- 3-methyl the preparation is similar to that of the bishydroxyphenyl alkanes except instead of phenol, orthocresol is used following the procedure of K Kakemi et al in Antioxidants III Yakuaku Zasshi 86 791-796 (1966). 3-hydroxy-4-methyl compounds of formula (II) are prepared by a similar method to that used to prepare a,wbis(3-hydroxyphenyl)alkanes following the procedure of K.
Kakemi et al. above except that a,w-bis(4-methylphenyl)a,w-alkanediones are used instead of a,w-bisphenyl-,walkanediones. 2,4-dihydroxy compounds of formula (II) are prepared by the reaction of resorcinol with the corresponding dicarboxylic acids in the presence of zinc chloride to give the intermediate alkanediones which are then reduced.
Compounds of formula (II) where R 2 is 2,6-dihydroxy- 4-methyl are prepared by a method similar to that used to prepare grifolin following the procedure of S Ohta et al A total synthesis of grifolin Chem.Pharm.Bull. 36 [6] 2239-2243 (1988).
The starting compound in step is obtained according to the procedure of S Ohta et al referred to above. The aldehyde at step (ii) is obtained by I d.
WO 94/28886 PCT/AU94/00297 16 alkylating commercially available compounds of formulae
X-CH
2
(CH
2 )r-6CH2-OR where R=H, X=C1 or Br with benzyl bromide (or iodide) to give an intermediace product where
R=CH
2 Ph followed by hydrolysis to give HOCH2(CH 2 )r- 6
CH
2
OCH
2 Ph followed by oxidation to give OHC(CH 2 r- 6
CH
2
OCH
2 Ph.
Compounds of formula (II) where R 2 is 4-methyl are prepared by a procedure similar to that described by K. Takahashi et al., J. Org. Chem. 1983, 48, 1909-1912.
Compounds of formula (II) where R 2 is 2,4-dihydroxy- 3-methyl are prepared by a procedure similar to that described by J. von Braun et al., Ber. (1941), 74B, 1772- 1783 except that 2-methylresorcinol is used instead of resorcinol.
Compounds of formula (III) are prepared by a method similar to that used to prepare a,w-bis(2hydroxyphenyl) alkanes following the procedure of K.
Kakemi et al. Yakugaku Zasshi (1966), 86, 793 ?9" Compounds of formula (IV) are prepared a method similar to that used to prepare 6-alkyl-7-hydroxy-4methylcoumarins following the procedure of S P Starkov, G A Goncharenko and A I Panasenko, Zh. Obshch. Khim (1993), 63 1111-3115.
Other compounds of formula were prepared according to literature procedures as follows: Bis-phenols 1,10-bis(2-hydroxphenyl)decane and 1,10-bis(3hydrox-yphenyl) decane Antioxidants III. K Kakemi, T Arita, R Hori, and H Takenaka Yakucaku Zasshi 86 (1966) 791-796 1,10-bis(4-hydroxyphenyl)decane w-di-p-hydroxyphenyl Alkanes E M Richardson and E E Reid J.Am.Chem.Soc. (1940) 62 413-415 Fries transformation of condensates of sebacic acid s ~b _ag WO 94/28886 WO 9428886PCT/A'U94/00297 17 with phenols: 1, 8-dibenzoyloctanes J P Varm~x and J S Aggarwal J.Indian Chem.Soc. (1959) 36 41-45 Synthesis of cu,wc-bis (p-hydroxyalkanes) Y E Doroshenko and V A Sergeev Zh. Org~an. Khirn. (1965) 1 1602-1604 1.,12-bis (4-hydroxvphenyl)dodecane Synthesis of a,w-bis (p-hydroxyalkanes) Y E Doroshenko and V A Sergeev Zh. Organ. Khim.
(1965) 1(9) 1602-1604 1. 14-bis (4-hydroxypmhenyl) tetradecane H Goldinann et al. J.Am.Chem.Soc. (1988) 110 6811-6817 1. 10-bis (4-hvdroxv-3-methylloheniyl) decane P Schlack and W Koller Ger. 1,086,711 Aug 11, 1960.
1, 1-bis (2-hydroxvhenyl) decane G Casiraghi et al. J. Chemn. Soc. Perkin Trans 1 (1982), 805-808.
Alkyliphenol s The synthesis of aromatic hydroxyketones. I. ortho and para-acylphenols with normal C 4
-C
9 chains.
G Sandulesco and A Girard Bull. Soc. Chim.Fr. (1930) 47 1300-1314 Fungicidal activity and chemical constitution, D Wood J.Chem.Soc. (1955) 4391-4393 Alkylation of phenol by 1-dodecane and 1-decanol. A literature correction.
B Campbell, S Donald et al. Bull.Chem.Soc. Japan (1990) 63 (12) 3665-3669 decylphenol and dodecylphenol WO 94/28886 PCT/AU94/00297 18 Cyclohexylphenols (ortho para) The direct alkylation of phenol by cyclohexene in the presence of boron trifluoride H Lejebure and E Levas Compt. Rend. (1945) 220 782- 784 and 826-827 H Lejebure and E Levas Compt. Rend. (1945) 221 301- 303 Syntheses of a,w-bis(2,4-dihydroxy) compounds.
Reaction of aliphatic dicarboxylic acids with resorcinol, J von Braun et al Ber. 74B 1772-1783 (1941).
Fries transformations of condensates of sebacic acid with phenols: 1,8-dibenzoyloctanes, J P Varma et al, J.Indian Chem.Soc. 36 41-45 (1959).
The Ca 2 stimulated, Mg 2 dependent, adenosine triphosphatase (Ca 2 +-ATPase) located on plasma membranes extrudes Ca 2 against its electrochemical Ca 2 gradient.
It has been reported that Ca2+-ATPase not only plays a fundamental role in regulating the total cellular Ca 2 concentration but also modulates or mediates the effects of Ca 2 mobilising hormones and neurotransmitters. (See Pripic V, Green K C, Blackmore P R and Exton J H Vasopressin-, angiotensin II-, and c-adrenergic -induced inhibition of Ca 2 +-transportation by rat liver plasma membrane vesicles, (1984) J.Biol.Chem. 259, 1382-1385 and Rega A F, Garahan P J, (1986) The Ca Pump of Plasma membranes, CRC Pres Inc., Florida.
Natural and synthetic compounds of formula were tested for their ability to influence human erythrocyte plasma membrane Ca 2 +-ATPase. The enzyme Ca2+-ATPase in the human red blood cell plasma membrane has previously been studied and its stimulation by calmodulin and activation by lipids and proteolysis and its primary structure have been published (see Carafoli E (1991) Calcium pump of the plasma membrane, Physiological Reviews 71, 129-153.
i' ~o~u WO 94/28886 IICTAU94/00297 19 The activity of compounds of formula in the inhibition of Ca 2 +-ATPase is shown in Table 1 and is interpreted graphically in figures 1, 2, 3 and 4.
Table 11 Structures and IC., values of natural and synthetic compounds of formula in the inhibition of Ca 2 +-ATPase inhibition was determined at a coicentration of 100 1
AM).
INHIBITION OF Ca 2 -ATPase COMPOUND SOURCE PERCENT(% I C 5 0 (pyM) 2-octylphenol b 100 32 2-nonylphenol b 95 2-nonanoylphenol b 32 >100 2-decylphenol b 85 42 nonylphenol a 100 27,5 (commercial) 4-octylphenol b 28 92 4-tert-octylphenol a 94 3-nonylphenol b 69 64 4-nonylphenol b 22 114 4-decyiphenol b 24 170 2-cyclohexylphenol h 28 186 4-cyclohexylphenol h 16 271 1,10-bis (2-hydroxyphenyl)- b 100i 7.6 decane 1,12-bis (2-hydroxyphenyl)- e 100- 8.3 dodecane 1, 14-bis (2-hydroxypheny.)- e 84 28 tetradecane 1- (2-hydroxyphenyl) -l0-(4-hydrox-yphenyl) e 1003 12 decane i at j at k at 37yuM WO 94/28886 'PCI/At194/00297 INHIBITION OF Ca 2 4 ATPase COMPOUND SOURCE PERCENT(-) IC 5 0 zm) 1 (2 hydroxyphenyl) 12 -hydroxyphenyl) e 99 18.8 dodecane 1- (2-hydroxyphenyl) -14-(4-hydroxyphenyl)- e 60 81 tetradecane 1, 10 -bis (4 -hydroxyphenyl) b 52 93 decane 1- D-dihydroxypheiayl', dihydroxy-4- c l00i 17 methyiphenyl) tetradecane (grebustol -k) 1, 14 -bi s(3, 5 -dihydroxy -4-me thylphenyl) f 98 16 tetradecane (striatol) striatol-B f100 norstriatol-B c 6k35 -nonylresorcinol f 44 108 1, 14-bis (4 -hydroxyphenyl) b 5 280 tetradecane 1, 14 bis 5 dihydroxyphenyl) tetradecane f 35 >100 (bisnorstriatol) 1,14-bis(3,5-dihydroxyphenyJ) tetradec -Z 6-ene c 90 (grebustol -B) ethyl 2,4di hydroxy 6- f 73 62 noriylbenzoate ethyl 2,4-dihydroxy-6- f95 44 nonylbenzoate grevillol c 44 143 5 -decylresorcinol f 30 135 ethyl 2,4dihydroxy-6- f 60 decylbenzoate ethyl 3,5-dibromo-2,4dihydroxy-6- f 73 69 decylbenzoate
I=
WO 94/28886 '/AJ94/00297 -221 INHIBITION O? Ca 2 ,+-ATPase COMPOUND SOURCE PERCENT(--) IC 5 0
([LM)
2-E rnethyl-resorcinol d100 22.5 (grifolin) rethyl-resorcinol d 100 23.3 (neogrifolin) 4-dodecyiresorcinol a 68 69 4-hexyiresorcino'L a 17 259 2,4,6-tri-tert- a 79 butyiphenol 3, 5 -di -tert- butyl a 76 84 catechol 2, 6- di -tert- butyl a 69 66 4 -methyiphenol (BHT) 2, 6 -di -tert a 61 79 bu tylphenol 2,6-di-tert-butyl- a 44 >400 4 -methoxyphenol (BHA) 2, 21-methylenebis (4-methyl-6-tert- g 74 butylphenol) The activity of the synthetic and natural compounds in the inhibition of Ca 2 +-ATPase in the presence (+CaM) and in the absence (-CaM) of Calmodulin (CaM) is shown in table 1A below and is interpreted graphically in the figures 5, 6, 7, 8, 9.
Table 1A SOURCE COMPOUND IC 50 (-CaM) (+CaM) b 2-nonylphenol 36 b 2-methyl-6-nonylphenol b3-methyl-6-nonylpchenol 52 b 4-methyl-6-nonylphenol 80 b 2-bromo-6-nonylphenol b4-brorao-6-noriylphenol 52 WO U/28886 WO ~4/8886 CT/A1194100297 22 SOURCE COMPOUND (-CaM) (+Cam) b 2,4-dibromo-6-nonylphenol b 2-nitro-6-nonylphenol b 4-nitro-6-nonylphenol 80 b 2-bromo-4-nitro-6-nonylphenol b 4-bromo-2-nitro-6-nonylphenol 65.5 b 4-nonylresorcinol 125 ND a 4 -dodecylresorcinol 80 100 b 2-bromo-6-nonylresorcinol 400 400 b 4-bromo-6-nonylresorcinol 400 400 b 2, 4-dibromo-6-nortylvesorcinol b 1,B-bis(2-hydroxyphenyl) 24 24 octane b 3.,9 -b is(2 -hydroxyphenyl) 13.5 14 nonane b 1,10-bis(2-hydroxyphenyl) 8.4 decane e1,12-bis(2-flydroxyphenyl) 12.5 13 dodecane b 1,10-bis(2-hydroxy-3- 50 methylphenyl) -decane b 1,10-bis(2-hydroxy-4- 29 29 methyiphenyl) -decane b 1,10-bis(2-hydroxy-5- 22 20.5 methyiphenyl) decane b 1,8-bis(2,4-dihydroxyphenyl) 50 48 L 11 octane 1 11 11AII RC- El E 2 POr IM 23 SOURCE COMPOUND
IIC
50 (-Cam) (+CaM) b 1, 10-bis 4-101 dihydroxyphenyl) decane 101 e 1,11-bis(2,4- 25 21.7 dihydroxyphenyl) undecane e 1, 12-bis 12 dihydroxyphenyl) dodecane e 1,10-bis(2,4-dihydroxy-3- 2.00 100 methylphenyl) decane b 1,10-bis(3-hydroxyphenyl) 30 32 decane e 1,10-bis(3-hydroxy-4- 51 48 methyiphenyl) -decane b 1,10-bis(4-hydroxy-3- 100 94 methyiphenyl) -decane b 1,1-bis(2~t-hydroxyphenyl) 74 68 decane e1,10-bis(2-hydroxy-l- 92 naphthyl)decane e 6-dodecyl-7-hydroxy-4- NA NA inethylcoumarin NA NA i5,7,21,61-tetrahydroxy-8- 20 ND lavandulyl flavatitone i5,7,2"-trihydroxy-8- 26.5 ND lavandulyl flavanone i5,2',El-trihydroxy-8- 45.3 ND lavandulyl -7 -methoxyflavnione £JAi no intibitory activity; INLu: not. d2etermied AMENDED SHEET
IPENIAU
Pcr/ 9 4 0 0 2 9 7 RECEIVED 0 2 MAR 1995 -24 Source a commercially available from Aldrich, Milwaukee, WI, USA b synthetic preparation from literature procedure c extracted from Grevillea d natural products provided by Prof Shibata [Misasa H, Matsue Y, Vehara H, Tanaka H, Ishihara M, Shibata H, (1992) Tyrosinase inhibitors from Albatrellus confluens, Biosci.Biotech.Biochem., 56 1660-1661] e novel compounds of formula (II) according to the present invention f natural and synthetic substances provided by Prof W C Tay2or [see references, page 9, lines 25-30 also Cleaver L, Croft J A, Ritchie E and Taylor W C (1976) Chemical studies of the Proteaceae IX Aust.J.Chem., 29, 1989-20011 g commercially available from Merck, Darmstardt, Germany h synthetic substance provided by Prof Kanzo Sakata, Shizuoka University, Shizuoka Japan [see reference for cyclohexylphenols (ortho para) on page 17 i natural substances provided by Assoc. Prof. C Chaichantipyuth, Chulalongkorn University of Thailand (see Ruangrungsi N et al, Phytochemistry, 31, 999-1001, 1992 and linuma M et al, Phytochemistry, 33, 203-208, 1993.
The potency of a compound for Ca 2 +-ATPase inhibition is characterised by either its inhibition at a single dose or more informatively as its ICso value from the dose-response curve. The most potent compounds have, therefore, curves located at the left hand side of the graphs. From figure 2, it can be seen that 1,10-bis(2hydroxyphenyl)decane and, to a lesser extent, 1,12-bis(2hydroxyphenyl) dodecane have the strongest Ca2+-ATPase Sinhibition among the synthesised phenolic compounds. The AMENDED SHEET
IPEAAU
WO 94/28886 PCT/AU94/00297 25 inhibitory activity is two-fold higher than that of the natural product 1,14-bis(3,5-dihydroxy-4methylphenyl)tetradecane (striatol) and is superior to the 2-alkylphenol series. Of the alkylphenols, the maximum activity appeared with 2-nonylphenol (IC 50 The results of nonylphenols and octylphenols showed that the potency order of alkylphenols was 2alkylphenols, 4-branched alkylphenols, and 4alkylphenols. Similarly, 4-dodecylresorcinol was the most potent among the alkylresorcinols tested and grifolin and neogrifolin the most potent among the alkyl and alkenyl resorcinols tested.
The results also show the importance of substitution at the 2- position of the phenol ring for strong Ca 2 ATPase inhibition. The results indicate that there is a significant difference in potency between ortho- and para- substituted compounds, whereas the activity of the meta- substituted compound is intermediate.
The results further indicate a structural preference for the inhibitory potency of the synthetic compounds and an optimal methylene chain length for the bis compounds is ten. 1,10-bis(2-hydroxyphenyl)decane and 1,10-bis(2,4dihydroxyphenyl)decane to lesser extent are the most potent inhibitors of plasma Ca2+-ATPase among the synthesised substances.
In the presence or absence of Calmodulin (CaM), in general, there is no significant difference in the inhibitory activity of the synthesised compounds (refer to table 1A). However, it is observed that at higher concentration of the test compounds, there was slightly higher inhibition of plasma membrane Ca2+-ATPase than in the absence of CaM (refer to figures This would indicate that the compounds may also inhibit CaM stimulant activity.
45 Ca efflux from cultured vascular smooth muscle cells consists of two major mechanisms; one is dependent on extracellular sodium, mediated by the Na+-Ca 2 exchanger (Na dependent Ca 2 efflux), and the other is lm WO 94/28886 PCT/AU94/00297 26 independent of extracellular sodium but is mediated by the Ca 2 pump independent Ca 2 efflux). The effect of striatol on the efflux of calcium 4 5Ca 2 from rat thoracic aorta smooth muscle cells in culture was studied. 45 Ca 2 efflux is a measure of plasma membrane Ca2+-ATPase (plus Na+: Ca 2 exchange) in intact (whole) cells. The results are as follows: 5 min partial inhibition 10 min partial inhibition 50AM 30 min complete inhibition Compounds of formula show significant inhibitory activity against plasma membrane Ca2+-ATPase and typically would be suitable for use in the treatment of cardiovascular disease. These compounds would be useful by their action on Ca2+-ATPase enzymes in general or on Na', K+-ATPase enzyme. In particular, compounds of formula may be suitable for the treatment or prophylaxis of chronic heart failure, angina, hypertension or arrhythmia.
Accordingly, in another aspect, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for the treatment or prophylaxis of cardiovascular disease.
The effective amount of the active compound required for use in the above conditions will vary both with the route of administration, the condition under treatment and the host undergoing treatment, and is ultimately at the discretion of the physician. In the above mentioned treatments, it is preferable to present the active compound as a pharmaceutical formulation. A pharmaceutical formulation of the present invention comprises the active compound together with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredient. The formulation may conveniently be prepared in unit dosage form and may be prepared according to conventional pharmaceutical techniques. Additionally, the formulations may include WO 94/28886 PCT/AU94/00297 27 one or more accessory ingredients, such as diluents, buffers, flavouring agents, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives and the like.
A typical tablet formulation comprises 1-50mg of the active constituent, 50-200mg of lactose, 7-28mg of maize starch and 0.25-1mg of magnesium stearate. Preferably, the tablet formulation comprises 1-50mg of the active constituent, about 97mg of lactose, about 14mg of maize starch and about 0.5mg of magnesium stearate.
Compounds of formula may also be useful for the treatment of ulcers (peptic ulcers) through H K+-ATPase inhibition (the proton pump in gastric parietal cells) or may act as depigmentation, antidiabetic, antithrombolytic, antiarteriosclerotic, antioxidant, anticancer, antiinflammatory or antiviral agents.
EXPERIMENTAL SYNTHESES Instrumentation Thin layer chromatographic plates were visualised by a UVSL-58 mineral-light lamp, multiband UV-254/366nm.
Precoated Si gel plates (Merck, Art. 5554) were utilised.
Analytical high-performance liquid chromatography were obtained with a Beckman 11OB solvent module with a PC-3800 controller.
Detection was achieved with a Spectra-Physics Spectra 100 variable wavelength detector.
Analytical column was an Activon exsil 100/10 ODS, 250 x 4.6mm i.d. (reversed-phase C 18 Preparative short column was 70 x 65 mm i.d.
connected with an one litre glass bell jar.
1H- and 3 C-NMR spectra were obtained on a Varian Gemini 300 and Joel FX90Q (for grevillol) spectrometer using CDC13 as solvent and referencing to tetramethylsilane.
UV spectra were measured on a Perkin-Elmer Lambda UV/VIS spectrophotometer.
Chemical ionisation and electron impact mass spectra were performed on a Finnigan TSQ46 spectrometer. All WO 94/28886 PCT/AU94/00297 28 chemical ionisation spectra were performed using methane as reagent gas.
Materials Ethyl acetate and petroleum (70-75 0 C) were distilled prior to use. Thionyl chloride was distilled at 75 0
C
Dichloromethane,, chloroform, methanol were HPLC grade. Merck silica gel 60H (Art. 7736) was used for preparative TLC and short column chromatography.
3,4,5-trimethoxybenzaldehyde, n-butyllithium, undecandioic acid, 1,6-dibromohexane, 1,8-dibromooctane, 1,10-dibromodecane, 1,12-dibromododecane, 2methylresorcinol, 1,10-decanedicarboxylic acid and 1,12dodecanedicarboxylic acid were purchased from Aldrich Chemical company.
I. Synthesis of alkylphenols 1.Preparation of 2- and 4-octylphenol 1.1 Preparation of phenyl octanoate The ester was prepared by slowly adding thionyl chloride (10ml) to a mixture of pure phenol(6.5g) and octanoic acid(l0g). The reaction mixture was warmed to drive off the sulfur dioxide and hydrogen chloride gases.
The crude mixture was distilled at 95-100 0 Phenyl nonanoate and phenyl decanoate were prepared similarly. The esters were distilled at 105-110 0 C and 120-125 oC/5mmHg respectively.
The yield of the esters was 81-84%.
1.2.Preparation of 2- and 4-octanoylphenol OCOC H OH OH 6KCS COC H CS7 t AICI1, COC H 7 Anhydrous aluminium chloride (7g) and carbon 3 disulfide(10ml) were placed in a three-necked round bottom flask fitted with a reflux condenser, a dropping funnel and a large magnetic stirrer. Phenyl was slowly added to the stirred suspension -I WO 94/28886 PCT/AU94/00297 29 through the dropping funnel. When all the ester was added, the mixture was further heated to gentle refluxing on a steam bath until the evolution of hydrogen chloride had almos- ceased (about 1/2hr). The reflux condenser was turned downward and carbon disulfide was distilled.
The steam bath was replaced by an oil bath which was heated to 140 0 C and maintained at 140-150 0 C for one hour.
The mixture thickened and finally congealed to a brown resinous mass. The solid was allowed to cool and the aluminium complex was decomposed by first slowly adding a mixture of concentrated hydrochloric acid(6ml) with water(6ml) and then water(10ml). The mixture was left overnight and large solid portion (mainly 4octanoylphenol) on the surface was collected. The liquid portion was extracted with ethyl acetate. The extract was combined with the solid portion and the resulting mixture was dried with anhydrous sodium sulfate, filtered and evaporated to obtain the crude mixture. The products, 2- and 4-octanoylphenols, were separated by short column vacuum chromatography based on the difference in their polarity.
The yield was 29-34% of 2-octanoylphenol and 46-50% of 4-octanoylphenol. The products were then characterised by NMR and CI-MS analysis (refer to tables 3 4).
Separation of the products using short column vacuum chromatography The crude mixture was dissolved firstly if dichloromethane (one-part) then petroleum (four-parts).
The resulting mixture (25ml) was loaded onto a diam.x 30mm) silica gel bed under vacuum and continuously washed with petroleum Eluent solvent mixtures used to isolate the products were a stepwise solvent gradient from petroleum spirit/ethyl acetate 9:1 to 1:1 (20 fractions).
Fractions were examined under UV light by TLC to determine the degree of separation between products. NMR and CI-MS analysis were performed on fractions #5-10 I WO 94/28886 PCT/AU94/00297 30 (identified as 2-octanoylphenol) and #14-20 (identified as 4-octanoylphenol).
1.3.PreDaration of 2- and 4-octylphenol OH OH COC H 1 CH C H Zn -2 7
HCI
Amalgamated zinc(10g) was placed in a 100ml roundbottom flask fitted with a stirrer and a reflux condenser. A mixture of acetic acid(10ml) and concentrated hydrochloric acid(10ml) was added and then a solution of 2-octanoylphenol (2g) (or 4-octanoylphenol) in acetic acid(5ml). The mixture was agitated and refluxed for 2 days. Aqueous 20% w/v NaCI was added and the mixture was extracted with The petroleum portion was dried with anhydrous sodium sulfate, filtered and purified by short column vacuum chromatography.
A yield of 82-84% was obtained.
The column chromatographic method used was the same as described above.
The products were characterised by NMR and CI-MS analysis (Refer to tables 3 4).
Note: 1. The zinc was amalgamated in the reaction flask by covering it with a solution of mercuric chloride(0.2g) in water(15ml) and occasionally agitated over 30 minutes.
The solution was poured off and the zinc was rinsed once with water.
2. Aqueous 20% w/v NaC1 was added to increase the ionic strength of the aqueous phase so that the octylphenol could be extracted into the organic phase.
Nonyl- and decylphenols were prepared as described for octylphenol. The products at each stage were also characterised by NMR and CI-MS analysis.(Refer to tables 3 4).
2. Preparation of 3-nonylphenol I WO 94/28886 PCT/AU94/00297 31 2.1 Preparation of 3-benzyloxvbenzaldehyde
CHO
CHO
K CO 2 3 OH CH Cl N 2C In a 100ml round-bottom flask were placed 3hydroxybenzaldehyde benzyl chloride(6g), sodium iodide(8g) and potassium carbonate(10g). The reaction mixture was sealed and stirred for one day. The crude product was extracted with ethyl acetate(20ml). The organic portion was washed twice with distilled to remove all aqueous soluble materials. The ethyl acetate portion was dried with anhydrous sodium sulfate and evaporated to obtain the crude solid. The solid was purified by the column chromatographic method described for octylphenol. The product was identified by NMR and CI-MS analysis (Refer to tables 3 4).
2.2 Preparation of 3-nonvlphenol CHO HCOMgBrC H r I8 17 OH2+ C H Br Mg OH l o^ i 1 oc 2 ether
OHC-
OH
I
HCC H CH CH OOH 2
C
0 10% Pd-C OH 1 1 Grignard reagent was prepared from magnesium(248mg) and l-bromooctane(l.8g) in dry diethyl ether(5ml). A tiny amount of solid iodine was added to initiate the reaction and ether(10ml) was further added. The reaction was stirred until all magnesium dissolved and 3benzyloxybenzaldehyde(10g) was then added to the reaction mixture. The reaction was refluxed at room temperature for 4 hours. Ice water was then added. The organic I- WO 94/28886 VCT/A'U94/029'7 32 phase was separated and washed with 1.5M sulfuric acid (2x25ml), 10% potassium carbonate (2x25ml), water 1M hydrochloric acid (20ml) in saturated sodium chloride, dried with anhydrous sodium sulfate and evaporated to obtain the product (79% of yield).
A portion of the product(500mg) was hydrogenated by catalytic reduction over 10% palladium-charcoal(130mg) in ethyl acetate (50ml) containing concentrated sulfuric drops). The reaction mixture was stirred at room temperature for one day. The mixture was then filtered, evaporated and purified by the column chromatographic method.
The product was identified by NMR and CI-MS analysis (Refer to tables 3 4).
II. Synthesis of ae,.-bis(hydroxvyhenyl)alkanes.
1. Preparation of bisphenyl decanedioate
SOCI
2 HOOC-(CH )--COOH CIOC (CH COCI
OH
CIOC-(C H2)-C CI OCO(CH2)COOhea heat The methods of preparation, isolation and purification were similar to those described for octylphenol except that the preparation of esters was done in two separate steps, as shown above, instead of one as described for phenyl octanoate.
Firstly, the acid dichloride was prepared by refluxing the mixture of dicarboxylic acid(5g) and thionyl chloride(20ml) at 60-70 0 C for two hours. Thionyl chloride was then removed by evaporation and the remaining product was dissolved in toluene(5ml). The toluene was evaporated to remove all traces of thionyl chloride.
Secondly, the phenol(2x moles of acid dichloride) was added to the acid dichloride. The mixture was warmed to drive off hydrogen chloride gas. The product s- WO 94/28886 PC'T/A(194/00297 33 solidified on cooling and was purified by the column chromatographic method.
The yield of the esters was 96-98-.
2. Preparation of 1, 10-bis (hydroxyphenyl) decane- 1,10-diones AICI .CS 3 -2-fi
OH
O C O(CH H 2 aOC c OH HO-- -CO(CH)O -&OH 3. Preiparation of 10O-bis (2 hdroxy~henvl) -de cane Zn HC1 The product was characterised by NMR and CI-MS analysis (Refer to NDMZ and CI-DIO tables 3 4).
111. Synthesis of ouw-bis[2-hvdroxv-(3-,4- or methyl) Dhenvll alkanes These were prepared according to K Kakemi et al. in Antioxidants III. K Kakemi, Y Arita, R Honi and H Takenaka. Yakugaku Zashi 86, 791-796 (1966) as follows: Aliphatic dicarboxylic acid chloride (1 mole) [for example sebacoyl chloride] was added to a solution of a phenol (2.2 moles) in tetrachloroethane containing WO 94/28886 PCT/0A194/00297 34 anhydrous AIC1 3 (2.5 moles). The mixture was stirred for 5-6 hrs at 70-80 0 C. The product was decomposed with icewater and concentrated HC1 The organic layer was separated and concentrated under reduced pressure. The residue was extracted with ethyl acetate and washed with water twice. After removal of the solvent the product was subjected to gradient chromatography to isolate the corresponding diketones which on Clemmensen reduction gave the title compounds.
The products were recrystallised from petroleum or petroleum ethyl acetate to give colourless crystalline solids.
The procedure was similarly applied to prepare bis(2-hydroxy-l-naphthyl)decane.
IV. 1,10-Bis(4-hydroxy-3-methylDhenvl)decane This was prepared by the method reported by Schlack and Koller in Aromatic aliphatic diketones. P Schlack and W Koller. Ger. 1,086,711 Aug. 11, 1960.
The title compound was prepare by treatment of orthocresol (2.2 moles) with sebacic acid (1 mole) .in the presence of polyphosphoric acid (3 moles). The mixture was stirred for 4 hrs at 80 0 C and poured to ice-water after cooling. The precipitant was filtered, dissolved in ethyl acetate and washed with water three times. The solvent was evaporated to yield a crude 1,10-b"-(4hydroxy-3-methylphenyl)decane-1,10-dione which was then subjected to Clemmensen reduction to give the title compound.
The product was recrystallised to yield a colourless crystalline white solid.
V. Synthesis of u,w-bis(2,4-dihydroxv-(3methb-) Dhenyl) alkanes These were prepared according the literature procedure in Reactions of aliphatic dicarboxylic acids with resorcinol. J von Braun, E Anton and F Meyer. Ber.
74B 1772-1783 (1941) as follows: The aliphatic dicarboxylic acid (1 mole) was heated with anhydrous ZnC12 (2 moles) at 140 0 C followed by added -L WO 94/28886 PCT/AU94/u0297 35 resorcino3s (10 moles) in portions. Stirring of the mixture way maintained for 4-5 hrs at 140-1600C (except that for 2-methylresorcinol which was stirred at 1700C). The products were decomposed with ice-water. The solid was collected, washed with 10% Na 2
CO
3 and then with water under vacuum.
The crude products were subjected to Clemmensen reduction to yield the title compounds and were recrystallised from ethanol water to give colourless crystalline solids.
VI. Synthesis of 1,10-bis[3-hydroxy-4methylphenylldecane.
1,10-Bis(4-methylphenyl)decane-l,10-dione was prepared according to (III) above. The diketone dissolved in concentrated sulfuric acid were gradually added to the mixture of fuming nitric acid and concentrated sulfuric acid (2:1 in volume) at -5 0 C. The mixture was stirred at 0°C for 30mins and poured in to ice-water. The precipitate was filtered off and recrystallised from ethyl acetate to give 1,10-bis(3-nitro-4which then underwent reduction with SnC1 2 .2H 2 0 in the presence of concentrated HC1 at elevated temperature of 90 0 C for 30mins. The precipitate that formed on cooling was collected, washed with HC1 and dissolved in dilute NaOH. The solid precipitating on the addition of dilute HC1 was collected and recrystallised from ethanol to yield 1,10-bis(3amino- 4 methylphenyl)decane-1,10-dione.
This product was diazotised with aq. NaN02 in 3M
H
2
SO
4 at 50C. The mixture was then hydrolysed with
H
2
SO
4 at 160°C. The resulting solution was extracted with ethyl acetate and the solvent was evaporated to yield 1,10- (bis(3-hydroxy-4-methylphenyl)decane-1,10-dione which on Clemmensen reduction and gave the title compound which was recrystallised from petroleum dichloromethane.
VII. Synthesis of u,w-bis(3,5-dihvdroxy-4-
~I
WO 94/28886 PCT/AUS194/00297 36 methyllpherv)vl) alkanes.
a. 3,5-dimethoxy-4-methylbenzaldehyde was prepared by a procedure published in Regioselective reduction alkylation of 3,4,5-trimethoxy-benzaldehydes and dimethylacetal. U Azzena, S Cossu, T Denurra, G Melloni and A M Piroddi. Synthesis 1990, 313 and Regioselective reduction electrophilic substitution of derivatives of 3,4,5-trimethoxybenzaldehyde. U Azzena, G Melloni, A M Piroddi, E Azara, S Contini and E Fenude.
J. Org. Chem. 57, 3101-3106 (1992).
The diacetal intermediate was distilled at 130 0 C 0.8 mmHg and 3,5-dimethoxy-4-methylbenzaldehyde (compound A) was recrystallised from petroleum.
b. Preparation of dimethoxy- 4 -methyl) benzyl idene (Compound B) (see Benzylation and related alkylation of u-dimethylaminophenylacetonitrile by mean of alkali. C R Hauser, H M Taylor and T G Ledford. J. Am. Chem. Soc. 82, 1786 (1960).
To a stirred solution of sodium bisulfite (1 mole) in 400 ml of water was added compound A (1 mole) in methanol followed by anhydrous dimethylamine (1.2 moles) in aq. methanol The reaction mixture was cooled prior the addition of aq. sodium cyanide (1.2 moles). The mixture was stirred at room temperature for 20 hrs and diethyl ether (50 ml) was then added. The ethereal layer was washed with water twice and evaporated to yield the product Compound B 90%) which was sufficiently pure for the next reaction.
c. Preparation of a, -bis(3,5-dimethoxy-4methylphenyl) aLane-, w-diones (see An efficient method for synthesis of symmetrical diketones via reaction of camino- arylacetonitriles with alkyl dibromides. K Takahashi, M Watsuzaki, K Ogura and H lida. J. Org. Chem.
48, 1909-1912 (1983).
Under dry nitrogen diisopropylamine (1,5 ml, 9 mmol) dissolved in a mixture of 5 ml each of dry THF and HMPA was treated with n-butyllithium (4 ml of 2.5 M solution
I
WO 94/28886 PCT/AU94/00297 37 in hexane, 10 mmol) at -78 0 C. Compound B (8 mmol) dissolved in THF (2 ml) was added and the reaction mixture was stirred for 15mins at -78 0 C and for Ihr at 0°C. To the mixture cooled to -20 0 C was added a,odibromoalkanes (4 mmol) dropwise. After the mixture was stirred for 20mins at -200C, the stirring was continue overnight at room temperature, The reaction mixture was poured into ice-water and extracted with diethyl ether (3x50 ml). The combined ethereal layers was washed with brine and concentrated under reduced pressure. The residue dissolved in a solution of 3 ml each of THF and 30% aq. oxalic acid was refluxed for 90mins then extracted with diethyl ether.
After the solvent was evaporated The product was recrystallised from ethanol to give the diketones (Compounds C).
d. Preparation of a,w-bis(3,5-dihydroxy-4methylphenyl)alkanes.
The Compounds r were subjected to Clemmensen reduction and then demethylation with 47% HBr in the presence of acetic acid at 130 0 C for 10 hrs. The product was subjected to gradient chromatography and recrystallised from benzene to give a colourless crystalline solid.
1,1-Bis(2-hydroxyphenyl)decane was prepared according to the literature procedure of G Casiraghi et al. in Regiospecificity in reactions of metal phenoxides.
Synthesis of 2,2-alkylidenebisphenols. G Casiraghi, G Casnati, A Pochini and R Ungaro. J. Chem. Soc.,Perkin Trans.l (1982), 3, 805-808. The product was distilled at 210OC/0.4mmHg.
6-dodecyl-7-hydroxy-4-methylcoumarin was prepared according to the literature procedure of S P Starkov et al. in Condensation of ethyl acetoacetate and ethyl benzoylacetate with 4-alkylresorcinols in the presence of boron trifluoride etherate. S P Starkov, G A Goncharenko and A I Panasenko. Zh. Obshch. Khim. (1993), 63(5), 1111-1115.
WO 94/28886 W'CO 94/CT/A94/00297 38 The product was recrystallised from ethanol.
I.omination of 2-nonylphenol was carried out according to D E Rearson et al.in The ortho bromination of phenols. D E Rearson, R D Wysong and C V Breder. J.
Org. Chem. (1967), 35(19), 3221-3231.
Nitration of 2-nonylphenol was carried out according to D S Ross et al. in Catalysis of aromatic nitration by the lower oxides of nitrogen. D S Ross, G P Hum and W G Blucher. J. Chem. Soc., Chem. Comm. 1980, 532-533.
Bromination of 4-dodecylresorcinol was carried out according to E Kiehlmann and R W Lauener in Bromophloroglucinols and their methyl ethers. E Kiehlmann and R W Lauener. Can. J. Chem. (1989), 67, 335-344.
4-bromo-6-dodecylresorcinol was prepared according to procedure 8 above in The ortho bromination of phenols.
D E Rearson, R D Wysong and C V Breder. J. Org. Chem.
(1967), 35(19), 3221-3231.
Clemmensen reduction was carried out as follows: The ketone compounds dissolved in toluene were added to the mixture of concentrated HC1 and acetic acid (1:1) containing amalgamated zinc. The reaction mixture was refluxed for 10hrs with vigorous stirring or stirred at room temperature for 2 days.
II I' WO 94128886 WO 94/2886 P(T/AU94/00297 39 Table Synthetic phenolic products COMPOUND (No) %YIELD 2-octylphenol (1) 19.8 -23.3 2-decylphenol (3) 4-octyiphenol m.p. 40-420C 4-nonylphenol m.p. 27-381C 31.5 -34.2 4-decylphenol m.P. 53-55'C 3-nonylphenol 10.0 20.8-23.3 1, 14-bis(2-hydroxyphenyl)tetradecane (14) 1-(2-hydroxyphenyl)-10- (4-hydroxyphenyl)decane (9) 1-(2-hydroxyphenyl)-12- (4-hydroxyphenyl)dodecane (12) 22.5 1-(2-hydroxyphenyl)- 14- (4-hydroxyphenyl) tetradecane 1,10-bis(4-hydroxyphenyl)decane 1, 12-bis(4-hydroxyphenyl)dodecane (13) 6.7 -8.3 1, 14-bis(4-hydroxyphenyl)tetradecane (16) WO 94/28886 WO 94/8886 'TAU94/00Z297 40 Table 2: continued C')MPOUNDS MELTING YIELD POINT OC I 2-nonyiphenol lig. 58-60 2-nethyl-6-nonylphenol lig. 56 3-methyl-6-nonylphenol lig. 52 4-methyl-6-nonylphenol lig. 2-bromo-6-nonylphenol lig. 4-brorno-6-nonylphenol 45-47 2 ,4-dibromo-6-nonylpheno. lg. 2--nitro-6-nonylphenol lig. 4-nitro-6-nonylphenol lig. 38 2-bromo-4-nitro-6-nonylphenol 64-65 9 0 a 4-bromo-2-nitro-6-nonylphenol 61-63 4-nonyiresorcinol 70-71 2 -bromo- 6- dodecylresorcinol 68-6970 4-bromo- 6-dodecyiresorcinol 61-62 82 2, 4-dibromo-6-dodecylresorcinol 58-59 6-dodecyl-7-hydroxy-±t-methyl- 135-136 couinarin 1, 8-bis (2-hydroxyphenyl)octane 74 1,9-bis (2-hydroxyphenyl)nonane 64-65 1,10-bis(2-hydroxyphenyl)decane 81-82 40-45 (8) 1, 12-bis (2-hydroxyphenyl) 88.5 dodecane (11) 1,10-bis(2-hydroxy-3- 82-83 38 methylphenyh) decane WO 94/28886 WO 94/2886 Pf /AU')4/0O297 41 COMPOUNDS MELTING %YIELD POINT OC 1,10-bisC2-hydroxy-4- 102-103 methyiphenyl) decane 1,10-bis(2-hydroxy-5- 86 methyiphenyl) decane 1,8-bis(2,4-dihydroxyphenyl) 167-168 octane 1,10-bis(2,4-dihydroxyphenyl) 155-156 decane -75-80 1,11-bis(2,4-dihydroxyphenyl)- 136-137 landecane 1,12-bis(2,4-dihydroxyphenyl) 146-147 dodecane 1,10-bis(2,4-dihydroxy-3- 138-1-10 methyiphenyl) decane 1, 10-bis (3 -hydroxyphenyl) decaqne 71-73 1,10-bis(3-lhydroxy-4- 100-102 182 methyiphenyl) decane 1,10-bis(4-hydroxy-3- 82-83 78 methyiphenvi) decane 1, 1-bis (2-hydroxyphenyl)decane liq. 1, 10-bis (2-hydroxy-1-naphthyl) 101-103 decane 91-92 7 0 1, 8-bis 5-dihydroxy-4methylphenyl) octane 173-175 1, 10-bis 5-dihydroxy-4methyiphenyl) decane 1 139-140 65-70 WO 94/28886 I)CT/AU94/00297 42
COMPOUNDS
1, 12-bis 5-dihydroxy-4methyiphenyl) dodecane 1, 14-bis (3,5-dihydroxy-4inethylphenyl) tetradecane I YIELD WO 94/28886 WO 94/8886 CT/AU94/00297 43 1 1
OH
Q
1 0H 2 1 6 2
HO
O CH 9 19 5 O C H 9 19 3 C9 19 4 21
OH
(CH -OH 12 HO (CH 2 -9OH 13 O H O ciOH 9 0 HO 0 (CH 2- 4
~..OH
H (GH2)4 OH 16 H OH Structures of synthetic alkcylphenols and a, w-bis(hydroxyphenyl)alkanes.
-44 Table 3: 1 H-NMR spectra of synthesised products (solvent CDC1 3 3 00 M~z, 6 (ppm)) Abbreviations are: s, singlet; d, doublet; t, triplet; m, rnultiplet; b, broad. Only coupling constants of aromatic protons are reported.
Intermediate products 2-alkanoyiphenols 2-octancyiphenol 6 7.77 (1H, dd, J 8, 2 Hz, 7.45 (1H, td, J 8, 2 Hz, 6.98 (lH, dd, J 2 Hz, 6.88 (1H, td, J 8, 2 Hz, H-4), 2.9 (2H, t, H-21), 1.7 (2H, H-31), 1.3 (8H, m(b), H-4' to H-71), 0.88 (3H, 2-nonanoylphenol 6 7.77 (1H, dd, J 8, 2 Hz, 7.45 (1H, td, J 8, 2 Hz, 6.98 (1H, dd, J 2 Hz, 6.88 (1H, td, J 8, 2 Hz, H- 4) 2. 9 (2H, t, H- 1. 7 (2H, m H- 1.3 (10H, H-4' to 0.88 (3H, H- 2-decanoylphenoi 6 7.77 (1H, dd, LT= 8, 2 Hz, H-3), 7.45 (1H, td, J 8, 2 Hz, 6.98 (1H, dd, J 8, 2 Hz, 6.88 (1H, td, J 2 Hz, 2.9 (2H, t, H- 1.7 (2H, 1.3 (12H, H-4' to 0.88 (3H, H-101).
4-alkagoyliphenols 4-octanoylphenol 6 7T. 8 0 (2 H, d, J 8 Hz, H- 3, 5) 6. 89 (2H, d, LT 8 Hz, H- 2, 6) 2. 90 (2H, t, 1.7 (2H, 1.3 (8H, H-4' to H- 0.88 (3H, 4-nonanoylphenol 6 7.80 (2H, d, J 8 Hz, H-3, 6.89 (2H, d, J =8 Hz, H-2, 6), 2.90 (2H, t, H-21), 1.7(2H, H-31), 1.3 (10H, m(b), H-4' to 0.88 (3H, H-91). 4-decanoylphenol 6 7.-G--42H, d, J 8 Hz, H-3, 5) 6.89 (2H. d, J 8 Hz, H- 2, 2.90 (2H, t, 1.7(2.H, H-31), 1.3 (12H, H-4' to 0.88 (3H, H-l0').
3-benzvloxybenzaldehyde 6 9.96 (lH, s, CHO), 7.3-7.5 (8H, m, Ar-H), 7.24 (1H, m, 5.11 (2H, s, diphenyl alkanedioates diphenyl decanedioate 6 7.36 (4H, t, J 8, 2 Hz, H-3, 59), 7.20 (2H, tt, J 8, 2 Hz; H-4) 7.08 (4H, dd, LT 8, 2 Hz, H-2, 6) 2.55 (4H, t, H- 1.75 (4H, rn(b), 1.33 (8H, H-4' to H-71). Bisphenyl decanedioate 6 7.36 (4H, t, J =8, 2 Hz, H-3, 7.20 (2H, tt, LJ 8, 2 Hz, 7.08 (4H, AMENDED SH&ET
IPEN/AU
RA
PC/A 9/ 9 97 RECEIVED 0291AR 1995 dd, J 8, 2 Hz, H-2, 2.55 (4H, t, H-2'I 1.75 (4H, mn(b), 1.33 (12H, H-4' to Bisphenyl tetradecanedicate 6 7.36 (4H, t, J= 8 Hz, H- 3, 7.20 (2H, tt, J 8, 2 Hz, 7.08 (4H, dd, J 8, 2 Hz, H-2, 6) 2.55 (4H, t, 1.75 (411, 1.33 (16H, H-4' to cc-bis(2-hvdroxynhenvl)alkanediones 1,10-bis(2hydroxyphenyl) decane-1,1Q--dione 6 7.76 (2H, da, J 8, 2 Hz, 7.46 (2H, td, J 8, 2 Hz, 6.98 (2H, dd, J 8, 2 Hz, 6.9 (2H, Ld, J 8, 2 Hz, 2.98 (4H, t, 1.75 (4H, 1.30 (8H, H-4' to 1, 12-bis(hydroxyphenyl)dodecane-1,12-dione 6 7.76 (2H, dd, J= 8, 2 Hz, 7.46 (2H, td, J 8, 2 Hz, 6.98 (2H, dd, J 8, 2 Hz, 6.9 (2H, td, J 8, 2 Hz, 2.98 (4H, t, 1.75 (4H, 1.30 (12H, H-4' to 1,14-bis(2hydroxyphenyl)tetradecane-1,14-dione 6 7.76 (2H, dd, J 8, 2 Hz, 7.46 (2H, td, J 8, 2 Hz, 6.98 (2H, da, J 8, 2 Hz, 6.9 (2H, td, J 8, 2 Hz, 2.98 (4H, t, 1.75 (4H, 1.30 (16H, H-4' to u-(2-hvdroxvphenvl)-w-(4-hvdroxvpohenvi)aikanediones 1- (2-OH-phenyl)-10-(4-OH-phenyl)decane-1, 10-dine 6 7.90 (2H, d, J 8, 2 Hz, 7.76 (1H, dd, J 8, 2 Hz, 7.45 (1H, td, J 8, 2 Hz, H-4) 6.96 (1H, dd, J 8, 2 Hz, 6.92 (2H, a, J 8, Hz, 6,jj~H, td, J 8, 2 Hz, 2.97 (2H, t, 2.92 (2H, t, 1.72 (4H, 1.30 (8H, m(b), H-4' to 1-(2-OH-phenyl)-12-(4-OH-phenyl)dodecane- 1,12-dione 6 7.90 (2H, d, J 8 Hz, 7.76 (1H, dd, J 8, 2 Hz, 7.45 (1H, td, J 8, 2 Hz, H-4), 6.96 (1H, d, J 8, 2 Hz, 6.92 (2H, d, J 8, Hz, 6.89 (1H, td, J 8, 2 Hz, 2.97 (2H, t,- 2.92 (2H, L, H-111) 1.72 (4H, m(b) 1.30 (12H, H-4' to 1-(2-OH-phenyl)-14-(4-OHphenyl)tetradecane-1,14-dione 6 7.90 (2H, d, J 8, 2 Hz, 7.76 (1H, dd, J 8, 2 Hz, 7.45 (1H, AMENDED SHEET
IPENAU
I -a rlC~LplB~ Par/l 9 4 0 02 9 7 RECEIVED 0 2 MAR 1995 46 td, J- 8, 2 Hz, H-4) 6.96 (1H1, dd, J 8, 2 Hz, H-3) 6. 92 (2 H, d, J 8, Hz ,H-3 113, 5"1) 6. 89 1H, t d, J= 8, 2 H z, H1- 5) 2. 97 (21H, t, H 2 .9 2 (2 H, t, H -131), 72 (4 H, m H1- 1. 30 (1611, m H 4' to H1-11' I) y~w-bis(4-hvdroxvphenvl)alkanediones 1,10-bis (4hydroxy-phenyl)decane-1,1Q-dione 6 7.9 (411, d, J =8 Hz, H-2, 6) 6.92 (4H, d, J 8 Hz, 11-3, 5) 2.92 (411, t, H1- 1.72 (411, rn(b), 11-3', 1.30 (811, H-4' to H- 1,12-bis(4-hydroxcy-phenyl)dodecane-1,12-dione 6 7.9 (411, d, J =8 Hz, 11-2, 6.92 (411, d, J =8 Hz, 11-3, 5) 2.92 (411, t, H-2'1, 11') 1.72 (411, m(b) 11-3' 10'1) 1. 30 (12H, 11-4' to H1- 1,14-bis (4-hydroxyphenyl)tetradecane-1,14-dione 6 7.9 (411, d, J 8 Hz, H- 2, 6.92 (411, d, J =8 Hz, H-3, 2.92 (411, t, H1-21, 13' 1. 72 (411, m 11-3', 1. 30 (16H1, m 11-4' to H1-11').
Alkyiphenols 2-alkylphenols 2-octyipheno. 6 7.09 (1H1, td, J 8, 2 Hz, 1-5) 7.05 (111, dd, LT 8, 2 Hz, 11-3) 6.86 (1H1, td, J 8, 2 Hz, 11-4), 6.74 (111, dd, J 2 Hz, H1-6), 2.6 to 0.88 (311, 2-nonyiphenol 6 7.09 (1H1, td, J 8, 2 Hz, 11-5), 7.05 (1H1, dd, J 2 Hz, H- 6.86 (1H1, td, J 2 Hz, 11-4), 6.74 (1H1, dd, J 8, 2 Hz, 11-6) 2. 6 (211, t, H11), 1.6 (211, 1.3 (1211, 11-3' to H1-81), 0.88 (311, 2decyiphenol 6 7.09 (111, td, J 8, 2 Hz, 7.05 (111, ddJ=8, 2 Hz, 11-3), 6.86 (111, td, LJ 8, 2 Hz, 11-4) 6.74 (1H1, dd, J 8, 2 Hz, H1-6), 2.6 (2H1, t, H11), 1.6 (211, 1.3 (1411, H1-3' to 0.88 4-alkylphenols 4-octyiphenol 6 7.02 (211, d, J 8 Hz, H1-3, 6.74 (211, d, J =8 Hz, 11-2, 2.52 (2H1, t, H- 1.56 (211, 1.28 (1011, 11-3' to H1- 0.88 (311, H1-81). 4-nonyiphenol 6 7.02 (211, d, J 8 Hz, 11-3, 6.74 (211, d, J 8 Hz, 11-2, 2.52 3' to 0.88 (311, 4-decyipheno. 6 7.02 AMENDED SHEET
IPEA/AU
of PGT/Au 9 4 0 02 9 7 RECEIVED 0 2 MAR 1995 -47 (2H, d, J =8 Hz, H1-3, 6.74 (211, d, J =8 Hz, 11-2, 2.52 (2H, t, 1.56 (211, H-21), 1.28 (1411, in(b), H-3' to H1-91), 0.88 (3H, 3-alkylphenols 3 -nonyiphenol1 6 7. 13 (1H1, td, J 8 Hz, 6.74 (111, dd, J 8 Hz, 11-4), 6.65 (1H1, d, J 2 Hz, 6.64 (111, dd, J 8 Hz, 11-6), 2.54 (211, t, H- 1.58 (211, in(b), 1.27 (1211, in(b), 11-3' to H- 0.88 (3H1, H-91).
2-nonxrlphenol derivatives 2-methyl.-6-nonylphenol 6 6.96 (2H, d, J 8 Hz, 11-3, 6.77 (111, t, J =8 Hz, 11-4), 2.6 (211, t, H11), 2.24 (3H, s, Ar-C1 3 1.6 (2H, m(b), H1-2') 1.3 (1211, rn(b), 11-3' to 11-81), 0.88 (311, t(b) H- 3-methyl-6-nonylphenol 6 7.02 (1H, d, J 8 Hz, H- 6. 72 (111, dd, J 8, 2 Hz, H1-4) 6. 62 (111, d, LTJ 2 Hz, H1-2) 2. 6 (211, t, 2.24 (311, s, Ar- C1 3 1. 6 (211, 1.3 (1211, 11-3' to 0.88 (311, 4-methyl-6-nonylphenol 6 6. 94 (111, d, J 8 Hz, H1-5), 6.89 (1H1, dd, J 8, 2 Hz. H1-3), 6.67 (111, d, J 8 Hz, 11-2) 2.0- (211, t, H1-I') 2.24 (311, s, Ar-CE 3 1.6 (211, in(b), 1.3 (1211, 11-3' to H- 0.88 (311, 2-bromo-6-nonylphenol 6 7.28 dd, J 8, 2 Hz, H1-3), 7.04 (11, mn, 11-5), 6.72 (111, t, J 8 Hz, H1-4), 2.6 (211, t, H11), 1.6 (211, in(b), 1.3 (1211, rn(b), 11-3' to 0.88 (311, H1- 4-bromo-6-nonylphenol 6 7.22 (111, d, J =2 Hz, H1- 7.15 (1H1, dd, J 8, 2 Hz, 11-3), 6.63 (1H1, d, J =8 (la,21i, 11-3' to 0.88 *(311, 11-9') 2,4dibromo-6-nonylphenol 6 7.42 (1H1, d, LTJ 2 Hz, 11-3) 7.17 (111, in, 11-5), 2.6 (211, t, H11), 1.6 (211, in(b), H- 1.3 (1211, in(b), 11-3' to 0.88 (311, H1- 2-nitro-6-nonylpheno. 6 7.95 (1H1, dd, J 8, 2 Hz, H1-3) 7.43 (111, in, 11-5) 6.89 (1H1, dd, J 8, 2 Hz, H1-4), 11-3' to 0.88 (317, H1-91). 4-nitro-6nonyiphenol 6 8.06 (1H1, d, J 2 Hz, 11-5) 8.00 (111, dd, 1 8, 2 Hz, 11-3), 6.84 (1H1, d, JT 8 Hz, H1-2), 2.6 (211, tH11), 1.6 (211, in(b), 1.3 (1211, in(b), 11-3' to AMENDED SHEET vi
IPEAVAU
48 0.88 (3H, 2-bromo-4-nitro-6nonylphenol 6 8.27 (1H, d, J 2 Hz, 8.01 (1H, d, J 2 Hz, 2.6 (2H, t, 1.6 (2H, 1.3 (12H, H-31 to 0.88 (3H, 2nitro-4-bromo-6-nonylphenol 6 8.27 (1H, d, J 2 Hz, H- 8.02 (1H, d, J 2 Hz, 2.6 (2H, t, 1.6 (2H, 1.3 (12H, H-3' to 0.88 (3H, 4-nonyiresorcinol 6 6.94 (1H, d, J 8 Hz, 6.35 (1H, dd, J 8, 2 Hz, 6.31 (1H, d, J 2 Hz, 2.6 (2H, t, 1.6 (2H, 1.3 (12H, H-3' to 0.88 (3H, o w-bis(2-hvdroxyvhenv1)alkanes 1,8-bis(2hydroxyphenyl)octane 6 7.10 (2H, td, J 8, 2 Hz, H-4), 7.07 (2H, dd, J= 8, 2 Hz, 6.86 (2H, td, J 8, 2 Hz, 6.75 (2H, dd, J 8, 2 Hz, 2.60 (4H, t, 1.60 (4H, 1.32 (8H, H- 3' to 1,9-bis(2-hydroxyphenyl)nonane 5 7.10 (2H, td, J 8, 2 Hz, 7.07 (2H, dd, J 8, 2 Hz, H-6), 6.86 (2H, td, J 8, 2 Hz, 6.75 (2H, dd, J= 8, 2 Hz, 2.60 (4H, t, 1.60 (4H, H-2', 1.32 (10H, H-3' to 1,1O-bis(2hydroxyphenyl)decane 6 7.10 (2H, td, J= 8, 2 Hz, H-4), 7.06 (2H, dd, J 8, 2 Hz, 6.86 (2H, td, J 8, 2 Hz, 6.75 (2H, dd, J 8, 2 Hz, H14-3), 2.60 (4H, t, 1.60 (4H, 1.28 (12H, mn(b), H-3' to 1,12-bis(2-hydroxyphenyl)dodecane 6 7.10 (2H, td, J 8, 2 Hz, 7.06 (2H, dd, J 8, 2 Hz, H- 6.,-X.-G86 (21, td., J 8, 2 Hz, 6.75 (2H, dd, J 8, 2 Hz, 2.60 (4H, t, 1.60 (4H, H- 1.28 (16H, H-3' to 1,14-bis(2hydroxyphenyl)tetradecane 6 7.10 (2H, td, J 8, 2 Hz, 7.06 (2H, dd, J 8, 2 Hz, 6.86 (2H, td, J 8, 2 Hz, 6.75 (2H, dd, J 8, 2 Hz, 2.60 (4H, t, 1.60 (4H, 1.28 H-3' to 1,10-bis(2-hydroxy-3methylphenyl)decane 6 7.07 (4H, d, J 8 Hz, H-4, 6), 6.88 (2H, t, J 8 Hz, 2.60 (4H, t, 2.30 (6H, s, CH 3 1.60 (4H, 1.32 d" LAMENDED SHEET
IPEAAU
-49- (12H, m(b) H-31 to 1,10-bis(2-hydroxy-4methyiphenyi)decane 6 7.01 (2H, d, J 8 Hz, H-6) 6.70 (2H, dd, J 8 Hz, H-5) 6.60 (2 H, d, J 2 Hz, H-3) 2.60 (4H, t, 10')N, 2.30 (611, s, CH 3 1.60 (4H, H-21, 1.32 (i2H, H-3' to 1,10- 6.91 (2H, di, J= 2 Hz, H-6) 6.86 (2H, dd, J 2 Hz, H-4) 6.63 (2H, ci, J 8 Hz, H1-3), 2.60 (4H, t, 2.30 (6H, s, CH 3 1.60 (4H, 1.32 (12H, rn(b) H-3' to u- (2-hydroxyohenvi) (4-hydroxvyphenvl)alkanes 1- (2hydroxyphenyl) 10 (4 -hydrox-yphenyl) decane 6 7.10 (iH, td, J 8, 2 Hz, 7.06 (1H, dci, J 2 Hz, H-6), 7.02 (2H, ci, J 8 Hz, H-2"1, 6.86 (1H, td, J 2 Hz, H-5) 6.75 (1H, dci, J 8, 2 Hz, H-3) 6.73 (2H, ci, J =8 Hz, H-311, 2.62 (2H, t, 2.55 (2H, t, H- 10'1) 1.60 (4H, m(b) H-2'1, 1.28 (12H, rn(b) H-3' to 1- (2-hydroxyphenyl) -12- (4-hydroxyphenyl) dodecane 6 7.10 (1H, td, J 8, 2 Hz, H-4) 7.06 (1H, dd, J 2 Hz, H-6) 7.02 (2H, ci, J 8 Hz, H-211, 6.86 (1H, td, J 8, 2 Hz, 6.75 (1H, cid, J 2 Hz, 6.73 (2H, ci, J 8 Hz, H- 2. 62 (2H, t, H-i'1) 2. (2H, t, H-121), 1.60 (4H, m(b) H-21, 111), 1.28 (16H, H-3' to I -hydroxyphenyl) 1,t-(4 hydroxcyphenyl)tetradecane 6 7. 10 (1H, td, J 8, 2 Hz, H 7. 06 (iH, dci, J 8, 2 Hz, 7.02 (2H, ci, J 8 Hz, H-21", 6.86 (1H, td, J 8, 2 Hz, 6.75 (1H, ciadr 8, 2 Hz, 6.73 (2H, ci, J 8 Hz, H-311, 5"1), 2.62 (2H, t, 2.55 (2H, t, H-141), 1.60 (4H, rn(b), H-21, 1.28 (20H, H-31 to H-12').
oe~w-bpis(4-hycroxvohenvl)alkanes 1,10-bis(4-hydroxy phenyl)decane 6 7.02 (4H, ci, J 8 Hz, H-2, 6) 6.74 (4H, di, J =8 Hz, H- 3, 5) 2.52 (4H, t, 10') 1. (4H, m H-2'1, 1. 28 (12H1, m H- 31 to H- 8') l,12-bis(4-hydroxy phenyl) dodecane 6 7. 02 (4H, di, LJ 8 Hz, H- 2, 6) 6. 74 (4H, ci, LT 8 Hz, H-3, 2.52 (4H, t, 1.60 (4H, m(b) H-2' 1.28 (16H, m(b) H-3' to H-i0').
AMENDED SHEET cT/AT) 09 ,l 9J5 RECEI ED I 19 50 1,14-bis(4-hydroxy phenyl)tetradecane 6 7.02 (4H, d, J 8 Hz, H-2, 6), 6. 74 (4H, d, J 8 Hz, H-3, 2.52 (4H, t, 141'), 1.60 (4H, 1.28 (20H, H-3' to H- 12').
bis(hydroxyarvl)alkanes and derivatives 1,10-bis(3-hydroxyphenyl)decane 6 7.13 (2H, t, J= 8 Hz, 6.75 (2H, dd, J 2 Khz, 6.64 (4H, m, H-2, 2.26 (4H, t, 1.53 (4H, m, 1.23 (12H, H-3' to H-81). 1,10-bis(3-hydroxy-4methyl)decane 6 7.01 (2H, d, J 8 Hz, 6.67 (2H, dd, J 2 Hz, 6.60 (2H, d, J 2 Hz, 2.26 (4H, t, 2.20 (6H, s, CH 3 1.53 (4H, m, H- 1.23 (12H, H-3' to 1,10-bis(4hydroxy-3-methylphenyl)decane 6 6.92 (2H, d, J 2 Hz, 6.87 (2H, dd, J 8, 2 Hz, 6.67 (2H, d, J 8, 2 Hz, 2.25 (4H, t, 2.,21 (6H, s, CH 3 1.53 (4H, mn, 1.23 (12H, rm(b), H-3' to H- 1,1-bis-(2-hydroxyphenyl)decane 6 7.30 (2H, dd, J= 8, 2 Hz, 7.03 (2H, td, J 8, 2 Hz, 6.90 (2H, td, J 8, 2 Hz, 6.79 (2H, dd, J 8, 2 Hz, 4.47 (1H, t, 2.12 (2H, m, 1.22 (14H, H-3' to 0.86 (3H, 1,10-bis(2-hydroxy-1-iiaphthyl)decane 6 7.91 (2H, d. J= 8, 2 Hz, 7.75 (2H, dd, J 8, 2 Hz, 7,61 (2H, d, J 8 Hz, 7.47 (2H, td, J 8, 2 Hz, 7.31 (2rr71 J 8, 2 Hz, 7.04 (2H, d, J 8 Hz, H-3), 3.01 (4H, t, 1.65 (4H, 1.46 (4H, 1.30 (8H, H-4' to Resorcinol and coumarin derivatives 6 6.24 (2H, s, H-2, 2.45 (2H, t, 2.01 (3H, s, CH 3 1.55 (2H, m, 1.26 (12H, H-3' to 0.88 (3H, 2bromo-6-dodecylresorcinoi 6' 6.95 (1H, d, L 8 Hz, 6.54 (1H, d, J 8 Hz, 2.58 (2H, t, 1,56 (2H, m, 1.260' (18H, H-3' to 0.88 (3H, J 8 Hz, 4-bromo-6-dodecylresorcinol 6 AMENDED SHEET
IPEA/AU
PC'rA 94 00207 RECEIVED 0 2 MAR 199 51 7.15 (1H, s, 6.49 (1H, s, 2.58 (2H, t, 1.56 (2H, m, 1.26 (18H, 1-3' to 0.88 (3H, tko), J 8 Hz, 2,4-dibromo-6dodecylresorcinol 6 7.18 (1H, s, 2.58 (2H, t, H- 1.56 (2H, m, 1.26 (18H, H-3' to 0.88 (3H, J 8 Hz, 6-dodecyl-7-hydroxy-4methylcoumarin 6 7.30 (1H, s, 7.16 (1H, s, H-3), 6.12 (14, s, 2.42 (31, s, CH3-4), 2.58 (2H, t, H- 1.56 (2H, m, 1.26 (18H, rn(b), 1-3' to 0.88 (3H, t(b) J 8 Hz, cw-bis(2,4-dihydroxvphenvl)alkanes 1,8-bis(2,4dihydroxyphenyl)octane 5 6.63 (2H, d, J 8 Hz, 11H-6), 6.17 (2H, d, J 2 Hz, 6.05 (2H, dd, J 8, 2 Hz, 2.26 (4H, t, 1.53 (4H, m, 1.23 (8H, H-3' to 1,10-bis(2,4dihydroxyphenyl)decane 6 6.63 (2H, d, J 8 Hz, 1-6), 6.17 (2H, d, J 2 Hz, 6.05 (2H, dd, J 8, 2 Hz, 2.26 (4H, t, 1.53 (4H, m, 1.23 (12H, H-3' to 1,11-bis(2,4 dihydroxyphenyl)undecane 6 6.63 (2H, d, J 8 Hz, H-6), 6.17 (2H, d, J 2 Hz, 6.05 (2H, dd, J 8, 2 Hz, 2.26 (41, t, 1.53 (4H, m, 1.23 (14H, H-3' to 1,12-bis(2,4dihydroxyphenyl)dodecane 6 6.63 (21H1, d, J 8 Hz, H-6), 6.17 (2H, d, J 2 Hz, 6.05 (2H, dd, J= 8, 2 Hz, 2.26 (4H, t, 1.53 (4H, m, 11'), 1.23 (16H, 1-3' to 1,10-bis(2,4-dihydroxy- 3 -ttiylphenyl) decane 6 6.75 (2H, d, J 8 Hz, 11-6), 6.38 (2H, d, J 8 Hz, 2.26 (4H, t, 2.14 (6H, s, CH3-3), 1.53 (4H, m, 1.23 (12H, H-3' to a,w-bis(3,5-dihydroxvDhenv1)alkanes 1,8-bis(3,5dihydroxy-4-methylphenyl)octane 6 6.24 (4H, s, 1-2, 6), 2.40 (4H, t, 2.04 (6H, s, CH 3 1.53 (411, m, 1.29 (81, 1-3' to 1,10-bis(3,5dihydroxy-4-methylphenyl)decane 6 6.24 (4H, s, H-2, 6), 2.40 (41, t, 2.04 (6H, s, CH 3 1.53 (41, n, 1.29 (12H, 1-3' to 1,12- AMENDED SHEET
IPENAU
PJ k 002 i R~ 02 MAR 19c 52 bis(3,5-dihydroxy-4-methylphenyl)dodecane 6 6.24 (4H, s, H-2, 2.40 (4H, t, H-11, 2.04 (6H, s, CH 3 1.53 (4H, m, H-21, 1.29 (16H, H-3' to 1,14-bis(3,5-dihydroxy-4-methylphenyl) etradecane (striatol) 6 6.24 (4H, s, H-2, 2.40 (4H, t, H-i', 2.04 (6H, s, CH 3 1.53 (4H, m, 1.29 H-3' to H-12').
Table 4: Chemical ionisation mass spectra of the synthesised alkylphenols and Uw bis(hydroxyphenyl)alkanes (reagent gas CH 4 m/z The intermediate products 2-octanoylphenol 261 249 (11), {M+11+ 221 (100) {M+1 C 6
H
4 -OHj+ 127 2noianoylphenol 275 263 235 (100) C6H 4 -OH} 141 2-decanoylphnno1 {M+41 289 {M+291+ 277 {M+1J+ 249 (100), {M1 C6H 4 -OH} 155 (15) {OH-benzoy1} 121 (16) 4octanoylphenol 221 (100), {M+1 C 6
H
4 127 (M+1 C 8
H
1 113 4-nonanoylphenol 235 (100), {M CH 3 221 IM+I C 6
H
4
-OH
4 141 (M+1
C
7
H
1 6 135 4-decanoylphenol (M+411+ 289 (M+291+ 277 {M+11+ 249 (100), (M+1 C 2
H
4 221 (M+1 C6H 4 155 {M+1 CSH 18 135 3benzyloxybenzaldehyde 253 241 (12), (M+11+ 213 (100), (M+1 C 6
H
6 135 diphenyl decanedioate (M+291+ 383 {M+11+ 355 (M+1
C
6
H
4 261 (100). diphenyl dodecanedioate (M+291+ 411 (2j1L){M+1}+ 383 (M+1 C 6
H
4 -PHI+ 289 (100). diphenyl tetradecanedioate 439 (M+11+ 411 (M C6H 4 -OHI+ 317 (100). 1,1O-bis(2-hydroxyphenyl)decane-1,10dione (M+291+ 383 355 (100), {M+1 C6H 4
-OH}+
261 1,12-bis(2-hydroxyphenyl)dodecane-1,12-dione (M+411" 423 (M+291+ 411 (M+11+ 383 (100), {M+1
C
6
H
4 289 1,14-bis(2-hydroxyphenyl)tetadecane-.
1,14-dione (M+41+ 451 439 411 (100), (M+1 C 6
H
4 317 1- (2-hydroxyphenyl) (4-hydroxyph-uyl decane-1, 10-dione 383 {M+11+ 355 (100) (M+1 281+ 327 (10) {M+1 C6H 4 261 4 OfAMENDED SHEET
IPEAIAU
PLC/AI(J 9 4 0 RECEIVEO C) 2 0 297 MAR 1995 (14) C 6
H
4 (OH) COCH 2 13 5 (2 {C H 4 (OH) 12 1 (15) 1 (2 hydroxyphenyl) 12 (4 -hydroxyphenyl) dodecane 12 dione 423 (M+291+ 411 {M+11+ 383 (100), {M±J.
C
6
H
4 289 {C H 4 (OH)CO)+ 121 1-2 hydroxyphenyl) 14 (4 -hydroxyphenyl) te tradecane 14 -di one {M+411+ 451 (M+291+ 439 (M+11+ 411 (100), {M+1
C
4
H
8 355 {M~1 C 6
H-
4 317 1,1O-bis(4hydroxyphenyl)decane-1,10-dione fM+41}+ 395 {M+291+ 383 (15) 355 (100) {M+1 C 6
H
4
-OH)
4 261
{C
6
H
4 (OH)CO)+ 121 1,12-bis(4-hydroxyphenyl)dodecane- 1,12-dionae 4-23 411 383 (100), {M+1 C6H 4 289 {C6H 4
(OH)C(OH)CH
3 137 f{C 6
H
4 (OH) C(OH) 123 1,14-bis(4hyfdroxyphenyl)tetradecane-1,14-dione {M 41)+ 451 {M+291+ 439 411 (100), {M+1 C 6
H
4 -OHI+ 317 {OH-benzoy1}+ 121 (12).
alkylphenols and u. w-biso(hydroxyp~henyl) alkanes 2-and 4-octylphenols {M+411+ 247 235 (16), 207 (100), {C 6
H
4
(OH)CH
2 107 3- and 4nonyiphenols 261 249 (12) 221 (100), {C 6
H
4
(OH)CH
2 107 2- and 4-decylpbhenol (M+411+ 275 (M+291+ 263 (M+11+ 235 (100), {%6H 4
(OH)CH
2 107 1,10-bis(hydroxyphenyl)decanes and 1- (2-hydroxyphenyl) -10- (4-hydroxyphenyl) decane {M+411+ 367 355 (20) 207 (100),
{C
6
H
4
(OI)CH
2 107 1,12-bis(hydroxyphenyl)dodecanes and 1- (hydroxyphenyl) -12- (4-hydroxyphenyl)dodecane 395 {M+291+ 383 (20) (M+11+ 355 (100),
{C
6
H
4
(OH)CH
2 107 1, 14 -bis (hydroxyphenyl) tetradecanes and 1-(2 7 hydroxyphenyl)-14-(4hydroxyphenyl)tetradecane 423 411 383 (100), {C 6
H
4
(OH)CH
2 107 anid 4-methyl-6-nonylphenol 275 263 235 (100), {M+1 CHX1 219 C 8
H
1 12-1 (10-20). 2- and 4-bromo-6-nonylphenol 327 299 (100), {M+1 C 8
H
1 185 {C 9
H
1 127 2,4-dibromo-6-nonylphenol 419 407 {M+11+ 377 (100), {M+1 CX 363 41-r O AMENDED SHEET PeTI1 94 0 02 7 RECEIVED 0 2 MAR 1995 54 2{M Br)+ 299 (18) {M+1 C 8
H,
8 265 (20) {M+1 C9H20)+ 127 (45) 2- and 4-nitro-6- nonylphenol {M+41)+ 306 294 266 (100), {M+1 CH4)+ 250 {M+1 C 2 H6)+ 236 (10-12) 2 -bromo-4-nitro-6nonylphenol/4-bromo-2-nitro-6-nonylpheno. {M+29) 372 (12) 344 (100) {M1 C 2 314 (10) {M Br)+ 266 {M+1 C 7
H
16 248 4-nonylresorcinol {M+411+ 277 265 (20) {M+1}j 237 (100),
{C
7
H
7
O
2 1+ 123 2- and 4-bromo-6-dodecylresorcinol {M+29+ 385 357 (100), (M Br)+ 279 (10-20), fM C 11
H
23 1+ 201 (15-25), (M C 8
H
7 02 135 2,4dibromo-6-dodecylresorcinol {M+411}. 477 465 437 (100), {M Br)+ 357 {M+1 CpIH 2 281 {M-2351+ 201 {C 8
H
7
O
2 135 6-dodecyl-7-hydroxy-4-methylcoumarin 385 373 {M+1)f 345 (100), {C 8
H
7
O
2 135 1, 8-bis (2-hydroxyphenyl) octane 339 {M+29} 327 299 (100), (C H 11
G
2 135 (30) 1,9bis(2-hydroxyphenyl)nonane 353 341 {M-i1} 313 (100) 1,10-bis[2-hydroxy-3 (4-and metbylphenyljdecanes {M+411+ 395 {M+291+ 383 (20-25), 355 (100). 1,8-bis (2,4-dihydroxyphenyl) octane 371 359 {M+11+ 331 (100), (M+1 CHX 315 1,10-bis(2,4-dihydroxyphenyl)d-cane 399 (M+291+ 387 359 (100).
bis(2,4-dihydroxyphenyl)undecane 413 {M+29}+ 401 373 (100), {C 7
H
7
O
2 123 1,12biw(2 r4-dihydroxyp heny1) dodecane {M+411+ 427 (M+291+ 415 {M+11+ 387 (100) 1,10-bis(2,4-dihydroxy-3methylphenyl) decane {M+411+ 427 {M+291+ 415 401 387 (100). l,10-bis(3hydroxyphenyl)decane 367 355 327 (100) 1,10-bis[3-hydroxy-4-methyl(and 4hydroxy-3-methyl)phenyl decanes 395 383 355 (100). 1, 1-bis (2hydroxyphenyl)decane {M+11+ 327 {M+1 C 6
H
4
-OH)+
233 (100) C 9
H
11 135 {C 7
H
7 107 1,10bis(2-hydroxy-l-naphthyl)decane (M+411+ 467 (M+29}+ "VT -r 0 "V2.:ED SHEET
PENAU
~1Y4 9400 2 RECEIVED 0 2 MAR 455 (15) 427 C 9
HE
1 135 (50) 119 (100) 291 (M+291+ 279 251 (100) {M+1 CH 4 235 fC 8
H
7
O
2 135 1,8-bis (3,5-dihyroxy-4-methylpbhenyl) octane 399 387 (25) 359 (100), (C H 7 O0} 135 1,iO-bis(3,5-dihydroxy-4methylphenyl)decane 427 415 387 (100), {CaH 7
O
2 135 1,12-bis(3,5dihydroxy-4-methylphenyl)dodecane {M-i41}+ 455 (M+i29)+ 443 415 (100) 1,14-bis(3,5-dihydroxy-4methylphenyl)tetradecane (striatol) 483 (M4-291+ 471 {M+1} 4 443 (100), {CBH 7
O
2 135 AMENDED SHEET cr/u 9 4 0 2 9 7 RECEIVED 0 2 MAR 1995 56 EXPERIMENTAL NATURAL PRODUCTS NOVEL RESORCINOLS FROM GREVILL3A ROBUSTA AND THEIR INHIBITORY ACTIVITY TO ERYTHROCYTE Ca2+-ATPase General experimental procedures for extraction and isolation The mobile phase was MeOH/H 2 0 (82:18). Flow rate was 1.0 ml/min; Preparative HPLC were carried out with a Altex 100 solvent delivery system, equipped with Altex UV detector at 254nm, a Activon Partisil ODS-3 column, 9x500mm was used. The mobile phase was a gradient of MeOH/H 2 0 to MeOH in 60 min. or an isocratic mobile phase 65% CH 3 CN in H 2 0, flow rate was ml/min. 6251, 6252 were separated by these systems.
Plant Material The stem of Grevillea robusta was collected in Sydney, Australia. A voucher specimen is available for inspection at the Department of Pharmacy, The University of Sydney. The stem wood, 5-10 cm in diameter, was sliced by an electrical plane and air dried.
Extraction and Isolation A sample of 2 kg was extracted by percolation with CHC1 3 /EtOH for 3 days twice. After concentration of the extract in vacuo, the residue (20g) was chromatographed by Silica gel short column vacuum chromatography collecting 250ml fractions.
The Ca2+-ATPase assay gave an inhibitory activity of at a concentration of 0.5mg/ml. Fractions 1 to 4 (3.82g) were eluced with petroleum/EtOAc Fractions 7-9 (11.91g), eluted from CHC1 3 /MeOH(2:1), were inactive in±jtheassay. They were not further studied. Fraction (2.78g), 6 (0.13g), 6b(1.38g) were eluted with CHC1 3 /EtOAc(2:1), CHC13/MeOH respectively and gave strong inhibitory activity. Fraction 5 was further short column vacuum chromatographed with CHC13/EtOH (95:5) and gave grevillol (0.74g). Fraction 6b was similarly chromatographed with CH 2 C1l/ EtOAc to give 6b2 (37.4mg). Fraction 6 was similarly separated into fractions (each 50"rl) with petroleum /EtOAc
CH
2 Cl 2 /EtOAc EtOAc, EtOAc/CH 3
CN
Only fraction 4-5 (63.4mg) from CH 2
CI
2 /EtOAc (2:1) 1 AMENDED SHEET T ro
IPEA/AU
IIOU I~"~CIIRII~ I~ IIIIC9RC~ PCr/AU 9 4 0 0 2 9 7 RECEIVED 0 2 MAR 1995 57 gave strong inhibitory activity, 74% inhibition at 0.04mg/ml. This fraction was then separated by gradient HPLC to give synapic aldehyde (623, 1.8mg), and a nonpolar fraction 625 which was finally separated into 6251 (6.6mg) and 6252 (1.3mg) by preparative TLC, with CHC13/EtOH (95:5) as a solvent.
The methylation and ozonolysis is a method from Barrow R A, Capon R J (1991) Alkyl and alkenyl resorcinols from an Australian marine sponge, Haliclona sp. Aust.J.Chem 44 1393-1409. The sample to be methylated (2-8 mg) was stirred in acetone (3 ml) with
K
2
CO
3 (200mg) and CH 3 I (0.5ml) at room temperature for h. The methylated products were isolated by preparative TLC with CHC13/EtOH for 6b2, petroleum EtOAc for 6251, 6252. The methylated compounds (0.5-1 mg in CS 2 (2ml) at -78 C were ozonolysed with a stream of 03 and then triphenylphosphine (2mg) added. The reaction mixtures were analysed directly by CI-MS.
The long chain resorcinols gave a characteristic purple colour on TLC after exposure to 12 vapour and being left on the bench overnight, while resorcinol, resorcinylic acid, orcinol gave a dust brown colour.
Grebustol-A (6251)- Colourless oil; Rf 0.51 (CHC1 3 /EtOH 1 H-NMR (CDC13, ppm) 1.26-1.35 12H,
CH
2 1.55 4H, ArCH 2
CH
2 2.00 4H, CH=CHCH 2 2.10 3H, ArCH 3 2.42-2.50 4H, ArCH 2 4.70 (br.s., OH), 5.32-5.39 2H, CH=CH), 6.17(t, J=2.0Hz, 1H, ArH), 6--2-44b-r.s., 4H, ArH); 13 C-NMR 156.5, 154.4, 142.6, 142.0, 130.0, 129.7, 108.1, 107.9, 100.2, 35.9, 35.6, 31.3, 31.1, 29.8, 29.7, 29.6, 29.4, 29.2, 29.1, 27.2, 7.9; CI- MS (CH 4 m/z 427{M+1}, 397, 285, 257, 229. 207; UV max(MeOH) 208.4 (loge 4.48), 274.4 279.4 (3.33).
Methylated Grebustol-A (6251m)- 1 H-NMR 1.26-1.38 (m, 12H, CH 2 1.55 4H, ArCCH2C 2 2.00 4H, =CHCH 2 2.06 3H, ArCH 3 2.55 4H, ArCH,), 3.78 6H, 2 X
OCH
3 3.81 6H, 2 X OCH 3 5.32-5.41 2H, CH=CH), 6.29 J=2.3Hz, 1H, 6.34 J=2.3Hz, 2H, 4, 6,- 6.36 2H, CI-MS 483, EI-MS 482{M} 7v o^ AMENDED SHEET
IPEAAU
9A 58- (32) 410 386 368 ,353 341 ,149 (12) 109 Ozonolysis of 6251mn, 'HNM 3. 81, 3. 78, 9. 76, 9.77 (CHO), Cl-MS 279, 237.
Norstriatol-B (6252)- colourless oil, Rf 0.55 (CHCl 3 /EtOH 9 1 1-NMR (CDCl 3 PPM) 1. 25 Cm, 1211, CH2) 1. 58 (mn, 4H1, CE 2 1.-9 1 (in, 411, =CH CE 2 2. 26 211, 1
CE
2 2. 60 (mn, 211, 14- CE 2 5. 30 (mn, 2H, CH=CH) 4.6 6 011), 6.45-6.49 (mn, 411, ArE) Cl-MS 411{M+1}+, 317, 285, 257; UIV max (MeOH) 206.4 (logE 4.61) 279.2 (3.49).
Methylated norstriatol-B (6252m) 1 11-NMP. 1.25 (in, 12H, CE 2 1.54 (in, 411, CE 2 1.87 Cm, 411, CH=CECH 2 2.23 (mn, 211, 1-CE 2 2.66 (mn, 211, 14-CE 2 3.68 J=3.O~z, 3H1, 22-0C1 3 3.69 Cd, J=1.40 Hz, 6H1, 17,190OCE 3 3.83 is J=J..4511z, 311, 24-0C1 3 5.30 (mn, 211, CE=CE) 6.42- 6.45 Cm, 411, ArH) Cl-MS 467 El-MS 466 (100) 451(2), 302(5), 149(10); 1 3 C_-NMR, 56.0, 56.1, 96.5, 104.8, 105.0; Methylated striatol-B- 1 3 C -NNR 56.0 ,56.1, 96.5, 105.0, 130.3, 145.5.
Results and Discussion Grebustol-A (6251) Compound (II) Grebustol-A (6251) (6.6 mng, 3.3ppm), molecular weight 426 (CI-MS), UV Max 274nm, is different from striatol in having a double bond in the alkyl chain and a single benzyl methyl group as indicated by the 1HN~ff signals at 5.32-5.39 ppm (mn, 2H) and 2.10 ppm 311,.
Th--mass spectrum and 3-H-NM spectrum of the methylated product revealed the presence of four OC11 3 groups (El-MS 482), 1 E-NMR 3.78 ppm 611) 3.81 611)), 1 11-ND4R H-H COSY found the coupling of the signals 1.6-1.55, 1.26- 2.00, 1.55-2.42, 2.00-5.35 ppm, but no coupling between the signals 1.55 and 2.00 ppm. This excludes the possibility of the double bond at C-10,11. 1 11-NMR dataindicated a non-symmetric structure. The Cl-MS of ozonolysis products indicated aldehydes of molecular weight 278 and 236, consistent with a C-8,9 or ,C-9,10 (mn=8, n=4) position for the double bond.
-1 AMENDED SHEET
IPEA/AU
PT/AU 9 4 0 0 2 9 7 RECEIVED 0 2 MAR 1995 59 Norstriatol-B (6252) Compound (III) Norstriatol-B (6252) 1.3mg, 0.65ppm) is a desmethyl product of striatol-B. The CI-MS (CH 4 reagent gas) revealed a molecular weight 410. UV Max 279nm. The 1 H-NMR UV, mass spectrum of norstriatol-B was consistent with the reported data for striatol-B [Ridley D D, et al (1970) Chemical studies of the Proteaceae IV Aust.J.Chem. 23, 147-183] and an authentic sample of striatol-B. It is a biphenyl derivative ra-'3r than a diphenyl ether derivative as with robustol [Cannon J R, et al (1973) Phenolic constituents of Grevillea robusta (Proteaceae). The structure of robustol, a novel macrocyclic phenol Aust.J.Chem. 26, 2257-2275]. The methylation of norstriatol-B gave tetramethyl norstriatol-B, which was identical to the substance formed by methylation of authentic striatol-B.
Table 1 shows that the inhibitory effect of grevillol to Ca2+-ATPase was weak. The most potent compounds are striatol and grebustol-A, with IC 50 of 16 and 17pM respectively. Without the benzylic methyl group, the activity was weaker, as in grebustol-B.
Bisnorstriatol gave only 69.1% inhibition at the concentration of 500 pM. After the methylation of the phenolic hydroxy group, the activity of grebustol-B was lost. The inhibitory activity of striatol has been confirmed with purified erythrocyte Ca2+-ATPase.
These results indicate the phenolic hydroxy groups were-recessary for the inhibition to occur. The benzylic methyl group between the hydroxy groups enhances the activity. Also the double bond in the alkyl chain increased the inhibitory activity to Ca2+-ATPase.
Erythrocyte membranes Calmodulin-depleted erythrocyte membranes were prepared by continuous filtration through a hollow fibre; Ashahi plasma separator as described by W S Price, B D Roufogalis, P W Kuchel (1989), A simple and inexpensive method for preparing erythrocyte membranes by filtration through a hollow-filter system, Anal. Biochem., 179, 190- AMENDED SHEET
IPEA/AU
lso- RA/,J 1 C., J^CIVED 1 7 JUL 193. Packed cells were obtained from the New South Wales Red Cross Trarnfusion Service, Sydney. The whole preparation was carried out at 4 0 C. 1 unit of packed red cells was washed 3 times with isotonic buffer, containing 130 mM KC1, 20 mM Tris-HC1 (pH and the cells were collected by centrifugation at 4,000 RPM. The cells were haemolysed by the buffer containing 1 mM EDTA, 10 mM Tris-Hcl (pH 0.5 mM PMSF (phenylmethyl sulfonyl fluoride). The haemolyzate mixture was passed through the hollow fibre system until the membrane appeared white, and then washed with 10 mM potassium Hepes (pH The membranes were collected by centr-fugation at 10,000 RPM for 20 min, the meibranes were resuspended in storage buffer containing 130 mM KC1, 2 mM dithiothreitol, 0.5 mM MgCl 2 20 mM potassium-Hepes (pH The membranes with protein concentration of 5.4 mg/ml were stored at -80 0 C until required.
Ca2+-ATPase assay Erythrocyte membranes (0.071-0.098 mg/ml) were incubated at 37 0 C for one hour in a total volume of 0.4 ml containing 65 mM KC1, 20 mM potassium-Hepes, 5 mM MgC1 2 150 AM CaCl 2 (50.4 AM calculated free Ca concentration), 0.1 AM calmodulin, 0.1 mM EGTA. The reaction was started by adding 2 mM ATP(pH The phosphate liberated to the medium was determined spectrometrically according to the procedure of B U Raess, F F Vincenzi (1980), A semi-automated method for the-determination of multiple membrane ATPase activities, J.Pharmacological Methods A, 273-283. The activity of the Mg2+-ATPase (assayed in the absence of added CaCl 2 was subtracted from the total activity assayed in the presence of Ca 2 Phenols were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the assay mixture was DMSO itself had no effect on.
ATPase activities. A concentrated solution of the test substances was added to the reaction medium before adding ATP. Protein concentration was determined according to the method of 0 H Lowry, N J Rosebrough, A L Farr, R J Randall (1951) J.Biol.Chem. 193, 265-275; bovine serum AMENDED SHEET
PEA/AU
L~ ~La 1Lls C_ 1 PeTrA r4 0029 I P 1O 61 albumin was used as a standard. The concentration of the free Ca 2 were calculated by computer using a program of D A Goldstein (1979) Calculation of the concentrations of free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands, Biophys.J., 26, 235-242.
Control specific activity of the ATPases were (unit: nmoles/mg protein/min) :Ca 2 +-ATPase, was 25.8±4.0 calmodulin stimulated Ca2+-ATPase was 63.5±7.9 (n=16), while the Mg2+-ATPase was 7.4±1.0 The enzyme was inhibited by NAP-taurine by 60% at a concentration of iM as shown by A Minocherhomjee, B D Roufogalis (1982), Selective antagonism of the Ca transport ATPase of the red cell membrane by N-(4-azido-2-nitrophenyl)-2aminoethylsulfonate (NAP-taurine), J.Biol.Chem. 257, 5426-5430.
Ca2+-ATPase assay (microplate method) Erythrocyte membranes were incubated at 37 0 C for one hour in a total volume of 60 Al containing 65 mM KC1, mM HEPES 5 mM MgCl 2 150 /M CaC12 (50.4 pM calculated free Ca++ concentration), 0.1 mM EGTA and in absence and presence of calmodulin (50 nM). Test compounds were dissolved in dimethyl sulfoxide DMSO and 2 ~l was added to the assay mixture prior the addition of ATP. The final concentration of DMSO in the assay mixture was The reaction was started by adding 2 mM ATP After 1 hour of incubation, colouring agent (180 1l) was added and incubated at 37 0 C for 1/2hrs. The phosphate liberated to the assay medium was determined spectrometrically using a microplate reader at 750nm. The activity of the Mg 2 -ATPase (assayed in the absence of added CaC1 2 was subtracted from the total activity assayed in the presence of Ca 2 The results are shown in Table 1A.
ABBREVIATIONS
ATP Adenosine triphosphate ATPase Adenosine triphosphatase BHT 2,6-di-tert-butyl-4-methylphenol CI-MS Chemical ionisation mass spectrometry I AMENDED SHEET
IPEA/AU
r r r~ r/AU 94 00 9 7 RECEIVED 0 2 MAR 1995 62 DMSO Dimethyl sulfoxide EI-MS Electron impact mass spectrometry EGTA ethylenebis(oxyethylenenitrilo)tetraacetic acid HEPES 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid HPLC High-performance liquid chromatography LDA Lithium diisopropylamide NAP N-(4-azido-2-nitrophenyl)-2aminoethanesulfonic acid NMR Nuclear magnetic resonance PMSF Phenylmethyl sulfonyl fluoride SDS Sodium dodecylsulfate TLC Thin layer chromatography TOXICITY BRINE SHRIMP ASSAY The brine shrimp assay procedure determines LC 50 values of active compounds. Activities of a broad range of known active compounds are manifested as toxicity to brine shrimp (Artemia salina Leach). There are many applications of the assay including analysis of toxic substances, anaesthetics, morphine-like substances and cocarcinogenicity of phorbol esters. The assay shows good correlation with some cytotoxicities and its utility as a prescreen for some antitumour activities has been recently confirmed.
DMSO (dimethyl sulfoxide) was the solvent of choice because of its good solubilising properties and also beiause the phenolic substances used in the Ca2+-ATPase inhibition study were already prepared with DMSO.
The method for testing solvent toxicity used was basically that reported by J L McLaughlin in Methods of Plant Biochemistry (1991), vol. 6 (K Hostettman, ed.), Academ Press, London, 1-32. DMSO solutions of the substances to be tested were added directly to the vials containing the brine shrimp. As the concentrati n of DMSO that we wished to use was higher than the reco ided 1% v/v testing of the toxicity of the DM80 was Lherefore necessary. The concentrations of DMO .rted on the AMENDED SHEET _IF PT/A 94 0 02 9 RECEIVED 0 2 MAR 19 63 shrimp, along with the results from the assay which was done in duplicate are listed in Table Table 5. Concentrations of DMSO tested.
Cone v/v) 0 1 2 3 Deaths 0 0 0 0 0 9 12 18 57 96 100 100 100 No toxicity towards brine shrimp was observed in a 24 hour period for concentrations of DMSO in brine up to 4% v/v.
Bioassay Brine shrimp toxicity was assayed, except for some minor modifications, according to the method of McLaughlin et al as reported in Brine Shrimp: A convenient general bioassay for active plant constituents, B N Meyer, N R Ferrigni, J E Putman, L B Jacobsen, D E Nhols and J L McLaughlin. Planta Medica (1982), 45, 31-34 and Crown gall tumours on potato discs and brine shrimp lethality: Two simple bioassay for higher plant screening and fractionation. J L McLaughlin.
Methods of Plant Biochemistry (1991), vol. 6 (K Hostettman, Academ Press, London, 1-32. Ten shrimp were added transferred to each of the vials and the volume adjusted to 4.9 mL. Each dose was performed in triplicate, including the control. In quick succession, the appropriate volume of additional DMSO for each dose,
SRA
4
IA
Arro AMENDED SHEET
IPEA/AU
IYlsU BI PI~C) ~YrlCL~d~ P~ /AU 94 002 9 RECEIVED 0 2 MAR 19 S64 required to achieve a final concentration of was added before the appropriate volume of test solution. The vials were gently mixed and the time noted. After 24 hours, the number of survivors were counted and mortality was determined. The test compounds were assayed at concentrations of 100 AM, 25 AM, 5 AM, 1 AM and 0.2 AM (and where appropriate concentrations of 0.04 pM and 0.008 IM).
The brine shrimp were able to survive without food in the vials over the 24 hour period and were therefore not fed.
The dose-response curves were constructed using the Sigmaplot computer program and the LC 50 value was calculated from the intersection point of the cur.'e and the 50 mortality line. The LC 50 values were expressed in both AM and Ag/mL.
Table 6. LC 5 0 values from brine shrimp bioassay MWt Compound
LC
5 0 pM Ag/mL 206 2-octylphenol 1.5 0.31 220 2-nonylphenol 0.48 0.11 234 2-decylphenol 0.68 0.16 220 3 nonylphenol 0.32 0.070 220 4-nonylphenol 0.40 0.088 234 4-decylphenol 0.27 0.064 234 2-nonanoylphenol 0.63 0.15 234 4-nonanoylphenol 0.10 0.024 176 2-cyclohexylphenol >25 >4.4 176 4- cyclohexylphenol >25 >4.4 298 1,8-bis(2-hydroxtbhenyl) octane 3 0.89 326 l,10-bis(2-hydroxyphenyl)decane 1.9 0.62 AMENDED SHE£T
IPEA/AU
9~fA 94 0 2 R[ECEIVL D 0 2 MAR IT 65 MWt Compound
LC
50 jam /ig/mL 36 1,l0-bis(3-hydroxyphenyl)decane 3 0.98 326 1,1-bis(2-hydroxyphenyl)decane 14 4.6 A~3 1,10-bis(2-hydroxy-4- 14 methyJlphenyl) decan3- 354 1,10-bis(2-hydroxy-3- 5.8 2.1 methyiphenyl) decane 34 1,10-bio(3-hydroxy-4- 11 3.9 methyiphenyl) decane 354 1,10-bis(4-hydroxy-3- 4.2 methyiphenyl) decane 354 t-(2-hydroxy-3-rnethylphenyl)-10-(4- 2.2 0.78 hydroxy- 3-methyiphenyl) decane 354 1,12-bis(2-hydroxyphenyl)dodecane 2.6 0.94 382 1,14-bis(2-hydroxyphenyl)tetradecane 1.9 0.72 326 i,10-bis(4-hydroxyphenyl)decane 0.81 0.26 354 1,12-bis(4-hydroxyphenyl)dodecane 3.8 1.4 382 1,14-bis(4-hydroxyphenyl)tetradecane 7.9 3.7 326 1-(2-hydroxyphenyl)-10-(4- 0.35 0.12.
hydroxyphenyl) decane 354 1-(2-hydroxy-phenyl)-12-(4- 0.054 0. 019 hydroxyphenyl) dode cane 382 1-(2-hydroxyphenyl)-14-(4- 3.7 1.4 hydroxyphenyl) tetradecane 426 1,10-bis(2-hydroxy-1-naphthyl)decane >25 >11 358 1,8-bis(3,5-dihydroxy-4- >25 methyiphenyl) octane- ,WiENDED SHEET
IPEA*AU
P'cr/AU9/ 0 R EC E IVA 'o0~129 66 MWt ICompound
LC
50 JLM /g/mL 386 1,10-bis(3,5-dihydroxy-4- >25 >9.7 methyiphenyl) decane 414 1,12-bis(3,5-dihydroxy-4- >25 methyiphenyl) dode cane 442 1,14-bis(3,5-dihydroxy-4- 11 4.9 methylplienyl) tetredecane (striatol) 414 1,14-bis(3,5-dihydroxyrphenyl) 40 17 tetradecane (bisnorstriatol) 330 1,8-bis(2,4-dihydroxypohenylloctane 74 24 358 1,10-bis(2,4-dihydroxyphenyl)decane 37 13 372 1',11-bis(2.4-dihydroxyphenyl)undecane >25 >9.3 386 1,12-bis(2,4-dihydroxyphenyl)dodecane 14 5.4 386 1,10-bis(2,4-dihydroxy-3- >25 >9.7 methyiphenyl) decane 194 4-hexyiresorcinol 62 12 236 4-nonyiresorcinol 10 2.4 278 4-dodecyiresorcinol 8.8 2.4 180 5-pentylresorcinol (olivetol) >100 >18 236 5-nonyiresorcinol >100 >18 250 2-methyl-5-nonylresorcino. 8.3 2.1 274 5-decyiresorcinol S4 292 5-tridecyiresorcinol (grevillol) 2.8 0.80 348 5-heptadecylrestrcinol >100 >18 308 ethyl 2,4-dihydroxy-6-nonylbenzoate 2.6 0.8 46 ethyl 3,5-dibromo-2,4-dihydroxy-6- 0.78 0.36 nonylbenze) ;e AMENDED SHEET
IPENIAU
PC/A'U 9 4 0 29 7 fl CEIVED 02 MAR 1995 67 MWt Compound
LC
50 PiM ptg/mL 322 ethyl 2,4-dihydroxy-6-decylbenzoate 2.3 0.75 480 ethyl 3,5-dibromo-2,4-dihydroxy-6- 0.78 0.37 decylbenzoate 324 6-dodecyl-7-hydroxy-4-rnethylcoumarin >25 >8.1 328 grifolin 2.3 0.75 328 neogrifolin 28 424 5,7,2',6'-tetrahydroxy-8- >100 >42 lavandulyl flavanone 414 podophyllotoxin 3.8 1.6 (2.4) solvent: dimethyl sulf oxide (DMSO) 11% v/v
LC
50 determined by Meyer et, al. Planta medica (1982), 31-34
DISCUSSION
In this bioassay, the estimated LC 5 0 value of the test compound indicates its toxicity to brine shrimp. A more useful comparison of potencies can be obtained by looking at the p.M instead of pzg/mL concentrations in Table 6.
Simple alkylphenols were very L.oxic with certain alkyl chain lengths. The most active the alkylphenols measured was 4-decylphenol (LC 5 Q 0.27 jiM) compared with the most active of all compounds measured 1- (2hydroxyphenol1) 12 (4 -hydroxyphenol1) dode cane (LC.
5 0 0.054 p4M) Ketophenols were found to be more toxic than simple alkylphenols e.g. 4-nonanoylphenol (LC 50 0.10 4iM). 4and 5-Alkylresorcinols showed low toxicity except for tridecylresorcinol (grevillol) (LC_9Q 2.8 AiM) which was moderately toxic. The u,c-bishydroxyphenylalkanes, for example 1,10 -bis (2 -hydroxyphenyl) decane (LC 5 Q 1. 9 am) AMENDED SHEET
IPEA/AU
68 were moderately toxic. l,10-Bis(2-hydroxy-4methylphenvl)decane (LC 50 14 M) and other substances with methyl groups on the phenyl groups showed reduced toxicity. The a,w-bisresorcinylalkanes, including, striatol (LC 50 11 bisnorstriatol (LC 50 40 iM) and 1,10-bis(2,4-dihydroxyphenyl)decane (LC50 37 iM) showed relatively low toxicity.
A significant difference in toxicity exists between grifolin and neogrifolin (LC50 2.3 AM and LC 5 o 28 iM respectively). Neogrifolin appears to be about ten-fold weaker than grifolin. Ca2+-ATPase inhibition by these two compounds, on the other hand, was relatively strong and identical (grifolin's ICso was 22.5 AM while neogrifolin's
IC
50 was 23.3 AM).
Podophyllotoxin was tested in order to check whether the bioassay's results were comparable with those of Meyer et al. The LC 50 from this study was 3.8 iM and is reasonably close to the LC 50 value of 5.8 iM determined by Meyer et al.
AMENDED SHEET
IPEA/AU
t II

Claims (18)

1. Use of a compound of formula (I) (Rem R -(OmH) n wherein Ar is a ring system comprising one or more optionally substituted phenyl rings optionally linked to and/or fused with one or more other optionally substituted phenyl rings or one or more 5 or 6-membered, optionally substituted heterocyclic rings wherein the heteroatom is oxygen; wherein Ar comprises 1-4 phenyl rings; wherein Ar can be linked to another via a group X or directly linked to another Ar, wherein the two Ar groups are independently selected; when Ar is linked to another Ar via a group X, the two Ar groups can also be directly linked to 15 each other; where X is optionally substituted C 1 20 alkylene, C 2 2 0 alkenylene or C2_ 2 0 alkynylene; when Ar is linked to another Ar via a group X, R is hydrogen; C1- 2 0 alky1, C 2 2 0 alkenyl, C2_ 20 alkynyl, C 2 20 20 alkanoyl, C2- 2 0 alkenoyl, C2- 2 0 alkynoyl, each of which can be optionally substituted; when Ar is not linked to another Ar via a group X, R is C5- 2 0 alkyl, C5- 2 0 alkenyl, C 5 _2alkynyl, C 5 2 0 alkanoyl, 20 alkenoyl, C 5 _2 0 alkynoyl, each of which can be optionally substituted; RI is independently selected and is hydrogen; optionally substituted C1- 1 2 alkyl, C2- 12 alkenyl, C 2 -12 alkynyl; -COOR' halogen, -COR', -CONR'R', =0, -SC 3 -SO 2 NR'R', -SOR', SO 2 -NO 2 -CM, glycoside, silyl; where R' is independently hydrogen; alkyl, alkenyl or alkynyl each optionally substituted; and where two groups R 1 can be joined; wherein the optional substituents are one or more RA,- f ^\33 5 independently selected from C1- 10 alkyl, C2_l 0 alkenyl, C 2 10 S n0 alkynyl; -COOR", halogen, -COR", -CONRR"",- a I I 70 SR", =S03R", -SO 2 NR"R", -SOR", -S02R", -NO 2 -CN; wherein R" is independently hydrogen, alkyl, alkenyl, or alkynyl; n 1, 2 or 3 m 1, 2, 3 or 4 or a pharmaceutically acceptable derivative thereof to inhibit the action of plasma membrane Ca2+-ATPase enzyme in a subject in need of such inhibition.
2. Use of a compound of formula as defined in claim 1 or a pharmaceutically acceptable derivative thereof in the treatment or prophylaxis of cardiovascular disease related to the action of plasma membrane Ca2+-ATPase enzyme in a subject in need of such treatment or propIwlaxis.
3. Use according to claim 1 or 2 wherein the compound of formula is a compound of formula (II).
4. Use according to claim 1 or 2 wherein the compound of formula is a compound of formula (III) or (IV)
5. Use according to claim 1 or 2 wherein the compound of formula is a compound of formula or (VI).
6. Use according to claim 1 or 2 wherein the compound of formula is 5,7,2',6'-tetrahydroxy-8-lavandulyl- S: flavanone, 5,7,2' -trihydroxy-8-lavandulylflavanone or trihydroxy-8-lavandulyl-7-methoxyflavanone or a pharmaceutically acceptable derivative thereof. 25 7. Compounds of formula (II) or pharmaceutically acceptable derivatives thereof: *CH)r II) where R 2 is 2-hydroxy 2-hydroxy and 4'-hydroxy 2-hydroxy-3-methyl 4-hydroxy-3-methyl 2,4-dihydroxy Spec: P04828ZT 3, -i71ro- 4-ety 3, 5-dihydroxy-4-methyl 2, 6-dihydroxy-4-methyl 2, -hydroxy--methyl when R2 LS 3bve -h=dro4y 4-eh; 2i wh~~boe r=8is 1)6bvr;-4 hnR 2 i 2- abovie rth-at proide th R s2hdoy hnri o 1) wn 13i; -ydoyte ri o whe10 n 13; -iyroy4mtlte whn 14i; ,-ihdoy4mehlte r3 isn 14; -ydoy3mthlte when 10i; -ydoy3mthlte ()weR2i2,4dhcrxthnris not whe0nd 13 isand hyfoytenri o w-10en 13 andhdrx--mt te wno 10i. -ydoy3mthlte nopudso otl 10. r hraeuial ac.eCompoudsofvformul (th )orphracutcal RC(CH 2 (R2 25 where R 2 is A -dihydroxy 3, 3-dihydroxy-4-methyl 2,6-dihydroxy-4-methyl, or 2, 4-dlihydroxy-3-methyl and where r 8-16; provided that when R 2 is 3, 5-dihydroxy-4-methyl then r is not 14; and when R 2 is 2, 4-dihydroxy then r is not 8- and 13. Spec: P04828ZT 72
9. Compounds of formulae (III), (VI) or pharmaceutically acceptable derivatives thereof: (III) where s 8-16 CH 3 a (C 2 )t-CH 3 0 0 OH (IV) where t =6-15 a a a a a CH=CH(0H 2 )q where p q 12 (VI) 0 H 2 )q CH=CH where p q 12 A method of preparing compounds of formula (IIA) as defined in claim 8 which comprises where R 2 is 2, 4-dihydroxy Spec: P04828ZT 73 Wi treating 'the corresponding ditacid with zinc chloride and resorcinol to give the diketo compound as follows: HOOC- (CH 2 r- 2 -COOH HO-- Co-(CH2)r-2O0 Q OH iU (ii) followed by reduction of the acyl groups to provide compounds of formula (IIA); where R 2 is 2, 6-dihydroxy-4-methyl treatment of a compound 6f formula CH 2 OCH 3 COOH CH00 OH 2 00 H 3 with LIDA to give the dianion followed by treatment with the desired protected alkane aldehyde of formula to give OCH 2 00H 3 OH 3 C;H 2 CHCH(0H 2 )rCH 2 00H 2 Ph OCH 2 00H 3 Spec: 204828ZT 74 (iii) dehydrative decarboxylation followed by reduction to give an intermediate product of formula PCH 2 OCH 3 -1CH 2 0H (iv) oxidation give OOH 2 0CH 3 OH 3 *rCH2)rCHO OCH 2 00H 3 *0 followed by treatment with the dianion f rom step to give OH 3 -OH 3 (C) CH 3 0CH 2 0 (vi) dehydrative decarboxylation followed by deprotection and reduction to give the desired product; where R 2 is 3,5-ciihydroxy-4-methyl treatment of cx-N,N-dimethylamino-ca-cyano- 5-direthoxy-4-methyl)benzylidene in tetrahydrofuran. and hexamethyiphosphoramide (HMlPA) with lithium diisopropylamide (LDA) to give the anion followed by Spec: P04828ZT 75 (ii) treatment with a,c-dibromoalkanes to give CH 3 0 OCH 3 CHzO CN NC OCH3 H 3 C C- (CH 2 C CH 3 CH 3 0 OCH 3 (iii) refluxing with 30% aqueous oxalic acid to .give the corresponding diacyl compound (iv) reduction of the acyl groups followed by demethylation with hydrogen bromide in acetic acid to provide compounds of formula (IIA); where R 2 is 2,4-dihydroxy-3-methl carrying out steps and (ii) in (a) except in resorcinol is replaced with 2-methylresorcinol. o 11. A method of preparing compounds of formula (III) as defined in claim 9 which comprises treating the corresponding diacid with a suitable 20 agent to provide the acid dichloride (ii) treating the corresponding acid dichloride with 2-naphthol followed by (iii)rearrangement of the diacyl groups and (iv) followed by reduction of the acyl groups to 25 provide compounds of formula (III).
12. A method of preparing compounds of formula (IV) as defined in claim 9 which comprises treatment of 4- alkylresorcinols with ethyl acetoacetate in the presence of an acid catalyst.
13. A method for inhibiting the action of plasma membrane Ca2-A'TPase enzymes in a subject in need of such inhibition which comprises administering to the subject an effective amount of a compound of formula as defined in claim 1 or a pharmaceutically acceptable derivative thereof.
14. A method of treatment or prophylaxis of cardiovascular disease related to the action of plasma membrane Ca 2 -ATPase Spec: P04828ZT 76 sass o sass a as as as a os a s us as sass a u as ass o o as as ~a -e 3 'Lg -7 C /tr enzyme in a subject in need of such treatment or prophylaxis which comprises administering to the subject an effective amount of a compound of formula as defined in claim 1 or a pharmaceutically acceptable derivative thereof. A method according to claim 13 or 14 wherein the compound of formula is a compound of formula (II) as defined in claim 7, or a compound of formula (III) or (IV) as derined in claim 9.
16. A method according to claim 13 or 14 wherein the compound of formula is a compound of formula as defined in claim 9.
17. A method according to claim 13 or 14.Wherein the compound of formula is a compound of formula (VI) as defined in claim 9.
18. A pharmaceutical formulation comprising a compound of formula (II) as defined in claim 7, or a compound of formula (III), or (VI) as defined in claim 9, or a pharmaceutically acceptable derivative thereof in a 20 pharmaceutically acceptable carrier.
19. A method according to claim 13 or 14 wherein the compound of formula is 5,7,2',6'-tetrahydroxy-8- lavandulylflavanone, 5,7,2'-trihydroxy-8- lavandulylflavanone or trihydroxy-8-lavandulyl-7- 25 methoxyflavanone or a pharmaceutically acceptable derivative thereof.
20. A compound of formula (IIA) as defined in claim 8, or a compound of formula (III), or (VI) as defined in claim 9, or a pharmaceutically acceptable derivative thereof, substantially as herein described.
21. A method of preparing compounds of formula (IIA) as defined in claim 8, or compounds of formula (III), (IV), or (VI) as defined in claim 9 substantially as herein described. 35 22. A method for inhibiting the action of plasma-membrane Ca 2 -ATPase enzymes in a subject in need of such inhibition .U Spec: P04828ZT I- 77 which comprises administering to the subject an effective amount of a compound of formula as defined in claim 1 or a pharmaceutically acceptable derivative thereof substantially as herein described.
23. A method of treatment or prophylaxis of cardiovascular disease related to the action of plasma-membrane Ca2+-ATPase enzyme in a subject in need of such treatment or prophylaxis which comprises administering to the subject an effective amount of a compound of formula as defined in claim 1 or a pharmaceutically acceptable derivative thereof substantially as herein described.
24. A pharmaceutical formulation comprising a compound of formula (II) as defined in claim 7, or a cqhpound of formula (III), or (VI) as defined in claim 9, or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier substantially as herein described. 20 Dated this 28th day of May 1998 THE UNIVERSITY OF SYDNEY By their Patent Attorneys *GRIFFITH HACK roo Spec. P04828ZT
AU68390/94A 1993-06-03 1994-06-03 Use of natural products and related synthetic compounds for the treatment of cardiovascular disease Ceased AU694428B2 (en)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
1001: CHEM ABS VOL 116 (1992) ABSTRCT NO 148483U *
PHYTOCHEMISTRY VOL 31 NO 3 (1992) RUANGRUAGSI N ET AL P999- *
PHYTOCHEMISTRY VOL 33 NO 1 (1993) INUMA M ET AL P203-8 *

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