Field of Invention

The present invention relates to 4-piperidone derivatives possessing

antineoplastic activities and their use in the treatment of cancer.

Background of the Invention

Various studies have shown that an initial chemical insult sensitizes tumour cells more than normal cells to a subsequent interaction with cellular constituents (Chen and Waxman, Biochem. Pharmacol. 1994, 47, 1079-1087; Tsutsui et al. Int. J. Radiat. Oncol. Biol. Phys. 1986, 72, 1 183-1 186). Therefore the development of compounds containing the 1 ,5-diaryl-3-oxo-1 ,4-pentadienyl group (dienone) as cytotoxic agents was undertaken; such dienones permit sequential alkylation to occur (Das et al., Bioorg. Med. Chem. 2009, 17, 3909-3915, Pati et al., Arch. Pharm. Chem. Life Sci. 2010, 9, 535-541 ; Dimmock et al., J. Med. Chem. 1999, 42, 1358-1366). The expression of thiol macromolecules e.g. glutathione, GSTs, and Trx is found to be elevated in a number of cancers compared to normal tissues (Blair et al., Cancer Res. 2003, 63, 8097-8102; Raffel et al., J. Lab. Clin. Med. 2003, 742, 46-51). Therefore the compounds that target cellular thiols could cause greater deleterious effects to tumour cells, thus sparing the normal cells; this approach has been used to develop novel antineoplastic agents.

Conjugated enones display a greater selective affinity for thiols than hydroxyl and amino groups (Mutus et al., Anal. Biochem.1989, 777, 237-243; Dimmock et al., Eur. J. Med. Chem. 1983, 78, 248-254), which are found in nucleic acids. Thus conjugated enones may not cause the genotoxic problems which occur with a number of anticancer drugs used today (Benvenuto et al., J. Pharm. Sci. 1993, 82, 988-991). A number of 1 ,5- diaryl-3-oxo-1 ,4-pentadienes have demonstrated greater toxicity to neoplasms than non- malignant cells (Das et al., J. Med. Chem. 201 1 , 54, 3445-3459; Das et al., Bioorg. Med. Chem. Lett. 2010, 20, 6464-6468). In addition, the 1 ,5-diaryl-3-oxo-1 ,4-pentadienes are structurally divergent from the anticancer drugs used today and therefore may be useful against tumours resistant to current drug therapy. Specifically the 1 ,5-diaryl-3-oxo-1 ,4- pentadienyl pharmacophore has been mounted on piperidinyl (Das et al., Eur. J. Med. Chem. 2007, 42, 71-80) and cyclohexyl (Das et al., Bioorg. Med. Chem. 2010, 78, 2219- 2224) scaffolds which led to various series of compounds which display high potency to malignant and transformed cells, e.g., the IC ₅₀ values are in the submicromolar or low micromolar (<10μΜ) range.

Nearly 50 per cent of human cancers are either completely resistant to

chemotherapy or respond only transiently, after which they are no longer affected by commonly used anticancer drugs. This phenomenon is referred to as multidrug resistance (MDR) and is inherently expressed by some tumour types while others acquire MDR after exposure to chemotherapy treatment. The majority of cases of drug resistance are caused by P-glycoprotein (P-gp) which is a member of ABC transporters. The sensitivity of tumours towards cytotoxic drugs can be enhanced by modulating P-gp. A number of piperidones possessing the 1 ,5-diaryl-3-oxo-1 ,4-pentadienyl pharmacophore demonstrate potent MDR- reversal properties (Das et al., Bioorg. Med. Chem. Lett. 2008, 18, 3484- 3487; Das et al., 2012, 51 , 193-199). The ability of a therapeutic to dampen or eliminate MDR in a cell is thus an important consideration.

Summary of the Invention

The synthesis of new compounds is disclosed herein, together with experiments demonstrating their activity in cytotoxicity (IC ₅₀ ) assays against cancer cell lines and their preferential toxicity toward cancerous cells (vs. non-malignant cells). The compounds are also useful in reversing MDR (multidrug resistance).

In one aspect, the present invention discloses the antineoplastic properties of a novel class of compounds represented by the formula I and acid addition salts thereof.

X is -(CH=CH)p- where p is 0 or 1 ,

Y is NH, S, SO, S0 ₂ , O, N(alkyl), N(aryl), N ⁽⁺⁾ (alkyl) ₂ , N-(alkyl)-OH, N-(alkyl)-SH,

N-alkyl-T-alkyl, N(alkyl)-T-(alkyl)-OH, -N-(alkyl)-T-(alkyl)-SH, N(alkyl)-T-(alkyl)0(alkyl), N(alkyl)-T-(alkyl)-S-(alkyl), CO(CH ₂ ) _b COOR, or CO(D)COOR; wherein b is an integer from 0 to 4; T is S, SO, S0 ₂ , or O; D is C-|. ₄ -alkenyl or C-|. ₄ -alkyne; and R is a straight or branched C-|. ₄ -alkyl,

R is an amino substituent selected from the group consisting of N(alkyl) ₂ ,

NH(alkyl), +N(alkyl) ₃ and Q, where Q is

(Q)

where n and m represent an integer from 1 to 4, and

Z is CH ₂ , O, S, SO, S0 ₂ , NH, N(alkyl), N(aryl), +N(aryl)(alkyl), or +N(alkyl) ₂ , and

R ² is H, CH ₃ , OCH ₃ , CI, F, I, Br, OH, N0 ₂ , or an amino substituent selected from the group consisting of -CH ₂ N(alkyl) ₂ , -CH ₂ NH(alkyl), -CH ₂ N ⁺ (alkyl) _3, and -CH ₂ Q.

In one aspect, the invention provides the proviso that when R ² is H or -CH ₂ NMe ₂ , R is not -NMe ₂ .

In another aspect, the present invention provides uses of the compounds and pharmaceutical compositions thereof for treating cancer and for reversing multidrug resistance.

Brief Description of the Drawings

FIG. 1 is a schematic representation of the synthesis of some of the compounds of the invention.

FIG. 2 is a schematic representation of the synthesis of intermediates required in the synthesis of compounds of the invention.

Detailed Description of the Invention

In one aspect, the present invention discloses the antineoplastic properties of a novel class of compounds n salts thereof.

wherein

X is -(CH=CH)p- where p is 0 or 1 ,

Y is NH, S, SO, S0 ₂ , O, N(alkyl), N(aryl), N ⁽⁺⁾ (alkyl) ₂ , N-(alkyl)-OH, N-(alkyl)-SH, N-alkyl-T-alkyl, N(alkyl)-T-(alkyl)-OH, -N-(alkyl)-T-(alkyl)-SH, N(alkyl)-T-(alkyl)0(alkyl), N(alkyl)-T-(alkyl)-S-(alkyl), CO(CH ₂ ) _b COOR, or CO(D)COOR; wherein b is an integer from 0 to 4; T is S, SO, S0 ₂ , or O; D is C-|. ₄ -alkenyl or C-|. ₄ -alkyne; and R is a straight or branched C-|. ₄ -alkyl,

R is an amino substituent selected from the group consisting of N(alkyl) ₂ , NH(alkyl), +N(alkyl) ₃ and Q, where Q is (Q). where

n and m represent an integer from 1 to 4, and Z is CH ₂ , O, S, SO, S0 ₂ , NH, N(alkyl), N(aryl), +N(aryl)(alkyl), or +N(alkyl) ₂ , and

R ² is H, CH ₃ , OCH ₃ , CI, F, I, Br, OH, N0 ₂ , or an amino substituent selected from the group consisting of -CH ₂ N(alkyl) ₂ , -CH ₂ NH(alkyl), -CH ₂ N ⁺ (alkyl) ₃ and -CH ₂ Q,

In one aspect there is a proviso that when R ² is H or -CH ₂ NMe ₂ , R is not -NMe ₂ . In one aspect, p is 0.

In one aspect Y is NH.

In one aspect Y is N(alkyl)OMe.

In one aspect, R is an amino substituent having the structure Q.

In one aspect, R is dimethylamino, diethylamino, 1 -pyrrolidinyl, 1-piperidinyl, 4- morpholinyl, or 4-methyl-1 -piperazinyl.

In one aspect, R ² is fluorine or chlorine.

In one aspect, R ² is H, dimethylaminomethyl, diethylaminomethyl, 1 - pyrrolidinylmethyl, 1 -piperidinylmethyl, 4-morpholinylmethyl, or 4-methyl-1 - piperazinylmethyl.

In one aspect, the alkyl group is substituted or unsubstituted, branched or unbranched C1 -C7, preferably C1 -5. Preferred alkyl groups are methyl or ethyl. Possible substituents are known in the art and include halogen, alkoxy, alkyl, phenyl, and/or nitro groups. Halogen may be fluorine, chlorine, bromine, or iodine, and preferably fluorine or chlorine. Preferably, the alkoxy groups are substituted or unsubstituted, branched or unbranched C1 -C7, preferably C1 -5. Preferred alkoxy groups are methoxy and ethoxy. Compounds of the invention include compounds of Formula (I) and acid addition salts thereof. Compounds of the invention include, but are not limited to, the following:

When Y = NH and p= 0 in Formula I

R = diethylamino, R ² = H (1 b)

R = 1 -pyrrolidinyl, R ² =H (1c)

R = 1 -piperidinyl, R ² =H (1d)

R = 4-morpholinyl, R ² =H (1e)

R = 4-methyl-1 -piperazinyl, R ² =H (1f)

R = diethylamino, R ² = diethylaminomethyl (2b)

R = 1 -pyrrolidinyl, R ² = 1 -pyrrolidinylmethyl (2c)

R = 1 -piperidinyl, R ² = 1 -piperidinylmethyl (2d)

R = 4-morpholinyl, R ² = 4-morpholinylmethyl (2e), and R = 4-methyl-1 -piperazinyl, R ² = 4-methyl-1 -piperizinylmethyl (2f).

In one aspect, compounds of the invention may include:

When Y = NH and p= 0 in Formula I

R = dimethylamino, R ² =H (1a), and

R = dimethylamino, R ² =dimethylaminomethyl (2a).

In one aspect, compounds 1a and 2a are excluded.

The compounds of formula (I) include acid addition salts thereof. By "acid addition salts" it is meant any salt which may be formed for the purpose of isolation, purification, and storage, such as the oxalate salt, and pharmaceutically acceptable salts meant for administration of the compound to a host, such the hydrochloride, sulfate, acetate, and citrate.

Other aspects of the invention include:

• A compound selected form the group consisting of:

3,5-bis(3 ^' -Diethylaminomethyl-4 ^' -hydroxybenzylidene)-4-piperidone trihydrochloride (1 b);

3,5-bis(4 ^' -Hydroxy-3 ^' -(pyrrolidin-1 -ylmethylbenzylidene))-4-piperidone trihydrochloride (1 c);

3,5-bis(4 ^' -Hydroxy-3 ^' -(piperidin-1 -ylmethyl)benzylidene)-4-piperidone

trihydrochloride (1 d);

3,5-bis(4 ^' -Hydroxy-3 ^' -(morpholin-4-ylmethyl)benzylidene)-4-piperidone trihydrochloride (1e);

3,5-bis(4 ^' -Hydroxy-3 ^' -(4-methylpiperazin-1ylmethyl)benzylidene)-4-piperidon e pentahydrochloride (1f);

3,5-bis(3 ^' ,5 ^' -bis(Diethylaminomethyl)-4 ^' -hydroxybenzylidene)-4-piperidone pentahydrochloride (2b);

3,5-bis(4 ^' -Hydroxy-3 ^' ,5 ^' -bis(pyrrolidin-1 -ylmethyl)benzylidene)-4-piperidone pentahydrochloride (2c);

3,5-bis(4 ^' -Hydroxy-3 ^' ,5 ^' -bis(piperidin-1 -ylmethyl)benzylidene)-4-piperidone pentahydrochloride (2d);

3,5-bis(4 ^' -Hydroxy-3 ^' ,5 ^' -bis(morpholin-4-ylmethyl)benzylidene)-4-piperidone pentahydrochloride (2e); and

3,5-bis(4 ^' -Hydroxy-3 ^' ,5 ^' -bis(4-methylpiperazin-1 -ylmethyl)benzylidene)-4-piperidone nonahydrochloride (2f).

• A compound selected from the group consisting of: 3,5-bis(3 ^' -Diethylaminomethyl-4 ^' -hydroxybenzylidene)-4-piperidone trihydrochloride (1a); and

3,5-bis(3 ^' ,5 ^' -bis(Dimethylaminomethyl)-4 ^' -hydroxybenzylidene)-4-piperidone pentahydrochloride (2a).

· A compound as described herein, with the proviso that 1a and 2a are excluded. • A method of preparing the compound of formula (I), comprising the steps of reacting a compound of formula (IV) with a compound of formula (III)

(IV)

wherein Y, R ² , R ¹ , have the meanings defined herein.

• A pharmaceutical composition comprising the compound described herein, together with a pharmaceutically acceptable diluent or carrier.

• The pharmaceutical composition, for the treatment of cancer.

• Use of the compound described herein for the manufacture of a medicament for the treatment of cancer.

• The use, wherein the cancer is breast cancer or leukemia.

• The pharmaceutical composition, for reversing multi-drug resistance.

• Use of the compound described herein for the manufacture of a medicament for the reversing multi-drug resistance.

· A method for the treatment of cancer or for reversing MDR, comprising

administering an effective amount of the compound described herein.

• A method for the treatment of cancer, comprising administering an effective amount of the compound described herein.

• A method for reversing MDR, comprising administering an effective amount of the compound described herein.

• A method for the treatment of malaria, comprising administering an effective amount of the compound described herein.

• A method for the treatment of a fungal infection, comprising administering an effective amount of the compound described herein. Preparation

The preparation of a compound of formula (I) can be according to the following r

where Y, R and R ² , has the same meaning as discussed earlier.

A compound of formula (I) can be obtained by reacting compound (III) with (IV) in the presence of dry hydrogen chloride gas in acetic acid or in a protic polar solvent such as ethanol and methanol. Acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with an equivalent amount of the chosen mineral or organic acid in an aqueous solvent or organic solvent medium such as methanol or ethanol. The desired solid acid salt is obtained upon evaporation of the solvents.

In a typical example, the synthesis of series 1 and 2 presented in Figure 1 has been carried out as follows. The syntheses of the intermediate aldehydes (IV) required in the syntheses of series 1 and 2 are indicated in Figure 2. The aryl aldehydes 4, 5 and 6 were used in the syntheses of 1a-f, 2a and 2b-f, respectively. The preparation of 1a-f was accomplished by heating a mixture of equimolar quantities of 4-hydroxybenzaldehyde, paraformaldehyde and the appropriate amine in methanol for 2 hours. The solvent was removed, the residue dissolved in ether and hydrogen chloride passed through the solution to yield the amine hydrochloride that was recrystallized from ethanol. Compound 5 was prepared by heating a mixture of 4-hydroxybenzaldehyde and a 2.5 molar excess of N,N-dimethyl-N-methyleneammonium chloride in acetonitrile for 24 hours. The precipitate was collected and recrystallized from ether-methanol. The diamines 6 were prepared in the same manner as series 4 from 4-hydroxybenzaldehyde except a threefold molar excess of paraformaldehyde and amine were employed. The products were recrystallized from ethanol or ether-ethanol.

The preparation of series 1 may be obtained in the following manner. Dry hydrogen chloride gas was passed into a suspension of 4-piperidone hydrochloride monohydrate and 4 in a 1 :2 molar ratio in acetic acid. After stirring at room temperature overnight, the resultant solid was collected and recrystallized from ethanol. Series 2 was prepared in an identical manner. Biological Activity

The compounds described in this invention may be used to treat cancer and some of these molecules have MDR-revertant properties.

The compounds in series 1-2 were evaluated against human Molt/4 and CEM T- lymphocytes in order to assess whether these molecules are toxic to human transformed cells. They were also screened against murine L1210 cells as a number of anticancer drugs are effective against this cell line. Hence bioactivity in this screen may reveal compounds with clinical potential. Table 1 indicates the result of this evaluation in which the designation IC ₅₀ means the concentrations of the compounds required to inhibit the growth of the cells by 50%. The biodata is compared to the potency of melphalan which is an anticancer drug which alkylates various cellular constituents. The results indicate that a number of compounds in series 1-2 are more potent or are equipotent with the reference drug melphalan. Structure-activity relationships (SAR) reveal that in general the presence of one aminoalkyl group in the aryl ring is preferable to two such substituents.

Evaluation of the selective cytotoxicity of these compounds towards neoplasms rather than nonmalignant cells was carried out. The compounds in series 1-2 were evaluated against human HSC-2, HSC-3 and HSC-4 oral squamous cell carcinomas and human HL-60 promyelocyte leukemia cells. In addition, evaluation of these compounds was also undertaken using human HGF gingival fibroblasts, HPC pulp cells and HPLF periodontal ligament fibroblasts which are nonmalignant cells. These results along with the biodata for melphalan are presented in Table 2, where

aThe CC ₅₀ values are the concentrations of the compounds required to kill 50% of the cells.

bThe letters SI refer to the selectivity index which is obtained by dividing the average CC ₅₀ value of the nonmalignant cells by the CC ₅₀ figure of a specific neoplastic cell line.

cThe letters PSE refer to the potency selectivity expression which is the product of the reciprocal of the average CC ₅₀ value towards the four tumour cell lines and the average SI figures multiplied by 100.

The biodata in Table 2 reveal the CC ₅₀ values of 1a-f, 2a-f and melphalan; the

CC ₅₀ figures being the concentrations of the compounds required to kill 50% of the cells. In series 1 , 58% of the CC ₅₀ values towards HSC-2, HSC-3, HSC-4 and HL-60 cells are submicromolar and the average CC ₅₀ values are in the range of 0.73-1.32 μΜ. By way of contrast, in series 2 only 2d and 2e have CC ₅₀ figures in the low micromolar range towards the neoplastic cell lines revealing that cytotoxic potencies are substantially lowered by the introduction of a second aminomethyl substituent into the aryl rings of the compounds in series 1.

In order to evaluate whether greater toxicity to neoplasms than nonmalignant cells takes place, selectivity index (SI) figures were computed. Since a neoplasm will be surrounded by a number of different types of nonmalignant cells, the SI values are the quotients of the average CC ₅₀ value towards HGF, HPC and HPLF cells and the CC ₅₀ figure towards a specific malignant cell line. The average SI values for 1a-f and 2d,e is 4- 5 and therefore these compounds may be viewed as lead molecules while the other compounds in series 2 do not present unequivocal evidence of such selectivity. A comparison of the CC ₅₀ data for 1a-f and 2a-f with melphalan revealed that 1a-f and 2d,e are significantly more potent towards HSC-2, HSC-3, HSC-4 and HL-60 cells than melphalan. In order to identify compounds with favourable CC ₅₀ and SI values, a potency selectivity expression (PSE) was developed for each compound. This result is the product of the recipricol of the average CC ₅₀ value towards the four tumour cell lines and the average SI figures for these compounds multiplied by 100. The results in Table 2 reveal that all of the compounds in series 1 have higher PSE figures than melphalan and the best result is displayed by 1 b.

A representative compound 1a was evaluated against 59 human tumour cell lines from 9 different neoplastic conditions. The data are summarized in Table 3. The average IC ₅₀ value against the 59 cell lines is 1.74 μΜ which compares favorably with melphalan in which the average IC ₅₀ figure in this assay comprising mainly the same cell lines is 24.5 μΜ. Of particular interest are the IC ₅₀ values of 0.60, 0.56 and 0.68 μΜ towards K-562 leukemic, U251 CNS and MCF7 breast cancer cell lines, respectively.

The compounds in series 1-2 were evaluated for their capacity to reverse multidrug resistance (MDR). The assay employed uses murine L-5178 leukemic cells transfected with the human MDR1 gene. The concentrations of rhodamine 123 in treated and untreated transfected and parental cells were measured and the ratios of the fluorescent intensities are referred to as fluorescence activity ratio (FAR) values. A FAR value of greater than 1 indicates that MDR reversal has occurred. Significant MDR- reversal was noted with the following compounds (FAR values are in parentheses) namely 1d (9.81), 1e (10.8), 1f (50.6), 2d (69.2) and 2e (50.1) which exceed or are similar to the data for an established MDR-revertant verapamil which has a FAR value of 12.9.

Compounds that display antineoplastic as well as multidrug reversal properties may have added advantage compared to those compounds which have only one of these properties. These compounds can be used against drug resistant tumours. The compounds of this invention can be used as either antineoplastics, MDR revertants, or for both effects.

Administration

The compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers. Thus, the active compounds of the invention may be formulated for oral, buccal, intranasal, parenteral (e.g., intravenous, intramuscular or subcutaneous), topical or rectal administration or in a form suitable for administration by inhalation or insufflation.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose,

microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.

The compounds of the invention can also be formulated for sustained delivery according to methods well known to those of ordinary skill in the art. Examples of such formulations can be found in U.S. Pat. Nos. 3,538,214, 4,060,598, 4,173,626, 3,1 19,742, and 3,492,397, which are herein incorporated by reference in their entirety.

The compounds of the invention may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion.

Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in a powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation, the active compounds of the invention are conveniently delivered in the form of a solution, dry powder formulation or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the active compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

The compounds of the invention including pharmaceutically acceptable salts and solvates thereof may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the compound (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Depending on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99% w (percent by weight), more preferably from 0.10 to 70% w, of active ingredient, and, from 1 to 99.95% w, more preferably from 30 to 99.90% w, of a pharmaceutically acceptable adjuvant, diluent or carrier, all percentages by weight being based on the total composition.

Synthesis

The synthetic chemical methods employed in preparing the compounds of series 1 and 2 are portrayed in Figure 1 . Melting points are uncorrected. The H NMR spectra (500 MHz) were obtained using a Bruker AMX 500 FT instrument while elemental analyses were obtained from an Elementer analyzer.

Synthesis of series 1 and 2: The compounds in series 1 and 2 were prepared as follows. Dry hydrogen chloride gas was passed into a suspension of 4-piperidone hydrochloride monohydrate (0.003 mol) and 3-aminomethyl-4-hydroxybenzaldehyde hydrochloride (0.006 mol) or 3,5-bis(aminomethyl)-4-hydroxybenzaldehyde dihydrochloride (0.006 mol) in glacial acetic acid (50 ml) and stirred overnight at room temperature. The solid which precipitated was filtered, washed with acetone, dried and recrystallized from ethanol.

3, 5-bis(3 ^' -Dimethylaminomethyl-4 ^' -hydroxybenzylidene)-4-piperidone

trihydrochloride (1a). Yield: 40 %; m.p. 218-222 °C; H NMR (D ₂ 0): δ 7.87 (s, 2H,

2x=CH), 7.40 (m, 4H, Ar-H), 6.95 (m, 2H, Ar-H), 4.49 (s, 4H, 2xNCH ₂ ), 4.23 (s, 4H, Ar- Chb-N), 2.77 (s, 12H, 4xNCH ₃ ); Found C, 54.00; H, 6.79; N, 7.45%. Anal. (C ₂₅ H ₃₄ CI ₃ N ₃ O ₃ 1.5H ₂ 0) requires C, 53.77; H, 6.63; N, 7.52%.

3,5-bis(3 ^' -Diethylaminomethyl-4 ^' -hydroxybenzylidene)-4-piperidone

trihydrochloride (1 b). Yield: 45 %; m.p. 208-215 °C; H NMR (D ₂ 0): δ 7.88 (s, 2H,

2x=CH), 7.39 (s, 4H, Ar), 6.99 (d, 2H, Ar-H), 4.53 (s, 4H, 2xNCH ₂ ), 4.24 (s, 4H, Ar-CH - N), 3.13(m, 8H, 4x NCH9CH3). 1.24-1.22 (m, 12H, 4XNCH9CH3V Found C, 54.63; H, 7.17; N, 6.33%. Anal. (C ₂₉ H ₄₂ Cl ₃ N ₃ 0 ₃ . 3H ₂ 0) requires C, 54.28; H, 7.49; N, 6.55:

3, 5-bis(4 ^' -Hydroxy-3 ^' -(pyrrolidin- 1 -ylmethylbenzylidene))-4-piperidone

trihydrochloride (1c). Yield: 40 %; m.p. 228-230 °C; H NMR (D ₂ 0): δ 7.92 (s, 2H,

2x=CH), 7.42 (s, 4H, Ar-H), 7.02 (d, 2H, Ar-H), 4.56 (s, 4H, 2xNCH ₂ ), 4.32 (s, 4H, Ar- CH9-N). 3.45-3.13 (m, 8H, 4x NCH9). 2.02 (m, 8H, 4*CH ; Found C, 54.72; H, 6.82; N, 6.52%. Anal. (C ₂₉ H ₃₈ Cl ₃ N ₃ 0 ₃ .3H ₂ 0) requires C, 54.63; H, 6.90; N, 6.59 %.

3,5-bis(4 ^' -Hydroxy-3 ^' -(piperidin-1-ylmethyl)benzylidene)-4-piperidone

trihydrochloride (1d). Yield: 53 %; m.p. 242-245 °C; H NMR (D ₂ 0): δ 7.90 (s, 2H,

2x=CH), 7.40 (s, 4H, Ar-H), 7.01 (d, 2H, Ar-H), 4.54 (s, 4H, 2xNCH ₂ ), 4.22 (s, 4H, 2xAr- Chb-N), 3.62 (d, 4H, 2xNCH _e ), 2.91 (t, 4H, 2xNCH _a ), 1.59 (m, 12H, 6xCH ₂ ). Found C, 57.00; H, 7.25; N, 6.32%. Anal. (C ₃₁ H ₄ 9CI ₃ N ₃ O ₃ . 2.5H ₂ 0) requires C, 56.70; H, 7.16; N, 6.40%.

3, 5-bis(4 ^' -Hydroxy-3 ^' -(morpholin-4-ylmethyl)benzylidene)-4-piperidone trihydrochloride (1e). Yield: 50 %; m.p. 224-226 °C; H NMR (D ₂ 0): δ 7.87 (s, 2H, 2x=CH), 7.40 (m, 4H, Ar-H), 7.00 (m, 2H, Ar-H), 4.55 (s, 4H, 2xNCH ₂ ), 4.31 (s, 4H, 2xAr- CH9-N). 3.99 (4H, m, 4xOCH _e ),3.72 (4H, m, 4xOCH _a ), 3.37 (4H, m, 4xNCH _e ), 3.21 (4H, m, 4xNCH _a ); Found C, 54.27; H, 6.36; N, 6.42. Anal. (C ₂₉ H ₃₈ Cl ₃ N ₃ 0 ₅ 1.5H ₂ 0) requires C, 54.20; H, 6.38; N, 6.54%.

3,5-bis(4 ^' -Hydroxy-3 ^' -(4-methylpiperazin-1ylmethyl)benzylidene)-4-piperidon e pentahydrochloride (1f). Yield: 55 %; m.p. 210-215 °C (dec); H NMR (D ₂ 0): δ 7.89 (s, 2H, 2x=CH), 7.44 (m, 4H, Ar-H), 7.03 (d, 2H, Ar-H), 4.57 (s, 4H, 2xNCH ₂ ), 4.40 (s, 4H, 2xAr-CH ₂ -N), 3.57-3.52 (m, 16H, 4xNCH ₂ CH ₂ N), 2.93 (s, 6H, 2xN-CH ₃ ). Found C, 46.42; H, 6.98; N, 8.89 %. Anal. C ₃ iH ₄₆ CI ₅ N ₅ 0 ₃ 5H ₂ 0 requires C, 46.27; H, 6.97; N, 8.71 %. 3,5-bis(3 ^' ,5 ^' -bis(Dimethylaminomethyl)-4 ^' -hydroxybenzylidene)-4-piperidone pentahydrochloride (2a). Yield: 65 %; m.p. 205-209 °C, H NMR (D ₂ 0): δ 7.90 (s, 2H, 2x=CH), 7.50 (s, 4H, Ar-H), 4.55 (s, 4H, 2xNCH ₂ ), 4.32 (s, 8H, 4xAr-CH ₉ -N). 2.78 (s, 24H, 8XNCH ₃ ); Found C, 49.89; H, 7.39; N, 9.32 %. Anal. (C ₃ iH ₅ oCI ₅ N ₅ 0 ₃ 1.5H ₂ 0) requires C, 49.93; H, 7.1 1 ; N, 9.39%.

3,5-bis(3 ^' ,5 ^' -bis(Diethylaminomethyl)-4 ^' -hydroxybenzylidene)-4-piperidone pentahydrochloride (2b). Yield: 55 %; m.p.88-90 °C (dec); H NMR (D ₂ 0): δ 7.92 (s, 2H, 2x=CH), 7.52 (s, 4H, Ar-H), 4.57 (s, 4H, 2xNCH ₂ ), 4.35 (s, 8H, 4xAr-CH ₉ -N). 3.15 (m, 16H, 8XNCH2CH ₃ ), 1.23 (t, 24H, 8xNCH ₉ CH ₃ ); Found C, 51.81 ; H, 8.47; N, 7.75%. Anal. (CsgHseClsNsOs 4H ₂ 0) requires C, 51.87; H, 8.20; N, 7.76%.

3, 5-bis(4 ^' -Hydroxy-3 ^' , 5 ^' -bis(pyrrolidin-1 -ylmethyl)benzylidene) -4-piperidone pentahydrochloride (2c). Yield: 50 %; m.p.172-175 °C (dec); H NMR (D ₂ 0): δ 7.92 (s, 2H, 2x=CH), 7.53 (s, 4H, Ar-H), 4.58 (s, 4H, 2xNCH ₂ ), 4.40 (s, 8H, 4xAr-CH ₉ -N). 3.46 (m, 8H, 8xNCH), 3.14 (m, 8H, 8xNCH), 2.08 (m, 8H, 8xCH), 1.91 (m, br, 8xCH); Calculated for C ₃₉ H ₅₈ CI ₅ N ₅ 0 ₃ .4H ₂ 0: C, 52.34; H, 7.38; N, 7.82: Found C, 52.47; H, 7.57; N, 7.77

3, 5-bis(4 ^' -Hydroxy-3 ^' , 5 ^' -bis(piperidin-1 -ylmethyl)benzylidene) -4-piperidone pentahydrochloride (2d).Yield: 53 %; m.p.223-336 °C (dec); H NMR (D ₂ 0): δ 7.91 (s, 2H, 2x=CH), 7.51 (s, 4H, Ar-H), 4.58 (s, 4H, 2xNCH ₂ ), 4.31 (s, 8H, 4xAr-CH -N). 3.40 (d, 8H, 8xNCH), 2.93 (t, 8H, 8xNCH), 1.83 (m, 8H, 8xCH), 1.70 (dd, 4H, 4xCH _e ), 1.62(q, 8H, 8xCH), 1.40 (m, 4H, 4xCH _a ); Found C, 53.75; H, 8.01 ; N, 7.19%. Anal, requires

(C ₄₃ H ₆₆ CI ₅ N ₅ 0 ₃ .5H ₂ 0) C, 53.29; H, 7.85; N, 7.22%.

3, 5-bis(4 ^' -Hydroxy-3 ^' , 5 ^' -bis(morpholin-4-ylmethyl)benzylidene) -4-piperidone pentahydrochloride (2e). Yield: 70 %; m.p. 228-230 °C (dec); H NMR (D ₂ 0): δ 7.91 (s, 2H, 2x=CH), 7.54 (s, 4H, Ar-H), 4.58 (s, 4H, 2xNCH ₉ ^' ), 4.39 (s, 8H, 4xAr-CH -N). 3.85 (m, 16H, 8xQCH ₉ ). 3.32 (m, 16H, 8xNCH ₉ ); Found C, 48.79; H, 7.16; N, 7.29%. Anal.

( C ₃₉ H ₅₈ CI ₅ N ₅ 0 ₇ 4H ₂ 0) requires C, 48.84; H, 6.88; N, 7.30%.

3, 5-bis(4 ^' -Hydroxy-3 ^' , 5 ^' -bis(4-methylpiperazin- 1 -ylmethyl)benzylidene) -4- piperidone nonahydrochloride (2f) . Yield: 60 %; m.p. 252-255 °C (dec); H NMR (D ₂ 0): δ 7.89 (s, 2H, 2x=CH), 7.45 (m, 4H, Ar-H), 4.59 (s, 4H, 2xNCHV). 4.27 (m, 8H, 4xAr-CH - N), 3.23(br m, 32H, 8xNCH ₉ CH N). 2.90 (s, 12H, 4xNCH ₃ ); Found C, 43.90; H, 7.30; N, 10.76%. Anal. 5H ₂ 0) requires C, 43.95; H, 7.15; N, 10.73%.

Synthesis of 4-hydroxy-3-(dimethylaminomethyl)benzaldehyde hydrochloride. A solution of 4-hydroxybenzaldehyde (0.05 mol) in methanol (50 ml) was added to a preheated mixture of paraformaldehyde (0.05mol) and dimethylamine (0.05 mol) in methanol (50 ml) at 70 °C for one hour. After addition of 4-hydroxybenzaldehyde the reaction mixture was heated at 70 °C for 2 hours. The solvent was removed under vacuo at 45-50 °C. The residue obtained was dissolved in ether and dry hydrogen chloride gas and was passed through the solution to form the hydrochloride salts. The precipitate obtained was filtered, dried and crystallized from ethanol to obtain the desired compound. Yield: 55 %; m.p. 209-21 1 °C; H NMR (D ₂ 0): δ 9.67 (s, 1 H, CHO), 7.88-7.85 (m, 2H, Ar- H), 7.06-7.04 (m, 1 H, Ar-H), 4.29 (s, 2H, Ar-Chb-N), 2.79 (s, 6H, 2xNCH ₃ ). Other 4- hydroxy-3-(aminomethyl)benzaldehyde hydrochlorides were prepared following the above general procudeure.

Synthesis of 3,5-bis(dimethylaminomethyl)-4-hydroxybenzaldehyde

dihydrochloride; A mixture of 4-hydroxybenzaldehyde (0.01 mol) and N,N-dimethyl-N- methylene- ammonium chloride (0.025 mol) was refluxed in dry acetonitrile (40 ml) for 24 hrs. The reaction mixture was cooled and the colorless solid precipitated was collected, washed with acetonitrile (2x5 ml) and recrystallized from ether-methanol to give 3,5- bis(dimethylaminomethyl)-4-hydroxybenzaldehyde dihydrochloride. Yield: 60 %; m.p.231- 232 °C; H NMR (D ₂ 0): δ 9.32 (s, 1 H, CHO), 7.66 (s, 2H, Ar-H), 3.97 (s, 4H, 2x Ar-CH ₂ - N), 2.61 (s, 12, 4xNCH ₃ ).

Other 3,5-bis(aminomethyl)-4-hydroxybenzaldehyde dihydrochloride were prepared following the general procedure as described for 4-hydroxy-3- (dimethylaminomethyl)benzaldehyde hydrochloride using a mixture of 4- hydroxybenzaldehyde (0.05 mol), amine (0.15 mol) and paraformaldeyde (0.15 mol) in methanol. The products were crystallized from a suitable solvent such as ethanol or ethanol-ether.

Biological Activity

Cytotoxicity assay: The compounds in series 1 , 2 and melphalan were evaluated against Molt4/C8, CEM and L1210 cells by a literature procedure (Baraldi et al., J. Med. Chem., 2004, 47, 2877-2886). In these assays, different concentrations of the compounds were incubated with the neoplastic cells at 37 °C for 48 hrs to determine the IC ₅₀ values. These data are presented in Table 1.

Cytotoxic evaluations towards malignant cells HSC-2,HSC-3, HSC-4, HL-60 and non-malignant cells HGF, HPC, HPLF were carried out using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay as per the literature method except for HL- 60 assay (Kawase et al., In Vivo 2005, 19, 705-712; Takeuchi et al., Anticancer Res. 2005, 25, 4037-4042). In the case of HL-60 assay, trypan blue exclusion method was used to determine cytotoxicity. For all the cell lines, varying concentrations of the compounds were incubated for 48 hrs. Representative piperidone derivatives were examined towards a panel of 59 human tumour cell lines using a literature method (Boyd and Paull, Drug. Dev. Res., 1995, 34, 91-109). In addition melphalan and 5-fluorouracil were evaluated towards 59 cell lines. In this assay, compounds were incubated for 48 hrs using five different concentrations to determine growth inhibitory effects on neoplastic cells. This data are presented in Table 3.

MDR-reversal assay: The ability of the compounds to reversal P-gp mediated multidrug resistance was examined according to the procedure described in the literature against murine lymphoma L-5178 cells transfected with human mdr1 genes using Rhodamine 123 fluorescent dye (Kawase et al., In Vivo 2005, 19, 705-712). The fluorescence of the cells was measured in treated MDR cells (F1), untreated MDR cells (F2), treated parental cells (F3) and the FAR values were obtained from the equation, viz (F1/F2)/(F3/F4).

All references mentioned herein are incorporated by reference.

Table 1. Evaluation of 1a-f, 2a-f and melphalan against human Molt 4/C8 and CEM T- lymphocytes and murine L1210 cells

IC ₅₀ (μΜ)

Compound Molt 4/C8 CEM L1210

1a 0.428±0.006 0.640±0.097 1.28±0.18

1 b 0.402±0.081 0.530±0.1 18 1.19±0.45

1c 0.334±0.049 0.384±0.142 0.774±0.281

1d 0.400±0.037 0.390±0.137 0.867±0.245

1e 1.60±0.18 1.67±0.35 7.78±1.74

1f 0.883±0.236 1.14±0.24 2.00±0.54

2a >500 >500 > 500

2b 158±37 141 ±41 223±42

2c 485±28 419±1 14 400±141

2d 6.78±0.12 5.05±1.92 7.94±0.25

2e 1.60±0.11 1.62±0.30 3.71 ±0.90

2f 85.4±53.2 121 ±67 330±1 16 melphalan 3.24±0.56 2.47±0.21 2.13±0.02 Table 2. Evaluation of series 1 and 2 and melphalan against human neoplastic and nonmalignant cell lines

Cmpd Human tumour cell lines, CC50 (μΜ) ³

HSC-2 SI" HSC-3 SI" HSC-4 SI" HL-60 SI" Ave Ave SI

CC50

1 a 0.57±0.017 6.84 1 .2±0.16 3.25 1.1 ±0.025 3.55 0.75±0.093 5.20 0.91 4.71

1 b 0.40±0.017 8.68 0.84±0.039 4.13 0.94±0.063 3.69 0.84±0.001 4.13 0.76 5.16

1 c 0.38±0.006 7.45 0.77±0.006 3.68 0.88±0.013 3.22 0.87±0.020 3.25 0.73 4.40

1 d 0.66±0.058 7.32 1 .4±0.089 3.45 1.6±0.040 3.02 1 .29±0.13 3.74 1.24 4.38

1 e 0.60±0.10 6.05 1 .0±0.15 3.63 1.1 ±0.093 3.30 0.71 ±0.070 5.1 1 0.85 4.52

1f 0.69±0.077 8.65 1 .7±0.060 3.51 1.8±0.10 3.32 1 .1 ±0.010 5.43 1.32 5.23

2a >200 — >200 — >200 — >200 — >200 —

2b 50±1 .5 >3.88 98±1.7 >1 .98 129±2.5 >1 .50 >200 -0.97 >1 19 -2.08

2c >200 -1 .00 >200 -1 .00 >200 -1 .00 >200 -1 .00 >200 -1 .00

2d 2.1 ±0.29 6.81 3.4±0.02 4.21 2.5±0.29 5.72 6.3±2.1 2.27 3.58 4.75

2e 1.5±0.39 7.27 3.2±0.34 4.47 2.8±0.53 5.1 1 4.0±1 .9 3.58 2.88 5.11

2f 66±5.9 >3.03 154±25 1.30 >200 -1 .00 >200 -1 .00 >155 -1 .58

Melph 8.7±4.2 24.1 25±7.7 8.40 32±8.8 6.56 1 .4±1 .2 150 16.8 47.3 alan

Compd Human nonmalignant cells, CC ₅₀ (μΜ) ³

HGF HPC HPLF Ave CC ₅₀ PSE ^C

1a 4.0±0.03 3.0±0.08 4.7±0.065 3.90 518

1 b 3.2±0.44 3.5±0.045 3.7±0.12 3.47 679

1c 2.9±0.12 2.4±0.67 3.2±0.43 2.83 603

1d 5.3±3.2 3.7±0.015 5.5±1.4 4.83 353

1e 3.7±1.7 3.4±0.09 3.8±0.66 3.63 532

1f 7.5±0.72 4.8±1.0 5.6±1.4 5.97 396

2a >200 >200 >200 >200 —

2b >200 183±6.5 >200 >194 -1.75

2c >200 >200 >200 >200 -0.50

2d 17+1.1 8.8±0.097 17±0.10 14.3 133

2e 15±1.5 6.7±0.58 1 1 ±2.1 10.9 177

2f >200 >200 >200 >200 -1.02

Melphalan 161 ±27 269±153 199±60 210 282 Table 3. Evaluation of 1a against a panel of 59 different tumour cell li

Subpanel Number of cell Average IC ₅₀

lines (μΜ)

Leukemia 5 1 .17

Non-small cell 9 2.19

Lung cancer

Colon 7 1 .26

CNS cancer 6 1 .66

Melanoma 9 2.04

Ovarian Cancer 7 2.34

Renal Cancer 8 1 .70

Prostate Cancer 2 1 .45

Breast Cancer 6 1 .38

Average IC ₅₀ values against 59 cells lines: melphalan (26.9 μΜ) and 5-fluorouracil (29.5 μΜ).

The following cell lines were used:

Leukemia Cells: CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR; Non-

Small Lung Cancer: A-549, EKVX, HOP-62 and 92, NCI-H226, H23, H322M, H460, H522; Colon Cancer: COLO-205, HCC-2998, HCT-1 16, HCT-15,HT-29, KM-12, SW-620; CNS Cancer: SF 268,295, 539 and 539, SNB-19 and75, U-251 ; Melanoma: LOX IMVI, MALME-3M, M 14, MDA-MB-435, SK-MEL-2, 28 and 28, UACC-257 and 62; Ovarian Cancer: IGROV1 , OVCAR-3,4,5 and 8, NCI/ADR-RES, SK-OV-3; Renal Cancer: 786-0, A498,ACHN,CAKI-1 ,RXF 393,SN12C,TK-10,UO-31 ; Prostate Cancer: PC-3, DU-145; Breast Cancer: MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T47D, MDA-MB-468