HK1030947B - N-benzyl-3-indenylacetamides derivatives for treating neoplasia - Google Patents

Description

N-benzyl-3-indenylacetamide derivatives for the treatment of tumors

Technical overview

The present invention relates to compounds and methodologies that direct and promote apoptosis and prevent the sudden proliferation of tumor cells. These methods are particularly useful for the treatment of tumors, including pre-cancer and cancer lesions.

Background

The fields of research and commercial development have increasingly focused on the study of effective drugs for the treatment of early stage tumors, which can delay or prevent the malignant transformation of precancerous lesions into cancer. In the united states, millions of people develop precancer each year, and these patients have a significant (statistical) trend to develop malignancies and cancers. These lesions are mainly included in breast (which may develop into breast cancer), skin (which may develop into malignant melanoma or basal cell tumors), adenomatous polyps of the colon (which may develop into colon tumors), cervical abnormalities (cervical cancer), and other tumor tissues.

Compounds that prevent or induce the presence of pre-cancer, cancer damage and tumor reversal can delay the onset of cancer and at least substantially reduce the pain and death of such diseases.

Such compounds and methodologies can benefit those patients with high probability of cancer with repetitive pre-cancer lesions. This patient population exists for many cancers (like breast, colon, prostate, etc.). An important example of this is the familial polypoid colon cancer, which patients are suffering from cancer if left untreated. Familial polyposis patients are characterized by many (thousands) of colonic polyps that grow at the age of the decade, and have been reported to be cancerous due to the fact that each colonic polyp population (whether familial or non-familial) has a life-long risk of becoming cancerous, until recently the treatment of this familial polyposis was limited to surgical resection at the age of twenty.

Many other tumor types also present a high risk of cancer and cancer recurrence in the early years, such as breast and colon cancer, compared to the population with the tumor as a whole. In colon cancer patients, resection of emerging polypoid tumors is the current treatment option. For non-familial patients, resection of polypectomy can be used with surgery or fiberendoscopic polypectomy. These procedures are uncomfortable, expensive (on the order of one thousand to fifteen hundred dollars per endoscopic polypectomy, and more expensive with surgical resection), and can pose a small but significant risk of colonic perforation.

Because chemotherapy and surgery are not very effective in treating cancer and the existing chemotherapy often has serious side effects, it is important to find effective drugs for treating and preventing early stage tumors, and more important to find effective compounds with the effect of resisting the loss of precancer but without the side effects of the conventional chemotherapy drugs. Since these compounds selectively induce apoptosis without damaging normal cells, they can be used for rehabilitation therapy of tumor patients with the possibility of tumor recurrence, and even general tumor patients can benefit from it.

Because the side effects (from the anti-tumor effect) possessed by chemotherapeutic drugs are often greater than their tumor-preventing effects, standard tumor chemotherapeutic drugs are not suitable for chemopreventive treatment of cancer. The carcinostatic effect of many standard chemotherapeutic drugs is now thought to be achieved by inducing apoptosis. Programmed cell death, which is naturally occurring in all tissues of the body, plays an important role in tissue balance in the body, and ensures an equal balance between the number of newly produced cells and the number of dead cells in the body. The role of apoptosis is particularly pronounced in self-renewing tissues such as bone marrow, immune cells, visceral and skin tissues. For example, the cells in the small intestine divide very rapidly and the body must clear these cells after three days to protect and prevent the proliferation of the small intestine wall.

Standard chemotherapeutic drugs cause programmed death not only of tumor cells but also of cells in normal human tissues, especially in normal tissues (like hair, intestine and skin) which divide rapidly in vivo and thus have serious side effects. Side effects on normal cells include hair loss, weight loss, vomiting and bone marrow immunosuppression, which are one reason standard chemotherapeutic drugs are not suitable for cancer prevention.

Unless cured once (e.g., gene therapy), cancer prevention always requires multiple long-term administrations to inhibit tumor formation, which is contraindicated for standard chemotherapeutic drugs because of their side effects as described above.

Deregulation of apoptosis can lead to precancerous lesions and tumors. Recent research has shown that defects in apoptosis play a significant role in diseases other than tumors. Therefore, drugs that modulate apoptosis may be useful not only in the prevention or control of cancer, but also in the treatment of other diseases.

Some non-steroidal anti-inflammatory drugs (NSAIDs), originally used for the treatment of arthritis, show some effect in the inhibition and elimination of colonic polyps. Polyps disappear when a patient takes sulindac, a non-steroidal anti-inflammatory drug. Unfortunately, due to the inhibition of prostate synthesis enzyme ("PGE-2") by NSAIDs, chronic use of existing NSAIDs can cause severe side effects in patients, particularly patients with polyposis syndrome, including gastrointestinal damage, perforation, ulceration, and renal toxicity. Since increased PGE-2 activity is associated with inflammation, inhibition of PGE-2 enzymes by NSAIDs is a prerequisite for their anti-inflammatory effects, and PGE-2 has a gastrointestinal protective effect, so that chronic treatment with NSAIDs causes gastrointestinal reactions. For arthritic patients, the gastrointestinal response of NSAIDs is not apparent due to short-term treatment. However, for patients with multiple polyps, chronic administration of sulindac causes gastrointestinal side effects in order to eliminate polyps and prevent regeneration of polyps. Patients with polyposis have to stop taking NSAIDs due to these side effects, but polyps recur.

Those compounds disclosed in us patent 5,643,959 have been shown to have the effect of causing apoptosis of tumor cells only without affecting normal cells in humans, and therefore these new therapeutic agents avoid the serious side effects of normal apoptosis caused by common chemotherapeutic agents, thus giving them the advantage of treating tumor loss. (Please see: phase I clinical trial of sulindac sulfolane for familial polypyristic sarcomaTesting: the applicable dosage and the safety are ensured,digestive diseases peri-meetingThe abstract number is as follows: 2457, months 5-10-16, 1997, gastrointestinal Association, et al). In addition, these drugs do not inhibit PGE-2 and thus do not produce gastrointestinal side effects as NSAIDs do. There is therefore a greater need for compounds that are more potent against tumors but without significant PGE-2 inhibition.

Summary of The Invention

The effective compound in the invention can trigger programmed death of tumor cells (but has no significant influence on normal cells), treat tumor injuries and does not significantly inhibit PGE-2 action. The invention also includes screening methods for assays for these compounds, which include determining whether the compounds can trigger programmed death of tumor cells in cell culture, determining an effective dose of the drug to treat the patient, and determining the modulation of pre-cancer lesions and programmed death and growth of tumors, and eliminating the side effects of conventional chemotherapeutic drugs and NSAIDs.

Detailed description of the invention

The present invention includes compounds (and their pharmaceutically acceptable salts) represented by formula I below for the treatment of tumors, particularly precancers.

Wherein, the substituent R₁Respectively represent hydrogen, halogen, low-carbon alkyl, low-carbon alkoxy, amino, low-carbon alkylamine, dual low-carbon alkylamine, low-carbon alkanethiol, low-carbon alkanesulfonyl, cyano, carboxyl amino, carboxylic acid, mercapto, sulfonic acid, xanthic acid and hydroxyl;

substituent R₂Represents hydrogen and lower alkyl;

R₃represents hydrogen, halogen, amino, hydroxyl, lower alkyl amine and lower dialkyl amine;

R₄represents hydrogen radical, or R₃And R₄Combining into oxygen;

R₅and R₆Each represents hydrogen, lower alkyl, hydroxy-substituted lower alkyl, amino lower alkyl, lower alkylaminoalkyl, lower alkylaminodilower alkyl, lower alkylnitrile, -CO₂H、-C(O)NH₂And two to six carbon amino acids;

R₇each represents hydrogen, amino lower alkyl, lower alkoxy, lower alkyl, hydroxy, amino, lower alkylamino, di-lower alkylamino, halogen, -COOH, -SO₃H、-SO₂NH₂and-SO₂(lower alkyl);

m and n represent any one integer between 0 and 3;

y represents quinolyl, isoquinolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, indolyl, benzimidazolyl, triazinyl, tetrazolyl, thiophenyl, furanyl, thialinyl, pyrazolyl, pyrrolyl, and these heterocyclic groups with substituents. These substituents may be halogen, lower alkyl, lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy, -SO₂(lower alkyl) and-SO₂NH₂One or two.

The invention also includes preferred compounds of formula I and methods for their screening and use, wherein:

R₁the groups represented by the general formula are halogen, lower alkoxy, amino, hydroxyl, lower alkylamino and double lower alkylamino, wherein the halogen, the lower alkoxy, the amino and the hydroxyl are preferred compounds;

R₂the group represented is a lower alkyl group;

R₃the groups represented by hydrogen, halogen, hydroxyl, amino, lower alkylamino and di-lower alkylamino, wherein hydrogen, hydroxyl and lower alkylamino are usedIs the first choice;

R₅and R₆Respectively represent hydrogen, hydroxy-substituted lower alkyl, amino-lower alkyl, lower alkylamine group disubstituted lower alkyl, -CO₂H、-C(O)NH₂Wherein lower alkyl, lower alkylamino-di-lower alkyl, -CO is substituted by hydrogen, hydroxy₂H and-C (O) NH₂Are preferred compounds;

R₇the groups represented by are hydrogen, lower alkoxy, hydroxyl, amino, lower alkylamino, di-lower alkylamino, halogen, -CO₂H、-SO₃H、-SO₂NH₂and-SO₂(lower alkyl) with hydrogen, lower alkoxy, hydroxy, amino lower alkyl, halogen, -CO₂H、-SO₃H、-SO₂NH₂and-SO₂(lower alkyl) is a preferred compound. At least R₇Any one of the substituents may be in a para, ortho or para position, most preferably the position is the ortho position;

y represents quinoline, isoquinoline, pyridine, pyrimidine, pyrazine or derivatives thereof with different substituents. Preferred substituents on the Y heterocyclic group are lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy, -SO₂(lower alkyl), and-SO₂NH₂Wherein the lower alkoxy, di-lower alkylamino, hydroxy, -SO₂(lower alkyl) and-SO₂NH₂And is most preferred.

The invention also includes all of the compounds of formula I above and their use in methods, pharmaceutically effective dosage forms and pharmaceutically effective dosages for treating neoplasms, with dosage forms that are free of NSAIDs therapeutic dosages being most preferred.

The invention also includes enteric coated tablets of the above compounds and methods of their use in treating patients having tumors.

The invention includes screening methods for inhibiting tumor cells using an effective amount of all compounds of formula I above.

In another embodiment, the invention includes a screening method for inducing apoptosis in a human in an amount effective to induce apoptosis in a human in all of the compounds of formula I above.

In another embodiment, the invention encompasses the use of an effective amount of all compounds of formula I above for the treatment of a disease associated with a disorder of apoptosis. Modulation of apoptosis plays an important role in diseases associated with dysregulated cell growth, like benign prostatic hyperplasia, neurodegenerative diseases (e.g. parkinson's disease), autoimmune diseases including multiple sclerosis and rheumatoid arthritis, infectious diseases like aids, and others.

The compounds of the invention are in turn inhibitors of cGMP-specific phosphodiesterase (cGMP-PDE) in tumor cells. This enzyme includes PED5 and the novel PDEs disclosed in U.S. patent No. 09/173.375. For convenience, the PDE inhibitory activity of these compounds can be tested as described in registered U.S. Pat. No. 09/046,739 (3/24/1998). Thus, the compounds of this invention are inhibitors of PDE5 and are useful in the treatment of diseases in which inhibition of this enzyme is desired.

The term "precancerous lesion" as used herein includes syndromes represented by abnormal neoplasia, including dysplasia, tissue alteration. Examples include dysplasia in colon, breast, prostate or lung tissue, or conditions such as dysplastic nevus syndrome, precursors to malignant melanoma of the skin, and the like. Examples include polyposis, colon polyps, precancerous lesions of the uterus (i.e. uterine dysplasia), oesophagus, lung, prostate dysplasia, intraprostate tumour, breast and/or skin and related diseases (e.g. active keratosis) in addition to dysplastic nevus syndrome, whether or not the lesions are clinically identifiable.

The term "halo" or "halogen" as used herein refers to chloro, bromo, fluoro, iodo. The term "alkyl" refers to straight, branched, and cyclic alkyl groups, as well as alternative arylalkyl groups. The term "lower alkyl" refers to a one to eight carbon alkyl group.

The term "hydroxy-substituted lower alkyl" refers to lower alkyl groups that are at least monohydroxy and preferably not more than trihydroxy substituted.

The term "-SO₂(lower alkyl) "means a lower alkyl-substituted sulfonyl group.

The term "lower alkoxy" refers to a straight, branched or cyclic alkoxy group having one to eight carbons.

The term "lower alkylmercapto" refers to a lower alkyl-substituted sulfide group. The term "lower alkylsulfonyl" refers to lower alkyl substituted sulfonyl.

The term "pharmaceutically acceptable salts" refers to salts of all compounds of formula I with non-toxic acids or bases and metal bases. These salts can be prepared directly at the final isolation and purification of the compound or indirectly by reaction with an organic acid or base. Representative acid salts are: hydrochloride, bromate, sulfate, sulfite, acetate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, mesylate, citrate, maleate, fumarate, succinate, tartrate, glucoheptonate, lactobionate, laurylsulfate, and the like. Representative bases and metal base salts are: sodium, calcium, potassium and magnesium salts.

For compounds of formula I containing asymmetric carbon atoms, their enantiomers may also be present. Unless otherwise specified, the present invention also includes these enantiomers and racemates. These enantiomeric compounds can be synthesized from chiral starting materials. The racemates can be separated by conventional chemical methods including chiral chromatography, fractional crystallization of diastereomeric salts, and the like.

The compounds represented in formula I also exist as stereoisomers (cis and trans), with trans (I) being preferred.

The compounds of the invention can be mixed with pharmaceutical carriers to make solid and liquid dosage forms for oral administration. Although these dosage forms are primarily oral, they may be administered rectally or epicutaneously.

Pharmaceutical carriers for oral use include capsules, tablets, pills, powders, lozenges and granules. The carrier for solid dosage forms may contain at least one inert diluent of the sucrose, lactose or starch type as conventionally used. These carriers may also be supplemented with other substances, for example, the lubricant magnesium stearate. The carriers for capsules, tablets, dragees, pills and granules may contain buffering agents. Tablets, pills and granules carriers may be prepared by coating tablets, pills and granules with an enteric coating, and the enteric coated compound may be compressed directly into tablets, pills and granules for administration to a patient. The enteric coating is preferably shellac or Eudragts which are easily soluble or decomposable at pH in the intestine.

Carriers for pharmaceuticals also include liquid dosage forms for oral administration, such as: pharmaceutical emulsions, solutions, suspensions, syrups and aqueous inert diluents for ordinary paintings, elixirs. In addition to inert diluents, the pharmaceutical compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents and flavoring agents.

Drug carriers for dermal administration include DMSO, alcohol or propylene glycol and other substances that can be used to mix or maintain moisture, and these carriers allow the drug to adhere to the skin without drying out.

Pharmaceutical carriers for rectal administration are preferably suppositories which can be formed by mixing the compound with excipients such as cocoa butter, suppository waxes, gels.

The compounds of the invention and pharmaceutical carriers can be formulated in unit dosage forms for administration to patients by any route (e.g., oral or rectal) and the amounts of active ingredients in the unit dosage can be adjusted depending on the desired dosage to be effective in the treatment of the injury. The dosage range may be selected based on the absorption properties of the active compound, the route of administration, the desired treatment time, and other factors. If desired, unit doses may be formulated as the amount of active compound required to be administered once a day, or as multiple doses (two to four times a day).

The pharmaceutical compositions of the invention are preferably packaged in containers (e.g., boxes, bottles) having appropriate instructions or labels indicating the particular composition, content, method of use, etc.

Several schemes for synthesizing these compounds are included in this invention. The first scheme (together with the list of other alternative pathways) represents an example of R₃And R₄Synthesis of compounds all of which are hydrogen. Please see flowchart I specifically. Other flow diagrams (and other paths listed) describe examples where R is₃And R₄Wherein at least one is not hydrogen but is still according to formula I. The second flowchart is listed in "flowchart II".

R₃And R₄The synthetic route to the compounds, all of which are hydrogen, is shown in scheme I, which is described in U.S. Pat. No. 3,312,730, incorporated herein by reference. In scheme I, R₁In addition to representing the groups contained in formula I, it may also be a nitro-like reactive group, so that a large number of other substituted indenyls can be synthesized from nitro-substituted indenyls.

Flow chart I

Flow chart I (continue)

In scheme I, several alternative approaches are also possible. One is the Knoevenagel condensation reaction of alternative benzaldehydes with ethyl acetate (equation 2), or the Reformatsky reaction with α -halopropynyl esters (equations 1 and 3). The synthesized unsaturated ester (c) is further hydrogenated and hydrolyzed to give the alternative benzylpropiolic acid (e) (reaction formulas 4 and 5). Alternatively, benzyl propiolic acid (e) can be obtained by hydrolysis with an alternative malonate and decarboxylation using typical malonate synthesis methods (equations 6 and 7). The latter is more suitable for the synthesis of nitro and alkylthio substituted benzene ring compounds.

The second step is the ring synthesis of indanone (h) from beta-arylpropiolic acid (e), which can be obtained by Friedel-Crafts reaction catalyzed by Lewis acid (refer to page 130 of the second volume of organic reactions) or by heating with polyphosphoric acid (equations 8 and 9). Indanone (h) reacts with the α -haloester to produce Reformatsky, which substitutes for the carboxyl group and then introduces an aliphatic acid (see equation 10). In addition, the reaction can be completed by a Wittig reaction, wherein the Wittig reagent and the carbonyl group on the alpha-triphenylphosphine ester substituted indanone (h) are double bonds (reaction formula 12) to obtain a product (i). The product (i) can be immediately rearranged to form the indeno compound (j) (see equation 13). If a Reformatsky reaction is chosen, the intermediate 3-hydroxy-3-aliphatic acid derivative (i) must be dehydrated to the indeno compound (j) (see equation 11).

The indenyl acetic acid (k) is evaporated with oxalyl chloride or thionyl chloride or other similar reagents in tetrahydrofuran to give the acid chloride compound (m) (see reaction formula 15). The addition reaction of the acid chloride (m) with the substituted benzylamine (n) can be carried out in two ways.

Method I

The substituted benzylamine (n) was slowly added to a solution of 5-fluoro-2-methyl-3-indenylacetyl chloride (k) in dichlorodimethane at room temperature, and the reaction mixture was distilled back overnight, followed by extraction with an aqueous solution of 10% hydrochloric acid and 5% sodium bicarbonate. The organic layer was dried over sodium sulfate and evaporated to give the amide compound (o).

Method II

Indenyl acetic acid (k) can be subjected to a dehydration condensation reaction with benzylamine catalyzed by a carbodiimide such as N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide. The reaction was continued at room temperature for two days using DMA as a solvent. The reaction mixture was then added dropwise to stirred ice water. The yellow precipitate is filtered, washed with water, vacuum dried and recrystallized to obtain pure product (o).

Compounds a' (scheme III), o (scheme I), t (scheme II) and y (scheme IIB) can be prepared by condensation reactions in scheme III.

Substituent(s):

x ═ halogen, typically Cl or Br.

E ═ methyl, ethyl, benzyl, or lower acyl.

R₁、R₂、R₆、R₅And R₇The same as described in formula I.

Y, n and m are the same as described in formula I.

Reagents and reaction conditions in scheme I (numbers represent reaction scheme)

(1) Zinc particles, anhydrous inert solvents like benzene, diethyl ether.

(2) Potassium hydrogen sulfate or p-toluenesulfonic acid.

(3) Sodium acetate, absolute ethyl alcohol, room temperature.

(4) Hydrogen, palladium-on-charcoal, 40 atmospheres, and room temperature.

(5) Sodium hydroxide, ethanol water solution, 20-100 degrees.

(6) Sodium acetate or other strong bases like sodium hydride or n-butyl potassium oxide.

(7) And (4) acid.

(8) Friedel-Crafts reaction, catalyzed by Lewis acid, is referred to in the second volume of organic reactions, page 130.

(9) Polyphosphoric acid, and heating.

(10) Reformatsky reaction: zinc, inert solvent and heating.

(11) P-toluenesulfonic acid and calcium chloride or iodine gas at 200 DEG C

(12) Wittig reaction, (C)₆H₅)₃P ═ C-COOE, temperature 20 to 80 ℃, ether or benzene.

(13)(a)NBS/CCl₄Benzoyl peroxide

(b)PtO₂/H₂(1 atm)/acetic acid

(14) (a) sodium hydroxide

(b) Hydrochloric acid

(15) Oxalyl chloride or thionyl chloride in a solvent of dichloromethane or tetrahydrofuran.

(16) The method I comprises the following steps: 2 times NH₂-C(R₅R₆)-Rh(R₇)_m

Method II: carbodiimides in tetrahydrofuran

(17)1N sodium methyl oxide, back-distilled in methanol

The known indanones contained in compound (h) in scheme I can be directly used as intermediates to synthesize the final product, thus avoiding equations 1-7. These known indanones include:

5-methoxyindanone

6-methoxyindanone

5-methylindenone

5-methyl-6-methoxyindanone

5-methyl-7-chloro-indanone

4-methoxy-7-chloro-indanone

4-isopropyl-2, 7-bismethylindenone

5,6, 7-trichloroindanone

2-n-butylindanone

5-methyl-thiaindanones

Scheme II is divided into two sub-diagrams: flow chart II a and flow chart II B. Scheme II A for when R₃Equal to hydroxy and R₄Is equal to hydrogen or R₃And R₄Synthesis of Compounds when oxygen bridge is formed, scheme II B for when R₃Equal to the synthesis of the compound with a lower alkylamino group.

Scheme II A

Similar to scheme I, in scheme IIA, the indenyl acetic acid (k) is dissolved in tetrahydrofuran and reacted with oxalyl chloride under reflux conditions to form the acid chloride (p) (see equation 18) product when the solvent evaporates. Then, mixing acyl chloride (p) dissolved in dichloromethane, benzylhydroxylamine hydrochloride (q) and triethylamine at 0 ℃ for 40-60 minutes, heating the reaction mixture to room temperature and continuing to stir for 1 hour, washing an organic layer formed after adding water with 1 equivalent of hydrochloric acid and brine, drying with magnesium sulfate, evaporating to obtain a crude product N-hydroxy-N-benzylacetamide (r), and finally purifying by a crystallization method or a flash chromatography. This reaction is described in Hoffman et al, JOC1992,57the 5700-5707 document is described in detail.

Next, the N-methanesulfonamide(s) was prepared (equation 20). This reaction is also described in Hoffman et al, JOC1992,575700-5707 are described. Specifically, triethylamine is added into a dichloromethane solution of hydroxamic acid which is precooled to zero, the reaction mixture is stirred for 10-12 minutes, and then methanesulfonyl chloride is added dropwise. The reaction was allowed to continue stirring at 0 ℃ for 2 hours, then warmed to room temperature and stirred for an additional 2 hours. The organic layer of the reaction is washed with water, 1 equivalent hydrochloric acid and brine, respectively, and finally dried over magnesium sulfate, and the product(s) is purified by crystallization or flash chromatography.

The preparation of N-benzyl-alpha- (hydroxy) amide (t) in equation 21 is also in Hoffman et al, JOC1992,575700-,604121-4125 is described in detail. Specifically, triethylamine dissolved in acetonitrile is slowly added (6-12 hours are needed) into a mixed solution of acetonitrile of N-methanesulfonamide(s) and water, and the reaction mixture is stirred overnight. After the solvent of the reaction was evaporated, the residue was dissolved in ethyl acetate, washed with water, 1N hydrochloric acid and brine, respectively, and the organic layer was dried over magnesium sulfate, and the solvent was evaporated to obtain a product (t). Purifying by crystallization.

The condensation reactions with certain aldehydes encompassed by equation 22 in scheme II A are described in detail in scheme III. Scheme III is generally used for the synthesis of the final products of schemes I, II a and II B.

Scheme IIA Final equation 23 is the preparation of the N-benzyl-alpha-ketoamide (v), which is the oxidation of the secondary alcohol (u) to a ketone. An oxidation reaction like Pfitzner-Moffatt, which can selectively oxidize alcohol groups without affecting Y groups, can be used. The compounds (u) and (v) may also be derivatized to R in formula I₃、R₄Compounds of different substituents.

Flow chart II B

As described above, scheme II B for R₃Equal to the synthesis of the compound with lower alkylamino. Similar to scheme II, scheme II B wherein indenyl acetic acid (k) is first dissolved in tetrahydrofuran solvent and evaporated under reflux with oxalyl chloride yields the acid chloride product (p) (see reaction 18), which is then subjected to equation 24. The acid chloride (p) dissolved in methylene chloride is mixed with an alkyl hydroxylamine hydrochloride (e.g. HO-NHR, R is a lower alkyl group, preferably isopropyl) and triethylamine at 0 ℃ for 40-60 minutes, the mixture is heated to room temperature, stirring is continued for 1 hour, then the mixture is diluted with water to form an organic layer, which is washed with 1 equivalent of hydrochloric acid and brine, respectively, and the organic layer is dried over magnesium sulfate and evaporated to dryness. The crude N-hydroxy-N-alkylacetamide (w) is crystallized orPurifying by flash chromatography. This reaction is also described in Hoffman et al, JOC1992,575700-.

The preparation of N-methanesulfonamide (x) in equation 25 is also described in Hoffman et al, JOC1992,575700-. Specifically, triethylamine is slowly added into a dichloromethane solution of hydroxamic acid which is precooled to zero, after the reaction mixture is stirred for 10-12 minutes, methanesulfonyl chloride is added dropwise, the reaction is continuously stirred for 2 hours at 0 ℃, then the reaction mixture is heated to room temperature, and the stirring is continuously carried out for 2 hours. The organic layer produced by the reaction was washed with water, 1 equivalent hydrochloric acid and brine, respectively, dried over magnesium sulfate and evaporated to give product (x). And finally purifying by crystallization or flash chromatography.

Scheme II B the preparation of N-benzylindenyl- α -lower alkylamine acetamide (y) is described in Hoffman et al, JOC 1995,604121-25 and J.Am.chem.Soc.1993,1155031-34. The N-methanesulfonamide (x) mixture was slowly added to the pre-cooled 0 ℃ benzylamine dichloromethane solution for 30 minutes, stirring was continued for 1 hour at 0 ℃, and after heating to room temperature, stirring was continued overnight. After evaporation of the solvent, 1 equivalent of sodium hydroxide is added to the residue, which is extracted with dichloromethane, washed with water and dried over magnesium sulfate, and after evaporation of the solvent, the product (y) is purified by crystallization or flash chromatography.

Scheme III

Scheme III includes the condensation reaction of a heterocyclic aldehyde (e.g., Y-CHO) with an indenylamide. The reaction yields the final product of formula I. This condensation method is used for both equation 17 in scheme I and equation 22 in scheme II A. This method is also useful for the synthesis of compounds y of scheme II B for conversion to the final products of formula I.

In scheme III, the amide (a'), N-heterocyclic aldehyde (z) and sodium methanoxide (1M in methanol) are reacted with mixing under nitrogen at 60 ℃ for 24 hours. After cooling, the reaction mixture was poured into ice water. The precipitate is filtered, washed with water, dried in vacuum, crystallized and purified to obtain the compound of formula I in schemes II and II B and the intermediate (u) in scheme IIA.

As mentioned above, various compounds of this invention are prepared starting preferentially with compounds substituted with the nitro group on the phenyl ring of indanones. As this route can produce a variety of alternative products. In this pathway, after reduction of the nitro group to an amino group, the Sandmeyer reaction is used to introduce chlorine, bromine, nitrile or xanthic acid instead of the amino group. The carbonyl amide and the carboxylic acid compound can be obtained by hydrolysis of the nitrile group substituted derivative. Other ester-like carboxyl derivatives may be further prepared. The xanthogen compound is hydrolyzed to obtain sulfhydryl compound, and oxidized to obtain sulfonic acid or alkylated to obtain alkyl sulfenyl and further nitrogenated to obtain alkyl sulfonic acid compound. These reactions may be carried out before or after the introduction of the substituent at position 1.

These reactions are illustrated in greater detail in the following specific examples, which are intended to better illustrate the invention and are not intended to limit the scope of the invention. In the following examples, like the substituents R₁、R₂Etc. refer to the corresponding substituents of formula I.

Example one

(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenylamide

(A)Para-fluoro-alpha-methyl cinnamic acid

P-fluorobenzaldehyde (200 g, 1.61 mol), propionic anhydride (3.5 g, 2.42 mol) and sodium propionate (155 g, 1.61 mol) were mixed in a nitrogen-filled three-necked flask, slowly heated to 140 ℃ with an oil bath, cooled to 100 ℃ after 20 hours, poured into 8 l of water, and the resulting precipitate was dissolved in 2 l of potassium hydroxide (302 g). Extracting the water solution with diethyl ether, and washing the diethyl ether extract with potassium hydroxide solution. Mixing potassium hydroxide aqueous solutions, filtering, acidifying with concentrated hydrochloric acid to produce precipitate, filtering to obtain p-fluoro-alpha-methyl cinnamic acid solid, washing with water, and drying.

(B)Para-fluoro-alpha-methyl hydrocinnamic acid

Adding 11 g of 5% Pd/C into an ethanol solution of p-fluoro-alpha-methyl cinnamic acid (177.9 g, 0.987 mol dissolved in 3.6L ethanol), introducing hydrogen (40 atm) for hydrogenation reduction at room temperature, filtering out the catalyst when the hydrogen is not consumed any more, and evaporating the solvent under reduced pressure to obtain the product of the p-fluoro-alpha-methyl hydrocinnamic acid. This product was used directly in the next reaction.

(C)6-fluoro-2-methylindenone

P-fluoro- α -methylhydrocinnamic acid (93.2 g, 0.5 mol) was slowly added to 932 g of polyphosphoric acid at 70 ℃ (vapor bath) with slow stirring, gradually warmed to 95 ℃, held at this temperature for 1 hour, cooled and 2 l of water was added. Extracting the aqueous suspension with diethyl ether, washing the diethyl ether extract with a saturated sodium chloride solution, a 5% sodium bicarbonate solution and an aqueous solution, drying, concentrating with 200 g of silica gel, adding the concentrate to a silica gel column (5 lbs) prepared with 5% diethyl ether-petroleum ether, and eluting with 5-10% diethyl ether-petroleum ether to obtain 6-fluoro-2-methylindenone. Thin layer chromatography was used to detect the elution process.

(D)5-fluoro-2-methylindenyl-3-acetic acid

6-fluoro-2-methylindenone (18.4 g, 0.112 mol), cyanoacetic acid (10.5 g, 0.123 mol), acetic acid (6.6 g) and ammonium acetate (1.7 g) in dry toluene (15.5 ml) were mixed, stirred, distilled back for 21 hours, and the water liberated by the reaction was collected with a Dean Stark trap. After evaporation of the toluene, the residue was dissolved in 60 ml of hot ethanol and 14 ml of 2.2 equivalents aqueous potassium hydroxide solution. 22 g of 85% aqueous potassium hydroxide solution (150 ml of water) are added and the mixture is distilled back under nitrogen for 13 hours. After ethanol was removed under reduced pressure, 500 ml of water was added. The aqueous solution was washed by extraction with ether, then boiled with activated carbon, filtered and the solution was acidified to pH2 with 50% glacial hydrochloric acid. The precipitate is filtered and dried to obtain the product 5-fluoro-2-methylindenyl-3-acetic acid (m.p. 164 ℃ -166 ℃).

(E)5-fluoro-2-methylindenyl-3-acetyl chloride

5-fluoro-2-methylindenyl-3-acetic acid (70 mmol) is dissolved in 70 ml of tetrahydrofuran, mixed with oxalyl chloride (70 mmol in 35 ml of dichloromethane, 2 mol equivalents) and distilled back for 24 hours, and the 5-fluoro-2-methylindenyl-3-acetyl chloride obtained after evaporation of the solvent is used directly in the next reaction.

(F)5-fluoro-2-methyl-3- (N-benzyl) -indenyl acetamide

Benzylamine (5 mmol) was slowly added to a solution of 5-fluoro-2-methylindenyl-3-acetyl chloride (2.5 mmol) in methylene chloride (10 ml) at room temperature, followed by reflux overnight. Washed with hydrochloric acid (10%), water, aqueous sodium bicarbonate (5%), respectively, the organic layer was dried over sodium sulfate and evaporated to give the product. Recrystallization from dichloromethane gave a white solid (m.p. 144 ℃).

(G)(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3-N-benzyl) -indenylacetamide

5-fluoro-2-methyl-3- (N-benzyl) - α -indenylacetamide (3.38 mmol), 4-pyridinecarboxyaldehyde (4 mmol) and sodium methoxide (1 mol in 30 ml of methanol) were mixed and heated under nitrogen, stirred for 24 hours, cooled, the reaction mixture was poured into 2000 ml of ice water, and the resulting precipitate was filtered, washed with water and dried in vacuo. Recrystallization from acetonitrile gave a yellow solid (melting point 202 ℃ C.) (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridine).

(H)(E) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

Compound 1G was recrystallized from acetonitrile in the mother liquor predominantly as the E-isomer. The pure E-isomer can be obtained by repeated crystallization in acetonitrile.

Example two

(Z) -5-fluoro-2-methyl- (3-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example-F) with 3-pyridinecarboxyaldehyde by the method of example one, the reaction procedure was the same as in example one G except that 3-pyridinecarboxyaldehyde was used instead of 4-pyridinecarboxyaldehyde, and the purified product (melting point 175 ℃ C.) (R) was recrystallized from acetonitrile₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-pyridyl).

EXAMPLE III

(Z) -5-fluoro-2-methyl- (2-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 2-pyridinecarboxyaldehyde in the same manner as in example G except that 2-pyridinecarboxyaldehyde was used instead of 4-pyridinecarboxyaldehyde, and recrystallizing from ethyl acetate to give the purified product (m.p. 218 ℃ C.) (R.₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-2-pyridyl).

Example four

(Z) -5-fluoro-2-methyl- (4-quinolinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 4-quinolinecarboxaldehyde according to the procedure of example I, which was identical to example G except that 4-quinolinecarboxaldehyde was used instead of 4-pyridinecarboxyaldehyde, and recrystallizing from ethyl acetate to give the purified product (m.p.239 ℃) (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-quinolinyl).

Example five

(Z) -5-fluoro-2-methyl- (4, 6 dimethyl-2-pyridylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 4, 6-dimethyl-2-pyridinecarboxaldehyde using the procedure of example G, and the purified product (R) was obtained by recrystallization₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4, 6-dimethyl-2-pyridyl).

Example six

(Z) -5-fluoro-2-methyl- (3-quinolinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 3-quinolinecarboxaldehyde according to the procedure of example I, and the purified product (R) was obtained by recrystallization in the same manner as in example G₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-quinolinyl).

Example seven

(Z) -5-fluoro-2-methyl- (2-quinolinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 2-quinolinecarboxaldehyde according to the procedure of example I, and the purified product (R) was obtained by recrystallization in the same manner as in example G₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-2-quinolinyl).

EXAMPLE VIII

(Z) -5-fluoro-2-methyl- (pyrazinylidene) -3- (N-benzyl) -indenyl-acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with pyrazinal according to the method of example one, the reaction procedure was the same as in example one G, and recrystallizing to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-pyrazinyl).

Example nine

(Z) -5-fluoro-2-methyl- (3-pyridazinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 3-pyridazinal according to the procedure of example I, the reaction procedure being the same as in example G, and recrystallization to give the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-pyridazinyl).

EXAMPLE ten

(Z) -5-fluoro-2-methyl- (4-pyrimidinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 4-pyrimidinaldehyde by the method of example one, the reaction procedure was the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyrimidinyl).

EXAMPLE eleven

(Z) -5-fluoro-2-methyl- (2-methyl-4-pyrimidinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 2-methyl-4-pyrimidinaldehyde by the method of example one, the reaction procedure was the same as in example one G, and recrystallizing to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-2-methyl-4-pyrimidinyl).

Example twelve

(Z) -5-fluoro-2-methyl- (4-pyridazinylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 4-pyridazinal according to the procedure of example I, the reaction procedure being the same as in example G, and recrystallization to give the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridazinyl).

EXAMPLE thirteen

(Z) -5-fluoro-2-methyl- (1-methyl-3-indolylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 1-methylindole-3-carboxaldehyde using the procedure of example one, the reaction procedure being the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇＝H，n-1, m-1, Y-1-methyl-3-indolyl).

Example fourteen

(Z) -5-fluoro-2-methyl- (1-acetyl-3-indolylidene) -3- (N-benzyl) -indenylacetamide

This compound was obtained by the reaction of 5-fluoro-2-methyl-3- (N-benzyl) -indenylacetamide (see example F) with 1-acetyl-3-indolecarboxaldehyde using the procedure of example one, the reaction procedure being the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-1-acetyl-3-indolyl).

Example fifteen

(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-2-fluorobenzyl) -indenylacetamide

(A)5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenyl acetamide

This compound was obtained by the reaction of 5-fluoro-2-methylindenyl-3-acetyl chloride (see example E) with 2-fluorobenzylamine using the procedure of example one, which was the same as in example one F except that aniline was replaced with 2-fluorobenzylamine.

(B)(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-2-fluorobenzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 4-pyridinecarboxaldehyde by the method of example one, the reaction procedure was the same as in example one G, and recrystallization was conducted to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridyl).

Example sixteen

(Z) -5-fluoro-2-methyl- (3-pyridylidene) -3- (N-2-fluorobenzyl) -indenyl acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 3-pyridinecarboxaldehyde in pentadeca A, the reaction procedure was the same as in example one G, and recrystallization was carried out to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-pyridyl).

Example seventeen

(Z) -5-fluoro-2-methyl- (2-pyridylidene) -3- (N-2-fluorobenzyl) -indenyl acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 2-pyridinecarboxaldehyde in pentadeca A, the reaction procedure was the same as in example one G, and recrystallization was carried out to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-2-pyridyl).

Example eighteen

(Z) -5-fluoro-2-methyl- (4-quinolinylidene) -3- (N-2-fluorobenzyli-ndenyl acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 4-quinolinecarboxaldehyde in pentadeca A, the reaction procedure was the same as in example one G, and recrystallization was carried out to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-quinolinyl).

Example nineteen

(Z) -5-fluoro-2-methyl- (3-pyrazinylidene-ylidene)) -3- (N-2-Fluorobenzyl) -indenyl-acetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 3-pyrazinal in pentadeca A, the reaction procedure was the same as in example one G, and recrystallizing to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-pyrazinyl).

Example twenty

(Z) -5-fluoro-2-methyl- (3-pyridazinylidene) -3- (N-2-fluorobenzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 3-pyridazinal in pentadeca A, the reaction procedure was the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-pyridazinyl).

Example twenty one

(Z) -5-fluoro-2-methyl- (3-pyrimidinylidene) -3- (N-2-fluorobenzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 3-pyrimidinaldehyde in pentadeca A, the reaction procedure was the same as in example one G, and recrystallizing to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-3-pyrimidinyl).

Example twenty two

(Z) -5-fluoro-2-methyl- (4-pyridazinylidene) -3- (N-2-fluorobenzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N-2-fluorobenzyl) -indenylacetamide with 4-pyridazinal in pentadeca A, the reaction procedure was the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridazinyl).

Example twenty three

(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenyl acetyl Amines as pesticides

(A)5-fluoro-2-methyl-3- (N- (S-. alpha. -hydroxymethyl) benzyl) -indenyl acetamide

5-fluoro-2-methylindenyl-3-acetic acid (prepared as in example one D) dissolved in DMA (2 ml) (2.6 mmol) was reacted with N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (4 mmol) and S-2-amino-2-phenylethanol (3.5 mmol) at room temperature for two days, the reaction mixture was added dropwise to stirred ice water (50 ml), the white precipitate was filtered off, washed with water, dried in vacuo and recrystallized from ethyl acetate to give the purified product.

(B)(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylethyl Amides of carboxylic acids

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (S-. alpha. -hydroxymethyl) benzyl) -indenylacetamide of A with 4-pyridinecarboxaldehyde by the method of example one G, the reaction procedure was the same as in example one G, and recrystallization was carried out to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-4-pyridyl).

Example twenty four

(Z)-5-fluoro-2-methyl- (3-pyridylidene) -3- (N- (S-. alpha. -hydroxymethyl) benzyl) -indenyl acetyl Amines as pesticides

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide with 3-pyridinecarboxaldehyde in the same manner as in example one G in the case of tricosane A, followed by recrystallization to give the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-3-pyridyl).

Example twenty-five

(Z) -5-fluoro-2-methyl- (2-pyridylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenyl acetyl Amines as pesticides

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide with 2-pyridinecarboxaldehyde in twenty-three A, the reaction procedure was the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-2-pyridyl).

Example twenty-six

(Z) -5-fluoro-2-methyl- (4-quinolinylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenyl acetyl Amines as pesticides

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (S-. alpha. -hydroxymethyl) benzyl) -indenylacetamide and 2-quinolinecarboxaldehyde in twenty-three A, the reaction procedure was the same as in example one G, and recrystallization was carried out to obtain a purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-4-quinolinyl).

Example twenty-seven

(Z) -5-fluoro-2-methyl- (pyrazinylidene) -3- (N- (S-. alpha. -hydroxymethyl) benzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide with pyrazinecarboxaldehyde in the same procedure as in example one G in twenty-three A, and recrystallizing to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-pyrazinyl).

Example twenty-eight

(Z) -5-fluoro-2-methyl- (3-pyridazinylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenyl-acetoacet-yl Amines as pesticides

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide with 3-pyridazinal in twenty-three A, the reaction procedure was the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-3-pyridazinyl).

Example twenty-nine

(Z) -5-fluoro-2-methyl- (4-pyrimidinylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenyl acetyl Amines as pesticides

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide with 4-pyrimidinaldehyde in twenty-three A, the reaction procedure was the same as in example one G, and recrystallizing to obtain the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇＝H，n＝1，m1, Y-4-pyrimidinyl).

Example thirty

(Z) -5-fluoro-2-methyl- (4-pyridazinylidene) -3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide

This compound was obtained by reacting 5-fluoro-2-methyl-3- (N- (. alpha. -hydroxymethyl) benzyl) -indenylacetamide with 4-pyridazinal in twenty-three A, the reaction procedure was the same as in example one G, and recrystallization gave the purified product (R)₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝CH₂OH，R₆＝H，R₇H, n-1, m-1, Y-4-pyridazinyl).

Example thirty-one

Rac- (Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl- α -hydroxyethane Amides of carboxylic acids

(A)5-fluoro-2-methyl-3- (N) -benzyl-N-hydroxy) -indenyl acetamide

5-fluoro-2-methylindenyl-3-acetyl chloride (10 mmol) (see example E) is dissolved in dichloromethane (75 ml), the cold solution is slowly added (45-60 min.) to a mixture of N-benzylhydroxylamine hydrochloride (12 mmol) and triethylamine (22 mmol) in dichloroethane (100 ml) at 0 ℃ and the mixture is warmed to room temperature and stirred for 1 h. The mixture was diluted with water (100 ml) and the organic layer was washed with hydrochloric acid (twice 100 ml each) and brine (twice 100 ml each), dried over magnesium sulfate and evaporated to dryness. The crude product is purified by flash chromatography to obtain the final product.

(B)5-fluoro-2-methyl-3- (N-benzyl-N-methylsulfonyloxy) -indenyl acetamide

Triethylamine (5 mmol) was added to a 0 ℃ solution of 5-fluoro-2-methyl-3- (N-benzyl-N-hydroxy) -indeneacetamide (5 mmol) in dichloroethane (25 ml), stirred for 10 minutes, methanesulfonyl chloride (5.5 mmol) was added dropwise, the solution was stirred at 0 ℃ for 2 hours, warmed to room temperature and stirred for 2 hours. The organic layer was washed with water (twice, 20 ml each), hydrochloric acid (15 ml) and brine (20 ml) and dried over magnesium sulfate. The product after rotary distillation was purified by flash chromatography to give the final product.

(C)Rac-5-fluoro-2-methyl-3- (N-benzyl) - α -hydroxyindenyl acetamide

Triethylamine (2.1 mmol) in acetonitrile (24 ml) was slowly added (over 6 hours) to a solution of 5-fluoro-2-methyl-3- (N-benzyl-N-methanesulfonyloxy) -indenylacetamide (2 mmol) in acetonitrile/water (12 ml each) and stirred overnight to remove the solvent, the residue was diluted with ethyl acetate (60 ml), washed with water (four times 20 ml each), hydrochloric acid (15 ml) and brine (20 ml), dried over magnesium sulfate and the product was rotary distilled and recrystallized to give the final product.

(D)Rac- (Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl- α -hydroxy Acetamide

This compound was obtained from rac-5-fluoro-2-methyl-3- (N-benzyl) - α -hydroxyindenyl-acetamide by the same procedure as in example one G (R₁＝F，R₂＝CH₃，R₃＝OH，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridyl).

Example thirty two

2- (Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-oxyacetamide

A solution of rac- (Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl- α -hydroxyacetamide (1 mmol) in DMSO (5 ml) was reacted with dicyclohexylcarbodiimide (3 mmol) according to Pfitzner-Moffatt oxidation, stirred overnight and the solvent evaporated to dryness. Purifying the crude product by flash chromatography to obtain the final product (R)₁＝F，R₂＝CH₃，R₃And R₄Combined to form C ═ O, R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridyl).

Example thirty-three

Rac- (Z) -5-fluoro-2-methyl- (4-pyridyl) -3 (N-benzyl) -indenyl-alpha- (2-propylamine) Acetamide

(A)5-fluoro-2-methyl-3- (N-2-propyl-N-hydroxy) -indenyl acetamideFrom 5-fluoro-2-methylindenyl-3-acetyl chloride (cf. example one E) in the same manner as in example thirty-one A except that N-benzylhydroxylamine hydrochloride is replaced by N-2-propylhydroxylamine hydrochloride.

(B)5-fluoro-2-methyl-3- (N-2-propyl-N-methylsulfonyloxy) -indenylacetamideWas prepared in the same manner as in the reaction procedure of example thirty-one B.

(C)Rac-5-fluoro-2-methyl-3- (N-benzyl) -alpha- (2-propylamino) -acetamide

A solution of benzylamine (4.4 mmol) in dichloromethane (15 ml) was slowly added (30 min) to a solution of 5-fluoro-2-methyl-3- (N-2-propyl-N-methylsulfonyloxy) -indenylacetamide (2 mmol) in dichloromethane (25 ml) at 0 ℃ and stirred at 0 ℃ for 1 hour and left overnight at room temperature. The residue after removal of the solvent was treated with 1 equivalent of sodium hydroxide and extracted with dichloromethane (100 ml). The extract was washed with water (twice, 10 ml each) and dried over magnesium sulfate. The product after rotary evaporation was purified by flash chromatography.

(D)Rac-5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-alpha- (2-propylamine Acyl) -acetamidesWas prepared from rac-5-fluoro-2-methyl-3- (N-benzyl) - α - (2-propylamino) -acetyl chloride by the same procedure as in example one G (R)₁＝F，R₂＝CH₃，R₃Is isopropylamino, R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridyl).

Example thirty-four

(Z) -6-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

(A)Ethyl-2-hydroxy-2- (p-methoxyphenol) -1-propanoic acid methyl ester

36.2 g of zinc granules (0.55 mol) are placed in a 500 ml three-necked flask, a 250 ml funnel is charged with 80 ml of anhydrous benzene, 20 ml of anhydrous ether, 80 g of p-methoxybenzaldehyde (0.58 mol) and 98 g (0.55 mol) of ethyl 2-bromopropionate solution, and about 10 ml of the solution is added to the zinc granules with vigorous stirring and slowly warmed to generate an exothermic reaction. The remaining solution was added dropwise to the mixture at such a rate that the reaction was gently refluxed (about 30-35 minutes). After the solution addition was complete, the mixture was placed on a water bath at reflux for 30 minutes. The mixture was cooled to 0 ℃ and 250 ml of a 10% sulfuric acid solution was added with vigorous stirring. The benzene layer was extracted twice with 50 ml of 5% sulfuric acid solution and washed twice with 50 ml of water. The acid solution was combined with the aqueous layer and extracted twice with 50 ml portions of ether. The ether solution was combined with the benzene layer and dried over sodium sulfate. The solvent was evaporated and fractionated using a 6 "Vigreux column to give 89 g of a crude ethyl-2-hydroxy-2- (p-methoxyphenol) -1-propanoic acid methyl ester (60%) boiling point 165-160 ℃ C (1.5 mm).

(B)6-methoxy-2-methyl-2, 3-dihydro-1-indanone

The compound of A is converted to 6-methoxy-2-methyl-2, 3-dihydro-1-indanone according to the method described by Vander Zanden, Rec. Trav. Chim., 68, 413 (1949).

This compound can furthermore be obtained by another process, namely: alpha-methyl-beta- (p-methoxyphenyl) propionic acid (15 g) was added to polyphosphoric acid (170 g) at 50 ℃, and the mixture was heated at 83-90 ℃ for 2 hours, poured into ice water, stirred for one and a half hours, and extracted three times with diethyl ether. The ether solution was washed twice with water and 5 times with 5% sodium bicarbonate until all acidic substances were removed, and then dried over sodium sulfate. The solution was evaporated to dryness to give 9.1 g of a pale yellow oil.

(C)(Z) -6-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

This compound was prepared as described in reaction steps D through G, substituting 6-methoxy-2-methyl-2, 3-dihydro-1-indanone for 6-fluoro-2-methyl-2, 3-dihydro-1-indanone in example D.

Example thirty-five

(Z) -5-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

(A)5-methoxy-2-methyl-3-indenyl acetic acid ethyl ester

13.4 g of 6-methoxy-2-methyl-indanone and 21 g of ethyl bromoacetate are dissolved in 40 ml of benzene and slowly added (5 minutes) to a solution of 110 ml of benzene in 21 g of zinc amalgam (see org. Syn. Coll. Vol.3) with 45 ml of dry ether as solvent. The reaction was started by the addition of a small amount of crystalline iodine and the mixture was kept at reflux temperature (about 65 ℃) by internal thermal energy. After three hours, 10 g of zinc amalgam and 10 g of ethyl bromide were added in two portions and refluxed for 8 hours. 30 ml of ethanol and 150 ml of acetic acid were added. The reaction was poured into 700 ml of 50% aqueous acetic acid, the organic layer was separated, and the aqueous layer was extracted twice with diethyl ether. The combined organic layers were washed with water, ammonium hydroxide and water, dried over sodium sulfate and the solvent was dried at 80 deg.C (bath temperature) (1-2 mm) under vacuum to give crude ethyl (1-hydroxy-2-methyl-6-methoxy-indenyl) acetate (18 g).

The above-mentioned hydroxy ester as a crude product, 20 g of p-toluenesulfonic acid monohydrate and 20 g of anhydrous calcium chloride were added to 250 ml of toluene and refluxed overnight. The filtered solid was washed with toluene. The toluene solutions were combined, washed with water, sodium bicarbonate and water, and then dried over sodium sulfate. The crude product, ethyl 5-methoxy-2-methyl-3-indenyl acetate, after evaporation to dryness, is chromatographed on an acid-washed alumina column, and eluted with petroleum ether-diethyl ether (50-100% by volume) to give a yellow oily product (11.8 g, 70%).

(B)(Z) -5-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl-acetamide

This compound was prepared as described in the reaction procedures of examples one E to G, substituting 5-methoxy-2-methyl-3-indenyl ethyl acetate for 5-fluoro-2-methylindenyl-3-acetic acid in example one E.

Example thirty-six

(Z) -alpha-5-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenylpropionamide

(A)Alpha- (5-methoxy-2-methyl-3-indenyl) propionic acid

Ethyl α - (1-hydroxy-6-methoxy-2-methyl-1-indanyl) bromopropionate was obtained by following the reaction procedure of example thirty-five A and replacing ethyl bromoacetate with the same amount of ethyl α -bromopropionate and was dehydrated in the same manner to ethyl α - (5-methoxy-2-methyl-3-indenyl) bromopropionate.

The above ethyl ester was saponified to give α - (5-methoxy-2-methyl-3-indenyl) propionic acid.

(B)(Z) -alpha-5-methoxy-2-methyl- (4-pyridyl) -3- (N-benzyl) -alpha-methylindenylpropan Amides of carboxylic acids

This compound was prepared as described in the reaction procedures of examples one E to G, replacing 5-fluoro-2-methylindenyl-3-acetic acid in example one E with α -5-methoxy-2-methyl- (3-indenyl) propionic acid.

Example thirty-seven

(Z) alpha-fluoro-5-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) indenyl acetamide

(A)5-methoxy-2-methyl-3-indenyl- α -fluoroacetic acid methyl ester

Potassium fluoride (0.1 mol) and methyl 5-methoxy-2-methyl-3-indenyl-. alpha. -toluenesulfonyloxyacetate (0.05 mol) were dissolved in 200 ml of a dimethylformamide solution, heated under reflux under nitrogen for 2 to 4 hours, cooled, poured into ice water, and extracted with diethyl ether. The ether solution was washed with water and sodium hydrogencarbonate, and then dried over sodium sulfate. Evaporating the solvent, separating by acid-washed alumina column chromatography, and eluting with diethyl ether-petroleum ether (volume ratio is 20-50%) to obtain the product, namely, methyl 5-methoxy-2-methyl-3-indenyl-alpha-fluoroacetate.

(B)(Z) -alpha-fluoro-5-methoxy-2-methyl- (4-pyridylidene) -3- (N-benzyl) -indenyl acetyl Amines as pesticides

This compound was prepared as described in the reaction procedures of examples one E to G, substituting 5-methoxy-2-methyl-3-indenyl- α -fluoroacetic acid methyl ester for 5-fluoro-2-methylindenyl-3-acetic acid in example one E.

As illustrated by the CH-Y moiety in scheme three, any suitable heterocyclic aldehyde can be used either directly in the base-catalyzed condensation reaction or in the Wittig reaction of another process. The heterocyclic aldehydes that may be used are listed in table one below:

watch 1

^*Available from Aldrich

The aldehydes described above in the preceding reaction schemes can be combined with various suitable amines to give compounds within the scope of the present invention. Examples of suitable amines are listed in table two below:

watch two

Example thirty-eight

(Z) -5-fluoro-2-methyl (4-pyri-dine)Pyridinylidene) -3- (N-benzyl) indenyl-acetamide hydrochloride

(Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) indenylacetamide from example one (1396 g; molecular weight 384.45; 3.63 mol) was dissolved in ethanol (28 l) at 45 ℃ and aqueous hydrochloric acid (12 mol; 363 ml) was added stepwise. The reaction mixture was heated to reflux for 1 hour, cooled to room temperature, and left at-10 ℃ for 3 hours. Washing the filtered solid with diethyl ether twice, each time 1.5L, air drying overnight, vacuum drying at 70 deg.C for three days to obtain the product (Z) -5-fluoro-2-methyl (4-pyridylidene) -3- (N-benzyl) indenyl acetamide hydrochloride with melting point 207-209 deg.C (R₁＝F，R₂＝CH₃，R₃＝H，R₄＝H，R₅＝H，R₆＝H，R₇H, n-1, m-1, Y-4-pyridyl hydrochloride). Yield: 1481 g (97%; 3.51 mol); molecular weight: 420.91 g/mole.

Nuclear magnetic resonance hydrogen spectrum (DMSO deuterizing agent): 2.18(s, 3, ═ C-CH)₃)；3.54(s，2，＝CH₂CO)；4.28(d，2，NCH₂) (ii) a 6.71(m, 1, ar.); 7.17(m, 8, ar.); 8.11(d, 2, ar., AB systems); 8.85(m, 1, NH); 8.95(d, 2, ar., AB systems); infrared spectrum (potassium bromide): 3432 NH; 1635C ═ O; 1598C ═ C.

Determination of biological effects

(A)Determination of growth inhibition

Example A compound was assayed for cell growth inhibitory activity using human colon tumor cell line SW-480 (cell source ATCC, Rockville, Md). The inhibition of growth of this cell by the compounds can be used as an indicator against precancerous lesions and tumors. The cell lines and methods for measuring growth inhibition are well established and can be used to evaluate the anti-tumor effects of NSAIDs. This approach has been used in the screening of new anti-tumor drugs for tumor studies in the united states.

The drug was first dissolved in 100% DMSO and then diluted with RPMI medium to the concentration required for the assay. All drugs were formulated on the day of testing. The cultured cells used were passage 99. The cells were cultured in RPMI medium containing 5% fetal bovine serum, 2mM glutamic acid, 100 units/ml penicillin, 100 units/ml streptomycin and 0.25. mu.g/ml amphotericin. The cells were placed in a 37 ℃ incubator with 95% air and 5% carbon dioxide and passaged by digestion with 0.05% pancreatin and 0.53 millimolar EDTA at approximately full surface density. Cells for the cytostatic assay were seeded in 96-well flat-bottom assay plates.

The inhibition of tumor cell growth was determined by using the Sulforhodamine B (SRB) protein binding method. Tumor cells were seeded in 96-well plates and then cultured for 6 days by adding culture medium containing the assay drug (continuous treatment). On each plate, 6 wells were used for a blank without drug treatment, 6 wells were used for a solvent control (0.1% DMSO), and the remaining wells were used for different drug concentrations, with triplicate wells for each drug concentration. Six days later, the cells were fixed, stained with Sulforhodamine B (a dye that binds to proteins), and after the dye that binds to cellular proteins was solubilized, the concentration was determined using a densitometer capable of reading 96-well plates. The inhibition of cell production by the drug can be determined by the quotient of the density (median) of the wells into which the drug was added divided by the density (median) of the solvent control (six wells). Since the density of staining is proportional to the number of cells or protein content per well. Thus, the percent inhibition value obtained may represent the extent to which the drug inhibits cell growth.

For each experiment, the half-maximal inhibition value measured was used to compare the effectiveness of the drug, which is equal to the drug concentration at which tumor cell growth is inhibited by 50%. The median inhibition values were plotted against the different drug concentrations. Log of drug concentration on X-axis. The half inhibition of SW480 cells for example compound was 0.724 micromolar.

(B)Inhibition of cyclooxygenase

Cyclooxygenase catalyzes the oxidation of arachidonic acid to prostaglandins and thromboxanes. This invention example compound, as well as the positive control compound, sulindac sulfide, were evaluated for inhibition of purified cyclooxygenase form I.

The compounds of this invention were evaluated for inhibition of purified cyclooxygenase. Cyclooxygenase was purified from stud vesicles, as described in detail in the literature by Boopathy, R and Balasubramnian, J, 1988. Measurement of cyclooxygenase activity is described in detail in "anti-inflammatory action of cannabis major component on arachidonic acid-metabolizing enzyme" by Evans, A.T., et al (biochem. Pharmacol., 36: 2035-Buchner 2037, 1987). Briefly, the purified cyclooxygenase and 100 micromolar arachidonic acid were incubated at 37 ℃ for 2 minutes with or without the test compound added, and then stopped by adding trichloroacetic acid. The epoxidase activity was determined by measuring the absorbance at 530 nm.

Watch III

Illustrative Compounds	Cyclooxygenase I% inhibition (100 micromolar)
			(-1000·M)
Sulindac sulfide	86
		1	＜25

(C)Cell processDeath by disorder

Apoptosis assays use a method of determining the morphological characteristics of dead cells, i.e., the morphological characteristics of apoptotic cells (e.g., chromosome condensation, etc.). The preparation of the drug solution and the conditions for cell culture were the same as the SRB method (for measuring cell growth inhibition) described above, except that HT-29 cells were used. Cells (5X 10)⁶Cells/dish) at 12.5cm²In the cell culture dish, programmed cell death was measured under a fluorescence microscope after staining with acridine orange and ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) when the cells were growing in the culture dish. Suspended and adherent cells were collected with pancreatin and pooled and washed three times with PBS. The cells were resuspended in 25. mu.l of culture medium and 1. mu.l of dye mixture containing 100. mu.l/ml acridine orange and 100. mu.g/ml ethidium bromide and shaken gently. 10 microliter of the mixture was placed on a microscope glass slide and 22mm of the mixture was used²The coverslip was covered and viewed under a filter lens with a 40-fold lens.

At least 100 cell groups are randomly selected under a microscope to be a sample, and the cell count of the nuclear chromosome concentration is programmed dead cells. The results of the experiment are shown in Table four.

Watch four

Apoptosis of compounds

Apoptosis can also be measured by the amount of fragmentation of cellular DNA. Briefly, SW480 colon tumor cells were plated in 96-well microplates (MTP for short) in an amount of 10,000 cells/well and 180. mu.l/well. After 24 hours of culture, 20. mu.l of a culture medium containing the drug was added, and the culture was continued for 48 hours.

After drug treatment, the assay samples were prepared as follows: after centrifugation in a 96-well microplate (1000 rpm, 15 minutes), the supernatant liquid was carefully and quickly decanted. The cell pellet was resuspended in 200. mu.l of cell lysate and incubated at room temperature for 45 minutes to allow complete lysis of the cells. After centrifugation (15 min, 1000 rpm) of the cell lysis mixture, 20. mu.l of supernatant (cytoplasmic fraction) was pipetted onto a streptavidin-coated microwell assay plate. In particular, the microplate containing cell debris is shaken, since the nuclei contain high molecular weight bulk DNA, which floats in the supernatant after shaking, thus affecting the experimental results. The samples were measured immediately after treatment. Storage at 4 ℃ or-20 ℃ for example, reduces the signal from the enzyme label.

The sample is then processed according to the procedure for DNA fragmentation determination. Optical density values can be used to plot dose response curves. The quantification of the DNA fragments was determined by the "enzyme assay for cell death" manufactured by Mannheim-Boehringer. The assay is based on the principle of quantifying sandwich-enzyme-immunoassay quantities, and is mainly performed with mouse monoclonal antibodies against DNA and histones. The method can specifically detect cytoplasmic fractions of single and oligo nucleosomes in cell lysates. Briefly, the assay procedure is as follows: samples (cell lysate, serum, cell culture supernatant, etc.) were placed in streptavidin-coated microplates, followed by sequential addition of a mixture of anti-histone-biotin and anti-DNA-POD antibodies and incubation at room temperature for 2 hours. During the incubation period, the anti-histone antibody binds to histone on nucleosomes while the immunocomplex is immobilized by biotin to a streptavidin-coated microplate, and then the anti-DNA-POD antibody binds to DNA on nucleosomes. After washing to remove excess unbound antibody, the amount of nucleosomes can be quantified from the POD bound to the immunocomplex body. POD activity of(2, 2' -azino-bis [ 3-ethylbenzthiazoline-sulfonic acid)]As a substrate, densitometry was used.

Fold stimulation (FS ═ OD max/OD solvent) is an indicator of apoptosis, and stimulation per compoundThe fold is determined by this method. Effective half doses were determined by specific data processing software or by effective concentration range (ECR-minimum effective dose-minimum peak dose) of each compound. Stimulation fold FS and effective half dose EC of the tested compounds₅₀The values are given in Table four.

In addition, dose response of the compounds can be obtained by DNA fragmentation assay. Specific data are listed in table five.

Watch five

The compounds of the invention may be formulated in conventional dosage forms with pharmaceutically acceptable carriers so that patients with pre-cancer may be administered periodically according to the method of the invention. The correct initial dose of the compound of the invention can be determined experimentally, with one criterion being that the initial dose should be sufficient to achieve a percentage of the blood concentration of the drug close to the half-maximal inhibitory value of the compound, the amount of this percentage being determined by the chemopreventive or chemotherapeutic index. The initial dose calculation may also take into account other factors, such as the dosage form of the drug, the route of administration (e.g., oral or intravenous). For example, if the mean circulatory volume of a patient is four liters, the IC based on the compounds of this invention₅₀(half the inhibitory dose), we can calculate an initial dose of about 0.6 mg to 4 g. Such as intravenous injection, the concentration of the drug in the circulatory system can reach IC₅₀And (4) concentration.

The compounds of the invention are in turn inhibitors of cGMP-specific phosphodiesterase. As described in U.S. patent application No. 09/046,739 (application date 3/24, 1998), the compounds can be assayed for their inhibitory activity against this enzyme by phosphodiesterases in tumor cells HT-29 or SW 480. Phosphodiesterase activity can be measured by known methods, e.g. by radioactivity³H-cGMP acts as a substrate for the PDE5 enzyme (Thompson, WJ., Teraski, W.L.E.pstein，PM，Strada，s.J.，Advances in Cyclic Nucleotide Research，10: 69-92, 1997, see reference). Briefly, comprise³A solution of H-cGMP substrate (0.2. mu. mol; 100,000cpm, containing 40 mmol Tris-hydrochloride (pH 8.0), 5 mmol magnesium chloride and 1 mg/ml albumin) and the drug to be tested were mixed in a total volume of 400. mu.l. The mixture was incubated with cGMP-specific phosphodiesterase partially purified from HT-29 cells at 30 ℃ for 10 minutes, and then the reaction was terminated by boiling for 75 seconds. After cooling on ice, 100. mu.l of 0.5 mg/ml snake toxin (O.Hannah toxin, Sigma Co.) was added and incubation continued at 30 ℃ for 10 minutes. The reaction was terminated by adding 1 ml of 100% methanol. The sample was passed through an anion exchange chromatography column (1 ml Dowex, Aldrich) and the column was eluted with 1 ml of 100% methanol, and the total amount of radioactivity eluted from the column was measured by a liquid scintillation counter. The degree of PDE5 inhibition was calculated by comparing the amount of radioactivity in the drug-added reaction to the amount of radioactivity in the control reaction (no drug added reaction).

IC was obtained by the procedure described above, using cGMP-specific phosphodiesterase (HT29) of one of the compounds₅₀The value was 0.68 micromolar.

Changes or modifications in the specific procedures, formulations and applications may be made without departing from the spirit of the invention. These are set forth with particularity in the claims that follow.

Claims (17)

1. A compound represented by the following formula

Wherein, the substituent R₁Independently selected from hydrogen and halogen;

substituent R₂Selected from hydrogen and C_1-8An alkyl group;

R₃selected from hydrogen, halogen, amino, hydroxy, C_1-8Alkyl radicalAmino and C_1-8A bisalkylamino group;

R₄represents hydrogen;

R₅and R₆Each independently selected from hydrogen;

R₇independently selected from hydrogen;

m and n represent any one integer between 0 and 3;

y is selected from the group consisting of quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, and these heterocyclic groups having one or two substituents selected from the group consisting of C_1-8Alkyl radical, C_1-8Alkoxy, -SO₂-C_1-8Alkyl and-SO₂NH₂；

Or a pharmaceutically acceptable salt of the above compound.

2. The compound of claim 1 wherein Y is pyridyl or quinolyl.

3. The compound of claim 1, wherein R₁Selected from halogens.

4. The compound of claim 1, wherein R₂Is C_1-8An alkyl group.

5. The compound of claim 1, wherein R₃Selected from hydrogen, hydroxy and C_1-8An alkylamino group.

6. A compound as claimed in claim 1, wherein Y represents quinolinyl, pyridinyl, pyrimidinyl, pyrazinyl or these heterocyclyl groups with one or two substituents selected from C_1-8Alkyl radical, C_1-8Alkoxy, -SO₂-C_1-8Alkyl and-SO₂NH₂。

7. The compound of claim 6 wherein one or two of the substituents on Y is selected from C_1-8Alkoxy, -SO₂-C_1-8Alkyl and-SO₂NH₂A group of (1).

8. The compound of claim 7 wherein the substituents on Y are one or two C_1-8An alkoxy group.

9. The compound of claim 1 which is (Z) -5-fluoro-2-methyl-1- (4-pyridyl) methano-inden-3-yl-N-benzylacetamide hydrochloride.

10. A pharmaceutical composition comprising an acceptable pharmaceutical carrier and a compound represented by the formula:

wherein, the substituent R₁Independently selected from hydrogen and halogen;

substituent R₂Selected from hydrogen and C_1-8An alkyl group;

R₃selected from hydrogen, halogen, amino, hydroxy, C_1-8Alkylamino radical and C_1-8A bisalkylamino group;

R₄represents hydrogen;

R₅and R₆Each independently selected from hydrogen;

R₇independently selected from hydrogen;

m and n represent any one integer between 0 and 3;

y is selected from the group consisting of quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, and these heterocyclic groups having one or two substituents selected from the group consisting of C_1-8Alkyl radical, C_1-8Alkoxy, -SO₂-C_1-8Alkyl and-SO₂NH₂；

Or a pharmaceutically acceptable salt of the above compound.

11. The pharmaceutical composition of claim 10 wherein Y is pyridyl or quinolyl.

12. The pharmaceutical composition of claim 10, wherein R₁Selected from halogens.

13. The pharmaceutical composition of claim 10, wherein R₂Is C_1-8An alkyl group.

14. The pharmaceutical composition of claim 10, wherein R₃Selected from hydrogen, hydroxy and C_1-8An alkylamino group.

15. A pharmaceutical composition according to claim 10 wherein Y represents quinolinyl, pyridinyl, pyrimidinyl, pyrazinyl or these heterocyclic groups bearing one or two substituents selected from C_1-8Alkyl radical, C_1-8Alkoxy, -SO₂-C_1-8Alkyl and-SO₂NH₂。

16. The pharmaceutical composition of claim 15 wherein the substituents on Y are one or two selected from C_1-8Alkoxy, -SO₂-C_1-8Alkyl and-SO₂NH₂A group of (1).

17. Use of a compound according to any one of claims 1 to 9 for the preparation of a pharmaceutical composition for the treatment of tumors.