WO1998007716A2 - Heteroarotinoids-anticancer agents with receptor specificity and tgase activity - Google Patents

Heteroarotinoids-anticancer agents with receptor specificity and tgase activity Download PDF

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WO1998007716A2
WO1998007716A2 PCT/US1997/014720 US9714720W WO9807716A2 WO 1998007716 A2 WO1998007716 A2 WO 1998007716A2 US 9714720 W US9714720 W US 9714720W WO 9807716 A2 WO9807716 A2 WO 9807716A2
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WO1998007716A3 (en
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Kenneth Darrel Berlin
Shanker Subramanian
Eldon Carl Nelson
Matora May Madler
Manford Kenneth Patterson, Jr.
Paul Joseph Birckbichler
Doris Mangiaracina Benbrook
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The Board Of Regents For Oklahoma State University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/94—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of polycyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/20—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 hydrogenated in the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/76—Benzo[c]pyrans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/60—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D319/18—Ethylenedioxybenzenes, not substituted on the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/02—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D335/06—Benzothiopyrans; Hydrogenated benzothiopyrans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • This invention relates to anticancer compositions in terms of receptor binding and activation as well as activity in stimulating formation of the enzyme transglutaminase as a marker for anticancer activity. Specifically, the invention relates to certain heteroarotinoids and derivatives thereof.
  • Retinoids (vitamin A and derivatives thereof) is a name associated with a family of compounds both of natural and synthetic origin. There is significant interest in such molecules because of the observed strong anticancer activity in a number of assays including the hamster tracheal organ culture (TOC) assay, the ornithine decarboxylase (ODC) assay, and with HL-60 cells (human leukemic cell line).
  • Heteroarotinoids are a group of derivatives which contain an aromatic ring and at least one heteroatom in a fused, partially saturated ring bonded to an aromatic ring. Examples of heteroarotinoids and the activity of such in the assays cited above are found in several papers such as in the Journal of Medicinal Chemistry, 1985, Vol.
  • TGase transglutaminase
  • RAR- ⁇ Certain human retinoic acid receptors have been isolated and are labelled RAR- ⁇ , RAR- ⁇ , and RAR- ⁇ , There have also been identified rather non-specific retinoid receptors designated as RXR- ⁇ , RXR- ⁇ , and RXR- ⁇ . These receptors are proteins which, when bound to an active agent, elicit a specific biological response. The exact tissue distribution of RARs and RXRs is still under investigation [See "Regulation of Gene Expression by Vitamin A. The Role of Nuclear Retinoic Acid Receptors", published in Annual Reviews of Nutrition, 1992, Vol. 12, pages 443-471 , by D. L. Hill and C. J. Grubbs].
  • the present invention involves certain unusual heteroarotinoids and the identification of receptor specificity of action as well as the induction of TGase activity by the retinoids.
  • the receptor specificity is very important for the inducement of specific biological reponses such as in development of normal versus cancerous conditions in specific tissues.
  • Table I contains representative structures for certain heteroarotinoids in the TGase assay along with data for tr ⁇ ns-retinoic acid (1) as the standard for each individual test compound. The greater the R value for the heteroarotinoid the greater the degree of activity
  • G symbolizes a propenyl type double bond, such as in systems 3, 4, 7, 9, 10, 12, 14, 16, 18, 19, 22, 24, 25, 27, 28, 30, 33, 34, 36, 42, 43, 45, 93, and 94, an amide type bond, such as in 2, 6, 21, 23, 26, 29, 31, 32, 35, 38, 39, 41, 123, and 124, an acryloyl type bond as in 5 (see Table I), and an oxycarbonyl or carbonyloxy group as in 96-102, 104, 107, 114, 122, 126, 134, and 138.
  • the X is O, S, NCH 3 , NC 2 H 5 , NCH2CH2CH3, or NCH(CH 3 ) 2 .
  • Compound 1 is ./' ⁇ /15-retinoic acid (7-RA) and is the standard used in the experiments.
  • HETEROAROTINOIDS DECREASING BINDING POTENCY WITH SPECIFIC HUMAN RETINOIC ACID RECEPTORS 3
  • the systems possess a fused five-six or a fused six-six ring unit with a heteroatom in the partially saturated ring.
  • novel heteroarotinoid compositions are heteronuclear rings, organic compounds shown below with the letters A, G, L, M, Q, R, T, X, and Y representing the groups illustrated. Although these compounds are structurally related to .ra/..s--retinoic acid (1), they are also unique in that they possess a heteroatom as well as a least one aromatic ring and in some cases two aromatic rings as illustrated.
  • the connecting group G in fused six-six-membered ring systems 2, 6, 26, 29, 32, 35, 39, 123, and 124 is somewhat flexible amide bridge as is true for 21, 23, 38, and 41 in the fused five-six-membered ring systems. Systems 3, 4, 7, 9, 10, 11, 12, 13, 14-20, 22, 24, 25, 27, 28, 30, 33, 34, 36, 37, 40, 42 ⁇ 5, 93, and 94 have a propenyl or polyene link or side chain for G.
  • Retinoic acid receptors are proteins which are known to be expressed in vivo as RAR ⁇ (ubiquitous), RAR ⁇ (kidney, heart, and skeletal muscle), and RAR ⁇ (skin and bones), respectively.
  • RAR ⁇ ubiquitous
  • RAR ⁇ kidney, heart, and skeletal muscle
  • RAR ⁇ skin and bones
  • Table II data are given in terms of potency of the minimum concentration of the heteroarotinoid required to bind to the specific receptor as compared to the concentration required of the standard 9- -retinoic acid.
  • the concentration required for a heteroarotinoid to bind to a specific receptor type the more active the heteroarotinoid for binding purposes and for inducing a specific biological response such as the differentiation of normal cell development in the production of normal skin cells, liver cells, etc.
  • Table III Data in Table III were obtained from an assay which involved the treatment of a CC-B cervical tumor cell line, which contained stabilized, integrated copies of RAR ⁇ - RARE-tk-CAT reporter plasmid, with the test heteroarotinoid with a separate experiment with /ra/.s-retinoic acid (1) as a control.
  • the values in Table III refer to percent of control in terms of ability to activate endogenous nuclear receptors.
  • Table III also contains data on ability of the heteroarotinoids to inhibit growth of cervical carcinoma cells (CC- 1 or CC-B) as percentage relative to that of the control, and the data are influenced by the same parameters as cited above for the receptor activation.
  • Table I demonstrates that the compounds induce transglutaminase enzyme in leukemia tumor cells.
  • Transglutaminase is a marker of differentiation and apoptosis, two activities that induce anticancer effects.
  • the heteroarotinoids reverse the loss of normal differentiation which is characteristic of tumor cells and thus restorethe cells to a more natural state and reduce their ability to spread within the body.
  • Human cells have a natural mechanism whereby the detect and eliminate abnormal cells via a self-induced suicide event called apoptosis.
  • Heteroarotinoids reduce tumor growth (Table III) via stimulation of apoptosis.
  • heteroarotinoids regulate biological activities to a lesser extent but are more selective than rran.s'-retinoic acid (1) and are therefore more likely to exhibit improved therapeutic ratios compared to 1.
  • Table II there is shown a strong selectivity of the heteroarotinoids for individual nuclear receptors. Therefore, by exhibiting a preference for one or two of the three retinoic acid receptors, heteroarototinoids will effect a different profile of biological activities that will in turn influence the chemotherapeutic ratio.
  • Ketone 48 is the key precursor of 19, 20, 34, and 37 via Scheme III. Intermediates 60-63 are also unknown , but these compounds were fully characterized.
  • the thiosemicarbazone derivative 97 is also the first example of such a derivative in the family of heteroarotinoids.
  • Certain thiosemicabazones have displayed useful anticancer activity as found in an article in the Journal of Medicinal Chemistry, 1995, vol. 38, pages 4234-4243, entitled "Synthesis and Antitumor Activity of 4- and 5-Substituted Derivatives of Isoquinoline- 1 -carboxyaldehyde Thiosemicarbazone" by M. C. Lium, T. P. Lim, P. Penketh, and A. C. Sartorelli.
  • Esters 98-102 in Scheme XII possess a small variations in terminal functional groups with the basic heterocyclic skeletal unit intact.
  • Example 102 allowed appraisal of the effect of a fused five-six-membered ring system on the activity while the other examples of 98-101 possessed the six-six-membered groups in the basic structure.
  • Examples 99 and 100 allow an assessment of two different heterocyclic rings on activity as well as hydrophilicity which can be very beneficial in agent formulation.
  • anticancer activity of the agents will be regulated.
  • Scheme XVIII outlines the synthesis of a novel heteroarotinoid in which the heteroatom (oxygen in this case, but the sulfur atom could also be utilized) is repositioned at a new location in the cyclohexyl ring unit.
  • the method involves 127 ⁇ 128-.129 ⁇ 130 ⁇ 131 ⁇ 132 ⁇ 133 ⁇ 134, the latter being the target molecule.
  • Methylation alpha to the nitrile group in 127 gave 128 which, when subjecte to basic hydrolysis, gave 129. Reduction of the carboxyl group in 129 produced alcohol 130.
  • the defense for this type of heteroarotinoid is that the new position of the heteroatom in the cyclohexyl ring and the flexible linker group will influence receptor binding and anticancer activity.
  • Target system 138 in Scheme XIX was designed as a model system for 134 since the former does not possess a heteroatom in the cyclohexyl ring unit.
  • Ether 135 was acetylated under standard conditions to yield ketone 136.
  • Haloform type conditions converted 136 to 137 although the reaction had to be done with care.
  • Esterification of 137 paralleled the method used to obtain 134 illustrated previously.
  • 138 allows a comparison of the influence on anticancer activity by the absence of the heteroatom.
  • Ester 26 (0.15 g, 0.39 mmol) in 95% ethanol (7 mL) was treated with 2 N NaO ⁇ (10 eq, 3.9 mmol). After stirring the solution for 4 h, the mixture was acidified with 2 N ⁇ C1 (30 mL) which produced a white foam that was filtered, washed ( ⁇ 2O), dried, and recrystallized (EtOAc:hexane; 2: 1). The yield of 2 was 0.1 g (68%) with a mp of 208-209.5
  • Ester 26 was prepared from amine 56 which was synthesized as follows. Nitro- substituted compound 79 (0.69 g, 2.7 mmol) was dissolved in acetic acid (25 mL) and water (5 mL) with vigorous stirring. Then TiCl3/HCl (28.06 g, 18 mmol) was added dropwise, and the resultant mixture was stirred at RT (2 h). After being cooled to 0 °C, the mixture was treated slowly with 30% NaOH (1 10 mL). Extracts (ethyl acetate followed by H 2 CCI2) of the aqueous phase were combined with the organic layer and washed with water and saturated NaHCO3.
  • Nitro-substituted compound 79 was obtained from the reaction involving dropwise addition of a solution of cold cone HNO 3 (3 mL) with acetic anhydride (9 mL) into a solution of ether 78 (4.35 g, 21.0 mmol) dissolved in acetic anhydride (8 mL) at 0 °C. After stirring 1 h, the solution was poured into a saturated solution (-100 mL) of NaHCO3, and the resulting mixture was extracted (H2CCI2). The organic layer was washed with water and brine and then dried (Na2SO 4 ).
  • ester 12 (0.22 g, 0.57 mmol), ethanol (10 mL), water (10 mL), and NaO ⁇ (0.04 g, 1.9 mmol) was boiled for 6 h and then cooled slowly to RT.
  • the new solution was chilled to 0 °C and was then treated with cone ⁇ C1 ( ⁇ 8 mL, p ⁇ ⁇ 2) to yield a solid.
  • the solid was washed well with water, air dried, and then dried under vacuum to give acid (E)-9 as needles (0.17 g, 0.49 mmol, 63%) with a mp of 171-172 °C.
  • ester 14 (0.32 g, 0.80 mmol), 95% ethanol (20 mL), water (5 mL), and
  • Salt 84 was prepared as follows. A solution of 4-thia- ⁇ -ionone (54, 8.0 g, 0.38
  • ester 37 (0.150 g, 0.48 mmol), KOH (1 mL, 35% aqueous), and ethanol
  • ester 23 (0.56 g, 1.61 mmol) in absolute ethanol (20 mL) was added dropwise 2 N ⁇ aOH (10 eq., 16.1 mmol), and the solution was boiled for 4 h. After acidification of this solution with HCl (2 N, 0 °C, -50 mL), the resulting mixture was stored in a freezer for 12 h. A white solid formed and was filtered off, washed with water, dried, and recrystallized (hexane:EtOAc, 2: 1). The crystals of 21 were dried under vacuum for 12 h and had a mp of 219-220 °C.
  • Acid 59 was prepared as follows: Ketone 47 (1.0 g, 5.26 mmol), dissolved in ethanol (17 mL), was treated with commercial clorox (50 mL), and the resultant solution 5 was boiled with stirring for 5 h and then was stirred at RT (5 h). The cooled (0 °C) reaction mixture was cautiously treated with a 25% solution of sodium metabisulfite (-50 mL) followed by cone HCl (-10 mL). A white solid formed in the mixture, after which another 70 mL of sodium metasulfite solution was added. The solid was filtered, dried, and recrystallized (ethanol) to yield 59 (0.71 g, 3.69 mmol) with a mp of 175-175 ⁇ C. IR (KBr)
  • Phosphonium salt 71 was prepared as follows. A mixture of thiochroman-4-one (3 g, 18 mmol), toluene (75 mL), water (120 mL), cone HCl (60 mL), and the Clemmenson- Martin amalgam (50 g) [E. L. Martin, Journal of the American Chemical Society, volume 58, pages, 1438-1432 (1936)] was boiled for 72 h with additional cone HCl (-20 mL) being added every 6 h to maintain a total volume of about 500 mL. After the mixture had cooled to RT, it was filtered, and the aqueous layer was extracted (toluene). The combined organic phases were washed with saturated NaHCO3, water, and brine.
  • ester 33 (0.25 g, 0.54 mmol) in ethanol (10 mL), water (10 mL), and
  • Ester 35 (0.4 g, 1.0 mmol), 95% ethanol (5 mL), and 2 N ⁇ aO ⁇ (10 eq) were boiled for 1 h and then stirred at RT for 48 h. Acidification of the solution with 2 N ⁇ C1 (-50 mL) produced a white solid which was filtered, washed with water, dried, and recrystallized (95% ethanol) to give a flaky solid 32 (0.26 g, 65%) with a mp of 331-331.8 °C.
  • IR (KBr) 3380, 3350, 1700 c ⁇ r 1 ; ! ⁇ ⁇ MR (DMSO-t/ 6 ) ⁇ 1.37 [s, 6 H], 1.46 [s, 6 H], 2.31 [s, 2 HJ,
  • Salt 61 was obtained as follows. Ketone 48 (2.0 g, 1 1.2 mmol) in dry THF (20 mL) was added dropwsie to a suspension of LiAlH 4 (1.266 g, 33.3 mmol) in dry THF ( 10 mL) over 10 min. The resultant mixture was boiled for 24 h with aliquots of dry ether ( 10 mL each) being added to maintain volume after 8 and 16 h. The mixture was allowed to cool to RT, and then it was chilled in ice. Extremely cautious addition of water was initiated to destroy excess LiAlH 4 over 0.5 h. A white precipitate formed, and the mixture, while still cool, was treated slowly with 5% HCl (80 mL) with stirring over 80 min.
  • Acid 76 was prepared as follows. To a solution of ketone 52 (2.0 g. 8.0 mmol) in ethanol (20 mL) was added commercial clorox (150 L), and the turbid mixure was stirred
  • Salt 63 was obtained as follows. Ketone 48 (4.0 g, 22.4 mmol) in dry THF (25 mL) was added dropwise over 0.25 h to a vinylmagnesium bromide [obtained from reaction of vinyl bromide (20 g, 187.9 mmol) in dry THF (35 mL) by standard procedures] using a dry- ice condenser. After being held at reflux for 2 h, the reaction mixture was stirred at RT (10 h). The mixture was then cooled in a water bath, and a saturated NH 4 CI (20 mL) was added dropwise. Two layers formed and the aqueous layer was extracted (ether), and the combined extracts were washed with brine and then dried (Na 2 SO 4 ). Evaporation left an oil 62 (-4.78 g, qt) which was used directly to prepare salt 63. Alcohol 62 (1.01 g, 4.92 mmol)
  • Acid 77 was prepared as follows. To a solution of ketone 53 ( 1.0 g, 6.1 mmol) in ethanol (35 L) was added a commercial clorox ( 140 mL), and the turbid mixture was boiled (24 h). After cooling (0 °C) the clear solution, a 25% solution of sodium metabisulfite (-30 mL) was added very slowly followed by cone HCl (-50 mL). Another 70 mL of the sodium metabisulfite solution was added to complete the precipitation of a solid. The white solid was filtered and recrystallized (95% ethanol) to give colorless needles of acid 77 (0.91 g, 81 %) with a mp of 285.5-286.4 °C. IR (KBr) 3500, 1710 cm 1 ;
  • Amine 69 was obtained as follows. To a solution of benzodioxan (67, 4.35 g, 31.85 mmol) in acetic anhydride (8 mL) was added dropwise a solution of HNO3 (3 mL) and acetic anhydride (9 mL) at 0 °C over 10 min. A thick yellow suspension formed and was stirred at RT (4 h). This mixture was poured slowly and cautiously into saturated NaHCO 3 (-250 mL), and the resultant mixture was extracted (H 2 CCI 2 ). The organic layer was separated, washed with water and brine, and then dried (MgSO4). Evaporation of the
  • Extracts (EtOAc and HCCI 3 ) of the aqueous layer were combined, washed with water and saturated NaHCO3, dried (MgSO 4 ), and evaporated to give an oil which was chromatographed over silica gel (H2CCl 2 :ethyl acetate, 3: 1 ) and gave oil 69 (2.08 g, 62%) that was used directly to prepare 39.
  • Properties of 69 were as follows: ⁇ NMR (DCC1 3 ) ⁇ 3.36 [bs, 2 H
  • Salt 65 was prepared as follows. Ketone 49 (2.00 g, 12.2 mmol) in dry THF (35 mL) was added dropwise to a cooled (0 °C) solution of freshly prepared vinylmagnesium bromide [obtained from vinyl bromide (4.9 g, 46 mmol) and Mg (0.90 g, 37 mmol) in THF (25 mL)] over 0.5 h. The cold bath was removed, and the resultant mixture was stirred at RT (1 h). The mixture was cooled again and then quenched by slow addition of water and then ether (-100 mL) was added to form two layers. Extracts (ether and H2CCI 2 ) of the aqueous layer were combined, dried (Na 2 SO 4 ), and evaporated to a yellow oil 64 (2.32 g,
  • Ketone 90 was prepared as follows. 2-Octylthiochroman (89, 1.5 g, 6 mmol) in CS2:H 3 CNO2 (1 :5, 25 mL) was added to a stirred suspension of acetic anhydride (0.5 mL, 6 mmol), AICI 3 (11.4 g, 9 mmol) in nitromethane (50 mL) at 0 °C. After 1 h, the suspension was allowed to warm to RT and then was stirred for 48 h. The resultant soution was cooled (0 °C) and then quenched with water (50 mL). Extracts (HCCI 3 ) of the aqueous layer were combined and washed with NaHCO3, water, and brine. The dried (Na 2 SO 4 ) solution was evaporated to an oil (quantitative yield) which was used directly to make alcohol 91.
  • ketone 90 Properties of ketone 90 were: IR (neat) 1680 cm 1 ; J H NMR (DCCI3) ⁇ 0.9 [t, 3 H], 1.35-
  • TFAA trifluoroacetic anhydride
  • ketone 90 Properties of 89 were: ⁇ NMR (DCCI3) ⁇ 0.88 [t, 3 HJ, 1.2-1.5 [bs, 12 H], 1.6-
  • Sulfide 88 was prepared as follows. 3,4-Dihydro-2-n-octyl-2H-l-benzothiopyran-l- oxide (87, 5.72 g, 30 mmol) in dry T ⁇ F (50 mL) was added to a solution of lithium diisopropylamide (LDA) in dry T ⁇ F (50 mL) at -78 °C over 15 min.
  • LDA lithium diisopropylamide
  • Intermediate 87 was prepared according to a literature procedure [T. Takata, Y Mayuni, K. Fujimori, H. K. Young, T. Iyangi, and S. Oae, Bull. Chem. Soc. Jpn., volume 15 56, pages 2300-2310 ( 1983)].
  • the preparation of intermediate 86 (3,4-dihydro-2H-l - benzothiopyran) was described under Example XVI and was a modification of a reported procedure [T. Takata, Y Mayuni, K. Fujimori, ⁇ . K. Young, T. Iyangi, and S. Oae, Bull. Chem. Soc. Jpn., volume 56, pages 2300-2310 (1983)J.
  • lactol 105 (0.100 g, 0.42 mmol)
  • a catalytic amount of p-toluenesulfonic acid 0.025 g
  • 4 A molecular seives 1.0 g
  • toluene 5 mL
  • the mixture was boiled for 2 h (showed completion by TLC).
  • the mixture was allowed to cool to RT and filtered.
  • the filtrate was washed with saturated Na ⁇ C ⁇ 3 (10 mL) and brine (10 mL) and then dried (Na2SO 4) .
  • the solvent was evaporated in vacuo to give 106 as a light
  • the tetrahydroquinoline 111 ( 2.5 g 0.01 mol), ⁇ aHCO 3 ( 1.51 g 0.018 mol), and H2O (2 mL) were placed in a standard system. The system was then cooled to 15-18 °C, and the dimethyl sulfate ( 1.64 g 0.013 mol) was added dropwise. The cloudy light yellow mixture was stirred at RT until the evolution of CO 2 ceased (1 h). The flask was then maintained at 50° C until additional CO2 ceased to be released ( 1 h). The flask was then allowed to cool to RT. Chloroform (25 mL) was added, and the layers were separated.
  • the carboxylic acid 113 (0.174 g, 0.794 mmol) and ethyl 4-hydroxybenzoate (0.198 g, 1.08 mmol) were mixed with CH2CI2 (6 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-0.025 g, 7.9 mmol) and a catalytic amount of DMAP (7 mg).
  • Acid 129 was prepared from 128 by the literature procedure [K. Nakatani, S. Numata, T. Inoue, K. K. Fujisawa, T. I. Kawasaki, T. Toyama, H. Tachibana, T. Udagawa, and M.Gohbara, Chemical Abstracts, volume 97, page 5964e ( 1982)], mp 79-80 °C.
  • Bioguard-LiOCl (5 g; LiOCl-29%, inert ingredients-71%), Clorox (20 mL] and 95% ethanol ( 10 mL). The resulting mixture was boiled (24 h), and it was then allowed to cool to RT. Ether extracts were washed with water and brine and then were dried (MgSO 4 ). Separate extracts (ether) of the acidified (2 N HCl, pH ⁇ 3) aqueous layer were washed with water and brine and then were dried (MgSO4). Evaporation of the solvent gave a solid which was recrystallized (EtOAc) to white 133 (0.420 g, 21 %), mp 147-149 °C. IR (KBr) 3443-2538, 1691 cm" 1 ; »H ⁇ MR (DCC1 3 ) ⁇ 1.30 [s, 6 HJ, 1.54 [s, 6 H], 3.61 Is, 2 HJ, 4.07
  • Ether 135 was prepared from a literature procedure [M. F. Boehm, L. Zhang, B. A. Badea, S. K. White, D. E. Mais, E. Berger, M. C. Suto, M. E. Goldman, and R. A. Heyman, Journal of Medicinal Chemistiy, volume 37, pages 2930-2941 (1994)].
  • ketone 136 (0.900 g, 3.46 mmol), a solution of Bioguard-LiOCl/Clorox (40 g of LiOCl and 80 mL of Clorox), and 95% ethanol (45 mL). After boiling the above mixture (24 h), it was allowed to cool to RT and was then treated with an aqueous solution of 25% Na2S2 ⁇ s (35 mL). Extracts (ether) of the acidified (6 M HCl, pH ⁇ 3) aqueous solution were combined, washed with water and brine and were finally dried (MgSO 4 ). Evaporation of the solvent gave a light yellow solid 137 (0.420 g, 44%),
  • t-RA t-RA
  • vehicle alone control
  • heteroarotinoid The r-RA (Sigma, St. Louis, MO) was added to one flask in all experiments to ensure that transglutaminase induction occurred, and it was also used as the standard to which the heteroarotinoid induction was compared.
  • Cultures were covered with aluminum foil to protect them from light and then incubated at 37 °C for 48 h. Cells ( 10-40 x 10 6 ) were collected by centrifugation at 2000 rpm for 5 min. The resulting cell pellet was resuspended in 10 mL of Ca,Mg-free Earles solution, and the cell suspension was centrifuged at 2000 rpm for 3 min.
  • Retinoic Acid is a High Affinity Ligand for RXR'" Cell, 1992, 68, 397-406. Data are in
  • DMSO in control and treated cultures was 0.1%.
  • cell extracts were prepared and CAT activity was assayed as previously described [Sleigh, M. J. "A Nonchromatographic Assay for Expression of the Chloramphenicol Acetyltransferase Gene in Eukaryotic Cells". Anal. Biochem. 1986, 156, 251-256] with the exception that 3 H acetyl-CoA was used instead of 14 C acetyl-CoA.
  • Protein concentrations of the extracts were determined using the Bio-Rad Protein Assay (Bio-Rad, Richmond, VA). Transactivation activities were derived by dividing CAT activity per milligram of protein in the drug-treated culture by the activity in the control culture. The results, presented as percentages in Table II and III, are the average of three experiments.
  • cervical carcinoma cells (CC-1 or CC-B) were treated with the heteroarotinoids in Table III for seven days.
  • Control cultures were treated with the same volume of solvent as used with the treated cultures. The effect of the agents on growth was as cited above

Abstract

Anticancer compositions having receptor specificity as well as activity in stimulating formation of the enzyme transglutaminase as a marker for anticancer activity. The compositions comprise certain heteroarotinoid structures partially related to trans-retinoic acid through the basic, fused-ring framework.

Description

HETEROAROTINOIDS-ANTICANCER AGENTS WITH RECEPTOR SPECIFICITY AND TGASE ACTIVITY
BACKGROUND OF INVENTION
1. Field of the Invention:
This invention relates to anticancer compositions in terms of receptor binding and activation as well as activity in stimulating formation of the enzyme transglutaminase as a marker for anticancer activity. Specifically, the invention relates to certain heteroarotinoids and derivatives thereof.
2. Description of Prior Art:
Retinoids (vitamin A and derivatives thereof) is a name associated with a family of compounds both of natural and synthetic origin. There is significant interest in such molecules because of the observed strong anticancer activity in a number of assays including the hamster tracheal organ culture (TOC) assay, the ornithine decarboxylase (ODC) assay, and with HL-60 cells (human leukemic cell line). Heteroarotinoids are a group of derivatives which contain an aromatic ring and at least one heteroatom in a fused, partially saturated ring bonded to an aromatic ring. Examples of heteroarotinoids and the activity of such in the assays cited above are found in several papers such as in the Journal of Medicinal Chemistry, 1985, Vol. 28, pages 1 16- 124, entitled "Synthesis and Characterization of Selected Heteroarotinoids. Pharmacological Activity as Assessed in Vitamin A Deficient Hamster Tracheal Organ Cultures. Single Crystal X-ray Diffraction Analysis of 4,4-Dimethylthiochrom-6-yl Methyl Ketone- 1,1 -Dioxide and Ethyl (E)-p-[2- (4,4-Dimethylthiochroma-6-yl)propenyl]benzoate", by K. M. Waugh, K. D. Berlin, W. T. Ford, E. M. Holt, J. P. Carrol, P. R. Schomber, and L. J. Schiff. Another pertinent paper is found in the Journal of Medicinal Chemistry, 1987, Vol. 30, pages 1 174- 1480, entitled "Heteroarotinoids. Synthesis, Characterization, and Biological Activity in Terms of an Assessment of these Systems to Inhibit the Induction of Ornithine Decarboxylase Activity and to Induce Terminal Differentiation of HL-60 cells", by L. W. Spruce, S. N. Rajadhyaksha, K. D. Berlin, J. B Gale, E. T. Miranda, W. T. Ford, E. C. Blossey, A. K. Verma, M. B. Hossain, D. van der Helm, and T. R. Breitman. Another paper has appeared in the Journal of Medicinal Chemistry, 1991, Vol. 34, pages 430-439, entitled "Novel Heteroarotinoids: Synthesis and Biological Activity", by L. W. Spruce, J. B. Gale, K. D. Berlin, A. K. Verma, T. R. Breitman, X. Ji, and D. van der Helm. Another report entitled "Heteroarotinoids; Crystal and Molecular Structure Analysis of Methyl (Z)- and Methyl (E)-4-[2-(4,4-Dimethylthiochroman-6-yl)- l -propenyl]benzoate", in the journal entitled Structural Chemistry, 1991, Vol. 2, pages 515-522, by W. J. Welsh, V. Cody, K. Suwinska, K. D. Berlin, S. N. Radjadhyaksha, S. Subramanian, and A. K. Verma, contains data pertinent to the invention as well. Other background information on heteroarotinoids is found in U.S. Patents 4,883,254 (May 23, 1989) and 4,997,276 (December 1 1 , 1990). Summaries of the biochemistry, chemistry, and prior biological activity of retinoids, including the few heteroarotinoids known, are found in four treatises, namely "Chemistry and Biology of Synthetic Retiniods", by M. 1. Dawson and W. H. Okamura, editors, CRC Press: Boca Raton, Florida, 1990, "The Retinoids", Volumes 1 and II, by M. B. Sporn, A. B. Roberts, and D. S. Goodman, editors, Academic Press: Orlando, Florida, 1984, and "The Retinoids", 2nd Edition, by M. B. Sporn, A. B. Roberts, and D. S. Goodman, editors, Raven Press: New York, N.Y., 1994.
In a recent study it was shown that cell differentiation of HEL (human erythroleukemia) cells by trα 15-retinoic acid (/-RA) was accompanied by an increase in the tissue concentration of the enzyme transglutaminase (TGase). For details, see "Differential Expression of Transglutaminase in Human Erythroleukemia Cells in Response to Retinoic Acid" in Cancer Research, 1990, Vol. 50, pages 7830-7834, by T. Suedhoff, P. J. Birckbichler, K. N. Lee, E. Conway, and M. K. Patterson, Jr. Thus, TGase can be used to predict the response of human myeloid leukemia cells to retinoids. The experimental procedure is given in the paper. Some of the biological data given in this invention disclosure utilizes this assay to verify useful anticancer activity of the heteroarotinoids delineated.
Certain human retinoic acid receptors have been isolated and are labelled RAR-α, RAR-β, and RAR-γ, There have also been identified rather non-specific retinoid receptors designated as RXR-α, RXR-β, and RXR-γ. These receptors are proteins which, when bound to an active agent, elicit a specific biological response. The exact tissue distribution of RARs and RXRs is still under investigation [See "Regulation of Gene Expression by Vitamin A. The Role of Nuclear Retinoic Acid Receptors", published in Annual Reviews of Nutrition, 1992, Vol. 12, pages 443-471 , by D. L. Hill and C. J. Grubbs]. Research to date indicates that RAR-α is ubiquitous in adult humans while RAR-β is found in heart, lung, and spleen, with RAR-γ apparently confined to lung and skin. Consequently, retinoids which are specific for interactions with RAR-α, RAR-β, or RAR-γ receptors could influence normal development in the tissues cited. The techniques employed for this work have been published and are found in two sources. These sources are: ( 1 ) the Journal of Steroid and Biochemical Molecular Biology, 1992, Vol. 41 , pages 733-738, and entitled "Interaction of Glucocorticoid Analogues with the Human Glucocorticoid Receptors", by T. Berger, Z. Parandoosh, B. W. Perry, and R. Stein, and (2) in the journal Cell, 1992, Vol. 68, pages 397-406, and entitled "9-α'._-Retinoic Acid is a High Affinity Ligand for RXR", by R. Heyman, P. J. Mangelsdorf, J. A. Dyke, R. B. Stien, G. Eichele, R. M. Evans, and C. Thaller.
SUMMARY OF THE INVENTION
The present invention involves certain unusual heteroarotinoids and the identification of receptor specificity of action as well as the induction of TGase activity by the retinoids. The receptor specificity is very important for the inducement of specific biological reponses such as in development of normal versus cancerous conditions in specific tissues.
Table I contains representative structures for certain heteroarotinoids in the TGase assay along with data for trαns-retinoic acid (1) as the standard for each individual test compound. The greater the R value for the heteroarotinoid the greater the degree of activity
(and therefore greater potential anticancer activity) when compared with that of trans- retinoic acid ( 1). It is clear that there exists a range of activity in compounds 2-41 in this assay. Structures of the heteroarotinoids are partially related to that of /rα.w-retinoic acid (1) through the basic, fused-ring framework. In this invention, the general formulas A, B, D,
Figure imgf000006_0001
.røπs-retinoic acid (1) = H and CH3
,
Figure imgf000006_0002
and E represent structures bridged by G which symbolizes a propenyl type double bond, such as in systems 3, 4, 7, 9, 10, 12, 14, 16, 18, 19, 22, 24, 25, 27, 28, 30, 33, 34, 36, 42, 43, 45, 93, and 94, an amide type bond, such as in 2, 6, 21, 23, 26, 29, 31, 32, 35, 38, 39, 41, 123, and 124, an acryloyl type bond as in 5 (see Table I), and an oxycarbonyl or carbonyloxy group as in 96-102, 104, 107, 114, 122, 126, 134, and 138. The X is O, S, NCH3, NC2H5, NCH2CH2CH3, or NCH(CH3)2.
Systems represented by F and H possess a polyene side chain bridged with a bridge G, which symbolizes a heptatrienyl system and occurs in 8, 11, 13, 17, 20, 37, 40, and 44, and with a bridge G as an amide bond as part of the polyene chain as in 6 and 31. The heteroatoms involved are S, O, and NR, and the meaning of a few substituents are symbolized by the letters shown in the drawings above and most are displayed in Tables I and II. Table II contains information which demonstrates the ability of the heteroarotinoids described herein to interact with retinoic acid receptors (RAR) which are special nuclear proteins. The lower the concentration required for binding of the heteroarotinoid with the receptor the greater the specificity for binding with the receptor in question. Moreover, the greater the specificity in binding the greater the propensity for inducement for production of a specific protein and therefore a normal specific tissue.
TABLE I EFFECT OF SELECTED HETEROAROTINOIDS ON TGase ACTIVITY"
HETEROAROTINOIDS RATIO SP. ACTIVITY'1 Rc
Figure imgf000008_0001
l [RA| 3.8 1.0
Figure imgf000008_0002
TABLE I (CONTINUED)
HETEROAROTINOIDS RATIO SP. ACTIVITY' Rl l[RA| 5.1 1.0
Figure imgf000009_0001
IΓRAJ 1.0
Figure imgf000009_0002
KRAI 3.8 1.0
Figure imgf000009_0003
-βcr^ :.s 0.54 TABLE I (CONTINUED)
HETEROAROTINOIDS RATIO SP. ACTIVITY1 Rl
Figure imgf000010_0001
18 1.3 0.47
Figure imgf000010_0002
7 TABLE I (CONTINUED)
HETEROAROTINOIDS RATIO SP. ACTIVITY' Rc l[RAj
Figure imgf000011_0001
1[RA] 5.7 1.0
Figure imgf000011_0002
10
TABLE I (CONTINUED)
HETEROAROTINOIDS RATIO SP. ACTIVITY Rc
Figure imgf000012_0001
1[RA[ 3.2 1.0
Figure imgf000012_0002
TABLE I (CONTINUED)
HETEROAROTINOIDS RATIO SP. ACTIVITY1 R
Figure imgf000013_0001
12
TABLE I (CONTINUED)
HETEROAROTINOIDS RATIO SP. ACTIVITY1 Rc
Figure imgf000014_0001
3.3 1.0
Figure imgf000014_0004
Figure imgf000014_0002
1 [RA] 5.1 1.0
Figure imgf000014_0003
Reference d below discusses the techniques involved in this assay. Compound 1 is ./'α/15-retinoic acid (7-RA) and is the standard used in the experiments. bActivity ratio = specific activity (dpm/mg/hr of test compound/specific activity (dpm/mg/hr) of the control ,-RA (1) [Dpm = disintegrations/min]. cActivity ratio of test heteroarotinoid/activity ratio of f-RA ( 1). dT. Suedhoff. P. J. Birc bichler, K. N. Lee, E. Conway, and M. K. Patterson. Jr. "Differential Expression of Transglutaminase in Human Erythroleukemia Cells in Response to Retinoic Acid", Cancer R search 1990.50, 7839-7834. TABLE II
HETEROAROTINOIDS: DECREASING BINDING POTENCY WITH SPECIFIC HUMAN RETINOIC ACID RECEPTORS3
Figure imgf000015_0001
TABLE II (continued)
Figure imgf000016_0001
TABLE II (continued)
Figure imgf000017_0001
TABLE II (continued)
Figure imgf000018_0001
aThe techniques involved for this assay are discussed in reference e below "Maximal response observed relative lo thai ot /rø.ι.-retιnoιc acid (1, r-RA) at 10 ' M cPotency [EC50 = concentration of heteroarotinoid to produce 50 t of the maximal observed response of f-RA
(1)) values for both reference compound l9-c.s-RA| and heteroarotinoids were calculated "Human retinoic acid receptors RAR-α. RAR- β and RAR-γ °V. Berger, Z Parandoosh, B. . Perry, R Stein "Interaction of Glucocorticoid Analogues with Human Glucocorticoid Receptors ". J Steroid Btachem. Molεc. Biol 1992. 41. 733-738 Sec also R Hcyman, P J Mangelsdorf, J A Dyke. R B Stein, G π.chele, R Evans, and C Thaller, "9-cύ-Retιnoιc Acid is a High Affinity l.igand for RXR'" Cell 1992. 68. 397-406
The case of I represents a simpler version of basic system in without the one aryl ring fused to the cycylohexyl system. In all of the systems A, B, D, E, F, H, and I, the common unit is the cyclohexyl ring or cyclopentyl type ring. Moreover, in all cases, except
I, the systems possess a fused five-six or a fused six-six ring unit with a heteroatom in the partially saturated ring.
It is the object of the present invention to provide novel heteroarotinoids which are structurally related to frøn.ϊ-retinoic acid (1) and which act as anticancer agents for the treatment of tumors, including skin tumors and related disorders. Fulfillment of this objective and the presence and fulfillment of other objects will be apparent upon complete reading of the specification and claims. There is no intuitively obvious manner whereby one skilled in the art of organic synthesis and structure-activity relationships could predict the receptor specificity or the TGase activity observed with the following heteroarotinoid which have flexible and nonflexible groups bridging the aryl rings or polyene side chains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The novel heteroarotinoid compositions according to the preferred embodiments of the present invention are heteronuclear rings, organic compounds shown below with the letters A, G, L, M, Q, R, T, X, and Y representing the groups illustrated. Although these compounds are structurally related to .ra/..s--retinoic acid (1), they are also unique in that they possess a heteroatom as well as a least one aromatic ring and in some cases two aromatic rings as illustrated. The connecting group G in fused six-six-membered ring systems 2, 6, 26, 29, 32, 35, 39, 123, and 124 is somewhat flexible amide bridge as is true for 21, 23, 38, and 41 in the fused five-six-membered ring systems. Systems 3, 4, 7, 9, 10, 11, 12, 13, 14-20, 22, 24, 25, 27, 28, 30, 33, 34, 36, 37, 40, 42^5, 93, and 94 have a propenyl or polyene link or side chain for G.
Figure imgf000019_0001
Note that (£)- and (Z)-isomers are possible with these rigid linking groups. Systems with a fused six-six-membered ring system and with G as a very flexible carbonyloxy or oxycarbonyl bridge are found in 96-102, 104, 107, 114, 122, 126, 134, and 138. One system with a fused five-five-membered ring with a flexible carbonyloxy bridge is 102. Thus, the invention allows assessment of the impact of flexible versus nonflexible linking groups in the heteroarotinoids on receptor activation and TGase stimulation.
It is clear that systems 2-41 (Table I) possess good activity with respect to normal cell differentiation in terms of transglutaminase production. Each and every experiment was performed using /rαn^-retinoic acid (1) as the standard and which is known to retard the development and formation of malignant cells. Members 2-41 appear to be less toxic than the standard 1. As can be seen, these compounds are either carboxylic acids or esters (R = H or CH or C2H5). In Table II are illustrated data for representative compounds in the invention which exhibit a wide range of capability to interact with specific retinoid receptors. Retinoic acid receptors are proteins which are known to be expressed in vivo as RARα (ubiquitous), RARβ (kidney, heart, and skeletal muscle), and RARγ (skin and bones), respectively. In Table II data are given in terms of potency of the minimum concentration of the heteroarotinoid required to bind to the specific receptor as compared to the concentration required of the standard 9- -retinoic acid. Thus, the lower the concentration required for a heteroarotinoid to bind to a specific receptor type the more active the heteroarotinoid for binding purposes and for inducing a specific biological response such as the differentiation of normal cell development in the production of normal skin cells, liver cells, etc.
Data in Table II are listed in terms of maximum potency initially [lowest concentration expressed as EC50] to minimum potency [highest concentration or high EC 50 value-minimum receptor specificity] for specific receptor interaction and therefore for specific biological response. It is clear that compound 17 has very high specificity for the RARγ receptor which is required for normal skin development. Heteroarotinoids 8, 11, 42, and 44, behave similarly although the specifity drops in the order given indicating a range of activity. Specificity for RARβ (for normal kidney, heart, and skeletal muscle development) is exhibited by heteroarotinoids 18, 9 , 7 , 43, 25, 45, and 14, and in descending order of activity. There is some specificity for RARα which is believed to be present in all or nearly all tissues of humans. Heteroarotinoids 10, 2, 24, and 38 exhibit some RARα activity. Consequently, there is ample evidence from these data that the heteroarotinoids synthesized have a gradient of receptor specificity of the three types described herein. The designation of receptor capability and specificity has been reviewed in an article entitled "Regulation of Gene Expression by Vitamins. The Role of Nuclear Retinoic Acid Receptors" by M. Petkovich, and published in The Annual Reviews of Nutrition, volume 12, pages 443-471, in 1992.
Data in Table III were obtained from an assay which involved the treatment of a CC-B cervical tumor cell line, which contained stabilized, integrated copies of RARβ- RARE-tk-CAT reporter plasmid, with the test heteroarotinoid with a separate experiment with /ra/.s-retinoic acid (1) as a control. Thus, the values in Table III refer to percent of control in terms of ability to activate endogenous nuclear receptors. The
TABLE III
RETINOIC ACID RECEPTOR ELEMENT (RARE) INDUCTION DATA AND GROWTH REGULATION | | FOR SELECTED HETEROAROTINOIDS IN TERMS OF PERCENT OF CONTROL
Figure imgf000021_0001
larger the numbers the greater the ability of the heteroarotinoid to activate the receptors in the tumor cell line. The techniques involved in this assay have just been published in an article entitled "A Biological Assay for Activity and Molecular Mechanism of Retinoids in a Cervical Tumor Cells." by D. M. Benbrook., S. Lu, C. Flanagan, J. Shen-Gunther, L. H. Angros, and S. A. Lightfoot, and published in Gynecological Oncology, volume 66, pages 1 14-121 , in 1997. It is clear that in the representative examples shown in Table III there exists a range of activation by the heteroarotinoids. The nature of the heteroatom (RN, O, or S), the various functional groups, and the linking groups appear to influence the receptor activation. Table III also contains data on ability of the heteroarotinoids to inhibit growth of cervical carcinoma cells (CC- 1 or CC-B) as percentage relative to that of the control, and the data are influenced by the same parameters as cited above for the receptor activation.
In summary, Table I demonstrates that the compounds induce transglutaminase enzyme in leukemia tumor cells. Transglutaminase is a marker of differentiation and apoptosis, two activities that induce anticancer effects. By inducing differentiation, the heteroarotinoids reverse the loss of normal differentiation which is characteristic of tumor cells and thus restorethe cells to a more natural state and reduce their ability to spread within the body. Human cells have a natural mechanism whereby the detect and eliminate abnormal cells via a self-induced suicide event called apoptosis. Heteroarotinoids reduce tumor growth (Table III) via stimulation of apoptosis. These biological actions occur through a mech-anism involving regulation of gene expression found in Table III. The heteroarotinoids regulate biological activities to a lesser extent but are more selective than rran.s'-retinoic acid (1) and are therefore more likely to exhibit improved therapeutic ratios compared to 1. For example, in Table II there is shown a strong selectivity of the heteroarotinoids for individual nuclear receptors. Therefore, by exhibiting a preference for one or two of the three retinoic acid receptors, heteroarototinoids will effect a different profile of biological activities that will in turn influence the chemotherapeutic ratio.
Methodology to obtain specific intermediates 46, 51, 57, 58, 72, and 73, and a few heteroarotinoids 3, 7, 16, 18, 25, 42, 43, and 45, are found in U.S. Patent 4,826,984. The preparations of 8, 11, and 44 are found in U.S. Patent 4,833,254. Compounds 17 and 44 are included in U.S. Patent 4,977,276. Reported intermediates are 52, 74, 75, and 78 [L. W. Spruce, J. B. Gale, K. D. Berlin, A. K. Verma, T. R. Breitman, X. Ji, and D. van der Helm, Journal of Medicinal Chemistry, volume 34, 1991 , pages 430-439], 54 [J. L. Bass, A. Davies-Fiddler, and H. O. Huisman, Tetrahedron, volume 22, pages 259-261, 1966], and 57 [K. U. Ingold, Journal of Organic Chemistry, volume 51 , pages 1700-1704, 1986]. All intermediates leading to the final products are new or have been published. Typically, the new compounds in Tables I and II can be prepared from rare but known ketones 46-49
Figure imgf000023_0003
Figure imgf000023_0001
and 51-54 and/or from the new and novel amines 55 and 56. Scheme I reveals the approach to obtain heteroarotinoids 4, 22, and 27 from ketone 46 with intermediates 57 and 58. A similar sequence of
SCHEME I
Figure imgf000023_0002
steps was used in Scheme II to prepare 21 and 23 as illustrated from ketone 47. The conditions for the conversion of 47 to 59 are reasonably unique in that the yield is high and the reagents inexpensive.
SCHEME II
Figure imgf000024_0001
Ketone 48 is the key precursor of 19, 20, 34, and 37 via Scheme III. Intermediates 60-63 are also unknown , but these compounds were fully characterized.
SCHEME III
Figure imgf000024_0002
Scheme IV outlines the formation of heteroarotinoid 66 from ketone 49 in a method reminiscent of that SCHEME IV
Figure imgf000025_0001
in Scheme III. Intermediates 64 and 65 are new and unreported as is the ester 40 leading to 66 Hetero-arotinoids 26 and 39 are accessible starting from 67 with intermediates 68 and 5 69 as shown in Scheme V. Heteroarotinoids 26 and 39 are novel in that they contain the slightly more flexible amide connecting group between the aryl rings
SCHEME V
Figure imgf000025_0002
The syntheses of structurally similar heteroarotinoids 5, 9, 10, 12, 14, 24, 28, 30, and 10 32-36 are delineated in Scheme VI using methodology like that found for the oxygen analogues in Schemes I-III. SCHEME VI
Figure imgf000026_0001
Again the chemistry is especially novel such as, for example, in the Wittig condensation involving 71→28 as well as the generation of chiral 36 from 28 and other transformations illustrated Intermediates 70, 71, and 76 are new and novel as well
In a manner similar to that used to prepare 32 and 35 in Scheme VI, heteroarotinoids 38 and 41 were obtained from ketone 53 as described in Scheme VII Intermediate 77 was heretofore unknown SCHEME VII
Figure imgf000027_0001
The preparations of 2, 6, 26, and 31 in Scheme VIII parallel to some degree the method used to obtain 29 and 39 in Scheme V. Intermediate 79 is new as are certain reaction conditions such as in 79→56.
SCHEMEVIII
Figure imgf000027_0002
In order to investigate the influence on biological activity, as described herein, of having one aryl ring in the side chain without a fused aryl-ring system, the synthesis of 15 was performed as in Scheme IX starting from the 54. Intermediates 84 and 85 are reported for the first time in addition to 15. SCHEME IX
Figure imgf000028_0001
SCHEME IX
Chiral heteroarotinoids have not been reported, and we describe here the first such synthesis. Such a system is potentially very important since one specific enantiomer is frequently more active than its counterpart in terms of biological activity. In Scheme X there is illustrated the conversion of 86→87→88→89→90→91→92→93→94, the sulfide 93 and sulfoxide 94 being chiral. The preparation of 86 from a commercial compound is given under the synthesis of 28 in Example XVI. Characterization of and confirmation of each intermediate illustrated in Scheme X was completed by spectral an elemental analyses. Heteroarotinoids 93 and 94 are the first reported chiral systems in this family of heterocycles.
In an effort to introduce flexibility into an oxygen-containing heteroarotinoid for potentially improved receptor binding, a series of compounds with an ester bridge were designed with a few examples in
SCHEME X
Figure imgf000029_0001
Schemes XI and XII in the conversion of 95→96→97 and 95→98-101 , and 59→102,
respectively. The reactions required a water absorbing compound (DCC) and a catalyst
(DMAP). The thiosemicarbazone derivative 97 is also the first example of such a derivative in the family of heteroarotinoids. Certain thiosemicabazones have displayed useful anticancer activity as found in an article in the Journal of Medicinal Chemistry, 1995, vol. 38, pages 4234-4243, entitled "Synthesis and Antitumor Activity of 4- and 5-Substituted Derivatives of Isoquinoline- 1 -carboxyaldehyde Thiosemicarbazone" by M. C. Lium, T. P. Lim, P. Penketh, and A. C. Sartorelli. Thus, the justification for 97 has validity in theory although not heretofore recognized with heteroarotinoids. SCHEME XI
Figure imgf000030_0001
Esters 98-102 in Scheme XII possess a small variations in terminal functional groups with the basic heterocyclic skeletal unit intact. Example 102 allowed appraisal of the effect of a fused five-six-membered ring system on the activity while the other examples of 98-101 possessed the six-six-membered groups in the basic structure. Examples 99 and 100 allow an assessment of two different heterocyclic rings on activity as well as hydrophilicity which can be very beneficial in agent formulation.
SCHEME XII
Figure imgf000031_0001
In Scheme XIII, starting material 103 was easily converted to oxygen-containing heteroarotinoids 104 and 107 via the route of 103→104 and 103→105→106→107 illustrated. The principle difference in these flexible systems is the presence of the C=O group adjacent to the oxygen atom in the six-membered ring in both 104 and 107. This stiffens the groups slightly making the ring more planar, but yet the flexible bridging ester group remains between both aryl groups. Such a slight difference in this fused, six-six- membered ring almost surely alters binding to the receptor which in turn influences anticancer properties of the agent. SCHEME XIII
Figure imgf000032_0001
In Schemes XIV and XV, a nitrogen atom has been introduced as the heteroatom in the heteroarotinoid system. In Scheme XIV, the objective was to prepare a small series of compounds to determine the influence of a small versus large group on nitrogen with respect to anticancer activity. Formation of the N-methyl analog is illustrated via 108→109→110→111→112→113→114, but an essentially identical procedure can be used to give other simple N-alkyl derivatives including the N-ethyl, N-propyl, and N-isopropyl analogs. Introduction of the larger groups, especially the isopropyl group, can give an indication as to the screening effect of the lone pair of electrons on nitrogen by the alkyl group as related to anticancer activity. Once again, the flexible bridging ester group is present in all of the systems. SCHEME XIV
Figure imgf000033_0001
In Scheme XV, one other parameter is varied, namely the inclusion of a methyl group bonded to the aryl ring in the fused six-six-membered ring unit of the heteroarotinoid. The conversion of 115→116→117→ 118→119→120→121→122 parallels to some degree that exhibited in Scheme XIV except slightly more severe conditions were required in certain steps of the synthesis of 122. Hydrolysis, for example, of the nitrile group in 120 required 6 days to give a moderate yield of intermediate 121. Presumably, this long reaction time was required because of the extreme hindrance to attack by hydroxide ion on the nitrile group which was partially shielded by the methyl group attached to the same ring in 120. The rationale for the presence of this methyl group was that it would alter the flexibility of the ester bridging group and thus could influence the binding of 122 to its receptor and in its overall anticancer activity. SCHEME XV
Figure imgf000034_0001
In Scheme XVI, acid 95 was converted to the corresponding acid chloride which, in the biphasic mixture, was treated with ethyl 4-hydroxyphenylbenzoate to give the hydroxamic acid derivative 123. O-Methylation of 123 led to the methoxy analog 124. The alteration in the flexibility of the linking amide type group in both 123 and 124 would expectedly influence binding to the receptors, and therefore the SCHEME XVI
Figure imgf000035_0001
anticancer activity of the agents will be regulated.
The addition of the methyl Grignard reagent to 103 in Scheme XVII produced the phenol derivative 125 which has a highly hindered ether group as well as a screened hydroxyl function. Esterification of 125 yielded the extremely hindered ester 126, but the system still retains a flexible ester linkage between the two aryl ring units. Thus, the presence of a flexible linking group, coupled with hindering methyl groups around the linking ester group, creates a unique agent for interaction with the receptors for enhanced activity.
SCHEME XVII
Figure imgf000035_0002
Scheme XVIII outlines the synthesis of a novel heteroarotinoid in which the heteroatom (oxygen in this case, but the sulfur atom could also be utilized) is repositioned at a new location in the cyclohexyl ring unit. The method involves 127→ 128-.129→130→131→132→133→134, the latter being the target molecule.
Methylation alpha to the nitrile group in 127 gave 128 which, when subjecte to basic hydrolysis, gave 129. Reduction of the carboxyl group in 129 produced alcohol 130.
Tandem etherification and ring closure occurred when 130 was treated with acetone in an acid medium to give 131. Acetylation of 131 led to 132 which, when treated with haloform type conditions, gave acid 133. E.sterification of 133 generated ester 134 which possesses the flexible internal ester link. The defense for this type of heteroarotinoid is that the new position of the heteroatom in the cyclohexyl ring and the flexible linker group will influence receptor binding and anticancer activity.
SCHEME XVIII
Figure imgf000036_0001
Target system 138 in Scheme XIX was designed as a model system for 134 since the former does not possess a heteroatom in the cyclohexyl ring unit. Ether 135 was acetylated under standard conditions to yield ketone 136. Haloform type conditions converted 136 to 137 although the reaction had to be done with care. Esterification of 137 paralleled the method used to obtain 134 illustrated previously. Thus, 138 allows a comparison of the influence on anticancer activity by the absence of the heteroatom. SCHEME XIX
Figure imgf000037_0001
Having thus described and examplified the preferred embodiments with a certain degree of particularity, it is manifest that many changes can be made within the details of the operation, operating parameters, and steps for preparing and utilizing the heteroarotinoids according to the present invention without deviating from the spirit and scope of the invention. Therefore, it should be understood that the invention is not limited to the embodiments set forth herein for the purposes of examplification, but it is limited only by the scope of the attached claims, including the full range of equivalents to which each step thereto is entitled.
EXAMPLE 1
4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H-l-benzothiopyran-6-yl)carbamoyl]benzoic Acid (2)
Ester 26 (0.15 g, 0.39 mmol) in 95% ethanol (7 mL) was treated with 2 N NaOΗ (10 eq, 3.9 mmol). After stirring the solution for 4 h, the mixture was acidified with 2 N ΗC1 (30 mL) which produced a white foam that was filtered, washed (Η2O), dried, and recrystallized (EtOAc:hexane; 2: 1). The yield of 2 was 0.1 g (68%) with a mp of 208-209.5
°C. IR (KBr) 3500, 3320, 1700, 1650 cm 1 ; »H ΝMR (DMSO- 6) δ 1.36 [s, 6 H], 1.37 [s, 6
H], 1.93 [s, 2 H], 7.04 [d, 1 H], 7.60 [d, 1 H], 7.88 [d, 1 H], 8.09 [s, 4 H], 10.32 [s, 1 H]. Mass spectral (El) data Calcd for C21H23O3SN m/z (M +): 369.1398. Found: 369.1384.
Anal. Calcd for C21H23O3SNO.5 H2O: C, 66.64; H, 6.39; N, 3.70. Found: C, 66.57; H, 6.47; N, 3.68.
Ester 26 was prepared from amine 56 which was synthesized as follows. Nitro- substituted compound 79 (0.69 g, 2.7 mmol) was dissolved in acetic acid (25 mL) and water (5 mL) with vigorous stirring. Then TiCl3/HCl (28.06 g, 18 mmol) was added dropwise, and the resultant mixture was stirred at RT (2 h). After being cooled to 0 °C, the mixture was treated slowly with 30% NaOH (1 10 mL). Extracts (ethyl acetate followed by H2CCI2) of the aqueous phase were combined with the organic layer and washed with water and saturated NaHCO3. After drying, the solvent was evaporated to an oil which was chromatographed on silica gel which yielded 56 as an oil (0.48 g, 80%) and was used directly to prepare 26. Properties of 56 were: IR (neat) 3600, 3450, cm 1; Η NMR (DCCI3) δ 1.36 [s, 6 H], 1.39 [s, 6 H], 1.90 [s, 2 H], 3.50 [bs, 2 H], 6.44 [d, 2 H], 6.75 [s, 1
H], 9.92 [d, 1 H].
Nitro-substituted compound 79 was obtained from the reaction involving dropwise addition of a solution of cold cone HNO3 (3 mL) with acetic anhydride (9 mL) into a solution of ether 78 (4.35 g, 21.0 mmol) dissolved in acetic anhydride (8 mL) at 0 °C. After stirring 1 h, the solution was poured into a saturated solution (-100 mL) of NaHCO3, and the resulting mixture was extracted (H2CCI2). The organic layer was washed with water and brine and then dried (Na2SO4). Evaporation of the solvent left a solid which was chromatographed over silica gel with H2CCI2 to give a mixture of 6-nitrochroman (79):8- nitrochroman (10: 1) (1.5 g, 28%) which had a mp of 106-109 °C. This mixture was used directly to prepare amine 56 since the 8-isomer did not prove a serious contaminant in the purification of 56. EXAMPLE II
(E)-4-[(2,3-Dihydro-4,4-dimethyl-2H- 1 -benzopyran-6-yl)- 1 -propenyl]-2-methylbenzoic Acid [(£)-4J
A solution of (£)-27:(Z)-27 isomers [9: 1 ; 0.50 g, 1.37 mmol], KOΗ (0.76 g, 13.7 mmol), 95% ethanol (20 mL), and water (5 mL) was boiled for 12 h and then cooled slowly
(30 min) to RT. After being chilled in an ice bath, the solution was treated dropwise with cone ΗC1 (20 mL, pΗ 2) which produced a white precipitate (0.34 g, 72%).
Recrystallization (95% ethanol) of the solid gave colorless crystals of (E)-4 with an mp of
167- 169°C. IR (KBr) 3500, 1725 cm 1; Η NMR (DMSO-d6) δ 1.34 [s, 6 Η], 1.79 ft, 2 Η],
2.23 [s, 3 Η], 2.56 [s, 3 Η], 4.16 [t, 2 Η], 6.70 [bs, 1 Η], 6.72 [d, 1 Η], 7.25 [m, 3 Η], 7.51 [s, 1 Η], 7.86 [d, 1 Η]. Mass spectral (El) data Calcd for C22Η24O3 mlz (M+): 336.1725. Found: 336.1729. Anal. Calcd for C22H24O3: C, 78.54; H, 7.19. Found: C, 78.45; H, 7.30.
EXAMPLE III
(£ 4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H- 1 -benzothiopyran-6-yl)-3-oxo- 1 - propenyl]benzoic Acid [(E)-5]
To a stirred solution of ketone 52 (0.92 g, 3.53 mmol) and terephthaldehyde acid methyl ester (0.61 g, 3.71 mmol) in methanol (30 L) was added dropwise 1 N ΝaOH (22 mL), and a turbid solution developed. After 5 h, the solution was clear and was poured into 1 N HC1 (100 mL) whereupon a solid formed. Extracts (ethyl acetate) of the aqueous layer were washed with water and brine and finally dried (Νa2Sθ4). Chromatrography over silica gel [HCCl3:H3COH = 3: 1, HCCl3:H3COH = 1 : 1 , and H3COH] resulted in the isolation of crude 5 which was recrystallized (hexane) to yield pure (E)-5 (0.61 g, 45%) with mp 198-
199 °C. IR (KBr) 3500, 1690, and 1680 cm 1; lH NMR (DCCI3) δ 1.45 [s, 12 H], 2.01 [s, 2
HI, 7.23 [d, 1 H], 7.61-8.10 [m, 7 H], 8.74 [d, 1 H]. Mass spectral (El) data Calcd for C23H24O3S mlz (M+): 3890.1446. Found: 380.1454. Anal. Calcd for C23H24O3S: C, 73.60; H, 6.35. Found: C, 74.02; H, 6.22.
EXAMPLE IV
4-[(2,2,4,4-Tetramethylthiochroman-6-yl)carbamoyl]muconic Acid (6)
To acid chloride-ester 31 (0.30 g, 0.83 mmol) in absolute ethanol (20 mL) was added 2 N ΝaOH (12 eq., 9.96 mmol). After stirring the solution for 3 h at RT, the mixture was acidified with 2 N HC1 (100 mL-0 °C). The solid was filtered, washed (H2O), dried, and recrystallized (EtOAc:hexane; 1:2) to give acid-amide 6 (0.21 g, 0.61 mmol-73%) with
a mp of 225-227.5 °C. IR (KBr) 3350, 3300, 1710, 1640 cm 1 ; lU ΝMR (DMSO- ) δ 1.33
[s, 6 H], 1.34 [s, 6 H], 1.91 [s, 2 H], 6.27 [d, 1 H], 6.28 [d, 2 H], 7.01 [d, 1 H], 7.45 [d, 1 H], 7.75 Is, 1 HJ, 7.29 [m, 2 H], 10.28 [s, 1 H]. Mass spectral (El) data Calcd for C 19H23ΝO3S
m/z (M+): 345.1399. Found: 345.1399. Anal. Calcd for C19H23O3SN: C, 66.06; H, 6.71 ; N. 4.05. Found: C, 66.02; H, 6.99; N, 3.92.
EXAMPLE V
(E)-4-[(2,3-Dihydro-4,4-dimethyl-2H-l-benzothiopyran-6-yI)-l-propenyl]-2-methylbenzoic Acid [(£ 9]
A solution of ester 12 (0.22 g, 0.57 mmol), ethanol (10 mL), water (10 mL), and NaOΗ (0.04 g, 1.9 mmol) was boiled for 6 h and then cooled slowly to RT. The new solution was chilled to 0 °C and was then treated with cone ΗC1 (~8 mL, pΗ ~ 2) to yield a solid. The solid was washed well with water, air dried, and then dried under vacuum to give acid (E)-9 as needles (0.17 g, 0.49 mmol, 63%) with a mp of 171-172 °C. IR (KBr) 3450,
1685 cm 1; tø NMR (DCCI3) δ 1.33 [s, 6 Η], 1.91 [t, 2 Η], 2.33 [s, 3 Η], 2.55 [s, 3 Ηl, 3.03
[t, 2 Η], 6.84 [bs, 1 Η], 7.04 [d, 1 Η], 7.29 [ , 3 Η], 7.59 [s, 1 Η], 7.87 [d, 1 Η]. Mass spectral (El) data Calcd for C22H24O2S m/z (M+): 352.1497. Found: 352.1498. Anal. Calcd for C22H24O2S: C, 74.96; H, 6.86. Found: C, 74.85; H, 6.90.
EXAMPLE VI
(E)-4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H-l-benzothiopyran-6-yl)-l-propenyl]-2- methylbenzoic Acid [(£)-10]
A solution of ester 14 (0.32 g, 0.80 mmol), 95% ethanol (20 mL), water (5 mL), and
KOΗ (0.45 g, 8.03 mmol) was boiled for 2 h and then stirred at RT for 12 h. The new solution was chilled to 0 °C and was then treated dropwise with 2 N ΗC1 (-20 mL) which precipitated acid (E)-10 as a white solid. Recrystal-lization (95% ethanol) gave colorless needles of (£)-10 with a mp of 178-180 °C. IR (KBr) 3450, 1690 cm 1 ; 'Η ΝMR (DMSO- d6) δ 1.39 [s, 12 ΗJ, 1.95 [s, 2 Η], 2.24 [s, 3 Η], 2.55 [s, 3 Η], 6.87 [bs, 1 Η], 7.06 [d, 1 Η],
7.31 [m, 3 Η], 7.64 [s, 1 Η], 7.86 [d, 1 Η]. Mass spectral (El) data Calcd for C24Η28θ2S m/z (M+): 380.1809. Found: 380.1810. Anal. Calcd for C24H28O2S: C, 75.75; H, 7.41. Found: C, 75.47; H, 7.49.
EXAMPLE VII
Ethyl(E)-4-[(2,3-Dihydro-4,4-dimethyI-2H-l-benzothiopyran-6-yl)- l-propenyl]-2- methylbenzoate [(£)-12]
A suspension of phosphonium salt 73 ( 1.17 g, 2.13 mmol) in dry ether (25 mL) was treated dropwise with a solution of M-butyllithium in hexane (10 , 0.22 mL, 2.26 mmol). This reddish solution was cooled to -78 °C, and then it was slowly treated with a solution of ethyl 2-methyl-4-formylbenzoate (0.37 g, 1.92 mmol) in ether (25 mL) with stirring for 0.5 h. After allowing this solution to warm to RT (1 h), it was stirred for 48 h and then filtered to remove triphenylphosphine oxide. The filtrate was dried, and the resulting solution was evaporated to a yellow oil. Chromatography of this oil on silica gel with hexane:ether (98:2) gave a mixture of esters [(E)-12:(Z)-12 = 8: 1 ] as a viscous oil which was treated with boling ethanol for 5 min. Chilling the resulting solution for 24 h produced crystals of (£)-
12 (0.22 g, 0.58 mmol-30%) with a mp of 60-62 °C. IR (KBr) 1710 cm 1 ; ] H NMR
(DCC13) δ 1.39 [s, 6 H], 1.43 [t, 3 H], 1.99 [t, 2 H], 2.27 |s, 3 H], 2.64 [s, 3 H], 3.05 [t, 2 H],
4.38 [q, 2 H], 6.75 [d, 1 H], 7.08 [d, 1 H], 7.23 [m, 3 H], 7.51 [s, 1 H], 7.93 [d, 1 HJ. Mass spectral (ΕI) data Calcd for C24H28O2S mlz (M+): 380.1809. Found: 380.1809. Anal. Calcd for C24H28θ2S: C, 75.75; H, 7.41. Found: C, 75.46; H, 7.48.
EXAMPLE VIII
Ethyl(E)-4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H-l-benzothiopyran-6-yl)-l-propenyl]-2- methylbenzoate [(£)-14]
A suspension of salt 75 (0.82 g, 1.43 mmol) in dry ether (25 mL) was treated dropwise with /.-butyl-lithium ( 10 M, 0.15 mL, 1.56 mmol) and then stirred at RT for 1 h. After being cooled to -78 °C, the resulting solution was treated slowly with ethyl 2-methyl- 4-formylbenzoate (0.25 g, 1.3 mmol) in dry ether (20 mL ).25 h). The solution was then allowed to warm to RT (~1 h) and was then stirred for 48 h. Filtra-tion removed triphenylphosphine oxide, and the filtrate was dried and then evaporated to a yellow oi! which was chromatrographed over silica gel with hexane:ether (98:2). A mixture of isomeric (£)-14 and (Z)-14 was treated with boiling ethanol, and, upon chilling, the solution deposited a solid which was recrystallized (ethanol) to give flakes of (£)-14 with a
mp of 77-79 °C. IR (KBr) 1705 cm 1 ; 'Η NMR (DCCI3) δ 1.44 [s, 12 Η], 1.40 [t, 3 Η],
1.98 [s, 2 Η], 2.28 [s, 3 Η], 2.63 [s, 3 Η], 4.36 [q, 2 Η], 6.77 [bs, 1 Η], 7.1 1 [d, 1 Η], 7.25 [m, 3 Η], 7.53 [s, 1 Η], 7.93 [d, 1 Η]. Mass spectral (El) data Calcd for C26Η32O2S mlz
(M+): 408.2123. Found: 408.21 14. Anal. Calcd for C26H32O2S: C, 76.43 H, 7.79. Found: C, 76.32; H, 7.96. EXAMPLE IX
4-f (AU-E)-2-methyl-4-(2,6,6-trimethyl-3-thia- 1 -cyclohexen- 1 -yl)- 1 ,3-butadienyl]benzoic Acid (15)
A suspension of phosphonium salt 84 (0.5 g, 0.93 mmol) in ether (10 mL) was treated dropwise with a solution of n-butyllithium ( 10 M, 0.1 mL, 1.0 mmol), and the resulting reddish solution was stirred at RT for 15 min. After the final solution had been cooled to -78 °C, ethyl 4-formylbenzoate (1.52 g, 0.93 mmol) was added dropwise and then the solution was stirred for 5 min. After allowing the solution to warm to RT (1 h), it was stirred for 24 h. Triphenylphosphine oxide was filtered from the mixture, and the solvent was evaporated to give the oil 85. The oil 85 was treated with absolute ethanol (10 mL) followed by KOH (0.2 g) in water (10 mL), and this solution was boiled for 1.5 h and then cooled to RT. Neutralization was effected with 5% H2SO4 (- 15 mL), and two layers formed. Ether extracts of the water layer were combined, washed with brine, and then concentrated to a yellow solid which was recrystallized (95% ethanol) to pure acid 15 with a mp of 186-187 "C. IR (KBr) 3500, 1680 cm 1; >H NMR (DCCI3) δ 1.1 [s, 6 H], 1.85 [m,
2 H], 1.9 [s, 3 HJ, 2.1 [s, 3 H], 2.85 [m, 2 H], 6.45-6.51 [m, 3 H], 7.35 [d, 2 H], 8.05 [d, 2 H]. Anal. Calcd for C20H24O2S: C, 72.74; H, 7.52. Found: C, 73.13; H, 7.52.
Salt 84 was prepared as follows. A solution of 4-thia-β-ionone (54, 8.0 g, 0.38
mmol) in ether (50 mL) was added slowly to a suspension of LiAlI j (3.0 g, 0.029 mmol) in ether (100 mL) at 0 °C over 0.5 h. After stirring for 5 h, the reaction mixture was poured slowly onto ice (200 g). Cautiously, 6 M HC1 (-300 mL) was added to make the solution acidic. Combined extracts (ether) of the aqueous layer were washed with brine, dried (Na2SO4), filtered, and concentrated to a brown liquid 83 (0.023 g, 62%). Properties of 83
were: IR (neat) 3700 cm 1; lH NMR (DCCI3) δ 1.05 [s, 6 H], 1.31 [d, 3 H], 1.85 [m, 2 H],
1.87 [s, 3 H], 2.83 [m, 2 H], 4.35 [m, 1 H], 5.4 [dd, 1 H], 6.04 [d, 1 H]. This 83 was used directly to prepare salt 84 as follows. Alcohol 83 (5.0 g, 0.023 mmol) in methanol was treated with Pr^P-HBr ( 14.0 g, 0.04 mmol) all at once, and the reaction mixture was stirred at RT (24 h). Evaporation of the methanol gave a thick oil which was dried under vacuum (25 °C/10 mm) for 2 h. Trituration of the oil with cold ether produced a light yellow solid
84 ( 13 g, 0.024 mmol). > H NMR (DCC13) δ 0.82 [s, 3 H], 0.91 [s, 3 HJ, 1.3 [m, 2 HJ, 1.49
[dd, 3 HI, 2.7 [m, 2 H], 3.2 [bs, 3 H], 5.0 [m, 1 H], 6.44 [m, 1 HJ, 6.81 [m, 1 H|, 7.2-8.0 [m, 1 5 HJ. This salt 84 was used directly to prepare ester 85 which was converted to acid 15.
EXAMPLE X
(E)-4-[2-(2,3-Dihydro- 1 ,4-benzodioxan-6-yl)- 1 -propenyljbenzoic Acid (19)
To ester 34 (0.2 g, 0.644 mmol) was added NaOH pellets (0.129 g, 3.23 mmol), absolute ethanol (3 mL), and water (9 mL), and the resulting mixture was boiled (5 h).
After cooling to RT (0.5 h), the solution was treated dropwise with 12 N HC1 with stirring until the solution became acidic to litmus. A solid formed and was filtered, washed with water, air dried, recrystallized (95% ethanol), and then dried under vacuum to yield a fluffy white solid 19 with mp 171-172 °C. IR (KBr) br 3400-2400 cm 1 ; »H (DCCI3) δ 2.26 [d, 3
HJ, 4.29 [s, 2 H], 6.80 [bs, 1 HJ, 6.89-8.12 [Ar-HJ. Mass spectral (ΕI) data Calcd for Cι8Hι6O4 mlz (M+): 296.1048. Found: 296.1048. Anal. Calcd for Cι8Hι6O4: C, 72.96; H, 5.44. Found: C, 73.07; H, 5.34.
EXAMPLE XI
(2E,4£,6E)-7-(2,3-Dihydro-l ,4-benzodioxan-6-yl)-3-methyl-2,4,6-octatrienoic Acid (20)
A solution of ester 37 (0.150 g, 0.48 mmol), KOH (1 mL, 35% aqueous), and ethanol
(2 mL) was boiled for 1 h and was then allowed to cool to RT. To this solution was added water (5 mL), ethyl acetate (50 mL), and H3CCO2H: water (1 : 1, 1 mL) in that order which resulted in two layers being formed. Extracts (ethyl acetate) of the aqueous layer were combined and dried (Na2SO4), filtered, and dried to give 20 (0.49 g, 35.8%) as yellow crystals with a mp of 181-182 °C. IR (KBr) br 3300-2400 cm 1; >H (DCC13) δ 2.21 [s, 3 H],
2.39 [s, 3 HJ, 4.27 [s, 4 HJ, 5.83 [bs, 1 H], 6.38 [d, 1 HJ, 6.53 [d, 1 HJ, 6.83-7.09 [m, 3 HJ. Mass spectral (El) data Calcd for Cι7Hi8θ4 m/z (M+): 286.1205. Found: 286.1203. Anal. Calcd for C17H18O4: C, 71.31 ; H, 6.34. Found: C, 71.29; H, 6.31.
EXAMPLE XII
4-[(2,3-Dihydro-3,3-dimethyl-5-benzofuranyl)carboxamido]benzoic Acid (21)
To a solution of ester 23 (0.56 g, 1.61 mmol) in absolute ethanol (20 mL) was added dropwise 2 N ΝaOH (10 eq., 16.1 mmol), and the solution was boiled for 4 h. After acidification of this solution with HCl (2 N, 0 °C, -50 mL), the resulting mixture was stored in a freezer for 12 h. A white solid formed and was filtered off, washed with water, dried, and recrystallized (hexane:EtOAc, 2: 1). The crystals of 21 were dried under vacuum for 12 h and had a mp of 219-220 °C. IR (KBr) 3500, 3340, 1700, 1660 cm 1; Η ΝMR (DCCI3) δ
1.36 [s, 6 HJ, 4.30 [s, 2 H], 6.89 [d, 1 HJ, 7.96 [m, 6 HJ, 10.30 [s, 1 HJ. Mass spectral (El) data Calcd for C 18H17O4Ν w/z (M+): 31 1.1 157. Found: 31 1.1 157. Anal. Calcd for
Cι8Hι7O N-0.1 H2O: C, 69.04; H, 5.54; N, 4.47. Found: C, 68.98; H, 5.74; N, 4.31.
EXAMPLE XIII
Ethyl(E)-3-[2,3-Dihydro-4,4-dimethyl-2H- 1 -benzopyran-6-yl)- 1 -propenyljbenzoate [(E)- 22]
A suspension of phosphonium salt 58 (1.29 g, 2.42 mmol) in ether (30 mL) was treated dropwise with a solution of n-butyllithium (0.90 M, 3.4 mL, 3.14 mmol) over a period of 5 min. The resulting reddish mixture was cooled to -78 °C, and a solution of ethyl
3-formylbenzoate (1.0 g, 5.61 mmol) in ether (25 mL) was added dropwise (10 min). After stirring for 30 min, the solution was allowed to arm to RT (1 h). After stirring for an additional 48 h, the yellow mixture was filtered, and the filtrate was dried (Na2SO4). Evaporation of the solvent gave a solid which was chromatographed over silica gel with hexane:ether (97:3). A mixture of (£)-22:(Z)-22 ( 10: 1 ) was treated with boiling ethanol for 5 min, and then the solution was chilled for 24 h. A white solid formed and was recrystallized (cold ethanol) to give 0.5 g ( 16.2%) of needle-like crystals of (E)-22 with mp of 42.5-44 °C. IR (KBr) 1725-1715 cm"1 ; Η NMR (DCCl3) δ 1.39 [s, 6 HJ, 1.40 [t, 3 HI,
1.86 [t, 3 HJ, 4.21 [t, 2 HJ, 4.40 [q, 2 HJ, 6.76 [s, 1 HJ, 6.80 [s, 1 HI, 7.25 hdd, 1 HJ, 7.42 [m, 2 HJ, 7.53 [dd, 1 HJ, 8.03 [dd, 1 HI. Mass spectral (El) data Calcd for C23H26O3 mlz
(M+): 350.1882. Found: 350.1882. Anal. Calcd for C23H26O2: C, 78.83; H, 7.48. Found: C, 79.12; H, 7.54.
EXAMPLE XIV
Ethyl 4-[(2,3-Dihydro-3,3-dimethyl-5-benzofuranyl)carboxamido]benzoate (23)
A mixture of acid 59 (0.71 g, 3.69 mmol), thionyl chloride (20 mL), and DMF (4 drops) was stirred at 0 °C for 12 h. Excess thionyl chloride was removed from the clear solution under aspirator pressure. Pyridine (35 mL) was added to the resultant solid, and this solution was added to a jacketed flask. To this system was added ethyl 4- aminobenzoate (0.67 g, 4.06 mmol), and the final solution was heated with stirring at the boiling point of acetone for 3 h. Water was added and the resulting mixture was extracted quickly with EtOAc. Combined organic layers/extracts were washed with 2 N HCl, saturated NaHCO3, water, and brine. After drying (Na2SO4), the solution was evaporated to a yellow solid which was dissolved in H2CCI2 (-2 mL), and this solution was chromatographed over silica gel with ^CChtEtOAc (3: 1 ) and 100: 1. Evaporation of the fractions gave a foamy white solid 23 (0.82 g, 2.42 mmol, 65%) with a mp of 51-55 °C. IR
(KBr) 3340, 1725, 1660 cm-'; *H NMR (DCCI3) δ 1.31 [s, 6 H], 1.38 [t, 3 H], 4.29 [s, 2 HJ,
4.33 [q, 2 HJ, 7.78 [m, 4 H], 8.01 [d, 2 HJ, 8.61 [s, 1 H]. Mass spectral (El) data Calcd for
C20H21O4N mlz (M+): 339.1470. Found: 339.1470. Anal. Calcd for C2()H2ιO4N-0.1 H O: C, 70.41 H. 6.26. Found: C, 70.35; H, 6.34. This ester was used directly to prepare acid 21.
Acid 59 was prepared as follows: Ketone 47 (1.0 g, 5.26 mmol), dissolved in ethanol (17 mL), was treated with commercial clorox (50 mL), and the resultant solution 5 was boiled with stirring for 5 h and then was stirred at RT (5 h). The cooled (0 °C) reaction mixture was cautiously treated with a 25% solution of sodium metabisulfite (-50 mL) followed by cone HCl (-10 mL). A white solid formed in the mixture, after which another 70 mL of sodium metasulfite solution was added. The solid was filtered, dried, and recrystallized (ethanol) to yield 59 (0.71 g, 3.69 mmol) with a mp of 175-175 βC. IR (KBr)
10 3500, 1680 cm-1; ]H NMR (DCC13) δ 1.38 [s, 6 H], 4.34 [s, 2 H], 6.18 [d, 1 HJ, 7.87 [s, 1
HJ, 7.99 [d, 1 HJ. Acid 59 was used directly to prepare 23.
EXAMPLE XV
Methyl (E)-4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H- 1 -benzothiopyran-6-yl)- 1 -propenyl]-3- 15 methyl benzoate [(E)-24J
To a stirred suspension of phosphonium salt 75 (3.29 g, 5.72 mmol) in dry ether (25 mL) was added drop-wise «-butyllithium (10 Λ , 0.18 mL, 1.79 mmol) over a period of 5 min at RT. The resulting reddish mixture was cooled to -78 °C, and methyl 3-methyl-4- formylbenzoate (0.85 g, 4.76 mmol) was added drop-wise (-10 min). The resultant solution 0 was stirred (0.5 h) at -78 °C, and then it was allowed to warm to RT. The new yellow solution was stirred for 48 h, was filtered to remove triphenylphosphine oxide, and was evaporated to a thick yellow oil. Chromatography over silica gel with hexane:ether (98:2 and 100: 1) gave a mixture of (E)-24:(Z)-24 (1 : 1) (0.75 g, 40%) as a clear viscous oil which
was used directly to prepare acid 10. IR (KBr) 1700 cm 1 ; >Η NMR (DCCI3) δ 1.45 [s, 12
5 ΗJ, 1.98 [s, 2 Η], 2.1 1 [s, 3 ΗJ, 2.34 [s, 3 Η], 3.92 [s, 3 Η], 6.80 [bs, 1 Η], 7.13-7.90 [m, 6 Ηl. Mass spectral (El) data Calcd for C25Η30O2S m/z (M+): 394.1966. Found: 394.1962. Anal. Calcd for C25H30O2S: C, 76.10 H, 7.66 Found: C, 76.39; H, 7.75. EXAMPLE XVI
Methyl 4-[(2,3-Dihydro-2,2,4,4-tetramethyl-2H-l-benzothiopyran-6-yl)carbamoylJbenzoate
(26)
Amine 56 (0.4 g, 1.8 mmol) in benzene (40 mL) and pyridine ( 2.3 mL) was treated with mono-mcthy\ terephthaloyl chloride (0.39 g, 2.16 mmol), and the mixture was vigorously stirred at RT for 12 h. The reaction mixture was poured into water, and the resultant mixture was quickly extracted with ethyl acetate. The combined organic layers were washed with 2 N ΗC1, water, ΝaΗCθ3, water, and brine. Drying (Na2SO4) followed by evaporation of the solvent, produced a yellow solid which was chromatographed over silica gel (H2CCI2) and recrystallized (EtOAc:hexane; 3: 1 ) to yield 26 (0.28 g, 40%) with a
mp of 162-164 °C. IR (KBr) 3395, 1725, 1685 cm 1 ; >H NMR (DCCI3) δ 1.41 [s, 6 HJ,
1.42 [s, 6 HJ, 1.96 [s, 2 H], 3.95 [s, 3 HJ, 7.1 1 [d, 1 HJ, 7.33 [d, 1 HJ, 7.76 |s, 1 H], 7.91 [d, 2 H], 7.93 [bs, 1 HJ, 8.12 [d, 2 HI, 8.13 Is, 1 HJ. Mass spectral (El) data Calcd for C22H25O3SN mlz (M+): 383.1555 Found: 383.1552. Anal. Calcd for C22H25O3SN: C, 68.90; H, 6.57; N, 3.65. Found: C, 68.88; H, 6.58; N, 3.64.
EXAMPLE XVII
Ethyl(E)-4-[(2,3-Dihydro-4,4-dimethyl-2H-l-benzopyran-6-yI)-l-propenylJ-2- methylbenzoate (27)
To a suspension of phosphonium salt 58 (0.60 g, 1.25 mmol) in dry ether (25 mL) was added dropwise n-butyllithium (10 M, 0.1 1 mL, 1.25 mmol) over a period of 10 min. After cooling to -78 °C, the reddish mixture was treated with a solution of ethyl 2-methyl-4- formylbenzoate (0.22 g, 1.14 mmol) in dry ether (20 L). After being stirred for 0.5 h at - 78 °C, the resultant mixture was allowed to warm to RT and then was stirred for 48 h. Filtration removed the triphenylphosphine oxide, and the filtrate was dried (Na2SO4) and evaporated to a yellow oil which was chromatographed over silica gel using hexane:ether (9: 1). The clear, viscous oil was composed of (E)-27:(Z)-27 (9: 1). IR (KBr) 1720 cm 1; iH
NMR (DCC13) δ 1.39 [s, 6 HJ, 1 :40 [t, 3 H], 1.86 [t, 2 H], 2.26 [s, 3 HJ, 2.64 [s, 3 HJ, 4.24
[t, 2 H], 435 [q, 2 H], 6.80 [d, 1 H], 7.22 [m, 3 H], 7.41 [s, 1 HJ, 7.93 Id, 1 HJ. Mass spectral (El) data Calcd for C24H28O3 mlz (M+): 369.2038. Found: 369.2049. Anal. Calcd for C24H28O3: C, 79.08; H, 7.74. Found: C, 78.74; H, 7.81.
EXAMPLE XVIII
Ethyl (£)-4-[2-(3,4-Dihydro-2H- 1 -benzothiopyran-6-yl)- 1 -propenyljbenzoate (28)
To a suspension of phosphonium salt 71 (16.7 g, 32 mmol) in dry TΗF (50 mL) was added K2CO3 (4.47 g, 32 mmol), 18-crown-6 (80 mg), and ethyl 4-formylbenzoate (4.8 g, 30 mmol), and the mixture was boiled for 48 h. Water (10 mL) and glacial acetic acid (15 mL) were added successively to the mixture which was then allowed to cool to RT. Filtration gave a filtrate which was washed with brine, dried (Na2SO4), and concentrated to a brown oil which was triturated with ether to yield 28 (4.3 g, 40%) as a white solid with mp of 78-79 °C. IR (KBr) 1710 cm 1 ; 'Η NMR (DCCI3) δ 1.4 [t, 3 Η], 2.15 [q, 2 Η], 2.25
[s, 3 Η], [t, 3 ΗJ, 3.05 [t, 2 Η], 4.4 [q, 2 Η], 6.8 [s, 1 ΗJ, 7.05-8.05 [m, 7 ΗJ. Mass spectral (El) data Calcd for C2ιΗ22θ2S m/z (M+): 338.1337; Found: 338.1339. Anal. Calcd for C21H22O2S: C, 74.52; H, 6.55; S, 9.45. Found: C, 74.29; H, 6.51; S, 9.57.
Phosphonium salt 71 was prepared as follows. A mixture of thiochroman-4-one (3 g, 18 mmol), toluene (75 mL), water (120 mL), cone HCl (60 mL), and the Clemmenson- Martin amalgam (50 g) [E. L. Martin, Journal of the American Chemical Society, volume 58, pages, 1438-1432 (1936)] was boiled for 72 h with additional cone HCl (-20 mL) being added every 6 h to maintain a total volume of about 500 mL. After the mixture had cooled to RT, it was filtered, and the aqueous layer was extracted (toluene). The combined organic phases were washed with saturated NaHCO3, water, and brine. After drying (MgSU4), solvent was evaporated to give 3,4-dihydro-2H-l-benzothiopyran 86 as an oil (2.7 g, 98%) which was used directly to prepare 50. Properties of 3,4-dihydro-2H-l-benzothiopyran were as follows: IR (neat) 1680 cm-'; JH NMR (DCC13) δ 2.1 [q, 2 H], 2.8 [t, 2 HI, 3.0 [t, 2
HJ, 7.05-7.20 [m, 4 HJ. A solution of 3,4-dihydro-2H-l -benzothiopyran (6.0 g, 40 mmol) in nitromethane (25 mL) was added dropwise to a suspension of acetic anhydride (4.1 g, 40 mmol), AICI3 (1 1.6 g, 88 mmol), and nitromethane (50 mL) at 0 °C with stirring over 15 min. Stirring was continued (1 h), and then the system was allowed to warm to RT with continued stirring for 48 h. After cooling to 0 °C, the reaction mixture was quenched ( 150 mL of water), and the aqueous layer was extracted (ΗCCI3). Combined organic phases were washed with saturated NaHCO3, water, and brine. Drying (N 2SO4) and evaporation of the solvent caused oil 50 (7.6 g, 98%) to be released, and it was used directly to prepare 70. Properties of 50 were: IR (neat) 1680 cm 1 ; 'H NMR (DCCl3) δ 1.9 [q, 2 HJ, 2.35 [s, 3
HJ, 2.65 [t, 2 H], 2.85 [t, 2 H], 6.9-7.5 [m, 3 HJ. The mp of the 2,4-DNP of 50 (prepared in standard fashion) was 243-245 °C.
A solution of 50 (7.0 g, 37 mmol) in dry THF (35 mL) was added slowly to a stirred suspension of LiAlH4 (2.1 g, 55 mmol) in dry THF (140 mL) at RT after which time the mixture was boiled (6 h). After cooling to 0 °C, the solution was treated with EtOAc (75 mL over 0.75 h) followed by 5% HCl (-100 L over 0.5 h). This mixture was stirred for 5 min, and then it was extracted (HCCI3). Combined organic phases were washed with saturated NaHCO3, water, and brine. After drying (MgSO4), the solution was evaporated to an oil which was chromatographed over silica gel (EtOAc:hexane, 4: 1) to give 70 (5.0 g, 72%). This alcohol was used directly to prepare salt 71. Properties of 70 were: IR (neat)
3650 cm 1; l NMR (DCCI3) δ 1.64 [d, 3 HJ, 1.9-2.05 [s, 1 H], 2.3 [q, 2 H], 3.0 [t, 2 HJ, 3.2
[t, 2 H], 4.9 [q, 1 H], 7.2-7.3 [m, 3 HJ.
A solution of alcohol 70 (4.0 g, 23 mmol) and triphenylphosphine hydrobromide
(7.8 g, 23 mmol) in H2CCI2 (25 mL) was stirred at RT (24 h) and then concentrated to a foam which was dried under vacuum to give salt 71 (11.5 g, 97%) with a mp of 68-70 °C.
This salt 71 was used directly to prepare 28. Properties of salt 71 were: 'H NMR (DCCI3)
δ 1.1.6 [dd, 3 HJ, 1.8 [q, 2 HJ, 2.4 [t, 2 HJ, 2.8 ft, 2 HJ, 6.4-6.5 [m, 1 HJ, 6.65-7.9 [m, 18 HJ. EXAMPLE XIX
4-[(2,3-Dihydro- 1 ,4-benzodioxan-6-yl)carbamoylJbenzoic Acid (29)
To ester 39 (0.5 g, 1.59 mmol) in 95% ethanol (20 mL) was added dropwise a 2 N ΝaOH (10 eq, 15.9 mmol), and the solution was stirred for 4 h at RT. Careful acidification of the solution with 2 N HCl (~ 30 mL) produced a white solid (0.466 g, 1.4 mmol, 96%) which was filtered washed (water), dried, and recrystallized (absolute ethanol) to yield flakes of 29 which were dried and had a mp of 289-191 *C. IR (KBr) 3600, 3340, 1690 cnr ' ; !H ΝMR δ 4.23 [bs, 4 HJ, 6.83 [d, 1 HJ, 7.22 [ d, 1 HJ, 7.40 [d, 1 H], 8.05 [m, 4 HJ, 10.27
[s, 1 HJ. Mass spectral (El) data Calcd for Cι6Hι3O5Ν mlz (M+): 299.0794. Found: 299.0796. Anal. Calcd for C16H13O5N: C, 64.21 ; H, 4.38, N, 4.68. Found: C, 63.89; H, 4.59; N, 4.69.
EXAMPLE XX
(£)-3-[(2,3-Dihydro-4,4-dimethyl-2H- 1 -benzothiopyran-6-yl)- 1 -propenyl Jbenzoic Acid (30)
A solution of ester 33 (0.25 g, 0.54 mmol) in ethanol (10 mL), water (10 mL), and
NaOΗ (0.76 g, 1.9 mmol) was boiled for 6 h and then cooled slowly (0.5 h) to RT. After being cooled to 0 °C, the solution was treated carefully with cone ΗC1 (-5 mL, pΗ - 2) whereupon a solid precipitated and was filtered, washed (water), air-dried, and then dried under vacuum. White solid 30 (0.66 g, 87%) had a mp of 217-218 "C. IR (KBr) 3440,
1690 cm 1; 'Η NMR (DMSO-d6) δ 1.34 [s, 6 Η], 1.91 [t, 2 Η], 2.22 [s, 3 Η], 3.03 [t, 2 ΗJ,
6.93 [s, 1 ΗJ, 7.05 [d, 1 Η], 7.28 [d, 1 ΗJ, 7.53 [m, 1 Η],' 7.62 [m, 2 Η], 7.82 [d, 1 Η], 7.99 [s, 1 ΗJ. Mass spectral (El) data Calcd for C21 Η22O3 mlz (M+): 338.1340 Found: 338.1343. Anal. Calcd for C21H22O3: C, 78.01; H, 6.91. Found: C, 78.23; H, 6.88. EXAMPLE XXI
Methyl 4-[(2,2,4,4-Tetramethylthiochroman-6-yl)carbamoyl]muconate (31)
To the stirred solution of 2,2,4,4-tetramethyl-6-aminothiochroman (56, 0.64 g, 2.86 mmol) in benzene (50 mL) and pyridine (2.5 mL) was added at RT mono-methyl muconyl chloride (82) [prepared fresh from stirring /nønσ-mcthyl muconate (81, 0.05 g, 3.15 mmol) in excess thionyl chloride (20 mL, RT, 12 h) via standard procedures], and the resulting solution was stirred for 14 h. The mixture was then poured into water, and the aqueous layer was extracted with EtOAc and H2CCI2. Combined organic layers were washed with 2 N HCl, water, NaHCO3, water, and brine. Drying (Na2SO4) the organic solution and evaporating it gave crude ester 31 which was chromatographed over silica gel with hexane:ethyl acetate (2: 1) to yield a light yellow solid 31 (0.74 g, 71 %) with a mp of 167-
168 °C. IR (KBr) 3380, 1700, 1680 cm 'H NMR (DCCl ) δ 1.37 [s, 6 H], 1.40 [s, 6 HJ,
1.93 [s, 2 HI, 3.79 [s, 3 HJ, 6.17 [d, 1 HJ, 6.32 [d, 1 HJ, 7.06-7.76 [m 3 HJ, 7.82 [s, 1 H] Mass spectral (El) data Calcd for C20H25NO3S mlz (M+): 359.1555. Found: 359.1555 Anal. Calcd for C20H25NO3S: C, 66.82 H, 7.00; N, 3.89. Found: C, 66.78; H, 7.20; N, 3.80.
EXAMPLE XXII
4-[(2,3-Dihydro-2,2,4,4-.etramethyl-l ,l-dioxy-2H-l-benzothiopyran-6- yl)carboxamidoJbenzoic Acid (32)
Ester 35 (0.4 g, 1.0 mmol), 95% ethanol (5 mL), and 2 N ΝaOΗ (10 eq) were boiled for 1 h and then stirred at RT for 48 h. Acidification of the solution with 2 N ΗC1 (-50 mL) produced a white solid which was filtered, washed with water, dried, and recrystallized (95% ethanol) to give a flaky solid 32 (0.26 g, 65%) with a mp of 331-331.8 °C. IR (KBr) 3380, 3350, 1700 cπr1 ; !Η ΝMR (DMSO-t/6) δ 1.37 [s, 6 H], 1.46 [s, 6 H], 2.31 [s, 2 HJ,
7.88-8.12 [m 7 HJ, 10.68 [s, 1 HJ. Mass spectral (El) data Calcd for C2 ] H23ΝO5S mlz (M+): 401.1296; Found: 401.1296. Anal. Calcd for C21H23NO5S: C, 62.82; H, 5.77; N, 3.49. Found: C, 62.88; H, 5.94; N, 3.50.
EXAMPLE XXIII
Ethyl(E)-3-[(2,3-Dihydro-4,4-dimethyl-2H-l-benzothiopyran-6-yl)-l-propenyl]benzoate (33)
To a suspension of phosphonium salt 73 (3.3 g, 6.03 mmol) in dry ether (20 mL) was added dropwise /.-butyllithium (10 M, 0.72 mL, 7.25 mmol) over 5 min at RT. After the reddish mixture was cooled to -78 °C, a solution of ethyl 3-formylbenzoate (0.98 g, 5.50 mmol) in ether (25 mL) was added dropwise over 10 min. After being stirred for 0.5 h at - 78 °C, the solution was allowed to warm to RT, and then it was stirred for 48 h. The mixture was filtered, and the filtrate was dried (Na2SO4), and the solvent was evaporated. Chromatography of the residual solids over silica gel with hexane:ether (4: 1) gave solid (£)- 33:(Z)-33 (10: 1) which was treated with boiling ethanol, and the resultant solution was chilled for 24 h. A white solid formed and was washed with cold 95% ethanol to give (E)- 33 (0.45 g, 25%) as needles with a mp of 72.5-74 °C. IR (KBr) 1725 cm 1 ; >Η NMR (DCCI3) δ 1.39 [s, 6 HJ, 1.40 [t, 3 H], 1.97 [t, 2 HJ, 2.26 [s, 3 HJ, 3.05 [t, 2 H], 4.40 [q, 2
H], 6.80 [s, 1 HJ7.11 [d, 1 HJ, 7.21 [d, 1 H], 7.43 [m, 2 H], 7.52 [d, 1 HJ, 7.92 [d, 1 HJ, 8.04
[s, 1 HJ. Mass spectral (El) data Calcd for C23H26O2S mlz (M+): 366.1653; Found: 366.1660. Anal. Calcd for C23H26O2S: C, 75.37; H, 7.15. Found: C, 75.13; H, 7.20.
EXAMPLE XXIV
Methyl (£)-4-[2-(2,3-Dihydro- 1 ,4-benzodioxan-6-yl)- 1 -propenyljbenzoate (34)
To a suspension of phosphonium salt 61 (4.83 g, 9.57 mmol) in dry ether (10 mL) was added dropwise n-butyllithium (0.906 M, 2 mL, 1.81 mmol) in hexanes over 2 min at RT. After being stirred at RT for 0.5 h, the mixture was chilled to -78 °C, and then methyl 4-formylbenzoate (1.57 g, 9.57 mmol) in dry ether (10 mL) was added over 3 min. The mixture was allowed to warm to RT with stirring over 48 h. Filtration of the contents of the flask gave a filtrate which was evaporated to an oil that solidified. Recrystallization (minimum of absolute alcohol) led to 34 (0.7476 g, 25.19%) with a mp of 91.5-92.5 °C. IR
(KBr) 1710 cm-1 ; Η NMR (DCCI3) δ2.24 [d, 3 HJ, 3.92 [s, 3 HI, 4.27 [s, 4 H], 6.78 [bs, 1
HJ, 6.85-8.04 [m, ArHJ. Mass spectral (El) data Calcd for C19H18O4 mlz (M+): 310.1205; Found: 310.1206 Anal. Calcd for Cι9H ι8O4: C, 73.53; H, 5.84. Found: C, 73.75; H, 5.82.
Salt 61 was obtained as follows. Ketone 48 (2.0 g, 1 1.2 mmol) in dry THF (20 mL) was added dropwsie to a suspension of LiAlH4 (1.266 g, 33.3 mmol) in dry THF ( 10 mL) over 10 min. The resultant mixture was boiled for 24 h with aliquots of dry ether ( 10 mL each) being added to maintain volume after 8 and 16 h. The mixture was allowed to cool to RT, and then it was chilled in ice. Extremely cautious addition of water was initiated to destroy excess LiAlH4 over 0.5 h. A white precipitate formed, and the mixture, while still cool, was treated slowly with 5% HCl (80 mL) with stirring over 80 min. Two layers formed with a suspension in the aqueous layer. The aqueous layer was extracted (ether), and the combined extracts were washed with 5% NaHCO3 and then brine. After being dried (Na2SO4), the solution was evaporated to a clear oil, namely alcohol 60 (2.00 g, 98.6%), which was used to prepare salt 61 in the next step. Alcohol 60 (2.81 g, 15.5 mmol)
in dry methanol (20 mL) was added dropwise to a suspension of Ph3P*HBr (5.33 g, 15.5
mmol) in dry methanol (25 mL) over 10 min. Additional methanol (60 mL) was added to create a homogeneous solution which was then stirred at RT for 24 h. Evaporation of the solvent left a foam which solidified. The solid was crushed and treated with dry ether which gave a suspension that was stirred (3 h) and then filtered and dried. After washing the solid with dry ether and drying it, the residual white material 61 (7.72 g, 98.3%) had a mp of 153-158 °C (dec). It was used directly to prepare 34. EXAMPLE XXV
Ethyl4-[(2,3-Dihydro-2,2,4,4-tetramethyl- 1 , 1 -dioxy-2H- 1 -benzothiopyran-6-yl) carboxamidol benzoate (35)
To acid 76 (1.1 g, 3.89 mmol) was added thionyl chloride (25 mL) and DMF (4 drops), and the mixture was stirred at 0 °C for 12 h. Excess thionyl chloride was removed (aspirator), and the white solid formed was dried under vacuum at RT until no detectable odor of thionyl chloride was observed. Pyridine (35 mL) was added and the resulting solution was treated with ethyl 4-aminobenzoate (0.76 g, 4.6 mmol) and DMAP (- 10 mg). The final solution was heated at the boiling point of acetone for 3 h and then was stirred at RT for 24 h. Water was added and the resulting mixture was quickly extracted with ethyl acetate. The combined organic phases were washed with 2 N ΗC1, saturated NaΗCθ3, water, and brine. After drying (Na2SO4), the solvent was evaporated and the residual solid was dissolved in a minimum of HCCI3 and chromatographed over silica gel (HCCl3:H COH-50: 1) to yield a white solid which recrystallized (ethanol) to give colorless needles of 35 (0.98 g, 59%) with a mp of 245-247 °C. IR (KBr) 3360, 1715, 1675 cm"1 ; IH NMR (DCCI3) δ 1.32-1.45 [m, 15 H], 2.29 [s, 2 HJ, 4.37 [q, 2 HI, 7.85 [m, 4 H], 8.05 [m, 4
H], 8.95 [s, 1 HJ. Mass spectral (El) data Calcd for C23H27NO5S mlz (M+): 429.1610; Found: 429.1619. Anal. Calcd for C23H27NO5S: C, 64.31 ; H, 6.39; N, 3.20. Found: C, 64.45; H, 6.69; N, 3.31.
Acid 76 was prepared as follows. To a solution of ketone 52 (2.0 g. 8.0 mmol) in ethanol (20 mL) was added commercial clorox (150 L), and the turbid mixure was stirred
(24 h). After cooling (0 °C) the clear solution, a 25% solution of sodium metabisulfite
(-100 mL) was added very slowly followed by cone HCl (-30 mL). The white solid formed was filterd and recrystallized (95% ethanol) to give colorless needles of 76 ( 1.1 g, 51 %)
with a mp of 256-259 °C. IR (KBr) 3500, 1710 cm 1 : 'H NMR (DCCI3) δ 1.45 [s, 6 H], 1.50 [s, 6 HJ, 2.36 [s, 2 HJ, 8.12 [s, 2 HJ, 8.20 [s, 1 HJ. This acid 76 was used directly to prepare amide 35.
EXAMPLE XXVI
Ethyl (E)-4-[2-(3,4-Dihydro-l-oxy-2H-l-benzothiopyran-6-yl)- l-propenylJbenzoate (36)
To a stirred mixture of Ti(O-i-Pr)4 (14.9 mL, 5 mmol) and (-t-)-diethyl -tartrate (17.1 mL, 10 mmol) in Η2CCI2 (50 mL) was added water (0.9 mL). After the mixture was stirred to a homogeneous solution, sulfide 28 (0.02 g, 5 mmol) was added, and the mixture was cooled to -20 °C after which time tertiary-butyl hydroperoxide (0.5 g, 5.5 mmol) in H2CCI2 (1.6 mL) was added. Stirring was continued for 4 h at -20 °C, and then water (50 mL) was added dropwise (10 min). Stirring was continued another 1 h at -20 °C and then at RT for 1 h. Filtration removed a white gel, and evaporation of the filtrate yielded 36 as a light yellow solid which recrystallized (HCCI3 and ethanol) to give pure 36 (0.01 g, 50%)
with mp 90-92 °C. JH NMR (DCCI3) δ 1.30 [t, 3 H }, 1.90-2.05 [ m, 1 HJ, 2.2 [s, 3 HJ, 2.3-
2.5 [m, 2 H], 2.7-3.25 [m, 4 HJ, 4.3 [q, 2 HI, 2.5-2.8 [m, 4 H], 6.8 [q, 2 HJ, 6.8 [s, 1 H], 7.25-8.05 [m, 7 HJ. Mass spectral (ΕI) data Calcd for C2.H22O3S mlz (M+): 354.1289; Found: 354.1289. Anal. Calcd for C21H22O3S: C, 71.76; H, 6.26; S, 9.02. Anal. Calcd for
C21H22O3SO.5 H2O: C, 69.39; H, 6.38. Found: C, 69.24; H, 6.16.
EXAMPLE XXVII
Ethyl (2£,4£,6£)-7-(2,3-Dihydro-] ,4-benzodioxan-6-yl)-3-methyl-2,4,6-octatrienoate (37)
To a suspension of phosphonium salt 63 (4.0 g, 7.53 mmol) in dry ether (40 mL) was added dropwise n-butyllithium (0.906 M, 14 mL, 21.7 mmol) over 5 min. After stirring for 0.5 h, the reddish solution was treated with ethyl 3-mcthyl-4-oxocrotonate (1.3 mL, 9.15 mmol) in dry ether (10 mL), and then the resultant mixture was stirred at RT ( 10 h). Hexanes (100 mL) were added followed by filtration and evaporation to give a yellow oil. Crystals began to form in the oil at RT, and the process was allowed to continue for 12 h. Recrystallization (minimum of absolute ethanol) gave light yellow needles of 37 (0.683 g,
28.8%) with mp of 86-87 °C. IR (KBr) 1699 cm 1 ; JH NMR (DCC13) δ 1.3 [t, 3 HJ, 2.20 [s,
3 HJ, 2.37 [d, 3 H], 4.17 [q, 2 H], 4.27 [s, 4 HJ, 5.78 [bs, 1 H], 6.33 [d, 1 HJ, 6.51 [d, 1 HJ, 6.87-7.04 [m, 3 HJ. Mass spectral (El) data Calcd for C19H22O4 mlz (M+): 314.1510; Found: 314.1518 Anal. Calcd for C19H22O4 C, 72.59; H, 7.05. Found: C, 72.62; H, 7.11.
Salt 63 was obtained as follows. Ketone 48 (4.0 g, 22.4 mmol) in dry THF (25 mL) was added dropwise over 0.25 h to a vinylmagnesium bromide [obtained from reaction of vinyl bromide (20 g, 187.9 mmol) in dry THF (35 mL) by standard procedures] using a dry- ice condenser. After being held at reflux for 2 h, the reaction mixture was stirred at RT (10 h). The mixture was then cooled in a water bath, and a saturated NH4CI (20 mL) was added dropwise. Two layers formed and the aqueous layer was extracted (ether), and the combined extracts were washed with brine and then dried (Na2SO4). Evaporation left an oil 62 (-4.78 g, qt) which was used directly to prepare salt 63. Alcohol 62 (1.01 g, 4.92 mmol)
in dry methanol (20 mL) was added dropwise to a suspension of Ph P-HBr (2.61 g, 4.92
mmol) in dry methanol (20 mL) over 10 min at RT. The resultant solution became clear and was stirred at RT (10 h) and was then concentrated to a volume of about 5 mL. Dry ether (-200 mL) was added with stirring to ensure complete precipitation. Filtration produced the solid which was washed with dry ether and then dissolved in methanol (- 15 mL). Dry ether was added, and the solution was allowed to stand in a freezer for 15 h. Filtration and drying of the solid yielded white crystals of salt 63 (2.22 g, 84.9%) with a mp of 234-235 °C. IR (KBr) 1 110 cm" 1; *H NMR (DCCI3) δ 1.58 [d, 3 H], 4.22 [s, 4 HJ, 4.84
[dd, 1 H], 5.57 [m, 1 H], 6.68 [m, 3 H], 7.65-7.93 [m, 15 H]. This salt was used directly to prepare 37. EXAMPLE XXVIII
Ethyl 4-[(2,3-Dihydro-3,3-dimethyl-l, l -dioxybenzo[blthienyl-5-yl)carboxamido]benzoate (38)
A solution of acid 77 (0.87 g, 3.62 mmol), thionyl chloride (25 L), and DMF (5 drops) was stirred at 0 °C for 12 h. Excess thionyl chloride was removed (aspirator) leaving a white solid which was vacuum dried to remove traces of thionyl chloride. Ethyl 4- aminobenzoate (0.75 g, 4.59 mmol) and DMAP (-10 mg) were added, and the resulting brown solution was heated at the boiling point of acetone for 3 h and then was stirred at RT for 24 h. Water was added and the mixture was quickly extracted with ethyl acetate. The combined organic phases were washed with 2 N HCl, saturated ΝaHCO^, water, and brine. After drying (Νa2Sθ4), the solution was evaporated to a white solid which was dissolved in a minimum of HCCI3. This new solution was chromatographed over silica gel using HCCl3:H3COH (100: 1 ) and led to a solid which was recrystallized (ethanol) to give coloress needles of 38 (0.6 g, 37%) with mp of 172.3- 174 °C. IR (KBr) 3340, 1710, 1680 cm 1; lH NMR (DCCl3) δ 1.40 [t, 3 H], 1.52 [s, 6 HJ, 3.37 [s, 2 H], 4.37 [q, 2 HJ, 7.53 [d, 1
HJ, 7.79 [d, 2 H], 7.89 [d, 1 HJ, 8.71 [s, 1 HJ. Mass spectral (El) data Calcd for
C2oH2iNO5S m/z (M+): 387.1 140 Found: 387.1 140. Anal. Calcd for C20H2.NO5SO.25
H2O: C, 61.28; H, 5.52 N, 3.61. Found: C, 61.40; H, 5.60; N, 3.51.
Acid 77 was prepared as follows. To a solution of ketone 53 ( 1.0 g, 6.1 mmol) in ethanol (35 L) was added a commercial clorox ( 140 mL), and the turbid mixture was boiled (24 h). After cooling (0 °C) the clear solution, a 25% solution of sodium metabisulfite (-30 mL) was added very slowly followed by cone HCl (-50 mL). Another 70 mL of the sodium metabisulfite solution was added to complete the precipitation of a solid. The white solid was filtered and recrystallized (95% ethanol) to give colorless needles of acid 77 (0.91 g, 81 %) with a mp of 285.5-286.4 °C. IR (KBr) 3500, 1710 cm 1;
»H NMR (DCCI3) δ 1.50 [s, 6 H], 3.58 [s, 2 H], 7.82 [d, 1 HJ, 8.08 [d, 1 H], 8.17 [s, 1 HJ.
Acid 77 was used directly to prepare 38. EXAMPLE XXIX
Methyl 4-[(2,3-Dihydro-l ,4-benzodioxan-6-yl)carbamoyl]benzoate (39)
A stirred solution of amine 69 (1.19 g, 12.63 mmol) in benzene ( 150 mL) and pyridine ( 12 mL) at RT was treated with monomethyl terephthaloyl chloride (2.8 g, 14.43 mmol), and the resultant suspension was stirred for 12 h. The reaction mixture was poured into water, and the mixture was quickly extracted with EtOAc. Combined organic phases were washed with 2 N HCl, water, saturated ΝaHCθ3, water, and brine. After drying (MgSO4), the solution was evaporated to an off-white solid which was recrystallized (ethanol:HCCl3, 2: 1 ) to give pure 39 (2.8 g, 71%) with mp of 203-205 βC. IR (KBr) 3300,
1720, 1650 cm-1 ; 'H NMR (DMSO- 6) δ 3.90 [s, 3 H], 4.24 [s, 4 H], 6.83 [d, 1 HJ, 7.23 [d,
1 HJ, 7.41 [s, 1 H], 8.10 [m, 4 H J, 10.30 [s, 1 HJ. Mass spectral (El) data Calcd for
C17H25NO5S mlz (M+): 313.0947; Found: 313.0950. Anal. Calcd for C17H25NO5SO.25
H2O: C, 64.24; H, 4.91 ; N, 4.40. Found: C, 64.28; H, 4.75; N, 4.09.
Amine 69 was obtained as follows. To a solution of benzodioxan (67, 4.35 g, 31.85 mmol) in acetic anhydride (8 mL) was added dropwise a solution of HNO3 (3 mL) and acetic anhydride (9 mL) at 0 °C over 10 min. A thick yellow suspension formed and was stirred at RT (4 h). This mixture was poured slowly and cautiously into saturated NaHCO3 (-250 mL), and the resultant mixture was extracted (H2CCI2). The organic layer was separated, washed with water and brine, and then dried (MgSO4). Evaporation of the
solvent left 68 (5.5 g, 94%) with a mp of 1 14-1 17 °C. IR (KBr) 1530, 350 cm 1 ; Η NMR
(DCCI3) δ 4.30-4.41 [ , 4 HJ, 6.91 [d, I H], 7.41 [s, 1 H], 7.77 [m, 1 HJ. This 68 was used
as follows to prepare amine 69. To ether 68 (4.0 g, 22.0 mmol) in acetic acid (1 10 mL) and water (5 mL) was added dropwise TiCl3/HCl (198.6 g, 15.46 mmol) at RT over a few min, and the resulting mixtrure was stirred at RT ( 12 h). After cooling (0 °C) the reaction mixture, 30% NaOH (-500 mL) was added slowly. Extracts (EtOAc and HCCI3) of the aqueous layer were combined, washed with water and saturated NaHCO3, dried (MgSO4), and evaporated to give an oil which was chromatographed over silica gel (H2CCl2:ethyl acetate, 3: 1 ) and gave oil 69 (2.08 g, 62%) that was used directly to prepare 39. Properties of 69 were as follows: Η NMR (DCC13) δ 3.36 [bs, 2 H|, 4.12-4.18 [m, 4 HJ, 6.17 [m, 2
HJ, 6.64 [d, 1 HJ.
EXAMPLE XXX
Ethyl (2£,4£,6£)-7-( 1 ,3-Benzodioxol-5-yl)-3-methyl-2 ,4,6-octatr ienoate (40)
To a suspension of phosphonium salt 65 (3.00 g, 5.8 mmol) in dry ether (40 mL) was added .. -butyl-lithium (1.6 , 3.7 mL, 5.9 mmol) in hexane over 2 min at RT. The dark mixture was stirred at RT for 0.5 h and then was cooled to -78 °C. A solution of ethyl
(£)-β-formylcrotonate (0.82 g, 5.8 mmol) in dry ether (10 mL) was added dropwise over 5
min after which time the mixture was allowed to warm to RT with stirring over 6 h. Hexanes (-20 mL) were added dropwise, and the resultant mixture was filtered. The isolated powder was washed with hexanes:ether and then with hexanes which were then combined with the filtrate and concentrated. Crystallization began upon standing, and crystals were collected and washed with cold hexanes and dried. Pale yellow crystals of 40
(0.25 g, 14%) were obtained and had a mp of 70-70.5 °C. IR (KBr) 1698 cπr1 ; ^ NMR
(DCCI3) δ 1.29 [t, 3 HJ, 2.20 [d, 3 HJ, 2.37 [d, 3 H], 4.18 lq, 2 HJ, 5.80 [bs, 1 HJ, 5.96 [s, 2
H], 6.35 [d, 1 HJ, 6.49 [m, 1 H], 6.79 [m, 1 H], 6.94-7.05 [m, 4 HJ. Anal. Calcd for C18H20O4: C, 71.98; H, 6.71. Found: C, 72.27; H, 6.71.
Salt 65 was prepared as follows. Ketone 49 (2.00 g, 12.2 mmol) in dry THF (35 mL) was added dropwise to a cooled (0 °C) solution of freshly prepared vinylmagnesium bromide [obtained from vinyl bromide (4.9 g, 46 mmol) and Mg (0.90 g, 37 mmol) in THF (25 mL)] over 0.5 h. The cold bath was removed, and the resultant mixture was stirred at RT (1 h). The mixture was cooled again and then quenched by slow addition of water and then ether (-100 mL) was added to form two layers. Extracts (ether and H2CCI2) of the aqueous layer were combined, dried (Na2SO4), and evaporated to a yellow oil 64 (2.32 g,
99%). IR (neat) 3650 cm-1.
This alcohol 64 was used directly to prepare salt 65 as follows. A solution of 64
(2.41 g, 12.5 mmol) and Pr^P-HBr (4.30 g, 12.5 mmol) in methanol (30 mL) was stirred at
RT (1 1 h). Filtering the solution and evaporating the solvent gave an oil which was triturated with cold ether to induce solid formation. The solid was filtered, washed (ether), dried, and recrystallized (HCCI3) to yield 65 (5.32 g, 82%) with a mp of 226.5-227.2 °C
(dec). IR (KBr) 1501, 1440 cm 1 ; »H NMR (DCCI3) δ 1.60 [d, 3 HJ, 4.87 [dd, 2 HJ, 5.57
[m, 1 HJ, 5.93 [s, 2 HJ, 6.6-6.9 [m, 3 HJ, 7.65-8.00 [ , 15 HJ. This salt 65 was used to prepare ester 40.
EXAMPLE XXXI
4-[(2,3-Dihydro-3,3-dimethyl-l ,l -dioxybenzo[b]thienyl-5-yl)carboxamidolbenzoic Acid (41)
A solution of ester 38 (0.2 g, 0.51 mmol) in absolute ethanol (8 mL) containing 2 N
NaOH (10 eq, 5.1 mmol) was stirred at RT for 12 h. Careful acidification of the solution with 2 N HCl (-50 mL) produced a white solid which was filtered, washed (water), dried, and recrystallized (ethanol) and led to flakes of 41 (0.12 g, 65%) precipitating from a chilled solution. The mp was 303.5-304.5 °C. IR (KBr) 3500, 3390, 1700, 1690 cm '; *H ΝMR (DMSO- ) δ 1.53 [s, 6 HJ, 3.60 [s, 2 H], 7.88-8.20 [m, 7 H], 10.72 [s, 1 HJ. Mass
spectra (El) data Calcd for Cι8H ι7O5SΝ mlz (M+): 359.0827; Found: 359.081 1 Anal. Calcd for C18H17O5NS: C, 60.15; H, 4.76; N, 3.89. Found: C, 60.00; H, 4.85; N, 3.78. EXAMPLE XXXII
(2£,4£,6£)-7-( 1 ,3-Benzodioxol-5-yl)-3-methyl-2,4,6-octatrienoic Acid (66)
A mixture of ester 40 ( 140 mg. 0.466 mmol), absolute ethanol (2 mL), and 35% aqueous KOH (0.5 mL) were heated and stirred at reflux in the dark for 1 h. After cooling to RT, the reaction mixture was treated with water (5 mL), EtOAc (50 mL), and AcOH acid:H2O ( 1 : 1, 0.8 mL). Two layers separated and the aqueous layer was extracted with EtOAc. Combined organic phases were dried (Na2SO4), filtered, and evaporated to a solid which was recrystallized (absolute ethanol) to a light yellow powder that was washed with cold ethanol and then hexanes. After drying, the yellow powder 66 (86 mg, 68%) had a mp of 199.5-200.0 °C. IR (KBr) 3150, 1680 cm 1 ; JH NMR (DCCI3) δ 2.21 [s, 3 HJ, 2.39 [s, 3
HJ, 5.83 [s, 1 H], 6.39 [d, 1 HJ, 6.51 [d, 1 HJ, 6.80 [m, 1 HJ, 6.95-7.12 [m, 3 HJ. Anal. Calcd for Cι6Hι6O4: C, 70.57; H, 5.92. Found: C, 70.26; H, 5.81.
EXAMPLE XXXIII
n-Octy l(£)-4-[2-(3,4-Dihydro-2-M-octyl- 1 -oxy-2H- 1 -benzothiopyran-6-yl)- 1 -propenylj benzoate (94)
A solution of sulfide 93 ( 1.02 g, 2.2 mmol, R = n-octyl) in H2CCI2 (15 mL) was
added dropwise a homogeneous solution of water (41 μL), Ti(/-OPr)4 (3.7 mL, 2.2 mmol),
and (+)-diethyl L-tartrate [(+)-DET] in H2CC12 (50 mL). This mixture was cooled (-20 °C), and tertiary-butyl hydroperoxide (TBHP) (0.19 g, 2.2 mmol) was added dropwise over 10 min. Stirring was continued for 1 h and then the temperature was allowed to rise to RT with stirring for 1 h. A suspension formed and was filtered to give a clear filtrate that was dried (Na2SO4) and concentrated to give 94 as a light yellow solid which was crystallized
(ethanol) to pure 94 (0.2 g, 30%) with mp of 122-124 °C. iH NMR (DCCI3) δ 0.9 [t, 3 H],
1.1-2.0 [m, 18 H], 2.2-2.3 [s, 3 H], 2.4-2.6 [m, 1 H], 2.7-3.2 [m, 4 H], 4.3 [q, 2 HJ, 6.8 [s, 1 HJ, 7.25-8.05 [ , 7 HJ. [α] = + 29.9° (acetone). Anal. Calcd for C29H38O3S -0.5 H2O: C,
73.22 H, 8.24.. Found: C, 73.09 H, 48.14.
The ethyl analog 93 was prepared in a manner similar to that outlined for 94 and had
a mp of 63-64 °C. ]H NMR (DCC1 ) δ 1.05 [t, 3 H], 1.40 [t, 3 HJ, 1.6-1.85 [m, 3 HJ, 2.25-
2.35 [m, 4 HJ, 2.8-2.9 [m, 2 H], 3.2-3.3 [m, 1 H], 4.4 [q, 2 H], 6.8 [s, 1 H], 7.05-8.15 [m 7 HJ. Mass spectral (El) data Calcd for C23H26O2S m/z (M+): 366.1653. Found: 366.1650. Anal. Calcd for C2 H26O2S: C, 74.64; H, 7.19. Found: C, 74.68; H, 7.30.
Sulfide 93 was prepared as follows. To a boiling mixture of phosphonium salt 92 (2.0 g, 3.0 mmol, R = n-octyl) in H2CCI2 (15 mL) containing 18-crown-6 (30 mg) and K2CO3 (0.4 g, 3.0 mmol) was added ethyl 4-formylbenzoate in a single portion. After boiling for 12 h, the solution was evaporated to an oil which was treated with hexane to generate a suspension. Filtration gave a filtrate that was washed with brine, dried (Na2SO4), and concentrated to a light yellow oil which was chromatographed on silica gel (hexane.EtOAc, 1 : 1 ) to yield 93 ( 1.1 g, 79%, R = n-octyl) with a mp of 72-73 "C. IR (KBr) 1750 cm 1 : JH NMR (DCCI3) δ 0.95 [t, 3 H], 1.1-1.7 [m, 17 H], 2.8-2.95 [m, 1 HJ, 2.15-2.3
[m, 4 H], 2.8-2.9 [m, 2 H], 3.1-3.2 [m, 1 H], 4.3 [q, 2 HJ, 6.8 [s, 1 H], 7.0-8.1 lm, 7 HJ. Mass spectral (El) data Calcd for C29H38O2S mlz (M+): 450.2593 Found: 450.2595. Anal. Calcd for C29H38O2S: C, 77.29 H, 8.51. Found: C, 77.61; H, 8.31.
Salt 92 was prepared as follows. A solution of alcohol 91 (4.4 g, 14.0 mmol, R = n- octyl) and triphenylphosphine hydrobromide (5.0 g, 14.0 mmol) in H2CCI2 (50 mL) was stirred at RT (24 h). The solvent was evaporated and the oil ws triturated with cold dry ether to yield white solid 92 (9.0 g, 98%), R = n-octyl) with a mp of 67-68 °C. Η NMR
(DCCI3) δ 0.9 [t, 3 H], 1.1-1.6 [m, 4 H], 1.7-1.95 [m, 4 H], 2.0-2.2 [ , 1 H], 2.5-2.7 [m, 1
HJ, 2.8 [m, 1 HJ, 3.0-3.1 [m, 1 H], 6.55 [m, 1 H], 6.9-7.1 [m, 3 HJ.
Alcohol 91 was prepared as follows. Ketone 90 (1.5 g, 6.0 mmol, R = n-octyl) in dry ether (15 mL) was added with stirring to a suspension of LiAlH4 (0.38 g, 9 mmol) in dry ether (10 mL), and the mixture was boiled for 6 h. After cooling (0 °C), the solution was treated with ETOAc (25 mL) followed by 5% HCl ( 10 mL, 10 min). Extracts (ether) of the aqueous layer were combined and washed with NaHCO3, water, and brine. After drying (MgSO4), the solution was evaporated to an oil which was chromatographed over silica gel (hexane:ether, 1 : 1 ) to yield alcohol 91 (1.2 g, 80%, R = n-octyl) which was used directly to make 92. Properties of 91 were: IR (neat) 3350 cm 1 ; 'H NMR (DCC13) δθ.9 [t, 3 HI, 1.1 -
1.6 [m, 17 H], 1.75-1.95 [m, 2 H], 2.0-2.2 [m, 1 HJ, 2.8 [m, 2 H|, 3.1-3.15 [m, 1 HJ, 4.65- 4.8 [m, 1 HJ, 6.9-7.1 [m, 3 HJ. This alcohol 91 was used directly to prepare salt 92.
Ketone 90 was prepared as follows. 2-Octylthiochroman (89, 1.5 g, 6 mmol) in CS2:H3CNO2 (1 :5, 25 mL) was added to a stirred suspension of acetic anhydride (0.5 mL, 6 mmol), AICI3 (11.4 g, 9 mmol) in nitromethane (50 mL) at 0 °C. After 1 h, the suspension was allowed to warm to RT and then was stirred for 48 h. The resultant soution was cooled (0 °C) and then quenched with water (50 mL). Extracts (HCCI3) of the aqueous layer were combined and washed with NaHCO3, water, and brine. The dried (Na2SO4) solution was evaporated to an oil (quantitative yield) which was used directly to make alcohol 91.
Properties of ketone 90 were: IR (neat) 1680 cm 1 ; JH NMR (DCCI3) δ 0.9 [t, 3 H], 1.35-
1.45 [m, 14 H], 2.0 [m, 1 HJ, 2.3 [m, 1 H], 2.7 [s, 3 HJ, 2.9-3.3 [m, 3 HJ, 7.2-7.7 [m, 3 HJ.
Thiochroman 89 was prepared as follows. Sulfoxide 88 (1.7 g, 6 mmol, R = n-octyl) and Nal (2.2 g, 15 mmol) in acetone (20 mL) at 0 °C was added with stirring to trifluoroacetic anhydride (TFAA, 2.5 mL, 18 mmol) in acetone (25 mL). After the mixture was stirred (1 h), the acetone was evaporated and water (50 mL) was added. Extracts (ether) of the aqueous layer were washed with saturated Na2S2θ3, water, and brine. The dried (MgSO4) solution was evaporated to an oil which was chromatographed over silica gel (hexane) to give sulfide 89 (1.5 g, 95%, R = n-octyl) which was used directly to make
ketone 90. Properties of 89 were: Η NMR (DCCI3) δ 0.88 [t, 3 HJ, 1.2-1.5 [bs, 12 H], 1.6-
1.8 [m, 3 HJ, 2.2-2.3 [m, 1 HJ, 2.75-2.9 [m, 2 H], 3.25-3.57 Lm, 1 HJ, 6.9-7.1 [m, 4 HJ. Sulfide 88 was prepared as follows. 3,4-Dihydro-2-n-octyl-2H-l-benzothiopyran-l- oxide (87, 5.72 g, 30 mmol) in dry TΗF (50 mL) was added to a solution of lithium diisopropylamide (LDA) in dry TΗF (50 mL) at -78 °C over 15 min. The solution was allowed to warm to -30 °C ( 1 h) and then was cooled to -78 °C whereupon n-octyl bromide 5 (5.4 mL, 34 mmol) was added in a single portion. After this solution was stirred for 12 h, cautious addition of 5% ΗC1 (-50 mL) followed. Extracts (ΗCCI3) of the aqueous phase were combined and washed with water, NaHCO3, and brine. When dried (Na2SO4), the solution was evaporated to an oil which was chromatographed over silica gel (hexane:HCCl3:EtOAc, 4: 1 : 1) to yield 88 (3.2 g, 40%, R = n-octyl) which was used directly
10 to prepare 89. Properties of 88 were: !H NMR (DCCI3) δ 0.9 [t, 3 H], 1.2- 1.4 [bs, 12 HJ,
1.4-1.7 [ , 2 HJ, 1.85 [m, 1 H], 1.85 [m, 1 H], 2.45 [m, 2 HJ, 2.8-3.1 [m, 3 H], 7.1-7.8 [m, 4 HJ.
Intermediate 87 was prepared according to a literature procedure [T. Takata, Y Mayuni, K. Fujimori, H. K. Young, T. Iyangi, and S. Oae, Bull. Chem. Soc. Jpn., volume 15 56, pages 2300-2310 ( 1983)]. The preparation of intermediate 86 (3,4-dihydro-2H-l - benzothiopyran) was described under Example XVI and was a modification of a reported procedure [T. Takata, Y Mayuni, K. Fujimori, Η. K. Young, T. Iyangi, and S. Oae, Bull. Chem. Soc. Jpn., volume 56, pages 2300-2310 (1983)J.
0 EXAMPLE XXXIV
4-[4,4-Dimethyl-3,4-dihydro-2H-benzo[bJpyran-6-yl)carbonyloxy]benzaldehyde (96).
In a standard system was placed the carboxylic acid 95 (0.300 g, 1.45 mmol) and 4- formylphenol (0.265 g, 2.17 mmol) in CΗ2CI2 (20 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-0.895 g, 4.33 mmol) and a catalytic amount of DMAP (10 mg). 5 The resulting cloudy solution was stirred at RT (48 h) and then filtered. The filtrate was cooled to 0 °C for 24 h and then filtered again. The solvent was then evaporated in vacuo, and the resulting oil was allowed to stand at RT for 24 h during which time crystallization took place. The resulting crystals were recrystallized from hexane:EtOAc (3: 1). The solid 96 was filtered and weighed 0.195 g, (43%), mp 132-133.5 °C. IR (KBr) 2724, 2850, 1700,
1730 cm 1 ; !H NMR (DCCl3) δ 1.40 [s, 6 H], 1.87 [t, 2 H], 4.30 [t, 2 HJ, 6.88 [d, 1 HJ, 7.39
[d, 2 HJ, 7.91 [d, 1 H], 7.97 [d, 2 H], 8.13 [d, 1 HJ, 10.02 [s, 1 H]. Mass spectral (FAB) data Calcd for Cι9Hι8O5:/n/z M(+): 310. Found: 31 1 (MH+).
EXAMPLE XXXV
4-[(4,4-Dimethyl-3,4-dihydro-2H-benzo[_'Jpyran-6-yl)carbonyloxy]benzaldehyde thiosemicarba-zone (97).
Thiosemicarbazide (0.017 0.184 mmol) was dissolved in Η2O (1 mL) and AcOH (I drop). Aldehyde 96 (0.050 0.161 mmol) was dissolved in hot EtOH (3 mL), and the solution was added hot to the thiosemicarbazide solution. Crystals began forming after 10 sec. The mixture was allowed to stand at RT for 8 h and then was cooled in the refrigerator for 12 h. The crystals were filtered and dried in the Abderhalden (P2O5, RT, 12 h) to give the thiosemicarbazone 97 in a yield of 81 % (0.050 g), mp 185- 186 °C. IR (KBr) 3436,
3403, 3266, 3164 (N-H) cm 1; lH NMR (DCCl3)δ 1.33 [s, 6 HJ, 1.82 [t, 2 H], 4.26 [t, 2 HJ,
6.90 [d, 1 H], 7.31 [d, 2 HJ, 7.82 [dd, 1 HJ, 7.84 [d, 2 HJ, 8.06 [d, 1 H], 8.21 [s, 2 H], 1 1.46 [s, 1 HJ. Anal. Calcd for C20H21N3O4S: C, 62.64; H, 5.52; N, 10.96. Found: C, 62.64; H, 5.44; N, 10.83.
EXAMPLE XXXVI
Ethyl 2-(4,4-Dimethyl-3,4-dihydro-2H-benzo[£]pyran-6-ylcarbonyloxy)indolate (99)
In a standard system was placed the carboxylic acid 95 (0.150 g, 0.73 mmol) and ethyl 5-hydroxyindole-2-carboxylate (0.179 g, 0.87 mmol) in CΗ2CI2 ( 15 mL), and the solution was cooled to 15 °C. To this solution was added dicyclohexylcarbodiimide
(-0.225 g, 1.09 mmol) and a catalytic amount of DMAP ( 10 mg). The resulting clear solution was stirred at RT (24 h) and then filtered. The filtrate was cooled to 0 °C for 24 h and filtered. The solvent was evaporated in vacuo, and the heavy oil (with a few solid particles) was subjected to circular chromatography (CH2θ2:MeOH, 100: 1). The off white solid was recrystallized (hexane:EtOAc, 3: 1) to give white crystals of 99 in a yield of 56% (0.160 g); mp 179- 180 °C. IR (KBr) 331 1, 1722, 1692 cm- > ; J H NMR (DCC13) δ 1.43 [t, 9
H], 1.89 [t, 2 H], 4.29 [t, 2 H], 4.42 [q, 2 H HzJ, 6.88 [d, 1 HJ, 7.16 [dd, IH], 7.22 [d, 1 H], 7.5 [d, 1 H], 7.95 [dd, 1 H], 8.18 [d, 1 HJ, 8.99 [s, 1 HJ. Anal. Calcd for C23H23NO5: C, 70.21 ; H, 5.89. Found: C, 69.94; H, 6.05.
EXAMPLE XXXVII
Ethyl 4-[(4,4-Dimethyl-3,4-dihydro-2H-benzo[fr]pyran-6-yl)carbonyloxy]nicotinate (100)
In a standard system was placed the carboxylic acid 95 (0.200 g, 0.97 mmol) and ethyl 2-hydroxynicotinate (0.209 g, 1.25 mmol) in CΗ2CI2 ( 15 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-0.75 g, 3.60 mmol) and a catalytic amount of DMAP (10 mg). The resulting clear solution was stirred at RT (24 h) and then filtered. The filtrate was cooled to 0 °C for 24 h and filtered. The solvent was evaporated in vacuo, and the heavy oil (with a few solid particles) was subjected to chromatography (CH2Ch:MeOH, 100: 1). The off white solid was recrystallized (hexane:EtOAc, 4: 1) to give white crystals of
100 in a yield of 43% (0.147 g); mp 52-53 °C. !H NMR (DCCI3) δ 1.39 [s, 6 H], 1.42 [t, 3
H], 1.88 [t, 2 H], 4.29 [t, 2 HJ, 4.43 [q, 2 H], 6.87 [d, 1 H], 7.29 [d, 1 H], 7.94 [dd, 1 H], 8.16 [d, 1 HJ, 8.43 [dd, 1 H], 9.07 [d, 1 HJ.
EXAMPLE XXXVIII
Ethyl 4-[(4,4-Dimethyl-2,3-dihydro-2H-benzo[b]furan-5-yl)carbonyloxyjbenzoate (102)
In a standard system was placed the carboxylic acid 59 (0.137 g, 0.71 mmol) and ethyl 4-hydroxybenzoate (0.130 g, 0.78 mmol) in CΗ2CI2 ( 15 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-.441 g, 2.14 mmol) and a catalytic amount of DMAP (10 mg). The resulting clear solution was stirred at RT (24 h) and then filtered. The filtrate was cooled to 0 °C and filtered again. The solvent was evaporated in vacuo, and the heavy oil (with a few solid particles) was subjected to chromatography (CH2CI2). The resulting oil was dissolved in EtOAc and placed in a hexane chamber where it crystallized to give 102 as a white solid in a yield of 43% (0.105); mp 89-90 °C; IR (KBr) cm 1 ; Η
NMR (DCCI3) δ 1.41 [t, 9 H], 4.38 [q, 4 H], 6.87 [d, 1 HJ, 7.27 [d, 2 HJ, 7.94 [d, 1 H], 8.05
[dd, 1 H], 8.12 [dd, 2 HJ. Anal. Calcd for C20H20O5: C, 70.57; H, 5.92. Found. C, 70.64; H. 6.1 1.
EXAMPLE XXXIX
Methyl 4-(4,4,5,7-Tetramethyl-6-coumarin)benzoate (104).
In a standard system was placed the coumarol 103 (0.125 g, 0.568 mmol) and mono- methylphthlalic acid (0.122 g, 0.682 mmol) in CH2CI2 (10 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-0.352 g, 1.7 mmol) and a catalytic amount of DMAP (10 mg). The resulting cloudy solution was stirred at RT (36 h) and then filtered. The solvent was evaporated in vacuo, and the heavy oil was subjected to chromatography (0.67: 1.33: 15, hexane:EtOAc:CH2Cl2). The white solid ester 104 was obtained in a yield of
51 % (0.109 g), mp 174-175 "C. IR (KBr) 1737, 1788 cm"1 ; lH NMR (DCCI3) δ 1.48 [s, 6
HJ, 2.16 [s, 3 H], 2.29 [s, 3 HJ, 2.62 [s, 2 HJ, 3.99 [s, 3 HJ, 6.87 [s, 1 H], 8.20 [d, 2 HJ, 8.30 [d, 2 HJ. Anal. Calcd for C22H22O : C, 69.15; H, 5.80. Found: C, 68.81 ; H, 5.74. Mass spectral (El) data Calcd for C22H22O6: 382.1416. Found: 382.141 1. EXAMPLE XL
3,4-Dihydro-2,6-dihydroxy-2,4,4,5,7-pentamethyl-2H- 1 -benzopyran (105).
In a standard system was placed 0.95 mL of a 3 M solution of methylmagnesium bromide (2.85 mmol) and ether (5 mL). The coumarol 103 (0.250, 1.1 mmol) in TΗF (2.5 mL) was added to the flask at 10 °C. The resulting solution was stirred for 24 h at RT during which time the solution turned to a cloudy yellow mixture. Ammonium chloride was slowly added to this mixture to destroy residual Grignard reagent. The layers were separated, and the aqueous layer was extracted with ether (5 x 10 mL). The combined organic layers were washed with Η2O (30 mL) and brine (30 mL) and then dried (MgSO4) overnight. The solvent was evaporated in vacuo and the resulting light yellow oil was dissolved in hexane:EtOAc (2: 1) and cooled to 0 °C where upon crystallization occurred to give the white solid 105 in a yield of 39% (0.100 g), mp 105.5-106.5 °C. IR (neat) 3472,
3408 cm 1 ; !H NMR (DCCI3) δ 1.41 [s, 3 H], 1.58 [s, 3 HJ, 1.62 [s, 3 HJ, 1.95 [dd, 2 H],
2.18 [s, 3 H], 2.38 [s, 3 H], 4.31 [s, 1 H], 6.52 [s, 1 HJ. The compound was use directly to prepare 106.
EXAMPLE XLI
6-Hydroxy-2,4,4,5,7-pentamethyl-3H-l -benzopyran (106)
In a standard system was placed lactol 105 (0.100 g, 0.42 mmol), a catalytic amount of p-toluenesulfonic acid (0.025 g), 4 A molecular seives ( 1.0 g), and toluene (5 mL). The mixture was boiled for 2 h (showed completion by TLC). The mixture was allowed to cool to RT and filtered. The filtrate was washed with saturated NaΗCθ3 (10 mL) and brine (10 mL) and then dried (Na2SO4). The solvent was evaporated in vacuo to give 106 as a light
yellow oil in a yield of 89% (0.082 g). IR (neat) 3576 cm ' ; 'H NMR (DCCI3) δ 1.43 [s, 6 HJ, 1.85 [s, 3 HJ, 2.19 [s, 3 HJ, 2.33 [s, 3 HJ, 4.33 [s, 1 H], 4.39 [s. 1 H], 6.60 [s, 1 H]. The compound was used directly to prepare 107.
EXAMPLE XLII
Methyl 4-(4,4,5,7-tetramethyl-6-coumarin)benzoate (107).
In a standard system was placed the chromenol 106 (0.150 g, 0.688 mmol) and mono-methylphthlalic acid (0.161 g, 0.894 mmol) in CH2CI2 (10 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-0.425 g, 2.06 mmol) and a catalytic amount of DMAP (7 mg). The resulting cloudy solution was stirred at RT (24 h) and then filtered. The filtrate was cooled (0 °C) overnight and filtered again. The solvent was evaporated in vacuo, and the heavy oil was subjected to chromatography (CH2CI2). The white solid ester
107 was obtained in a yield of 50% (0.130 g), mp 142- 143 °C. IR (KBr) 1736 cm-1 ; > H NMR (DCCI3) δ 1.44 [s, 3 H], 1.57 [s, 3 H], 2.12 [s, 3 HJ, 2.27 [s, 3 HJ, 3.98 [s, 3 H], 4.39
[s, 1 H], 6.72 [s, 1 H], 8.18 [dd, 2 HJ, 8.30 [dd, 2 HJ. Mass spectral (El) data Calcd for C22H22O6WΛ (M+): 382.1416. Found: 382.141 1. Anal. Calcd for C22H22O6: C, 69.15; H, 5.80. Found: C, 68.81 ; H, 5.74.
EXAMPLE XLII1
N-(4-BromophenyI)-3-methyl-2-butenamide (109).
4-BromoaniIine (108) (6.13 g, 0.035 mol) in 225 mL of HCCI3 was placed in a standard system. 3,3-Dimethylacryloyl chloride (2.06 g, 0.017 mol) in HCCI3 (10 mL) was then slowly added to the brown solution. The resulting cloudy, tan mixture was then boiled for 5 h. After cooling to RT, the precipitate was removed by vacuum filtration. The chloroform solution was washed with 2 M HCl (2 x 50 mL), satd NaHCO3 (2 x 50 mL), and brine. After drying (MgSO4) overnight, the solution was concentrated in vacuo. Recrystalliztion (95% ethanol) of the product 109 gave off-white flaky crystals (mp 118- 1 19 °C) in a yield of 87% (7.70 g). IR (KBr) 3320, 1680 cm ' ; l NMR (DCC13) δ 1.88 [s,
3 HJ, 2.21 [s, 3 H], 5.69 [t, 1 H], 7.26-7.45 [m, 4 HJ. Mass spectral (FAB) data Calcd for CnHi2BrNO /z (M+): 253. Found: 254 (MH+). The compound was used directly to make 110.
EXAMPLE XLIV
6-Bromo-4,4-dimethyl-2-oxo- 1,2,3,4-tetrahydroquinoline (110).
In a standard system was placed the anilide 109 (2.01 g, 7.94 mmol). The system was then heated to 130- 140 °C, and then AICI3 (1.57 g 0.012 mol) was added portion wise over 1 h. The flask was the allowed to cool to 80° C, and one final portion of AICI3 (0.2 g) was added. The mixture was then stirred at that temperature for 0.5 h. The flask was then allowed to cool to RT, and 20 mL of ice water was added to the dark brown solid. Chloroform (50 mL) was then added to dissolve the solid. The solution was then stirred for 15 minutes, and 10 mL of 2 M HCl was added. The layers were separated, and the aqueous layer was extracted with HCCI3 (3 x 40 mL). The combined organic layers were washed with satd NaHCO3 (40 mL) and brine (40 mL). After drying (MgSO4) overnight, the solution was concentrated in vacuo to give a yield of 78% (1.74 g) of crude orange solid. Recrystallization (95% ethanol) of the product gave white crystals of 110 (mp 174-175 °C) in a yield of 65% (1.30 g). IR (KBr) 3240, 1700 cm 1 ; >H NMR (DCCI3) δ 1.6 [s, 6 HJ,
2.48 [s, 2 H], 6.7-7.40 [m, 3 HJ. Mass spectral (FAB) data Calcd for CnH^BrNO mlz
(M+): 253. Found: 254 (MH+). The lactam was used directly to make 111.
EXAMPLE XLV
6-Bromo-4,4-dimethyl- 1,2,3,4-tetrahydroquinoline (111).
In a standard system was placed the lactam 110 (1.0 g, 3.95 mmol) in 10 mL of dry, distilled toluene. The system was cooled in an ice bath to 0-4 °C, and the 10 M borane- dimethylsulfide (0.31 g, 4.10 mmol, 5% excess) was added dropwise from a syringe. The reaction mixture was stirred at 0 °C for 15 minutes and then was boiled for 5 h. The flask was allowed to cool to RT, and then 15 mL of 10% Na2CO3 was added. The solution was stirred at RT for 30 minutes. The toluene layer was separated and dried (MgSU4) overnight. The solution was concentrated in vacuo to give the quinoline 111 as a light yellow oil (0.85 g, 90%) which was used without further purification to make 112. IR
(neat) 3420 cm"1 ; >H NMR (DCC13) δ 1.26 [s, 6 HJ 1.70 [t, 2 HJ, 3.26 [t, 2 H], 6.35 [s, 1
HJ, 7.26 [s, 1 HJ. Mass spectral (FAB) data Calcd for iH l4BrN mlz (M+): 239. Found: 239.
EXAMPLE XLVI
6-Bromo-N-4,4-trimethyl- 1,2,3,4-tetrahydroquinoline (112).
The tetrahydroquinoline 111 ( 2.5 g 0.01 mol), ΝaHCO3 ( 1.51 g 0.018 mol), and H2O (2 mL) were placed in a standard system. The system was then cooled to 15-18 °C, and the dimethyl sulfate ( 1.64 g 0.013 mol) was added dropwise. The cloudy light yellow mixture was stirred at RT until the evolution of CO2 ceased (1 h). The flask was then maintained at 50° C until additional CO2 ceased to be released ( 1 h). The flask was then allowed to cool to RT. Chloroform (25 mL) was added, and the layers were separated. The aqueous layer was extracted with HCCI3 (4 x 40 mL). The combined organic layers were washed with H2O (40 mL) and brine (40 mL). After drying (MgSO4) overnight, the solution was concentrate in vacuo. The product was then cooled to 0 °C overnight during which time solid impurities precipitated. The mixture was vacuum filtered and washed with hexane. The solution was concentrated in vacuo to give the N-methylated product 112 as a
yellow oil (2.18 g, 86%) which was used directly to make 113. Η ΝMR (DCCI3) δ 1.25 [s,
6 HJ, 1.73 [t, 2 H], 2.86 [s, 3 H], 3.20 [t, 2 HJ, 6.42 [d, 1 H], 7.13 [dd, 1 HJ, 7.23 [d, 1 H,J. Mass spectral (FAB) Calcd for Cι2H ι6BrΝ m/z (M+): 253. found: 253. EXAMPLE XLVII
N,4,4-Trimethyl-l,2,3,4-tetrahydroquinolinecarboxylic Acid (113).
In a standard system was placed 1.6 M -BuLi (4.90 mL, 784 mmol). The tetrahydroquinoline 112 ( 1.0 g, 4.1 1 mmol) in ether (5 mL) was then added dropwise via a syringe. The solution was stirred at RT for 24 h. The resulting cloudy orange mixture was poured over solid CO2 in excess ether. The mixture was stirred until it warmed to RT (-2 h), and then H2O (20 mL) was added. The layers were separated, and the aqueous layer was acidified with 2 M HCl to ~pH 3. The resulting mixture was extracted with CH2CI2 (5 x 30 mL). After drying (MgSO4) overnight, the solution was concentrated in vacuo and recrystallized (95% ethanol). White flaky crystals (mp 223-225 °C) of the carboxylic acid 113 were obtained in a yield of 30% (0.270 g). It was used directly to make 114. IR (KBr)
3500-2400 cm-1; >H ΝMR (DCCI3) δ 1.30 [s, 6 H], 1.75 [t, 2 HJ, 3.00 [s, 3 HJ, 3.70 [t, 2 H],
6.53 [d, 1 HJ, 7.82 [dd, 1 HJ, 7.92 [d, 1 HJ. Mass spectral (FAB) data Calcd for C13H17ΝO2 WZ (M+): 219: Found: 219.
EXAMPLE XLVIII
Ethyl 4-(N,4,4-Trimethyl-l,2,3,4-tetrahydroquinoline)benzoate (114).
The carboxylic acid 113 (0.174 g, 0.794 mmol) and ethyl 4-hydroxybenzoate (0.198 g, 1.08 mmol) were mixed with CH2CI2 (6 mL). To this cloudy mixture was added dicyclohexylcarbodiimide (-0.025 g, 7.9 mmol) and a catalytic amount of DMAP (7 mg).
The resulting clear yellow solution was stirred at RT (48 h) and then filtered. The solvent was evaporated in vacuo ,and the product was subjected to chromatography (HCCl3:MeOH,
50: 1) to give the solid 114 (mp 116-1 18 °C), 0.105 g (43%). IR (KBr) 1720 cm 1 ; >H ΝMR
(DCCI3) δ 1.32 [s. 6 HJ, 1.40 [t, 3 H], 1.76 [t, 2 HJ, 3.03 [s, 3 H], 3.39 [t, 2 HJ, 4.38 [q, 2
H], 6.56 [d, 1 HJ, 7.27 [d, 2 H], 7.90 [dd, 2 H], 7.98 [d, 1 HJ, 8.10 [dd, 1 HJ. Anal. Calcd for C22H25NO4: C, 71.91 ; H, 6.86; N, 3.81. Found: C, 71.79; H, 6.60; N, 3.83. By similar technology, the N-methyl, N-n-propyl, and N-.-propyl analogs can be obtained.
EXAMPLE XLIX
N-(4-Bromo-3-methylphenyl)-3-methyl-2-butenamide (116).
In a standard system was placed the aniline 115 (15.00 g, 0.081 mol), 10% ΝaOH (80 mL), and CH2CI2 (50 mL). To the biphasic solution was added slowly (2 h) 3,3- dimethylacryloyl chloride ( 1 1.47 g, 0.097 mol) in CH2CI2 (20 mL). The resulting solution was stirred at RT (16 h) and diluted with H2O (30 mL), and the aqueous layer was extracted with CH2CI2 (4 x 30 mL) and dried (Νa2Sθ4). The solvent was evaporated in vacuo and the resulting dark brown solid was recrystallized (hexane:EtOAc, 4:1) to give 116 in a yield of 65% (14.12 g), mp 97-98 °C. Η NMR (DCCI3) δ 1.89 [s, 3 H], 2.21 |s, 3 H], 2.35 [s, 3
HJ, 5.68 [m, 1 H], 7.17-7.20 [m, 2 H], 7.41 [d, 2 HJ, 7.51 [s, 1 HJ.
EXAMPLE L
6-Bromo-4,4,7-trimethyI-2-oxo- 1,2,3,4-tetrahydroquinoline (117).
In a standard system was placed the anilide 116 (6.77 g, 0.025 mol). The flask was then heated to 1 10-120 °C, and then AICI3 (5.05 g 0.038 mol) was added portionwise over 1 h. The mixture was then stirred at that temperature for 0.5 h. The flask was then allowed to cool to RT, and 45 mL of ice water was added to the dark brown solid. Chloroform (30 mL) was then added to dissolve the solid. The solution was then stirred for 15 minutes, and 30 mL of 2 M HCl was added. The layers were separated, and the aqueous layer was extracted with HCCI3 (5 x 20 mL). The combined organic layers were washed with satd NaHCO3 (30 mL) and brine (30 mL). After drying (MgSO4), the solution was concentrated in vacuo to give a dark brown solid. Recrystallization (95% ethanol) of the product gave white crystals of 117 in a yield of 50% (3.48 g), mp 179-180 °C. IR (KBr) 3240, 1700 cm- ' ; 'H NMR (DCCI3) δ 1.31 [s, 6 HJ, 2.33 [s, 3 H], 2.48 [s, 2 HJ, 6.78 [s, 1 H], 7.40 [s, 1 HJ.
9.93 [s, 1 H].
EXAMPLE LI
6-Bromo-N-4,4-trimethyl-2-oxo-l ,2,3,4-tetrahydroquinoline (118).
In a standard system was placed DMSO (15 mL), and powdered KOH (0.732 g, 0.013 mol). The mixture was stirred for 5 min. and then the lactam (3.51 g, 0.013 mol) 117 was added, followed immediately by CH3I (4.79 g, 0.034 mol). Stirrring was continued for 30 min. The resulting mixture was poured into H2O (20 mL) and extracted with CH2CI2 (3 x 20 mL). The combined organic layers were washed with H2O (20 mL) and brine (20 mL) and dried (MgSO4). The solvent was evaporated in vacuo to give the N-methyllactam 118 in a yield of 73% (2.7 g), mp 69-71 °C and IR 1672 cm-1 [C=Ol, which was used directly to make 119.
EXAMPLE LII
6-Bromo-N-4,4,7-tetramethyl-l ,2,3,4-tetrahydroquinoline (119).
In a standard system was placed the lactam 118 (4.0 g, 0.014 mol) in 25 mL of dry, distilled toluene. The flask was cooled in an ice bath to 0-4 °C, and the 10 M borane- dimethylsulfide (2.8 mL, 0.028 mol,) was added dropwise from a syringe. The reaction mixture was stirred at 0 °C for 15 minutes and then was boiled for 2 h during which time the solution turned to a grey mixture. The flask was allowed to cool to RT, and then 20 mL of 10% Νa2Cθ3 was added. The solution was stirred at RT for 30 minutes The toluene layer was separated and dried (MgSO4) overnight. The solution was concentrated in vacuo to give the quinoline 119 as a light yellow oil which was placed in 2 mL of EtOAc, and cooled at 0 °C to crystallize. The pure quinoline 119 was obtained in a yield of 82% (3.06 g), p 56-58 °C. It was used directly to make 120. JH NMR (DCCI3) δ 1.25 [s, 6 HJ, 1.73 [t, 2
HJ, 2.30 [s, 3 H], 2.87 [s, 3 H], 3.19 [t, 2 H], 6.43 [s, 1 HJ, 7.25 [s, 1 HJ.
EXAMPLE LIII
6-Cyano-N,4,4,7-trimethyl- 1,2,3,4-tetrahydroquinoline (120)
In a standard system was added the tetrahydroquinoline 119, (2.90 g, 0.01 1 mol), copper (I) cyanide (1.34 g, 0.022 mol), and DMF (50 mL). The resulting mixture was boiled vigorously for 6 h (until complete by TLC) during which time the mixture changed from nearly black to dark yellow with dark brown solid particles. The mixture was allowed to to cool to RT and was then diluted with 30% NaCN (50 mL). The resulting solution was extracted with benzene, and the combined organic layers were dried Na2SO4. The solvent was evaporated in vacuo, to give the light orange solid 120 in a yield of 92% (2.13 g), mp
135-137 °C. It was used directly to make 121. IR (KBr) 2205 cm 1 ; ]H NMR (DCCI3) δ
1.24 [s, 6 HJ, 1.70 [t, 2 H], 2.41 [s, 3 H], 2.96 [s, 3 H], 3.34 [t, 2 HJ, 6.34 [s, 1 HJ, 7.32 [s, 1 HJ.
EXAMPLE LIV
N,4,4,7-Tetramethyl- 1 ,2,3,4-tetrahydroquinoline-6-carboxylic Acid (121)
In a standard system was placed nitrile 120 (0.150 g, 0.070 mmol), KOH ( 1.0 g), H2O (1 mL), MeOH (3 mL), and DEG (5 mL). The resulting solution was boiled for six days (until complete by TLC), during which time H2O (2 mL) was added to maintain volume. The solution was then allowed to cool to RT, diluted with H2O (5 mL), and acidified with cone HCl (pH~ 4). The aqueous solution was extracted with CH2CI2, and the combined organic layers were washed with H2O and saturated Ν HC03 and then dried Na2SU4. The solvent was evaporated in vacuo to give the white solid 121 in a yield of 38% (0.062 g), mp 191-192 °C. IR 3472-2483, 1663 cm '. It was used directly to make 122. EXAMPLE LV
Ethyl 4-(N,4,4,7-Tetramethyl- 1 ,2,3,4-tetrahydroquinoline)benzoate (122)
The procedure was essentially identical to that used to obtain 114. IR for 122 had a band at 1729 (C=O) cm 1. The ΝC2H5, NCH2CH2CH3, and NCH(CH3)2 analogs can be obtained similiarly.
EXAMPLE LVI
N-(4-Methoxyoxophenyl)-4,4-dimethyl-3,4-dihydro-2H-benzo[_>Jpyran-6-yl Hydroxamic Acid (123)
In a standard system were placed acid 95 (0.300 g, 1.40 mmol) and thionyl chloride
(15 mL), and the mixture was stirred (16 h) at RT. Excess thionyl chloride was removed by evaporation, and the residual oil was subjected to high vacuum to remove traces of thionyl chloride. A separate solution of ethyl 4-hydroxylaminebenzoate (0.253 g, 1.4 mmol) in THF ( 10 mL) was treated with ΝaHCU3 (0.176 g, 2.1 mmol) was prepared. To this preparation at 0 °C was added slowly the slightly crude acid chloride dissolved in dry ether, and the resulting mixture was stirred at RT for 18 h. The mixture was then filtered and the solvent was evaporated to yield a solid. Chromatography (silica gel) of the solid with hexanes:EtOAc (1 : 1) gave 123 (0.250 g, 50%), mp 70-71 °C. IR 3428, 1634 cm 1 ; !H
NMR (DCCI3) δ 1.13 [s, 6 H], 1.78 [t, 2 H], 3.90 [s, 3 H], 4.91 [t, 2 H], 6.68 [d, 2 H], 7.20
[dd, 1 HJ, 7.25 [dd, 1 H], 7.34 [d, 1 HJ, 7.96 [d, 2 HJ, 0.18 [s, 1 HJ. Anal. Calcd for C20H21NO5: C, 67.59; H, 5.96; N, 3.94. Found: C, 67.34; H, 6.05; N, 4.03.
EXAMPLE LVII
MethylN-(4-Methoxyoxophenyl)-4,4-dimethyl-3,4-dihydro-2H-benzo[b]pyran-6- ylhydroxamate (124) In a standard system was placed DMSO ( 1 mL) and powdered KOH (0.022 g, 0.38 mmol), and the solution was stirred for 5 min. The hydroxaminc acid 123 was then added, followed quickly by the addition of H3CI (0.031 mL, 0.51 mmol). The mixture was stirred for 0.5 h, and the the mixture was poured into water. Extracts (H2CCI2) were combined and washed with water and brine. The organic layer was dried (MgSO4) and then evaporated to a solid which was recrystallized (hexane:EtOAc, 4: 1 ) to give colorless 124 (0.045 g, 48%), p 104-106 °C. IR 1670 cm 1 ; Η NMR (DCCl3) δ 1.29 [s, 6 HJ, 1.83 [t, 2 HJ. 3.71 [s, 3
H], 3.92 [s, 3 HI, 4.23 [t, 2 HJ, 6.77 [d, 1 HJ, 8.04 [dd, 2 HJ. Anal. Calcd for C21H23NO5: C, 68.28; H, 6.28; N, 3.79. Found: C, 68.00; H, 6.24; N, 3.72.
EXAMPLE LVIII
2,2,4,4,5,7-Hexamethyl-6-hydoxy-3,4-dihydro-2H-benzo[ ]pyran (125)
In a standard system was placed 1.50 mL of a 3 M solution of methylmagnesium bromide (4.50 mmol) and TΗF (5 mL). The phenol 103 (0.100 g, 0.45 mmol) in TΗF (5 mL) was slowly added to the system at RT, and the resulting solution was stirred at reflux (4 days). After the system was allowed to cool to RT, A saturated solution of ammonium chloride was added. The aqueous layer was extracted (ether), and the original organic layer and extracts were combined and washed with water and brine. The dried (MgSO4) organic solution was evaporated to give a colorless solid 125 (0.086 g, 76%), mp 62.5-64.5 °C. IR
(KBr) 3458 cm-' ; 'Η NMR (DCCI3) δ 1.31 [s, 6 ΗJ, 1.47 [s, 6 Η], 1.84 [s, 2 Η], 2.17 [s, 3
Η], 2.37 [s, 3 Η], 4.25 [s, 1 ΗJ, 6.50 [s, 1 ΗJ. Mass spectral (El) data Calcd for C 15Η22O2
mlz (M+): 234. Found: 234. EXAMPLE LIX
Methyl4-[(2,2,4,4,5,7-Hexamethyl-3,4-dihydro-2H-benzo[b]pyran-6-yI)carbonyloxy] benzoate (126)
In a standard system was placed 125 (0.085 g, 0.336 mmol) and monomethylphthalic acid (0.1 15 g, 0.641 mmol) in Η2CCI2 (10 mL). To the cloudly mixture was added dicyclohexylcarbodiimide (DCC, 0.132 g, 0.641 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP, 4 mg). The resulting mixture was stirred at RT (18 h) and filtered. Evaporation of the solvent gave an oil which was chromatographed over silica gel (H2CCI2). A light yellow solid was crystallized (hexane) to give pure 126
(0.075 g, 52%), mp 95-96.5 "C. IR (KBr) 1730 cm 1 ; ]H NMR (DCCI3) δ 1.35 [s, 6 H],
1.48 [s, 6 H], 1.87 [s, 2 HJ, 2.08 [s, 3 H], 2.29 [s, 3 HJ, 3.98 [s, 3 HJ, 6.61 [s, 1 H], 8.18 [d, 2 H], 8.29 [d, 2 HJ. Anal. Calcd for C24H28O5: C, 72.70; H, 7.12. Found: C, 72.81 ; H, 7.15.
EXAMPLE LX
2-(3-Methoxylphenyl)-2-methylpropanenitrile (128)
In a standard system were placed KOH (20 g, 0.356 mmol), H2O (20 mL), and triethylbenzylammonium bromide (TEBA, 2 g, 7.46 mmol). 3-Methoxyphenyl)acetonitrile (127, 20 g, 0.135 mmol) was added slowly, and the solution turned an orange color. The system was heated to 80 °C (-0.5 h), and then H3CI (1 1.50 g, 0.081 mmol) was added slowly ( 2 h). Additional KOH (20 g, 0.356 mmol) and TEBA (2.0 g, 7.46 mmol) was added, and stirring was continued (0.5 h). Additional H3CI (1 1.50 g, 0.081 mmol) was then added, with stirring, over 1 h. The resulting mixture was stirred for 5 h at 80-100 °C. The mixture was allowed to cool to RT, and it was then extracted (toluene). The organic layer and extracts were combined, washed with water and brine, and then dried (MgSU4). Evaporation of the solvent gave an oil which was distilled (100-101 "C/2 mm) to yield a
colorless oil 128 ( 13.4 g, 57%). IR (neat) 2242 cm-' (ON); ]H NMR (DCCI3) δ 1.58 [s, 6 HJ, 3.80 [s, 3 HJ, 6.80 [ddd, 1 H], 6.95 [t, 1 H], 6.99 [ddd, 1 HJ, 7.25 [t, 1 HJ. Mas spectral (El) data calcd for CπHι4θ3 /n/z (M+): 194. Found: 194.
EXAMPLE LXI
2_(3-Methoxyphenyl)-2-methylpropanoic Acid (129)
Acid 129 was prepared from 128 by the literature procedure [K. Nakatani, S. Numata, T. Inoue, K. K. Fujisawa, T. I. Kawasaki, T. Toyama, H. Tachibana, T. Udagawa, and M.Gohbara, Chemical Abstracts, volume 97, page 5964e ( 1982)], mp 79-80 °C.
EXAMPLE LXI
2-(3-Methoxyphenyl)-2-methyl-lpropanol (130)
In a standard system was placed lithium aluminum hydride (2.23 g. 58.9 mmol) in dry THF (10 mL). Acid 129 (3.81 g, 19.6 mmol) in dry THF (5 mL) was added dropwise (5 min). After heating at reflux for 48 h, the system was allowed to cool to RT, and residual LiAIH4 was cautiously destroyed with ethanol/water. A solid formed during the water addition and was filtered off. Extracts (ether) of the aqueous layer were washed with brine and then dried (MgSO4). Evaporation of the solvent gave a colorless oil 130 (2.6 g, 75%).
IR (neat) 3392 cm-' ; JH NMR (DCC13) δ 1.32 [s, 6 H], 3.59 [s, 2 H], 3.81 [s. 3 H], 6.75 [td,
1 H], 6.93 [t, 1 HJ, 6.97 [td, 1 HJ. 7.27 [s, 1 H, Ar-HJ. Mass spectral (El) data calcd for CuHl602 mlz (M+): 180. Found: 180.
EXAMPLE LXII
l,l,4,4-Tetramethyl-6-methoxy-3,4-dihydro-lH-2-benzopyran (131)
In a standard system alcohol 130 (0.989 g, 5.6 mmol), acteone (30 mL), and cone ΗC1 (10 mL) were heated at 40-50 °C. The system was allowed to cool to RT. and then the contents were poured into ice-water. Extracts (ether) of the aqueous layer were washed with water, saturated NaHCO3, and brine and then were dried (MgSO4). Evaporation of the solvent gave an oil 131 (0.92 g, 75%) which was used directly to prepare 132. 'H NMR
(DCC13) δ 1.26 [ 1.26 [s, 6 H], 1.51 [s, 6 H], 3.58 [s, 2 H], 3.80 [s, 3 HJ, 6.73 [dd, 1 HJ, 6.82
[d, 1 HJ, 6.99 [d, 1 HJ. Mass spectral (El) data calcd for C14H20O2 mlz ( M+): 220. Found: 220.
EXAMPLE LXIII
7-Aceto- 1 , 1 ,4,4-Tetramethyl-6-methoxy-3,4-dihydro- 1 H-2-benzopyran (132)
In a standard system were placed ether 131 (0.989 g, 4.18 mmol), AcCl (0.74 mL,
10.0 mmol), and Η3CNO2 (15 mL). The AICI3 (1.33 g, 10.0 mmol) was added portionwise (1 h). The brown solution was stirred (12 h), and then it was poured slowly into ice-water. Dilute HCl (2 ) was added slowly to destroy excess AICI3. Extracts (H2CCI2) and the original organic layer were combined, washed with water, saturated NaHCO3, and brine. Evaporation of the solvent gave 132 and side products. Chromatography of the crude product over silica gel gave a colorless, solid 132, mp 1 15-1 17 °C. The material was used
directly to prepare 133. IR (KBr) 1630 cm 1 ; 'H NMR (DCCl3) δ 1.29 [s, 6 H], 1.52 [s, 6
H], 2.60 [s, 3 H], 3.59 [s, 2 HJ, 3.91 [s, 3 H], 6.85 [s, 1 H], 7.50 [s, 1 HJ. Mass spectral (El) data calcd for Ci6H22θ3 /z (M+): 262. Found: 262.
EXAMPLE LXIV
l,l,4,4-Tetramethyl-6-methoxy-3,4-dihydro-lH-2-benzopyran-7-carboxylic Acid (133)
In a standard system was placed ketone 132 (0.560 g, 2.13 mmol), a solution of
Bioguard-LiOCl (5 g; LiOCl-29%, inert ingredients-71%), Clorox (20 mL] and 95% ethanol ( 10 mL). The resulting mixture was boiled (24 h), and it was then allowed to cool to RT. Ether extracts were washed with water and brine and then were dried (MgSO4). Separate extracts (ether) of the acidified (2 N HCl, pH~ 3) aqueous layer were washed with water and brine and then were dried (MgSO4). Evaporation of the solvent gave a solid which was recrystallized (EtOAc) to white 133 (0.420 g, 21 %), mp 147-149 °C. IR (KBr) 3443-2538, 1691 cm"1 ; »H ΝMR (DCC13) δ 1.30 [s, 6 HJ, 1.54 [s, 6 H], 3.61 Is, 2 HJ, 4.07
[s, 2 HJ, 6.93 [s, 1 H], 7.89 [s, 1 HJ. Mass spectral (El) data calcd for C15H20O4 mlz (M+): 264. Found: 264. This acid 133 was used directly to prepare ester 134.
EXAMPLE LXV
Ethyl4-[(l , l ,4,4-Tetramethyl-7-methoxy-3,4-dihydro-lH-2-benzo[bJpyran-6-yI) carbonyloxy] benzoate (134)
In a standard system was placed acid 133 (0.100 g, 0.382 mmol), ethyl 4- hydroxybenzoate (0.095 g, 0.573 mmol), and Η2CC12 ( 10 mL). The DCC (0.1 18 g, 0.573 mmol) and DMAP (7 mg) were added carefully to the mixture which was then stirred at RT (48 h). Evaporation of the solvent gave an oil which was chromatographed over silica gel (hexane:EtOAc, 3: 1 ). To the resulting oil was added 0.5 mL of a solution of hexane:EtOAc (4: 1 ) the solution of which, upon cooling (0 °C), produced crystals of 134 (0.1 10 g, 70%), mp 106.5-108 °C. IR (KBr) 1722 cm-'; 'H ΝMR (DCCI3) δ 1.32 [s, 6 HJ, 1.40 [t, 3 H],
1.56 [s, 6 HJ, 3.62 [s, 2 H], 3.94 [s, 3 H], 4.40 [q, 2 H, 7.29 [d, 2 H], 7.73 [s, 1 H], 8.12 [d, 1 HJ. Anal. Calcd for C24H28O6: C, 69.88; H, 6.84. Found: C, 69.89; H, 6.94.
EXAMPLE LXVI
1 ,,2,3,4-Tetrahydro- 1 , 1 ,4,4-tetramethy-6-methoxynaphthalene ( 135)
Ether 135 was prepared from a literature procedure [M. F. Boehm, L. Zhang, B. A. Badea, S. K. White, D. E. Mais, E. Berger, M. C. Suto, M. E. Goldman, and R. A. Heyman, Journal of Medicinal Chemistiy, volume 37, pages 2930-2941 (1994)]. EXAMPLE LXVII
l -(3-Methoxyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)ethanone (136)
In a standard system was placed ether 135 (0.050 g, 0.23 mmol), AcCl (0.049 g,
0.69 mmol), and H3CNO2 (5 mL). The AICI3 (0.092 g, 0.69 mmol) was added slowly, and the resulting mixture was stirred at RT (4 h). The mixture was poured into ice-water, and extracts (ether) of the aqueous layer were combined, washed with water, saturated
NaHCO3, and brine and then were finally dried (MgSO4). Evaporation of the solvent gave a light yellow solid 136 (0.045 g, 75%), mp 96-98 °C. IR (KBr) 1664 cm 1 ; ! H NMR
(DCCI3) δ 1.27 [s, 6 H], 1.30 [s, 6 H], 1.68 [2t, 4 H], 2.59 [3 HJ, 3.89 [s, 3 H], 6.85 [s, 1 HJ,
7.73 [s, 1 HJ, 1 1.87 [s, 1 HJ. Mass spectral (El) data calcd for C17H24O2 mlz (M+) : 260. Found: 260. Ketone 136 was used directly to make acid 137.
EXAMPLE LXVIII
l -(3-Methoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)-2-carboxylic Acid (137)
In a standard system was placed ketone 136 (0.900 g, 3.46 mmol), a solution of Bioguard-LiOCl/Clorox (40 g of LiOCl and 80 mL of Clorox), and 95% ethanol (45 mL). After boiling the above mixture (24 h), it was allowed to cool to RT and was then treated with an aqueous solution of 25% Na2S2θs (35 mL). Extracts (ether) of the acidified (6 M HCl, pH~3) aqueous solution were combined, washed with water and brine and were finally dried (MgSO4). Evaporation of the solvent gave a light yellow solid 137 (0.420 g, 44%),
mp 136.5-138 °C. IR (KBr) 3269, 1731 cm"1 ; >H NMR (DCCI3) δ 1.28 [s, 6 HJ, 1.31 [s, 6
H, 1.69 [t, 4 HJ, 1.69 [t, 4 H], 4.06 [s, 3 H], 6.93 [s, 1 H], 8.12 [s, 1 HJ. Mass spectral (El) data calcd for C16H22O3 mlz (M+): 262. Found: 262. Acid 137 was used directly to prepare ester 138. EXAMPLE LXIX
Ethyl4-[(3-Methoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)carbonyloxyJ benzoate (138)
In a standard system was placed acid 137 (0.300 g, 1.14 mmol), ethyl 4- hydroxybenzoate (0.300 g, 1.8 mmol), and H2CCI2 ( 15 mL). To the mixture was added
DCC (0.750 g, 3.6 mmol) and DMAP (10 mg). After stirring at RT (48 h) and being filtered, the resulting solution was evaporated in vacuo to an oil which was chromatographed over silica gel (hexane:EtOAc, 4: 1 ). White crystals of 138 (0.312 g,
67%) were collected, mp 104- 106 °C. IR (KBr) 1722 cm 1 ; JH NMR (DCCl3) δ 1.28 [s, 6
HJ, 1.30 [s, 6 H], 1.39 [t, 2 HJ, 1.68 [2t, 4 H], 3.89 [s, 3 HJ, 4.37 [q, 2 HI, 6.90 [s, 1 H], 7.26 d, 2 HJ, 7.93 [s, 1 H], 8.07 [d, 2 HJ. Anal. Calcd for C23H23NO5: C, 73.24; H, 7.37. Found: C, 73.22; H, 7.49.
EXAMPLE LXX
In order to assess the biological activity in terms of transglutaminase activity, the following procedure was utilized. Data are in Table I.
General Method for Assaying Transglutaminase Activity. Human erythroleukemia cells (GMO6141A, Human Genetic Mutant Cell Respository, Camden, NJ) were inoculated into T-25 flasks (Corning, Corning, NY) containing McCoy's Medium 5a (GIBCO/BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Intergen, Purchase, NY) at a density of 2 x 105 cells/mL. The heteroarotinoids were dissolved either in ethanol or
DMSO. Twenty-four hours after subculture, the cells were treated with a 10 μM solution
of either t-RA, the vehicle alone (control), or the heteroarotinoid. The r-RA (Sigma, St. Louis, MO) was added to one flask in all experiments to ensure that transglutaminase induction occurred, and it was also used as the standard to which the heteroarotinoid induction was compared. Cultures were covered with aluminum foil to protect them from light and then incubated at 37 °C for 48 h. Cells ( 10-40 x 106) were collected by centrifugation at 2000 rpm for 5 min. The resulting cell pellet was resuspended in 10 mL of Ca,Mg-free Earles solution, and the cell suspension was centrifuged at 2000 rpm for 3 min. These cells were twice washed again. The final washed cell pellet was resuspended in 1 mL of Tris-buffered saline (150 m NaCl- 1 mM EDTA-20 mΛ Tris-HCl, pH 7.4), and the cell suspension was disrupted by a 10-second sonication. Cell lysates were quantitated for transglutaminase activity by measuring the covalent incorporation of l 4C-labelled putrescine into dimethylcasein in a calcium-dependent manner as previously described in the Proceedings of the National Academy of Science, U.S.A., Vol. 78, 1981, pages 5005- 5008 and entitled "Increase in Proliferative Markers After Inhibition of Transglutaminase" by P. J. Birckbichler, G. R. Orr, M. K. Patterson, Jr., E. Conway, and H. A. Carter. Protein was estimated using the Bio-Rad procedure in the Journal of Biological Chemistry, Vol. 193, 1951 , pages 265-275 and entitled "Protein Measurement With The Folin Phenol Reagent" by O. H. Lowry, N. J. Rosebrough, A. L Farr, and R. J. Randall. The values for the transglutaminase activity in Table I represent the mean of duplicate assays performed on replicate samples.
EXAMPLE LXXI
In order to assess the receptor binding potency, the procedure used can be found in the literature: . Steroid Biochem. Molec. Biol. 1992, 41, 733-738. See also R. Heyman, P. J. Mangelsdorf, J. A. Dyke, R. B. Stein, G. Eichele, R. Evans, and C. Thaller, "9-cis-
Retinoic Acid is a High Affinity Ligand for RXR'" Cell, 1992, 68, 397-406. Data are in
Table II.
EXAMPLE LXXII
In order to assess the transactivation ability of the heteroarotinoids, the following procedure was employed. Data are in Table III. Transactivation Assay. The CC-B cervical tumor cell line [D. M. Benbrook,S. Lu,
C. W. Flanagan, J. Shen-Gunther, L. H. Angros, and S. A. Lightfoot. "A Biological Assay and Molecular Mechanism of Retinoids in Cervical Tumor Cells". Gynecological Oncology
, 1997, 66, pages 1 14-121 ] which contains stablized integrated copies of RARβ-RARE-tk- CAT reporter plasmid, was used to evaluate heteroarotinoid activation of endogenous nuclear receptors. A concentration of 4 x 105 cells per well was used to inoculate 6-well culture dishes containing Eagles Minimal Essential Media which contained Earle's Salts and L-glutamine (Cellgro Mediatech, Herndon, VA) supplemented with nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum (FBS). Only lots of FBS that contain negligible quantities of /-RA as determined by HPLC analysis were used. Triplicate cultures were treated with 1 μM agent dissolved in DMSO. The final concentration in
DMSO in control and treated cultures was 0.1%. After 48 h, cell extracts were prepared and CAT activity was assayed as previously described [Sleigh, M. J. "A Nonchromatographic Assay for Expression of the Chloramphenicol Acetyltransferase Gene in Eukaryotic Cells". Anal. Biochem. 1986, 156, 251-256] with the exception that 3H acetyl-CoA was used instead of 14C acetyl-CoA. Protein concentrations of the extracts were determined using the Bio-Rad Protein Assay (Bio-Rad, Richmond, VA). Transactivation activities were derived by dividing CAT activity per milligram of protein in the drug-treated culture by the activity in the control culture. The results, presented as percentages in Table II and III, are the average of three experiments.
EXAMPLE LXXIII
In order to assess the growth inhibitory properties of representatives of the heteroarotinoids, the following procedure was developed. Data are in Table III.
Growth Assay. In order to assess the growth inhibitory properties of the heteroarotinoids, the following procedure was developed. Data are in Table III. Growth Assay. Six-well tissue cultures dishes were inoculated with a concentration of 20,000 CC-1 or CC-B cells per dish. Twenty-four hours after plating, the media was replenished and treatment was initiated. After seven days, the number of cells/well in the treated and control cultures was determined using a Particle Counter (Coulter ZM, Miami, FL). At this time, neither the cells in the treated or control cultures had reached saturation density. The percent growth was determined by dividing the number of cells in the treated cultures by the number of cells in the control cultures and multiplying by 100. The results presented are the average of three independent experiments. Proliferation of cultures treated with /røns-retinoic acid compared to control cultures were statistically evaluated with a one-way analysis of variance using SPSS version of 6.1 software. The trans-retinoic acid served as the standard for comparision purposes.
In summary, cervical carcinoma cells (CC-1 or CC-B) were treated with the heteroarotinoids in Table III for seven days. Control cultures were treated with the same volume of solvent as used with the treated cultures. The effect of the agents on growth was as cited above

Claims

We claim:
1. Heteroarotinoids characterized by the formulae:
Figure imgf000088_0001
where A = H or CH3;
G = C(O)O, OC(O), C(O)NH, C(O)NOH, C(O)NOCH3, NHC(O), C(CH3)=CH or C(O)CH=CH;
L = H, CH or OCH3;
M = H or CH3;
Q = H or CH3;
R = H, CH3, C2H5, n-C3H7 or -C3H7;
T = C6H4-4-CO2R, C6H4-3-CO2R, C6H3-3-CH3-4-CO2R, C6H3-2-CH3- 4-CO2R, CH=CH-CH=CH-CO2R or CH=CH-C(CH3)=CH-CO2R;
X = O, S, S→O, SO , NCH3, NC2H5, NCH2CH2CH3, NCH(CH3)2 or C(CH3)2 ; and
Y = CH2, O or S.
2. The compound according to claim 1 wherein A = CH3, G = NHC(O), L = Q = H,
M = CH3, T = C6H4-4-CO2R, X = S, and Y = CH2.
The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = M = Q = H, T = C6H3-3-CH3-4-CO2R, X = O, S or S→O, and Y = CH2.
The compound according to claim 1 wherein A = CH3, G = C(O)CH=CH, L = Q = H, M = CH3, T = C6H4-4-CO2R, X = O, S or S→O, and Y = CH2.
The compound according to claim 1 wherein A = CH3, G = NHC(O), L = Q = H, M = CH3, T = CH=CH-CH=CH-CO2R, X = S, and Y = CH2.
6. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = M
Q = H, T = C6H3-3-CH3-4-CO2R, X = S, and Y = CH2.
The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = Q H, M = CH , T = C6H3-3-CH3-4-CO2R, X = S, and Y = CH2.
8. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = Q
H, M = CH3, T = C6H3-3-CH3-4-CO2R, X = S, and Y = CH2.
9. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = Q
H, M = CH3, T = C6H3-3-CH3-4-CO2R, X = O or S, and Y = CH2 "
10. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = M =
Q = H, T = C6H4-3-CO2R, X = O or S, and Y = CH2.
1 1. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = Q = H, M = CH , T = C6H3-2-CH3-4-CO2R, X = S, and Y = CH2.
12. The compound according to claim 1 wherein A = CH3, G = NHC(O), L = Q = H,
M = CH3, T = C6H4-4-CO2R, X = S, and Y = CH2.
13. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH. L = Q
M = H, T = C6H3-3-CH3-4-Cθ2R, X = O or S, and Y = CH2.
14. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = Q H, M = CH3, T = C6H4CO2R, X = O, S or NR, and Y = CH2.
15. The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = M =
Q = H, T = C6H4-3-CO2R, X = O or S, and Y = CH2.
16. The compound according to claim 1 wherein A = CH3, G = NHC(O), L = Q = H,
M = CH3, T = CH=CH-CH=CH-CO2R, X = O or S, and Y = CH2.
17. The compound according to claim 1 wherein A = CH3, G = C(O)NH, L = Q = H, M = CH3,T = C6H4-4-CO2R, X = S, S→O or SO2, and Y = CH2.
18 The compound according to claim 1 wherein A = CH3, G = C(CH3)=CH, L = M =
Q = H, T = C6H4-3-CO2R, X = O or S, and Y = CH2.
19. The compound according to claim 1 wherein A = CH3, G = C(O)NH, L = Q = H, M = CH3, T = C6H4-4-CO2R, X - S, S→O or SO2, and Y = CH2.
20. The compound according to claim 1 wherein A = H, G = C(CH3)=CH, L = M = Q = H, T = C6H4CO2R, X = O, S, S→O or NR, and Y = CH2.
21. The compound according to claim 1 wherein A = H or CH3, G = C(CH3)=CH, L =
Q = H, M = H or n-C8Hι7, T = C6H4-4-CO2R, X = O, S, S→O or NR, and Y = CH2.
22. The compound according to claim 1 wherein A = H or CH3, G = C(CH3)=CH, L = Q = H, M = H or C2H5, T = C6H4-4-CO2R, X = O, S, S→O or NR, and Y = CH2.
23. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = M = Q = H, T = 2-ethyl 5-indolate, X = O or S, and Y = CH2.
24. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = M = Q = H, T = 5-ethyl 2-nicotinate, X = O or S, and Y = CH2.
25. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = M = Q =
H, T = C6H4-4-CO2R, X = NCH3, NC2H5, NCH2CH2CH3 or NCH(CH3)2, and Y = CH2.
26. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = CH3, M =
CH2, Q = H, T = C6H 4-4-CO2R, X = NCH3, NC2H5, NCH2CH9CH3 or NCH(CH3)2, and Y = CH2.
27. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = H or
OCH3, M = Q = H, T = C6H4-4-CO2R, X = C(CH3)2, and Y = CH2.
28. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = H or OCH3, M = CH2, Q = H, T = C6H4-4-CO2R, X = C(CH3)2, and Y = O.
29. The compound according to claim 1 wherein A = M = CH3, G = OC(O), L = Q CH3, T = C6H4-4-CO2R, X = O or S, and Y = CH2.
30. The compound according to claim 1 wherein A = CH3, G = C(O)NOH or C(O)NOCH3, L = Q = H, M = CH2, T = C6H4-4-CO2R, X = O or S, and Y = CH2.
31. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = Q = H, M= CH2, T = C6H4-4-C(O)H, X = O or S, and Y = CH .
32. The compound according to claim 1 wherein A = CH3, G = C(O)O, L = H or CH3, M = Q = H, T = C6H -4-CH=NNHC(S)NH2, X = O or S, and Y = CH2.
33. Heteroarotinoids characterized by the formulae:
Figure imgf000091_0001
where G = C(CH3)=CH or NHC(O); T = C6H4-4-CO2R or CH=CH-C(CH )=CH- CO2R; and R = H, CH3C2H5; with the proviso that when G = NHC(O) T = C6H4-4-CO2R.
34. Heteroarotinoids characterized by the formulae:
Figure imgf000092_0001
where G = C(O)NH, T = C6H4-4-CO2R, R = H, CH or C2H5, and X = O, S, S→O or SO2.
35. Heteroarotinoids characterized by the formulae:
Figure imgf000092_0002
where G = C(CH3)=CH, T = CH=CH-C(CH3)=CH-CO2R, and R = H, CH3 or C2H5.
36. Heteroarotinoids characterized by the formulae:
Figure imgf000092_0003
where G = C(CH3)=CH; T = C6H4-4-CO2R, and R = H, CH3 or C2H5.
37. Heteroarotinoids characterized by the formulae:
Figure imgf000092_0004
where G = C(O)O, T = C6H4-4-CO2R, and R = H, CH3 or C2H5.
38. Heteroarotinoids characterized by the formulae:
Figure imgf000093_0001
where G = C(O)O; T = C6H4-4-CO2R, and R = H, CH3 or C2H5.
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